Bacterial Genetics in Meningitis: Associating Meningococcal and Pneumococcal Genes with Clinical Outcome

UvA-DARE (Digital Academic Repository)

Bacterial genetics in meningitis: Associating meningococcal and pneumococcal genes with clinical outcome

Piet, J.R.

Publication date 2016 Document Version Final published version

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Citation for published version (APA): Piet, J. R. (2016). Bacterial genetics in meningitis: Associating meningococcal and pneumococcal genes with clinical outcome.

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Bacterial genetics in meningitis

Associating meningococcal and pneumococcal genes with clinical outcome

Jurgen R. Piet

40049 Piet, Jurgen.indd 1 26-04-16 15:08

Bacterial genetics in meningitis

Associating meningococcal and pneumococcal genes with clinical outcome

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties

Colofon ingestelde commissie,

ISBN 978-‐94-‐6332-‐027-‐6 in het openbaar te verdedigen in de Aula der Universiteit Cover Jurgen R. Piet & Ferdinand van -‐ Nispen Citroenvlinder DTP & Vormgeving Lay-‐out Jurgen R. Piet op woensdag 22 juni 2016, te 11:00 uur Printed by GVO drukkers & vormgevers B.V.

The research described in this thesis was financially supported by an Academic Medical Center PhD Scholarship awarded to the author by the AMC Graduate School.

Printing of this thesis was financially supported by the department of neurology of the Academic Medical Center, University of Amsterdam, de Nederlandse Meningitis Stichting, Chipsoft, , Pfizer Roche and TAKE2Development. door

© 2016 Jurgen R. Piet Jurgen Rogier Piet All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means without the permission of the author. geboren te Hoorn

40049 Piet, Jurgen.indd 2 26-04-16 15:08

Bacterial genetics in meningitis

Associating meningococcal and pneumococcal genes with clinical outcome

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het College voor Promoties ingestelde commissie,

in het openbaar te verdedigen in de Aula der Universiteit Colofon

op woensdag 22 juni 2016, te 11:00 uur ISBN 978-‐94-‐6332-‐027-‐6 Cover Jurgen R. Piet & Ferdinand van -‐ Nispen Citroenvlinder DTP & Vormgeving

Lay-‐out Jurgen R. Piet Printed by GVO drukkers & vormgevers B.V.

The research described in this thesis was financially supported by an Academic Medical Center PhD Scholarship awarded to the author by the AMC Graduate School.

© 2016 Jurgen R. Piet Jurgen Rogier Piet All rights reserved. No part of this publication may be reproduced or transmitted in any form or by means without the permission of the author. geboren te Hoorn

40049 Piet, Jurgen.indd 3 02-05-16 17:38 Promotiecommissie:

Promotor: Prof. dr. D. van de Beek Universiteit van Amsterdam

Copromotor: Dr. A. van der Ende Universiteit van Amsterdam

Overige leden: Prof. dr. S.D. Bentley Wellcome Trust Sanger Institute

Prof. dr. M.D. de Jong Universiteit van Amsterdam

Prof. dr. P. Portegies Universiteit van Amsterdam

Prof. dr. I.N. van Schaik Universiteit van Amsterdam

Prof. dr. J.W. Veening Rijksuniversiteit Groningen

Prof. dr. A.H. Zwinderman Universiteit van Amsterdam

Faculteit der Geneeskunde

40049 Piet, Jurgen.indd 4 26-04-16 15:08 Promotiecommissie:

Promotor: Prof. dr. D. van de Beek Universiteit van Amsterdam

Copromotor: Dr. A. van der Ende Universiteit van Amsterdam

Overige leden: Prof. dr. S.D. Bentley Wellcome Trust Sanger Institute

Prof. dr. M.D. de Jong Universiteit van Amsterdam

Prof. dr. P. Portegies Universiteit van Amsterdam

Prof. dr. I.N. van Schaik Universiteit van Amsterdam

Prof. dr. J.W. Veening Rijksuniversiteit Groningen

Prof. dr. A.H. Zwinderman Universiteit van Amsterdam

Faculteit der Geneeskunde

40049 Piet, Jurgen.indd 5 26-04-16 15:08 Contents Nederlandse samenvatting 177 Dankwoord 191 Chapter 1 General introduction 9 List of abbreviations 194 Part I. Neisseria meningitidis Contributing authors and affiliations 198 Chapter 2 Clinical features, outcome, and meningococcal genotype in 258 17 adults with meningococcal meningitis: a prospective cohort study About the author 203 Medicine (Baltimore) 2008;87:185-192 Portfolio 204

Chapter 3 Meningococcal factor H binding protein fHbpD184 polymorphism 39 List of publications 206 influences clinical course of meningococcal meningitis PLoS One 2012;7:e47973

Chapter 4 Meningococcal Two-Partner Secretion systems and outcome in 61 patients with meningitis Submitted

Chapter 5 An analysis of the sequence variability of meningococcal fHbp, 85 NadA and NHBA over a 50-year period in the Netherlands PLoS One 2013;8:e65043

Chapter 6 Genome sequence of Neisseria meningitidis serogroup B strain 113 H44/76 J Bacteriol 2011;193:2371-2372

Chapter 7 Meningitis caused by a lipopolysaccharide deficient Neisseria 121 meningitidis J Infect 2014;69:352-357

Part II. Streptococcus pneumoniae

Chapter 8 Streptococcus pneumoniae arginine synthesis genes promote 135 growth and virulence in pneumococcal meningitis J Infect Dis 2014;209:1781-1791

Chapter 9 Summary and general discussion 161

40049 Piet, Jurgen.indd 6 26-04-16 15:08 Contents Nederlandse samenvatting 177 Dankwoord 191 Chapter 1 General introduction 9 List of abbreviations 194 Part I. Neisseria meningitidis Contributing authors and affiliations 198 Chapter 2 Clinical features, outcome, and meningococcal genotype in 258 17 adults with meningococcal meningitis: a prospective cohort study About the author 203 Medicine (Baltimore) 2008;87:185-192 Portfolio 204

Chapter 3 Meningococcal factor H binding protein fHbpD184 polymorphism 39 List of publications 206 influences clinical course of meningococcal meningitis PLoS One 2012;7:e47973

Chapter 4 Meningococcal Two-Partner Secretion systems and outcome in 61 patients with meningitis Submitted

Chapter 5 An analysis of the sequence variability of meningococcal fHbp, 85 NadA and NHBA over a 50-year period in the Netherlands PLoS One 2013;8:e65043

Chapter 6 Genome sequence of Neisseria meningitidis serogroup B strain 113 H44/76 J Bacteriol 2011;193:2371-2372

Chapter 7 Meningitis caused by a lipopolysaccharide deficient Neisseria 121 meningitidis J Infect 2014;69:352-357

Part II. Streptococcus pneumoniae

Chapter 8 Streptococcus pneumoniae arginine synthesis genes promote 135 growth and virulence in pneumococcal meningitis J Infect Dis 2014;209:1781-1791

Chapter 9 Summary and general discussion 161

40049 Piet, Jurgen.indd 7 26-04-16 15:08

CHAPTER 1:

General introduction

40049 Piet, Jurgen.indd 8 26-04-16 15:08

CHAPTER 1: CHAPTER 1: General introduction

General introduction

40049 Piet, Jurgen.indd 9 27-04-16 10:57 Chapter 1 General introduction

Bacterial meningitis is the disease that occurs when bacteria infect the protective host immune system. It is also used for phenotypical characterization. In meningococci, only membranes that envelop the brain and spinal cord. Bacteria can reach the subarachnoid 6 of 12 serogroups (A, B, C, W, X, Y) cause the vast majority of invasive disease. space between these membranes through the bloodstream or through spread of infection The last decade, some genetic loci have been identified in the meningococcal and from contiguous sites (for example, infection in paranasal sinuses or mastoid). Blood-borne pneumococcal genome, which are possibly associated with virulence, although the clinical pathogen invasion is assumed to be the main route of subarachnoid space entry; it relevance of these loci is often not clear. Investigating the relationship between bacterial represents a multistep process involving mucosal colonization, invasion into, survival and genetics and clinical outcome requires a large collection of well-characterized clinical replication within the bloodstream, and traversal of the blood-cerebrospinal fluid (CSF) isolates. The Netherlands Reference Laboratory for Bacterial Meningitis was established in barrier. The result of the infection and the subsequent inflammatory response of the 1958, collecting meningococcal isolates from patients with meningitis and extended the meninges and brain is a life-threatening disease and a century ago, this disease was nearly surveillance in 1975 to survey all cases of bacterial meningitis. Nowadays, it collects and always fatal. In adults, the most common organisms causing meningitis are Neisseria stores ~90% of all isolates that cause bacterial meningitis in the Netherlands. In this meningitidis (meningococcus) and Streptococcus pneumonia (pneumococcus), accounting for laboratory, strains are typed either by culture-based methods and examination of capsular 85% of cases.1,2 For these bacteria, meningitis is an evolutionary dead end, since they cannot polysaccharides and outer membrane compounds, or by DNA sequencing methods. The be easily transmitted to other hosts once they have invaded the bloodstream. In the normal most commonly employed method for typing meningococci and streptococci, is multilocus population, asymptomatic colonization of the upper respiratory tract occurs in up to 18 for sequence typing (MLST) in which parts of several house-keeping genes are sequenced. meningococci and 40% for pneumococci.3-5 Besides bacterial meningitis, the meningococcus Obtaining the DNA sequence of whole bacterial genomes is a novel approach that allows can cause severe sepsis and septic shock. The case fatality rate in patients with more detailed characterization of bacterial isolates. meningococcal meningitis is 5-10%.6 The pneumococcus causes worldwide a range of invasive infections, including meningitis, with devastating consequences. The case fatality Aim and outline of this thesis rate in patients with pneumococcal meningitis is up to 30%, and long-term sequelae, The aim of the present thesis is to investigate the association of bacterial genetic diversity including hearing loss, focal neurological deficits, and cognitive impairment, develop in with clinical characteristics of patients with bacterial meningitis. In part I of this thesis we about half of survivors.1,7 Bacterial meningitis can be treated with antibiotics, and the discuss bacterial meningitis caused by N. meningitidis. In chapter 2 we describe a nationwide inflammatory response with adjunctive dexamethasone,2,8,9 but because of the rapid cohort study of adults with meningococcal meningitis and evaluate the correlation of the progression of the illness, early diagnosis is essential for successful treatment. meningococcal genotype, acquired by MLST, with patient characteristics. In chapter 3 and 4 In patients with meningococcal meningitis, hypervirulent bacterial clones are more we evaluate the distribution of 2 virulence factors in the meningococcal isolates from the 10 frequently found than in asymptomatic carriers. This suggests increased virulence in these same clinical cohort. In chapter 3 we focus on the role of Meningococcal fH binding protein strains. Virulence factors are bacterial molecules that play a role in the pathogenesis of (fHbp) and clinical outcome. This protein binds human complement downregulator factor H, disease and are encoded in the bacterial DNA. The pneumococcal and meningococcal thus inhibiting complement activation and hampering complement-mediated lysis.11,12 We genome each consists of 2.1 million nucleotides that contain around 2000-2500 genes of an discuss its genetic diversity and its role with clinical characteristics. In chapter 4 the average size of 900 nucleotides. In comparison, the human genome is 1500 times as large, distribution of meningococcal two-partner secretion systems, involved in intracellular but contains only 10 times as much encoding genes. In both meningococci and pneumococci, survival and adhesion to host cells13,14, and their association with clinical outcome is a main determinant of virulence is their polysaccharide capsule, which shields it from the discussed.

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40049 Piet, Jurgen.indd 10 26-04-16 15:08 Chapter 1 General introduction

Bacterial meningitis is the disease that occurs when bacteria infect the protective host immune system. It is also used for phenotypical characterization. In meningococci, only membranes that envelop the brain and spinal cord. Bacteria can reach the subarachnoid 6 of 12 serogroups (A, B, C, W, X, Y) cause the vast majority of invasive disease. 1 space between these membranes through the bloodstream or through spread of infection The last decade, some genetic loci have been identified in the meningococcal and from contiguous sites (for example, infection in paranasal sinuses or mastoid). Blood-borne pneumococcal genome, which are possibly associated with virulence, although the clinical pathogen invasion is assumed to be the main route of subarachnoid space entry; it relevance of these loci is often not clear. Investigating the relationship between bacterial represents a multistep process involving mucosal colonization, invasion into, survival and genetics and clinical outcome requires a large collection of well-characterized clinical replication within the bloodstream, and traversal of the blood-cerebrospinal fluid (CSF) isolates. The Netherlands Reference Laboratory for Bacterial Meningitis was established in barrier. The result of the infection and the subsequent inflammatory response of the 1958, collecting meningococcal isolates from patients with meningitis and extended the meninges and brain is a life-threatening disease and a century ago, this disease was nearly surveillance in 1975 to survey all cases of bacterial meningitis. Nowadays, it collects and always fatal. In adults, the most common organisms causing meningitis are Neisseria stores ~90% of all isolates that cause bacterial meningitis in the Netherlands. In this meningitidis (meningococcus) and Streptococcus pneumonia (pneumococcus), accounting for laboratory, strains are typed either by culture-based methods and examination of capsular 85% of cases.1,2 For these bacteria, meningitis is an evolutionary dead end, since they cannot polysaccharides and outer membrane compounds, or by DNA sequencing methods. The be easily transmitted to other hosts once they have invaded the bloodstream. In the normal most commonly employed method for typing meningococci and streptococci, is multilocus population, asymptomatic colonization of the upper respiratory tract occurs in up to 18 for sequence typing (MLST) in which parts of several house-keeping genes are sequenced. meningococci and 40% for pneumococci.3-5 Besides bacterial meningitis, the meningococcus Obtaining the DNA sequence of whole bacterial genomes is a novel approach that allows can cause severe sepsis and septic shock. The case fatality rate in patients with more detailed characterization of bacterial isolates. meningococcal meningitis is 5-10%.6 The pneumococcus causes worldwide a range of invasive infections, including meningitis, with devastating consequences. The case fatality Aim and outline of this thesis rate in patients with pneumococcal meningitis is up to 30%, and long-term sequelae, The aim of the present thesis is to investigate the association of bacterial genetic diversity including hearing loss, focal neurological deficits, and cognitive impairment, develop in with clinical characteristics of patients with bacterial meningitis. In part I of this thesis we about half of survivors.1,7 Bacterial meningitis can be treated with antibiotics, and the discuss bacterial meningitis caused by N. meningitidis. In chapter 2 we describe a nationwide inflammatory response with adjunctive dexamethasone,2,8,9 but because of the rapid cohort study of adults with meningococcal meningitis and evaluate the correlation of the progression of the illness, early diagnosis is essential for successful treatment. meningococcal genotype, acquired by MLST, with patient characteristics. In chapter 3 and 4 In patients with meningococcal meningitis, hypervirulent bacterial clones are more we evaluate the distribution of 2 virulence factors in the meningococcal isolates from the 10 frequently found than in asymptomatic carriers. This suggests increased virulence in these same clinical cohort. In chapter 3 we focus on the role of Meningococcal fH binding protein strains. Virulence factors are bacterial molecules that play a role in the pathogenesis of (fHbp) and clinical outcome. This protein binds human complement downregulator factor H, disease and are encoded in the bacterial DNA. The pneumococcal and meningococcal thus inhibiting complement activation and hampering complement-mediated lysis.11,12 We genome each consists of 2.1 million nucleotides that contain around 2000-2500 genes of an discuss its genetic diversity and its role with clinical characteristics. In chapter 4 the average size of 900 nucleotides. In comparison, the human genome is 1500 times as large, distribution of meningococcal two-partner secretion systems, involved in intracellular but contains only 10 times as much encoding genes. In both meningococci and pneumococci, survival and adhesion to host cells13,14, and their association with clinical outcome is a main determinant of virulence is their polysaccharide capsule, which shields it from the discussed.

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40049 Piet, Jurgen.indd 11 26-04-16 15:08 Chapter 1 General introduction

Studies of meningococcal evolution and genetic population structure are essential for REFERENCES vaccine development. In chapter 5 we study the genetic variability in a period of 50 years of 1. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. 3 antigens, acquired by reverse vaccinology: factor H-binding protein (fHbp), Neisserial Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Heparin-Binding Antigen (NHBA) and Neisseria adhesion A (NadA). These antigens are Med 2004;351:1849-1859. included in the multicomponent meningococcal serogroup B vaccine 4CMenB. 2. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial meningitis in adults. N Engl J Med 2006;354:44-53. In chapters 4, 6 and 7 we perform next generation whole genome sequencing on 3. Maiden MC, Stuart JM, Group UKMC. Carriage of serogroup C meningococci 1 year meningococcal isolates. The genome of a commonly used laboratory serogroup B strain is after meningococcal C conjugate polysaccharide vaccination. Lancet 2002;359:1829- sequenced de novo and analyzed in chapter 6. The hallmark of meningococcal disease is the 1831. excessive stimulation of the immune system by LPS.15 In chapter 7 we discuss the whole 4. Mook-Kanamori BB, Geldhoff M, van der Poll T, van de Beek D. Pathogenesis and pathophysiology of pneumococcal meningitis. Clin Microbiol Rev 2011;24:557-591. genome sequence of a naturally occurring LPS-deficient meningococcal isolate that caused invasive meningococcal disease and evaluate the genetic background of its LPS-deficiency. 5. Greenfield S, Sheehe PR, Feldman HA. Meningococcal carriage in a population of "normal" families. J Infect Dis 1971;123:67-73.

In part II of this thesis we focus on the genetics of S. pneumoniae. In a clinical phenotype 6. Heckenberg SG, de Gans J, Brouwer MC, et al. Clinical features, outcome, and based approach, combined with bacterial whole-genome sequencing, we identify the meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study. Medicine (Baltimore) 2008;87:185-192. association of pneumococcal genes with outcome in patients with pneumococcal meningitis in chapter 8. The results of the different studies are summarized and discussed in chapter 9. 7. Hoogman M, van de Beek D, Weisfelt M, de Gans J, Schmand B. Cognitive outcome in adults after bacterial meningitis. J Neurol Neurosurg Psychiatry 2007;78:1092- 1096.

8. de Gans J, van de Beek D. Dexamethasone in adults with bacterial meningitis. N Engl J Med 2002;347:1549-1556.

9. van de Beek D, Brouwer MC, Thwaites GE, Tunkel AR. Advances in treatment of bacterial meningitis. Lancet 2012;380:1693-1702.

10. Caugant DA, Tzanakaki G, Kriz P. Lessons from meningococcal carriage studies. FEMS Microbiol Rev 2007;31:52-63.

11. Madico G, Welsch JA, Lewis LA, et al. The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance. J Immunol 2006;177:501-510.

12. Schneider MC, Prosser BE, Caesar JJ, et al. Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates. Nature 2009;458:890-893.

13. Tala A, Progida C, De SM, et al. The HrpB-HrpA two-partner secretion system is essential for intracellular survival of Neisseria meningitidis. Cell Microbiol 2008;10:2461-2482.

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40049 Piet, Jurgen.indd 12 26-04-16 15:08 Chapter 1 General introduction

Studies of meningococcal evolution and genetic population structure are essential for REFERENCES vaccine development. In chapter 5 we study the genetic variability in a period of 50 years of 1. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. 1 3 antigens, acquired by reverse vaccinology: factor H-binding protein (fHbp), Neisserial Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Heparin-Binding Antigen (NHBA) and Neisseria adhesion A (NadA). These antigens are Med 2004;351:1849-1859. included in the multicomponent meningococcal serogroup B vaccine 4CMenB. 2. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial meningitis in adults. N Engl J Med 2006;354:44-53. In chapters 4, 6 and 7 we perform next generation whole genome sequencing on 3. Maiden MC, Stuart JM, Group UKMC. Carriage of serogroup C meningococci 1 year meningococcal isolates. The genome of a commonly used laboratory serogroup B strain is after meningococcal C conjugate polysaccharide vaccination. Lancet 2002;359:1829- sequenced de novo and analyzed in chapter 6. The hallmark of meningococcal disease is the 1831. excessive stimulation of the immune system by LPS.15 In chapter 7 we discuss the whole 4. Mook-Kanamori BB, Geldhoff M, van der Poll T, van de Beek D. Pathogenesis and pathophysiology of pneumococcal meningitis. Clin Microbiol Rev 2011;24:557-591. genome sequence of a naturally occurring LPS-deficient meningococcal isolate that caused invasive meningococcal disease and evaluate the genetic background of its LPS-deficiency. 5. Greenfield S, Sheehe PR, Feldman HA. Meningococcal carriage in a population of "normal" families. J Infect Dis 1971;123:67-73.

8. de Gans J, van de Beek D. Dexamethasone in adults with bacterial meningitis. N Engl J Med 2002;347:1549-1556.

9. van de Beek D, Brouwer MC, Thwaites GE, Tunkel AR. Advances in treatment of bacterial meningitis. Lancet 2012;380:1693-1702.

10. Caugant DA, Tzanakaki G, Kriz P. Lessons from meningococcal carriage studies. FEMS Microbiol Rev 2007;31:52-63.

11. Madico G, Welsch JA, Lewis LA, et al. The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance. J Immunol 2006;177:501-510.

12. Schneider MC, Prosser BE, Caesar JJ, et al. Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates. Nature 2009;458:890-893.

13. Tala A, Progida C, De SM, et al. The HrpB-HrpA two-partner secretion system is essential for intracellular survival of Neisseria meningitidis. Cell Microbiol 2008;10:2461-2482.

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40049 Piet, Jurgen.indd 13 26-04-16 15:08 Chapter 1

14. Schmitt C, Turner D, Boesl M, Abele M, Frosch M, Kurzai O. A functional two-partner secretion system contributes to adhesion of Neisseria meningitidis to epithelial cells. J Bacteriol 2007;189:7968-7976.

15. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 2007;369:2196-2210.

40049 Piet, Jurgen.indd 14 26-04-16 15:08 Chapter 1

15. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 2007;369:2196-2210.

40049 Piet, Jurgen.indd 15 26-04-16 15:08

CHAPTER 2: Clinical features, outcome, and meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study

Sebastiaan G.B. Heckenberg Jan de Gans Matthijs C. Brouwer Martijn Weisfelt Jurgen R. Piet Lodewijk Spanjaard Arie van der Ende Diederik van de Beek

Medicine (Baltimore) 2008;87:185-192

40049 Piet, Jurgen.indd 16 26-04-16 15:08

CHAPTER 2: CHAPTER 2: Clinical features, outcome, and meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study

Clinical features, outcome, Sebastiaan G.B. Heckenbergand meningococcal Jan de Gans Matthijs C. Brouwergenotype in 258 adults with Martijn Weisfelt Jurgen R. Piet meningococcal meningitis: a Lodewijk Spanjaardprospective cohort study Arie van der Ende Diederik van de Beek

Sebastiaan G.B. Heckenberg

Jan de Gans Matthijs C. Brouwer

Martijn Weisfelt Jurgen R. Piet Lodewijk Spanjaard Arie van der Ende Diederik van de Beek

Medicine (Baltimore) 2008;87:185-192 Medicine (Baltimore) 2008;87:185-192

40049 Piet, Jurgen.indd 17 26-04-16 15:08 Chapter 2 Meningococcal meningitis cohort study

ABSTRACT INTRODUCTION

Meningococcal meningitis remains a life-threatening disease. Neisseria meningitidis is the Bacterial meningitis is a life-threatening infectious disease. The estimated incidence of leading cause of meningitis and septicemia in young adults and is a major cause of endemic bacterial meningitis is 2.6–6.0 cases per 100,000 adults per year in developed countries and bacterial meningitis worldwide. The Meningitis Cohort Study was a Dutch nationwide is up to 10 times higher in less developed countries.1 The predominant causative pathogens prospective observational cohort study of adults with community-acquired bacterial in adults are Streptococcus pneumoniae (pneumococcus) and Neisseria meningitidis meningitis, confirmed by culture of cerebrospinal fluid, from October 1998 to April 2002. (meningococcus), which are responsible for 80%–85% of all cases.2 Patients underwent a neurologic examination at discharge, and outcome was graded with The meningococcus is the leading cause of meningitis and septicemia in young adults and is the Glasgow Outcome Scale. Serogrouping, multilocus sequence typing, and susceptibility a major cause of endemic bacterial meningitis worldwide.3 The epidemiology of testing of meningococcal isolates were performed. meningococcal disease is important for vaccination strategies.3,4 Whereas meningococci of The study identified 258 episodes of meningococcal meningitis in 258 patients. The serogroups B, C, and Y were responsible for several recent outbreaks of invasive disease in prevalence of the classical triad of fever, neck stiffness, and change in mental status was low the United States and other developed countries, serogroup A meningococci are the primary (70/258, 27%). When rash was added to the classical triad, 229 of 258 (89%) patients had at cause of endemic disease in developing countries.3 Current meningococcal vaccines are least 2 of 4 signs. Systolic hypotension was associated with rash (22/23 vs. 137/222, based on the capsular polysaccharides of serogroup A, C, W, and Y meningococci.4 p=0.002) and absence of neck stiffness (6/23 vs. 21/220, p=0.05). Neuroimaging before Multilocus sequence typing (MLST) is considered the gold standard for genotyping of lumbar puncture was an important cause of delay of therapy: antibiotics were not initiated meningococci and can be used to study epidemiology.4,5 before computed tomography (CT) scan in 85% of patients who underwent CT scan before We previously described a prospective cohort of 696 adult patients with community- lumbar puncture. Unfavorable outcome occurred in 30 of 258 (12%) patients, including a acquired bacterial meningitis.2 We now provide a detailed description of clinical features, mortality rate of 7%. Neurologic sequelae occurred in 28 of 238 (12%) patients, particularly prognostic factors, outcome, and MLST in the subset of 258 adults with meningococcal hearing loss (8%). Factors associated with sepsis and infection with meningococci of clonal meningitis. complex 11 (cc11) are related with unfavorable outcome.

MATERIALS AND METHODS

The Dutch Meningitis Cohort Study was a prospective nationwide observational cohort study of adults with community-acquired bacterial meningitis in the Netherlands. Inclusion and exclusion criteria have been described extensively elsewhere.2 In summary, eligible patients were older than 16 years, had bacterial meningitis confirmed by culture of cerebrospinal fluid (CSF), and were listed in the database of the Netherlands Reference Laboratory for Bacterial Meningitis from October 1998 to April 2002. This laboratory receives CSF isolates from about 85% of all patients with bacterial meningitis in the Netherlands. The treating

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ABSTRACT INTRODUCTION

Meningococcal meningitis remains a life-threatening disease. Neisseria meningitidis is the Bacterial meningitis is a life-threatening infectious disease. The estimated incidence of leading cause of meningitis and septicemia in young adults and is a major cause of endemic bacterial meningitis is 2.6–6.0 cases per 100,000 adults per year in developed countries and bacterial meningitis worldwide. The Meningitis Cohort Study was a Dutch nationwide is up to 10 times higher in less developed countries.1 The predominant causative pathogens prospective observational cohort study of adults with community-acquired bacterial in adults are Streptococcus pneumoniae (pneumococcus) and Neisseria meningitidis 2 meningitis, confirmed by culture of cerebrospinal fluid, from October 1998 to April 2002. (meningococcus), which are responsible for 80%–85% of all cases.2 Patients underwent a neurologic examination at discharge, and outcome was graded with The meningococcus is the leading cause of meningitis and septicemia in young adults and is the Glasgow Outcome Scale. Serogrouping, multilocus sequence typing, and susceptibility a major cause of endemic bacterial meningitis worldwide.3 The epidemiology of testing of meningococcal isolates were performed. meningococcal disease is important for vaccination strategies.3,4 Whereas meningococci of The study identified 258 episodes of meningococcal meningitis in 258 patients. The serogroups B, C, and Y were responsible for several recent outbreaks of invasive disease in prevalence of the classical triad of fever, neck stiffness, and change in mental status was low the United States and other developed countries, serogroup A meningococci are the primary (70/258, 27%). When rash was added to the classical triad, 229 of 258 (89%) patients had at cause of endemic disease in developing countries.3 Current meningococcal vaccines are least 2 of 4 signs. Systolic hypotension was associated with rash (22/23 vs. 137/222, based on the capsular polysaccharides of serogroup A, C, W, and Y meningococci.4 p=0.002) and absence of neck stiffness (6/23 vs. 21/220, p=0.05). Neuroimaging before Multilocus sequence typing (MLST) is considered the gold standard for genotyping of lumbar puncture was an important cause of delay of therapy: antibiotics were not initiated meningococci and can be used to study epidemiology.4,5 before computed tomography (CT) scan in 85% of patients who underwent CT scan before We previously described a prospective cohort of 696 adult patients with community- lumbar puncture. Unfavorable outcome occurred in 30 of 258 (12%) patients, including a acquired bacterial meningitis.2 We now provide a detailed description of clinical features, mortality rate of 7%. Neurologic sequelae occurred in 28 of 238 (12%) patients, particularly prognostic factors, outcome, and MLST in the subset of 258 adults with meningococcal hearing loss (8%). Factors associated with sepsis and infection with meningococci of clonal meningitis. complex 11 (cc11) are related with unfavorable outcome.

MATERIALS AND METHODS

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divided into focal cerebral deficits (aphasia, monoparesis, or hemiparesis) and cranial nerve palsies. Whenever audiometry was done, hearing loss was classified as normal (<30 decibels RESULTS [dB]), mild (30–55 dB), moderate (55–70 dB), severe (70–90 dB), or profound (>90 dB).6 A total of 258 episodes of meningococcal meningitis occurred in 258 patients; in 1 patient Patients using immunosuppressive drugs and those with diabetes mellitus, alcoholism, CSF cultures yielded both N. meningitidis and group B streptococcus. The calculated annual asplenia, or human immunodeficiency virus (HIV) infection were considered to be incidence of community-acquired meningococcal meningitis was approximately 1 case per immunocompromised. 100,000 adults. Causes of death were independently classified in 2 categories by 2 clinicians as described Patient characteristics are presented in Table 1. The median age was 36 years (interquartile previously.7 The 2 categories were 1) systemic causes, including septic shock, respiratory range [IQR], 19–50 yr), and 52% were male. Two patients had a family history of bacterial failure, multiple-organ dysfunction, cardiac ischemia; and 2) neurologic causes, including meningitis. Otitis or sinusitis was present in 9 of 258 (4%) patients, and pneumonia in 13 of brain herniation, cerebrovascular complications, intractable seizures, and withdrawal of care 258 (5%) patients. Seizures before admission were present in only 2 of 255 patients. Acute because of poor neurologic prognosis. Only patients who died within 14 days after admission onset of illness, defined as duration of symptoms before admission less than 24 hours, was were classified, because death within this period is probably caused by direct consequences present in 123 of 251 (49%) patients. Six patients were treated with antibiotics before of the meningitis.8 Differences in classification between the 2 clinicians were resolved by presentation in the emergency department (4 orally, 2 intravenously). discussion.

The prevalence of the triad of fever, neck stiffness, and change in mental status was low Serogrouping, MLST, and susceptibility testing of meningococcal isolates were performed by (27%; Table 1). Relatively small proportions of patients had fever (temperature >38.0 °C, the Netherlands Reference Laboratory for Bacterial Meningitis. Serogrouping and penicillin- 64%) or change in mental status (defined as a score on the Glasgow Coma Scale (GCS) <14; susceptibility testing were performed as described elsewhere.2 MLST was performed on all 51%); 246 of 258 (95%) patients had at least 2 of 4 signs (classic triad plus headache). Focal available strains (n = 254) as described by Maiden et al;5 4 meningococcal strains were not neurologic abnormalities were present in 56 of 256 (22%) patients, including aphasia in 29 of available for analyses. MLST uses sequence data obtained from 7 housekeeping genes.5 The 256 (11%) patients. Cranial nerve palsy was present in 18 of 256 (7%) patients; N.III in 1 alleles from these housekeeping genes are assigned allele numbers, and the combination of patient, N.VI in 6, N.VII in 3, N.VIII in 9 patients; 1 patient had a palsy of both N.VI and N.VIII. these allele numbers makes up a sequence type. Clonal complexes were allocated according

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physician was contacted, and informed consent was obtained from all participating patients to the online MLST-database (available at http://pubmlst.org/neisseria/, accessed 3 October or their legally authorized representatives. Compared with the original dataset, 1 additional 2007. patient with meningococcal meningitis was identified.2 This observational study with Statistical Analysis anonymous patient data was carried out in accordance with the Dutch privacy legislation. The Mann-Whitney U test was used to identify differences between groups in continuous Procedures 2 variables, and dichotomous variables were compared by the chi-square or Fisher exact test. Patients underwent a neurologic examination at discharge, and outcome was graded with We did not perform multivariate analysis since the number of patients with unfavorable the Glasgow Outcome Scale. This measurement scale is well validated with scores varying outcome was limited. All statistical tests were 2-tailed, and a p value less than 0.05 was from 1 (indicating death) to 5 (good recovery). A favorable outcome was defined as a score regarded as significant. of 5, and an unfavorable outcome as a score of 1–4. Focal neurologic abnormalities were divided into focal cerebral deficits (aphasia, monoparesis, or hemiparesis) and cranial nerve palsies. Whenever audiometry was done, hearing loss was classified as normal (<30 decibels RESULTS [dB]), mild (30–55 dB), moderate (55–70 dB), severe (70–90 dB), or profound (>90 dB).6 A total of 258 episodes of meningococcal meningitis occurred in 258 patients; in 1 patient Patients using immunosuppressive drugs and those with diabetes mellitus, alcoholism, CSF cultures yielded both N. meningitidis and group B streptococcus. The calculated annual asplenia, or human immunodeficiency virus (HIV) infection were considered to be incidence of community-acquired meningococcal meningitis was approximately 1 case per immunocompromised. 100,000 adults. Causes of death were independently classified in 2 categories by 2 clinicians as described Patient characteristics are presented in Table 1. The median age was 36 years (interquartile previously.7 The 2 categories were 1) systemic causes, including septic shock, respiratory range [IQR], 19–50 yr), and 52% were male. Two patients had a family history of bacterial failure, multiple-organ dysfunction, cardiac ischemia; and 2) neurologic causes, including meningitis. Otitis or sinusitis was present in 9 of 258 (4%) patients, and pneumonia in 13 of brain herniation, cerebrovascular complications, intractable seizures, and withdrawal of care 258 (5%) patients. Seizures before admission were present in only 2 of 255 patients. Acute because of poor neurologic prognosis. Only patients who died within 14 days after admission onset of illness, defined as duration of symptoms before admission less than 24 hours, was were classified, because death within this period is probably caused by direct consequences present in 123 of 251 (49%) patients. Six patients were treated with antibiotics before of the meningitis.8 Differences in classification between the 2 clinicians were resolved by presentation in the emergency department (4 orally, 2 intravenously). discussion.

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At presentation, signs of septic shock (defined as diastolic blood pressure <60 mm Hg, blood: erythrocyte sedimentation rate (ESR) (0.16, p=0.03), and C-reactive protein (CRP) systolic blood pressure ≤90 mm Hg and/or heart rate ≥120/min) were present in 74 of 241 (0.40, p<0.0001). Blood WBCC was significantly associated with blood thrombocyte count (31%) evaluated patients. Rash was present in 164 (64%) patients and was characterized as (0.34, p<0.0001), but not with ESR (0.12, p=0.10) or CRP (0.15, p=0.08). petechial in 145 of 160 (91%) patients; 41 also had purpura and/or ecchymoses. In 15 of 160

(9%) patients, only purpura or ecchymoses were noted; rash was not specified in 4 patients. Systolic hypotension was associated with presence of rash (22/23 [96%] vs. 137/222 [62%], p=0.002) and absence of neck stiffness (6/23 [26%] vs. 21/220 [10%], p=0.05). When rash was added to the classical triad of fever, neck stiffness, and change in mental status, 229 of 258 (89%) patients had at least 2 of 4 signs. Of the 12 patients without any signs of the classical triad plus headache, 10 (83%) did present with rash.

Lumbar puncture was performed in all patients. Opening pressures were measured with a water-manometer in 76 of 258 (29%) patients. The median pressure was 36 cm water (IQR, 22–50 cm); very high pressures (>40 cm water) were found in 33 (43%) patients. In 214 of 248 (86%) patients, at least 1 individual CSF finding predictive of bacterial meningitis was present (glucose concentration <1.9 mmol/L, ratio of CSF glucose to blood glucose <0.23, protein concentration >2.20 g/L, CSF white blood cell count [WBCC] >2000/mm3, or CSF neutrophil count >1180/mm3).9 A CSF WBCC of <1000/mm3 was found in 47/242 (19%) patients; in these patients, systolic hypotension was more common (12/42 vs. 8/188, p<0.0001).

Five patients had a normal initial CSF analysis (defined as CSF WBCC ≤5/mm3, CSF protein ≤0.50 g/L, and ratio of CSF glucose to blood glucose ≥0.40); all of these 5 patients presented with rash, and Gram staining of CSF showed bacteria in 3. Gram staining of CSF was done for 244 of 258 (95%) patients and showed bacteria in 216 (89%) patients. Findings of Gram staining were indicative of N. meningitidis in 209 of 244 (86%) patients. In 5 patients, findings were interpreted as pneumococci; all of these patients presented without rash.

Routine blood examination was performed in all patients. To explore indexes of inflammation in CSF and blood we performed an analysis of correlations. There was a significant correlation between low WBCC in CSF and blood (Spearman r 0.28, p<0.0001). Low CSF WBCC was also significantly associated with low CSF protein level (0.52, p<0.0001), low blood thrombocyte count (0.19, p=0.004), and lower indexes of inflammation in the

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(9%) patients, only purpura or ecchymoses were noted; rash was not specified in 4 patients. 2 Systolic hypotension was associated with presence of rash (22/23 [96%] vs. 137/222 [62%], p=0.002) and absence of neck stiffness (6/23 [26%] vs. 21/220 [10%], p=0.05). When rash was added to the classical triad of fever, neck stiffness, and change in mental status, 229 of 258 (89%) patients had at least 2 of 4 signs. Of the 12 patients without any signs of the classical triad plus headache, 10 (83%) did present with rash.

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Table 1. Clinical and laboratory characteristics of 258 episodes of meningococcal meningitis. Neuroimaging was performed in 133 of 258 (52%) patients and consisted of computed Characteristic Demographics tomography (CT) in all patients. Cranial CT was done on presentation in 92 of 133 (69%) Age – yr (mean±SD) 36±19 patients. CT abnormalities were found in 15 of these patients and consisted of cerebral Male sex 133 (52) Duration of symptoms <24 hr 123/251 (49) edema in 8 (9%) and signs of hydrocephalus in 4 (4%). Neuroimaging preceded lumbar Pretreated with antibiotics 6/257 (2) puncture in 85 of 92 (92%) patients; antibiotics were administered before CT in 15 of 88 Pneumonia 13/258 (5) (17%) of these patients. Indications for performing CT before lumbar puncture (defined as a Immunocompromised 10/258 (4) Symptoms and signs at presentation score on the GCS <10, focal cerebral deficits, new onset seizures, or papilledema) were Headache 223/247 (90) present in 90 of 258 (35%) patients. Of these 90 patients who met these criteria of CT before Nausea 194/247 (79) Neck stiffness 226/255 (89) lumbar puncture, 44 (49%) underwent CT before lumbar puncture. During the clinical Rash 164/256 (64) course, cranial CT was performed in 41 additional patients. CT was prompted by a decline in Systolic blood pressure (BP) – mmHg 125 (110-140) Diastolic BP – mmHg 71 (60-80) level of consciousness in 10 patients, focal neurologic abnormalities in 14 patients, and Diastolic BP <60 mmHg 37/246 (15) persistent fever in 11 patients. Cerebral edema was found in 4 patients and subdural Heart rate – beats/minute 95 (80-110) Fever (T ≥38.0 °C) 161/250 (64) empyema, slight hydrocephalus and multiple abscesses were found in 1 scan each. Other Impaired consciousness (GCS <14) 131/257 (51) scans were reported as normal. Coma (GCS <8) 19/257 (7) Focal neurologic deficits Initial antimicrobial treatment consisted of monotherapy penicillin or amoxicillin in 92 (36%), Cranial nerve palsy 18/258 (7) Cerebral palsy 32/258 (12) monotherapy third-generation cephalosporin in 21 (8%), penicillin or amoxicillin plus third- Triad of fever, neck stiffness and change in mental status 70/258 (27) generation cephalosporin in 77 (30%), and another regimen in 63 of 253 (25%) patients. Cerebrospinal fluid parameters Opening pressure – cm of water 40 (22-50) Adjunctive steroid treatment was administered to 43 patients; the regimen was specified in White cell count – cells/mm³ 5328 (1590-12433) 37 of those. In 12 of 37 patients, 10 mg of dexamethasone was administered before or with <100 cells/mm³ 21/242 (9) 100-999 cells/mm³ 26/242 (11) the first dose of antibiotics and given every 6 hours for 4 days. In the remaining 25 patients, >999 cells/mm³ 195/242 (80) various steroid regimens were administered after antibiotic treatment had started. Steroids Protein – g/L 4.5 (2.2-7.0) CSF/Blood glucose ratio 0.08 (0.01-0.30) used were hydrocortisone, dexamethasone, prednisone, and prednisolone; the median daily Blood parameters dose equivalent to dexamethasone was 15 mg (range, 3–100 mg), and duration of treatment Positive blood culture 129/227 (57) Erythrocyte sedimentation rate (ESR) – mm/hr 40±38 varied between 1 and 7 days. C-reactive protein – mg/L 240±114 Sodium – mmol/L 137±4 During the clinical course, neurologic or systemic complications developed in 113 of 258 Thrombocyte count – 109/L 180±89 (44%) patients. Cardiorespiratory failure occurred in 44 of 258 (17%) patients, requiring Creatinin – μmol / L 113±61 Data are number/number assessed (%) or median (IQR) unless otherwise stated. Heart rate was evaluated in mechanical ventilation in 35. Patients who developed cardiorespiratory failure during the 240 episodes. Opening pressure was evaluated in 76 episodes. White cell count was evaluated in 242 episodes. CSF Protein was evaluated in 238 episodes. CSF/Blood glucose ratio was evaluated in 230 episodes. ESR was clinical course were more likely to have systolic hypotension on admission (18/23 [78%] vs. evaluated in 200 episodes. C-reactive protein was evaluated in 150 episodes. Sodium was evaluated in 255 22/223 [10%], p<0.0001), CSF WBCC <1000/mm3 (21/41 [51%] vs. 26/201 [13%], p<0.0001), episodes. Thrombocyte count was evaluated in 245 episodes. Creatinin was evaluated in 251 episodes. and rash (40/44 [91%] vs. 124/212 [58%], p<0.0001). One or more neurologic complications

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Table 1. Clinical and laboratory characteristics of 258 episodes of meningococcal meningitis. Neuroimaging was performed in 133 of 258 (52%) patients and consisted of computed Characteristic Demographics tomography (CT) in all patients. Cranial CT was done on presentation in 92 of 133 (69%) Age – yr (mean±SD) 36±19 patients. CT abnormalities were found in 15 of these patients and consisted of cerebral Male sex 133 (52) Duration of symptoms <24 hr 123/251 (49) edema in 8 (9%) and signs of hydrocephalus in 4 (4%). Neuroimaging preceded lumbar Pretreated with antibiotics 6/257 (2) puncture in 85 of 92 (92%) patients; antibiotics were administered before CT in 15 of 88 Pneumonia 13/258 (5) 2 (17%) of these patients. Indications for performing CT before lumbar puncture (defined as a Immunocompromised 10/258 (4) Symptoms and signs at presentation score on the GCS <10, focal cerebral deficits, new onset seizures, or papilledema) were Headache 223/247 (90) present in 90 of 258 (35%) patients. Of these 90 patients who met these criteria of CT before Nausea 194/247 (79) Neck stiffness 226/255 (89) lumbar puncture, 44 (49%) underwent CT before lumbar puncture. During the clinical Rash 164/256 (64) course, cranial CT was performed in 41 additional patients. CT was prompted by a decline in Systolic blood pressure (BP) – mmHg 125 (110-140) Diastolic BP – mmHg 71 (60-80) level of consciousness in 10 patients, focal neurologic abnormalities in 14 patients, and Diastolic BP <60 mmHg 37/246 (15) persistent fever in 11 patients. Cerebral edema was found in 4 patients and subdural Heart rate – beats/minute 95 (80-110) Fever (T ≥38.0 °C) 161/250 (64) empyema, slight hydrocephalus and multiple abscesses were found in 1 scan each. Other Impaired consciousness (GCS <14) 131/257 (51) scans were reported as normal. Coma (GCS <8) 19/257 (7) Focal neurologic deficits Initial antimicrobial treatment consisted of monotherapy penicillin or amoxicillin in 92 (36%), Cranial nerve palsy 18/258 (7) Cerebral palsy 32/258 (12) monotherapy third-generation cephalosporin in 21 (8%), penicillin or amoxicillin plus third- Triad of fever, neck stiffness and change in mental status 70/258 (27) generation cephalosporin in 77 (30%), and another regimen in 63 of 253 (25%) patients. Cerebrospinal fluid parameters Opening pressure – cm of water 40 (22-50) Adjunctive steroid treatment was administered to 43 patients; the regimen was specified in White cell count – cells/mm³ 5328 (1590-12433) 37 of those. In 12 of 37 patients, 10 mg of dexamethasone was administered before or with <100 cells/mm³ 21/242 (9) 100-999 cells/mm³ 26/242 (11) the first dose of antibiotics and given every 6 hours for 4 days. In the remaining 25 patients, >999 cells/mm³ 195/242 (80) various steroid regimens were administered after antibiotic treatment had started. Steroids Protein – g/L 4.5 (2.2-7.0) CSF/Blood glucose ratio 0.08 (0.01-0.30) used were hydrocortisone, dexamethasone, prednisone, and prednisolone; the median daily Blood parameters dose equivalent to dexamethasone was 15 mg (range, 3–100 mg), and duration of treatment Positive blood culture 129/227 (57) Erythrocyte sedimentation rate (ESR) – mm/hr 40±38 varied between 1 and 7 days. C-reactive protein – mg/L 240±114 Sodium – mmol/L 137±4 During the clinical course, neurologic or systemic complications developed in 113 of 258 Thrombocyte count – 109/L 180±89 (44%) patients. Cardiorespiratory failure occurred in 44 of 258 (17%) patients, requiring Creatinin – μmol / L 113±61 Data are number/number assessed (%) or median (IQR) unless otherwise stated. Heart rate was evaluated in mechanical ventilation in 35. Patients who developed cardiorespiratory failure during the 240 episodes. Opening pressure was evaluated in 76 episodes. White cell count was evaluated in 242 episodes. CSF Protein was evaluated in 238 episodes. CSF/Blood glucose ratio was evaluated in 230 episodes. ESR was clinical course were more likely to have systolic hypotension on admission (18/23 [78%] vs. evaluated in 200 episodes. C-reactive protein was evaluated in 150 episodes. Sodium was evaluated in 255 22/223 [10%], p<0.0001), CSF WBCC <1000/mm3 (21/41 [51%] vs. 26/201 [13%], p<0.0001), episodes. Thrombocyte count was evaluated in 245 episodes. Creatinin was evaluated in 251 episodes. and rash (40/44 [91%] vs. 124/212 [58%], p<0.0001). One or more neurologic complications

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(impairment of consciousness, seizures, or focal neurologic abnormalities) developed in 105 (cardiorespiratory failure in 2 patients and multi-organ failure in 1 patient). At discharge, of 258 (41%) patients. neurologic examination was performed in 238 of 239 (99%) patients and revealed focal neurologic abnormalities in 28 (12%); hearing loss was the most common neurologic CSF culture yielded N. meningitidis in all patients. Blood samples were cultured in 227 sequelae in 19 (8%) (Table 4). Hearing loss appeared after a median of 2 days (range, 0–24 patients, of which 129 (57%) were positive. Positive blood cultures were related with d), and results of audiometry were available for 9 patients. Hearing loss was unilateral in 3 presence of rash (103/128 [80%] vs. 48/98 [49%], p<0.0001). Antibiotic susceptibility was (33%) and bilateral in 6 (67%) patients. The severity of hearing loss was classified as tested in 256 strains; 252 strains were sensitive to penicillin, and 4 strains showed profound in 3 (33%), severe in 1 (11%), moderate in 1 (11%), mild in 3 (33%), and normal in 1 intermediate susceptibility to penicillin. Initial antimicrobial therapy was judged to be (11%).6 Outcome was graded as favorable in 228 (88%) and unfavorable in 30 (12%) patients microbiologically adequate in 249 of 253 (98%) patients. All 4 patients with inadequate (see Table 4). therapy were infected with intermediately susceptible strains and were treated with monotherapy amoxicillin or penicillin; outcome was favorable in all 4 patients. Antimicrobial therapy was stepped down to monotherapy penicillin or amoxicillin within 3 days in 179 of Figure 1. Number of cases due to meningococci of cc11 during study period. 253 (71%) cases.

20 The serogrouping result was available for 256 meningococcal strains. Of these, 173 (68%) were of serogroup B, 79 (31%) of serogroup C, 3 (1%) of serogroup Y, and 1 (<1%) was of serogroup W. Of 258 isolates, 254 (98%) were analyzed by MLST (Table 2). MLST analysis of 15 254 isolates showed 91 unique sequence types. The most prevalent clonal complexes (cc) were cc41/44 (41%), cc11 (24%), and cc32 (16%). All cc11 strains were serogroup C. During the study period there was an increase in disease caused by meningococci belonging to cc11 (Figure 1). 10 Number of cases of Number In a univariate analysis we explored relations between different clonal complexes and clinical characteristics. Table 3 represents univariate testing characteristics of infection by 5 meningococci of 1 clonal complex versus infection by meningococci of all other complexes. Results should be interpreted with caution because of the explorative nature and multiple relations tested. The main finding of this exploration is the relation between infection by

0 meningococci of cc11, disease severity, and characteristics related with sepsis (low level of jan-99 jul-99 jan-00 jul-00 jan-01 jul-01 jan-02 3 Time consciousness, p=0.02; CSF WBCC <1000/mm , p=0.01; low blood WBCC, p=0.03; high serum creatinine, p=0.003; positive blood culture, p=0.03). Blood pressure was not related with

clonal complex.

Of 258 patients, 19 (7%) died; 13 of the 19 (68%) within 24 hours. Sepsis was the leading cause of mortality (14 of 19 fatalities, 73%); 3 additional patients died of systemic causes

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(impairment of consciousness, seizures, or focal neurologic abnormalities) developed in 105 (cardiorespiratory failure in 2 patients and multi-organ failure in 1 patient). At discharge, of 258 (41%) patients. neurologic examination was performed in 238 of 239 (99%) patients and revealed focal neurologic abnormalities in 28 (12%); hearing loss was the most common neurologic CSF culture yielded N. meningitidis in all patients. Blood samples were cultured in 227 sequelae in 19 (8%) (Table 4). Hearing loss appeared after a median of 2 days (range, 0–24 patients, of which 129 (57%) were positive. Positive blood cultures were related with d), and results of audiometry were available for 9 patients. Hearing loss was unilateral in 3 presence of rash (103/128 [80%] vs. 48/98 [49%], p<0.0001). Antibiotic susceptibility was 2 (33%) and bilateral in 6 (67%) patients. The severity of hearing loss was classified as tested in 256 strains; 252 strains were sensitive to penicillin, and 4 strains showed profound in 3 (33%), severe in 1 (11%), moderate in 1 (11%), mild in 3 (33%), and normal in 1 intermediate susceptibility to penicillin. Initial antimicrobial therapy was judged to be (11%).6 Outcome was graded as favorable in 228 (88%) and unfavorable in 30 (12%) patients microbiologically adequate in 249 of 253 (98%) patients. All 4 patients with inadequate (see Table 4). therapy were infected with intermediately susceptible strains and were treated with monotherapy amoxicillin or penicillin; outcome was favorable in all 4 patients. Antimicrobial therapy was stepped down to monotherapy penicillin or amoxicillin within 3 days in 179 of Figure 1. Number of cases due to meningococci of cc11 during study period. 253 (71%) cases.

Of 258 patients, 19 (7%) died; 13 of the 19 (68%) within 24 hours. Sepsis was the leading cause of mortality (14 of 19 fatalities, 73%); 3 additional patients died of systemic causes

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Table 2. Frequency of clonal complexes and serogroups.

Clonal complex No (%) Serogroup B Serogroup C Serogroup Y .11 .24 .00 .72 .50 .23 .73 .30 .85 .77 .25 .52 .84 .35 .95 p .96 .41

cc41/44 104 (41) 103 1 -

------12 18 21 18 n 20 cc11 62 (24) - 62 - 19

cc32 41 (16) 41 - -

cc269 16 (6) 14 2 - 5/21 (24) 6/21 (29) 7/21 (33) 2/21 (10) 2/20 (10) 13/21 (81) 59±78 3/18 (17) 20/21 (95) 3/19 (16) 1/20 (5) 7/18 (39) 7.2±5.5 13±3 172±63 Other ST n=21

21±9 cc8 10 (4) - 10 - 101±49

cc60 4 (2) 3 1 -

.47 .01 .29 .36 .55 .10 .56 .43 .86 .81 .66 .33 .73 .95 p .68 .03 .77 cc461 2 (1) 1 1 -

- - - 10 ------10 10 n 10 10 cc18 2 (1) 1 1 - 10 Othera 7 (3) 3 - 3

No cc 6 (2) 6 - - Total 254 172 78 3 2/10 (20) 5/10 (50) 8/10 (80) 33±26 1/10 (10) 9/10 (90) 0/10 (0) 2/9 (22) 2/9 (22) 7/10 (70) 1/9 (11) 1/9 (11) 9.8±9.6 12±3 cc8 176±89 n=10

14±9 98±28 acc35, cc167, cc213, cc1286 (serogroup W), cc3544, cc5457, cc5453.

.99 .50 .01 .67 .98 .48 .08 .75 .83 .85 .54 .62 .80 .87 .57 p .32 ST-212, ST-2700, ST-3549, ST 3621, ST-4258, ST-5408. .49

- - - 14 ------15 16 n 15 16 16

2/16 (13) 2/16 (13) 15/16 (94) 34±26 3/15 (20) 15/16 (94) 3/16 (19) 2/16 (13) 7/15 (47) 7/14 (50) 3/16 (19) 2/16 (13) 203±114 cc269 n=16 11.5±23.6

12±4 96±32 22±9

.10 .04 .23 .28 .34 .66 .18 .97 .34 .57 .43 .80 .82 .70 .25 p .09 .80

- - - 31 ------40 36 40 - - n 40 41

2/41 (5) 3/41 (7) 23/41 (56) 46±35 5/36 (14) 37/41 (90) 1/40 (2) 6/40 (15) 17/40 (43) 20/34 (59) 97±52 185±88 cc32 n=41 17.3±61.5 13±3 8/40 (20) 4/40 (10)

20±7

.07 .39 .81 .86 .01 .01 .18 .24 .80 .03 .00 .40 .11 .02 .83 .44 p .03

- - - 42 ------61 59 59 61 - - n 61

12/62 (19) 14/62 (23) 36±28 40/61 (66) 7/61 (12) 177±109 19/59 (32) 49/62 (79) 9/57 (16) 12/60 (20) 8/60 (13) 31/61(51) 39/59 (66) 138±80 cc11 n=62 10.2±17.3 11±3 18±10

.12 .59 .64 .46 .38 .63 .22 .37 .86 .48 .39 .58 .34 .64 p .12 .55 .04

- - 87 - - 101 99 - - n 100 104 - - - - 104 -

9/104 (9) 18/104 (17) 38±39 69/103 (67) 6/104 (6) 111±57 178±77 cc41/44 n=104 16/100 (16) 11.6±19.8 94/104 (90) 12±3 17/96 (16) 13/98 (13) 7/98 (7) 52/101 (52) 22±9 48/88 (55)

9 - –

mol / L μ

mm/hr

–

Cerebral palsy Cranial nerve palsyCranial nerve Rash ESR ESR Coma (GCS <8) Creatinin - Table 3. Clonal complexes and clinical characteristics. CSF white cell count 10³cells/mm³ Thrombocyte count platelets/mm³ Favorable outcome (GOS 5) Data are number/number assessed (%) or mean±SD. Blood white cells - CSF white cell count <1000 cells/mm³ Glasgow Coma Scale score Heart rate >120 beats/minute Diastolic blood pressure mmHg <60 Systolic blood pressure mmHg <90 Duration of Duration symptoms hr <24 Positive blood culture

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Table 2. Frequency of clonal complexes and serogroups.

Clonal complex No (%) Serogroup B Serogroup C Serogroup Y .11 .24 .00 .72 .50 .23 .73 .30 .85 .77 .25 .52 .84 .35 .95 p .96 .41

cc41/44 104 (41) 103 1 -

------12 18 21 18 n 20 cc11 62 (24) - 62 - 19

cc32 41 (16) 41 - -

cc269 16 (6) 14 2 - 5/21 (24) 6/21 (29) 7/21 (33) 2/21 (10) 2/20 (10) 13/21 (81) 59±78 3/18 (17) 20/21 (95) 3/19 (16) 1/20 (5) 7/18 (39) 7.2±5.5 13±3 172±63 Other ST n=21

21±9 cc8 10 (4) - 10 - 101±49

cc60 4 (2) 3 1 - 2 .47 .01 .29 .36 .55 .10 .56 .43 .86 .81 .66 .33 .73 .95 p .68 .03 .77 cc461 2 (1) 1 1 -

- - - 10 ------10 10 n 10 10 cc18 2 (1) 1 1 - 10 Othera 7 (3) 3 - 3

No cc 6 (2) 6 - - Total 254 172 78 3 2/10 (20) 5/10 (50) 8/10 (80) 33±26 1/10 (10) 9/10 (90) 0/10 (0) 2/9 (22) 2/9 (22) 7/10 (70) 1/9 (11) 1/9 (11) 9.8±9.6 12±3 cc8 176±89 n=10

14±9 98±28 acc35, cc167, cc213, cc1286 (serogroup W), cc3544, cc5457, cc5453.

.99 .50 .01 .67 .98 .48 .08 .75 .83 .85 .54 .62 .80 .87 .57 p .32 ST-212, ST-2700, ST-3549, ST 3621, ST-4258, ST-5408. .49

- - - 14 ------15 16 n 15 16 16

2/16 (13) 2/16 (13) 15/16 (94) 34±26 3/15 (20) 15/16 (94) 3/16 (19) 2/16 (13) 7/15 (47) 7/14 (50) 3/16 (19) 2/16 (13) 203±114 cc269 n=16 11.5±23.6

12±4 96±32 22±9

.10 .04 .23 .28 .34 .66 .18 .97 .34 .57 .43 .80 .82 .70 .25 p .09 .80

- - - 31 ------40 36 40 - - n 40 41

2/41 (5) 3/41 (7) 23/41 (56) 46±35 5/36 (14) 37/41 (90) 1/40 (2) 6/40 (15) 17/40 (43) 20/34 (59) 97±52 185±88 cc32 n=41 17.3±61.5 13±3 8/40 (20) 4/40 (10)

20±7

.07 .39 .81 .86 .01 .01 .18 .24 .80 .03 .00 .40 .11 .02 .83 .44 p .03

- - - 42 ------61 59 59 61 - - n 61

12/62 (19) 14/62 (23) 36±28 40/61 (66) 7/61 (12) 177±109 19/59 (32) 49/62 (79) 9/57 (16) 12/60 (20) 8/60 (13) 31/61(51) 39/59 (66) 138±80 cc11 n=62 10.2±17.3 11±3 18±10

.12 .59 .64 .46 .38 .63 .22 .37 .86 .48 .39 .58 .34 .64 p .12 .55 .04

- - 87 - - 101 99 - - n 100 104 - - - - 104 -

9/104 (9) 18/104 (17) 38±39 69/103 (67) 6/104 (6) 111±57 178±77 cc41/44 n=104 16/100 (16) 11.6±19.8 94/104 (90) 12±3 17/96 (16) 13/98 (13) 7/98 (7) 52/101 (52) 22±9 48/88 (55)

9 - –

mol / L μ

mm/hr

–

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Table 4. Patient outcome. Table 5. Univariate analysis of predictors of unfavorable outcome in 258 episodes of meningococcal Neurological deficits at discharge No./No. assessed (%) meningitis. Hearing loss (Eighth cranial nerve palsy) 19/237 (8) Unfavorable Favorable p Other cranial nerve palsy 6/238 (3) Demographics Aphasia 1/238 (0.4) Age - yr (mean ±SD) 46±22 35±18 .13 Monoparesis 1/238 (0.4) Duration of symptoms <24 hr 16/30 (53) 107/221 (48) .61 Hemiparesis 2/238 (1) Symptoms at presentation Quadriparesis 2/238 (1) Neck stiffness 22/29 (76) 204/226 (90) .02 Glasgow outcome scale Rash 25/30 (83) 139/226 (61) .02 1 (death) 19/258 (7) Systolic blood pressure <90 mmHg 12/28 (43) 11/218 (5) <.0001 2 (vegetative state) 0/258 (0) Diastolic Blood Pressure <60 mmHg 12/28 (43) 25/218 (11) <.0001 3 (severe disability) 4/258 (2) Heart rate >120 bpm 11/28 (40) 30/212 (14) .004 4 (moderate disability) 7/258 (3) Fever (T ≥38.0º C) 19/28 (68) 142/222 (64) .69 5 (mild/no disability) 228/258 (88) Impaired consciousness (GCS Score <14) 19/30 (63) 112/227 (49) .15 Coma (GCS <8) 4/30 (13) 15/227 (7) .19 Focal neurologic deficits - Cranial nerve palsy 8/30 (27) 40/228 (18) .23 Factors associated with unfavorable outcome (Table 5) were advanced age, absence of neck - Cerebral palsy 5/30 (17) 27/228 (12) .45 stiffness, presence of rash, systolic or diastolic hypotension, tachycardia, low CSF WBCC, low Cerebrospinal fluid parameters Opening pressure – cm of water 34 (14-50) 40 (24-40) .58 CSF protein level, high CSF/blood glucose ratio, positive blood culture, high serum creatinine White cell count <1000/mm3 – no. (%) 15/27 (56) 32/215 (15) <.0001 level, and low level of thrombocytes. The proportion of patients who died was significantly Protein – g/L 2.9 (0.6-4.9) 4.9 (2.4-7.3) .006 CSF/Blood glucose ratio 0.26 (0.01-0.53) 0.07 (0.01-0.27) .04 higher in patients without a CSF predictor for meningitis than in those with at least 1 CSF Blood tests finding considered predictive for bacterial meningitis (10/33 [30%] vs. 8/214 [4%], Positive Blood Culture - no. (%) 25/30 (83) 104/197 (53) <.0001 ESR – mm/hr 19 (5-69) 32 (15-58) .24 p<0.0001). Risk for unfavorable outcome was significantly higher in patients infected by C-reactive protein – mg/L 193 (137-272) 231 (173-317) .25 meningococci of cc11 (all of which were group C meningococci) compared to patients Creatinin – μmol/L 228 (95-285) 91 (75-114) <.0001 Sodium – mmol/L 138 (136-140 137 (135-139) .10 infected by meningococci of other clonal complexes (13/62 [21%] vs. 17/192 [9%], p=0.01). Thrombocyte count – 109/L 120 (93-149) 173 (139-225) .004 Although serogroup was not significantly associated with outcome in univariate analysis, the Data are number/number assessed (%) or median (IQR) unless otherwise stated. CSF white cell count was determined in 242 episodes. CSF protein level was determined in 238 episodes. CSF/blood glucose level was risk for an unfavorable outcome tended to be higher in patients with serogroup C disease, determined in 230 episodes. ESR was determined in 200 episodes. C-reactive protein was determined in 150 episodes. Creatinin was determined in 251 episodes. Sodium was determined in 255 episodes. Thrombocyte compared with those with non-serogroup C disease (14/79 [18%] vs. 16/177 [9%], p=0.06). count was determined in 245 episodes.

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Table 4. Patient outcome. Table 5. Univariate analysis of predictors of unfavorable outcome in 258 episodes of meningococcal Neurological deficits at discharge No./No. assessed (%) meningitis. Hearing loss (Eighth cranial nerve palsy) 19/237 (8) Unfavorable Favorable p Other cranial nerve palsy 6/238 (3) Demographics Aphasia 1/238 (0.4) Age - yr (mean ±SD) 46±22 35±18 .13 Monoparesis 1/238 (0.4) Duration of symptoms <24 hr 16/30 (53) 107/221 (48) .61 Hemiparesis 2/238 (1) Symptoms at presentation Quadriparesis 2/238 (1) Neck stiffness 22/29 (76) 204/226 (90) .02 2 Glasgow outcome scale Rash 25/30 (83) 139/226 (61) .02 1 (death) 19/258 (7) Systolic blood pressure <90 mmHg 12/28 (43) 11/218 (5) <.0001 2 (vegetative state) 0/258 (0) Diastolic Blood Pressure <60 mmHg 12/28 (43) 25/218 (11) <.0001 3 (severe disability) 4/258 (2) Heart rate >120 bpm 11/28 (40) 30/212 (14) .004 4 (moderate disability) 7/258 (3) Fever (T ≥38.0º C) 19/28 (68) 142/222 (64) .69 5 (mild/no disability) 228/258 (88) Impaired consciousness (GCS Score <14) 19/30 (63) 112/227 (49) .15 Coma (GCS <8) 4/30 (13) 15/227 (7) .19 Focal neurologic deficits - Cranial nerve palsy 8/30 (27) 40/228 (18) .23 Factors associated with unfavorable outcome (Table 5) were advanced age, absence of neck - Cerebral palsy 5/30 (17) 27/228 (12) .45 stiffness, presence of rash, systolic or diastolic hypotension, tachycardia, low CSF WBCC, low Cerebrospinal fluid parameters Opening pressure – cm of water 34 (14-50) 40 (24-40) .58 CSF protein level, high CSF/blood glucose ratio, positive blood culture, high serum creatinine White cell count <1000/mm3 – no. (%) 15/27 (56) 32/215 (15) <.0001 level, and low level of thrombocytes. The proportion of patients who died was significantly Protein – g/L 2.9 (0.6-4.9) 4.9 (2.4-7.3) .006 CSF/Blood glucose ratio 0.26 (0.01-0.53) 0.07 (0.01-0.27) .04 higher in patients without a CSF predictor for meningitis than in those with at least 1 CSF Blood tests finding considered predictive for bacterial meningitis (10/33 [30%] vs. 8/214 [4%], Positive Blood Culture - no. (%) 25/30 (83) 104/197 (53) <.0001 ESR – mm/hr 19 (5-69) 32 (15-58) .24 p<0.0001). Risk for unfavorable outcome was significantly higher in patients infected by C-reactive protein – mg/L 193 (137-272) 231 (173-317) .25 meningococci of cc11 (all of which were group C meningococci) compared to patients Creatinin – μmol/L 228 (95-285) 91 (75-114) <.0001 Sodium – mmol/L 138 (136-140 137 (135-139) .10 infected by meningococci of other clonal complexes (13/62 [21%] vs. 17/192 [9%], p=0.01). Thrombocyte count – 109/L 120 (93-149) 173 (139-225) .004 Although serogroup was not significantly associated with outcome in univariate analysis, the Data are number/number assessed (%) or median (IQR) unless otherwise stated. CSF white cell count was determined in 242 episodes. CSF protein level was determined in 238 episodes. CSF/blood glucose level was risk for an unfavorable outcome tended to be higher in patients with serogroup C disease, determined in 230 episodes. ESR was determined in 200 episodes. C-reactive protein was determined in 150 episodes. Creatinin was determined in 251 episodes. Sodium was determined in 255 episodes. Thrombocyte compared with those with non-serogroup C disease (14/79 [18%] vs. 16/177 [9%], p=0.06). count was determined in 245 episodes.

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DISCUSSION in the Netherlands.3,11 Two problems arise with treatment before admission to the hospital. First, identification of patients with meningococcal meningitis by observation of symptoms The current study provides a detailed description of meningococcal meningitis from a large alone is difficult.10,12 Presenting symptoms of meningococcal disease are often nonspecific, prospective cohort study in the Netherlands, aiming to correlate bacterial genotype, clinical and the current data show that typical signs and symptoms often do not develop at all.10 features, prognostic factors, and outcome. Our findings indicate that meningococcal Second, it remains unclear whether patients benefit from such prehospital treatment. meningitis remains a serious and life-threatening disease. The rate of unfavorable outcome Although retrospective data from the United Kingdom showed a favorable outcome for of meningococcal disease remains substantial at 12%, including a mortality rate of 7%. patients who were treated early with parenteral antibiotics, prehospital antibiotic treatment Neurologic sequelae in survivors are common (12%), most frequently hearing loss (8%). of such patients remains controversial.13 A case-control study of children suspected of

A broad spectrum of meningococcal disease was observed in our patients, ranging from having meningococcal disease treated with parenteral antibiotics before admission showed 14 sepsis to meningitis. Signs of systemic disease as indicated by rash, hypotension, tachycardia, an association between early treatment and poor outcome. While confounding by severity and positive blood cultures occurred frequently in our patients, frequently resulted in the is a possible explanation for this relation, it has added to the controversy concerning the 15 necessity for cardiopulmonary support in an intensive care unit, and were associated with strategy of prehospital treatment of suspected meningococcal disease. unfavorable outcome. Patients on the meningitis side of the spectrum had a better outcome Cranial CT was a major cause of delay of inhospital administration of therapy. Antibiotics compared with those on the sepsis side. In a categorization of the cause of death, sepsis was were not initiated before CT in 83% of patients who underwent CT before lumbar puncture. the leading cause of death, emphasizing the need for prompt treatment of systemic We did not specifically record time of delay of administration of antibiotics, precluding complications and the development of new adjunctive therapies against the septic conclusions about a causal relation between possible delay of treatment and outcome. component of this disease. Nevertheless, the association between delay in the inhospital administration of antibiotics 16,17 Classic symptoms and signs of bacterial meningitis such as headache, fever, neck stiffness and adverse outcome has been shown in previous studies. If cranial CT is to precede and decreased level of consciousness were absent in many patients. The classic triad as lumbar puncture, we recommend that appropriate treatment (antibiotics with adjunctive described in textbooks of fever, neck stiffness, and change in mental status was not found in dexamethasone) be initiated first. two-thirds of patients. This is important information for physicians who are involved in the Dexamethasone was administered in a minority of patients in the current study. In 2002, a identification and treatment of patients with meningococcal meningitis, and is in line with European randomized clinical trial showed that treatment with adjunctive dexamethasone previous research.10 Initial CSF examination was suspect for bacterial meningitis in most started before or with the first dose of antibiotics reduces unfavorable outcome and patients.9 Nevertheless, low CSF white cell counts (<100/mm3) were present in ~10% and mortality in adults with bacterial meningitis.18 This reduction was most obvious in patients were associated with signs of sepsis and unfavorable outcome. In 5 patients, initial CSF with pneumococcal meningitis. A subsequent meta-analysis including 5 trials involving 623 examination of leukocytes, protein, and glucose was entirely normal. However, patients (pneumococcal meningitis = 234, meningococcal meningitis = 232, other = 127, meningococcal disease could be identified in all 5 patients through the presence of rash or unknown = 30) showed a reduction of mortality and neurologic sequelae associated with the presence of bacteria on Gram staining. dexamethasone.19 In meningococcal meningitis, the point estimate for risk reduction in this Intravenous antibiotics were started before transportation to the hospital in 2 patients. In meta-analysis was low and not statistically significant (0.9, confidence interval 0.3–2.1, 19 the United Kingdom, family doctors are advised to give (parenteral) antibiotics before p=0.7). Guidelines differ in their advice about whether patients with meningococcal transferring the patient to the hospital if meningococcal meningitis is suspected, but not so meningitis should receive steroids; some advise the use of steroids in all patients with

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DISCUSSION in the Netherlands.3,11 Two problems arise with treatment before admission to the hospital. First, identification of patients with meningococcal meningitis by observation of symptoms The current study provides a detailed description of meningococcal meningitis from a large alone is difficult.10,12 Presenting symptoms of meningococcal disease are often nonspecific, prospective cohort study in the Netherlands, aiming to correlate bacterial genotype, clinical and the current data show that typical signs and symptoms often do not develop at all.10 features, prognostic factors, and outcome. Our findings indicate that meningococcal Second, it remains unclear whether patients benefit from such prehospital treatment. meningitis remains a serious and life-threatening disease. The rate of unfavorable outcome 2 Although retrospective data from the United Kingdom showed a favorable outcome for of meningococcal disease remains substantial at 12%, including a mortality rate of 7%. patients who were treated early with parenteral antibiotics, prehospital antibiotic treatment Neurologic sequelae in survivors are common (12%), most frequently hearing loss (8%). of such patients remains controversial.13 A case-control study of children suspected of

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outcome. We found no additional relations between clinical features and MLST genotypes. 3. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, The cc11 was strongly related to the phenotype serogroup C and has been associated with and Neisseria meningitidis. Lancet 2007;369:2196-2210. elevated levels of disease spreading across several continents.3,4 During the study period we 4. Snape MD, Pollard AJ. Meningococcal polysaccharide-protein conjugate vaccines. Lancet Infect Dis 2005;5:21-30. noticed an increase in disease caused by meningococci of cc11 (Figure 1). Since 2002, routine vaccination with a single dose of conjugated meningococcal C vaccine at 14 months and a 5. Maiden MC, Bygraves JA, Feil E, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic catch-up campaign have reduced the incidence of meningococcal serogroup C disease in the microorganisms. Proc Natl Acad Sci U S A 1998;95:3140-3145. Netherlands.22 6. Dodge PR, Davis H, Feigin RD, et al. Prospective evaluation of hearing impairment as a sequela of acute bacterial meningitis. N Engl J Med 1984;311:869-874. The current study has several limitations. First, only patients with positive CSF cultures were van de Beek D, de Gans J Ann Intern included. Negative CSF cultures occur in 11%–30% of patients with bacterial meningitis. 7. . Dexamethasone and pneumococcal meningitis. Med 2004;141:327. Patients in severe septic shock may not undergo lumbar puncture, as meningococcal sepsis 8. McMillan DA, Lin CY, Aronin SI, Quagliarello VJ. Community-acquired bacterial is frequently associated with coagulation disorders such as disseminated intravascular meningitis in adults: categorization of causes and timing of death. Clin Infect Dis coagulation.3 In those patients lumbar puncture may not be performed.1 Therefore, these 2001;33:969-975.

patients are probably only partly represented in our cohort, probably causing an 9. Spanos A, Harrell FE, Jr., Durack DT. Differential diagnosis of acute meningitis. An underestimation of the rate of sepsis and unfavorable outcome among our population. analysis of the predictive value of initial observations. JAMA 1989;262:2700-2707. Second, a substantial proportion of identified patients with bacterial meningitis (32%) were 10. Thompson MJ, Ninis N, Perera R, et al. Clinical recognition of meningococcal disease 2 in children and adolescents. Lancet 2006;367:397-403. not included in our Dutch Meningitis Cohort, which may also have resulted in selection bias. 11. Heyderman RS, British Infection S. Early management of suspected bacterial In conclusion, meningococcal meningitis is still a serious and life-threatening disease. meningitis and meningococcal septicaemia in immunocompetent adults--second edition. J Infect 2005;50:373-374. Neuroimaging before lumbar puncture is an important cause of delay in the administration of antibiotics. Infection with meningococci of cc11 is related to factors associated with sepsis 12. Riordan FA, Thomson AP, Sills JA, Hart CA. Who spots the spots? Diagnosis and treatment of early meningococcal disease in children. BMJ 1996;313:1255-1256. and to unfavorable outcome. 13. Cartwright K, Reilly S, White D, Stuart J. Early treatment with parenteral penicillin in meningococcal disease. BMJ 1992;305:143-147.

14. Harnden A, Ninis N, Thompson M, et al. Parenteral penicillin for children with meningococcal disease before hospital admission: case-control study. BMJ 2006;332:1295-1298.

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bacterial meningitis, others advise discontinuing steroid treatment if the causative pathogen REFERENCES 1,20 is not S. pneumoniae. Current guidelines by the British Infection Society support the use 1. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial of steroids in patients with suspected meningococcal meningitis.11 The use of high-dose meningitis in adults. N Engl J Med 2006;354:44-53. 21 steroids in patients with septic shock may be harmful, and is therefore not recommended. 2. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Infection with meningococci belonging to cc11 was associated with sepsis and poor Med 2004;351:1849-1859. 2 outcome. We found no additional relations between clinical features and MLST genotypes. 3. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, The cc11 was strongly related to the phenotype serogroup C and has been associated with and Neisseria meningitidis. Lancet 2007;369:2196-2210. elevated levels of disease spreading across several continents.3,4 During the study period we 4. Snape MD, Pollard AJ. Meningococcal polysaccharide-protein conjugate vaccines. Lancet Infect Dis 2005;5:21-30. noticed an increase in disease caused by meningococci of cc11 (Figure 1). Since 2002, routine vaccination with a single dose of conjugated meningococcal C vaccine at 14 months and a 5. Maiden MC, Bygraves JA, Feil E, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic catch-up campaign have reduced the incidence of meningococcal serogroup C disease in the microorganisms. Proc Natl Acad Sci U S A 1998;95:3140-3145. Netherlands.22 6. Dodge PR, Davis H, Feigin RD, et al. Prospective evaluation of hearing impairment as a sequela of acute bacterial meningitis. N Engl J Med 1984;311:869-874. The current study has several limitations. First, only patients with positive CSF cultures were van de Beek D, de Gans J Ann Intern included. Negative CSF cultures occur in 11%–30% of patients with bacterial meningitis. 7. . Dexamethasone and pneumococcal meningitis. Med 2004;141:327. Patients in severe septic shock may not undergo lumbar puncture, as meningococcal sepsis 8. McMillan DA, Lin CY, Aronin SI, Quagliarello VJ. Community-acquired bacterial is frequently associated with coagulation disorders such as disseminated intravascular meningitis in adults: categorization of causes and timing of death. Clin Infect Dis coagulation.3 In those patients lumbar puncture may not be performed.1 Therefore, these 2001;33:969-975. patients are probably only partly represented in our cohort, probably causing an 9. Spanos A, Harrell FE, Jr., Durack DT. Differential diagnosis of acute meningitis. An underestimation of the rate of sepsis and unfavorable outcome among our population. analysis of the predictive value of initial observations. JAMA 1989;262:2700-2707. Second, a substantial proportion of identified patients with bacterial meningitis (32%) were 10. Thompson MJ, Ninis N, Perera R, et al. Clinical recognition of meningococcal disease 2 in children and adolescents. Lancet 2006;367:397-403. not included in our Dutch Meningitis Cohort, which may also have resulted in selection bias. 11. Heyderman RS, British Infection S. Early management of suspected bacterial In conclusion, meningococcal meningitis is still a serious and life-threatening disease. meningitis and meningococcal septicaemia in immunocompetent adults--second edition. J Infect 2005;50:373-374. Neuroimaging before lumbar puncture is an important cause of delay in the administration of antibiotics. Infection with meningococci of cc11 is related to factors associated with sepsis 12. Riordan FA, Thomson AP, Sills JA, Hart CA. Who spots the spots? Diagnosis and treatment of early meningococcal disease in children. BMJ 1996;313:1255-1256. and to unfavorable outcome. 13. Cartwright K, Reilly S, White D, Stuart J. Early treatment with parenteral penicillin in meningococcal disease. BMJ 1992;305:143-147.

14. Harnden A, Ninis N, Thompson M, et al. Parenteral penicillin for children with meningococcal disease before hospital admission: case-control study. BMJ 2006;332:1295-1298.

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15. Hahne SJ, Charlett A, Purcell B, et al. Effectiveness of antibiotics given before admission in reducing mortality from meningococcal disease: systematic review. BMJ 2006;332:1299-1303.

16. Aronin SI, Peduzzi P, Quagliarello VJ. Community-acquired bacterial meningitis: risk stratification for adverse clinical outcome and effect of antibiotic timing. Ann Intern Med 1998;129:862-869.

17. Proulx N, Frechette D, Toye B, Chan J, Kravcik S. Delays in the administration of antibiotics are associated with mortality from adult acute bacterial meningitis. QJM 2005;98:291-298.

18. de Gans J, van de Beek D. Dexamethasone in adults with bacterial meningitis. N Engl J Med 2002;347:1549-1556.

19. van de Beek D, de Gans J, McIntyre P, Prasad K. Steroids in adults with acute bacterial meningitis: a systematic review. Lancet Infect Dis 2004;4:139-143.

20. Tunkel AR, Hartman BJ, Kaplan SL, et al. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis 2004;39:1267-1284.

21. Cooper MS, Stewart PM. Corticosteroid insufficiency in acutely ill patients. N Engl J Med 2003;348:727-734.

22. de Greeff SC, de Melker HE, Spanjaard L, Schouls LM, van der Ende A. Protection from routine vaccination at the age of 14 months with meningococcal serogroup C conjugate vaccine in the Netherlands. Pediatr Infect Dis J 2006;25:79-80.

40049 Piet, Jurgen.indd 36 26-04-16 15:08 Chapter 2

15. Hahne SJ, Charlett A, Purcell B, et al. Effectiveness of antibiotics given before admission in reducing mortality from meningococcal disease: systematic review. BMJ 2006;332:1299-1303.

16. Aronin SI, Peduzzi P, Quagliarello VJ. Community-acquired bacterial meningitis: risk stratification for adverse clinical outcome and effect of antibiotic timing. Ann Intern Med 1998;129:862-869.

17. Proulx N, Frechette D, Toye B, Chan J, Kravcik S. Delays in the administration of antibiotics are associated with mortality from adult acute bacterial meningitis. QJM 2005;98:291-298.

18. de Gans J, van de Beek D. Dexamethasone in adults with bacterial meningitis. N Engl J Med 2002;347:1549-1556.

19. van de Beek D, de Gans J, McIntyre P, Prasad K. Steroids in adults with acute bacterial meningitis: a systematic review. Lancet Infect Dis 2004;4:139-143.

20. Tunkel AR, Hartman BJ, Kaplan SL, et al. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis 2004;39:1267-1284.

21. Cooper MS, Stewart PM. Corticosteroid insufficiency in acutely ill patients. N Engl J Med 2003;348:727-734.

40049 Piet, Jurgen.indd 37 26-04-16 15:08

CHAPTER 3:

Meningococcal factor H binding protein fHbpD184 polymorphism influences clinical course of meningococcal meningitis

Jurgen R. Piet Matthijs C. Brouwer Rachel Exley Stijn van der Veen Diederik van de Beek* Arie van der Ende*

*Both authors contributed equally

PLoS One 2012;7:e47973

40049 Piet, Jurgen.indd 38 26-04-16 15:08

CHAPTER 3: CHAPTER 3:

Meningococcal factor H binding protein fHbpD184 polymorphism influences clinical course of meningococcal meningitis

Meningococcal factor H

binding protein fHbp Jurgen R. Piet D184 Matthijs C. Brouwerpolymorphism influences Rachel Exley Stijn van der Veen clinical course of Diederik van de Beek*meningococcal meningitis Arie van der Ende*

*Both authors contributed equally

Jurgen R. Piet Matthijs C. Brouwer Rachel Exley Stijn van der Veen Diederik van de Beek* Arie van der Ende*

*Both authors contributed equally

PLoS One 2012;7:e47973 PLoS One 2012;7:e47973

40049 Piet, Jurgen.indd 39 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

ABSTRACT INTRODUCTION

severity and outcome. All fHbp of subfamily A had E304 substituted with T304. Of the 15 amino illustrated by a higher abundance of hyper-virulent clones among isolates from patients as acids in fHbp making hydrogen bonds to fH, 3 were conserved, 11 show a similar distribution compared to asymptomatic carriers.11 The distribution of meningococcal housekeeping between the two fHbp subfamilies as the polymorphism at position 304. The proportion of genes has also been described to influence disease course in patients with meningococcal patients infected with meningococci with fHbp of subfamily A with unfavorable outcome meningitis. Meningococci of clonal complex 11 (cc11) were associated with sepsis and was 2.5-fold lower than that of patients infected with meningococci with fHbp of subfamily B unfavorable outcome.2 Furthermore, meningococci with penta-acylated instead of hexa- (2 of 40 (5%) vs. 27 of 213 (13%) (p=0.28). The charge of 2 of 15 amino acids (at position 184 acylated lipid A (due to a mutation in the lpxL1 gene) were found to activate TLR4 less 12 and 306) forming hydrogen bonds was either basic or acidic. The affinity of fHbpK184 and of efficiently, leading to reduced inflammation and coagulopathy. On the host side, case

fHbpD184 for recombinant purified human fH was assessed by Surface Plasmon Resonance control studies found single nucleotide polymorphisms (SNPs) influencing susceptibility and -8 -8 13,14 and showed average KD of 2.60 x 10 and 1.74 x 10 , respectively (not significant). Patients outcome of meningococcal disease. A genome wide association study has recently

infected with meningococci with fHbpD184 were more likely to develop septic shock during identified an SNP in human complement downregulator factor H (fH) to be associated with admission (11 of 42 [26%] vs. 19 of 211 [9%]; p=0.002) resulting in more frequent susceptibility.9 unfavorable outcome (9 of 42 [21%] vs. 20 of 211 [10%]; p=0.026). In conclusion, we Activation of the complement cascade is a critical host defense against meningococcal identified fHBPD184 to be associated with septic shock in patients with meningococcal disease.15 Many pathogenic organisms have found ways to elude complement attack.16 meningitis. Meningococcal fH binding protein (fHbp) binds fH, thus inhibiting complement activation and hampering complement-mediated lysis in human plasma.17,18 This 27 kDa lipoprotein, formerly named genome-derived neisserial antigen 1870 (GNA1870)19 or lipoprotein 208620, is present on the surface of all meningococcal strains.20 It acts as a receptor for fH, although fHbp is not the only factor determining the amount of fH bound.21,22 High levels of fHbp has been found in hyper-virulent meningococcal strains.19 Experiments with fHbp knockout meningococcal strains showed a normal growth in broth, but no survival in blood.23 Some strains have incomplete (truncated) forms of fHbp, but the functional relevance is unknown.24 Inhibiting the binding of fH to the neisserial surface by vaccine derived fHbp

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ABSTRACT INTRODUCTION

Factor H binding protein (fHbp) is an important meningococcal virulence factor, enabling the Neisseria meningitidis (the meningococcus) is the leading cause of meningitis and septicemia meningococcus to evade the complement system, and a main target for vaccination. in young adults, and is associated with substantial mortality and morbidity.1-3 The Recently, the structure of fHBP complexed with factor H (fH) was published. Two fHbp meningococcus is a commensal of the human upper respiratory tract with asymptomatic 4-7 glutamic acids, E283 and E304, form salt bridges with fH, influencing interaction between fHbp colonization occurring in up to 30% of healthy individuals. Invasive meningococcal disease and fH. Fifteen amino acids were identified forming hydrogen bonds with fH. We sequenced has been associated with environmental factors like smoking, living in the same household fHbp of 254 meningococcal isolates from adults with meningococcal meningitis included in a as a patient and disease in proxies.8 Other important risk factors for developing disease can prospective clinical cohort to study the effect of fHbp variants on meningococcal disease be found in bacterial and host genetic factors.9,10 The influence of genetic bacterial factors is 3 severity and outcome. All fHbp of subfamily A had E304 substituted with T304. Of the 15 amino illustrated by a higher abundance of hyper-virulent clones among isolates from patients as acids in fHbp making hydrogen bonds to fH, 3 were conserved, 11 show a similar distribution compared to asymptomatic carriers.11 The distribution of meningococcal housekeeping between the two fHbp subfamilies as the polymorphism at position 304. The proportion of genes has also been described to influence disease course in patients with meningococcal patients infected with meningococci with fHbp of subfamily A with unfavorable outcome meningitis. Meningococci of clonal complex 11 (cc11) were associated with sepsis and was 2.5-fold lower than that of patients infected with meningococci with fHbp of subfamily B unfavorable outcome.2 Furthermore, meningococci with penta-acylated instead of hexa- (2 of 40 (5%) vs. 27 of 213 (13%) (p=0.28). The charge of 2 of 15 amino acids (at position 184 acylated lipid A (due to a mutation in the lpxL1 gene) were found to activate TLR4 less 12 and 306) forming hydrogen bonds was either basic or acidic. The affinity of fHbpK184 and of efficiently, leading to reduced inflammation and coagulopathy. On the host side, case fHbpD184 for recombinant purified human fH was assessed by Surface Plasmon Resonance control studies found single nucleotide polymorphisms (SNPs) influencing susceptibility and -8 -8 13,14 and showed average KD of 2.60 x 10 and 1.74 x 10 , respectively (not significant). Patients outcome of meningococcal disease. A genome wide association study has recently infected with meningococci with fHbpD184 were more likely to develop septic shock during identified an SNP in human complement downregulator factor H (fH) to be associated with admission (11 of 42 [26%] vs. 19 of 211 [9%]; p=0.002) resulting in more frequent susceptibility.9 unfavorable outcome (9 of 42 [21%] vs. 20 of 211 [10%]; p=0.026). In conclusion, we Activation of the complement cascade is a critical host defense against meningococcal identified fHBPD184 to be associated with septic shock in patients with meningococcal disease.15 Many pathogenic organisms have found ways to elude complement attack.16 meningitis. Meningococcal fH binding protein (fHbp) binds fH, thus inhibiting complement activation and hampering complement-mediated lysis in human plasma.17,18 This 27 kDa lipoprotein, formerly named genome-derived neisserial antigen 1870 (GNA1870)19 or lipoprotein 208620, is present on the surface of all meningococcal strains.20 It acts as a receptor for fH, although fHbp is not the only factor determining the amount of fH bound.21,22 High levels of fHbp has been found in hyper-virulent meningococcal strains.19 Experiments with fHbp knockout meningococcal strains showed a normal growth in broth, but no survival in blood.23 Some strains have incomplete (truncated) forms of fHbp, but the functional relevance is unknown.24 Inhibiting the binding of fH to the neisserial surface by vaccine derived fHbp

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antibodies may enhance bactericidal activity of the complement system, resulting in two Meningitis cases distinct mechanisms of antibody-mediated vaccine efficacy.17 The Dutch Meningitis Cohort Study from 1998-2002 was a prospective nationwide

Epidemiological surveys showed a high fHbp sequence diversity leading to great variations in observational cohort study of adults with community-acquired bacterial meningitis in the 26 fHbp structure. Currently, over 550 fHbp protein types have been identified Netherlands. Inclusion and exclusion criteria have been described extensively 2,26 (http://pubmlst.org/neisseria/fHbp/). These protein types can be subdivided in 3 major fHbp elsewhere. In short, patients older than 16 years, who had bacterial meningitis confirmed subgroups: variant 1 (subfamily B), and variants 2 and 3 (collectively called subfamily A).19,20 by culture of cerebrospinal fluid (CSF), were included from October 1998 to April 2002 after The overall fHbp architecture appears to be modular, consisting of combinations of five identification by the Netherlands Reference Laboratory for Bacterial Meningitis. This modular variable segments, each flanked by blocks of two to five conserved amino acids.25 laboratory received CSF isolates from 85% of all patients with bacterial meningitis in the Each of the modular segments is derived from one of two lineages and based on the Netherlands. The treating physician was contacted, and informed consent was obtained modular classification fHbp can be classified in six modular groups (I-VI). Recently, the from all participating patients or their legally authorized representatives. structure of fHbp of subfamily B in complex with fH was published.18 Two fHbp amino acids, Case-record forms were used to collect data on patients’ history, symptoms and signs on E and E , were found to be important for the interaction with fH, via salt bridges. A 283 304 admission, laboratory findings at admission, clinical course, outcome and neurologic findings further fifteen amino acids were identified hydrogen bonding to fH. at discharge, and treatment. Outcome was graded according to the Glasgow Outcome Scale. A score of one on this scale indicates death; a score of two a vegetative state (the patient is Results of studies of fHbp variance among patients’ isolates can have important unable to interact with the environment); a score of three severe disability (the patient is consequences for further vaccine development as inclusion of more fHbp proteins could unable to live independently but can follow commands); a score of four moderate disability improve vaccine efficacy. Furthermore, it is unclear if the variation in fHbp and its encoded (the patient is capable of living independently but unable to return to work or school); and a protein influences disease characteristics and outcome. Therefore we investigated the score of five mild or no disability (the patient is able to return to work or school). A favorable distribution of fHbp protein variants among the meningococcal isolates of 254 patients with outcome was defined as a score of five, and an unfavorable outcome as a score below. The meningitis from a nation-wide prospective cohort study and correlate protein types with Glasgow Outcome Scale is a well-validated instrument with good inter-observer clinical characteristics. agreement.27 At discharge, all surviving patients underwent a neurologic examination performed by a neurologist, which included the assessment of the Glasgow Outcome Scale.

MATERIALS AND METHODS Bacterial strains

Ethics statement Upon reception of bacterial isolates in the Netherlands Reference Laboratory for Bacterial Meningitis, a monoculture of the causative isolate was frozen and stored at –80° C. All This observational study with anonymous patient data was carried out in accordance with isolates were passaged less than 5 times. Serogrouping and genotyping by Multilocus the Dutch privacy legislation. Written informed consent to use data made anonymous was Sequence Typing (MLST) have previously been described.2 obtained from the patient (if possible) or from the patient's legal representative. The Dutch

Meningitis Cohort Study was approved by the ethics committee of the Academic Medical Center in Amsterdam.

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Epidemiological surveys showed a high fHbp sequence diversity leading to great variations in observational cohort study of adults with community-acquired bacterial meningitis in the 26 fHbp structure. Currently, over 550 fHbp protein types have been identified Netherlands. Inclusion and exclusion criteria have been described extensively 2,26 (http://pubmlst.org/neisseria/fHbp/). These protein types can be subdivided in 3 major fHbp elsewhere. In short, patients older than 16 years, who had bacterial meningitis confirmed subgroups: variant 1 (subfamily B), and variants 2 and 3 (collectively called subfamily A).19,20 by culture of cerebrospinal fluid (CSF), were included from October 1998 to April 2002 after The overall fHbp architecture appears to be modular, consisting of combinations of five identification by the Netherlands Reference Laboratory for Bacterial Meningitis. This modular variable segments, each flanked by blocks of two to five conserved amino acids.25 laboratory received CSF isolates from 85% of all patients with bacterial meningitis in the 3 Each of the modular segments is derived from one of two lineages and based on the Netherlands. The treating physician was contacted, and informed consent was obtained modular classification fHbp can be classified in six modular groups (I-VI). Recently, the from all participating patients or their legally authorized representatives. structure of fHbp of subfamily B in complex with fH was published.18 Two fHbp amino acids, Case-record forms were used to collect data on patients’ history, symptoms and signs on E and E , were found to be important for the interaction with fH, via salt bridges. A 283 304 admission, laboratory findings at admission, clinical course, outcome and neurologic findings further fifteen amino acids were identified hydrogen bonding to fH. at discharge, and treatment. Outcome was graded according to the Glasgow Outcome Scale. A score of one on this scale indicates death; a score of two a vegetative state (the patient is Results of studies of fHbp variance among patients’ isolates can have important unable to interact with the environment); a score of three severe disability (the patient is consequences for further vaccine development as inclusion of more fHbp proteins could unable to live independently but can follow commands); a score of four moderate disability improve vaccine efficacy. Furthermore, it is unclear if the variation in fHbp and its encoded (the patient is capable of living independently but unable to return to work or school); and a protein influences disease characteristics and outcome. Therefore we investigated the score of five mild or no disability (the patient is able to return to work or school). A favorable distribution of fHbp protein variants among the meningococcal isolates of 254 patients with outcome was defined as a score of five, and an unfavorable outcome as a score below. The meningitis from a nation-wide prospective cohort study and correlate protein types with Glasgow Outcome Scale is a well-validated instrument with good inter-observer clinical characteristics. agreement.27 At discharge, all surviving patients underwent a neurologic examination performed by a neurologist, which included the assessment of the Glasgow Outcome Scale.

MATERIALS AND METHODS Bacterial strains

Meningitis Cohort Study was approved by the ethics committee of the Academic Medical Center in Amsterdam.

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Bioinformatic and phylogenetic analysis The Mann-Whitney U test was used to identify differences between groups with respect to continuous variables, and dichotomous variables were compared with use of the χ2 test or Sequences were assembled and analyzed using the Codoncode aligner software suite. Fischer’s Exact test. All statistical tests were two-tailed, with p<0.05 regarded as significant. Evolutionary history was inferred using the Neighbor-Joining method.28 All positions Analyses were undertaken with PASW Statistics 18.0. We used logistic regression analysis to containing gaps and missing data were eliminated from the dataset (Complete deletion calculate odds ratios and 95% CI to assess the strength of the association between potential option). There were a total of 251 positions in the final dataset. Phylogenetic analyses were prognostic factors and the probability of an unfavorable outcome. conducted in MEGA4.29

was replaced by codon AAA encoding basic residue, lysine (K184). All sequences were checked Multilocus Sequence Typing and fHbp sequencing by sequencing. Serogrouping results were available for all 254 meningococcal strains and have been described previously.2 Of these, 172 (68%) were of serogroup B, 78 (31%) of serogroup C, 3 fHbp purification and interaction with fH (1%) of serogroup Y, and 1 (<1%) was of serogroup W. MLST data of 254 isolates showed 91 Following growth of B834(DE3) strains harboring pET21fHbp1P10 or pET21fHbp1P10D184K unique sequence types (ST). The most prevalent clonal complexes (cc) were cc41/44 (41%), overnight at 21°C in LB containing 1mM IPTG fHbp, recombinant fHbp was purified by cc11 (24%), and cc32 (16%). All cc11 strains were serogroup C.2 One isolate contained an 143

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PCR and sequencing affinity chromatography using Ni-NTA Magnetic Agarose Beads (Qiagen). Interaction with recombinant purified human fH (SCR67) was assessed by Surface Plasmon Resonance using a PCR templates were prepared by boiling ~100 N. meningitidis colonies from chocolate agar ProteOn XPR36 (BioRad). fHbp was immobilized on a ProteOn GLM sensor chip and plates in 200 µl of distilled H2O for 5 min, and then centrifuged. 1 µl of the supernatant was increasing concentrations of fH67 were injected over the flow channels at 40 µl/min and used in the PCR reaction (10 µl total volume). The primers used for PCR and sequencing are allowed to dissociate for 300 seconds. ProteOn manager software was used to calculate the listed in Supplementary Table 4. The amplification steps were 95 °C for 5 min, 30 cycles of 95 KD. °C for 30 s, 58 °C for 30 s, and 72 °C for 90 s, followed by a final extension step at 72 °C for 5 min. Amplicons were 1:9 diluted and 1 µl was used in the sequencing reaction. Statistics 3 Bioinformatic and phylogenetic analysis The Mann-Whitney U test was used to identify differences between groups with respect to continuous variables, and dichotomous variables were compared with use of the χ2 test or Sequences were assembled and analyzed using the Codoncode aligner software suite. Fischer’s Exact test. All statistical tests were two-tailed, with p<0.05 regarded as significant. Evolutionary history was inferred using the Neighbor-Joining method.28 All positions Analyses were undertaken with PASW Statistics 18.0. We used logistic regression analysis to containing gaps and missing data were eliminated from the dataset (Complete deletion calculate odds ratios and 95% CI to assess the strength of the association between potential option). There were a total of 251 positions in the final dataset. Phylogenetic analyses were prognostic factors and the probability of an unfavorable outcome. conducted in MEGA4.29

In vitro mutagenesis and cloning of fHbp RESULTS fHbp_10 (1.P10) was chosen as a representative fHbp having an acidic residue (D, aspartic acid) at position 184. The gene encoding this protein was amplified from N. meningitidis Cohort of patients with meningococcal meningitis strain M01 240185 (serogroup B, clonal complex ST-11; kindly provided by dr. R. Borrow, From October 1998 to April 2002, 258 episodes of community-acquired meningococcal Meningococcal Reference Unit, Health Protection Agency North West Laboratory, meningitis in 258 patients were included. Detailed clinical and microbiological characteristics Manchester) using primers NG2145 and NG2143 (Supplementary Table 4) and cloned into have been described previously.2,26 Patient characteristics, clinical course and the causative pET21b at NdeI-XhoI restriction sites for expression as a C-terminally 6xHIS tagged fusion bacterial strain isolated from the cerebrospinal fluid (CSF) were available for 254 (98.4%) of protein lacking the N-terminal sequence required for lipid attachment. This plasmid was the 258 meningococcal meningitis episodes. The case fatality rate was 7%, and 12% patients subjected to mutagenesis using the QuikChange site directed mutagenesis kit (Agilent had an unfavorable outcome, defined as a score from one to four on the Glasgow outcome Technologies) with primers 1P10DKF and 1P10DKR (Supplementary Table 4) to produce scale.30 plasmid pET21fHbp1P10D184K, in which codon GAT encoding acidic residue aspartic acid (D184) was replaced by codon AAA encoding basic residue, lysine (K184). All sequences were checked Multilocus Sequence Typing and fHbp sequencing by sequencing. Serogrouping results were available for all 254 meningococcal strains and have been described previously.2 Of these, 172 (68%) were of serogroup B, 78 (31%) of serogroup C, 3 fHbp purification and interaction with fH (1%) of serogroup Y, and 1 (<1%) was of serogroup W. MLST data of 254 isolates showed 91 Following growth of B834(DE3) strains harboring pET21fHbp1P10 or pET21fHbp1P10D184K unique sequence types (ST). The most prevalent clonal complexes (cc) were cc41/44 (41%), overnight at 21°C in LB containing 1mM IPTG fHbp, recombinant fHbp was purified by cc11 (24%), and cc32 (16%). All cc11 strains were serogroup C.2 One isolate contained an 143

44 45

40049 Piet, Jurgen.indd 45 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

bp duplication in fHbp resulting in a premature stop codon and was excluded as non- Table 1. Distribution of meningococcal fHbp subfamilies and variants among clonal complexes. fHbp subfamilya functional. Full-length fHbp sequences could be obtained from the 253 meningococcal A B A/B hybrid variantb variant strains. DNA and protein allele numbers were assigned by the Neisseria Factor H binding clonal complex 2 3 1 protein sequence typing website (http://pubmlst.org/neisseria/fHbp). All meningococcal cc11 10 2 50 cc167 1 serogroup data, sequence types, clonal complexes, and fHbp DNA/protein allele are shown cc174 1 cc18 2 in Supplementary Table 1. cc213 1 cc22 1 fHbp protein type prevalence cc269 2 1 13 cc32 1 39 Among 253 isolates, 42 unique fHbp sequences were found, of which 40 encoded unique cc35 1 cc364 1 protein sequences of mature fHbp. Of 253 fHbp protein sequences, 212 (84%) could be cc41/44 4 1 98 1 assigned to subfamily B (nomenclature according to Fletcher et al.20) or variant 1 cc461 1 1 cc60 4 (nomenclature according to Masignani et al.).19 Of these 212 isolates 201 were of modular cc8 9 1

25 cc92 1 group I (nomenclature according to Beernink and Granhoff ) and 11 of modular group IV. singleton 1 1 4 all 32 8 212 1 Forty isolates (16%) belonged to subfamily A of which 32 belong to variant 2 (13%) and 8 to a 20 Nomenclature according to Fletcher et al. b 19 variant 3 (3%; Table 1). The isolates with subfamily A fHbp comprised modular groups II, III, V Nomenclature according to Masignani et al. and VI, with 3, 12, 5 and 20 isolates, respectively. Strain 2011148 contained an intermediate fHbp with sequences from subfamily A and B. Of 161 serogroup B isolates, 15 had subfamily A fHbp, 155 had subfamily B and one had a hybrid A/b fHbp. Of 78 serogroup C isolates, 21 had subfamily A fHbp and 57 had subfamily B fHbp. The one serogroup W and the three serogroup Y isolates had subfamily A fHbp (Supplementary Table 1). The major hyper- virulent clonal complexes cc41/44, cc32 and cc11 had predominantly subfamily B fHbp or variant 1: 98 of 104 (94%), 39 of 40 (98%) and 50 of 62 (81%), respectively. Isolates of cc8 had predominantly fHbp of subfamily A: 9 of 10 (90%; all variant 2) (Table 1). The most prevalent fHbp protein types (protein type from the fHbp database at http://pubmlst.org/neisseria/fHbp/) were 14 (35%), 1 (15%) and 10 (10%). They were strongly associated with meningococcal sequence type: Of 89 isolates with fHbp protein type 14, 87 (98%) were cc 41/44, 38 of 38 (100%) of fHbp type 1 were cc32 and 25 of 26 fHbp

type 10 isolates were from cc11 (Table 2).

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40049 Piet, Jurgen.indd 46 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

bp duplication in fHbp resulting in a premature stop codon and was excluded as non- Table 1. Distribution of meningococcal fHbp subfamilies and variants among clonal complexes. fHbp subfamilya functional. Full-length fHbp sequences could be obtained from the 253 meningococcal A B A/B hybrid variantb variant strains. DNA and protein allele numbers were assigned by the Neisseria Factor H binding clonal complex 2 3 1 protein sequence typing website (http://pubmlst.org/neisseria/fHbp). All meningococcal cc11 10 2 50 cc167 1 serogroup data, sequence types, clonal complexes, and fHbp DNA/protein allele are shown cc174 1 cc18 2 in Supplementary Table 1. cc213 1 cc22 1 fHbp protein type prevalence cc269 2 1 13 cc32 1 39 Among 253 isolates, 42 unique fHbp sequences were found, of which 40 encoded unique cc35 1 3 cc364 1 protein sequences of mature fHbp. Of 253 fHbp protein sequences, 212 (84%) could be cc41/44 4 1 98 1 assigned to subfamily B (nomenclature according to Fletcher et al.20) or variant 1 cc461 1 1 cc60 4 (nomenclature according to Masignani et al.).19 Of these 212 isolates 201 were of modular cc8 9 1

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40049 Piet, Jurgen.indd 47 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

Associations of fHbp polymorphism with clinical characteristics

9 1

31 (13)

2 2 10 7 Other

Of the two residues in fHbp, E283 and E304, which are involved in salt bridges to fH and found

3 (1)

131 to be important for the interaction with fH , E283 is conserved among fHbp of all 253

isolates. All fHbp of subfamily A (variant 2 and 3) had E304 substituted with T304. Of the 15

11 (4)

129 18

amino acids in fHbp making hydrogen bonds to fH , 3 were conserved: K264 (all except one

3 (1)

89 isolate), D266 and V272. Of the 12 amino acids changed, 11 show a similar distribution

between the two fHbp subfamilies as the polymorphism at position 304 if change in charge

3 (1)

or size of side groups were considered (Supplementary Table 2). The hybrid fHbp of strain

5 (2)

2011148 contained fHbpE304 and 3 of 10 of the other subfamily A polymorphisms. The results

7 (3) 1

1 3 19 by Seib and colleagues who investigated the binding of fH to purified fHbp of 12 different

protein types (6 of each subfamily A and B) showed a 5-fold higher thermodynamic

2 11 (4)

dissociation constant KD for the interaction between fH and fHbp of subfamily B than for that (4)

15 between fH and fHbp of subfamily A (Supplementary Table 3). The average KD's were 153 nM and 29 nM, respectively (p=0.02, calculated from Table 3 in22), demonstrating that subfamily

89 (35) 1

87 14 B fHbp has a lower affinity for fH. However, physiological fH concentration is in the order of

31 23

. 2 μM , which would give rise to saturated fH-fHbp binding. The difference in KD is mainly 8 (3) 2 4

1 13

caused by a 14-fold higher dissociation constant in case of fHbp of subfamily B (p=0.03)

a (Supplementary Table 3). Interestingly, the association constant of the fH-fHbp binding is 26 (10)

25 1 10 2.5-fold lower in case of fHbp of subfamily A (p=0.04), indicating a slightly reduced on-rate of

7 (3)

1 5 4

fH to fHbp of subfamily A. Together, these differences between fHbp of subfamily A and B protein type protein

prompt us to evaluate the relation between clinical course and infection with meningococci 38 (15)

1 fHbp fHbp

with either subfamily fHbp. Surprisingly, the proportion of patients infected with

meningococci with fHbp of subfamily A (fHbp of strain 2011148 was considered to be of 253 (100) 11 (4) 6 (2) 4 (2) 10 (4) 16 (6) 40 (16) 62 (24) 104 (41) No. (%) subfamily B) with unfavorable outcome was 2.5-fold lower than that of patients infected

database at http://pubmlst.org/neisseria/fHbp/ at database with meningococci with fHbp of subfamily B (2 of 40 (5%) vs. 27 of 213 (13%), though not

reaching statistical significance (p=0.28). Including strain 2011148 in the subfamily A group

did not affect these percentages. 5 complex 37 complex - -

Of note, in subfamily B fHbp proteins, the residue at position 184 and 306 was either basic or

acidic, whereas subfamily A fHbp proteins were basic in these positions. At position 306 of

Table 2. fHbp protein type and clonal complexes. complexes. clonal and type protein fHbp 2. Table a Changing lysine at position 306 in fHbp to a glutamic acid in the fHbp-fH model structure

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40049 Piet, Jurgen.indd 48 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

Associations of fHbp polymorphism with clinical characteristics

9 1

31 (13)

2 2 10 7 Other

Of the two residues in fHbp, E283 and E304, which are involved in salt bridges to fH and found

3 (1)

131 to be important for the interaction with fH , E283 is conserved among fHbp of all 253

isolates. All fHbp of subfamily A (variant 2 and 3) had E304 substituted with T304. Of the 15

11 (4)

129 18

amino acids in fHbp making hydrogen bonds to fH , 3 were conserved: K264 (all except one

3 (1)

89 isolate), D266 and V272. Of the 12 amino acids changed, 11 show a similar distribution

between the two fHbp subfamilies as the polymorphism at position 304 if change in charge

3 (1)

or size of side groups were considered (Supplementary Table 2). The hybrid fHbp of strain

5 (2)

2011148 contained fHbpE304 and 3 of 10 of the other subfamily A polymorphisms. The results

7 (3) 1

1 3 19 by Seib and colleagues who investigated the binding of fH to purified fHbp of 12 different

protein types (6 of each subfamily A and B) showed a 5-fold higher thermodynamic

2 11 (4)

dissociation constant KD for the interaction between fH and fHbp of subfamily B than for that (4)

15 between fH and fHbp of subfamily A (Supplementary Table 3). The average KD's were 153 nM and 29 nM, respectively (p=0.02, calculated from Table 3 in22), demonstrating that subfamily

89 (35) 1

87 14 B fHbp has a lower affinity for fH. However, physiological fH concentration is in the order of

31 23

. 2 μM , which would give rise to saturated fH-fHbp binding. The difference in KD is mainly 8 (3) 2 4

1 13

caused by a 14-fold higher dissociation constant in case of fHbp of subfamily B (p=0.03)

a (Supplementary Table 3). Interestingly, the association constant of the fH-fHbp binding is 26 (10)

25 1 10 2.5-fold lower in case of fHbp of subfamily A (p=0.04), indicating a slightly reduced on-rate of

7 (3)

1 5 4

fH to fHbp of subfamily A. Together, these differences between fHbp of subfamily A and B protein type protein

prompt us to evaluate the relation between clinical course and infection with meningococci 38 (15)

1 fHbp fHbp

with either subfamily fHbp. Surprisingly, the proportion of patients infected with

reaching statistical significance (p=0.28). Including strain 2011148 in the subfamily A group

did not affect these percentages. 5 complex 37 complex - -

Of note, in subfamily B fHbp proteins, the residue at position 184 and 306 was either basic or

acidic, whereas subfamily A fHbp proteins were basic in these positions. At position 306 of

60 complex 8 complex/Cluster A4 269 complex 32 complex/ET 11 complex/ET 41/44 complex/Lineage 3 fHbp in meningococci from patients we found either a lysine (basic) or a glutamine (acidic). ------database at http://pubmlst.org/neisseria/fHbp/ at fHbp database the from type protein fHbp Total (%) Total Other Non typable Non ST ST ST ST ST ST Clonal complex Table 2. fHbp protein type and clonal complexes. complexes. clonal and type protein fHbp 2. Table a Changing lysine at position 306 in fHbp to a glutamic acid in the fHbp-fH model structure

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40049 Piet, Jurgen.indd 49 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

18 a reported by Schneider and colleagues may only weakly enhance the interaction between Table 3. Associations of meningococci containing fHbpD184 with clinical characteristics . fHbpD184 (n=42) fHbpH184 or fHbpK184 (n=211) P-value fhbp and fH through the interaction with a tyrosine at 352 in fH. Of 253 isolates, 154 had an Demographics acidic instead of a basic residue at position 306, without a correlation with the course of Age - yr (Mean±SD) 33 (21) 37 (±18) 0.016 Duration of symptoms <24 hr 21/41 (51) 101/205 (49) 0.82 disease. Symptoms at presentation Rash 26/42 (62) 136/209 (65) 0.70 At position 184 of fHbp in patients’ meningococcal isolates an histidine (basic), a lysine Focal neurological deficitsb 11/42 (26) 42/211 (20) 0.36

(basic) or a aspartic acid (acidic) was found. In the published fHbp-fH model structure18 the Diastolic BP (n=242) 73 (60-80) 66 (60-90) 0.66 Blood parameters histidine at position 184 in fHbp is close to a loop of fH with His402, Gly403, Arg404, Lys405, Positive blood culture 22/41 (54) 106/181 (59) 0.57 Thrombocyte count, 109/L (n=240) 166 (125-226) 151 (116-185) 0.102 Phe406, Val407. Changing Lys184 in fHbp to Asp184 may improve binding between fHbp and fH Creatinin µmol/L (n=246) 95 (75-123) 94 (79-171) 0.258 due to the interaction between the acidic Asp184 in fHbp with the basic residues Arg404 and Cerebrospinal fluid parameters 3 e or Lys405 in fH. We assessed the interaction between fHbpK184 or fHbpD184 with recombinant CSF leukocyte count (cells/mm , n=237) 5419 (1676-12478) 3500 (552-12307) 0.25 CSF/blood glucose ratio (n=246) 0.088 (0.01-0.31) 0.02 (0.009-0.29) 0.10 purified human fH (SCR67) by Surface Plasmon Resonance. fHbp_10 (1.P10) was chosen as a Outcome parameters representative fHbp having an acidic residue (D, aspartic acid) at position 184. The average Sepsis 11/42 (26) 19/211 (9) 0.002 -8 -8 Unfavorable outcome (GOS = 1-4) 9/42 (21) 20/211 (10) 0.026 K was 1.74 x 10 M and 2.60 x 10 M for fHbp and fHbp , respectively, which was not a D D184 K184 Data are number/number assessed (%) or median (25th–75th percentile), unless otherwise stated. b significantly different. Defined as aphasia, monoparesis, or hemiparesis) and cranial nerve palsies

Of 253 isolates, 42 (41 (98%) of serogroup B and 1 (2%) of serogroup C) had a fHbpD184 DISCUSSION (acidic residue at position 184), while 211 had fHbpH184 or fHbpK184 (both with a basic residue

at position 184). Of the 42 isolates with fHbpD184, 28 (67%) belonged to cc11, while the Over the last decade, fHbp has become of substantial interest: it is a key component in two remaining 14 isolates belonged to 6 clonal complexes; 2 were singletons. There were clear forthcoming vaccines. Subvariant fHbp-1.1 (subfamily B), is included in the vaccine 4CMenB

correlations with disease progression. Patients infected with meningococci with fHbpD184 of Novartis, while fHbp-1.55 (subfamily B) and fHbp-3.45 (subfamily A) are included in the were more likely to develop septic shock during admission (11 of 42 [26%] vs. 19 of 211 [9%]; vaccine rLP2086 developed by Pfizer. In addition, it is recognized as an important virulence p=0.002) contributing to a higher rate of unfavorable outcome (9 of 42 [21%] vs. 20 of 211 factor, essential for survival in human blood.23 Here we show that variance in meningococcal

[10%]; p=0.026; Table 3). Of 34 patients infected with cc11 meningococci with fHbpH184 or fHbp sequences cultured from 254 meningitis patients included in a prospective nationwide

fHbpK184 (all of subfamily B), 4 of 34 (12%) developed sepsis during clinical course as cohort study was associated with disease severity and outcome. Of 254 strains evaluated in

compared to 10 of 28 (36%) among the cc11 isolates with fHbpD184 (P=0.034). In a this study, only 1 did not contain functional fHbp, consistent with the attributed important multivariate regression analysis including age, admission score on the Glasgow coma scale, role in survival of the organism in its host.17 One hybrid variant containing sequences from

and focal neurological deficits, the association fHbpD184 with unfavorable outcome was both subfamily A and B was detected. An A/B hybrid isolated in 1960 in the Netherlands has preserved (odds ratio = 2.89; 95% confidence interval 1.15-7.25; p=0.024). been shown to bind fH.22 The proportion of patients with unfavorable outcome was lower among patients infected with meningococci containing fHbp of subfamily A, although this did

not reach significance. Patients infected with meningococci with fHbpD184 were more likely to develop septic shock during admission resulting in a higher rate of unfavorable outcome.

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40049 Piet, Jurgen.indd 50 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

(basic) or a aspartic acid (acidic) was found. In the published fHbp-fH model structure18 the Diastolic BP (n=242) 73 (60-80) 66 (60-90) 0.66 Blood parameters histidine at position 184 in fHbp is close to a loop of fH with His402, Gly403, Arg404, Lys405, Positive blood culture 22/41 (54) 106/181 (59) 0.57 Thrombocyte count, 109/L (n=240) 166 (125-226) 151 (116-185) 0.102 Phe406, Val407. Changing Lys184 in fHbp to Asp184 may improve binding between fHbp and fH 3 Creatinin µmol/L (n=246) 95 (75-123) 94 (79-171) 0.258 due to the interaction between the acidic Asp184 in fHbp with the basic residues Arg404 and Cerebrospinal fluid parameters 3 e or Lys405 in fH. We assessed the interaction between fHbpK184 or fHbpD184 with recombinant CSF leukocyte count (cells/mm , n=237) 5419 (1676-12478) 3500 (552-12307) 0.25 CSF/blood glucose ratio (n=246) 0.088 (0.01-0.31) 0.02 (0.009-0.29) 0.10 purified human fH (SCR67) by Surface Plasmon Resonance. fHbp_10 (1.P10) was chosen as a Outcome parameters representative fHbp having an acidic residue (D, aspartic acid) at position 184. The average Sepsis 11/42 (26) 19/211 (9) 0.002 -8 -8 Unfavorable outcome (GOS = 1-4) 9/42 (21) 20/211 (10) 0.026 K was 1.74 x 10 M and 2.60 x 10 M for fHbp and fHbp , respectively, which was not a D D184 K184 Data are number/number assessed (%) or median (25th–75th percentile), unless otherwise stated. b significantly different. Defined as aphasia, monoparesis, or hemiparesis) and cranial nerve palsies

Of 253 isolates, 42 (41 (98%) of serogroup B and 1 (2%) of serogroup C) had a fHbpD184 DISCUSSION (acidic residue at position 184), while 211 had fHbpH184 or fHbpK184 (both with a basic residue at position 184). Of the 42 isolates with fHbpD184, 28 (67%) belonged to cc11, while the Over the last decade, fHbp has become of substantial interest: it is a key component in two remaining 14 isolates belonged to 6 clonal complexes; 2 were singletons. There were clear forthcoming vaccines. Subvariant fHbp-1.1 (subfamily B), is included in the vaccine 4CMenB correlations with disease progression. Patients infected with meningococci with fHbpD184 of Novartis, while fHbp-1.55 (subfamily B) and fHbp-3.45 (subfamily A) are included in the were more likely to develop septic shock during admission (11 of 42 [26%] vs. 19 of 211 [9%]; vaccine rLP2086 developed by Pfizer. In addition, it is recognized as an important virulence p=0.002) contributing to a higher rate of unfavorable outcome (9 of 42 [21%] vs. 20 of 211 factor, essential for survival in human blood.23 Here we show that variance in meningococcal

[10%]; p=0.026; Table 3). Of 34 patients infected with cc11 meningococci with fHbpH184 or fHbp sequences cultured from 254 meningitis patients included in a prospective nationwide fHbpK184 (all of subfamily B), 4 of 34 (12%) developed sepsis during clinical course as cohort study was associated with disease severity and outcome. Of 254 strains evaluated in compared to 10 of 28 (36%) among the cc11 isolates with fHbpD184 (P=0.034). In a this study, only 1 did not contain functional fHbp, consistent with the attributed important multivariate regression analysis including age, admission score on the Glasgow coma scale, role in survival of the organism in its host.17 One hybrid variant containing sequences from and focal neurological deficits, the association fHbpD184 with unfavorable outcome was both subfamily A and B was detected. An A/B hybrid isolated in 1960 in the Netherlands has preserved (odds ratio = 2.89; 95% confidence interval 1.15-7.25; p=0.024). been shown to bind fH.22 The proportion of patients with unfavorable outcome was lower among patients infected with meningococci containing fHbp of subfamily A, although this did

not reach significance. Patients infected with meningococci with fHbpD184 were more likely to develop septic shock during admission resulting in a higher rate of unfavorable outcome.

50 51

40049 Piet, Jurgen.indd 51 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

Compared to the published structure of a subfamily B fHbp18, fHbp of subfamily A isolates in expression levels of fHbp, physiological conditions of the host that may affect factor H this study had a number of substitutions at positions important for the interaction with fH. concentrations) than simply the affinity of fHbp with factor H.

E304, one of the two residues involved in salt bridges to fH, is substituted by T304 in subfamily In our study, fHbp of subfamily B was found to be dominant among all major hyper-virulent A fHbp. Importance of these salt bridge forming amino acids in binding to fH was clonal complexes, indicating that disease is caused mainly by meningococci with fHbp of demonstrated with the double mutant fHbpE283A,E304A, having a more than two orders of subfamily B. Patients infected with meningococci with fHbpD184 (acidic residue substituting a magnitude reduced affinity for fH, with almost no interaction at analyte concentrations 18 basic residue at position 184) were more likely to develop septic shock during admission around ten times the wild-type KD. However, single mutants were not investigated. Of 15 resulting in a higher rate of unfavorable outcome. Substitution of H184 to D184 in the fHbp-fH amino acids making hydrogen bonds to fH, 11 are substituted with an amino acid with a model structure18 suggest a stronger interaction between fHbp and fH. Results of binding different charge or a different size (mostly smaller) of the side chain in subfamily A fHbp. experiments however did not show significant differences in binding affinity between fH and This also suggests that fHbp of subfamily A might have a lower affinity for fH than fHbp of the different fHbp184 variants. Possibly, the binding assay might not be sensitive enough to subfamily B. Hence, meningococci with subfamily A fHbp will be less protected in blood of detect differences that are biologically relevant. Alternatively, fHbp might have other still the host, resulting in less severe disease. This seems to be contradicted by the results by Seib unknown functions which are modified by this substitution. A stronger interaction between and colleagues22 who investigated the binding of fH to purified fHbp of 12 different protein fHbpD184 and fH would lead to better protection of meningococci with fHbpD184 in the blood types (6 of each subfamily A and B). They state that the interaction between fH and fHbp of stream, which is consistent with more severe disease in patients infected with these subfamily B on average had a 5-fold higher dissociation constant (KD) than that of fH and meningococci. Of these patients, 67% were infected with meningococci belonging to cc11, fHbp of subfamily A, mainly due to a 13-fold higher off-rate (kd). It is unclear if this is but the predictive effect of fHbpD184 on outcome was preserved. Among patients infected compatible with the published binding structure. Moreover, under physiological conditions, with cc11 isolates, patients infected with meningococci with fHbpD184 were more likely to i.e., about 2 μM fH31, binding of fH to fHbp is saturated and without multiplying develop sepsis than those infected with meningococci with fHbpH184 or fHbpK184. Previously, meningococci virtually all fHbps would be occupied by fH in blood.23 However, when we reported cc11 meningococcal isolates to be associated with sepsis and unfavorable meningococci divide, non-ligand bound fHbp will arise and the on-rate (ka) determines the outcome.2 The present results, indicate that part of the virulence of cc11 isolates might be speed with which fHbp will be occupied. Interestingly, the ka of fH to fHbp binding seems to attributed to fHbpD184. be reduced in case of subfamily A fHbp. This might reduce survival of meningococci with subfamily A fHbp in the blood. Meningococci with subfamily B fHbp will be more protected During our inclusion period, routine serogroup C vaccination had not yet started in the by fH binding. However, we did not observe a significant difference between the rate of Netherlands. Vaccinating all children and adolescents from 14 months to 19 years -starting in unfavorable outcome in patients infected with meningococci with fHbp of subfamily A and June 2002- has dramatically reduced serogroup C disease in the Netherlands from 31% in our that of patients infected with meningococci with fHbp of subfamily B. In addition, the level cohort to an incidence of 6.5% in 2009 (http://www.amc.nl/upload/teksten/medical of survival in human sera of recombinant MC58 strains expressing the diverse subvariants microbiology/nrlbm jv/jv2009site.pdf).32 Therefore fHbp protein type distribution will most 22 did not correlate with levels of fH binding. Similar results were obtained by Dunphy and likely have changed, warranting epidemiological surveys of a more recent period. colleagues showing that expression by strain H44/76 of two natural fHbp sequence variants In conclusion, we found fHbp to be associated with severe disease and unfavorable with lower fH affinity had minimal or no effect on non-immune clearance in blood or D184 outcome. This study provides insights regarding the clinical relevance of this potent vaccine plasma.23 Most likely, the outcome of the interaction between meningococcal fHbp and target that is included in two forthcoming serogroup B vaccines. Because of the natural factor H on the survival of meningococci in blood depends on a multiple factors, like

52 53

40049 Piet, Jurgen.indd 52 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

E304, one of the two residues involved in salt bridges to fH, is substituted by T304 in subfamily In our study, fHbp of subfamily B was found to be dominant among all major hyper-virulent A fHbp. Importance of these salt bridge forming amino acids in binding to fH was clonal complexes, indicating that disease is caused mainly by meningococci with fHbp of demonstrated with the double mutant fHbpE283A,E304A, having a more than two orders of subfamily B. Patients infected with meningococci with fHbpD184 (acidic residue substituting a magnitude reduced affinity for fH, with almost no interaction at analyte concentrations 18 basic residue at position 184) were more likely to develop septic shock during admission around ten times the wild-type KD. However, single mutants were not investigated. Of 15 resulting in a higher rate of unfavorable outcome. Substitution of H184 to D184 in the fHbp-fH amino acids making hydrogen bonds to fH, 11 are substituted with an amino acid with a model structure18 suggest a stronger interaction between fHbp and fH. Results of binding 3 different charge or a different size (mostly smaller) of the side chain in subfamily A fHbp. experiments however did not show significant differences in binding affinity between fH and This also suggests that fHbp of subfamily A might have a lower affinity for fH than fHbp of the different fHbp184 variants. Possibly, the binding assay might not be sensitive enough to subfamily B. Hence, meningococci with subfamily A fHbp will be less protected in blood of detect differences that are biologically relevant. Alternatively, fHbp might have other still the host, resulting in less severe disease. This seems to be contradicted by the results by Seib unknown functions which are modified by this substitution. A stronger interaction between and colleagues22 who investigated the binding of fH to purified fHbp of 12 different protein fHbpD184 and fH would lead to better protection of meningococci with fHbpD184 in the blood types (6 of each subfamily A and B). They state that the interaction between fH and fHbp of stream, which is consistent with more severe disease in patients infected with these subfamily B on average had a 5-fold higher dissociation constant (KD) than that of fH and meningococci. Of these patients, 67% were infected with meningococci belonging to cc11, fHbp of subfamily A, mainly due to a 13-fold higher off-rate (kd). It is unclear if this is but the predictive effect of fHbpD184 on outcome was preserved. Among patients infected compatible with the published binding structure. Moreover, under physiological conditions, with cc11 isolates, patients infected with meningococci with fHbpD184 were more likely to i.e., about 2 μM fH31, binding of fH to fHbp is saturated and without multiplying develop sepsis than those infected with meningococci with fHbpH184 or fHbpK184. Previously, meningococci virtually all fHbps would be occupied by fH in blood.23 However, when we reported cc11 meningococcal isolates to be associated with sepsis and unfavorable meningococci divide, non-ligand bound fHbp will arise and the on-rate (ka) determines the outcome.2 The present results, indicate that part of the virulence of cc11 isolates might be speed with which fHbp will be occupied. Interestingly, the ka of fH to fHbp binding seems to attributed to fHbpD184. be reduced in case of subfamily A fHbp. This might reduce survival of meningococci with subfamily A fHbp in the blood. Meningococci with subfamily B fHbp will be more protected During our inclusion period, routine serogroup C vaccination had not yet started in the by fH binding. However, we did not observe a significant difference between the rate of Netherlands. Vaccinating all children and adolescents from 14 months to 19 years -starting in unfavorable outcome in patients infected with meningococci with fHbp of subfamily A and June 2002- has dramatically reduced serogroup C disease in the Netherlands from 31% in our that of patients infected with meningococci with fHbp of subfamily B. In addition, the level cohort to an incidence of 6.5% in 2009 (http://www.amc.nl/upload/teksten/medical of survival in human sera of recombinant MC58 strains expressing the diverse subvariants microbiology/nrlbm jv/jv2009site.pdf).32 Therefore fHbp protein type distribution will most 22 did not correlate with levels of fH binding. Similar results were obtained by Dunphy and likely have changed, warranting epidemiological surveys of a more recent period. colleagues showing that expression by strain H44/76 of two natural fHbp sequence variants In conclusion, we found fHbp to be associated with severe disease and unfavorable with lower fH affinity had minimal or no effect on non-immune clearance in blood or D184 outcome. This study provides insights regarding the clinical relevance of this potent vaccine plasma.23 Most likely, the outcome of the interaction between meningococcal fHbp and target that is included in two forthcoming serogroup B vaccines. Because of the natural factor H on the survival of meningococci in blood depends on a multiple factors, like

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occurring strain variation of fHbp, further epidemiological surveys of sequence variation of REFERENCES

this vaccine component remains warranted. 1. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 2007;369:2196-2210.

2. Heckenberg SG, de Gans J, Brouwer MC, et al. Clinical features, outcome, and meningococcal genotype in 258 adults with meningococcal meningitis: a prospective ACKNOWLEDGMENTS cohort study. Medicine (Baltimore) 2008;87:185-192.

The authors acknowledge dr. D. Speijer for stimulating dicussions and critically reading of 3. Rosenstein NE, Perkins BA, Stephens DS, Popovic T, Hughes JM. Meningococcal disease. N Engl J Med 2001;344:1378-1388. the manuscript. Dr. P. Gros is acknowledged for his expert help in evaluating the fHbp-fH 4. Claus H, Maiden MC, Wilson DJ, et al. Genetic analysis of meningococci carried by model structure. D. van de Beek and A. van der Ende participate in the Seventh Framework children and young adults. J Infect Dis 2005;191:1263-1271. Programme of the European Commission, Proposal/Contract no.: 279185 (EUCLIDS -The 5. Kellerman SE, McCombs K, Ray M, et al. Genotype-specific carriage of Neisseria genetic basis of meningococcal and other life threatening bacterial infections of childhood), meningitidis in Georgia counties with hyper- and hyposporadic rates of J Infect Dis A. van der Ende participated in the Sixth Framework Programme of the European meningococcal disease. 2002;186:40-48. Commission, Proposal/Contract no.: 512061 (Network of Excellence “European Virtual 6. Maclennan J, Kafatos G, Neal K, et al. Social behavior and meningococcal carriage in British teenagers. Emerg Infect Dis 2006;12:950-957. Institute for Functional Genomics of Bacterial Pathogens”.) This publication made use of the 7. Christensen H, May M, Bowen L, Hickman M, Trotter CL. Meningococcal carriage by Neisseria Multilocus Sequence Typing website (http://pubmlst.org/neisseria/) developed by age: a systematic review and meta-analysis. Lancet Infect Dis 2010;10:853-861. Keith Jolley and sited at the University of Oxford33: Development of this site has been funded 8. Gardner P. Clinical practice. Prevention of meningococcal disease. N Engl J Med by the Wellcome Trust and European Union. 2006;355:1466-1473.

9. Davila S, Wright VJ, Khor CC, et al. Genome-wide association study identifies variants in the CFH region associated with host susceptibility to meningococcal disease. Nat Genet 2010;42:772-776.

10. Brouwer MC, van de Beek D. Genetics in meningococcal disease: one step beyond. Clin Infect Dis 2009;48:595-597.

11. Caugant DA, Tzanakaki G, Kriz P. Lessons from meningococcal carriage studies. FEMS Microbiol Rev 2007;31:52-63.

12. Fransen F, Heckenberg SG, Hamstra HJ, et al. Naturally occurring lipid A mutants in Neisseria meningitidis from patients with invasive meningococcal disease are associated with reduced coagulopathy. PLoS Pathog 2009;5:e1000396.

13. Brouwer MC, de Gans J, Heckenberg SG, Zwinderman AH, van der Poll T, van de Beek D. Host genetic susceptibility to pneumococcal and meningococcal disease: a systematic review and meta-analysis. Lancet Infect Dis 2009;9:31-44.

14. Brouwer MC, Read RC, van de Beek D. Host genetics and outcome in meningococcal disease: a systematic review and meta-analysis. Lancet Infect Dis 2010;10:262-274.

15. Lo H, Tang CM, Exley RM. Mechanisms of avoidance of host immunity by Neisseria meningitidis and its effect on vaccine development. Lancet Infect Dis 2009;9:418-427.

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40049 Piet, Jurgen.indd 54 26-04-16 15:08 Chapter 3 Meningococcal Factor H binding protein and clinical outcome

occurring strain variation of fHbp, further epidemiological surveys of sequence variation of REFERENCES this vaccine component remains warranted. 1. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 2007;369:2196-2210.

The authors acknowledge dr. D. Speijer for stimulating dicussions and critically reading of 3. Rosenstein NE, Perkins BA, Stephens DS, Popovic T, Hughes JM. Meningococcal disease. N Engl J Med 2001;344:1378-1388. the manuscript. Dr. P. Gros is acknowledged for his expert help in evaluating the fHbp-fH 4. Claus H, Maiden MC, Wilson DJ, et al. Genetic analysis of meningococci carried by model structure. D. van de Beek and A. van der Ende participate in the Seventh Framework 3 children and young adults. J Infect Dis 2005;191:1263-1271. Programme of the European Commission, Proposal/Contract no.: 279185 (EUCLIDS -The 5. Kellerman SE, McCombs K, Ray M, et al. Genotype-specific carriage of Neisseria genetic basis of meningococcal and other life threatening bacterial infections of childhood), meningitidis in Georgia counties with hyper- and hyposporadic rates of J Infect Dis A. van der Ende participated in the Sixth Framework Programme of the European meningococcal disease. 2002;186:40-48. Commission, Proposal/Contract no.: 512061 (Network of Excellence “European Virtual 6. Maclennan J, Kafatos G, Neal K, et al. Social behavior and meningococcal carriage in British teenagers. Emerg Infect Dis 2006;12:950-957. Institute for Functional Genomics of Bacterial Pathogens”.) This publication made use of the 7. Christensen H, May M, Bowen L, Hickman M, Trotter CL. Meningococcal carriage by Neisseria Multilocus Sequence Typing website (http://pubmlst.org/neisseria/) developed by age: a systematic review and meta-analysis. Lancet Infect Dis 2010;10:853-861. Keith Jolley and sited at the University of Oxford33: Development of this site has been funded 8. Gardner P. Clinical practice. Prevention of meningococcal disease. N Engl J Med by the Wellcome Trust and European Union. 2006;355:1466-1473.

9. Davila S, Wright VJ, Khor CC, et al. Genome-wide association study identifies variants in the CFH region associated with host susceptibility to meningococcal disease. Nat Genet 2010;42:772-776.

10. Brouwer MC, van de Beek D. Genetics in meningococcal disease: one step beyond. Clin Infect Dis 2009;48:595-597.

11. Caugant DA, Tzanakaki G, Kriz P. Lessons from meningococcal carriage studies. FEMS Microbiol Rev 2007;31:52-63.

14. Brouwer MC, Read RC, van de Beek D. Host genetics and outcome in meningococcal disease: a systematic review and meta-analysis. Lancet Infect Dis 2010;10:262-274.

15. Lo H, Tang CM, Exley RM. Mechanisms of avoidance of host immunity by Neisseria meningitidis and its effect on vaccine development. Lancet Infect Dis 2009;9:418-427.

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16. Lambris JD, Ricklin D, Geisbrecht BV. Complement evasion by human pathogens. Nat 31. Ingram G, Hakobyan S, Hirst CL, et al. Complement regulator factor H as a serum Rev Microbiol 2008;6:132-142. biomarker of multiple sclerosis disease state. Brain 2010;133:1602-1611.

17. Madico G, Welsch JA, Lewis LA, et al. The meningococcal vaccine candidate 32. de Greeff SC, de Melker HE, Spanjaard L, Schouls LM, van der Ende A. Protection GNA1870 binds the complement regulatory protein factor H and enhances serum from routine vaccination at the age of 14 months with meningococcal serogroup C resistance. J Immunol 2006;177:501-510. conjugate vaccine in the Netherlands. Pediatr Infect Dis J 2006;25:79-80.

18. Schneider MC, Prosser BE, Caesar JJ, et al. Neisseria meningitidis recruits factor H 33. Jolley KA, Chan MS, Maiden MC. mlstdbNet - distributed multi-locus sequence typing using protein mimicry of host carbohydrates. Nature 2009;458:890-893. (MLST) databases. BMC Bioinformatics 2004;5:86.

19. Masignani V, Comanducci M, Giuliani MM, et al. Vaccination against Neisseria meningitidis using three variants of the lipoprotein GNA1870. J Exp Med 2003;197:789-799.

20. Fletcher LD, Bernfield L, Barniak V, et al. Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect Immun 2004;72:2088-2100.

21. Schneider MC, Exley RM, Chan H, et al. Functional significance of factor H binding to Neisseria meningitidis. J Immunol 2006;176:7566-7575.

22. Seib KL, Brunelli B, Brogioni B, et al. Characterization of diverse subvariants of the meningococcal factor H (fH) binding protein for their ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies. Infect Immun 2011;79:970- 981.

23. Dunphy KY, Beernink PT, Brogioni B, Granoff DM. Effect of Factor H-Binding Protein Sequence Variation on Factor H Binding and Survival of Neisseria meningitidis in Human Blood. Infect Immun 2011;79:353-359.

24. Murphy E, Andrew L, Lee KL, et al. Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria meningitidis. J Infect Dis 2009;200:379-389.

25. Beernink PT, Granoff DM. The modular architecture of meningococcal factor H- binding protein. Microbiology 2009;155:2873-2883.

26. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Med 2004;351:1849-1859.

27. Jennett B, Teasdale G. Management of head injuries. 2 ed. Philadelphia: F.A.Davis; 1981.

28. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-425.

29. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-1599.

30. Jennett B, Bond M. Assessment of outcome after severe brain damage. Lancet 1975;1:480-484.

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19. Masignani V, Comanducci M, Giuliani MM, et al. Vaccination against Neisseria meningitidis using three variants of the lipoprotein GNA1870. J Exp Med 2003;197:789-799. 3 20. Fletcher LD, Bernfield L, Barniak V, et al. Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect Immun 2004;72:2088-2100.

21. Schneider MC, Exley RM, Chan H, et al. Functional significance of factor H binding to Neisseria meningitidis. J Immunol 2006;176:7566-7575.

25. Beernink PT, Granoff DM. The modular architecture of meningococcal factor H- binding protein. Microbiology 2009;155:2873-2883.

26. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Med 2004;351:1849-1859.

27. Jennett B, Teasdale G. Management of head injuries. 2 ed. Philadelphia: F.A.Davis; 1981.

28. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-425.

29. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-1599.

30. Jennett B, Bond M. Assessment of outcome after severe brain damage. Lancet 1975;1:480-484.

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SUPPLEMENTARY MATERIAL Supplementary Table 3. The association constant (ka), dissociation constant (kd) and the thermodynamic dissociation constants (KD) of the binding between fH and fHbp, according to protein family. a Supplementary Table 1. All meningococcal isolates used in the study with serogroup data, sequence types, Average binding values fHbp - fH clonal complexes, and fHbp DNA/protein allele. fHbp family A fHbp family B Pb -1 -1 5 5 ka (M s ) 0.80 x 10 1.92 x 10 0.04 -1 -2 -2 kd (s ) 0.21 x10 2.87 x10 0.03 Due to size not printable. Online accessible at KD (nM) 29 153 0.02 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0047973 aAverage values were calculated from Table 3 of Seib et al.22 bStudent T-test; two sided.

Supplementary Table 2. fHbp polymorphism of the 17 amino acids showing interaction with human factor H, Supplementary Table 4. Primers. according to fHbp subfamilies. Primers used for fHbp sequencing a position fHbp subfamily fHbp subfamily CH1870-1 TGACCTGCCTCATTGATGC B (212 isolates) A (40 isolates) CH1870-2 CGGTAAATTATCGTGTTCGGACGGC Making salt CH1870-3 CAAATCGAAGTGGACGGGCAG bridge to fH CH1870-4 TGTTCGATTTTGCCGTTTCCCTG

CH1870-5 GAAGTGGACGGACAAACCATC 304 E (acidic) T (hydrophilic neutral; smaller side chain) Primers used for amplification and mutagenesis of fHbp_10 Making 1P10DK F hydrogen GCAAGTACAAGACTCGGAGAAATCCGGGAAGATGGTTGCG bonds to fH 1P10DK R CGCAACCATCTTCCCGGATTTCTCCGAGTCTTGTACTTGC NG2143 CGCGGATCCCATATGGTTGCCGCCGACATCG 180 Q (hydrophilic neutral) N (hydrophilic neutral; smaller side chain) NG2145 CCCGCTCGAGCTGCTTGGCGGCAAGAC 181 D (acidic) N (hydrophilic neutral) 183 E (acidic) D (acidic; smaller side chain) 184 H (basic; strongly polar) or D (acidic) K (basic; strongly polar) 185 S (hydrophilic neutral) I (hydrophobic; larger side chain) 191 K (basic; strongly polar) Q (hydrophilic neutral; smaller side chain) S (hydrophilic neutral; smaller side chain than Q 193 Q (hydrophilic neutral) or R (basic) and R) 195 R (basic) L (hydrophobic neutral; smaller side chain) 262 Y (hydrophobic, weak acidic) or D (acidic) E (acidic; smaller side chain than Y) 274 S (hydrophilic neutral) L (hydrophobic neutral; larger side chain) 286 S (hydrophilic neutral) T (hydrophilic neutral) 306 K (basic; strongly polar) or E (acidic) K (basic; strongly polar) aFour of 17 aminoacids were conserved.

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Supplementary Table 2. fHbp polymorphism of the 17 amino acids showing interaction with human factor H, Supplementary Table 4. Primers. according to fHbp subfamilies. Primers used for fHbp sequencing 3 a position fHbp subfamily fHbp subfamily CH1870-1 TGACCTGCCTCATTGATGC B (212 isolates) A (40 isolates) CH1870-2 CGGTAAATTATCGTGTTCGGACGGC Making salt CH1870-3 CAAATCGAAGTGGACGGGCAG bridge to fH CH1870-4 TGTTCGATTTTGCCGTTTCCCTG

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CHAPTER 4:

Meningococcal two-partner secretion systems and outcome in patients with meningitis

Jurgen R. Piet Peter van Ulsen Sadeeq ur Rahman Sandra Bovenkerk Stephen D. Bentley Diederik van de Beek* Arie van der Ende*

*Both authors contributed equally

Submitted

40049 Piet, Jurgen.indd 60 26-04-16 15:08

CHAPTER 4: CHAPTER 4: Meningococcal two-partner secretion systems and outcome in patients with meningitis

Jurgen R. Piet Meningococcal two-partner Peter van Ulsen Sadeeq ur Rahman secretion systems and Sandra Bovenkerk outcome in patients with Stephen D. Bentley Diederik van de Beek* meningitis Arie van der Ende*

*Both authors contributed equally

Jurgen R. Piet Peter van Ulsen

Sadeeq ur Rahman Sandra Bovenkerk

Stephen D. Bentley Diederik van de Beek*

Arie van der Ende*

*Both authors contributed equally

Submitted Submitted

40049 Piet, Jurgen.indd 61 26-04-16 15:08 Chapter 4 Meningococcal two-partner secretion systems and outcome

ABSTRACT INTRODUCTION

Two-partner secretion (TPS) systems export large TpsA proteins to the surface and The two-partner secretion (TPS) pathway is a widespread protein secretion route in Gram- extracellular milieu. In meningococci, three different TPS systems exist of which systems 2 negative bacteria consisting of a large and secreted exoprotein (TpsA) of typically more than and 3 can be detected by the host’s immune system. Meningococcal TPS systems are 100 kDa and a ~ 60 kDa transporter protein (TpsB).1 TpsA and TpsB translocate across the thought to be involved in intracellular survival and adhesion. We evaluated the distribution inner cytoplasmic membrane through the general secretion system.2 Subsequently, the TpsB of TPS systems among clinical isolates from two prospective cohort studies comprising 373 inserts into the outer membrane and facilitates the export of TpsA. The TpsA targets the patients with meningococcal meningitis. TPS system 1 was present in 91% of isolates and TpsB using the so-called TPS domain, which is located at the N terminus of the periplasmic system 2 and/or 3 were present in 67%. TPS system distribution was related to clonal intermediate. Functions of TPS systems vary among different bacterial species and include complexes. Infection with TPS2 and/or TPS3 strains resulted in less severe disease and better hemolysis, cytotoxicity and iron acquisition.3,4 Neisseria meningitidis (meningococcus) is a outcome, as compared to infection without these systems. Using whole-blood stimulation Gram-negative diplococcus and a major cause of bacterial meningitis and sepsis worldwide.5 experiments, we found no differences in cytokine host response between TPS systems 2 and Secreted products are considered to be important in the pathogenesis of meningococcal 3 knockout strains and a wild-type strain harboring all TPS systems. In conclusion, the invasive disease.6 The TPS pathway is one of many protein export mechanism in meningococcal TPS system 2 and/or 3 is associated with disease severity and outcome in meningococci and one TPS system (system 1, see below) had initially been indicated to be patients with meningitis. We could not prove a causal relationship between different TPS involved in intracellular survival and adhesion to host cells.6-8 This TPS system was more systems and cytokine response in vitro. recently also shown to play a role in meningococcal biofilms9 and to act in bacterial fratricide.10,11

Three TPS systems are known in the meningococcal genome: TPS1 encoded by tpsA1a, tpsA1b, tpsB1 and tpsB1tr, TPS2 encoded by tpsA2a, tpsA2 and tpsB2 and TPS3 encoded by tpsA3 (the numbers in the gene/protein names refer to the systems).12 However, the TPS domains of TpsA proteins of all three systems can be exported by TpsB2, whereas TpsB1 is only capable of exporting TpsA1.1 During meningococcal infection, components of TPS2 and TPS3 can elicit an immune response, which suggests that antibodies against TPS systems may be potential vaccination components.12 Most TPS genes are unique except for tpsA1b, a duplicate of tpsA1a, and tpsB1tr, a truncated and non-functional duplicate of tpsB1. TPS genes are located within genomic islands.12 Downstream of tpsA1a, tpsA1b and tpsA2a genes, repetitive putative open reading frames (ORFs) encoding for the C-terminal end of full-length TpsA proteins (tpsC cassettes) are present, possibly to enable variation of tpsA by recombination between these cassettes and the full-length tpsA.11,13

We previously reported the frequency of tps genes in 88 clinical invasive N. meningitidis isolates of both sepsis and meningitis patients.12 With the current knowledge on the TpsA-

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ABSTRACT INTRODUCTION

Two-partner secretion (TPS) systems export large TpsA proteins to the surface and The two-partner secretion (TPS) pathway is a widespread protein secretion route in Gram- extracellular milieu. In meningococci, three different TPS systems exist of which systems 2 negative bacteria consisting of a large and secreted exoprotein (TpsA) of typically more than and 3 can be detected by the host’s immune system. Meningococcal TPS systems are 100 kDa and a ~ 60 kDa transporter protein (TpsB).1 TpsA and TpsB translocate across the thought to be involved in intracellular survival and adhesion. We evaluated the distribution inner cytoplasmic membrane through the general secretion system.2 Subsequently, the TpsB of TPS systems among clinical isolates from two prospective cohort studies comprising 373 inserts into the outer membrane and facilitates the export of TpsA. The TpsA targets the patients with meningococcal meningitis. TPS system 1 was present in 91% of isolates and TpsB using the so-called TPS domain, which is located at the N terminus of the periplasmic system 2 and/or 3 were present in 67%. TPS system distribution was related to clonal intermediate. Functions of TPS systems vary among different bacterial species and include complexes. Infection with TPS2 and/or TPS3 strains resulted in less severe disease and better hemolysis, cytotoxicity and iron acquisition.3,4 Neisseria meningitidis (meningococcus) is a outcome, as compared to infection without these systems. Using whole-blood stimulation Gram-negative diplococcus and a major cause of bacterial meningitis and sepsis worldwide.5 experiments, we found no differences in cytokine host response between TPS systems 2 and Secreted products are considered to be important in the pathogenesis of meningococcal 4 3 knockout strains and a wild-type strain harboring all TPS systems. In conclusion, the invasive disease.6 The TPS pathway is one of many protein export mechanism in meningococcal TPS system 2 and/or 3 is associated with disease severity and outcome in meningococci and one TPS system (system 1, see below) had initially been indicated to be patients with meningitis. We could not prove a causal relationship between different TPS involved in intracellular survival and adhesion to host cells.6-8 This TPS system was more systems and cytokine response in vitro. recently also shown to play a role in meningococcal biofilms9 and to act in bacterial fratricide.10,11

We previously reported the frequency of tps genes in 88 clinical invasive N. meningitidis isolates of both sepsis and meningitis patients.12 With the current knowledge on the TpsA-

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TpsB specificity,1 82 % harbored a functional TPS1, 47% TPS2, and 20% TPS3. Another study patients’ conscious state on admission. Outcome was graded according to the Glasgow reported frequency of TPS genes in 822 meningococcal carrier isolates using dot blot Outcome Scale (GOS).14 A favorable outcome was defined as a score of five, and an hybridization of tpsA genes: 77 % harbored an intact TPS1, 40% TPS2, and 15% TPS3.8 In both unfavorable outcome as a score lower than five. studies, isolates with TPS2 and/or TPS3 were linked to hyper-invasive clonal complexes (cc, Meningococcal typing defined as cc8, cc11, cc32, cc41/44 and cc269). Although these studies used different selections of isolates, results suggest that meningococcal TPS systems are related to N. meningitidis strains of patients with meningococcal meningitis are collected and stored at bacterial invasiveness or virulence. However, it does not indicate whether the TPS systems the NRLBM. Serogrouping and Multilocus Sequence Typing (MLST), was performed at the play a role in the severity of infection or the disease outcome. To establish such a link, we NRLBM as previously described 15 and have been published previously.16,17 The clonal evaluated the distribution of TPS systems among clinical isolates from two prospective complexes (cc) were assigned according to the MLST-database available at cohorts of bacterial meningitis patients and evaluated the association between these http://pubmlst.org/neisseria/. systems and meningococcal disease severity. Subsequently, we evaluated our findings with PCR analysis of strains in the 1998-2002 cohort in-vitro experiments using tps knockout meningococcal strains and complemented knockout

strains. The distribution of genes encoding for TpsA (tpsA1, tpsA2a, tpsA2b, tpsA3) and genes encoding for TpsB (tpsB1, tpsB2) among the meningococcal isolates in the 1998-2002 cohort

(n=254) was determined by 6 different PCR’s. Primer pairs used are displayed in MATERIALS AND METHODS Supplementary Table 1. The primers targeting the tpsB genes were designed to produce full- length tpsB genes, meaning that the truncated form will not be amplified in these PCR’s. The Meningitis cases tpsA1 PCR did not discriminate between a possible duplication of this ORF as is present in The Dutch Meningitis Cohort Study (October 1998 to April 2002) and the MeninGene Study the genome of N. meningitidis MC58 (tpsA1b). (January 2006 to July 2014) were prospective nationwide observational cohort study of Whole-genome sequencing of strains in the 2006-2014 cohort adults with community-acquired bacterial meningitis in the Netherlands. Patients were included with the help of the Netherlands Reference Laboratory for Bacterial Meningitis An in house adapted version of the Wizard® Genomic DNA Purification Kit (Promega) was (NRLBM). This laboratory received CSF isolates from 90% of all patients with bacterial used to isolate chromosomal DNA. Meningococcal genomes of the isolates in the 2006-2014 meningitis in the Netherlands. Written informed consent was obtained from all patients or cohort (n=115) were sequenced at the Welcome Trust Sanger Institute using Illumina HiSeq their legally authorized representatives. The study was approved by the Medical Ethics 2000 analyzers as was previously described.18 Committee of the Academic Medical Center, Amsterdam, the Netherlands. TPS system definition

In the 1998-2002 cohort, TPS system 1 was defined as isolates yielding an amplicon in the Case-record forms were used to collect data on patients’ history, symptoms and signs on tpsA1 and either the tpsB1 or the tpsB2 PCR. TPS system 2 was defined as isolates yielding an admission, laboratory findings at admission, clinical course, outcome and neurologic findings amplicon in either the tpsA2a and/or tpsA2b in combination with the tpsB2 PCR. TPS system at discharge, and treatment. The Glasgow Coma Scale (GCS) was used to determine the 3 was defined as isolates yielding an amplicon in the tpsA3 and the tpsB2 PCR. In the 2006-

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TpsB specificity,1 82 % harbored a functional TPS1, 47% TPS2, and 20% TPS3. Another study patients’ conscious state on admission. Outcome was graded according to the Glasgow reported frequency of TPS genes in 822 meningococcal carrier isolates using dot blot Outcome Scale (GOS).14 A favorable outcome was defined as a score of five, and an hybridization of tpsA genes: 77 % harbored an intact TPS1, 40% TPS2, and 15% TPS3.8 In both unfavorable outcome as a score lower than five. studies, isolates with TPS2 and/or TPS3 were linked to hyper-invasive clonal complexes (cc, Meningococcal typing defined as cc8, cc11, cc32, cc41/44 and cc269). Although these studies used different selections of isolates, results suggest that meningococcal TPS systems are related to N. meningitidis strains of patients with meningococcal meningitis are collected and stored at bacterial invasiveness or virulence. However, it does not indicate whether the TPS systems the NRLBM. Serogrouping and Multilocus Sequence Typing (MLST), was performed at the play a role in the severity of infection or the disease outcome. To establish such a link, we NRLBM as previously described 15 and have been published previously.16,17 The clonal evaluated the distribution of TPS systems among clinical isolates from two prospective complexes (cc) were assigned according to the MLST-database available at cohorts of bacterial meningitis patients and evaluated the association between these http://pubmlst.org/neisseria/. systems and meningococcal disease severity. Subsequently, we evaluated our findings with PCR analysis of strains in the 1998-2002 cohort 4 in-vitro experiments using tps knockout meningococcal strains and complemented knockout strains. The distribution of genes encoding for TpsA (tpsA1, tpsA2a, tpsA2b, tpsA3) and genes encoding for TpsB (tpsB1, tpsB2) among the meningococcal isolates in the 1998-2002 cohort

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present. polypropylene tubes and grown for 4 hours at 37°C in 5% CO2, with or without the presence of 1mM IPTG. Colony forming units (CFU’s) were determined in triplo at T = 0 and T = 4 Meningococcal knockout and complemented knockout strains hours. IL-6 was measured in duplo by enzyme-linked immunosorbent assay (ELISA, Biosource N. meningitidis knockout strain H44/76 (B:15:P1.7,16; cc 32; 19) tpsB2::ery lacks a functional - Invitrogen). TPS2 due to replacement of the tpsB2 gene by an erythromycin-resistance cassette (ery). Statistics Replacement of tpsB2 resulted from allelic exchange through homologous recombination using plasmid pKO-tpsB2::ery. This plasmid was constructed by replacing the kan cassette in In the analysis of the association between the patient characteristics and the TPS system pKO-tpsB212for the ery cassette of plasmid pFLOB430020 using the PstI restriction sites presence, we used the Mann-Whitney U test to identify differences between patient groups present. The replacement resulted in strain H44/76 tpsB2::ery. The knockout strain H44/76 with respect to continuous variables. Dichotomous variables were compared with use of the tpsA3::kan lacks a functional TPS3 due to replacement of the of the tpsA3 gene by a χ2 test and statistical tests were two-tailed, with p<0.05 regarded as significant. kanamycin-resistance cassette (kan).12 The knockout strain H44/76 tpsA3::kan tpsB2::ery

(H44/76∆tpsA3,tpsB2) lacks both a functional TPS2 and TPS3. In the H44/76 tpsB2::ery and the H44/76 tpsA3::kan tpsB2::ery strain, tpsB2 was complemented by transforming the RESULTS knockout strain with plasmid pEN-TpsB2. The tpsB2 ORF was obtained by PCR using primers Patient cohorts TCCGTGTATTGAATGCCCATTGATGA and GAGATCTGAATTCCGTTGATTTGACTATGCCGTTTTA.1

The resulting amplicon was digested by the restriction enzymes BamHi and EcoRI and ligated This study used meningococcal strains from two time periods. From the first period, October into pET11a (Invitrogen) digested with the same enzymes. The ORF was subsequently 1998 to April 2002, 258 episodes of community-acquired meningococcal meningitis in 258 21 inserted into the neisserial expression vector pEN300 using restriction enzymes NdeI and patients were included. Detailed clinical and microbiological characteristics have been AatII. The resulting plasmid (pEN-TpsB2) encodes tpsB2 under control of an IPTG-inducible described previously.16,22 Patient characteristics, clinical course and the causative bacterial 1,21 lac promoter and was introduced in strain H44/76 and derivates by transformation. strain isolated from the cerebrospinal fluid (CSF) were available for 254 (98.4%) of the 258 meningococcal meningitis episodes. Most prevalent clonal complexes were cc41/44 (41%; all Western Blot analysis but one serogroup B), cc11 (24%; all serogroup C), and cc32 (16%; all serogroup B). These 1 All procedures were carried out as described earlier. Blots were incubated with antisera clonal complexes are all considered to be hyper-invasive because they are more frequently (αTPS1, αTPS2 and αTPSB2). isolated from patients than from carriers.23 The case fatality rate was 7% and 12% patients had an unfavorable outcome. Blood stimulation experiments

In the second period analyzed, from 2006 to 2014, N. meningitidis was the causative N. meningitidis colonies were suspended in RPMI medium with 2% glucose and diluted to an organism in 150 of 1412 episodes of community-acquired meningitis. The case fatality rate optical density (OD530) of 0.05 in 40 ml RPMI. Bacteria were grown for 2.5 hours to a mid-log was 3% and 13% of patients had an unfavorable outcome.17 In our analysis we included 115 growth phase, washed with 25 ml RPMI and resuspended in 10 ml RPMI. Venous blood was meningococcal isolates of which whole genomes were sequenced. Most prevalent clonal

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2014 cohort, TPS system genes were defined by BLASTing the tps genes from meningococcal drawn from seven healthy subjects, stored in Sodium-heparin tubes and used directly. 6 strain MC58 and TPS systems were identified if the necessary tpsA and tpsB genes were Meningococcal cultures of OD530 0.01 (10 bacteria/ml) were added to blood in a 1:1 ratio in present. polypropylene tubes and grown for 4 hours at 37°C in 5% CO2, with or without the presence of 1mM IPTG. Colony forming units (CFU’s) were determined in triplo at T = 0 and T = 4 Meningococcal knockout and complemented knockout strains hours. IL-6 was measured in duplo by enzyme-linked immunosorbent assay (ELISA, Biosource N. meningitidis knockout strain H44/76 (B:15:P1.7,16; cc 32; 19) tpsB2::ery lacks a functional - Invitrogen). TPS2 due to replacement of the tpsB2 gene by an erythromycin-resistance cassette (ery). Statistics Replacement of tpsB2 resulted from allelic exchange through homologous recombination using plasmid pKO-tpsB2::ery. This plasmid was constructed by replacing the kan cassette in In the analysis of the association between the patient characteristics and the TPS system pKO-tpsB212for the ery cassette of plasmid pFLOB430020 using the PstI restriction sites presence, we used the Mann-Whitney U test to identify differences between patient groups present. The replacement resulted in strain H44/76 tpsB2::ery. The knockout strain H44/76 with respect to continuous variables. Dichotomous variables were compared with use of the 4 tpsA3::kan lacks a functional TPS3 due to replacement of the of the tpsA3 gene by a χ2 test and statistical tests were two-tailed, with p<0.05 regarded as significant. kanamycin-resistance cassette (kan).12 The knockout strain H44/76 tpsA3::kan tpsB2::ery

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(tpsB2 and either tpsA2a or tpsA2b, or both). In 52 isolates (20%) a functional TPS3 was Table 1. Associations with clinical characteristics of meningitis patients and meningococcal TPS system present (tpsB2 and tpsA3). Of the 87 strains that harbored TPS1 only, 71 (82%) were distribution.a serogroup C strains, 59 (68%) belonged to the hyper-invasive cc11 and 10 (11%) belonged to Isolates without an Isolates with an P-Value immunogenic TPS system immunogenic TPS system the hyper-invasive cc8. Of note, none of the tested cc11 and cc8 isolates did contain a TPS2 (without 2 and/or 3) n=123 (either2 and/or 3) n=246 Demographics or TPS3 system. Of the 162 strains containing either TPS system 2 and/or 3, 158 (98%) were Age, yr (Mean ±SD) 38 ±21; n=123 36 ±18; n=246 0.63 serogroup B strains. In this subgroup, the most prevalent clonal complexes were hyper- Duration of symptoms <24 hrs 61/121 (50%) 115/236 (49%) 0.76 Symptoms at presentation invasive cc41/44 (n=99), cc32 (n=36) and cc269 (n=16). The 52 isolates harboring all 3 Glasgow Coma Scale score 12 (9-15; n=121) 14 (11-15; n=245) 0.006 systems belonged to hyper-invasive cc32 or cc269. Rash 71/123 (58%) 146/246 (59%) 0.77 Focal neurological deficitsb 31/122 (25%) 32/246 (13%) 0.003 In the 2006-2014 cohort, 89 of 115 (77%; p<0.0001 compared to 1998-2002 cohort) Diastolic BP 70 (60-84; n=120) 70 (62-80; n=235) 0.77 Blood parameters harbored an intact TPS 1 system. In 84 of 115 (73%) a TPS 2 and in 33 of 115 (29%) a TPS 3 Positive blood culture 75/116 (65%) 102/209 (49%) 0.006 system was present. Only 8 of 115 (7%) isolates were serogroup C strains, of which 6 (all Thrombocyte count, 109/L 166 (122-213; n=118) 173 (139-225; n=232) 0.14 Cerebrospinal fluid parameters hyper-invasive cc11) did not contain any TPS systems. Non-hyper-invasive clonal complexes CSF leukocyte count (cells/mm3) 4998 (1100-12000; n=115) 5600 (1742-13325; n=233) 0.29 detected that did not contain any TPS system were cc22 (n=5), cc23 (n=4) and cc174 (n=3). CSF/blood glucose ratio 0.081 (0.01-0.35; n=112) 0.80 (0.01-0.31; n=219) 0.84 Outcome parameters These clonal complexes were absent in the first cohort. In 84/115 (73%) of strains either a Any systemic complication 31/121 (26%) 34/245 (14%) 0.006 TPS2 or a TPS 3 system was present. Of the 31 isolates harboring all TPS systems isolates, 30 Unfavorable outcome (GOS = 1-4) 21/123 (17%) 23/246 (9,3%) 0.031 aData are number/number assessed (%) or median (25th–75th percentile; number assessed), unless otherwise belonged to hyper-invasive cc32 or cc269. stated. bDefined as aphasia, monoparesis, hemiparesis) or cranial nerve palsies.

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complexes were cc41/44 (32%), cc32 (23%), cc269 (9%) and cc213 (9%). In this cohort, the TPS systems and clinical outcome proportion of cc11 isolates dropped to 5%, due to a national serogroup C vaccination Patients infected with meningococci without TPS systems or harboring solely TPS1 presented campaign in 2002. with a lower GCS score (median 12 [IQR 9-15] vs 14 [IQR 11-15]; p=0.006) and more often Tps genes in clinical isolates in the two patient cohorts had focal neurological deficits upon presentation (31 of 122 [25%] vs. 32 of 246 [13%]; p=0.003) as compared to patients infected with isolates that contained multiple TPS systems Meningococcal serogroup data, sequence types, clonal complexes, and tps gene/TPS system (Table 1). The proportion of patients with systemic complications (31 of 121 [26%] vs. 34 of distribution in both cohorts are shown in Supplementary Table 2 and Supplementary Figure 245 [14%]; p=0.006) and unfavorable outcome (21 of 123 [17%] vs. 23 of 246 [9%]; p=0.031) 1. A summary of TPS system distribution in all available cohorts is shown in Supplementary was higher in the patients infected with meningococci without TPS or harboring solely TPS1 Table 3. In the 1998-2002 cohort, 246 of 254 isolates (97%) harbored an intact TPS1 system. as compared to patients infected with isolates containing TPS systems 2 and/or 3. The tpsA1 was absent in five isolates and three isolates contained an insertion sequence (IS1301) in tpsB1 (all ST2704 from cc11). In 162 (64%) isolates a complete TPS2 was present 4 (tpsB2 and either tpsA2a or tpsA2b, or both). In 52 isolates (20%) a functional TPS3 was Table 1. Associations with clinical characteristics of meningitis patients and meningococcal TPS system present (tpsB2 and tpsA3). Of the 87 strains that harbored TPS1 only, 71 (82%) were distribution.a serogroup C strains, 59 (68%) belonged to the hyper-invasive cc11 and 10 (11%) belonged to Isolates without an Isolates with an P-Value immunogenic TPS system immunogenic TPS system the hyper-invasive cc8. Of note, none of the tested cc11 and cc8 isolates did contain a TPS2 (without 2 and/or 3) n=123 (either2 and/or 3) n=246 Demographics or TPS3 system. Of the 162 strains containing either TPS system 2 and/or 3, 158 (98%) were Age, yr (Mean ±SD) 38 ±21; n=123 36 ±18; n=246 0.63 serogroup B strains. In this subgroup, the most prevalent clonal complexes were hyper- Duration of symptoms <24 hrs 61/121 (50%) 115/236 (49%) 0.76 Symptoms at presentation invasive cc41/44 (n=99), cc32 (n=36) and cc269 (n=16). The 52 isolates harboring all 3 Glasgow Coma Scale score 12 (9-15; n=121) 14 (11-15; n=245) 0.006 systems belonged to hyper-invasive cc32 or cc269. Rash 71/123 (58%) 146/246 (59%) 0.77 Focal neurological deficitsb 31/122 (25%) 32/246 (13%) 0.003 In the 2006-2014 cohort, 89 of 115 (77%; p<0.0001 compared to 1998-2002 cohort) Diastolic BP 70 (60-84; n=120) 70 (62-80; n=235) 0.77 Blood parameters harbored an intact TPS 1 system. In 84 of 115 (73%) a TPS 2 and in 33 of 115 (29%) a TPS 3 Positive blood culture 75/116 (65%) 102/209 (49%) 0.006 system was present. Only 8 of 115 (7%) isolates were serogroup C strains, of which 6 (all Thrombocyte count, 109/L 166 (122-213; n=118) 173 (139-225; n=232) 0.14 Cerebrospinal fluid parameters hyper-invasive cc11) did not contain any TPS systems. Non-hyper-invasive clonal complexes CSF leukocyte count (cells/mm3) 4998 (1100-12000; n=115) 5600 (1742-13325; n=233) 0.29 detected that did not contain any TPS system were cc22 (n=5), cc23 (n=4) and cc174 (n=3). CSF/blood glucose ratio 0.081 (0.01-0.35; n=112) 0.80 (0.01-0.31; n=219) 0.84 Outcome parameters These clonal complexes were absent in the first cohort. In 84/115 (73%) of strains either a Any systemic complication 31/121 (26%) 34/245 (14%) 0.006 TPS2 or a TPS 3 system was present. Of the 31 isolates harboring all TPS systems isolates, 30 Unfavorable outcome (GOS = 1-4) 21/123 (17%) 23/246 (9,3%) 0.031 aData are number/number assessed (%) or median (25th–75th percentile; number assessed), unless otherwise belonged to hyper-invasive cc32 or cc269. stated. bDefined as aphasia, monoparesis, hemiparesis) or cranial nerve palsies.

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In the 1998-2002 cohort, we previously described a polymorphism at position 184 in the Tps knockout strain and cytokine induction meningococcal factor H binding protein (fHbp) to be associated with cc11 and unfavorable Next, we performed whole-blood stimulation experiments in which we compared bacterial outcome in this cohort.24 There is considerable overlap in distribution between cc11, the growth and IL-6 (one of the acute phase mediators of the immune response to infection) of factor fHbpD184 polymorphism, and TPS systems, raising the possibility that rather this meningococcal strains H44/76, which has all three TPS systems intact, and isogenic TPS polymorphism than the TPS systems is important for clinical outcome (Figure 1). However, in system 2 and 3 knockout strains (Figure 2). We also included strains in which the knocked- analyses that excluded the patients who were infected with meningococci with fHbpD184 the out gene was complemented by the original gene on a plasmid. The TpsA protein expression difference in proportion for meningococci with no TPS or solely TPS1 and meningococci profile of all created strains was tested by Western blot analysis and was consistent with the infected with multiple TPS remained detectable between patients with a GCS score GCS < 14 genetic background (Supplementary Figure 2). Bacterial growth in RPMI medium of all tested or GCS ≥ 14: 38 of 60 (63%) vs 68 of 151 (45%; p=0.016). The difference in proportion of strains was similar (Supplementary Figure 3). We observed no difference in growth in whole patients with any systemic complication showed a similar trend: 14 of 60 (23.3%) vs. 19 of blood after 4 hours between the tested knockout mutants and the wild-type, or 151 (12.6%; p=0.052). complemented strains for each of the blood samples of the seven donors. However, bacterial growth results were different between donors: in samples of three persons all strains grew exponentially. In samples of three other persons no growth was observed and Figure 1. Venn diagram of meningococcal genetic factors associated with clinical outcome. the number of viable bacteria at the inoculum and after 4 hours of incubation remained equal. In samples of one person all strains were killed and IL-6 concentration in the blood of this person was therefore not measured. In the blood samples of the six remaining test subjects, the induced IL-6 concentrations after 4 hours of growth showed no differences among the tested strains. There was no correlation between CFU’s and IL-6.

In our 1998-2002 cohort of 254 patients with bacterial meningitis, the overlap of isolates from clonal complex 11, isolates with the fHbpD184 polymorphism and isolates without immunogenic TPS systems is shown (total n=102).

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In the 1998-2002 cohort, we previously described a polymorphism at position 184 in the Tps knockout strain and cytokine induction meningococcal factor H binding protein (fHbp) to be associated with cc11 and unfavorable Next, we performed whole-blood stimulation experiments in which we compared bacterial outcome in this cohort.24 There is considerable overlap in distribution between cc11, the growth and IL-6 (one of the acute phase mediators of the immune response to infection) of factor fHbpD184 polymorphism, and TPS systems, raising the possibility that rather this meningococcal strains H44/76, which has all three TPS systems intact, and isogenic TPS polymorphism than the TPS systems is important for clinical outcome (Figure 1). However, in system 2 and 3 knockout strains (Figure 2). We also included strains in which the knocked- analyses that excluded the patients who were infected with meningococci with fHbpD184 the out gene was complemented by the original gene on a plasmid. The TpsA protein expression difference in proportion for meningococci with no TPS or solely TPS1 and meningococci profile of all created strains was tested by Western blot analysis and was consistent with the infected with multiple TPS remained detectable between patients with a GCS score GCS < 14 genetic background (Supplementary Figure 2). Bacterial growth in RPMI medium of all tested or GCS ≥ 14: 38 of 60 (63%) vs 68 of 151 (45%; p=0.016). The difference in proportion of strains was similar (Supplementary Figure 3). We observed no difference in growth in whole patients with any systemic complication showed a similar trend: 14 of 60 (23.3%) vs. 19 of blood after 4 hours between the tested knockout mutants and the wild-type, or 151 (12.6%; p=0.052). complemented strains for each of the blood samples of the seven donors. However, 4 bacterial growth results were different between donors: in samples of three persons all strains grew exponentially. In samples of three other persons no growth was observed and Figure 1. Venn diagram of meningococcal genetic factors associated with clinical outcome. the number of viable bacteria at the inoculum and after 4 hours of incubation remained equal. In samples of one person all strains were killed and IL-6 concentration in the blood of this person was therefore not measured. In the blood samples of the six remaining test subjects, the induced IL-6 concentrations after 4 hours of growth showed no differences among the tested strains. There was no correlation between CFU’s and IL-6.

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Figure 2. Meningococcal blood stimulation experiments. Tps genome signature

The TPS systems 1 and 2 are encoded within genomic islands that carry several traits that suggest influence of recombination events that may influence strain to strain variation; e.g. the presence of the tpsC cassettes. Horizontally acquired genes can be detected by the aberrant dinucleotide composition (genome signature) when compared to the rest of the genome.25 We characterized the genome signature of all tps genes by determining the dinucleotide relative abundance (δ*) in the genome of N. meningitidis strain MC58 (cc32).26 The genome signature of tpsA3 and all tpsB genes where similar to the rest of the genome, whereas all tpsA genes from system 1 and 2 (tpsA1a, tpsA1b, tpsA2a, tpsA2b) were highly dissimilar from the rest of the genome: over 96% of the genome fragments yielded a lower δ* score than tpsA1a, tpsA1b, tpsA2a or tpsA2b (Table 2). This supports a role for

recombination with heterologous DNA fragments to vary the tpsA sequences.

Table 2. Genome signature of tps genes. Gene Percentage of genomic fragments with a lower δ*

tpsB1 49.4 tpsB1btr 41.9 tpsA1 96.0 tpsA1b 98.4 tpsB2 64.5 tpsA2a 99.3 tpsA2b 97.9 tpsA3 44.1 The percentage of genomic fragments with lower dinucleotide relative abundance (δ*, as described in 26) of all tps genes in the Neisseria meningitidis MC58 strain (NCBI reference sequence NC_003112) is shown.

DISCUSSION

Our results show that the presence of functional meningococcal TPS 2 and 3 systems is associated with a better clinical outcome in meningococcal meningitis. Patients infected with

(A) Growth of N. meningitidis H44/76 wild-type (WT) and several TPS system 2&3 knockout and meningococci harboring no TPS or solely TPS1 were admitted to the hospital with more complemented knockout strains in human blood samples. The arrow indicates the start inoculum. Between persons, 3 different growth patterns were observed. Between meningococcal strains, no differences in growth severe disease and had a higher risk of unfavorable outcome, as compared with patients were observed. Negative controls without bacteria (only RPMI) showed no growth. (B) IL-6 induction in human infected with meningococci harboring TPS 2 and/or 3 systems. This contrasts findings that blood samples of 6 persons after 4 hours of growth. Wild-type, several TPS system 2&3 knockout and complemented knockout strains were compared. We observed no difference in IL-6 induction. these systems are immunogenic in humans and thought to contribute to virulence, since

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Figure 2. Meningococcal blood stimulation experiments. Tps genome signature

Table 2. Genome signature of tps genes. Gene Percentage of genomic fragments with a lower δ*

DISCUSSION

Our results show that the presence of functional meningococcal TPS 2 and 3 systems is associated with a better clinical outcome in meningococcal meningitis. Patients infected with

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they are more often found in hyper-invasive strains, as described earlier12 and corroborated the genomic islands of both TPS system 1 and 2 contain tpsC cassettes that appear to be by the results presented here. Unfortunately, we were not able to provide an underlying silent variants of the 3’ end of the functional tpsA’s.6,11 biological mechanism in our blood stimulation experiments since growth characteristics and All tested meningococcal WT and knockout strains induced a similar IL-6 response in our IL-6 responses were similar between isogenic strains that either carried or lacked the three blood-stimulation experiments. Remarkably, the size of the response did not correlate with TPS systems. bacterial growth, even though there was a 1,000-fold difference in CFU’s at 4h between In our collection of clinical CSF isolates, TPS1 was almost uniformly present in the first cohort samples of different donors. Therefore it is likely that in our experiments the IL-6 immune (97%), whereas the presence decreased to 77% in the second cohort, due to the higher response is unrelated to presence of TpsA but is already maximally stimulated by other prevalence (17% vs 4%) of strains without TPS systems (Supplementary Figure 1C). The high meningococcal factors. Previously, TPS system components elicited an antibody response in rate of TPS1 in our first cohort is in contrast with the frequency for TPS1 (82%) that was 4 out of 17 human blood sera from patients who were recovering from a meningococcal reported in a previous study of meningococcal disease isolates covering strains from 2001- infection.12 Probably, only a small portion of the population is capable of an antibody 2005.12 This might be explained by the higher proportion of serogroup Y and W in that response against TPS system components or responses are masked by immunodominant collection (7 of 13 strains were from one of these serogroups as compared to 2 of 8 strains in antigens that are more strongly expressed. the 1998-2002 cohort). Previously, an association between cc11 and outcome was observed, although at that time In the same study, the prevalence of TPS systems capable of inducing an immune response the reason remained unknown.16 Due to serogroup C vaccination in the Netherlands in 2002, (TPS system 2 or 3) was 47%, which is markedly lower than the 64% found in the 1998-2002 the incidence of cc11 meningitis has decreased from 24 to 5%. More recently, the cohort and the 73% found in the 2006-2014 cohort. In our 2 cohorts, 92% and 69% of the meningococcal factor H binding protein D184 polymorphism was associated with CSF isolates were from hyper-virulent clonal complexes, respectively. Remarkably, we could unfavorable outcome.24 The considerable overlap of these traits with TPS system distribution not detect TPS systems 2 and/or 3 in hyper-virulent cc11 and cc8, whereas the prevalence of might suggest that the observed association with outcome is a genetic linkage effect. these systems in the other hyper-virulent clonal complexes was 81-100%. The combined However, when we re-analyzed our data, excluding the patients who were infected with data of the available studies shows that cc8 isolates (n=17) never contained TPS2 and/or 3, meningococci with fHbpD184, the difference in proportion of patients with a GCS score < 14 whereas in cc11 isolates these systems appeared rare (2 isolates positive out of 81 total).7,12 remained between patients infected with meningococci without TPS systems or harboring Overall, the TPS system distribution is highly related to specific clonal complexes. In the first solely TPS1 versus those infected with multiple TPS. cohort, of 235 isolates belonging to the 5 most prevalent clonal complexes, only 13 (6%) Our study provides insights regarding the presence of the types of TPS system and their have an aberrant distribution of TPS systems. In the second cohort, this proportion was 16%. effect on disease outcome and reveals a high frequency of certain TPS system types in The genes encoding TPS systems are located on genomic islands. This is in line with the hyper-invasive clonal complexes, but also a less severe disease outcome when these are observed genomic dissimilarity of the tpsA genes of TPS system 1 and 2, suggesting present. This is relevant to the discussion on the hyper-invasiveness of some clonal horizontal gene transfer as the mechanism of TPS system acquisition. However, the genome complexes. Because of the natural variation in distribution of these systems among strains, signature of the two tpsB’s and the tpsA3 was relatively normal, which would not support further epidemiological surveys as well as analysis of the biological role of the systems such an explanation. On the other hand, the tpsA genes vary in sequence at their 3’ end and remain warranted.

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they are more often found in hyper-invasive strains, as described earlier12 and corroborated the genomic islands of both TPS system 1 and 2 contain tpsC cassettes that appear to be by the results presented here. Unfortunately, we were not able to provide an underlying silent variants of the 3’ end of the functional tpsA’s.6,11 biological mechanism in our blood stimulation experiments since growth characteristics and All tested meningococcal WT and knockout strains induced a similar IL-6 response in our IL-6 responses were similar between isogenic strains that either carried or lacked the three blood-stimulation experiments. Remarkably, the size of the response did not correlate with TPS systems. bacterial growth, even though there was a 1,000-fold difference in CFU’s at 4h between In our collection of clinical CSF isolates, TPS1 was almost uniformly present in the first cohort samples of different donors. Therefore it is likely that in our experiments the IL-6 immune (97%), whereas the presence decreased to 77% in the second cohort, due to the higher response is unrelated to presence of TpsA but is already maximally stimulated by other prevalence (17% vs 4%) of strains without TPS systems (Supplementary Figure 1C). The high meningococcal factors. Previously, TPS system components elicited an antibody response in rate of TPS1 in our first cohort is in contrast with the frequency for TPS1 (82%) that was 4 out of 17 human blood sera from patients who were recovering from a meningococcal reported in a previous study of meningococcal disease isolates covering strains from 2001- infection.12 Probably, only a small portion of the population is capable of an antibody 2005.12 This might be explained by the higher proportion of serogroup Y and W in that response against TPS system components or responses are masked by immunodominant 4 collection (7 of 13 strains were from one of these serogroups as compared to 2 of 8 strains in antigens that are more strongly expressed. the 1998-2002 cohort). Previously, an association between cc11 and outcome was observed, although at that time In the same study, the prevalence of TPS systems capable of inducing an immune response the reason remained unknown.16 Due to serogroup C vaccination in the Netherlands in 2002, (TPS system 2 or 3) was 47%, which is markedly lower than the 64% found in the 1998-2002 the incidence of cc11 meningitis has decreased from 24 to 5%. More recently, the cohort and the 73% found in the 2006-2014 cohort. In our 2 cohorts, 92% and 69% of the meningococcal factor H binding protein D184 polymorphism was associated with CSF isolates were from hyper-virulent clonal complexes, respectively. Remarkably, we could unfavorable outcome.24 The considerable overlap of these traits with TPS system distribution not detect TPS systems 2 and/or 3 in hyper-virulent cc11 and cc8, whereas the prevalence of might suggest that the observed association with outcome is a genetic linkage effect. these systems in the other hyper-virulent clonal complexes was 81-100%. The combined However, when we re-analyzed our data, excluding the patients who were infected with data of the available studies shows that cc8 isolates (n=17) never contained TPS2 and/or 3, meningococci with fHbpD184, the difference in proportion of patients with a GCS score < 14 whereas in cc11 isolates these systems appeared rare (2 isolates positive out of 81 total).7,12 remained between patients infected with meningococci without TPS systems or harboring Overall, the TPS system distribution is highly related to specific clonal complexes. In the first solely TPS1 versus those infected with multiple TPS. cohort, of 235 isolates belonging to the 5 most prevalent clonal complexes, only 13 (6%) Our study provides insights regarding the presence of the types of TPS system and their have an aberrant distribution of TPS systems. In the second cohort, this proportion was 16%. effect on disease outcome and reveals a high frequency of certain TPS system types in The genes encoding TPS systems are located on genomic islands. This is in line with the hyper-invasive clonal complexes, but also a less severe disease outcome when these are observed genomic dissimilarity of the tpsA genes of TPS system 1 and 2, suggesting present. This is relevant to the discussion on the hyper-invasiveness of some clonal horizontal gene transfer as the mechanism of TPS system acquisition. However, the genome complexes. Because of the natural variation in distribution of these systems among strains, signature of the two tpsB’s and the tpsA3 was relatively normal, which would not support further epidemiological surveys as well as analysis of the biological role of the systems such an explanation. On the other hand, the tpsA genes vary in sequence at their 3’ end and remain warranted.

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ACKNOWLEDGMENTS REFERENCES

meningococcal and other life threatening bacterial infections of childhood), A. van der Ende 2. Chevalier N, Moser M, Koch HG, et al. Membrane targeting of a bacterial virulence participated in the Sixth Framework Programme of the European Commission, factor harbouring an extended signal peptide. J Mol Microbiol Biotechnol 2004;8:7- 18. Proposal/Contract no.: 512061 (Network of Excellence “European Virtual Institute for Functional Genomics of Bacterial Pathogens”.) This publication made use of the Neisseria 3. Jacob-Dubuisson F, Guerin J, Baelen S, Clantin B. Two-partner secretion: as simple as it sounds? Research in microbiology 2013;164:583-595. Multilocus Sequence Typing website (http://pubmlst.org/neisseria/) developed by Keith 4. van Ulsen P, Rahman S, Jong WS, Daleke-Schermerhorn MH, Luirink J. Type V Jolley and sited at the University of Oxford: Development of this site has been funded by the secretion: from biogenesis to biotechnology. Biochim Biophys Acta 2014;1843:1592- Wellcome Trust and European Union. 1611.

5. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia,

and Neisseria meningitidis. Lancet 2007;369:2196-2210.

FUNDING 6. van Ulsen P, Tommassen J. Protein secretion and secreted proteins in pathogenic Neisseriaceae. FEMS Microbiol Rev 2006;30:292-319. This study has been funded by grants from the Netherlands Organization for Health Research 7. Tala A, Progida C, De SM, et al. The HrpB-HrpA two-partner secretion system is and Development (ZonMw; NWO-Veni grant 2006 [916.76.023], NWO-Vidi grant 2010 essential for intracellular survival of Neisseria meningitidis. Cell Microbiol 2008;10:2461-2482. [016.116.358], both to D. van de Beek), the Academic Medical Center (AMC) Fellowship 2008 to D. van de Beek and AMC PhD scholarship 2008 to J. Piet. The funders had no role in study 8. Schmitt C, Turner D, Boesl M, Abele M, Frosch M, Kurzai O. A functional two-partner secretion system contributes to adhesion of Neisseria meningitidis to epithelial cells. design, data collection and interpretation, or the decision to submit the work for publication. J Bacteriol 2007;189:7968-7976.

9. Neil RB, Apicella MA. Role of HrpA in biofilm formation of Neisseria meningitidis and regulation of the hrpBAS transcripts. Infect Immun 2009;77:2285-2293.

10. Poole SJ, Diner EJ, Aoki SK, et al. Identification of functional toxin/immunity genes linked to contact-dependent growth inhibition (CDI) and rearrangement hotspot (Rhs) systems. PLoS Genet 2011;7:e1002217.

11. Arenas J, Schipper K, van Ulsen P, van der Ende A, Tommassen J. Domain exchange at the 3' end of the gene encoding the fratricide meningococcal two-partner secretion protein A. BMC Genomics 2013;14:622.

12. van Ulsen P, Rutten L, Feller M, Tommassen J, van der Ende A. Two-partner secretion systems of Neisseria meningitidis associated with invasive clonal complexes. Infect Immun 2008;76:4649-4658.

13. Bentley SD, Vernikos GS, Snyder LA, et al. Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18. PLoS Genet 2007;3:e23.

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1. ur Rahman S, van Ulsen P. System specificity of the TpsB transporters of coexpressed D. van de Beek and A. van der Ende participate in the Seventh Framework Programme of the two-partner secretion systems of Neisseria meningitidis. J Bacteriol 2013;195:788- European Commission, Proposal/Contract no.: 279185 (EUCLIDS -The genetic basis of 797. meningococcal and other life threatening bacterial infections of childhood), A. van der Ende 2. Chevalier N, Moser M, Koch HG, et al. Membrane targeting of a bacterial virulence participated in the Sixth Framework Programme of the European Commission, factor harbouring an extended signal peptide. J Mol Microbiol Biotechnol 2004;8:7- 18. Proposal/Contract no.: 512061 (Network of Excellence “European Virtual Institute for Functional Genomics of Bacterial Pathogens”.) This publication made use of the Neisseria 3. Jacob-Dubuisson F, Guerin J, Baelen S, Clantin B. Two-partner secretion: as simple as it sounds? Research in microbiology 2013;164:583-595. Multilocus Sequence Typing website (http://pubmlst.org/neisseria/) developed by Keith 4. van Ulsen P, Rahman S, Jong WS, Daleke-Schermerhorn MH, Luirink J. Type V Jolley and sited at the University of Oxford: Development of this site has been funded by the secretion: from biogenesis to biotechnology. Biochim Biophys Acta 2014;1843:1592- Wellcome Trust and European Union. 1611. 5. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, 4

and Neisseria meningitidis. Lancet 2007;369:2196-2210.

9. Neil RB, Apicella MA. Role of HrpA in biofilm formation of Neisseria meningitidis and regulation of the hrpBAS transcripts. Infect Immun 2009;77:2285-2293.

12. van Ulsen P, Rutten L, Feller M, Tommassen J, van der Ende A. Two-partner secretion systems of Neisseria meningitidis associated with invasive clonal complexes. Infect Immun 2008;76:4649-4658.

13. Bentley SD, Vernikos GS, Snyder LA, et al. Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18. PLoS Genet 2007;3:e23.

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14. Jennett B, Teasdale G. Management of head injuries. 2 ed. Philadelphia: F.A.Davis; SUPPLEMENTARY MATERIAL 1981. Supplementary Figure 1. TPS system distribution by cohort, serogroup and clonal complex. 15. Maiden MC, Bygraves JA, Feil E, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 1998;95:3140-3145.

16. Heckenberg SG, de Gans J, Brouwer MC, et al. Clinical features, outcome, and meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study. Medicine (Baltimore) 2008;87:185-192.

17. Bijlsma MW, Brouwer MC, Kasanmoentalib ES, et al. Community-acquired bacterial meningitis in adults in the Netherlands, 2006-14: a prospective cohort study. Lancet Infect Dis 2015.

18. Chewapreecha C, Harris SR, Croucher NJ, et al. Dense genomic sampling identifies highways of pneumococcal recombination. Nat Genet 2014;46:305-309.

19. Holten E. Serotypes of Neisseria meningitidis isolated from patients in Norway during the first six months of 1978. J Clin Microbiol 1979;9:186-188.

20. Johnston DM, Cannon JG. Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic resistance markers using a two gene cassette with positive and negative selection. Gene 1999;236:179-184.

21. van Ulsen P, van Alphen L, ten Hove J, Fransen F, van der Ley P, Tommassen J. A Neisserial autotransporter NalP modulating the processing of other autotransporters. Mol Microbiol 2003;50:1017-1030.

22. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Med 2004;351:1849-1859.

23. Caugant DA. Population genetics and molecular epidemiology of Neisseria meningitidis. APMIS 1998;106:505-525. The proportion of TPS system distribution by cohort (A), serogroup (B) and clonal complex (cc; C) in both 24. Piet JR, Brouwer MC, Exley R, van der Veen S, van de Beek D, van der Ende A. cohorts is shown. ND = not determined. Each isolate could only contribute to one of the 6 TPS distribution Meningococcal Factor H Binding Protein fHbpd184 Polymorphism Influences Clinical categories. Course of Meningococcal Meningitis. PLoS One 2012;7:e47973.

25. van Passel MW, Bart A, Thygesen HH, Luyf AC, van Kampen AH, van der Ende A. An acquisition account of genomic islands based on genome signature comparisons. BMC Genomics 2005;6:163.

26. van Passel MW, Luyf AC, van Kampen AH, Bart A, van der Ende A. Deltarho-web, an online tool to assess composition similarity of individual nucleic acid sequences. Bioinformatics 2005;21:3053-3055.

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17. Bijlsma MW, Brouwer MC, Kasanmoentalib ES, et al. Community-acquired bacterial meningitis in adults in the Netherlands, 2006-14: a prospective cohort study. Lancet Infect Dis 2015.

18. Chewapreecha C, Harris SR, Croucher NJ, et al. Dense genomic sampling identifies highways of pneumococcal recombination. Nat Genet 2014;46:305-309. 4 19. Holten E. Serotypes of Neisseria meningitidis isolated from patients in Norway during the first six months of 1978. J Clin Microbiol 1979;9:186-188.

22. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J Med 2004;351:1849-1859.

25. van Passel MW, Bart A, Thygesen HH, Luyf AC, van Kampen AH, van der Ende A. An acquisition account of genomic islands based on genome signature comparisons. BMC Genomics 2005;6:163.

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Supplementary Figure 2. Western Blot analysis of TpsA proteins. Supplementary Figure 3. N. meningitidis growth curve in RPMI.

Growth of N. meningitidis H44/76 wild-type (WT) and several TPS system 2&3 knockout and complemented knockout strains in RPMI medium.

Immunoblots of whole cell lysates (P) and concentrated culture supernatants (S) of cultures of H44/76, H44/76 ΔtpsB2, H44/76 ΔtpsB2/tpsA3, H44/76 ΔtpsB2 with pEN-TpsB2 and H44/76 ΔtpsB2/tpsA3 with pEN-TpsB2, the Supplementary Table 1. TPS system primers. latter grown in the presence or absence of 0.5 mM IPTG to induce expression of the tpsB2 gene, as indicated Primer name Target gene DNA sequence above the lanes. The blots were incubated with αTPS2 (top),αTpsB2 (middle), and αTPS1 (bottom) as indicated LR1 tpsB1 AATTTTTTCCTGCTCCATGTCTGT on the left. The blots show secretion of the TPS1 system in all strains, indicating that it was not affected by the LR2 knockout mutations in systems 2 and 3. Furthermore, the TpsB2 is only present in the wild-type and TTAGAAACTGTAATTCAAGTTGAAGCCGTAA complemented strains. When present it resulted in the secretion of ~145 and ~130 kDa TpsA protein variants of tps1 tpsA1 ATGAATAAAGGTTTACATCGCATTATCTTTAGT systems 2 and 3. tps2 AAGCATTTAATACCGTGGTAGCTGGGCGA LR6 tpsB2 TCCCCCAACCCTGCCGAAATCCG LR8 TTAAAACGTATAGCCTACCTGAAAACCGCT tps4 tpsA2a ATGGAATAAAACTCTCTATCGTGTAATTTTCAA tps5 AGTGTTTTGAATCGACTGACTGTGAATT tps9 tpsA2b TATGAACCGCACCCTGTACAAAGTTGT tps10 GCCGGCAGATACCAAATCCAATGCT tps7 tpsA3 ATGAACAAGCGATGCTACAAGGTTATCT

tps8 GCGTCCGGCTTCAATATCGCGT

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Supplementary Figure 2. Western Blot analysis of TpsA proteins. Supplementary Figure 3. N. meningitidis growth curve in RPMI.

4 Growth of N. meningitidis H44/76 wild-type (WT) and several TPS system 2&3 knockout and complemented knockout strains in RPMI medium.

tps8 GCGTCCGGCTTCAATATCGCGT

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Supplementary Table 2 (online available after publication). Meningococcal serogroup data, sequence types, Supplementary Table 3. Summary of TPS system distribution in all available cohorts. clonal complexes, and tps gene/TPS system distribution in both cohorts.( Isolates with Isolates with TPS system 2 and/or 3

TPS1 Cohort 1998-2002 Cohort 2006-2014 LR1-LR2 tps1-tps2 LR6-LR8 tps4-tps5 tps9-tps10 tps7-tps8 Strain number serogroup Sequence type Clonal complex Strain number serogroup Sequence type Clonal complex tpsB1 tpsA 1a tpsA 1b tpsB2 tpsA 2a tpsA 2b tpsA 3 12,a (tpsB1 ) (tpsA 1 ) (tpsB2 ) (tpsA 2a ) (tpsA 2b ) (tpsA 3 ) 981781 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2060092 W 22 ST-22 complex 0 1 1 0 0 0 0 Van Ulsen et al. (invasive disease isolates) 72/88 (82%) 41/88 (47%) 981784 C 60 ST-60 complex 1 1 0 0 0 0 2060516 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 981826 B 5444 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2060603 B 5548 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 981827 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2060640 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 981871 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2060716 B 41 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 8,b 981915 B 2203 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2060737 B 1403 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 981936 B 41 ST-41/44 complex/Lineage 3 1 1 0 0 0 0 2060979 B 4484 ST-22 complex 0 1 1 0 0 0 0 Schmitt et al. (carrier isolates) 485/632 (77%) 328/822 (40%) 981987 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2061025 B 467 ST-269 complex 1 0 0 1 1 1 1 982029 B 1374 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061079 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 982084 C 66 ST-8 complex/Cluster A4 1 1 0 0 0 0 2061250 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 982102 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061255 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 982144 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061370 B 269 ST-269 complex 1 1 1 1 1 1 1 982153 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061400 B 42 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 982199 B 269 ST-269 complex 1 1 1 1 1 1 2061481 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 982200 B 5445 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061515 Y 167 ST-167 complex 1 1 1 0 0 0 0 982245 B 5098 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061596 B 154 ST-41/44 complex/Lineage 3 1 1 1 0 0 0 0 982340 B 5446 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061655 B 2707 ST-32 complex/ET-5 complex 0 1 1 1 1 1 1 982347 B 269 ST-269 complex 1 1 1 1 1 1 2070069 Y 167 ST-167 complex 1 1 1 0 0 0 0 990001 B 1163 ST-269 complex 1 1 1 1 1 1 2070093 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 Current study 990005 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2070150 B 6287 NA 0 1 1 1 1 1 0 990007 B 2080 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070151 B 1255 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 990030 B 2286 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070189 Y 1466 ST-174 complex 0 1 1 0 0 0 0 990056 C 51 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2070215 W 6288 ST-22 complex 0 1 1 0 0 0 0 990062 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070308 B 6289 NA 1 1 1 1 1 0 0 990069 B 2016 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070339 B 1273 ST-269 complex 1 1 1 1 1 1 1 1998-2002 cohort 246/254 (97%) 162/254 (64%) 990082 B 2016 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070395 B 3754 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 990092 B 159 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070406 B 269 ST-269 complex 1 1 1 1 1 1 1 990104 B 5447 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070506 B 1239 ST-60 complex 1 1 1 0 0 0 0 990121 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2070591 B 1374 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 990134 B 5448 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070737 X 5063 NA 0 1 1 1 1 0 0 990135 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070862 B 6297 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 2006-2014 cohort 89/115 (77%) 84/115 (73%) 990146 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070929 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 990149 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071109 B 213 ST-213 complex 1 1 1 0 1 1 0 990328 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071236 B 1161 ST-269 complex 1 1 1 1 1 1 1 990334 B 5449 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071276 B 2080 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 a 990492 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2071282 B 259 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 990502 B 4100 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071283 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 Positive isolates are redefined as stated in the material and methods section of this article. 990576 B 571 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071328 B 4172 ST-41/44 complex/Lineage 3 1 0 0 1 1 1 0 990601 B 212 - 1 1 1 1 1 0 2071344 B 42 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 b 990602 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2071345 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 990615 W 1286 ST-22 complex 1 0 0 0 0 0 2071354 B 40 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 tpsA1, tpsA2a, 990653 B 259 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2071442 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 Positive isolates are defined as giving a positive result in the dot plot with specific probes for 990738 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2071538 B 461 ST-461 complex 1 0 0 1 1 1 0 990797 C 5450 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2071749 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 990808 B 259 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2071759 B 213 ST-213 complex 1 1 1 1 1 1 0 990815 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071794 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 tpsA2B or tpsA3 genes (the corresponding information on the tpsB status of each strain was not reported). 990907 B 2707 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2071864 B 154 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 990947 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2071909 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 990975 B 269 ST-269 complex 1 1 1 1 1 1 2080098 C 211 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 991027 B 4065 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080184 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 991044 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2080418 B 41 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991056 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2080520 B 1374 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991093 B 5451 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080543 B 7883 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 991097 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080556 B 4923 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991117 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080584 C 1031 ST-334 complex 1 1 1 1 1 1 0 991159 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080856 B 303 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991174 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2081063 B 1161 ST-269 complex 1 1 1 1 1 1 1 991192 B 1403 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2081105 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 991208 B 283 ST-269 complex 0 1 1 1 1 1 2081595 B 213 ST-213 complex 1 1 1 1 1 1 0 991210 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2081653 B 41 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991247 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2081656 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 991275 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2081977 B 2136 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991344 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2082123 B 40 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991379 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2082183 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 991382 B 191 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2082258 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 991397 B 5452 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2090237 B 213 ST-213 complex 1 1 1 1 1 1 0 991511 B 3754 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2090353 B 2925 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 991598 Y 167 ST-167 complex 1 1 0 0 0 0 2090516 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 991625 B 1374 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2090911 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 991642 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2091136 B 5118 NA 1 1 1 1 1 1 1 991661 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2091153 B 7741 ST-213 complex 1 1 1 1 1 1 0 991705 B 1960 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2091198 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 991712 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2091661 B 213 ST-213 complex 1 1 1 1 1 1 0 991722 B 3621 - 1 1 1 1 1 0 2091724 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 991765 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2092127 B 7737 ST-269 complex 0 0 0 1 1 1 1 991774 B 1788 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2093373 B 40 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991853 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2094485 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 991930 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2100125 B 2925 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991932 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2100157 B 2925 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 992008 B 146 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2100282 B 269 ST-269 complex 1 1 1 1 1 1 1 992062 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2100381 B 9072 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 992073 Y 5453 ST-92 complex 1 0 0 0 0 0 2102065 B 2925 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 992076 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2102083 Y 23 ST-23 complex/Cluster A3 0 1 1 0 0 0 0 2000020 B 4586 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103113 B 136 ST-41/44 complex/Lineage 3 0 0 0 1 1 1 0 2000024 B 32 ST-32 complex/ET-5 complex 1 1 0 0 0 1 2103119 Y 6288 ST-22 complex 0 1 1 0 0 0 0 2000041 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103251 B 2578 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 2000070 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103596 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000100 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103742 B 2945 NA 1 1 1 1 1 0 0 2000101 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103747 B 213 ST-213 complex 1 0 0 1 1 1 0 2000131 B 269 ST-269 complex 1 1 1 1 1 1 2104366 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000136 B 5454 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2110092 B 9073 ST-213 complex 1 1 1 1 1 1 0 2000149 B 269 ST-269 complex 1 1 1 1 1 1 2110538 B 5266 ST-269 complex 1 1 1 1 1 1 1 2000151 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2110842 B ND ND 1 0 0 1 1 1 0 2000194 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2110915 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000201 B 2708 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2110947 B 41 ST-41/44 complex/Lineage 3 1 1 1 0 0 0 0 2000234 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2111171 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000236 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2111581 C 5133 ST-103 complex 1 1 1 0 0 0 0 2000250 B 32 ST-32 complex/ET-5 complex 1 1 0 0 0 1 2120056 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 2000297 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2120194 Y 1466 ST-174 complex 0 1 1 0 0 0 0 2000311 B 461 ST-461 complex 1 1 1 1 1 0 2120265 Y 1466 ST-174 complex 0 1 1 0 0 0 0 2000345 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2120394 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000373 C 461 ST-461 complex 1 1 1 1 1 0 2120492 B 1157 ST-1157 complex 0 1 1 0 0 0 0 2000384 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2120610 Y 23 ST-23 complex/Cluster A3 0 1 1 0 0 0 0 2000488 B 2631 ST-41/44 complex/Lineage 3 1 1 0 0 0 0 2120785 W 1158 ST-22 complex 0 1 1 0 0 0 0 2000500 B 269 ST-269 complex 1 1 1 1 1 1 2120864 Y 4272 ST-23 complex/Cluster A3 0 1 1 0 0 0 0 2000589 B 4257 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2120910 B 2314 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 2000594 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2120945 B 2827 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 2000607 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2121404 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 2000622 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2121460 B 213 ST-213 complex 1 1 1 1 1 1 0 2000709 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2121477 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000726 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2121528 B 213 ST-213 complex 1 1 1 1 1 1 0 2000732 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2130047 Y 23 ST-23 complex/Cluster A3 0 1 1 0 0 0 0 2000739 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2130367 B 9808 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 2000749 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2130695 B 1163 ST-269 complex 1 1 1 1 1 1 1 2000760 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2130749 B 571 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 2000779 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2000803 B 3488 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000804 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000869 B 5455 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2000880 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000881 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000885 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000948 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2000974 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2000987 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001043 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001212 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2001256 B 1374 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001297 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2001318 B 269 ST-269 complex 1 1 1 1 1 1 2001329 C 2704 ST-11 complex/ET-37 complex insertion 1 0 0 0 0 2001346 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2001394 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001396 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001449 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001474 B 4018 ST-41/44 complex/Lineage 3 1 1 0 0 0 0 2001477 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2001533 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2001553 B 2916 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001556 B 1163 ST-269 complex 1 1 1 1 1 1 2001573 C 483 ST-18 complex 1 1 1 1 1 0 2001615 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001632 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001633 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2001717 C 2704 ST-11 complex/ET-37 complex insertion 1 0 0 0 0 2001718 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2001782 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2001862 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001915 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001970 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001976 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2002042 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2002060 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2002091 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2002154 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2002173 C 3456 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010002 B 2696 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2010024 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010080 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010082 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010115 C 2699 ST-8 complex/Cluster A4 1 1 0 0 0 0 2010178 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010216 B 3546 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010221 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2010259 B 269 ST-269 complex 1 1 1 1 1 1 2010277 B 5406 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010306 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2010321 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010353 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010450 B 60 ST-60 complex 1 1 0 0 0 0 2010513 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010640 B 1475 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010688 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2010699 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010749 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010760 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010903 B 3547 ST-60 complex 1 1 1 1 0 0 2010904 B 3548 ST-18 complex 1 1 1 1 1 0 2010939 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010999 B 3549 - 1 0 1 1 0 0 2011023 B 3550 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011029 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011043 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011060 C 2699 ST-8 complex/Cluster A4 1 1 0 0 0 0 2011098 B 4258 - 1 0 1 1 1 0 2011148 B 3551 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011150 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011212 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011215 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011233 B 3552 ST-32 complex/ET-5 complex 1 1 0 0 0 1 2011246 B 3552 ST-32 complex/ET-5 complex 1 1 0 0 0 1 2011332 B 274 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011334 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011337 B 259 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2011528 C 2704 ST-11 complex/ET-37 complex insertion 1 0 0 0 0 2011564 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2011595 B 5456 ST-269 complex 1 1 1 1 1 1 2011728 C 4283 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011745 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2011764 B 303 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011814 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011831 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011832 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011833 C 3553 ST-269 complex 1 1 1 1 1 1 2011851 B 3554 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011929 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011973 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2011979 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2012202 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2012239 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2012278 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012280 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012303 C 269 ST-269 complex 1 1 1 1 1 1 2012326 B 5408 - 1 1 1 1 1 0 2012431 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012531 B 2700 - 1 0 1 1 0 0 2012552 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2012598 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012602 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012620 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012640 B 2708 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2012655 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012673 C 2709 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020047 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020094 B 269 ST-269 complex 1 1 1 1 1 1 2020149 B 60 ST-60 complex 1 1 0 0 0 0 2020150 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020151 C 3486 ST-8 complex/Cluster A4 1 1 0 0 0 0 2020165 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020193 Y 3544 ST-174 complex 1 1 0 0 0 0 2020207 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020226 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020276 B 3608 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020324 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020328 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020383 B 213 ST-213 complex 1 1 0 1 1 0 2020416 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020417 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020434 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020435 C 3298 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020449 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020473 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020503 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020546 B 1374 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020547 B 5457 ST-364 complex 1 1 0 1 1 0 2020561 C 1374 ST-41/44 complex/Lineage 3 1 1 0 0 0 0 2020622 B 5458 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020707 C 8 ST-8 complex/Cluster A4 1 1 0 0 0 0 2020745 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2020786 B 3515 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020798 B 275 ST-269 complex 1 1 1 1 1 1 2020799 B 35 ST-35 complex 1 1 0 0 0 1 2020811 B 1960 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020843 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0

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40049 Piet, Jurgen.indd 82 26-04-16 15:08 Chapter 4 Meningococcal two-partner secretion systems and outcome

TPS1 Cohort 1998-2002 Cohort 2006-2014 LR1-LR2 tps1-tps2 LR6-LR8 tps4-tps5 tps9-tps10 tps7-tps8 Strain number serogroup Sequence type Clonal complex Strain number serogroup Sequence type Clonal complex tpsB1 tpsA 1a tpsA 1b tpsB2 tpsA 2a tpsA 2b tpsA 3 12,a (tpsB1 ) (tpsA 1 ) (tpsB2 ) (tpsA 2a ) (tpsA 2b ) (tpsA 3 ) 981781 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2060092 W 22 ST-22 complex 0 1 1 0 0 0 0 Van Ulsen et al. (invasive disease isolates) 72/88 (82%) 41/88 (47%) 981784 C 60 ST-60 complex 1 1 0 0 0 0 2060516 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 981826 B 5444 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2060603 B 5548 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 981827 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2060640 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 981871 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2060716 B 41 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 8,b 981915 B 2203 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2060737 B 1403 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 981936 B 41 ST-41/44 complex/Lineage 3 1 1 0 0 0 0 2060979 B 4484 ST-22 complex 0 1 1 0 0 0 0 Schmitt et al. (carrier isolates) 485/632 (77%) 328/822 (40%) 981987 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2061025 B 467 ST-269 complex 1 0 0 1 1 1 1 982029 B 1374 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061079 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 982084 C 66 ST-8 complex/Cluster A4 1 1 0 0 0 0 2061250 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 982102 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061255 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 982144 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061370 B 269 ST-269 complex 1 1 1 1 1 1 1 982153 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061400 B 42 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 982199 B 269 ST-269 complex 1 1 1 1 1 1 2061481 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 982200 B 5445 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061515 Y 167 ST-167 complex 1 1 1 0 0 0 0 982245 B 5098 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061596 B 154 ST-41/44 complex/Lineage 3 1 1 1 0 0 0 0 982340 B 5446 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2061655 B 2707 ST-32 complex/ET-5 complex 0 1 1 1 1 1 1 982347 B 269 ST-269 complex 1 1 1 1 1 1 2070069 Y 167 ST-167 complex 1 1 1 0 0 0 0 990001 B 1163 ST-269 complex 1 1 1 1 1 1 2070093 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 Current study 990005 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2070150 B 6287 NA 0 1 1 1 1 1 0 990007 B 2080 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070151 B 1255 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 990030 B 2286 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070189 Y 1466 ST-174 complex 0 1 1 0 0 0 0 990056 C 51 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2070215 W 6288 ST-22 complex 0 1 1 0 0 0 0 990062 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070308 B 6289 NA 1 1 1 1 1 0 0 990069 B 2016 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070339 B 1273 ST-269 complex 1 1 1 1 1 1 1 1998-2002 cohort 246/254 (97%) 162/254 (64%) 990082 B 2016 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070395 B 3754 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 990092 B 159 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070406 B 269 ST-269 complex 1 1 1 1 1 1 1 990104 B 5447 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070506 B 1239 ST-60 complex 1 1 1 0 0 0 0 990121 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2070591 B 1374 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 990134 B 5448 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070737 X 5063 NA 0 1 1 1 1 0 0 990135 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070862 B 6297 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 2006-2014 cohort 89/115 (77%) 84/115 (73%) 990146 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2070929 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 990149 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071109 B 213 ST-213 complex 1 1 1 0 1 1 0 990328 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071236 B 1161 ST-269 complex 1 1 1 1 1 1 1 990334 B 5449 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071276 B 2080 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 a 990492 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2071282 B 259 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 990502 B 4100 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071283 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 Positive isolates are redefined as stated in the material and methods section of this article. 990576 B 571 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071328 B 4172 ST-41/44 complex/Lineage 3 1 0 0 1 1 1 0 990601 B 212 - 1 1 1 1 1 0 2071344 B 42 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 b 990602 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2071345 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 990615 W 1286 ST-22 complex 1 0 0 0 0 0 2071354 B 40 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 tpsA1, tpsA2a, 990653 B 259 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2071442 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 Positive isolates are defined as giving a positive result in the dot plot with specific probes for 990738 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2071538 B 461 ST-461 complex 1 0 0 1 1 1 0 990797 C 5450 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2071749 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 990808 B 259 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2071759 B 213 ST-213 complex 1 1 1 1 1 1 0 990815 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2071794 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 tpsA2B or tpsA3 genes (the corresponding information on the tpsB status of each strain was not reported). 990907 B 2707 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2071864 B 154 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 990947 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2071909 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 990975 B 269 ST-269 complex 1 1 1 1 1 1 2080098 C 211 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 991027 B 4065 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080184 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 991044 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2080418 B 41 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991056 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2080520 B 1374 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991093 B 5451 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080543 B 7883 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 991097 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080556 B 4923 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991117 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080584 C 1031 ST-334 complex 1 1 1 1 1 1 0 991159 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2080856 B 303 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991174 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2081063 B 1161 ST-269 complex 1 1 1 1 1 1 1 991192 B 1403 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2081105 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 991208 B 283 ST-269 complex 0 1 1 1 1 1 2081595 B 213 ST-213 complex 1 1 1 1 1 1 0 991210 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2081653 B 41 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991247 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2081656 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 991275 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2081977 B 2136 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991344 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2082123 B 40 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991379 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2082183 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 991382 B 191 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2082258 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 991397 B 5452 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2090237 B 213 ST-213 complex 1 1 1 1 1 1 0 991511 B 3754 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2090353 B 2925 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 991598 Y 167 ST-167 complex 1 1 0 0 0 0 2090516 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 991625 B 1374 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2090911 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 991642 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2091136 B 5118 NA 1 1 1 1 1 1 1 991661 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2091153 B 7741 ST-213 complex 1 1 1 1 1 1 0 991705 B 1960 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2091198 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 991712 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2091661 B 213 ST-213 complex 1 1 1 1 1 1 0 991722 B 3621 - 1 1 1 1 1 0 2091724 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 991765 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2092127 B 7737 ST-269 complex 0 0 0 1 1 1 1 991774 B 1788 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2093373 B 40 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991853 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2094485 C 11 ST-11 complex/ET-37 complex 1 0 0 0 0 0 0 991930 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2100125 B 2925 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 991932 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2100157 B 2925 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 992008 B 146 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2100282 B 269 ST-269 complex 1 1 1 1 1 1 1 992062 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2100381 B 9072 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 4 992073 Y 5453 ST-92 complex 1 0 0 0 0 0 2102065 B 2925 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 992076 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2102083 Y 23 ST-23 complex/Cluster A3 0 1 1 0 0 0 0 2000020 B 4586 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103113 B 136 ST-41/44 complex/Lineage 3 0 0 0 1 1 1 0 2000024 B 32 ST-32 complex/ET-5 complex 1 1 0 0 0 1 2103119 Y 6288 ST-22 complex 0 1 1 0 0 0 0 2000041 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103251 B 2578 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 2000070 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103596 B 3720 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000100 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103742 B 2945 NA 1 1 1 1 1 0 0 2000101 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2103747 B 213 ST-213 complex 1 0 0 1 1 1 0 2000131 B 269 ST-269 complex 1 1 1 1 1 1 2104366 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000136 B 5454 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2110092 B 9073 ST-213 complex 1 1 1 1 1 1 0 2000149 B 269 ST-269 complex 1 1 1 1 1 1 2110538 B 5266 ST-269 complex 1 1 1 1 1 1 1 2000151 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2110842 B ND ND 1 0 0 1 1 1 0 2000194 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2110915 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000201 B 2708 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2110947 B 41 ST-41/44 complex/Lineage 3 1 1 1 0 0 0 0 2000234 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2111171 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000236 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2111581 C 5133 ST-103 complex 1 1 1 0 0 0 0 2000250 B 32 ST-32 complex/ET-5 complex 1 1 0 0 0 1 2120056 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 2000297 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2120194 Y 1466 ST-174 complex 0 1 1 0 0 0 0 2000311 B 461 ST-461 complex 1 1 1 1 1 0 2120265 Y 1466 ST-174 complex 0 1 1 0 0 0 0 2000345 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2120394 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000373 C 461 ST-461 complex 1 1 1 1 1 0 2120492 B 1157 ST-1157 complex 0 1 1 0 0 0 0 2000384 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2120610 Y 23 ST-23 complex/Cluster A3 0 1 1 0 0 0 0 2000488 B 2631 ST-41/44 complex/Lineage 3 1 1 0 0 0 0 2120785 W 1158 ST-22 complex 0 1 1 0 0 0 0 2000500 B 269 ST-269 complex 1 1 1 1 1 1 2120864 Y 4272 ST-23 complex/Cluster A3 0 1 1 0 0 0 0 2000589 B 4257 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2120910 B 2314 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 2000594 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2120945 B 2827 ST-41/44 complex/Lineage 3 1 1 1 1 1 1 0 2000607 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2121404 B 32 ST-32 complex/ET-5 complex 1 1 1 0 0 0 1 2000622 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2121460 B 213 ST-213 complex 1 1 1 1 1 1 0 2000709 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2121477 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 1 2000726 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2121528 B 213 ST-213 complex 1 1 1 1 1 1 0 2000732 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2130047 Y 23 ST-23 complex/Cluster A3 0 1 1 0 0 0 0 2000739 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2130367 B 9808 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 2000749 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2130695 B 1163 ST-269 complex 1 1 1 1 1 1 1 2000760 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2130749 B 571 ST-41/44 complex/Lineage 3 0 1 1 1 1 1 0 2000779 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2000803 B 3488 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000804 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000869 B 5455 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2000880 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000881 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000885 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2000948 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2000974 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2000987 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001043 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001212 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2001256 B 1374 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001297 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2001318 B 269 ST-269 complex 1 1 1 1 1 1 2001329 C 2704 ST-11 complex/ET-37 complex insertion 1 0 0 0 0 2001346 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2001394 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001396 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001449 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001474 B 4018 ST-41/44 complex/Lineage 3 1 1 0 0 0 0 2001477 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2001533 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2001553 B 2916 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001556 B 1163 ST-269 complex 1 1 1 1 1 1 2001573 C 483 ST-18 complex 1 1 1 1 1 0 2001615 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001632 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001633 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2001717 C 2704 ST-11 complex/ET-37 complex insertion 1 0 0 0 0 2001718 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2001782 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2001862 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001915 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001970 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2001976 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2002042 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2002060 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2002091 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2002154 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2002173 C 3456 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010002 B 2696 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2010024 B 40 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010080 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010082 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010115 C 2699 ST-8 complex/Cluster A4 1 1 0 0 0 0 2010178 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010216 B 3546 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010221 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2010259 B 269 ST-269 complex 1 1 1 1 1 1 2010277 B 5406 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010306 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2010321 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010353 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010450 B 60 ST-60 complex 1 1 0 0 0 0 2010513 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010640 B 1475 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010688 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2010699 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010749 B 42 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2010760 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010903 B 3547 ST-60 complex 1 1 1 1 0 0 2010904 B 3548 ST-18 complex 1 1 1 1 1 0 2010939 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2010999 B 3549 - 1 0 1 1 0 0 2011023 B 3550 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011029 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011043 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011060 C 2699 ST-8 complex/Cluster A4 1 1 0 0 0 0 2011098 B 4258 - 1 0 1 1 1 0 2011148 B 3551 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011150 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011212 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011215 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011233 B 3552 ST-32 complex/ET-5 complex 1 1 0 0 0 1 2011246 B 3552 ST-32 complex/ET-5 complex 1 1 0 0 0 1 2011332 B 274 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011334 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011337 B 259 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2011528 C 2704 ST-11 complex/ET-37 complex insertion 1 0 0 0 0 2011564 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2011595 B 5456 ST-269 complex 1 1 1 1 1 1 2011728 C 4283 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011745 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2011764 B 303 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011814 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011831 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011832 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011833 C 3553 ST-269 complex 1 1 1 1 1 1 2011851 B 3554 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2011929 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2011973 B 32 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2011979 B 33 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2012202 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2012239 B 34 ST-32 complex/ET-5 complex 1 1 1 1 1 1 2012278 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012280 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012303 C 269 ST-269 complex 1 1 1 1 1 1 2012326 B 5408 - 1 1 1 1 1 0 2012431 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012531 B 2700 - 1 0 1 1 0 0 2012552 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2012598 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012602 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012620 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012640 B 2708 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2012655 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2012673 C 2709 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020047 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020094 B 269 ST-269 complex 1 1 1 1 1 1 2020149 B 60 ST-60 complex 1 1 0 0 0 0 2020150 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020151 C 3486 ST-8 complex/Cluster A4 1 1 0 0 0 0 2020165 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020193 Y 3544 ST-174 complex 1 1 0 0 0 0 2020207 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020226 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020276 B 3608 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020324 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020328 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020383 B 213 ST-213 complex 1 1 0 1 1 0 2020416 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020417 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020434 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020435 C 3298 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020449 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020473 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020503 C 11 ST-11 complex/ET-37 complex 1 1 0 0 0 0 2020546 B 1374 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020547 B 5457 ST-364 complex 1 1 0 1 1 0 2020561 C 1374 ST-41/44 complex/Lineage 3 1 1 0 0 0 0 2020622 B 5458 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020707 C 8 ST-8 complex/Cluster A4 1 1 0 0 0 0 2020745 C 2680 ST-8 complex/Cluster A4 1 1 0 0 0 0 2020786 B 3515 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020798 B 275 ST-269 complex 1 1 1 1 1 1 2020799 B 35 ST-35 complex 1 1 0 0 0 1 2020811 B 1960 ST-41/44 complex/Lineage 3 1 1 1 1 1 0 2020843 B 41 ST-41/44 complex/Lineage 3 1 1 1 1 1 0

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CHAPTER 5:

An analysis of the sequence variability of meningococcal fHbp, NadA and NHBA over a 50-year period in the Netherlands

Stefania Bambini Jurgen R. Piet Alessandro Muzzi Wendy Keijzers Sara Comandi Lisa De Tora Mariagrazia Pizza Rino Rappuoli Diederik van de Beek Arie van der Ende Maurizio Comanducci

PLoS One 2013;8:e65043

40049 Piet, Jurgen.indd 84 26-04-16 15:08 Meningococcal sequence variability of fHbp, NadA and NHBA

CHAPTER 5: CHAPTER 5: An analysis of the sequence variability of meningococcal fHbp, NadA and NHBA over a 50-year period in the Netherlands

An analysis of the sequence

Stefania Bambinivariability of meningococcal Jurgen R. Piet Alessandro Muzzi fHbp, NadA and NHBA Wendy Keijzersover a 50-year period in the Sara Comandi Lisa De Tora Netherlands Mariagrazia Pizza Rino Rappuoli Diederik van de Beek Arie van der Ende Maurizio Comanducci

Stefania Bambini Jurgen R. Piet Alessandro Muzzi Wendy Keijzers Sara Comandi

Lisa De Tora Mariagrazia Pizza

Rino Rappuoli Diederik van de Beek Arie van der Ende PLoS One 2013;8:e65043 Maurizio Comanducci

PLoS One 2013;8:e65043

40049 Piet, Jurgen.indd 85 26-04-16 15:08 Chapter 5 Meningococcal sequence variability of fHbp, NadA and NHBA

ABSTRACT INTRODUCTION

Studies of meningococcal evolution and genetic population structure, including the long- Neisseria meningitidis is a major cause of invasive bacterial meningitis and septicemia term stability of non-random associations between variants of surface proteins, are essential worldwide. Meningococcal populations are highly diverse, and engage in extensive genetic for vaccine development. We analyzed the sequence variability of factor H-binding protein exchange.1,2 Studies of genetic variation, by MultiLocus Enzyme Electrophoresis (MLEE) 3 and (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisseria adhesin A (NadA), three subsequently by MultiLocus Sequence Typing (MLST)4, show that meningococci are major antigens in the multicomponent meningococcal serogroup B vaccine 4CMenB. A panel structured into clonal complexes.5,6 Isolates belonging to the different clonal complexes of invasive isolates collected in the Netherlands over a period of 50 years was used. To our exhibit different phenotypes and vary in their propensity to cause disease.7-9 Meningococcal knowledge, this strain collection covers the longest time period of any collection available isolates belonging to the hyper-virulent clonal complexes (cc)32, cc11, cc8, cc41/44, cc1, cc4 worldwide. Long-term persistence of several antigen sub/variants and of non-overlapping and cc5, have a high capacity to cause invasive disease. Also cc269 could be considered antigen sub/variant combinations was observed. Our data suggest that certain antigen hyper-virulent.10 It is genetically related to cc32, as shown by whole genome analysis.11 sub/variants including those used in 4CMenB are conserved over time and promoted by Strains belonging to the hyper-virulent clonal complexes are over-represented in collections selection. of pathogenic isolates. Clonal complexes are relatively genetically stable over time, despite high rates of recombination.4 In contrast, strains associated with asymptomatic carriage exhibit more extensive genetic diversity.12

The paradoxical persistence of clonal complex structure despite high levels of recombination can be explained by evolutionary models that invoke positive selection13-15, which has important implications for the design of protein-based vaccines against meningococcal serogroup B (MenB). Outer membrane vesicle (OMV) vaccines that rely on the immunogenic properties of PorA16-19 have been used: though, those vaccines only provide protection for homologous strains carrying the same PorA.20-23 To overcome antigenic variability, vaccines based on multiple outer membrane proteins have been proposed to provide protection against a broad range of meningococcal isolates. Novel antigens were identified by Reverse Vaccinology20-22 and combined into a multicomponent vaccine against MenB, 4CMenB (Bexsero®), which has been recently licensed in the Europe.23-25 4CMenB includes OMV from the New Zealand MeNZB® vaccine26 and three major protein antigens: factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisseria adhesin A (NadA).

fHbp (genome-derived Neisseria antigen [GNA] 1870 or LP2086)27,28 binds human factor H29,30, enhancing the ability of the bacterium to resist complement-mediated killing. Its expression enables survival in ex vivo human blood and serum.31-33 As different fHbp classification schemes have been proposed, a dedicated database is available

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ABSTRACT INTRODUCTION

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nomenclature scheme, the sub/variants are grouped into subfamily A (corresponding to 27 variants 2 and 3) and subfamily B (corresponding to variant 1) based on sequence diversity. MATERIALS AND METHODS Bactericidal activity is variant specific; antibodies raised against one variant are not cross- Bacterial isolates, PCR amplification and gene sequencing protective against other variants, even if some cross-reactivity has been described between 28 fHbp-2 and -3. Antibodies raised against sub/variant fHbp-1.1, included in 4CMenB vaccine, One hundred sixty-five meningococcal isolates collected from clinical cases (from blood or 34 are highly cross-reactive with fHbp-1 and poorly cross-reactive with fHbp-2 and -3. Clinical cerebrospinal fluid) in the Netherlands were randomly selected at the Netherlands 35,36 trials are also ongoing for a different vaccine including fHbp-1.55 and fHbp-3.45. Reference Laboratory for Bacterial Meningitis (NRLBM). Approximately 30 isolates were included from each decade as follows: every 2nd, 5th, 10th, 20th, 20th and 10th isolate were NHBA (GNA2132) is a heparin-binding protein37 which shows sufficient sequence diversity to chosen of the years 1960, 1970, 1980, 1990, 2000 and 2008-2009, respectively. prevent a subdivision into a small number of variant families.38,39 For this reason NHBA

nomenclature is based on the assignment of a unique identifier x to each specific peptide Upon receipt of bacterial isolates in the NRLBM, a monoculture of the causative isolate was sub/variant, which was indicated as NHBA-x. The 4CMenB vaccine contains NHBA peptide 2 frozen and stored at –80º C. All isolates were passaged fewer than 5 times. All isolates were (NHBA-2), which elicited antibodies that were cross-reactive with heterologous sub/variants characterized by serogroup, PorA, FetA and MLST (http://pubmlst.org/neisseria/).6,46,47 The 40,41 and cross-protective in the infant rat model. complete characterization of isolates is reported in Supplementary Table 1. PCR templates were prepared by boiling ~100 N. meningitidis colonies from chocolate agar plates in 200 µl NadA (GNA1994) plays a role in cell adhesion and invasion.42 The nadA gene is present in of distilled H O for 5 min, and subsequent centrifugation. 1 µl of the supernatant was used in almost all isolates belonging to the hyper-virulent lineages cc8, cc11 and cc32, and in all 2 the PCR reaction (10 µl total volume). The amplification enzyme used was AccuPrime Taq strains so far isolated belonging to cc213 and cc1157.46-48 Conversely, it is absent in almost DNA Polymerase System (Invitrogen). All genes were amplified using primers external to the all strains belonging to cc41/44 and cc269. NadA protein is present in five variants. NadA-1, coding region. The primers used for PCR and sequencing are in Supplementary Table 2. The NadA-2 and NadA-3 elicit cross-reactive bactericidal antibodies.43 NadA is usually absent in fHbp gene was amplified using primers A1 and B2. PCR conditions were: 30 cycles of carrier isolates, although a small proportion express NadA-4, which does not induce cross- denaturation at 94 °C for 30s, annealing at 58 °C for 30s, elongation at 68 °C for 1min. reactive bactericidal antibodies to NadA-1, NadA-2 and NadA-3.44 NadA-5 occurs mostly in Sequences were performed using forward primers A1, 22, and reverse primers B2, 32. The cc213 strains.38,45 The NadA protein included in 4CMenB is NadA-3 sub/variant 8 (NadA-3.8), nhba gene was amplified using primers 1 and 6. PCR conditions were: 94 °C for 30 s, 55 °C for which induces antibodies cross-reactive with NadA-1, NadA-2 and NadA-3.43 30 s, 68 °C for 1min, 30 cycles. Forward sequencing primers were 1, 22, 23, 3, 84, reverse Statistical association studies indicate that the repertoire of fHbp, NHBA and NadA is primers were 5, 6, 7, 85, 93. nadA PCR primers were A1 and B. PCR conditions were: 94 °C structured among hyper-invasive lineages. Isolates from the same clonal complexes have for 30s, 56 °C for 30s, 68 °C for 3min, 30 cycles. Forward sequencing primers were A1, 1, 2, 3, similar profiles for each antigen, even when derived from disparate geographical locations reverse primers were B, C, 5, 6. PCR products were purified with QIAquik PCR purification kit 38 and time periods. (QIAGEN) and sequenced using the ABI 377 automatic sequencer (Applied Biosystems).

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(http://neisseria.org/nm/typing/fhbp), with a unified fHbp nomenclature for the assignment We investigated the prevalence and sequence variation of fHbp, NHBA and NadA in a panel of new sub-variants. fHbp has been classified into three (main) variants 1, 2 and 3, described of 165 pathogenic meningococcal isolates randomly selected from those collected in the here as fHbp-1, fHbp-2 and fHbp-328, which were further divided into sub/variants fHbp-1.x, Netherlands over a period of 50 years. fHbp-2.x and fHbp-3.x, where x denotes the specific peptide sub/variant. In a different nomenclature scheme, the sub/variants are grouped into subfamily A (corresponding to 27 variants 2 and 3) and subfamily B (corresponding to variant 1) based on sequence diversity. MATERIALS AND METHODS Bactericidal activity is variant specific; antibodies raised against one variant are not cross- Bacterial isolates, PCR amplification and gene sequencing protective against other variants, even if some cross-reactivity has been described between 28 fHbp-2 and -3. Antibodies raised against sub/variant fHbp-1.1, included in 4CMenB vaccine, One hundred sixty-five meningococcal isolates collected from clinical cases (from blood or 34 are highly cross-reactive with fHbp-1 and poorly cross-reactive with fHbp-2 and -3. Clinical cerebrospinal fluid) in the Netherlands were randomly selected at the Netherlands 35,36 trials are also ongoing for a different vaccine including fHbp-1.55 and fHbp-3.45. Reference Laboratory for Bacterial Meningitis (NRLBM). Approximately 30 isolates were included from each decade as follows: every 2nd, 5th, 10th, 20th, 20th and 10th isolate were NHBA (GNA2132) is a heparin-binding protein37 which shows sufficient sequence diversity to chosen of the years 1960, 1970, 1980, 1990, 2000 and 2008-2009, respectively. prevent a subdivision into a small number of variant families.38,39 For this reason NHBA nomenclature is based on the assignment of a unique identifier x to each specific peptide Upon receipt of bacterial isolates in the NRLBM, a monoculture of the causative isolate was 5 sub/variant, which was indicated as NHBA-x. The 4CMenB vaccine contains NHBA peptide 2 frozen and stored at –80º C. All isolates were passaged fewer than 5 times. All isolates were (NHBA-2), which elicited antibodies that were cross-reactive with heterologous sub/variants characterized by serogroup, PorA, FetA and MLST (http://pubmlst.org/neisseria/).6,46,47 The 40,41 and cross-protective in the infant rat model. complete characterization of isolates is reported in Supplementary Table 1. PCR templates were prepared by boiling ~100 N. meningitidis colonies from chocolate agar plates in 200 µl NadA (GNA1994) plays a role in cell adhesion and invasion.42 The nadA gene is present in of distilled H O for 5 min, and subsequent centrifugation. 1 µl of the supernatant was used in almost all isolates belonging to the hyper-virulent lineages cc8, cc11 and cc32, and in all 2 the PCR reaction (10 µl total volume). The amplification enzyme used was AccuPrime Taq strains so far isolated belonging to cc213 and cc1157.46-48 Conversely, it is absent in almost DNA Polymerase System (Invitrogen). All genes were amplified using primers external to the all strains belonging to cc41/44 and cc269. NadA protein is present in five variants. NadA-1, coding region. The primers used for PCR and sequencing are in Supplementary Table 2. The NadA-2 and NadA-3 elicit cross-reactive bactericidal antibodies.43 NadA is usually absent in fHbp gene was amplified using primers A1 and B2. PCR conditions were: 30 cycles of carrier isolates, although a small proportion express NadA-4, which does not induce cross- denaturation at 94 °C for 30s, annealing at 58 °C for 30s, elongation at 68 °C for 1min. reactive bactericidal antibodies to NadA-1, NadA-2 and NadA-3.44 NadA-5 occurs mostly in Sequences were performed using forward primers A1, 22, and reverse primers B2, 32. The cc213 strains.38,45 The NadA protein included in 4CMenB is NadA-3 sub/variant 8 (NadA-3.8), nhba gene was amplified using primers 1 and 6. PCR conditions were: 94 °C for 30 s, 55 °C for which induces antibodies cross-reactive with NadA-1, NadA-2 and NadA-3.43 30 s, 68 °C for 1min, 30 cycles. Forward sequencing primers were 1, 22, 23, 3, 84, reverse Statistical association studies indicate that the repertoire of fHbp, NHBA and NadA is primers were 5, 6, 7, 85, 93. nadA PCR primers were A1 and B. PCR conditions were: 94 °C structured among hyper-invasive lineages. Isolates from the same clonal complexes have for 30s, 56 °C for 30s, 68 °C for 3min, 30 cycles. Forward sequencing primers were A1, 1, 2, 3, similar profiles for each antigen, even when derived from disparate geographical locations reverse primers were B, C, 5, 6. PCR products were purified with QIAquik PCR purification kit 38 and time periods. (QIAGEN) and sequenced using the ABI 377 automatic sequencer (Applied Biosystems).

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Discrimination between ST44 and ST41 subcomplexes of clonal complex41/44 was assessed (3%) in two or three non-consecutive decades. Twenty-seven percent of the isolates by PCRs targeting MoxR like AAA ATPase and nmeSI, respectively. PCR to assess nmeSI was belonged to cc41/44, 13% to cc8, 9% to cc11, 8% to cc32 and 7% to cc269. The remaining performed as previously described50,52 using primers AVDE 0712 and AVDE 0716. Part of the 36% of isolates included 17 different clonal complexes. Ten clonal complexes were observed gene encoding the MoxR like AAA ATPase was amplified using primers AVDE 0701F and only once, 3 were present twice, whereas 9 persisted for more than two decades (Figure 1). AVDE 0710. PCR conditions were: 95 °C for 1 min, 58 °C for 1 min, 72 °C for 2 min, 30 cycles.

Sequence analysis and measure of the associations between different loci Figure 1. Distribution of the MLST clonal complexes from 1960 to 2008-2009 in the Netherlands.

DNA sequences were assembled and analyzed using Sequencher version 4.10.1 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI USA, http://www.genecodes.com), BioEdit (developed by Tom Hall, Ibis Biosciences), Molecular Evolutionary Genetics Analysis (MEGA) software version 4.048 and Jalview.49 Molecular typing of fHbp, NHBA and NadA sub/variants was based on amino acid sequence determination. fHbp sub/variants were designated as variants.oxford.peptide (http://pubmlst.org/neisseria/fHbp/). NHBA sub/variants were designated as peptides. NadA sub/variants were designated as variant.peptide. The structure of MLST clonal complexes was analyzed with PHYLOViZ 1.0 based on the goeBURST algorithm.50 The Cramer’s V coefficient51 was used to measure association statistics with clonal complexes. The S 52 Standardized Index of Association IA was used to test the stability of associations between different loci. The non-overlapping structure of antigen variant combinations was measured 13 S by f* metrics. The three statistical parameters V, IA and f* are based on the frequency of the alleles and vary between 0 (random distribution) and 1 (perfect association or non- overlapping distribution in the case of f*).

RESULTS

Prevalence and distribution of sequence types (STs) and MLST clonal complexes over time

Ninety-five different STs and 22 clonal complexes were found (Supplementary Table 1). ST-8 (9%), ST-11 (8%), ST-41 (8%), ST-1 (5%) were the most commonly represented STs. ST-1, ST- Clonal complexes (cc)1, cc11, cc32, cc231, cc254, cc269, cc334, cc41/44 and cc8 persisted for more than two decades. cc41/44 (27%), cc8 (13%), cc11(9%), cc32(8%) and cc269 (7%) were the most frequent. cc41/44, which 41 and ST-11 were the only STs detected in more than two consecutive decades based on was present in all time-periods considered, was predominant in 1960, 1990, 2000 and 2008-2009, cc8 was predominant in 1970 and cc1 in 1980. cc5, cc22, cc35, cc37, cc60, cc103, cc162, cc167, cc213, cc1157 were the sample studied. In contrast, 82 (86%) STs were observed only once, 7 (7%) twice and 3 observed only once, cc18, cc364, cc461 were present twice. Over time, there were several ccs that disappeared, such as cc37 and cc167,which were present in 1960 only, and on the other new ccs emerged.

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Sequence analysis and measure of the associations between different loci Figure 1. Distribution of the MLST clonal complexes from 1960 to 2008-2009 in the Netherlands.

DNA sequences were assembled and analyzed using Sequencher version 4.10.1 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI USA, http://www.genecodes.com), BioEdit (developed by Tom Hall, Ibis Biosciences), Molecular Evolutionary Genetics Analysis (MEGA) software version 4.048 and Jalview.49 Molecular typing of fHbp, NHBA and NadA sub/variants was based on amino acid sequence determination. fHbp sub/variants were designated as variants.oxford.peptide (http://pubmlst.org/neisseria/fHbp/). NHBA sub/variants were designated as peptides. NadA 5 sub/variants were designated as variant.peptide. The structure of MLST clonal complexes was analyzed with PHYLOViZ 1.0 based on the goeBURST algorithm.50 The Cramer’s V coefficient51 was used to measure association statistics with clonal complexes. The S 52 Standardized Index of Association IA was used to test the stability of associations between different loci. The non-overlapping structure of antigen variant combinations was measured 13 S by f* metrics. The three statistical parameters V, IA and f* are based on the frequency of the alleles and vary between 0 (random distribution) and 1 (perfect association or non- overlapping distribution in the case of f*).

RESULTS

Prevalence and distribution of sequence types (STs) and MLST clonal complexes over time

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Diversity and distribution of fHbp Figure 3. Distribution of antigens from 1960 to 2008-2009.

fHbp-2 predominated in 1960-1980, whereas fHbp-1 became more prevalent thereafter (Figure 2). fHbp-1.14 (16%), 2.16 (13%), 2.24 (8%), 1.4 (8%), 1.1 (8 %; included in 4CMenB), 1.13 (5%), 2.22 (4%), 2.19 (4%) and 3.45 (2%) were the most frequent sub-variants (Figure 3A).

Figure 2. Distribution of the three fHbp variants over time.

(A) fHbp variants and sub/variants. Out of the 45 sub/variants in frame identified from 1960 to 2008-2009, fHbp-1.14 (16%), 2.16 (13%), 1.1 (8%), 1.4 (8%), and 2.24 (8%) were the most frequent and persisting. fHbp- 2.24 was predominant in 1960, 2.16 in 1970, 1.4 in 1980, 1.14 in 1990, 2000 and 2008-2009, together with 1.1. (B) NHBA peptides. Out of the 43 peptides identified from 1960 to 2008-2009, NHBA- 2 (24%) and 20 (22%) were the most frequent and persisting. NHBA-2 was predominant in 1960, 1990, 2000 and 2008-2009, NHBA- 20 in 1970 and 1980 (this latter case, together with NHBA-29). (C) NadA presence, variants and sub/variants. nadA gene was absent or presented frame-shift mutations or insertion sequences in 115 out of 165 isolates. Out of the 11 sub/variants identified from 1960 to 2008-2009 in the 50 isolates with the gene, NadA-3.8 (40%) and NadA-1.1 (28%) were the most frequent and persisting. NadA-3.8 was present from 1960 to 1990, NadA- fHbp-2 predominated in 1960-1980 and was subsequently substituted by fHbp-1. The presence of fHbp-3 in 1.1 from 1980 to 2008-2009. 2008-2009 was mainly related with the emergence of the cc213.

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Diversity and distribution of fHbp Figure 3. Distribution of antigens from 1960 to 2008-2009.

The fHbp gene was present in all isolates. Forty-nine different nucleotide sequences were identified. Forty-five out of the 47 different amino acid sequences were in frame. 98% of amino acid sequences belonged to the three variants: fHbp-1 (52%), fHbp-2 (39%) and fHbp- 3 (7%). Concerning the remaining 2%, in one case the fHbp variant was a natural chimera between fHbp-1 and fHbp-3, which was recently described 30; one sequence presented a frame-shift, causing a premature end of the encoded peptide; moreover, in one case the gene sequence was not obtained (Supplementary Table 1). fHbp-2 predominated in 1960-1980, whereas fHbp-1 became more prevalent thereafter (Figure 2). fHbp-1.14 (16%), 2.16 (13%), 2.24 (8%), 1.4 (8%), 1.1 (8 %; included in 4CMenB), 1.13 (5%), 2.22 (4%), 2.19 (4%) and 3.45 (2%) were the most frequent sub-variants (Figure 3A). 5

Figure 2. Distribution of the three fHbp variants over time.

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Diversity and distribution of NHBA Table 1. Associations between different loci. Measure of association Measure of the association between Measure of non-overlapping with clonal complexes two different loci (Standardized structure of combinations (f* § § S § The nhba gene was found in all isolates considered. In one case the gene presented a frame- (Cramer’s V ) Index of Association , IA ) metrics ) Gene Housekeeping genes fHbp nhba nadA fHbp nhba nadA shift mutation, probably causing a lack of protein expression. Forty-eight different fHbp 0.705 0.371 0.667 0.284 0.601 nucleotide sequences, corresponding to 43 different amino acid sequences, were identified nhba 0.769 0.371 0.512 0.284 0.462 (Supplementary Table 1). NHBA-2 (24 %), 20 (22%), 3 (6%), 29 (6%), 6 (5%), 12 (4%), 21 (4%), nadA 0.582 0.667 0.512 0.601 0.462 The Cramer’s V coefficient was used to measure association statistics with clonal complexes. The values 18 (3%) and 24 (3%) were the most frequently occurring (Figure 3B). NHBA-2 (included in indicated a strong association between antigen alleles and clonal complexes. The Standardized Index of S Association IA was used to test the stability of associations between different loci. The antigen pairs showed S 4CMenB) was predominant in 1960 (34%), 1990 (33%), 2000 (43%) and 2008-2009 (20%), relatively high IA values, indicative of strong, stable associations between the different loci over time. Moreover, the combinations showed a non-overlapping structure (measured by f* metrics) that suggested the whereas NHBA-20 was predominant in 1970 (53%) and NHBA-29 in 1980 (28%). § S immune selection maintaining antigenic combinations. The three statistical parameters V, IA and f* are based on the frequency of the alleles and vary between 0 (random distribution) and 1 (perfect association or non- Diversity and distribution of NadA overlapping distribution in the case of f*).

Of 165 isolates, 60 (36%) had the nadA gene. Eight isolates had the nadA gene interrupted by the insertion sequence IS1301. Therefore, they most likely did not express the protein.43 Several sub/variants were associated with specific clonal complexes. For example, fHbp-1.1 The gene of two isolates presented a frame-shift mutation. Therefore, 50/165 (30%) isolates was always present in cc32 isolates. Twenty-five out of 27 fHbp-1.14 were present in were considered positive for NadA. The gene was absent in cc41/44 and cc269 with one cc41/44 (92%). fHbp-2.16 was almost always represented in cc8 isolates (91%). fHbp-1.15 exception in both clonal complexes, strain 600091 and 701844, respectively (Supplementary and fHbp-3.45 were found mostly in cc269 (80%) and cc213 (75%), respectively. However, Table 1). Variants NadA-1, NadA-2, NadA-3 and NadA-5, which are generally present in eleven different fHbp sub/variants were found in cc41/44, the most frequent being fHbp- pathogenic strains, accounted for 28%, 16%, 42% and 8% of the positive isolates, 1.14 (55%). fHbp-2.16 was almost always represented in cc8 isolates. Seven and 9 different respectively. NadA-4, the variant described in carrier strains, was found in 6% of the positive sub/variants were represented within cc11 and cc269, respectively. isolates. NadA-3 and NadA-4 were most frequent in 1960-1970, NadA-1 and NadA-2 were Similar association with clonal complexes was observed for NHBA peptides. NHBA-3 was prevalent in 1990-2000, NadA-1 and NadA-5 in 2008-2009. Eleven sub/variants were always present (100%) in cc32, NHBA-12 in cc37, NHBA-18 in cc213 and NHBA-21 in cc269. identified (Supplementary Table 1), the most frequent being NadA-3.8, the NadA sub-variant NHBA-2 was predominant in cc41/44 (37 out of 39 isolates, 95%). NHBA-29 was found in 4CMenB, present in 20 out of the 50 isolates with the nadA gene (40%), which was mostly in cc1 (80%). NHBA-20 was the most frequent peptide in cc8 (57%), even if it was also predominant in 1960-’70-’80. NadA-1.1 (28%) has been prevalent after 1990 (Figure 3C). present in cc11 (32%). Association of fHbp, NadA, and NHBA diversity with MLST clonal complexes Also NadA variants were associated with clonal complexes. NadA-1 was solely present in We observed clear associations between fHbp, NadA, and NHBA unique sequences with cc32 strains. NadA-2 (7 out of 8 isolates, 88%) was mostly present in cc11 strains. NadA-3 clonal complexes. These results, also obtained in other strain panels38, were based on a (81%) was mostly present in cc8 strains. NadA-5 was solely present in cc213 strains. standardized measure of association, the Cramer’s V coefficient (Table 1). The value of this

coefficient of correlation is high, in particular for fHbp and NHBA (VNHBA=0.769, VfHbp=0.705

and VNadA= 0.582).

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Of 165 isolates, 60 (36%) had the nadA gene. Eight isolates had the nadA gene interrupted by the insertion sequence IS1301. Therefore, they most likely did not express the protein.43 Several sub/variants were associated with specific clonal complexes. For example, fHbp-1.1 The gene of two isolates presented a frame-shift mutation. Therefore, 50/165 (30%) isolates was always present in cc32 isolates. Twenty-five out of 27 fHbp-1.14 were present in were considered positive for NadA. The gene was absent in cc41/44 and cc269 with one cc41/44 (92%). fHbp-2.16 was almost always represented in cc8 isolates (91%). fHbp-1.15 5 exception in both clonal complexes, strain 600091 and 701844, respectively (Supplementary and fHbp-3.45 were found mostly in cc269 (80%) and cc213 (75%), respectively. However, Table 1). Variants NadA-1, NadA-2, NadA-3 and NadA-5, which are generally present in eleven different fHbp sub/variants were found in cc41/44, the most frequent being fHbp- pathogenic strains, accounted for 28%, 16%, 42% and 8% of the positive isolates, 1.14 (55%). fHbp-2.16 was almost always represented in cc8 isolates. Seven and 9 different respectively. NadA-4, the variant described in carrier strains, was found in 6% of the positive sub/variants were represented within cc11 and cc269, respectively. isolates. NadA-3 and NadA-4 were most frequent in 1960-1970, NadA-1 and NadA-2 were Similar association with clonal complexes was observed for NHBA peptides. NHBA-3 was prevalent in 1990-2000, NadA-1 and NadA-5 in 2008-2009. Eleven sub/variants were always present (100%) in cc32, NHBA-12 in cc37, NHBA-18 in cc213 and NHBA-21 in cc269. identified (Supplementary Table 1), the most frequent being NadA-3.8, the NadA sub-variant NHBA-2 was predominant in cc41/44 (37 out of 39 isolates, 95%). NHBA-29 was found in 4CMenB, present in 20 out of the 50 isolates with the nadA gene (40%), which was mostly in cc1 (80%). NHBA-20 was the most frequent peptide in cc8 (57%), even if it was also predominant in 1960-’70-’80. NadA-1.1 (28%) has been prevalent after 1990 (Figure 3C). present in cc11 (32%). Association of fHbp, NadA, and NHBA diversity with MLST clonal complexes Also NadA variants were associated with clonal complexes. NadA-1 was solely present in We observed clear associations between fHbp, NadA, and NHBA unique sequences with cc32 strains. NadA-2 (7 out of 8 isolates, 88%) was mostly present in cc11 strains. NadA-3 clonal complexes. These results, also obtained in other strain panels38, were based on a (81%) was mostly present in cc8 strains. NadA-5 was solely present in cc213 strains. standardized measure of association, the Cramer’s V coefficient (Table 1). The value of this coefficient of correlation is high, in particular for fHbp and NHBA (VNHBA=0.769, VfHbp=0.705 and VNadA= 0.582).

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Figure 4. The evolution of cc41/44 over 50 years. Evolution of cc41/44

The only clonal complex present at all observed time points in the sample was cc41/44. It showed a shift over time between its two central STs6 (Figure 4). The ST-44 sub-complex was predominant in 1960-1970, and ST-41 was prevalent after 1980.

Still, due to association between (any) protein diversity and MLST, a trend in the ratio of fHbp variants was observed to follow changes in the central genotypes (Figure 4A). From 1960 to 1980, when the ST-44 sub-complex was predominant, fHbp-2 was most represented, and sub/variants fHbp-2.24 and 2.19 were predominant. After 1980, as sub-complex ST-41 became predominant, fHbp-1 became the most frequent variant and sub/variant fHbp-1.14 predominated after 1990 (Supplementary Table 1 and Figure 3A).

Similar associations with each of the two sub-complexes of cc41/44 were also observed for PorA and FetA variants. PorA P1.7-2, 4 and FetA F1-5 were associated with the ST-41 sub- complex isolates collected in 1990 and afterward, while this combination was not observed among the ST-44 sub-complex isolates. The latter was more heterogeneous with PorA P1.18, 25-7 prevalent.

In contrast, NHBA-2 was the most frequently occurring peptide in both cc41/44 sub- complexes across the 50-year period examined (Figure 4B). As for all clonal complexes, the association with ST-41 and ST-44 sub-complexes, within cc41/44, was evaluated using Cramer’s V coefficient. NHBA-2 was predominant but evenly distributed between the two sub-complexes and consequently NHBA did not show a relevant enrichment of specific

peptides in one of the sub-complexes (VNHBA=0.298). In contrast, the overall distribution of the fHbp sub/variants in cc41/44 was not uniform and showed a relevant association with

sub-complexes (VfHbp=0.683).

Long-term persistence of antigen sub/variants

A number of antigen sub/variants appeared in the dataset only once, indicating a low The structure of cc41/44 was analyzed with phyloviz based on the goeBURST algorithm.50 Each number correspond to a different ST, the sizes of circles are proportional to the number of isolates. (A) fHbp. In 1960- frequency in the population. This might suggest that they were less fit and excluded by 1970, when the ST-44 sub-complex was predominant, fHbp-2 and fHbp-3 were most represented. After 1980, when the ST-41 sub-complex was prevalent, fHbp-1 became the most frequent. Of note, independently from its selection over time, while other sub/variants persisted over decades. Of the 45 fHbp prevalence, fHbp-1 was the only variant that was present in both sub-complexes at all time periods. (B) NHBA. NHBA-2, the most frequent in cc41/44 (82%), was equally shared by the two sub-complexes from 1960 to 2008- sub/variants identified in the current sample, 30 were observed once, and 10 were seen 2009.

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Figure 4. The evolution of cc41/44 over 50 years. Evolution of cc41/44

Similar associations with each of the two sub-complexes of cc41/44 were also observed for PorA and FetA variants. PorA P1.7-2, 4 and FetA F1-5 were associated with the ST-41 sub- complex isolates collected in 1990 and afterward, while this combination was not observed 5 among the ST-44 sub-complex isolates. The latter was more heterogeneous with PorA P1.18, 25-7 prevalent.

peptides in one of the sub-complexes (VNHBA=0.298). In contrast, the overall distribution of the fHbp sub/variants in cc41/44 was not uniform and showed a relevant association with

sub-complexes (VfHbp=0.683).

Long-term persistence of antigen sub/variants

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over a period of at least 20 years. Among the 43 NHBA peptides observed, 30 occurred once, In general, sub/variants that were found persisting over decades in this present study were and 7 have been encountered over at least 20 years. Eleven NadA sub/variants were found also in other strain panels from different time periods.38,39,45 Moreover, several identified, of which 5 were observed once and 2 were present over at least 20 years. The sub/variants have been associated over time also with different clonal complexes. For proteins included in 4CMenB (fHbp-1.1 and NadA-3.8) were observed over thirty years, while instance fHbp-1.13 was identified in cc254 strains collected during 1970, 1980, 1990 and NHBA-2 was observed over fifty years (Figure 5). 2008-2009, and in cc60 strains in 2000. fHbp-1.14 was identified in cc41/44 isolates in 1990, 2000 and 2008-2009, and in cc254 isolates collected in 2008-2009. fHbp-2.16 was found in Figure 5. Persistence of the fHbp, NHBA and NadA sub/variants included in 4CMenB. cc8 isolates collected between 1970 and 2000, and in cc22 isolates from 2008-2009.NHBA-20 was identified in cc11 isolates in 1960, 1990, 2000 and 2008-2009, while in 1970 and 1980 it was prevalent in cc8.

Long-term persistence of antigen sub/variant combinations

Of 83 combinations of fHbp, NadA, and NHBA sub/variants, most were found only once, yet

certain combinations were stable and persisted over time. Examples include: fHbp-1.1:NadA-

1.1, which persisted for 30 years; and fHbp-2.16:NHBA-20, which persisted for 40 years (Figure 6). The relative presence of several combinations changed over time. For example, fHbp-2.16:NHBA-20 was the most frequent in 1970, fHbp-1.4:NHBA-29 was more commonly found in 1980, and fHbp-1.14:NHBA-2 was predominant after 1990.

fHbp-1.1 and NadA-3.8 persisted for thirty years, NHBA-2 for fifty years.

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Long-term persistence of antigen sub/variant combinations

Of 83 combinations of fHbp, NadA, and NHBA sub/variants, most were found only once, yet

certain combinations were stable and persisted over time. Examples include: fHbp-1.1:NadA- 1.1, which persisted for 30 years; and fHbp-2.16:NHBA-20, which persisted for 40 years 5 (Figure 6). The relative presence of several combinations changed over time. For example, fHbp-2.16:NHBA-20 was the most frequent in 1970, fHbp-1.4:NHBA-29 was more commonly found in 1980, and fHbp-1.14:NHBA-2 was predominant after 1990.

fHbp-1.1 and NadA-3.8 persisted for thirty years, NHBA-2 for fifty years.

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Figure 6. Longevity of the most frequent combinations of two and three antigen sub/variants. belonging to the same gene. The linkage disequilibrium between the fHbp, nhba and nadA loci showed also a non-overlapping structure (measured by f*metrics, Table 1). The non- overlapping structure, in particular between fHbp and nadA (f*metrics = 0.601) can be interpreted as the result of the pressure of selection exerted by the immune system in maintaining antigenic combinations in the neisserial population.13

DISCUSSION

Among the meningococci isolated in a 50 year period many sub/variants of each antigen fHbp, NHBA and NadA were identified. Although most sub/variants were observed only Out of the 9 most frequent combinations observed, several persisted for at least twenty years. fHbp-1.1:NadA- 1.1 and fHbp-2.16:NHBA-20 were very stable, persisting for thirty and forty years, respectively. once, or persistent over a period of a few years, a significant number, such as those used in *Combinations generated by fHbp-2.16, NHBA-20 and NadA-3.8. §Combinations generated by fHbp-1.1, NHBA-3 and NadA-1.1 . 4CMenB, were observed to persist over time. Of note, the antigen sub/variants we found to be stable and conserved over at least twenty years have been identified as the most frequent in other panels of invasive isolates.38-42, 47, 48 Short-lived sub/variants were likely In addition to association of antigens sub/variants with clonal complexes, several antigen less fit or occurred too infrequently to be observed, which may suggest that they were sub/variant combinations were maintained in the same clonal complex. The fHbp-1.1:NadA- excluded by immune selection. 1.1 combination, first observed in 1980, persisted in all isolates of cc32 for thirty years. The

fHbp-1.14:NHBA-2 combination was found in cc41\44 isolates in 1990 and persisted for 20 Associations between antigen sub/variants and clonal complexes measured by the Cramer’s years. The fHbp-1.4:NHBA-29 combination was found in cc1 isolates from 1960 to 1980. The V coefficient, a standardized measure of association, were also identified and were fHbp-2.16:NHBA-20:NadA-3.8 combination was observed in cc8 isolates in 1970 and in 1980. consistent with previous studies in other strain panels.38,45

S 52 The Standardized Index of Association IA was used to test the stability of associations The three protein sub/variants chosen for inclusion in 4CMenB (fHbp-1.1, NHBA-2 and NadA- S between pairs of loci, i.e. the presence of linkage disequilibrium (Table 1). The IA of the fHbp 3.8) were the most common worldwide and were expected to provide cross- with respect to nhba and nadA was 0.371 and 0.667 respectively, the index between nhba reactivity.31,33,37,41 An interesting and unexpected finding of this study is their long-term and nadA was 0.512. These three values showed a particular faculty of nadA to be associated persistence in the current strain panel, which was thirty years for fHbp-1.1 and NadA-3.8, with fHbp and nhba. For example, these rates were comparable with the same index and fifty years for NHBA-2. S calculated for the association between porA VR1 and VR2 (IA = 0.566), two very close loci

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DISCUSSION

The sequence variability of fHbp, NHBA and NadA has been examined in several strain panels worldwide and investigations are still ongoing.38,39,45 We evaluated prevalence and sequence variations of fHbp, NHBA and NadA in a panel of invasive meningococcal isolates collected in the Netherlands from 1960 to 2009, which, is to our knowledge, the widest time frame of any collection worldwide. 5 Among the meningococci isolated in a 50 year period many sub/variants of each antigen fHbp, NHBA and NadA were identified. Although most sub/variants were observed only Out of the 9 most frequent combinations observed, several persisted for at least twenty years. fHbp-1.1:NadA- 1.1 and fHbp-2.16:NHBA-20 were very stable, persisting for thirty and forty years, respectively. once, or persistent over a period of a few years, a significant number, such as those used in *Combinations generated by fHbp-2.16, NHBA-20 and NadA-3.8. §Combinations generated by fHbp-1.1, NHBA-3 and NadA-1.1 . 4CMenB, were observed to persist over time. Of note, the antigen sub/variants we found to be stable and conserved over at least twenty years have been identified as the most frequent in other panels of invasive isolates.38-42, 47, 48 Short-lived sub/variants were likely In addition to association of antigens sub/variants with clonal complexes, several antigen less fit or occurred too infrequently to be observed, which may suggest that they were sub/variant combinations were maintained in the same clonal complex. The fHbp-1.1:NadA- excluded by immune selection. 1.1 combination, first observed in 1980, persisted in all isolates of cc32 for thirty years. The fHbp-1.14:NHBA-2 combination was found in cc41\44 isolates in 1990 and persisted for 20 Associations between antigen sub/variants and clonal complexes measured by the Cramer’s years. The fHbp-1.4:NHBA-29 combination was found in cc1 isolates from 1960 to 1980. The V coefficient, a standardized measure of association, were also identified and were fHbp-2.16:NHBA-20:NadA-3.8 combination was observed in cc8 isolates in 1970 and in 1980. consistent with previous studies in other strain panels.38,45

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Long term stability and persistence was observed also for several combinations of protein New Zealand55 from 1990 onwards. As previously published, cc41/44 isolates collected sub/variants. Antigen combinations showed a non-overlapping structure. As reported in during 1980 and later harbored the restriction modification system nmeSI.58 In contrast, 80% literature, if immune selection was absent or neutral, then all antigen variants and of the isolates of the ST44 sub-complex appeared to have two different genes at the sub/variants would occur for similar time spans. On the contrary, it has been suggested that genomic position of the nmeSI system encoding a MoxR like AAA ATPase and a protein with selection may cause strong linkage disequilibrium (or non-random association of alleles at a Von Willenbrand domain, respectively (data not shown). In eBURST analyses, fHbp variant the different loci) between some locus pairs.14,15,53 As observed in this study, the stability distribution changed with the relative predominance of the ST-44 and ST-41 sub-complexes. and persistence over decades of discrete fHbp, NHBA and NadA sub/variants and non- In 1960-1970, when the ST-44 sub-complex was predominant, fHbp-2 and fHbp-3 were more overlapping antigen sub/variant combinations suggest they were maintained by natural common in the strain collection. After 1980 ST-41 and ST-41 associated fHbp-1 became most selection. In the case of antigen sub/variants, the persistence was noticed also in different prevalent. Although it increased in prevalence over time, fHbp-1 was the only variant that genetic environments such as different clonal complexes. Persistent antigen combinations in was present in both sub-complexes at all time periods. Two additional surface proteins, PorA association with certain clonal complexes may indicate that acquisition of new alleles and FetA showed a similar trend to fHbp. NHBA-2, the most frequent sub/variant in cc41/44, encoding antigen variants may impair fitness of that clonal complex.52 Our observation of was also observed in both sub-complexes at all time periods. Given the intrinsic potential the persistence of the antigen sub/variants in 4CMenB may also indicate that the vaccine will variability of NHBA, the maintenance of the same sub/variant in both sub-complexes over be able to provide protection against populations of meningococci over time, as the antigens fifty years, despite changes in the genotype and in the predominance of the other protein they target have tended to persist. variants, could be a consequence of selective pressure or fitness constraints.

S We used the Standardized Index of Association IA to quantify the extent of allele association We examined the broadest collection of pathogenic meningococcal isolates over the longest and long-term stability and f* metrics to evidence a non-overlapping structure of antigen time span available globally. Significantly, we confirmed that the sequence conservation of combinations. Buckee and colleagues first used this parameter to evaluate the long-term specific fHbp, NadA, and NHBA sub/variants observed across strains and geographic regions stability of FetA and PorA combinations on carried meningococci.13 Given the novelty of the in recent years has also been present over the last several decades. Thus, the selection of Buckee study, our results contribute not only to an understanding of invasive meningococcal fHbp, NadA, and NHBA as antigens in 4CMenB is supported by current and past molecular strains but also to the development of approaches for evaluating long-term stability in epidemiology. The hypothesis that the stability of certain sub/variants and combinations of bacterial populations over time. fHbp, NadA, and NHBA likely results from natural selection also supports earlier interpretations that these proteins contribute to meningococcal survival and pathogenesis In the cc41/44 complex, the predominance of antigen sub/variants and STs shifted over or fitness.37,42,59-61 Further, our results may help to support the long-term validity of fHbp, time. This clonal complex is of additional public health interest because cc41/44 strains are NHBA and NadA characterization and additional typing systems for meningococci currently almost equally often isolated from healthy carriers and from cases of invasive disease.54-57 being implemented. Recently, a cc41/44 outbreak in the city of Aachen, Germany and 3 neighboring counties (Greater Aachen) has been described.56 Strains of cc41/44 are also among the most Further studies are needed in order to verify whether the observations in this strain panel important causes of serogroup B disease in the USA.57 The central STs of cc41/44, that is to are generalizable. To our knowledge, no similar panels, composed of invasive meningococcal say ST-41 and ST-44, are putative ‘ancestral genotypes’.6 In the Netherlands strain collection, isolates collected over a so long time period, exist in other countries. However, molecular the ST-44 sub-complex was predominant during the 1960s, whereas the ST-41 sub-complex typing data suggest that the distribution of clonal complexes in Europe shows only limited became predominant from 1980 onwards, a circumstance also observed in Belgium54 and variation between individual countries.9 Further work will also be needed to articulate the

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results of the present study with ongoing efforts to evaluate the clinical effects and Stability and longevity suggest that several fHbp, NHBA and NadA sub/variants are evaluation of the strain coverage of 4CMenB.62 The Meningococcal Antigen Typing System maintained by selection despite the fact that recombination continuously generates new (MATS) has been recently described as a qualitative and quantitative assay to predict sub/variants. In particular, the long-term persistence of the three antigen sub/variants 4CMenB vaccine coverage. This assay measures for each strain the expression level and the included in the vaccine, fHbp-1.1, NHBA-2 and NadA-3.8, may be indicative for long term cross-reactivity of each vaccine antigen.23 It will be important in future to apply MATS to old broad coverage of 4CMenB. and new isolates to evaluate the temporal dynamics of changes in epidemiology and of

potential antigenic shift for the vaccine-target antigens in normal condition and following vaccine introduction. ACKNOWLEDGEMENTS

is important, even if as an initial step. Also, protein expression was evaluated for genes that have an insertion or a frame shift mutation, only. As protein expression would yield even greater differences in bactericidal titer, the evaluation of MATS results in this panel would be very interesting.

Another limitation is that only 165 invasive isolates randomly selected were tested over a period of 50 years, and how representative are the selected isolates could appear to be actually doubtful. However, the number of isolates for each clonal complex indeed reflects the relative incidence of clonal complexes and represents a substantial spectrum of different serogroup B meningococci in the Netherlands over the last decades. In addition, carriage isolates were not included. Limiting selection to pathogenic isolates may have resulted in the over-representation of hyper-virulent lineages, instead of a more even balance of all meningococcal clones.63 Moreover, we cannot comment on the two-variant fHbp vaccine currently in clinical trials because only one of the two variants, fHbp-3.45, was identified in this study. Surprisingly, only four isolates at one time point harbored this fHbp variant, whereas fHbp-1.55, was not found in the current strain panel.

Data obtained in this study highlight the importance of monitoring over time the evolutionary pattern of surface proteins included as vaccine antigens. The stability of certain sub/variants was of course observed in a pre-vaccination era, therefore in the absence of a strong immune selection against the three antigen sub/variants. It will be interesting to monitor the long-term persistence even after the introduction of the vaccine.

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A limitation of the present study is that it comprises genetic data only. The study of temporal We are grateful to Giorgio Corsi for artwork. patterns of genetic associations among vaccine protein variants and MLST clonal complexes is important, even if as an initial step. Also, protein expression was evaluated for genes that have an insertion or a frame shift mutation, only. As protein expression would yield even greater differences in bactericidal titer, the evaluation of MATS results in this panel would be very interesting. 5

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REFERENCES 15. Caugant DA, Maiden MC. Meningococcal carriage and disease--population biology and evolution. Vaccine 2009;27 Suppl 2:B64-70. 1. Jolley KA, Wilson DJ, Kriz P, McVean G, Maiden MC. The influence of mutation, recombination, population history, and selection on patterns of genetic diversity in 16. Jodar L, Feavers IM, Salisbury D, Granoff DM. Development of vaccines against Neisseria meningitidis. Mol Biol Evol 2005;22:562-569. meningococcal disease. Lancet 2002;359:1499-1508.

2. Harrison OB, Evans NJ, Blair JM, et al. Epidemiological evidence for the role of the 17. Van Der Ley P, Poolman JT. Construction of a multivalent meningococcal vaccine hemoglobin receptor, hmbR, in meningococcal virulence. J Infect Dis 2009;200:94-98. strain based on the class 1 outer membrane protein. Infect Immun 1992;60:3156- 3161. 3. Caugant DA, Mocca LF, Frasch CE, Froholm LO, Zollinger WD, Selander RK. Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, 18. Peeters CC, Rumke HC, Sundermann LC, et al. Phase I clinical trial with a hexavalent and outer membrane protein pattern. J Bacteriol 1987;169:2781-2792. PorA containing meningococcal outer membrane vesicle vaccine. Vaccine 1996;14:1009-1015. 4. Maiden MC, Bygraves JA, Feil E, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic 19. Cartwright K, Morris R, Rumke H, et al. Immunogenicity and reactogenicity in UK microorganisms. Proc Natl Acad Sci U S A 1998;95:3140-3145. infants of a novel meningococcal vesicle vaccine containing multiple class 1 (PorA) outer membrane proteins. Vaccine 1999;17:2612-2619. 5. Maiden MC. Multilocus sequence typing of bacteria. Annu Rev Microbiol 2006;60:561-588. 20. Rappuoli R. Reverse vaccinology, a genome-based approach to vaccine development. Vaccine 2001;19:2688-2691. 6. Brehony C, Jolley KA, Maiden MC. Multilocus sequence typing for global surveillance of meningococcal disease. FEMS Microbiol Rev 2007;31:15-26. 21. Tettelin H, Saunders NJ, Heidelberg J, et al. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 2000;287:1809-1815. 7. Achtman M, ed. Meningococcal disease. New York: Wiley1995. 22. Pizza M, Scarlato V, Masignani V, et al. Identification of vaccine candidates against 8. Callaghan MJ, Jolley KA, Maiden MC. Opacity-associated adhesin repertoire in serogroup B meningococcus by whole-genome sequencing. Science 2000;287:1816- hyperinvasive Neisseria meningitidis. Infect Immun 2006;74:5085-5094. 1820.

9. Yazdankhah SP, Kriz P, Tzanakaki G, et al. Distribution of serogroups and genotypes 23. Vogel U, Taha MK, Vazquez JA, et al. Predicted strain coverage of a meningococcal among disease-associated and carried isolates of Neisseria meningitidis from the multicomponent vaccine (4CMenB) in Europe: a qualitative and quantitative Czech Republic, Greece, and Norway. J Clin Microbiol 2004;42:5146-5153. assessment. Lancet Infect Dis 2013.

10. Law DK, Lorange M, Ringuette L, et al. Invasive meningococcal disease in Quebec, 24. Kimura A, Toneatto D, Kleinschmidt A, Wang H, Dull P. Immunogenicity and safety Canada, due to an emerging clone of ST-269 serogroup B meningococci with serotype of a multicomponent meningococcal serogroup B vaccine and a quadrivalent antigen 17 and serosubtype antigen P1.19 (B:17:P1.19). J Clin Microbiol meningococcal CRM197 conjugate vaccine against serogroups A, C, W-135, and Y in 2006;44:2743-2749. adults who are at increased risk for occupational exposure to meningococcal isolates. Clin Vaccine Immunol 2011;18:483-486. 11. Budroni S, Siena E, Dunning Hotopp JC, et al. Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous 25. Bai X, Findlow J, Borrow R. Recombinant protein meningococcal serogroup B vaccine recombination. Proc Natl Acad Sci U S A 2011;108:4494-4499. combined with outer membrane vesicles. Expert Opin Biol Ther 2011;11:969-985.

12. Caugant DA. Genetics and evolution of Neisseria meningitidis: importance for the 26. Sexton K, Lennon D, Oster P, et al. Proceedings of the Meningococcal Vaccine epidemiology of meningococcal disease. Infect Genet Evol 2008;8:558-565. Strategy World Health Organization satellite meeting, 10 March 2004, Auckland, New Zealand. N Z Med J 2004;117:1 p preceding U1027. 13. Buckee CO, Gupta S, Kriz P, Maiden MC, Jolley KA. Long-term evolution of antigen repertoires among carried meningococci. Proc Biol Sci 2010;277:1635-1641. 27. Fletcher LD, Bernfield L, Barniak V, et al. Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect Immun 2004;72:2088-2100. 14. Buckee CO, Jolley KA, Recker M, et al. Role of selection in the emergence of lineages and the evolution of virulence in Neisseria meningitidis. Proc Natl Acad Sci U S A 2008;105:15082-15087.

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2. Harrison OB, Evans NJ, Blair JM, et al. Epidemiological evidence for the role of the 17. Van Der Ley P, Poolman JT. Construction of a multivalent meningococcal vaccine hemoglobin receptor, hmbR, in meningococcal virulence. J Infect Dis 2009;200:94-98. strain based on the class 1 outer membrane protein. Infect Immun 1992;60:3156- 3161. 3. Caugant DA, Mocca LF, Frasch CE, Froholm LO, Zollinger WD, Selander RK. Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, 18. Peeters CC, Rumke HC, Sundermann LC, et al. Phase I clinical trial with a hexavalent and outer membrane protein pattern. J Bacteriol 1987;169:2781-2792. PorA containing meningococcal outer membrane vesicle vaccine. Vaccine 1996;14:1009-1015. 4. Maiden MC, Bygraves JA, Feil E, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic 19. Cartwright K, Morris R, Rumke H, et al. Immunogenicity and reactogenicity in UK microorganisms. Proc Natl Acad Sci U S A 1998;95:3140-3145. infants of a novel meningococcal vesicle vaccine containing multiple class 1 (PorA) outer membrane proteins. Vaccine 1999;17:2612-2619. 5. Maiden MC. Multilocus sequence typing of bacteria. Annu Rev Microbiol 2006;60:561-588. 20. Rappuoli R. Reverse vaccinology, a genome-based approach to vaccine development. Vaccine 2001;19:2688-2691. 6. Brehony C, Jolley KA, Maiden MC. Multilocus sequence typing for global surveillance of meningococcal disease. FEMS Microbiol Rev 2007;31:15-26. 21. Tettelin H, Saunders NJ, Heidelberg J, et al. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 2000;287:1809-1815. 5 7. Achtman M, ed. Meningococcal disease. New York: Wiley1995. 22. Pizza M, Scarlato V, Masignani V, et al. Identification of vaccine candidates against 8. Callaghan MJ, Jolley KA, Maiden MC. Opacity-associated adhesin repertoire in serogroup B meningococcus by whole-genome sequencing. Science 2000;287:1816- hyperinvasive Neisseria meningitidis. Infect Immun 2006;74:5085-5094. 1820.

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28. Masignani V, Comanducci M, Giuliani MM, et al. Vaccination against Neisseria 40. Giuliani MM, Adu-Bobie J, Comanducci M, et al. A universal vaccine for serogroup B meningitidis using three variants of the lipoprotein GNA1870. J Exp Med meningococcus. Proc Natl Acad Sci U S A 2006;103:10834-10839. 2003;197:789-799. 41. Welsch JA, Moe GR, Rossi R, Adu-Bobie J, Rappuoli R, Granoff DM. Antibody to 29. Madico G, Welsch JA, Lewis LA, et al. The meningococcal vaccine candidate genome-derived neisserial antigen 2132, a Neisseria meningitidis candidate vaccine, GNA1870 binds the complement regulatory protein factor H and enhances serum confers protection against bacteremia in the absence of complement-mediated resistance. J Immunol 2006;177:501-510. bactericidal activity. J Infect Dis 2003;188:1730-1740.

30. Seib KL, Brunelli B, Brogioni B, et al. Characterization of diverse subvariants of the 42. Capecchi B, Adu-Bobie J, Di Marcello F, et al. Neisseria meningitidis NadA is a new meningococcal factor H (fH) binding protein for their ability to bind fH, to mediate invasin which promotes bacterial adhesion to and penetration into human epithelial serum resistance, and to induce bactericidal antibodies. Infect Immun 2011;79:970- cells. Mol Microbiol 2005;55:687-698. 981. 43. Comanducci M, Bambini S, Brunelli B, et al. NadA, a novel vaccine candidate of 31. Plested JS, Welsch JA, Granoff DM. Ex vivo model of meningococcal bacteremia Neisseria meningitidis. J Exp Med 2002;195:1445-1454. using human blood for measuring vaccine-induced serum passive protective activity. Clin Vaccine Immunol 2009;16:785-791. 44. Comanducci M, Bambini S, Caugant DA, et al. NadA diversity and carriage in Neisseria meningitidis. Infect Immun 2004;72:4217-4223. 32. Seib KL, Serruto D, Oriente F, et al. Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the 45. Lucidarme J, Comanducci M, Findlow J, et al. Characterization of fHbp, nhba antimicrobial peptide LL-37. Infect Immun 2009;77:292-299. (gna2132), nadA, porA, sequence type (ST), and genomic presence of IS1301 in group B meningococcal ST269 clonal complex isolates from England and Wales. J Clin 33. Welsch JA, Ram S, Koeberling O, Granoff DM. Complement-dependent synergistic Microbiol 2009;47:3577-3585. bactericidal activity of antibodies against factor H-binding protein, a sparsely distributed meningococcal vaccine antigen. J Infect Dis 2008;197:1053-1061. 46. Jolley KA, Chan MS, Maiden MC. mlstdbNet - distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics 2004;5:86. 34. Brunelli B, Del Tordello E, Palumbo E, et al. Influence of sequence variability on bactericidal activity sera induced by Factor H binding protein variant 1.1. Vaccine 47. Heckenberg SG, de Gans J, Brouwer MC, et al. Clinical features, outcome, and 2011;29:1072-1081. meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study. Medicine (Baltimore) 2008;87:185-192. 35. Murphy E, Andrew L, Lee KL, et al. Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria 48. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics meningitidis. J Infect Dis 2009;200:379-389. Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-1599.

36. Marsh JW, Shutt KA, Pajon R, et al. Diversity of factor H-binding protein in Neisseria 49. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ. Jalview Version 2--a meningitidis carriage isolates. Vaccine 2011;29:6049-6058. multiple sequence alignment editor and analysis workbench. Bioinformatics 2009;25:1189-1191. 37. Serruto D, Spadafina T, Ciucchi L, et al. Neisseria meningitidis GNA2132, a heparin- binding protein that induces protective immunity in humans. Proc Natl Acad Sci U S A 50. Francisco AP, Bugalho M, Ramirez M, Carrico JA. Global optimal eBURST analysis of 2010;107:3770-3775. multilocus typing data using a graphic matroid approach. BMC Bioinformatics 2009;10:152. 38. Bambini S, Muzzi A, Olcen P, Rappuoli R, Pizza M, Comanducci M. Distribution and genetic variability of three vaccine components in a panel of strains representative of 51. Cramér H, ed. Mathematical Methods of Statistics1946. the diversity of serogroup B meningococcus. Vaccine 2009;27:2794-2803. 52. Haubold B, Hudson RR. LIAN 3.0: detecting linkage disequilibrium in multilocus data. 39. Jacobsson S, Hedberg ST, Molling P, et al. Prevalence and sequence variations of the Linkage Analysis. Bioinformatics 2000;16:847-848. genes encoding the five antigens included in the novel 5CVMB vaccine covering group B meningococcal disease. Vaccine 2009;27:1579-1584. 53. Gupta S, Maiden MC, Feavers IM, Nee S, May RM, Anderson RM. The maintenance of strain structure in populations of recombining infectious agents. Nat Med 1996;2:437-442.

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30. Seib KL, Brunelli B, Brogioni B, et al. Characterization of diverse subvariants of the 42. Capecchi B, Adu-Bobie J, Di Marcello F, et al. Neisseria meningitidis NadA is a new meningococcal factor H (fH) binding protein for their ability to bind fH, to mediate invasin which promotes bacterial adhesion to and penetration into human epithelial serum resistance, and to induce bactericidal antibodies. Infect Immun 2011;79:970- cells. Mol Microbiol 2005;55:687-698. 981. 43. Comanducci M, Bambini S, Brunelli B, et al. NadA, a novel vaccine candidate of 31. Plested JS, Welsch JA, Granoff DM. Ex vivo model of meningococcal bacteremia Neisseria meningitidis. J Exp Med 2002;195:1445-1454. using human blood for measuring vaccine-induced serum passive protective activity. Clin Vaccine Immunol 2009;16:785-791. 44. Comanducci M, Bambini S, Caugant DA, et al. NadA diversity and carriage in Neisseria meningitidis. Infect Immun 2004;72:4217-4223. 32. Seib KL, Serruto D, Oriente F, et al. Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the 45. Lucidarme J, Comanducci M, Findlow J, et al. Characterization of fHbp, nhba antimicrobial peptide LL-37. Infect Immun 2009;77:292-299. (gna2132), nadA, porA, sequence type (ST), and genomic presence of IS1301 in group B meningococcal ST269 clonal complex isolates from England and Wales. J Clin 33. Welsch JA, Ram S, Koeberling O, Granoff DM. Complement-dependent synergistic Microbiol 2009;47:3577-3585. 5 bactericidal activity of antibodies against factor H-binding protein, a sparsely distributed meningococcal vaccine antigen. J Infect Dis 2008;197:1053-1061. 46. Jolley KA, Chan MS, Maiden MC. mlstdbNet - distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics 2004;5:86. 34. Brunelli B, Del Tordello E, Palumbo E, et al. Influence of sequence variability on bactericidal activity sera induced by Factor H binding protein variant 1.1. Vaccine 47. Heckenberg SG, de Gans J, Brouwer MC, et al. Clinical features, outcome, and 2011;29:1072-1081. meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study. Medicine (Baltimore) 2008;87:185-192. 35. Murphy E, Andrew L, Lee KL, et al. Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria 48. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics meningitidis. J Infect Dis 2009;200:379-389. Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-1599.

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54. Van Looveren M, Vandamme P, Hauchecorne M, et al. Molecular epidemiology of SUPPLEMENTARY MATERIAL recent belgian isolates of Neisseria meningitidis serogroup B. J Clin Microbiol 1998;36:2828-2834. Supplementary Table 1. Characterization of the 165 clinical isolates selected for this study

55. Martin DR, Walker SJ, Baker MG, Lennon DR. New Zealand epidemic of Due to size, only online accessible at: meningococcal disease identified by a strain with phenotype B:4:P1.4. J Infect Dis http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0065043. For each isolate, year of 1998;177:497-500. isolation, serogroup, FetA, PorA, sequence type (ST), clonal complex, fHbp (allele, sub/variant and variant), NHBA (peptide) and NadA (gene presence, sub/variant and variant) are indicated. fHbp sub/variants were 56. Elias J, Schouls LM, van de Pol I, et al. Vaccine preventability of meningococcal clone, designated as reported in the database (http://pubmlst.org/neisseria/fHbp/). For NHBA and NadA, an internal Greater Aachen Region, Germany. Emerg Infect Dis 2010;16:465-472. nomenclature was used. FS: frame-shift mutation: INS: insertion sequence IS1301, causing no expression of the protein. 57. Anne Whitney LM, Elizabeth Mothershed, Claudio Sacchi, Susanna Schmink, Jordan

Theodore, Jane Marsh, Kathleen Shutt, Nancy Messonier, Patricia Wilkins, Lee Harrison, and the Active Bacterial Core Surveillance Team (ABCs). Distribution of Supplementary Table 2. Primers used for PCR and sequencing. multi-locus sequence types of N. meningitidis serogroup B in the US, 2000-2005. Forward Reverse Fifteenth International Pathogenic Conference 2006. Cairns, North Queensland, primer Sequence 5'-3' Primer Sequence 5'-3' a a Australia2006. fHbp A1 GACCTGCCTCATTGATG B2 CGGTAAATTATCGTGTTCGGACGGC 22 CAAATCGAAGTGGACGGGCAG 32 TGTTCGATTTTGCCGTTTCCCTG 58. Bart A, Dankert J, van der Ende A. Representational difference analysis of Neisseria a a meningitidis identifies sequences that are specific for the hyper-virulent lineage III NHBA 1 GGCGTTCAGACGGCATATTTTTACA 6 GGTTTATCAACTGATGCGGACTTGA clone. FEMS Microbiol Lett 2000;188:111-114. 22 GCGGACACGCTGTCAAAACC 5 ATGGGTACGCAAAAATTCAA

23 GAAAATACAGGCAATGGCGG 7 AATGCAGTACTTCGCCGTTGT 59. Seib KL, Oriente F, Adu-Bobie J, et al. Influence of serogroup B meningococcal 3 GCAAAATGCCGGCAATACGC 85 CAGCGTATCCGCCTGATTG vaccine antigens on growth and survival of the meningococcus in vitro and in ex vivo 84 CAATCAGGCGGATACGCTG 93 CCGCCATTGCCTGTATTTTC and in vivo models of infection. Vaccine 2010;28:2416-2427. a a 60. Dunphy KY, Beernink PT, Brogioni B, Granoff DM. Effect of factor H-binding protein NadA A1 GTGGACGTACTCGACTACGAAGG B CGAGGCGATTGTCAAACCGTTC sequence variation on factor H binding and survival of Neisseria meningitidis in 1 TATGTAAACAAACTTGGTGGGG C TTTCGAGGTGGCGCGTTCGGG

human blood. Infect Immun 2011;79:353-359. 2 GAAATAGAAAAGTTAACAACCAAGTT 5 GTAGCGATATCAGCTTTGATGTC

3 GACATCAAAGCTGATATCGCTAC 6 AACTTGGTTGTTAACTTTTCTATTTC 61. Echenique-Rivera H, Muzzi A, Del Tordello E, et al. Transcriptome analysis of

Neisseria meningitidis in human whole blood and mutagenesis studies identify nmeSI AVDE 0712 ATCAATTTGTGGATTTTGG AVDE 0716 CACATCTCCACCATACAATATTGG virulence factors involved in blood survival. PLoS Pathog 2011;7:e1002027.

moxR AVDE 0701F GAAAGAAGACCGATATCTCC AVDE 0710 GCGTCCATTGCAAAAAGCAG 62. Donnelly J, Medini D, Boccadifuoco G, et al. Qualitative and quantitative assessment aThese primers were also used for locus amplification of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines. Proc Natl Acad Sci U S A 2010;107:19490-19495.

63. Didelot X, Maiden MC. Impact of recombination on bacterial evolution. Trends Microbiol 2010;18:315-322.

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23 GAAAATACAGGCAATGGCGG 7 AATGCAGTACTTCGCCGTTGT 5 59. Seib KL, Oriente F, Adu-Bobie J, et al. Influence of serogroup B meningococcal 3 GCAAAATGCCGGCAATACGC 85 CAGCGTATCCGCCTGATTG vaccine antigens on growth and survival of the meningococcus in vitro and in ex vivo 84 CAATCAGGCGGATACGCTG 93 CCGCCATTGCCTGTATTTTC and in vivo models of infection. Vaccine 2010;28:2416-2427. a a 60. Dunphy KY, Beernink PT, Brogioni B, Granoff DM. Effect of factor H-binding protein NadA A1 GTGGACGTACTCGACTACGAAGG B CGAGGCGATTGTCAAACCGTTC sequence variation on factor H binding and survival of Neisseria meningitidis in 1 TATGTAAACAAACTTGGTGGGG C TTTCGAGGTGGCGCGTTCGGG human blood. Infect Immun 2011;79:353-359. 2 GAAATAGAAAAGTTAACAACCAAGTT 5 GTAGCGATATCAGCTTTGATGTC

3 GACATCAAAGCTGATATCGCTAC 6 AACTTGGTTGTTAACTTTTCTATTTC 61. Echenique-Rivera H, Muzzi A, Del Tordello E, et al. Transcriptome analysis of

63. Didelot X, Maiden MC. Impact of recombination on bacterial evolution. Trends Microbiol 2010;18:315-322.

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CHAPTER 6:

Genome sequence of Neisseria meningitidis serogroup B strain H44/76

Jurgen R. Piet* Robert A.G. Huis in ’t Veld* Barbara D.C. van Schaik Antoine H.C. van Kampen Frank Baas Diederik van de Beek Yvonne Pannekoek Arie van der Ende

*Both authors contributed equally

J Bacteriol 2011;193:2371-2372

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CHAPTER 6: CHAPTER 6: Genome sequence of Neisseria meningitidis serogroup B strain H44/76

Jurgen R. Piet* Robert A.G. Huis in ’t Veld* Barbara D.C. van SchaiGenomek sequence of Antoine H.C. van KampenNeisseria meningitidis Frank Baas Diederik van de Beekserogroup B strain H44/76 Yvonne Pannekoek Arie van der Ende

*Both authors contributed equally

Jurgen R. Piet* Robert A.G. Huis in ’t Veld* Barbara D.C. van Schaik Antoine H.C. van Kampen Frank Baas Diederik van de Beek Yvonne Pannekoek

Arie van der Ende

*Both authors contributed equally J Bacteriol 2011;193:2371-2372

J Bacteriol 2011;193:2371-2372

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ABSTRACT Neisseria meningitidis resides in the nasopharynx of approximately 10 to 30% of the human population. However, in some individuals this pathogen results in invasive disease leading to Neisseria meningitidis is an obligate human pathogen. While it is a frequent commensal of life-threatening septicemia and meningitis.1,2 The reasons triggering meningococcal the upper respiratory tract, in some individuals the bacterium spreads to the bloodstream pathogenic outbreaks have not yet been elucidated, but several host and bacterium factors causing meningitis and/or sepsis, serious conditions with high morbidity and mortality. Here have been proposed.3-5 we report the availability of the genome sequence of the widely used serogroup B laboratory strain H44/76. To date 7 complete N. meningitidis genomes are deposited at GenBank6-10 of which only one is serogroup B. Here we present the sequence of N. meningitidis serogroup B strain H44/76, related to the sequenced serogroup B strain MC58 (GenBank accession no. AE002098), belonging to the same clonal complex 32.11 N. meningitidis H44/76 is widely used in molecular genetic studies, including those related to serogroup B vaccine development, because of its favorable natural transformation efficiency.

Whole genomic DNA of a H44/76 culture that only had 6 plate-culture passages since its isolation from a patient in Norway in 1976 was sequenced.12 Sequencing was performed using shotgun 454 Titanium (Roche) pyrosequencing, according to the manufacturer’s recommendations. The coverage was 20-fold with a median read length of 403 nucleotides. De novo and reference mapping assemblies were performed with Newbler (version 2.3; 454 Life Sciences, Branford, CT). The final assembly, containing 46 contigs, was obtained by combining both assemblies using CodonCode Aligner software (version 3.5, CodonCode Corporation, Dedham, MA). Investigation of the regions surrounding contig boundaries showed that further reduction of the number of contigs was hampered due to misassembly of repetitive sequences and duplicated genes like transposases, rRNA/tRNA regions, two partner secretion systems, and Type IV pili.13

Annotation was performed using the annotation engine at the Institute for Genome Sciences (University of Maryland, Baltimore, MD [http://ae.igs.umaryland.edu/cgi/index.cgi]). We compared the sequences of 21 gene fragments of abcZ, adk, aroE, fumC, gdh, pdhC, pgm, aspA, carB, dhpS, glnA, gpm, mtgA, pilA, pip, ppk, pykA, rpiA, serC, talA, porA and porB of the currently sequenced H44/76 genome with those of H44/76 previously submitted to the Neisseria Multilocus Sequence Typing database.14 All 11,549 compared nucleotides were identical, indicating a high quality of our H44/76 genome. Comparative genome analysis of

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Whole genomic DNA of a H44/76 culture that only had 6 plate-culture passages since its isolation from a patient in Norway in 1976 was sequenced.12 Sequencing was performed using shotgun 454 Titanium (Roche) pyrosequencing, according to the manufacturer’s recommendations. The coverage was 20-fold with a median read length of 403 nucleotides. De novo and reference mapping assemblies were performed with Newbler (version 2.3; 454 6 Life Sciences, Branford, CT). The final assembly, containing 46 contigs, was obtained by combining both assemblies using CodonCode Aligner software (version 3.5, CodonCode Corporation, Dedham, MA). Investigation of the regions surrounding contig boundaries showed that further reduction of the number of contigs was hampered due to misassembly of repetitive sequences and duplicated genes like transposases, rRNA/tRNA regions, two partner secretion systems, and Type IV pili.13

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strain H44/76 and MC58 (recently re-annotated8) was performed using BLAT15 and REFERENCES 16 progressiveMauve. 1. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 2007;369:2196-2210. The genome size of H44/76 is 2.18 Mb and 2480 ORFs were annotated, compared to 2.27 2. de Souza AL, Seguro AC. Two centuries of meningococcal infection: from Vieusseux Mb and 2465 ORFs in MC58. Both strains have a GC content of 51.5%. Comparative analysis to the cellular and molecular basis of disease. J Med Microbiol 2008;57:1313-1321. showed that 4 genes are uniquely present in H44/76 and 9 genes are only present in MC58. 3. Lo H, Tang CM, Exley RM. Mechanisms of avoidance of host immunity by Neisseria Of all ORFs in H44/76, 2317 (93%) show more than 99% sequence identity. Of the 18 least meningitidis and its effect on vaccine development. Lancet Infect Dis 2009;9:418-427. similar genes (90-95% sequence identity), 3 (hmbR, tbp1 and an iron(III) ABC transporter) are 4. Serruto D, Rappuoli R, Scarselli M, Gros P, van Strijp JA. Molecular mechanisms of associated with iron acquisition from the host. complement evasion: learning from staphylococci and meningococci. Nat Rev Microbiol 2010;8:393-399. In the genome sequence of MC58, a ~32 Kb region (NMB1124 to NMB1159), is duplicated 5. Brouwer MC, de Gans J, Heckenberg SG, Zwinderman AH, van der Poll T, van de (NMB1162 to NMB1197) while this region occurs only once in H44/76 (verified by PCR and Beek D. Host genetic susceptibility to pneumococcal and meningococcal disease: a systematic review and meta-analysis. Lancet Infect Dis 2009;9:31-44. 454 read coverage analysis). Obviously, the erythromycin cassette that truncates the siaD gene in MC5817 is not present in H44/76. 6. Tettelin H, Saunders NJ, Heidelberg J, et al. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 2000;287:1809-1815.

Nucleotide sequence accession numbers 7. Parkhill J, Achtman M, James KD, et al. Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 2000;404:502-506. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the 8. Rusniok C, Vallenet D, Floquet S, et al. NeMeSys: a biological resource for narrowing accession AEQZ00000000. the gap between sequence and function in the human pathogen Neisseria meningitidis. Genome Biol 2009;10:R110. ACKNOWLEDGMENTS 9. Bentley SD, Vernikos GS, Snyder LA, et al. Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18. PLoS This publication made use of the Neisseria Multilocus Sequence Typing website Genet 2007;3:e23. (http://pubmlst.org/neisseria) developed by Keith Jolley and Man-Suen Chan and sited at 14 10. Peng J, Yang L, Yang F, et al. Characterization of ST-4821 complex, a unique Neisseria the University of Oxford. The development of this site has been funded by the Wellcome meningitidis clone. Genomics 2008;91:78-87. Trust and the European Union. We would like to thank Michelle Giglio and Sean Daugherty 11. van der Ende A, Hopman CT, Dankert J. Deletion of porA by recombination between at the Institute of Genome Sciences for their support in annotating the genome and clusters of repetitive extragenic palindromic sequences in Neisseria meningitidis. Infect Immun 1999 depositing it at GenBank. We also thank Marja Jakobs for her technical assistance with ;67:2928-2934. genome sequencing. 12. Frasch CE, Zollinger WD, Poolman JT. Serotype antigens of Neisseria meningitidis and a proposed scheme for designation of serotypes. Rev Infect Dis 1985;7:504-510.

13. Marri PR, Paniscus M, Weyand NJ, et al. Genome sequencing reveals widespread virulence gene exchange among human Neisseria species. PLoS One 2010;5:e11835.

14. Jolley KA, Chan MS, Maiden MC. mlstdbNet - distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics 2004;5:86.

15. Kent WJ. BLAT--the BLAST-like alignment tool. Genome Res 2002;12:656-664.

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Nucleotide sequence accession numbers 7. Parkhill J, Achtman M, James KD, et al. Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 2000;404:502-506. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the 8. Rusniok C, Vallenet D, Floquet S, et al. NeMeSys: a biological resource for narrowing accession AEQZ00000000. the gap between sequence and function in the human pathogen Neisseria 6 meningitidis. Genome Biol 2009;10:R110. ACKNOWLEDGMENTS 9. Bentley SD, Vernikos GS, Snyder LA, et al. Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18. PLoS This publication made use of the Neisseria Multilocus Sequence Typing website Genet 2007;3:e23. (http://pubmlst.org/neisseria) developed by Keith Jolley and Man-Suen Chan and sited at 14 10. Peng J, Yang L, Yang F, et al. Characterization of ST-4821 complex, a unique Neisseria the University of Oxford. The development of this site has been funded by the Wellcome meningitidis clone. Genomics 2008;91:78-87. Trust and the European Union. We would like to thank Michelle Giglio and Sean Daugherty 11. van der Ende A, Hopman CT, Dankert J. Deletion of porA by recombination between at the Institute of Genome Sciences for their support in annotating the genome and clusters of repetitive extragenic palindromic sequences in Neisseria meningitidis. Infect Immun 1999 depositing it at GenBank. We also thank Marja Jakobs for her technical assistance with ;67:2928-2934. genome sequencing. 12. Frasch CE, Zollinger WD, Poolman JT. Serotype antigens of Neisseria meningitidis and a proposed scheme for designation of serotypes. Rev Infect Dis 1985;7:504-510.

13. Marri PR, Paniscus M, Weyand NJ, et al. Genome sequencing reveals widespread virulence gene exchange among human Neisseria species. PLoS One 2010;5:e11835.

14. Jolley KA, Chan MS, Maiden MC. mlstdbNet - distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics 2004;5:86.

15. Kent WJ. BLAT--the BLAST-like alignment tool. Genome Res 2002;12:656-664.

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16. Darling AE, Mau B, Perna NT. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 2010;5:e11147.

17. van Passel M, Bart A, Pannekoek Y, van der Ende A. Phylogenetic validation of horizontal gene transfer? Nat Genet 2004;36:1028.

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16. Darling AE, Mau B, Perna NT. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 2010;5:e11147.

17. van Passel M, Bart A, Pannekoek Y, van der Ende A. Phylogenetic validation of horizontal gene transfer? Nat Genet 2004;36:1028.

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CHAPTER 7:

Meningitis caused by a lipopolysaccharide deficient Neisseria meningitidis

Jurgen R. Piet* Afshin Zariri* Floris Fransen Kim Schipper Peter van der Ley Diederik van de Beek Arie van der Ende

*Both authors contributed equally

J Infect 2014;69:352-357

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CHAPTER 7: CHAPTER 7: Meningitis caused by a lipopolysaccharide deficient Neisseria meningitidis

Jurgen R. Piet* Afshin Zariri* Floris Fransen Meningitis caused by a Kim Schipper lipopolysaccharide deficient Peter van der Ley Diederik van de Beek Neisseria meningitidis Arie van der Ende

*Both authors contributed equally

Jurgen R. Piet* Afshin Zariri*

Floris Fransen Kim Schipper

Peter van der Ley

Diederik van de Beek Arie van der Ende

*Both authors contributed equally

J Infect 2014;69:352-357 J Infect 2014;69:352-357

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ABSTRACT INTRODUCTION

Lipopolysaccharide (LPS) is a major component of the Neisseria meningitidis outer Neisseria meningitidis is an important cause of meningitis and sepsis worldwide.1 A major membrane. Here we report a patient with meningococcal meningitis of which the causative component of the meningococcal outer membrane is lipopolysaccharide (LPS) consisting of a isolate lacked LPS. Thus far, no naturally occurring LPS-deficient meningococcal isolate has core oligosaccharide and a membrane-anchoring lipid A part which is recognized by been known to cause clinical disease. We used SDS-PAGE, silver staining and LPS-specific mammalian immune cells through the TLR4/MD-2 receptor complex in the initiation of an antibodies in whole cell ELISA to determine LPS presence in the causative isolate. inflammatory response.2 N. meningitidis is one of the few Gram-negative bacteria which can Meningococcal whole genome sequencing was performed using Roche 454-sequencing. The grow without LPS in vitro.3 The hallmark of meningococcal disease is the excessive N. meningitidis strain MC58 was used to compare all LPS biosynthesis associated genes. We stimulation of the immune system by LPS.4 We previously described that about 12% of compared growth characteristics of Escherichia coli transformed with a plasmid containing 2 meningococcal isolates causing meningitis induced a reduced inflammatory response and lpxH types. The patient presented with isolated thunderclap headache. Analysis of the patients infected with these strains had less severe disease.5 Further analysis showed that all causative N. meningitidis showed no LPS. Whole-genome sequencing revealed a mutation but one of these isolates had a mutation in lpxL1, one of the 9 genes involved in lipid A located in lpxH explaining LPS-deficiency. Expression of this lpxH variant in E. coli resulted in biosynthesis, resulting in a penta-acylated lipid A structure which has strongly reduced TLR4 growth impairment compared to E. coli expressing the meningococcal wild-type lpxH variant. activation capacity.5 One isolate (strain 992008) induced a low inflammatory response but In addition, inactivating lpxH in N. meningitidis H44/76 by insertional inactivation with a still harbored an intact lpxL1 gene. Currently, we show that this meningococcal strain lacks kanamycin cassette resulted in a LPS-deficient phenotype. Concluding, we describe invasive LPS in its outer membrane. We report the uncommon clinical presentation of a patient that meningococcal disease caused by a naturally occurring LPS-deficient meningococcal isolate. was infected by this meningococcal strain and provide evidence that the responsible mutation is located in lpxH, encoding the enzyme UDP-2,3-diacylglucosamine hydrolase required for the fourth step in the Raetz pathway for lipid A biosynthesis.6

MATERIALS AND METHODS

Meningococcal typing

Serogrouping and Multilocus Sequence Typing (MLST), was performed by the Netherlands Reference Laboratory for Bacterial Meningitis. MLST was performed as previously described.7 The clonal complex was allocated according to the online MLST-database (available at http://pubmlst.org/neisseria/).

LPS detection using SDS polyacrylamide gel electrophoresis

To prepare bacteria for LPS analysis by SDS polyacrylamide gel electrophoresis, Tricine-SDS sample buffer (Novex, Invitrogen) was added to whole cell bacteria and incubated at 100ºC

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ABSTRACT INTRODUCTION

Meningococcal typing

LPS detection using SDS polyacrylamide gel electrophoresis

To prepare bacteria for LPS analysis by SDS polyacrylamide gel electrophoresis, Tricine-SDS sample buffer (Novex, Invitrogen) was added to whole cell bacteria and incubated at 100ºC

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for 5 minutes. After addition of 5µl proteinase K (20 mg/ml), the lysate was incubated at in E. coli was induced using the LacZ promoter of pGEM-T easy plasmids by addition of 56ºC overnight. Lysates were incubated at 100ºC for 10 minutes and loaded onto a 16% isopropyl-β-D-thioglactopyranoside (IPTG, 1µM). An lpxH knockout mutant was created in N. polyacrylamide gel (Novex® Tricine Gel, Invitrogen). After electrophoresis, LPS bands were meningitidis H44/76. For this pGEM-T easy plasmid harboring the lpxH gene was used as visualized by silver staining. template in a PCR using primers 5’-GGATCCAAGCCCGCCGATATTATG-3’ and 5’- GGATCCCGCCTTTGTCGGACAATTTC-3’. After digestion of the amplicon with BamH1, a LPS detection using monoclonal antibodies kanamycin cassette was ligated to the product, resulting in replacement of a 300 bp deletion

containing meningococcal strains. Binding of monoclonal antibodies was detected using an in a humidified atmosphere containing 5% CO2. Human IL-6 cytokine was quantified in the anti-mouse IgG antibody conjugated to horseradish peroxidase (Southern Biotechnology culture supernatants using the PeliPairTM reagent set (Sanquin). Associates, Inc.). 3,3’,5,5’-Tetramethylbenzidine (TMB 10 µg/ml) was used as a substrate and Role of the funding source the reaction was stopped by the addition of 2 M H2SO4. This study has been funded by grants from the Netherlands Organization for Health Research Bacterial whole-genome sequencing, annotation and LPS biosynthesis gene analysis and Development (ZonMw; NWO-Vidi grant 2010 [016.116.358], to D. van de Beek), the The whole genome of isolate 992008 was sequenced using Roche 454-Titanium sequencing. European Research Council (ERC Starting Grant [Proposal/Contract number 281156] to D. We annotated the genome using the Annotation Engine at the Institute for Genome Sciences van de Beek), the Academic Medical Center (AMC Fellowship 2008 to D. van de Beek, AMC of the University of Maryland School of Medicine. The N. meningitidis strain MC58 was used PhD scholarship 2008 to J. Piet). A. van der Ende participated in the Sixth Framework to compare all LPS biosynthesis associated genes. The information reported here from the Programme of the European Commission, Proposal/Contract no.: 512061 (Network of Whole Genome Shotgun project has been deposited with DDBJ/EMBL/GenBank under Excellence “European Virtual Institute for Functional Genomics of Bacterial Pathogens”.) The BioProject Id: PRJNA178630. funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Growth experiment in Escherichia. coli and meningococcal lpxH knockout

The lpxH gene was amplified by PCR from N. meningitidis H44/76 and 992008 strains using primers 5’-TTGAAGCCGGAGAGTTTG-3’ and 3’-CCCGTGGTTTCCATCATAC-5’. The lpxH gene was ligated into pGEM-T easy (Promega) plasmid and cloned in JM109 E. coli competent cells (Promega). Expression of lpxH from N. meningitidis H44/76 and from N. meningitidis 992008

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LPS was detected by whole cell ELISA. Briefly, 96-well plates were coated overnight at 37 ºC of lpxH by the kanamycin cassette. with whole cells of various heat inactivated (1h at 56 ºC) meningococcal strains. The Induction of IL-6 in MM6 cells presence of LPS was assessed using three different monoclonal antibodies produced in house, named 29 A1.7, 31.G9.19 and 4A8-B2. Monoclonal antibody 4A8-B2 recognizes Mono-mac-6 (MM6) cells were seeded at 1.105 cells in 200µl IMDM (Gibco BRL) medium immunotypes L3,7 and 9 of N. meningitidis.8 Monoclonal antibody 29 A1.7 is reactive for supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 300 µg/ml l-glutamine immunotypes L2-L9, and 31.G9.19 recognizes L1,3,5,7-12 (unpublished data). Taken (Gibco BRL), and 10% heat-inactivated fetal calf serum (FCS) (Gibco BRL) per well in 96-well together, these three monoclonals react against all immunotypes and against all LPS plates. Cells were stimulated with heat-inactivated (56 °C for 1h) bacteria overnight at 37°C containing meningococcal strains. Binding of monoclonal antibodies was detected using an in a humidified atmosphere containing 5% CO2. Human IL-6 cytokine was quantified in the anti-mouse IgG antibody conjugated to horseradish peroxidase (Southern Biotechnology culture supernatants using the PeliPairTM reagent set (Sanquin). Associates, Inc.). 3,3’,5,5’-Tetramethylbenzidine (TMB 10 µg/ml) was used as a substrate and Role of the funding source the reaction was stopped by the addition of 2 M H2SO4. This study has been funded by grants from the Netherlands Organization for Health Research Bacterial whole-genome sequencing, annotation and LPS biosynthesis gene analysis and Development (ZonMw; NWO-Vidi grant 2010 [016.116.358], to D. van de Beek), the The whole genome of isolate 992008 was sequenced using Roche 454-Titanium sequencing. European Research Council (ERC Starting Grant [Proposal/Contract number 281156] to D. 7 We annotated the genome using the Annotation Engine at the Institute for Genome Sciences van de Beek), the Academic Medical Center (AMC Fellowship 2008 to D. van de Beek, AMC of the University of Maryland School of Medicine. The N. meningitidis strain MC58 was used PhD scholarship 2008 to J. Piet). A. van der Ende participated in the Sixth Framework to compare all LPS biosynthesis associated genes. The information reported here from the Programme of the European Commission, Proposal/Contract no.: 512061 (Network of Whole Genome Shotgun project has been deposited with DDBJ/EMBL/GenBank under Excellence “European Virtual Institute for Functional Genomics of Bacterial Pathogens”.) The BioProject Id: PRJNA178630. funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Growth experiment in Escherichia. coli and meningococcal lpxH knockout

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RESULTS detectable binding, whereas all other strains included for comparison bound at least one of the LPS specific monoclonal antibodies. Therefore in both these LPS analyses, isolate 992008 Case report was indistinguishable from the LPS-deficient strain PLAK33 (Figure 1).3 The 992008 strain was isolated from a 19-year-old man with no apparent medical history, Figure 1. LPS detection in meningococcal strains. particularly no recurrent infections, who presented to the emergency room with sudden headache. He was agitated and confused, opened his eyes and bent his arms on painful stimuli (score on Glasgow Coma Scale, 11). Neurologic examination was otherwise normal: there was no fever, no neck stiffness and fundoscopy was normal. Cranial CT scan performed to detect subarachnoidal hemorrhage showed no abnormalities. The patient was admitted to the hospital and the next day a lumbar puncture was performed to rule out subarachnoidal hemorrhage. Cerebrospinal fluid (CSF) was cloudy and examination revealed a leukocyte count of 4620 per ml (84% neutrophils), a protein level of 3.81 g/l, and a glucose level of 1.0 mmol/l. Subsequently, neck stiffness and a low grade fever developed (temperature 38.8°C). Blood tests revealed a serum leukocytosis of 27.5 x 10^9/l. Bacterial (A) Silver-stained Tricine-SDS-PAGE LPS gel of proteinase K-treated whole-cell of meningococcal isolates. meningitis was diagnosed and empirical therapy was started with penicillin (24 million IU/24 H44/76 HB-1: capsule-deficient H44/76; 992008: LPS-deficient patient strain isolated from blood or cerebral hours). CSF Gram staining showed Gram-negative cocci and cultures from CSF and blood spinal fluid; pLAK33: LPS-deficient lpxA mutant. (B) LPS detection in several meningococcal strains using monoclonal antibodies (29.A1.7, 31.G9.19, 4A8-B2) that bind different parts of LPS. PLAK33 represents the LPS grew N. meningitidis. During hospital stay the patient gradually improved. Neurological deficient lpxA mutant and 992008 the LPS deficient patient isolate. A set of different patient isolates and lab strains is included for comparison. The results are expressed as OD 450nm values. examination at discharge (17 days after admission) showed no abnormalities.

LPS-deficient meningococcal patient isolate Whole-genome sequencing and analysis of LPS biosynthesis genes N. meningitidis strain 992008 was serogrouped as serogroup B and Sequence Type (ST) 146, The 992008 genome was sequenced using Roche 454-Titanium sequencing, resulting in 139 belonging to the ST-41/44 complex/Lineage 3 clonal complex. Isolates from CSF and blood contigs. The genome consisted of 2.18 Megabases and annotation resulted in 2578 predicted were available. In vitro growth characteristics were similar when compared with N. open reading frames. All lpx genes known to be required for lipid A biosynthesis were meningitidis H44/76, and Gram staining showed Gram-negative cocci. It was shown to be a manually screened for mutations that could be responsible for the LPS-deficient phenotype. low inducer of IL-6 in MM6 cells during a screen of whole cell suspensions from a large Except for lpxH, none contained polymorphisms that would be able to affect the function of number of meningococcal clinical isolates, but in contrast to all other low IL-6 inducers, it the encoded proteins. The putative amino acid sequence of LpxH in 992008 contained showed no inactivating mutation in its lpxL1 gene.5 When proteinase K-treated cell lysates glycine to aspartic acid substitution at position 95, which was not observed in any of the were subjected to Tricine-SDS-PAGE followed by silver staining, no LPS was detectable in the publicly accessible LpxH sequences of pathogenic (meningococci and gonococci) and non- previously constructed LPS-deficient H44/76 lpxA mutant strain PLAK33 and in the patient’s pathogenic Neisseria species, where glycine-95 is always conserved. In addition, BLAST isolate 992008 cultured from either CSF or blood (Figure 1a). In a whole cell ELISA, LPS screening against LpxH from other bacterial species showed a universal presence of glycine specific monoclonal antibodies together capable of binding all 12 LPS immunotypes did not at this position, making it likely that it is essential for normal functioning of the LpxH show any detectable binding to PLAK33 (Figure 1b). Isolate 992008 also showed no

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LPS-deficient meningococcal patient isolate Whole-genome sequencing and analysis of LPS biosynthesis genes 7 N. meningitidis strain 992008 was serogrouped as serogroup B and Sequence Type (ST) 146, The 992008 genome was sequenced using Roche 454-Titanium sequencing, resulting in 139 belonging to the ST-41/44 complex/Lineage 3 clonal complex. Isolates from CSF and blood contigs. The genome consisted of 2.18 Megabases and annotation resulted in 2578 predicted were available. In vitro growth characteristics were similar when compared with N. open reading frames. All lpx genes known to be required for lipid A biosynthesis were meningitidis H44/76, and Gram staining showed Gram-negative cocci. It was shown to be a manually screened for mutations that could be responsible for the LPS-deficient phenotype. low inducer of IL-6 in MM6 cells during a screen of whole cell suspensions from a large Except for lpxH, none contained polymorphisms that would be able to affect the function of number of meningococcal clinical isolates, but in contrast to all other low IL-6 inducers, it the encoded proteins. The putative amino acid sequence of LpxH in 992008 contained showed no inactivating mutation in its lpxL1 gene.5 When proteinase K-treated cell lysates glycine to aspartic acid substitution at position 95, which was not observed in any of the were subjected to Tricine-SDS-PAGE followed by silver staining, no LPS was detectable in the publicly accessible LpxH sequences of pathogenic (meningococci and gonococci) and non- previously constructed LPS-deficient H44/76 lpxA mutant strain PLAK33 and in the patient’s pathogenic Neisseria species, where glycine-95 is always conserved. In addition, BLAST isolate 992008 cultured from either CSF or blood (Figure 1a). In a whole cell ELISA, LPS screening against LpxH from other bacterial species showed a universal presence of glycine specific monoclonal antibodies together capable of binding all 12 LPS immunotypes did not at this position, making it likely that it is essential for normal functioning of the LpxH show any detectable binding to PLAK33 (Figure 1b). Isolate 992008 also showed no

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enzyme. The mutation in both blood and CSF isolates of strain 992008 was confirmed by Induction of pro-inflammatory cytokine IL-6 by LPS-deficient N. meningitidis strains conventional sequencing. This mutation was not found among 1392 isolates in the Bacterial LPS is the main inducer of an inflammatory response by most gram-negative bacteria and Isolate Genome Sequence Database (BIGSdb at http://pubmlst.org/neisseria/). especially N. meningitidis. To investigate whether the LPS-deficient patient strain 992008 Expression of lpxH in E. coli and construction of an lpxH knockout N. meningitidis and the constructed lpxH mutant have reduced inflammatory responses they were compared to wild-type meningococcal strains and the previously constructed lpxA and lpxL1 To further explore the implications on LPS biosynthesis of the observed amino acid change at mutant strains in their ability to induce the pro-inflammatory cytokine IL-6. MM6 cells were position 95, we compared growth characteristics of E. coli transformed with a plasmid stimulated with titrations of bacterial suspensions mentioned above and IL-6 was quantified containing either meningococcal lpxHH44/76 or lpxH992008. Expression of the lpxH992008 variant by ELISA. Earlier studies using the lpxA mutant strain devoid of LPS and an lpxL1 mutant with in E. coli resulted in growth impairment compared to the lpxHH44/76 variant (Figure 2a). penta-acylated LPS showed highly reduced induction of IL-6.5 Strain 992008 and the lpxH Furthermore, engineering a meningococcal lpxH knockout strain by insertional inactivation knockout mutant were found to be much less potent in inducing IL-6 in MM6 cells compared with a kanamycin cassette resulted in a similar LPS-deficient phenotype in strain H44/76 as to the H44/76 wild-type strain and its capsule-deficient HB-1derivative. This reduced level of found for the lpxA knockout (Figure 2b). As a positive control H44/76 wild-type and HB-1 IL-6 induction was similar to what was found for lpxA and lpxL1 mutants (Figure 3). (H44/76 capsule-deficient mutant) both show presence of LPS. The HB-1 strain shows a lower LPS band because deletion of the B-polysaccharide has also removed the galE gene. Deletion of galE results in the loss of three terminal sugar moieties from L3 wild-type LPS. Figure 3. Comparison of IL-6 induction in MM6 cells of LPS-deficient patient strain. Knockout of lpxH in H44/76 resulted in a reduced growth rate in vitro compared to H44/76 wild-type, as was also previously reported for the lpxA mutant strain plak33.3

Figure 2. E. coli growth and LPS detection in meningococcal strains.

MM6 cells were stimulated overnight with indicated strains and IL-6 production was quantified by ELISA. H44/76: wild-type strain; HB-1; capsule-deficient H44/76; pLAK33: LPS-deficient lpxA mutant; 992008: LPS- (A) Measurement of the growth of E. coli, expressing either H44/76 (wild-type) lpxH or 992008 lpxH, is depicted deficient patient strain isolated from blood or cerebral spinal fluid; LpxL1 is a constructed LpxL1 mutant; by showing the OD at 600 nm. (B) Silver-stained Tricine-SDS-PAGE LPS gel of proteinase K-treated whole-cells of lpxHKO; LPS deficient lpxH knockout derivative of H44/76. Error bars indicate SEM of duplicate. meningococcal strains. HB-1: capsule-deficient H44/76; 992008: LPS-deficient patient strain; pLAK33: LPS- deficient lpxA mutant; lpxHKO; LPS deficient lpxH knockout derivative of H44/76; H44/76: wild-type strain.

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Figure 2. E. coli growth and LPS detection in meningococcal strains. 7

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DISCUSSION antibiotic targets lipid A. The LPS deficiency was due to point mutations, deletions or insertion sequences in lipid A biosynthesis associated genes (lpxA, lpxC and lpxD).14 To our knowledge, we describe the first naturally occurring LPS-deficient meningococcal isolate causing clinical disease. Previously, we identified this isolate as one with a reduced We were able to generate an lpxH knockout mutation in N. meningitidis strain H44/76 by cytokine response in vitro, but in contrast to all other such meningococcal isolates, no allelic exchange, which resulted in an LPS-deficient phenotype similar to the 992008 patient mutation was seen in lpxL1.5 We now show that the defective cytokine response is due to isolate. In addition, when the lpxH992008 variant was expressed in E. coli, growth was absence of LPS that is caused by a missense mutation in lpxH. impaired, compared with expression of wild-type meningococcal lpxH. Possibly, dysfunctional LpxH992008 competes with E. coli LpxH, disturbing LPS synthesis and thereby The strain was isolated from a patient with meningococcal meningitis presenting with slowing its growth. Indeed, LpxH function is essential in E. coli, as lpxH-deficient strains were isolated thunderclap headache, a very uncommon clinical presentation of meningococcal not viable.15 Taken together, these results indicate that the lack of LPS in 992008 is most meningitis.9 In a consecutive study of 137 patients presenting with thunderclap headache, 4 likely caused by the G95D mutation in lpxH. patients had aseptic meningitis and no patients had bacterial meningitis.10 Patients with meningococcal meningitis commonly present with headache, nausea, neck stiffness, rash It is unknown how the meningococcus compensates for the lack of LPS and it is unclear why and fever.11 In our patient the diagnosis of bacterial meningitis was initially not suspected LPS-deficient meningococci are able to survive. In A. baumannii, an LPS-deficient mutant and a lumbar puncture was performed to rule out subarachnoidal hemorrhage. Fever and (with mutation in lpxA) showed increased in vitro expression of many genes involved in cell neck stiffness developed, but only 12 hours after clinical admission. As LPS is considered to envelope biogenesis. Genes involved in fimbrial construction and a type VI secretion system be the main vehicle of immune system activation on Gram-negative bacterial invasion4, the showed reduced expression, thereby modulating the composition and structure of the absence of LPS on meningococcal isolate 992008 most likely caused the inflammatory bacterial surface.16 If strain 992008 would show a similar altered gene expression profile, reaction to develop less effectively. An alternative explanation could be pre-existing needs to be investigated. In any case, the strongly reduced TLR4 activation caused by the immunocompromise of our patient, but there was no history of (opportunistic) infection.12 absence of LPS may be the main in vivo selection advantage conferred by this mutation.

The LPS-deficient phenotype was due to an lpxH mutation. This gene encodes the enzyme UDP-2,3-diacylglucosamine hydrolase, which is required for the fourth step in the Raetz ACKNOWLEDGMENTS pathway for lipid A biosynthesis.6 LpxH is located in the inner membrane and cleaves UDP- 13 2,3-diacylglucosamine to lipid X, a lipid A precursor . Lipid A is essential for the viability of This publication made use of the Neisseria Multilocus Sequence Typing website almost all Gram-negative bacteria; however, a viable LPS-deficient phenotype could be (http://pubmlst.org/neisseria/) developed by Keith Jolley and sited at the University of 3 constructed in vitro in N. meningitidis by mutating lpxA. Oxford. The development of this site has been funded by the Wellcome Trust and European Union. To our knowledge, only a single other instance of an LPS-deficient phenotype in a Gram- negative bacterial patient isolate has been reported previously. Acinetobacter baumannii strains were the first Gram-negative bacteria reported to spontaneously lose the ability to produce lipid A. A clinical isolate of this species was recovered from a patient with an insertion in lpxD, leading to an LPS-deficient phenotype.14 By selecting for colistin resistance, LPS-deficient A. baumannii strains could also be selected in vitro, presumably because this

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The LPS-deficient phenotype was due to an lpxH mutation. This gene encodes the enzyme 7 UDP-2,3-diacylglucosamine hydrolase, which is required for the fourth step in the Raetz ACKNOWLEDGMENTS pathway for lipid A biosynthesis.6 LpxH is located in the inner membrane and cleaves UDP- 13 2,3-diacylglucosamine to lipid X, a lipid A precursor . Lipid A is essential for the viability of This publication made use of the Neisseria Multilocus Sequence Typing website almost all Gram-negative bacteria; however, a viable LPS-deficient phenotype could be (http://pubmlst.org/neisseria/) developed by Keith Jolley and sited at the University of 3 constructed in vitro in N. meningitidis by mutating lpxA. Oxford. The development of this site has been funded by the Wellcome Trust and European Union. To our knowledge, only a single other instance of an LPS-deficient phenotype in a Gram- negative bacterial patient isolate has been reported previously. Acinetobacter baumannii strains were the first Gram-negative bacteria reported to spontaneously lose the ability to produce lipid A. A clinical isolate of this species was recovered from a patient with an insertion in lpxD, leading to an LPS-deficient phenotype.14 By selecting for colistin resistance, LPS-deficient A. baumannii strains could also be selected in vitro, presumably because this

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REFERENCES 14. Moffatt JH, Harper M, Harrison P, et al. Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production. 1. McIntyre PB, O'Brien KL, Greenwood B, van de Beek D. Effect of vaccines on Antimicrob Agents Chemother 2010;54:4971-4977. bacterial meningitis worldwide. Lancet 2012;380:1703-1711. 15. Metzger LE, Raetz CR. An alternative route for UDP-diacylglucosamine hydrolysis in 2. Beutler B, Rietschel ET. Innate immune sensing and its roots: the story of endotoxin. bacterial lipid A biosynthesis. Biochemistry 2010;49:6715-6726. Nat Rev Immunol 2003;3:169-176. 16. Henry R, Vithanage N, Harrison P, et al. Colistin-resistant, lipopolysaccharide- 3. Steeghs L, den Hartog R, den Boer A, Zomer B, Roholl P, van der Ley P. Meningitis deficient Acinetobacter baumannii responds to lipopolysaccharide loss through bacterium is viable without endotoxin. Nature 1998;392:449-450. increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids, and poly-beta-1,6-N-acetylglucosamine. Antimicrob Agents 4. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, Chemother 2012;56:59-69. and Neisseria meningitidis. Lancet 2007;369:2196-2210.

5. Fransen F, Heckenberg SG, Hamstra HJ, et al. Naturally occurring lipid A mutants in Neisseria meningitidis from patients with invasive meningococcal disease are associated with reduced coagulopathy. PLoS Pathog 2009;5:e1000396.

6. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem 2002;71:635-700.

7. Maiden MC, Bygraves JA, Feil E, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 1998;95:3140-3145.

8. Verheul AF, Kuipers AJ, Braat AK, et al. Development, characterization, and biological properties of meningococcal immunotype L3,7,(8),9-specific monoclonal antibodies. Clin Diagn Lab Immunol 1994;1:729-736.

9. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial meningitis in adults. N Engl J Med 2006;354:44-53.

10. Landtblom AM, Fridriksson S, Boivie J, Hillman J, Johansson G, Johansson I. Sudden onset headache: a prospective study of features, incidence and causes. Cephalalgia 2002;22:354-360.

11. Heckenberg SG, de Gans J, Brouwer MC, et al. Clinical features, outcome, and meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study. Medicine (Baltimore) 2008;87:185-192.

12. Brouwer MC, de Gans J, Heckenberg SG, Zwinderman AH, van der Poll T, van de Beek D. Host genetic susceptibility to pneumococcal and meningococcal disease: a systematic review and meta-analysis. Lancet Infect Dis 2009;9:31-44.

13. Bulawa CE, Raetz CR. The biosynthesis of gram-negative endotoxin. Identification and function of UDP-2,3-diacylglucosamine in Escherichia coli. J Biol Chem 1984;259:4846-4851.

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6. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem 2002;71:635-700.

9. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial meningitis in adults. N Engl J Med 2006;354:44-53. 7 10. Landtblom AM, Fridriksson S, Boivie J, Hillman J, Johansson G, Johansson I. Sudden onset headache: a prospective study of features, incidence and causes. Cephalalgia 2002;22:354-360.

13. Bulawa CE, Raetz CR. The biosynthesis of gram-negative endotoxin. Identification and function of UDP-2,3-diacylglucosamine in Escherichia coli. J Biol Chem 1984;259:4846-4851.

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CHAPTER 8:

Streptococcus pneumoniae arginine synthesis genes promote growth and virulence in pneumococcal meningitis

Jurgen R. Piet* Madelijn Geldhoff* Barbera D.C. van Schaik Matthijs C. Brouwer Mercedes Valls Seron Marja E. Jakobs Kim Schipper Yvonne Pannekoek Aeilko H. Zwinderman Tom van der Poll Antoine H.C. van Kampen Frank Baas Arie van der Ende‡ Diederik van de Beek‡

*‡Authors contributed equally

J Infect Dis 2014;209:1781-1791

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CHAPTER 8: CHAPTER 8: Streptococcus pneumoniae arginine synthesis genes promote growth and virulence in pneumococcal meningitis

Jurgen R. Piet* Streptococcus pneumoniae Madelijn Geldhoff*arginine synthesis genes Barbera D.C. van Schaik Matthijs C. Brouwerpromote growth and virulence Mercedes Valls Seron Marja E. Jakobsin pneumococcal meningitis Kim Schipper Yvonne Pannekoek Aeilko H. Zwinderman Tom van der Poll Antoine H.C. van Kampen Frank Baas Arie van der Ende‡ Jurgen R. Piet* Diederik van de Beek‡ Madelijn Geldhoff* Barbera D.C. van Schaik Matthijs C. Brouwer Mercedes Valls Seron *‡Authors contributed equally Marja E. Jakobs Kim Schipper Yvonne Pannekoek Aeilko H. Zwinderman Tom van der Poll Antoine H.C. van Kampen Frank Baas Arie van der Ende‡ Diederik van de Beek‡ *‡Authors contributed equally

J Infect Dis 2014;209:1781-1791 J Infect Dis 2014;209:1781-1791

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ABSTRACT INTRODUCTION

Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, Streptococcus pneumoniae can cause a range of invasive infections, such as pneumonia and sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial meningitis, with devastating consequences worldwide.1-3 Pneumococcal meningitis whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be continues to exact a heavy toll despite the implementation of childhood vaccination associated with outcome in patients with pneumococcal meningitis. Pneumococci harboring programs.4,5 The fatality rate in patients with pneumococcal meningitis is substantial, at these genes show increased growth in human blood and cerebrospinal fluid (CSF). Mouse 30%, and long-term sequelae, including hearing loss, focal neurological deficit, and cognitive models of meningitis and pneumonia showed that pneumococcal strains without arginine impairment, develop in about half of survivors.3,6 biosynthesis genes were attenuated in growth or cleared, from lung, blood and CSF. Thus, S. Invasive disease caused by S. pneumoniae is seen at the beginning and end of life or in pneumoniae arginine synthesis genes promote growth and virulence in invasive patients with underlying conditions.1,4 Several studies of extreme phenotypes have identified pneumococcal disease. genetic defects in the complement system and intracellular signaling proteins to be associated with increased susceptibility.7 A common variant in complement component 5 has been associated with unfavorable outcome in pneumococcal meningitis.8 Bacterial genetic factors may also determine susceptibility and disease severity. Few studies have investigated the role of bacterial factors in invasive pneumococcal disease, including meningitis. Pneumococcal capsular polysaccharides have been associated with disease severity and are used for phenotypical characterization.9 The pneumococcal capsular phenotype has been linked to genotype but the mechanisms of virulence have not been elucidated.10 S. pneumoniae contains an inducible system for the uptake of DNA from the environment that readily allows for horizontal gene transfer.11 The pneumococcal pan- genome has been estimated at 5,000 genes, but less than 50% of these genes are shared by all pneumococcal strains (forming the pneumococcal core genome).12

Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. We found that pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid. Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth and/or cleared from lung, blood and cerebrospinal fluid.

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ABSTRACT INTRODUCTION

Using a clinical phenotype based approach with bacterial whole-genome sequencing we 8 identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. We found that pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid. Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth and/or cleared from lung, blood and cerebrospinal fluid.

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MATERIALS AND METHODS boundary regions in 8 pneumococcal genomes, 21 (70%) encoded genes that occurred multiple times in the pneumococcal genome. The median contig-number produced by Clinical cohort genome assembly was 27 (range, 13-66). The GC content of sequenced genomes was highly The Dutch Meningitis Cohort Studies from 1998-2002 and 2006-2009 are prospective similar, ranging from 39.5% to 39.8%, and genome sizes ranged from 1.98-2.15Mb. nationwide cohort studies of adults with community-acquired bacterial meningitis in the The genomes were annotated using Annotation Engine at the Institute for Genome Sciences Netherlands.3,8 Patients older than 16 years with positive cerebrospinal fluid (CSF) cultures of the University of Maryland School of Medicine (http://ae.igs.umaryland.edu/cgi). To were included after identification by the Netherlands Reference Laboratory for Bacterial compare gene content of bacterial isolates from patients with favorable outcome vs. isolates Meningitis (NRLBM). The NRLBM receives isolates from CSF of 90% of all patients with from patients who died, we first build an extended genome that represents a non-redundant bacterial meningitis in the Netherlands.3 Written informed consent was obtained from all collection of all genes occurring at least once in our 20 genomes. We started collection with patients or their legally authorized representatives. Details on the data collection of the genes of genome AMCSP01 and aligned all genes of all genomes (one by one) to this cohorts were presented elsewhere.3,8 The studies were approved by the Medical Ethics collection using Blast Like Alignment Tool (BLAT).14 Genes were added to the extended Committee, Academic Medical Center (AMC). genome when they did not match to the extended genome that was built so far. The Bacterial isolates matching criteria were set to 90% identity and 90% query coverage.

Upon receipt by the NRLBM, isolates were serotyped and stored at -80°C. Patients’ isolates Genome comparison and validation had less than 5 passages before storage. Pneumococci were recultured on Trypticase Soya Genes of the extended genome were aligned to genomes of all bacteria. We scored per agar (TSA) (Difco Laboratories, Inc., Detroit, MI), with 5% defibrinated sheep blood. bacteria which genes were present with similar BLAT matching criteria. As positive control, Whole-genome sequencing and annotation we used an artificial gene that was correctly identified in all genomes (row 2 Supplementary Table 2). Candidate gene clusters were evaluated in duplicate by PCR with primers specific Bacterial genome Roche/454 shotgun sequencing was performed according to the for one gene in the cluster (Supplementary Table 3). The argGH locus was determined by manufacturer's instructions. The mean genomic coverage was 25-fold (range 14-63; Table 1). PCR using 2 primer sets specific for either the argG or argH gene (Supplementary Table 3). Based on de novo assemblies, a median of 88 contigs was produced. Sequence reads were sorted by sample and assembled using the Roche GS De Novo Assembler (Newbler) v 2.3 and Pneumococcal knockout strain the Roche GS Reference Mapper v 2.0.0.12. In the de novo assemblies, we allowed 0 and 2 The D39 argGH knockout mutant was constructed as described by Trzciński.15 A Janus-type errors in the MID extraction settings. Each of the 20 genomes was also assembled in a cassette with kanamycin resistance and streptomycin sensitivity alleles with flanking DNA reference mapping assembly using 11 publically available pneumococcal reference genomes sequences corresponding to sequences that flank the argGH locus, was constructed using a (Supplementary Table 1). In the Reference Mapper assemblies, 0 errors were allowed in the 1,368-bp fragment containing aphIII-rpsL amplified from chromosomal DNA of TIGR4ΔpspA MID calling parameters. Alignments of the contigs were created with Codoncode Aligner (Supplementary Table 3).16,17 Flanking sequences, one with a BamHI 3’ terminus and one software (version 3.5.1, CodonCode Corporation, Dedham, MA). All contigs were put in a with an ApaI 5’ terminus, were amplified using chromosomal DNA of D39 as template and more correct order by rearranging them with the ‘move contigs’ option in Mauve software primer pairs KSPNArg2061617F8 / KSPNArg2061617R6 and KSPNArg2061617F7 / using S. pneumoniae TIGR4 strain as a reference.13 In an exploratory analysis of 30 contig KSPNArg2061617R9_1. The aphIII-rpsL and flanking sequence PCR products were digested

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Upon receipt by the NRLBM, isolates were serotyped and stored at -80°C. Patients’ isolates Genome comparison and validation had less than 5 passages before storage. Pneumococci were recultured on Trypticase Soya Genes of the extended genome were aligned to genomes of all bacteria. We scored per agar (TSA) (Difco Laboratories, Inc., Detroit, MI), with 5% defibrinated sheep blood. bacteria which genes were present with similar BLAT matching criteria. As positive control, Whole-genome sequencing and annotation we used an artificial gene that was correctly identified in all genomes (row 2 Supplementary Table 2). Candidate gene clusters were evaluated in duplicate by PCR with primers specific Bacterial genome Roche/454 shotgun sequencing was performed according to the for one gene in the cluster (Supplementary Table 3). The argGH locus was determined by manufacturer's instructions. The mean genomic coverage was 25-fold (range 14-63; Table 1). PCR using 2 primer sets specific for either the argG or argH gene (Supplementary Table 3). Based on de novo assemblies, a median of 88 contigs was produced. Sequence reads were sorted by sample and assembled using the Roche GS De Novo Assembler (Newbler) v 2.3 and Pneumococcal knockout strain 8 the Roche GS Reference Mapper v 2.0.0.12. In the de novo assemblies, we allowed 0 and 2 The D39 argGH knockout mutant was constructed as described by Trzciński.15 A Janus-type errors in the MID extraction settings. Each of the 20 genomes was also assembled in a cassette with kanamycin resistance and streptomycin sensitivity alleles with flanking DNA reference mapping assembly using 11 publically available pneumococcal reference genomes sequences corresponding to sequences that flank the argGH locus, was constructed using a (Supplementary Table 1). In the Reference Mapper assemblies, 0 errors were allowed in the 1,368-bp fragment containing aphIII-rpsL amplified from chromosomal DNA of TIGR4ΔpspA MID calling parameters. Alignments of the contigs were created with Codoncode Aligner (Supplementary Table 3).16,17 Flanking sequences, one with a BamHI 3’ terminus and one software (version 3.5.1, CodonCode Corporation, Dedham, MA). All contigs were put in a with an ApaI 5’ terminus, were amplified using chromosomal DNA of D39 as template and more correct order by rearranging them with the ‘move contigs’ option in Mauve software primer pairs KSPNArg2061617F8 / KSPNArg2061617R6 and KSPNArg2061617F7 / using S. pneumoniae TIGR4 strain as a reference.13 In an exploratory analysis of 30 contig KSPNArg2061617R9_1. The aphIII-rpsL and flanking sequence PCR products were digested

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with BamHI and ApaI, and purified with a GeneJET Gel Extraction Kit (Fermentas). The PCR System for First-Strand cDNA Synthesis kit (Life technologies). Primers are shown in digested PCR products were ligated using T4 DNA ligase (Roche) and the ligation mixture was Supplementary Table 3. used as template in a PCR with primers KSPNArg2061617F1 and KSPN2061617ArgR9_1. The Mouse models of meningitis and pneumonia 2,525 bp PCR product was used to transform D39 with selection for resistance to kanamycin R (Km ) to create D39 ∆argGH. The structure of the insertion was confirmed by PCR and Mouse experiments were conducted with specific pathogen-free male C57BL/6 mice sequencing. (Charles River, Wilmington, MN, USA) aged 8-12 weeks. Experiments were approved by the Institutional Animal Care and Use Committee, AMC. Bacterial growth in blood

incubated for 6 hours at 37°C in a humidified atmosphere of 5% CO2 in air. For the induction of pneumonia D39 and D39 ∆argGH were grown to mid log phase at 37°C in Todd-Hewitt broth supplemented with yeast (Difco, Detroit, MI) and harvested by Exposure to oxidative stress centrifugation. Eight mice per group were intranasally inoculated with D39 or D39 ∆argGH 8 Bacteria were grown to an OD450 of 0.15 in THY. Cultures were washed with THY and 100- by putting a 50μL of 5x10 CFU per mL droplet of bacterial suspension on the nose under 20 fold diluted in THY or in THY with 0.04% H2O2. Cultures were incubated without agitation at isoflurane anesthesia, as described previously.

Bacteria were grown to an OD450 of 0.2 in THY. Bacteria were collected by centrifugation and variables, and dichotomous variables were compared with use of the χ2 test. We used resuspended in PBS and thereafter an enzyme cocktail of Achromopeptidase (1000 U/ml), logistic regression analysis to calculate odds ratios (OR) and 95% confidence intervals (CI). All Mutanolysine (100 U/ml) and Lysozym (10 mg/ml) was added and incubated for 30 minutes statistical tests were two-tailed; p<0.05 was regarded significant. at 37 ºC. Bacteria were collected by centrifugation and the pellet was resuspended in Tris- EDTA buffer with 1 mg/ml lysozyme and RNA was isolated using the Rneasy midikit (QIAgen), according to the manufacturer’s protocol. RNA was treated with the Turbo DNAse (AMBION) according to the manufacturer’s protocol and cDNA was made with the ThermoScript™ RT-

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For the induction of meningitis, D39 and D39 ∆argGH were grown to mid log phase at 37°C Five pneumococcal colonies from an overnight culture on TSA were collected and in Todd-Hewitt broth supplemented with yeast (THY, Difco, Detroit, MI) and harvested by resuspended in 5 ml Todd–Hewitt broth supplemented with 0.5% yeast extract (THY) and centrifugation. Pneumococci were diluted with sterile saline to an OD620 of 0.3 grown to optical density at 450 nm (OD450) between 0.25 and 0.35. Bacteria were washed corresponding to 1x108 CFU mL-1. Inoculum dose was based on a previous study.18 Twelve with nutrient mixture F-10 Ham (Sigma-Aldrich) and resuspended in 5ml F-10 Ham mice per group were inoculated into the cisterna magna under isoflurane anesthesia, with 1 supplemented with citrulline or arginine where appropriate to OD at 620 nm of 0.01. µl bacterial suspension containing D39 or D39∆argGH, as described earlier.19 Hundred µl bacterial suspension was mixed with 100µl citrate blood or 100µl CSF and incubated for 6 hours at 37°C in a humidified atmosphere of 5% CO2 in air. For the induction of pneumonia D39 and D39 ∆argGH were grown to mid log phase at 37°C in Todd-Hewitt broth supplemented with yeast (Difco, Detroit, MI) and harvested by Exposure to oxidative stress centrifugation. Eight mice per group were intranasally inoculated with D39 or D39 ∆argGH 8 Bacteria were grown to an OD450 of 0.15 in THY. Cultures were washed with THY and 100- by putting a 50μL of 5x10 CFU per mL droplet of bacterial suspension on the nose under 20 fold diluted in THY or in THY with 0.04% H2O2. Cultures were incubated without agitation at isoflurane anesthesia, as described previously.

37°C in a humidified atmosphere of 5% CO2 in air. At t=0, 30, 60, 90 and 120 minutes, Statistics samples were taken and serially diluted for CFU count. For analysis of survival data the Log-rank test was used. The Mann-Whitney U test was used RT PCR to identify differences between patient and animal groups with respect to continuous 8 Bacteria were grown to an OD450 of 0.2 in THY. Bacteria were collected by centrifugation and variables, and dichotomous variables were compared with use of the χ2 test. We used resuspended in PBS and thereafter an enzyme cocktail of Achromopeptidase (1000 U/ml), logistic regression analysis to calculate odds ratios (OR) and 95% confidence intervals (CI). All Mutanolysine (100 U/ml) and Lysozym (10 mg/ml) was added and incubated for 30 minutes statistical tests were two-tailed; p<0.05 was regarded significant. at 37 ºC. Bacteria were collected by centrifugation and the pellet was resuspended in Tris- EDTA buffer with 1 mg/ml lysozyme and RNA was isolated using the Rneasy midikit (QIAgen), according to the manufacturer’s protocol. RNA was treated with the Turbo DNAse (AMBION) according to the manufacturer’s protocol and cDNA was made with the ThermoScript™ RT-

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RESULTS Table 1. Whole-genome sequencing results for 20 pneumococcal strains from patients with community- acquired bacterial meningitis. Bacterial Sequenced Sequence Coding Genome GC No. Contigs Pneumococcal gene cluster associated with outcome in patients with meningitis genome nucleotides (Mb) deptha sequence (%) size (Mb) content (%) genes AMCSP01 35 17 86.4 28 2.07 39.5 2,196 From 2006 through 2009, we included 642 episodes of community-acquired bacterial AMCSP02 72 35 86.2 23 2.08 39.6 2,175 AMCSP03 45 22 86.3 24 2.05 39.6 2,133 8 meningitis in a prospective nationwide cohort. Overall, case fatality rate was 19% and 39% AMCSP04 62 30 86.1 31 2.00 39.7 2,074 patients had an unfavorable outcome, defined as a score of 1-4 on the Glasgow outcome AMCSP05 33 16 86.0 66 2.15 39.5 2,280 AMCSP06 60 29 85.4 33 2.05 39.6 2,308 scale. 427 of these patients had pneumococcal meningitis. In an extreme phenotype AMCSP07 68 33 86.4 25 2.11 39.6 2,215 approach, we sequenced genomes of 10 randomly selected pneumococcal isolates from AMCSP08 32 16 85.2 37 2.07 39.7 2,437 AMCSP09 28 14 86.1 26 2.06 39.6 2,234 immunocompetent patients who died and 10 isolates from matched patients with favorable AMCSP10 48 24 86.2 13 1.98 39.7 2,054 outcome (patients were matched for serotype, age, absence of predisposing factors; Table AMCSP11 39 19 86.0 37 2.04 39.6 2,151 AMCSP12 65 32 85.1 31 2.00 39.8 2,160 1). Genomes were annotated resulting in a median of 2,190 open reading frames per AMCSP13 84 41 82.7 41 2.16 39.6 2,882 genome (range, 2,054 to 2,882; Table 2 and Supplementary Table 1). The number of genes AMCSP14 57 28 86.1 53 2.14 39.5 2,275 AMCSP15 30 15 86.0 22 2.03 39.7 2,161 present in all sequenced genomes, the pneumococcal core genome, consisted of 1,981 of AMCSP16 54 26 86.3 27 2.06 39.6 2,168 12 3,453 genes (57%; 1.55Mb, Supplementary Table 2), is consistent with previous work. AMCSP17 130 63 86.2 22 2.07 39.7 2,183 Genes (n=2,652) that were equally represented in both groups, and genes found only once AMCSP18 48 24 86.2 26 2.14 39.5 2,312 AMCSP19 57 28 85.9 27 2.05 39.5 2,160 per group were not further analyzed (Figure 1A; 642 genes were included; 273 AMCSP20 37 18 86.4 44 2.14 39.5 2,382 a pneumococcal genes associated with death, and 369 associated with favorable outcome). Sequence depth: the n-fold genome coverage is shown as defined by the sequenced nucleotides divided by the average genome size (2.1 Mb). Next, four genes were excluded from the analysis since only a partial sequence was available. Finally, 86 of 155 (55%) genes with a group frequency difference ≥2 were located in 14 gene clusters: 5 clusters including 28 genes were associated with favorable outcome, and 9 clusters including 58 genes were associated with unfavorable outcome (Figure 1B).

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Table 2. 20 Pneumococcal strains from patients with pneumococcal meningitis. Figure 1. Genome comparisons. Clinical characteristics Pneumococcal typing CSF WBC GOS Sequence Clonal Strain Sex Age (year) GCS score Serotype (cells/mm3) score type complex AMCSP01 M 76 13 46 1 22F 433 433 AMCSP02 M 68 10 1,877 1 20 1030 235 AMCSP03 M 64 11 5,200 1 10A 97 460 AMCSP04 F 67 9 267 1 7F 191 191 AMCSP05 M 60 10 40 1 9V 2726 156 AMCSP06 M 57 15 7,230 1 23F 507 439 AMCSP07 M 58 13 12,400 1 23F 440 156 AMCSP08 F 30 14 2,000 1 18C 1233 1381 AMCSP09 M 61 11 592 1 4 247 246 AMCSP10 M 82 9 2,810 1 8 53 53 AMCSP11 F 60 8 2,220 5 23F 507 439 AMCSP12 M 62 8 3,307 5 7F 191 191 AMCSP13 M 37 15 2,747 5 18C 113 113 AMCSP14 M 60 15 3,960 5 9V 162 156 AMCSP15 M 88 15 1,700 5 8 53 53 AMCSP16 F 69 13 5,000 5 10A 1551 460 AMCSP17 M 54 9 35,000 5 23F 36 439 AMCSP18 F 52 7 12 5 4 205 205 AMCSP19 M 50 15 3,000 5 20 235 235 AMCSP20 M 67 12 48 5 22F 433 433 CSF WBC denotes cerebrospinal fluid white blood cells, GCS Glasgow Coma Scale, GOS Glasgow Outcome Scale. Sequence Type was based on Multilocus Sequence Typing (MLST).10

(A) Genomic comparison in a clinical-based approach with whole bacterial genome sequencing of 20 S. pneumoniae clinical isolates. Differences in gene content between extreme phenotype groups. 328 genes were relatively more present in strains from patients who died (striped) and 473 genes were more present in strains from survivors with favorable outcome (white); 2,652 genes were equally represented in both groups. (B) Genome comparisons of gene cluster frequency among 20 pneumococcal strains. Gray boxes indicate presence of all genes in the gene cluster to be present in that genome. Per outcome group, the frequency of each gene cluster is shown, as are the group-differences. aThese genes were present twice in the genome and count as 1 in for the group total.

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(A) Genomic comparison in a clinical-based approach with whole bacterial genome sequencing of 20 S. pneumoniae clinical isolates. Differences in gene content between extreme phenotype groups. 328 genes were relatively more present in strains from patients who died (striped) and 473 genes were more present in strains from survivors with favorable outcome (white); 2,652 genes were equally represented in both groups. (B) Genome comparisons of gene cluster frequency among 20 pneumococcal strains. Gray boxes indicate 8 presence of all genes in the gene cluster to be present in that genome. Per outcome group, the frequency of each gene cluster is shown, as are the group-differences. aThese genes were present twice in the genome and count as 1 in for the group total.

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Subsequently, we evaluated associations between 14 gene clusters and clinical outcome in Genes involved in conversion of citrulline to arginine contribute to pneumococcal growth 748 other new pneumococcal meningitis patients included in two prospective nationwide Arginine biosynthesis is linked to the biosynthesis of polyamines (Figure 2A), which are cohort studies.3,8 In the first cohort (321 pneumococcal strains, Table 3) three gene clusters implicated to play a role in the resistance to oxidative stress.25 To gain further insight in the were significantly associated with favorable outcome. In the second cohort (427 role of the argGH locus we used S. pneumoniae D39 that contains the argGH locus (Genbank pneumococcal strains), used as a subsequent validation cohort, Cluster L remained accession number NC_008533.1) and constructed an argGH knockout.26 In standard rich significantly associated with favorable outcome (p=0.027). laboratory medium THY D39 and D39ΔargGH showed equal growth rates (Figure 2B).

Cluster L (4.4 kb) includes 6 genes that all encode membrane proteins.21 BLASTX analysis of (encoding the housekeeping protein glucose kinase). Transcription from the gene upstream these genes showed similarities to pneumococcal proteins. The GC content of cluster L was of argGH (SPD_109 encoding anamino acid ABC transporter periplasmic amino acid-binding lower than the average of the whole genome (30% vs. 40%) and the dinucleotide protein) was detectable in both D39 wt and D39ΔargGH, albeit lower in the argGH knockout composition (δ*, genome signature) was highly dissimilar as compared to similar sized strain (results not shown). fragments of the whole genome (98% of the genome fragments yielded a lower δ* than cluster L),22 suggesting acquisition by horizontal gene transfer.23 Cluster L replaced or truncated an arg locus comprising genes encoding argininosuccinate synthase (ArgG) and argininosuccinate lyase (ArgH). ArgG converts citrulline together with aspartate to argininosuccinate that is converted to arginine and fumarate by ArgH.24

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Table 3. Validation of associations between identified gene clusters and outcome in two nationwide cohort However, D39 wt was more resistant to 0.04% H2O2 compared to D39ΔargGH (Figure 2C). In studies (n = 748). Cohort 1 Cohort 2 blood CFUs of D39 increased 300-fold after 6 hours incubation, but CFUs of D39ΔargGH Cluster Favorable Unfavorable P Favorable Unfavorable P remained at the level of the start inoculum in non-supplemented blood (Figure 2D). In blood outcome outcome outcome outcome n = 160 (%) n = 161 (%) n = 261 (%) n = 166 (%) supplemented with 0.25% citrulline, CFUs of D39 were 2-fold higher than without citrulline A 100 (63%) 104 (65%) 0.70 B 44 (28%) 37 (23%) 0.35 (p=0.004), but CFUs of D39ΔargGH remained similar as compared to growth in non- C 23 (14%) 28 (17%) 0.46 supplemented blood (Figure 2D). Both D39 wt and D39ΔargGH showed less growth in blood D 47 (29%) 56 (35%) 0.30 E 75 (47%) 71 (44%) 0.62 supplemented with 0.25% arginine although this did not reach significance (Figure 2D). F 56 (35%) 46 (29%) 0.22 Relative bacterial outgrowth was similar in CSF when compared to blood, although bacterial G 32 (20%) 18 (11%) 0.029 35 (13%) 24 (15%) 0.76 H 36 (23%) 35 (22%) 0.87 growth of D39 and D39ΔargGH did not increase in CSF when 0.25% citrulline was added I 19 (12%) 29 (18%) 0.12 (Figure 2E). To investigate the role of NO we repeated growth experiments with NO J 54 (34%) 45 (28%) 0.26 argGH K 81 (51%) 64 (40%) 0.050 83 (32%) 50 (30%) 0.72 synthetase blockers, but this did not influence growth. knockouts obtained in two L 145 (91%) 132 (82%) 0.024 227 (87%) 131 (79%) 0.027 independent transformation had the same phenotype. We did not succeed to complement M 73 (46%) 86 (53%) 0.16 D39ΔargGH. In this strain, we assessed transcription of the genes upstream and downstream N 25 (16%) 28 (17%) 0.67 of the argGH locus by RT-PCR, to exclude potential polar effect of its replacement by the Janus cassette. In both D39 wt and D39ΔargGH, transcription from gene SP_0112 (encoding Genomic locus is variable region for genes encoding pneumococcal arginine synthesis a hypothetical protein) downstream of argGH was high and comparable to that of gki Cluster L (4.4 kb) includes 6 genes that all encode membrane proteins.21 BLASTX analysis of (encoding the housekeeping protein glucose kinase). Transcription from the gene upstream 8 these genes showed similarities to pneumococcal proteins. The GC content of cluster L was of argGH (SPD_109 encoding anamino acid ABC transporter periplasmic amino acid-binding lower than the average of the whole genome (30% vs. 40%) and the dinucleotide protein) was detectable in both D39 wt and D39ΔargGH, albeit lower in the argGH knockout composition (δ*, genome signature) was highly dissimilar as compared to similar sized strain (results not shown). fragments of the whole genome (98% of the genome fragments yielded a lower δ* than cluster L),22 suggesting acquisition by horizontal gene transfer.23 Cluster L replaced or truncated an arg locus comprising genes encoding argininosuccinate synthase (ArgG) and argininosuccinate lyase (ArgH). ArgG converts citrulline together with aspartate to argininosuccinate that is converted to arginine and fumarate by ArgH.24

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Figure 2. Spermidine biosynthesis pathway and bacterial growth. Pneumococcal arginine synthesis contribute to bacterial growth and virulence in mouse models of meningitis and pneumonia

To gain further insight into the role that genes involved in conversion of citrulline to arginine plays in the infectivity and virulence of S. pneumoniae, C57BL/6 mice were injected intracisternally with 1x105 CFU D39 or D39ΔargGH. Mice with pneumococcal meningitis caused by D39 died within 4 days, whereas all mice infected with D39ΔargGH survived (log- rank test, p<0.001; Figure 3A). Subsequently, groups of mice were infected and sacrificed at 6h and 40 h after infection. Organs from both groups of mice were surveyed for viable S. pneumoniae CFUs. At 6h, CFUs in CSF, blood, and lung were higher in the group infected with D39 compared to the group infected with D39ΔargGH (p<0.01; Figure 3B). At 40h post- infection, CFUs were higher CSF, brain, blood, and lung in the group infected with wild-type bacteria (p<0.001; Figure 3B). To investigate the role of arginine synthesis in pneumococcal pneumonia, we inoculated mice intranasally with 2.5x107 CFU D39 or D39ΔargGH. At 6 h post-infection, CFUs were higher in the lung in the group infected with wild-type bacteria (p<0.001; Figure 3C). At later time points (24 and 48h) both D39 and D39ΔargGH bacteria were cleared from all organs in all infected mice.

(A) Spermidine biosynthesis pathway in S. pneumoniae D39. (B-E) Bacterial growth of S. pneumoniae D39 wild- type or arginine biosynthesis genes knockout strains. S. pneumoniae D39 (striped) or D39ΔargGH (black) were grown in standard rich laboratory medium THY (B and C), or in blood for 6 hours (D) or in CSF (E). Growth is expressed as the ratio of the number of CFUs of the start inoculum and that after 6 hours growth (CFU fold difference).

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Figure 2. Spermidine biosynthesis pathway and bacterial growth. Pneumococcal arginine synthesis contribute to bacterial growth and virulence in mouse models of meningitis and pneumonia

(A) Spermidine biosynthesis pathway in S. pneumoniae D39. (B-E) Bacterial growth of S. pneumoniae D39 wild- type or arginine biosynthesis genes knockout strains. S. pneumoniae D39 (striped) or D39ΔargGH (black) were 8 grown in standard rich laboratory medium THY (B and C), or in blood for 6 hours (D) or in CSF (E). Growth is expressed as the ratio of the number of CFUs of the start inoculum and that after 6 hours growth (CFU fold difference).

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Figure 3. Pneumococcal meningitis mouse model, patient survival and pneumococcal serotype distribution. Pneumococcal arginine synthesis genes are associated with disease outcome and infection with pneumococcal serotype 7F in meningitis patients

In an explorative analysis we evaluated the association between the argGH locus and clinical findings in 748 patients with pneumococcal meningitis (Table 4). On admission, patients infected with argGH positive pneumococci more often had pneumonia (p=0.037), and presented with more severe disease, as reflected by a higher proportion of patients in coma (p=0.002) and focal neurologic deficits (p=0.042), which resulted in a higher mortality rate (p=0.003; Figure 3D). In a multivariate regression analysis including pneumonia on admission, admission score on the Glasgow coma scale, and focal neurological deficits, the association of the argGH locus with unfavorable outcome was preserved (odds ratio [OR] = 1.62; 95%CI 1.08-2.42; p=0.019).

Table 4. Associations of pneumococci argGH locus with clinical characteristics, disease severity and outcome. Patient Characteristic S. pneumoniae S. pneumoniae argGH locus argGH positive truncated or replaced by p (n = 132) cluster L (n = 616) Age – yr 61 (48-72) 60 (48-70) 0.16 Predisposing conditions (A) Effect of infection with D39 or D39ΔargGH on survival in mice with pneumococcal meningitis. (B) CFUs at 6 and 40 h in CSF, brain, blood, and lung in mice with pneumococcal meningitis caused by D39 or D39ΔargGH. (C) Otitis/sinusitis 54/132 (41%) 267/615 (43%) 0.60 CFUs at 6 and 24 h in lung and blood in mice with pneumococcal pneumonia caused by D39 or D39ΔargGH. (D) Pneumonia 33/132 (25%) 106/616 (17%) 0.037 Kaplan–Meier survival curve of patients with community-acquired meningitis infected with arg positive (black) Immuncompromised stateb 33/132 (25%) 138/615 (22%) 0.53 and arg negative (striped) S. pneumoniae. (E) Pneumococcal serotype distribution of arg positive (black) and Signs and symptoms on admission arg negative (gray) pneumococci. Glasgow coma scale score < 8 37/132 (28%) 101/612 (17%) 0.002 Focal neurologic deficitsc 58/131 (44%) 213/611 (35%) 0.042 Laboratory examination CSF white blood cell count/mm3 d 2,899 (416-7,862) 2,500 (575-6,934) 0.97 CSF protein – g/Le 5.2 (3.4-7.6) 4.0 (2.5-6.1) < 0.001 Systemic complicationsf 73/132 (55%) 239/606 (39%) 0.001 Neurologic complicationsg 96/125 (77%) 396/585 (68%) 0.045 Death 46/132 (35%) 138/616 (22%) 0.003 Unfavorable outcomeh 76/132 (58%) 251/616 (41%) < 0.001 aData are number/number assessed (%) or median (25th–75th percentile). bDefined by the use of immunosuppressive drugs, a history of splenectomy, the presence of diabetes mellitus, alcoholism, and patients infected with HIV. cDefined as aphasia, monoparesis, hemiparesis and cranial nerve palsies. dCSF leukocyte count was determined in 122 patients infected with argH positive and 572 with argH negative pneumococci. eCSF protein levels were determined in 119 patients infected with argH positive and 576 patients with argH negative pneumococci. fDefined as septic shock, respiratory failure, multiple-organ dysfunction, and cardiac ischemia. gDefined as brain herniation, cerebrovascular complications, intractable seizures and withdrawal of care because of poor neurological prognosis. hDefined as a score of one to four on the Glasgow outcome scale.

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Table 4. Associations of pneumococci argGH locus with clinical characteristics, disease severity and outcome. Patient Characteristic S. pneumoniae S. pneumoniae argGH locus argGH positive truncated or replaced by p (n = 132) cluster L (n = 616) Age – yr 61 (48-72) 60 (48-70) 0.16 Predisposing conditions (A) Effect of infection with D39 or D39ΔargGH on survival in mice with pneumococcal meningitis. (B) CFUs at 6 and 40 h in CSF, brain, blood, and lung in mice with pneumococcal meningitis caused by D39 or D39ΔargGH. (C) Otitis/sinusitis 54/132 (41%) 267/615 (43%) 0.60 CFUs at 6 and 24 h in lung and blood in mice with pneumococcal pneumonia caused by D39 or D39ΔargGH. (D) Pneumonia 33/132 (25%) 106/616 (17%) 0.037 Kaplan–Meier survival curve of patients with community-acquired meningitis infected with arg positive (black) Immuncompromised stateb 33/132 (25%) 138/615 (22%) 0.53 and arg negative (striped) S. pneumoniae. (E) Pneumococcal serotype distribution of arg positive (black) and Signs and symptoms on admission arg negative (gray) pneumococci. Glasgow coma scale score < 8 37/132 (28%) 101/612 (17%) 0.002 Focal neurologic deficitsc 58/131 (44%) 213/611 (35%) 0.042 Laboratory examination CSF white blood cell count/mm3 d 2,899 (416-7,862) 2,500 (575-6,934) 0.97 CSF protein – g/Le 5.2 (3.4-7.6) 4.0 (2.5-6.1) < 0.001 Systemic complicationsf 73/132 (55%) 239/606 (39%) 0.001 Neurologic complicationsg 96/125 (77%) 396/585 (68%) 0.045 8 Death 46/132 (35%) 138/616 (22%) 0.003 Unfavorable outcomeh 76/132 (58%) 251/616 (41%) < 0.001 aData are number/number assessed (%) or median (25th–75th percentile). bDefined by the use of immunosuppressive drugs, a history of splenectomy, the presence of diabetes mellitus, alcoholism, and patients infected with HIV. cDefined as aphasia, monoparesis, hemiparesis and cranial nerve palsies. dCSF leukocyte count was determined in 122 patients infected with argH positive and 572 with argH negative pneumococci. eCSF protein levels were determined in 119 patients infected with argH positive and 576 patients with argH negative pneumococci. fDefined as septic shock, respiratory failure, multiple-organ dysfunction, and cardiac ischemia. gDefined as brain herniation, cerebrovascular complications, intractable seizures and withdrawal of care because of poor neurological prognosis. hDefined as a score of one to four on the Glasgow outcome scale.

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Presence of argGH was associated with serotype 7F (60 of 71 [85%] serotype 7F strains were arginine is a substrate of nitric oxide (NO) synthase resulting in the production of citrulline argGH positive vs. 72 of 677 [11%] non-serotype 7F strains; p<0.0001; Figure 3E). In a and NO. Citrulline, through the reactions catalyzed by argininosuccinate synthetase and multivariate regression analysis including age, admission score on the Glasgow coma scale, argininosuccinate lyase may cycle back to arginine, constituting an arginine–citrulline cycle.30 and infection by pneumococcal serotype 7F, the predictive effect of argGH on outcome was The synthetase ArgG is considered to catalyze the rate-limiting step in NO production and preserved (OR 1.65; 95%CI 1.02-2.67; p=0.040). withdrawal of citrulline by pneumococci may reduce NO production, which could be advantageous for bacteria. However, repeated growth experiments with NO synthetase

blockers did not influence growth, implying that pneumococcal arginine biosynthesis genes DISCUSSION contribute to virulence by influencing bacterial growth only. Arginine is a precursor in the biosynthesis of polyamines, which have been implicated in oxidative stress responses and We identified pneumococcal arginine biosynthesis genes associated with outcome in protection against free radicals.31 The natural polyamine spermine functions directly as a patients with pneumococcal meningitis. Clustered genes were always present on similar free radical scavenger 25 and linked to the fitness, survival and pathogenesis of the genomic loci, implicating regions of plasticity acquired by horizontal gene transfer.27 Such pneumococcus in host microenvironments.32 We showed the ΔargGH strain being less plasticity regions have been associated with pneumococcal virulence.21 The region we resistant to oxidative stress. Polar effects by the knockout mutation on transcription from identified to be associated with disease outcome in our clinical cohort has been identified genes flanking argGH was excluded by RT-PCR. Transcription from SP_0109 (encoding ApbA) previously as a plasticity region with a complex history of transposition and integration was lower in the argGH mutant, but a deletion of apbA had no effect on growth in low events.24 In this region, genes encoding argininosuccinate synthase (argG) and arginine concentration.29 In addition, in D39 the products of abpA, artP and abpB can argininosuccinate lyase (argH) were replaced or truncated by a cluster of genes acquired by substitute for each other.29 Antibiotics selectively blocking bacterial argininosuccinate horizontal gene transfer. Subsequently, we showed that pneumococci harboring these synthetase and argininosuccinate lyase could inhibit pneumococcal growth, reducing disease arginine biosynthesis genes show increased resistance to oxidative stress and increased severity. growth in human blood and cerebrospinal fluid. Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in Presence of arginine biosynthesis genes was associated with pneumococcal serotype 7F, but growth and/or cleared from lung, blood and cerebrospinal fluid. The results of our the association between these genes and clinical outcome even remained robust in a pneumonia model imply that our findings are not limited to pneumococcal meningitis and multivariate analysis including pneumococcal serotype. Individual serotypes and major may well apply to other invasive pneumococcal infections, such as sepsis and pneumonia. clones of S. pneumoniae appear to differ markedly in their potential to cause invasive disease (80–120-fold variation).9 Serotype 7F has previously been associated with higher Pneumococci lack a complete arginine biosynthesis pathway. argGH positive pneumococci invasive disease potential, but with lower case-fatality rates.33,34 In the Netherlands, 7F has convert host’s citrulline to arginine, which promotes bacterial growth and contributes to increased during the last decade, being the most frequent serotype in meningitis patients virulence. Arginine biosynthesis genes have been associated with bacterial growth and from 2007 onwards.35 Because of lack of heterogeneity between the OR for invasive disease virulence in other pathogens.24,28 An auxotrophic mutant of Mycobacterium tuberculosis of different clones of the same serotype, and analysis of isolates of the same genotype, but defective in L-arginine biosynthesis displayed reduced virulence suggesting that L-arginine different serotype, it has been suggested that capsular serotype may be more important availability was restricted in vivo.24 An Actinobacillus pleuropneumoniae transposon mutant than genotype in the ability of pneumococci to cause invasive disease. 9 The effect of argGH of argG was attenuated in growth in an experimental pig infection model.28 Previous in vitro data suggested that arginine uptake is essential for growth of S. pneumoniae.29 In humans,

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blockers did not influence growth, implying that pneumococcal arginine biosynthesis genes DISCUSSION contribute to virulence by influencing bacterial growth only. Arginine is a precursor in the biosynthesis of polyamines, which have been implicated in oxidative stress responses and We identified pneumococcal arginine biosynthesis genes associated with outcome in protection against free radicals.31 The natural polyamine spermine functions directly as a patients with pneumococcal meningitis. Clustered genes were always present on similar free radical scavenger 25 and linked to the fitness, survival and pathogenesis of the genomic loci, implicating regions of plasticity acquired by horizontal gene transfer.27 Such pneumococcus in host microenvironments.32 We showed the ΔargGH strain being less plasticity regions have been associated with pneumococcal virulence.21 The region we resistant to oxidative stress. Polar effects by the knockout mutation on transcription from identified to be associated with disease outcome in our clinical cohort has been identified genes flanking argGH was excluded by RT-PCR. Transcription from SP_0109 (encoding ApbA) previously as a plasticity region with a complex history of transposition and integration was lower in the argGH mutant, but a deletion of apbA had no effect on growth in low events.24 In this region, genes encoding argininosuccinate synthase (argG) and arginine concentration.29 In addition, in D39 the products of abpA, artP and abpB can argininosuccinate lyase (argH) were replaced or truncated by a cluster of genes acquired by substitute for each other.29 Antibiotics selectively blocking bacterial argininosuccinate horizontal gene transfer. Subsequently, we showed that pneumococci harboring these synthetase and argininosuccinate lyase could inhibit pneumococcal growth, reducing disease arginine biosynthesis genes show increased resistance to oxidative stress and increased severity. growth in human blood and cerebrospinal fluid. Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in Presence of arginine biosynthesis genes was associated with pneumococcal serotype 7F, but growth and/or cleared from lung, blood and cerebrospinal fluid. The results of our the association between these genes and clinical outcome even remained robust in a pneumonia model imply that our findings are not limited to pneumococcal meningitis and multivariate analysis including pneumococcal serotype. Individual serotypes and major may well apply to other invasive pneumococcal infections, such as sepsis and pneumonia. clones of S. pneumoniae appear to differ markedly in their potential to cause invasive 8 disease (80–120-fold variation).9 Serotype 7F has previously been associated with higher Pneumococci lack a complete arginine biosynthesis pathway. argGH positive pneumococci invasive disease potential, but with lower case-fatality rates.33,34 In the Netherlands, 7F has convert host’s citrulline to arginine, which promotes bacterial growth and contributes to increased during the last decade, being the most frequent serotype in meningitis patients virulence. Arginine biosynthesis genes have been associated with bacterial growth and from 2007 onwards.35 Because of lack of heterogeneity between the OR for invasive disease virulence in other pathogens.24,28 An auxotrophic mutant of Mycobacterium tuberculosis of different clones of the same serotype, and analysis of isolates of the same genotype, but defective in L-arginine biosynthesis displayed reduced virulence suggesting that L-arginine different serotype, it has been suggested that capsular serotype may be more important availability was restricted in vivo.24 An Actinobacillus pleuropneumoniae transposon mutant than genotype in the ability of pneumococci to cause invasive disease. 9 The effect of argGH of argG was attenuated in growth in an experimental pig infection model.28 Previous in vitro data suggested that arginine uptake is essential for growth of S. pneumoniae.29 In humans,

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on disease outcome in patients with pneumococcal meningitis remained robust in a REFERENCES

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6. Hoogman M, van de Beek D, Weisfelt M, de Gans J, Schmand B. Cognitive outcome ACKNOWLEDGMENTS in adults after bacterial meningitis. J Neurol Neurosurg Psychiatry 2007;78:1092- 1096. We are indebted to all Dutch physicians who participated in the study. We thank R.A.G. Huis 7. Brouwer MC, de Gans J, Heckenberg SG, Zwinderman AH, van der Poll T, van de in 't Veld for his technical assistance. We also would like to thank M. Giglio and S. Daugherty Beek D. Host genetic susceptibility to pneumococcal and meningococcal disease: a at the Institute of Genome Sciences for their support in annotating the genomes and systematic review and meta-analysis. Lancet Infect Dis 2009;9:31-44.

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on disease outcome in patients with pneumococcal meningitis remained robust in a REFERENCES multivariate analysis including serotype. 1. van der Poll T, Opal SM. Pathogenesis, treatment, and prevention of pneumococcal pneumonia. Lancet 2009;374:1543-1556. Our findings are in line with the “avirulence hypothesis” assuming parasites (bacteria, 36 2. McIntyre PB, O'Brien KL, Greenwood B, van de Beek D. Effect of vaccines on viruses, protozoa and helminths) will evolve to become avirulent. Pneumococci with the bacterial meningitis worldwide. Lancet 2012;380:1703-1711. argGH locus truncated or replaced by cluster L are less fit and virulent. Virulence is 3. van de Beek D, de Gans J, Spanjaard L, Weisfelt M, Reitsma JB, Vermeulen M. 36 considered as a maladaptation of new associations between parasites and hosts. During Clinical features and prognostic factors in adults with bacterial meningitis. N Engl J co-evolution with their host, microorganism may acquire functions that make them more fit Med 2004;351:1849-1859. in the interaction with their host.36 Genetic exchange with species sharing the same 4. Brouwer MC, Tunkel AR, van de Beek D. Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin Microbiol Rev 2010;23:467-492. ecological niche is the main mechanism of evolution of S. pneumoniae.37 5. van de Beek D. Progress and challenges in bacterial meningitis. Lancet 2012;380:1623-1624.

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16. Sung CK, Li H, Claverys JP, Morrison DA. An rpsL cassette, janus, for gene 29. Kloosterman TG, Kuipers OP. Regulation of arginine acquisition and virulence gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ expression in the human pathogen Streptococcus pneumoniae by transcription Microbiol 2001;67:5190-5196. regulators ArgR1 and AhrC. J Biol Chem 2011;286:44594-44605.

17. Trzcinski K, Thompson CM, Srivastava A, Basset A, Malley R, Lipsitch M. Protection 30. Haines RJ, Pendleton LC, Eichler DC. Argininosuccinate synthase: at the center of against nasopharyngeal colonization by Streptococcus pneumoniae is mediated by arginine metabolism. Int J Biochem Mol Biol 2011;2:8-23. antigen-specific CD4+ T cells. Infect Immun 2008;76:2678-2684. 31. Ha HC, Sirisoma NS, Kuppusamy P, Zweier JL, Woster PM, Casero RA, Jr. The natural 18. Hartel T, Klein M, Koedel U, Rohde M, Petruschka L, Hammerschmidt S. Impact of polyamine spermine functions directly as a free radical scavenger. Proc Natl Acad Sci glutamine transporters on pneumococcal fitness under infection-related conditions. U S A 1998;95:11140-11145. Infect Immun 2011;79:44-58. 32. Shah P, Nanduri B, Swiatlo E, Ma Y, Pendarvis K. Polyamine biosynthesis and 19. Mook-Kanamori B, Geldhoff M, Troost D, van der Poll T, van de Beek D. transport mechanisms are crucial for fitness and pathogenesis of Streptococcus Characterization of a pneumococcal meningitis mouse model. BMC Infect Dis pneumoniae. Microbiology 2011;157:504-515. 2012;12:71. 33. Jansen AG, Rodenburg GD, van der Ende A, et al. Invasive pneumococcal disease 20. Dessing MC, Florquin S, Paton JC, van der Poll T. Toll-like receptor 2 contributes to among adults: associations among serotypes, disease characteristics, and outcome. antibacterial defence against pneumolysin-deficient pneumococci. Cell Microbiol Clin Infect Dis 2009;49:e23-e29. 2008;10:237-246. 34. Weinberger DM, Harboe ZB, Sanders EA, et al. Association of serotype with risk of 21. Krogh A, Larsson B, von HG, Sonnhammer EL. Predicting transmembrane protein death due to pneumococcal pneumonia: a meta-analysis. Clin Infect Dis 2010;51:692- topology with a hidden Markov model: application to complete genomes. J Mol Biol 699. 2001;305:567-580. 35. Rodenburg GD, de Greeff SC, Jansen AG, et al. Effects of pneumococcal conjugate 22. Lawrence JG, Ochman H. Amelioration of bacterial genomes: rates of change and vaccine 2 years after its introduction, the Netherlands. Emerg Infect Dis 2010;16:816- exchange. J Mol Evol 1997;44:383-397. 823.

23. Bruckner R, Nuhn M, Reichmann P, Weber B, Hakenbeck R. Mosaic genes and 36. Adiba S, Nizak C, van BM, Denamur E, Depaulis F. From grazing resistance to mosaic chromosomes-genomic variation in Streptococcus pneumoniae. Int J Med pathogenesis: the coincidental evolution of virulence factors. PLoS One Microbiol 2004;294:157-168. 2010;5:e11882.

24. Gordhan BG, Smith DA, Alderton H, McAdam RA, Bancroft GJ, Mizrahi V. 37. Croucher NJ, Walker D, Romero P, et al. Role of conjugative elements in the Construction and phenotypic characterization of an auxotrophic mutant of evolution of the multidrug-resistant pandemic clone Streptococcus Mycobacterium tuberculosis defective in L-arginine biosynthesis. Infect Immun pneumoniaeSpain23F ST81. J Bacteriol 2009;191:1480-1489. 2002;70:3080-3084.

25. Chattopadhyay MK, Tabor CW, Tabor H. Polyamines protect Escherichia coli cells from the toxic effect of oxygen. Proc Natl Acad Sci U S A 2003;100:2261-2265.

26. Pozzi G, Masala L, Iannelli F, et al. Competence for genetic transformation in encapsulated strains of Streptococcus pneumoniae: two allelic variants of the peptide pheromone. J Bacteriol 1996;178:6087-6090.

27. Burrus V, Pavlovic G, Decaris B, Guedon G. Conjugative transposons: the tip of the iceberg. Mol Microbiol 2002;46:601-610.

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24. Gordhan BG, Smith DA, Alderton H, McAdam RA, Bancroft GJ, Mizrahi V. 37. Croucher NJ, Walker D, Romero P, et al. Role of conjugative elements in the Construction and phenotypic characterization of an auxotrophic mutant of evolution of the multidrug-resistant pandemic clone Streptococcus 8 Mycobacterium tuberculosis defective in L-arginine biosynthesis. Infect Immun pneumoniaeSpain23F ST81. J Bacteriol 2009;191:1480-1489. 2002;70:3080-3084.

25. Chattopadhyay MK, Tabor CW, Tabor H. Polyamines protect Escherichia coli cells from the toxic effect of oxygen. Proc Natl Acad Sci U S A 2003;100:2261-2265.

27. Burrus V, Pavlovic G, Decaris B, Guedon G. Conjugative transposons: the tip of the iceberg. Mol Microbiol 2002;46:601-610.

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SUPPLEMENTARY MATERIAL Supplementary Table 3. Primers used for cluster determination and constructing bacterial knockouts. Primer name Orientation DNA sequence Target Cluster Cluster determination Supplementary Table 1. Genome assembly strategies. JP_pneu_01 Forward A Bacterial genome de novo 0 errors de novo 2 errors Reference mapping Cabog CCCTGACCCTGCTAGCTCTT JP_pneu_02 Reverse A AMCSP01 93 80 136 (NC_008533) 80 CTGACGGCCTTACTGATTGG JP_pneu_07 Forward GATTGGGCTACCCTTTGACA F AMCSP02 56 61 105 (NC_011072) 65 JP_pneu_08 Reverse TGGATAATCAAGTCCATGCTC F AMCSP03 75 72 227 (NC_003098) 280 JP_pneu_11 Forward ATGTCGCAAAAAGAGGGTCA G AMCSP04 98 87 128 (NC_003098) 112 JP_pneu_12 Reverse TTTCAACATCTCTGACAAAGTCAA G AMCSP05 155 148 198 (NC_008533) 240 JP_pneu_15 Forward CTATTGCTGGTTTGGGTGGT H AMCSP06 83 90 118 (NC_008533) 75 JP_pneu_16 Reverse CAATTGGCATTCCAAAAGAAA H AMCSP07 66 70 108 (NC_008533) 92 JP_pneu_17 Forward ATGATTTGGAAATCTTAGCGAAA I AMCSP08 149 110 119 (NC_003098) 81 JP_pneu_18 Reverse TCATTGATCGTTCCCCATTT I AMCSP09 144 139 72 (NC_003028) 83 JP_pneu_21 Forward TGGCAGCCACTTGTTTATCC B AMCSP10 47 46 98 (NC_011072) 72 JP_pneu_22 Reverse TGAACGCGAGTAAATGCTTG B AMCSP11 88 86 117 (NC_003098) 70 JP_pneu_23 Forward TCCATGGCATTACACCTGAA C AMCSP12 87 90 131 (NC_011072) 90 JP_pneu_24 Reverse GCTTGCTCGTGCATCTGATA C AMCSP13 141 149 85 (NC_003098) 100 JP_pneu_25 Forward AATCTTGGAGTGGCAACAGG D AMCSP14 136 133 150 (NC_008533) 95 JP_pneu_26 Reverse TCCTTTTGGAGGACGACTGT D JP_pneu_27 Forward J AMCSP15 64 81 101 (NC_011072) 83 ACAAAGTGGCCGACTACACC JP_pneu_28 Reverse J AMCSP16 60 57 105 (NC_008533) 81 CAGTGAACTGCCCAATAGCA JP_pneu_31 Forward K AMCSP17 41 49 100 (NC_008533) 102 TGCTTCGTTTTTGTTGGTTAAA JP_pneu_32 Reverse K AMCSP18 104 a 33 (NC_003028) 144 CTTGTCACCGGAATCAAACA JP_pneu_33 Forward GAACTCCCTCTCATGCCATC L AMCSP19 59 65 113 (NC_008533) 86 JP_pneu_34 Reverse AGAGGCAAAAGCAACGGTTT L AMCSP20 84 82 135 (NC_008533) 92 JP_pneu_35 Forward CACTACCAGGGAGGGAAACA M The number of contigs is shown per assembly strategy: Newbler de novo assembler with 0 and 2 errors allowed JP_pneu_36 Reverse M in the MID calling, a Newbler Reference Mapper assembly (all reference strain GenBank accession numbers ATCGCAAGTCCCATCACTTC producing the least amounts of contigs in brackets: S. pneumoniae TIGR4 (NC_003028), S. pneumoniae R6 JP_pneu_37 Forward TTTGTTGTGGTCGATTGTGG E (NC_003098), S. pneumoniae D39 (NC_008533), S. pneumoniae Hungary 19A-6 (NC_010380), S. pneumoniae JP_pneu_38 Reverse GTTGCAGAATCTCCCAAGGA E CGSP14 (NC_010582), S. pneumoniae G54 (NC_011072), S. pneumoniae Spain23F (NC_011900) S. pneumoniae JP_pneu_39 Forward TCTGAGGGACAACGTCAGC N JJA (NC_012466), S. pneumoniae P1031 (NC_012467), S. pneumoniae 70585 (NC_012468) and S. pneumoniae JP_pneu_40 Reverse CCGTACCATCACGTCTTCCT N Taiwan 19F-14 (NC_012469), and a Cabog assembly. argH a JP_pneu_43 Forward TGGGTAGAGCGCTTTGGTGCG Because this strain was sequenced with MID 16 (TCACGTACTA) which has close resemblance to MID 18, no de JP_pneu_44 Reverse argH novo assembly with 2 errors allowed in the MID calling procedure was made. GCCCATAAACTCTGCCAGTCTTTCC JP_pneu_45 Forward TCTGCCTTGAGCCGCCCTCT argG Supplementary Table 2. Pneumococcal extended genome. JP_pneu_48 Reverse TTTGCAGTCCCATTGACAAC argG Constructing bacterial knockouts A full list of the genes in the pneumococcal extended genome is accessible online at KSPNArg2061617F8 GGCTGGAATTGAAGCC http://jid.oxfordjournals.org/content/209/11/1781/suppl/DC1. For each gene (rows), the frequency per KSPNArg2061617R6 CGAAACCGCCACAATAGGATCCAAAATTAC genome is scored (columns). KSPNArg2061617F7 ATTAAAGGGCCCATGATGCATATGAGTCG KSPNArg2061617R9_1 CGACTTGGTCAAACC KSPNArg2061617F1 GCTATCGCAACAGAACTAGG KSjanuskanaR1 AAAGATTATATCACATTATCCATT RT PCR KSABCF GAACGGAGCAAGGTGTTTG KSABCR AAATAACGGCATCCACTTGC KSarghypF AGATCGTGGTGGGTCTTGAG KsarghypR GGCAAGACAACCCCAAAATA Gki up GGCATTGGAATGGGATCACC Gki dn TCTCCCGCAGCTGACAC

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SUPPLEMENTARY MATERIAL Supplementary Table 3. Primers used for cluster determination and constructing bacterial knockouts. Primer name Orientation DNA sequence Target Cluster Cluster determination Supplementary Table 1. Genome assembly strategies. JP_pneu_01 Forward A Bacterial genome de novo 0 errors de novo 2 errors Reference mapping Cabog CCCTGACCCTGCTAGCTCTT JP_pneu_02 Reverse A AMCSP01 93 80 136 (NC_008533) 80 CTGACGGCCTTACTGATTGG JP_pneu_07 Forward GATTGGGCTACCCTTTGACA F AMCSP02 56 61 105 (NC_011072) 65 JP_pneu_08 Reverse TGGATAATCAAGTCCATGCTC F AMCSP03 75 72 227 (NC_003098) 280 JP_pneu_11 Forward ATGTCGCAAAAAGAGGGTCA G AMCSP04 98 87 128 (NC_003098) 112 JP_pneu_12 Reverse TTTCAACATCTCTGACAAAGTCAA G AMCSP05 155 148 198 (NC_008533) 240 JP_pneu_15 Forward CTATTGCTGGTTTGGGTGGT H AMCSP06 83 90 118 (NC_008533) 75 JP_pneu_16 Reverse CAATTGGCATTCCAAAAGAAA H AMCSP07 66 70 108 (NC_008533) 92 JP_pneu_17 Forward ATGATTTGGAAATCTTAGCGAAA I AMCSP08 149 110 119 (NC_003098) 81 JP_pneu_18 Reverse TCATTGATCGTTCCCCATTT I AMCSP09 144 139 72 (NC_003028) 83 JP_pneu_21 Forward TGGCAGCCACTTGTTTATCC B AMCSP10 47 46 98 (NC_011072) 72 JP_pneu_22 Reverse TGAACGCGAGTAAATGCTTG B AMCSP11 88 86 117 (NC_003098) 70 JP_pneu_23 Forward TCCATGGCATTACACCTGAA C AMCSP12 87 90 131 (NC_011072) 90 JP_pneu_24 Reverse GCTTGCTCGTGCATCTGATA C AMCSP13 141 149 85 (NC_003098) 100 JP_pneu_25 Forward AATCTTGGAGTGGCAACAGG D AMCSP14 136 133 150 (NC_008533) 95 JP_pneu_26 Reverse TCCTTTTGGAGGACGACTGT D JP_pneu_27 Forward J AMCSP15 64 81 101 (NC_011072) 83 ACAAAGTGGCCGACTACACC JP_pneu_28 Reverse J AMCSP16 60 57 105 (NC_008533) 81 CAGTGAACTGCCCAATAGCA JP_pneu_31 Forward K AMCSP17 41 49 100 (NC_008533) 102 TGCTTCGTTTTTGTTGGTTAAA JP_pneu_32 Reverse K AMCSP18 104 a 33 (NC_003028) 144 CTTGTCACCGGAATCAAACA JP_pneu_33 Forward GAACTCCCTCTCATGCCATC L AMCSP19 59 65 113 (NC_008533) 86 JP_pneu_34 Reverse AGAGGCAAAAGCAACGGTTT L AMCSP20 84 82 135 (NC_008533) 92 JP_pneu_35 Forward CACTACCAGGGAGGGAAACA M The number of contigs is shown per assembly strategy: Newbler de novo assembler with 0 and 2 errors allowed JP_pneu_36 Reverse M in the MID calling, a Newbler Reference Mapper assembly (all reference strain GenBank accession numbers ATCGCAAGTCCCATCACTTC producing the least amounts of contigs in brackets: S. pneumoniae TIGR4 (NC_003028), S. pneumoniae R6 JP_pneu_37 Forward TTTGTTGTGGTCGATTGTGG E (NC_003098), S. pneumoniae D39 (NC_008533), S. pneumoniae Hungary 19A-6 (NC_010380), S. pneumoniae JP_pneu_38 Reverse GTTGCAGAATCTCCCAAGGA E CGSP14 (NC_010582), S. pneumoniae G54 (NC_011072), S. pneumoniae Spain23F (NC_011900) S. pneumoniae JP_pneu_39 Forward TCTGAGGGACAACGTCAGC N JJA (NC_012466), S. pneumoniae P1031 (NC_012467), S. pneumoniae 70585 (NC_012468) and S. pneumoniae JP_pneu_40 Reverse CCGTACCATCACGTCTTCCT N Taiwan 19F-14 (NC_012469), and a Cabog assembly. argH a JP_pneu_43 Forward TGGGTAGAGCGCTTTGGTGCG Because this strain was sequenced with MID 16 (TCACGTACTA) which has close resemblance to MID 18, no de JP_pneu_44 Reverse argH novo assembly with 2 errors allowed in the MID calling procedure was made. GCCCATAAACTCTGCCAGTCTTTCC JP_pneu_45 Forward TCTGCCTTGAGCCGCCCTCT argG Supplementary Table 2. Pneumococcal extended genome. JP_pneu_48 Reverse TTTGCAGTCCCATTGACAAC argG Constructing bacterial knockouts 8 A full list of the genes in the pneumococcal extended genome is accessible online at KSPNArg2061617F8 GGCTGGAATTGAAGCC http://jid.oxfordjournals.org/content/209/11/1781/suppl/DC1. For each gene (rows), the frequency per KSPNArg2061617R6 CGAAACCGCCACAATAGGATCCAAAATTAC genome is scored (columns). KSPNArg2061617F7 ATTAAAGGGCCCATGATGCATATGAGTCG KSPNArg2061617R9_1 CGACTTGGTCAAACC KSPNArg2061617F1 GCTATCGCAACAGAACTAGG KSjanuskanaR1 AAAGATTATATCACATTATCCATT RT PCR KSABCF GAACGGAGCAAGGTGTTTG KSABCR AAATAACGGCATCCACTTGC KSarghypF AGATCGTGGTGGGTCTTGAG KsarghypR GGCAAGACAACCCCAAAATA Gki up GGCATTGGAATGGGATCACC Gki dn TCTCCCGCAGCTGACAC

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Summary and general discussion

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Summary and general discussion

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alleles to 7 housekeeping genes. The combination of alleles makes up a sequence type and fHbpD184 (acidic residue substituting a basic residue at position 184 in the protein) were when they exhibit a high degree of similarity, these sequence types can be grouped in clonal more likely to develop septic shock during admission, resulting in a higher rate of complexes (cc). Hyper-invasive clonal complexes (defined as cc8, cc11, cc32, cc41/44 and unfavorable outcome. Of these patients, 67% were infected with meningococci belonging to 2 cc269) are types of meningococci that more often cause invasive disease than other clonal cc11. Substitution of H184 to D184 in the fHbp-fH model structure suggest a stronger complexes. In our cohort, infection with meningococci of cc11 (all serogroup C) was related interaction between fHbp and fH. In theory, this could lead to better protection of

to unfavorable outcome. The most common serogroup B ccs (cc41/44 [60%], cc41 [24%] and meningococci with fHbpD184 in the bloodstream, which is consistent with more severe cc269 [8%]) showed no association with disease severity. disease in patients infected with these meningococci. This theory would contradict, however, with a previous study suggesting that more severe disease is caused by This data provides evidence that in meningococci causing invasive disease, bacterial gene meningococcal strains with lower expression of fHbp or lower fHbp affinity.6 Results of our content influences disease outcome. Since genes investigated by MLST are non-virulent binding experiments did not show significant differences in binding affinity between fH and housekeeping genes, it is likely that other genetic factors cause the association of clonal the different fHbp184 variants, but this could be due to low sensitivity of our assay or complexes and outcome. Many investigations have aimed to identify these meningococcal another yet unexplained altered function of fHbp which is modified by this substitution. virulence factors in vitro, but the clinical relevance of these factors is often not clear. Ideally, such possible factors would be investigated in vivo in animal models of meningococcal In chapter 4 we investigated meningococcal two-partner secretion (TPS) systems. These meningitis, but meningococcal strict adaptation to the human host has thus far made the protein systems export large TpsA proteins to the surface and extracellular milieu. In development of such models impossible. Therefore, we tried to associate known meningococci, three different TPS systems exist of which systems 2 and 3, each with a

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Introduction meningococcal virulence factors with clinical outcome in the next chapters of part I of this thesis. The objective of this thesis is to provide more insight in the association of bacterial genetics Factor H binding protein (fHbp) is a meningococcal virulence factor which we investigated in with clinical characteristics of patients with bacterial meningitis. This chapter summarizes chapter 3. This factor binds human complement downregulator factor H (fH), thereby the main results of our studies on bacterial genetics and clinical outcome, places the results inhibiting complement activation and hampering complement-mediated lysis.1,2 Over the in a broader perspective and provides heading for future research. last decade, fHbp has become of substantial interest: it is a key component in two Part I: Neisseria meningitidis forthcoming vaccines. High levels of fHbp have been found in hyper-virulent meningococcal strains and invasive isolates have been shown to express higher levels of fHbp than carrier Investigations on meningococcal virulence factors in clinical cohort studies isolates.3,4 In addition, fHbp is essential for meningococcal survival in human blood.5 Results In chapter 2 of this thesis we describe a Dutch nationwide prospective cohort from 1998- of studies of fHbp variance among patients’ isolates can have important consequences for 2002 of 258 adults with community acquired meningococcal meningitis, together with the further vaccine development, as inclusion of the right fHbp protein types could improve bacterial genotype of the causative isolate. Unfavorable outcome occurred in 30 of 258 vaccine efficacy. Furthermore, it is unclear if the variation in fHbp and its encoded protein (12%) patients, including a mortality rate of 7%. Neisseria meningitidis can be classified into influences disease characteristics and outcome. Therefore, we investigated the distribution serogroups based on the immunologic reactivity of their capsular polysaccharides. The of fHbp protein variants among the meningococcal isolates in our 1998-2002 cohort study, proportion serogroup B meningococci was 68% and serogroup C 31%. Meningococci were and correlated fHbp protein types with clinical characteristics. In the fHbp-fH complex, typed by multilocus sequence typing (MLST), which uses sequence data and designates seventeen fHbp amino acids interact with fH.2 Patients infected with meningococci with alleles to 7 housekeeping genes. The combination of alleles makes up a sequence type and fHbpD184 (acidic residue substituting a basic residue at position 184 in the protein) were when they exhibit a high degree of similarity, these sequence types can be grouped in clonal more likely to develop septic shock during admission, resulting in a higher rate of complexes (cc). Hyper-invasive clonal complexes (defined as cc8, cc11, cc32, cc41/44 and unfavorable outcome. Of these patients, 67% were infected with meningococci belonging to 2 cc269) are types of meningococci that more often cause invasive disease than other clonal cc11. Substitution of H184 to D184 in the fHbp-fH model structure suggest a stronger complexes. In our cohort, infection with meningococci of cc11 (all serogroup C) was related interaction between fHbp and fH. In theory, this could lead to better protection of to unfavorable outcome. The most common serogroup B ccs (cc41/44 [60%], cc41 [24%] and meningococci with fHbpD184 in the bloodstream, which is consistent with more severe cc269 [8%]) showed no association with disease severity. disease in patients infected with these meningococci. This theory would contradict, however, with a previous study suggesting that more severe disease is caused by This data provides evidence that in meningococci causing invasive disease, bacterial gene meningococcal strains with lower expression of fHbp or lower fHbp affinity.6 Results of our content influences disease outcome. Since genes investigated by MLST are non-virulent binding experiments did not show significant differences in binding affinity between fH and housekeeping genes, it is likely that other genetic factors cause the association of clonal 9 the different fHbp184 variants, but this could be due to low sensitivity of our assay or complexes and outcome. Many investigations have aimed to identify these meningococcal another yet unexplained altered function of fHbp which is modified by this substitution. virulence factors in vitro, but the clinical relevance of these factors is often not clear. Ideally, such possible factors would be investigated in vivo in animal models of meningococcal In chapter 4 we investigated meningococcal two-partner secretion (TPS) systems. These meningitis, but meningococcal strict adaptation to the human host has thus far made the protein systems export large TpsA proteins to the surface and extracellular milieu. In development of such models impossible. Therefore, we tried to associate known meningococci, three different TPS systems exist of which systems 2 and 3, each with a

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isolates investigated) using PCR and whole-genome sequencing. TPS system 1 was present in 91% of isolates and TPS system 2 and/or 3 was present in 67% of isolates. TPS system Figure 1. Venn diagram of meningococcal genetic factors associated with clinical outcome. distribution was highly related to clonal complexes. Of strains that exclusively harbored TPS1, 82% were serogroup C strains and 68% belonged to the hyper-invasive cc11. Next, we explored the association between infection with strains harboring immunogenic TPS systems and disease severity. Patients infected with meningococcal isolates with either TPS 2 and/or TPS3 presented with less severe disease and were more likely to have favorable outcome, as compared to patients infected with isolates without these systems. A plausible explanation for this finding is that isolates exclusively harboring TPS system 1 might have a survival advantage by evading the immune system, thereby causing more severe disease. To test this hypothesis we evaluated our findings with in vitro experiments using tps knockout meningococcal strains and complemented knockout strains. Using whole-blood stimulation experiments, we found no differences in host cytokine response between TPS systems 2 and 3 knockout strains and a wild-type strain harboring all TPS systems.

background provides an ideal situation to investigate a virulence factor, have failed to Contributing to vaccine research provide an explanation between the observed association with outcome. As is shown in Polysaccharide vaccines and conjugate vaccines (in which the polysaccharide is attached to a figure 1, there is considerable overlap between isolates with the fHbpD184 protein type, isolates lacking an immunogenic TPS system and the cc11 genetic make-up. Possibly, the carrier protein) can offer protection against meningococcal serogroups A, C, W and Y. observed associations with outcome are the result of a genetic linkage effect, suggesting Serogroup C conjugate vaccine has proven to be highly effective and have almost eradicated that there are other, currently unknown virulence factors present in these strains. It is not serogroup C disease in countries where they have been implemented in routine vaccination 11 likely that these factors are only present in cc11, because after a nationwide meningococcal schedules. In contrast, developing vaccines against serogroup B strains remains difficult. serogroup C vaccination campaign in 2002 had almost completely eradicated cc11 The capsule polysaccharide of serogroup B meningococci does not trigger an immune

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different TpsA type, are detected by the immune system.7 Meningococcal TPS systems are meningitis, mortality and unfavorable outcome rates have remained similar. Together, these thought to be involved in intracellular survival and adhesion.8-10 We evaluated the chapters provide insight regarding the relevance of meningococcal virulence factors and distribution of TPS systems among clinical isolates from our 1998-2002 and 2006-2014 contribute to the discussion of the hyper-invasiveness of some clonal complexes. prospective cohort studies of bacterial meningitis patients (total of 369 meningococcal isolates investigated) using PCR and whole-genome sequencing. TPS system 1 was present in 91% of isolates and TPS system 2 and/or 3 was present in 67% of isolates. TPS system Figure 1. Venn diagram of meningococcal genetic factors associated with clinical outcome. distribution was highly related to clonal complexes. Of strains that exclusively harbored TPS1, 82% were serogroup C strains and 68% belonged to the hyper-invasive cc11. Next, we explored the association between infection with strains harboring immunogenic TPS systems and disease severity. Patients infected with meningococcal isolates with either TPS 2 and/or TPS3 presented with less severe disease and were more likely to have favorable outcome, as compared to patients infected with isolates without these systems. A plausible explanation for this finding is that isolates exclusively harboring TPS system 1 might have a survival advantage by evading the immune system, thereby causing more severe disease. To test this hypothesis we evaluated our findings with in vitro experiments using tps knockout meningococcal strains and complemented knockout strains. Using whole-blood stimulation experiments, we found no differences in host cytokine response between TPS systems 2 and 3 knockout strains and a wild-type strain harboring all TPS systems.

Our results from chapters 2, 3 and 4 regarding the clinical associations between clonal complexes, fHbp type and TPS system distribution profile, show a clear association between In our cohort of 254 isolates from patients with meningococcal meningitis, the overlap of isolates from clonal bacterial genetic make-up and disease outcome in meningococcal meningitis. Unfortunately, complex 11, isolates with the fHbpD184 polymorphism and isolates without immunogenic TPS systems is shown we have thus far not been able to prove a causal relationship of these virulence factors and (total n=102). clinical outcome. Also our bacterial knockout studies, in which the common genetic background provides an ideal situation to investigate a virulence factor, have failed to Contributing to vaccine research provide an explanation between the observed association with outcome. As is shown in Polysaccharide vaccines and conjugate vaccines (in which the polysaccharide is attached to a figure 1, there is considerable overlap between isolates with the fHbpD184 protein type, 9 isolates lacking an immunogenic TPS system and the cc11 genetic make-up. Possibly, the carrier protein) can offer protection against meningococcal serogroups A, C, W and Y. observed associations with outcome are the result of a genetic linkage effect, suggesting Serogroup C conjugate vaccine has proven to be highly effective and have almost eradicated that there are other, currently unknown virulence factors present in these strains. It is not serogroup C disease in countries where they have been implemented in routine vaccination 11 likely that these factors are only present in cc11, because after a nationwide meningococcal schedules. In contrast, developing vaccines against serogroup B strains remains difficult. serogroup C vaccination campaign in 2002 had almost completely eradicated cc11 The capsule polysaccharide of serogroup B meningococci does not trigger an immune

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response, because it is similar in structure to components of human cells. Consequently, the Studies on bacterial whole genome sequences immune system does not recognize the polysaccharide as a foreign molecule, thereby Research in the field of bacterial genetics is changing rapidly, as PCR-based sequencing preventing the production of protecting antibodies. Outer membrane vesicle vaccines have methods are being replaced by new ultra-high throughput sequencing techniques. At the been used to control serogroup B epidemic outbreaks.12 However, these vaccines offer time of writing the cost of sequencing a complete bacterial genome is less then €50, protection only to specific meningococcal types.13 Therefore, other strategies are necessary depending on the sequencing technique, and whole-genome sequencing is readily available for the development of vaccines targeting serogroup B meningococci. It is an important worldwide. In chapters 4, 6, 7 and 8 we have used this technique as a method in our question which vaccination targets to include in future vaccines, since changing bacterial investigations on bacterial virulence factors. The genome sequences analyzed in chapters 6, gene content can have an effect on the (duration of) effectiveness of vaccines. Recently, 7 and 8 were sequenced using shotgun 454 Titanium (Roche) pyrosequencing in the using reverse vaccinology to identify novel antigens,14-16 4CMenB (Bexsero® - Novartis) was Academic Medical Center and genome assemblies were performed using an in-house developed and is currently licensed in Europe, Canada, and Australia. This multicomponent developed pipeline. vaccine can reduce meningococcal carriage by 18%.17 The vaccine includes outer membrane vesicles and three major protein antigens variants: fHbp variant 1.1, Neisserial Heparin- In 2010, only seven complete N. meningitidis genomes had been sequenced, of which only Binding Antigen (NHBA) variant 2 and Neisseria adhesion A (NadA) variant 3.8. one was serogroup B. In chapter 6 we report the availability of the N. meningitidis serogroup B H44/76 genome sequence. This strain was originally isolated from a patient in Norway in In chapter 5 we describe the distribution of fHbp, NadA and NHBA variants of meningococcal 1976 and is widely used in molecular genetics studies, including those related to serogroup B isolates collected in the Netherlands over a period of 50 years. This collection of isolates vaccine development. We compared ~11,500 nucleotides of the obtained genome sequence covers a longer time period than any other collection available worldwide. Two of three with closely related strain MC58,20 and found zero mismatches. This indicates a very high investigated antigens were present in all isolates. Most variants were observed only once, quality of our whole-genome sequencing strategies used in chapters 6, 7 and 8. The suggesting that these are likely less fit and thereby excluded from the population by availability of the H44/76 genome helps future research that involves this laboratory strain. selection. The annual incidence of the fHbp, NadA and NHBA variants included in the 4CMenB vaccine, ranged from 0 to 20%, 0 to 50% and 5 to 40%, respectively. The varying In chapter 7, the last meningococcal virulence factor we investigated was lipopolysaccharide incidence over time of these variants could be of concern, as the effectiveness of the vaccine (LPS), a major component of the meningococcal outer membrane. The hallmark of could change as a result. The 4CMenB variants persisted during thirty, thirty and fifty years, meningococcal disease is the excessive stimulation of the immune system by LPS. Thus far, respectively. These results may be indicative for long-term broad coverage of 4CMenB, as no naturally occurring LPS-deficient meningococcal isolate has been known to cause clinical the antigens it targets have tended to persist. Moreover, the NHBA variant included in the disease. Our research group previously described that 12% of meningococcal isolates causing 18 vaccine could also provide cross protection to other NHBA variants , but cross protection of meningitis induce a reduced inflammatory response. All but one of these isolates had a 11 the included fHbp variant is expected to be poor. To predict the 4CMenB vaccine mutation in lpxL1, one of the 9 genes involved in lipid A biosynthesis, resulting in an altered effectiveness, the meningococcal antigen typing system (MATS), which combines PorA LPS structure. In chapter 7 we report on the isolate that caused the reduced inflammatory genotyping with an enzyme-linked immunosorbent assay [ELISA] to quantify the expression response, but had no mutations in lpxL1. The infected patient presented with isolated 19 and the cross-reactivity of antigenic variants, can be used. Moreover, further studies of thunderclap headache, a very uncommon clinical presentation of meningococcal meningococcal population structure and evolution are essential in predicting vaccine meningitis.21 It appeared that the causative isolate lacked LPS completely. Whole-genome coverage. sequencing revealed a mutation located in lpxH, which encodes an enzyme in the lipid A

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response, because it is similar in structure to components of human cells. Consequently, the Studies on bacterial whole genome sequences immune system does not recognize the polysaccharide as a foreign molecule, thereby Research in the field of bacterial genetics is changing rapidly, as PCR-based sequencing preventing the production of protecting antibodies. Outer membrane vesicle vaccines have methods are being replaced by new ultra-high throughput sequencing techniques. At the been used to control serogroup B epidemic outbreaks.12 However, these vaccines offer time of writing the cost of sequencing a complete bacterial genome is less then €50, protection only to specific meningococcal types.13 Therefore, other strategies are necessary depending on the sequencing technique, and whole-genome sequencing is readily available for the development of vaccines targeting serogroup B meningococci. It is an important worldwide. In chapters 4, 6, 7 and 8 we have used this technique as a method in our question which vaccination targets to include in future vaccines, since changing bacterial investigations on bacterial virulence factors. The genome sequences analyzed in chapters 6, gene content can have an effect on the (duration of) effectiveness of vaccines. Recently, 7 and 8 were sequenced using shotgun 454 Titanium (Roche) pyrosequencing in the using reverse vaccinology to identify novel antigens,14-16 4CMenB (Bexsero® - Novartis) was Academic Medical Center and genome assemblies were performed using an in-house developed and is currently licensed in Europe, Canada, and Australia. This multicomponent developed pipeline. vaccine can reduce meningococcal carriage by 18%.17 The vaccine includes outer membrane vesicles and three major protein antigens variants: fHbp variant 1.1, Neisserial Heparin- In 2010, only seven complete N. meningitidis genomes had been sequenced, of which only Binding Antigen (NHBA) variant 2 and Neisseria adhesion A (NadA) variant 3.8. one was serogroup B. In chapter 6 we report the availability of the N. meningitidis serogroup B H44/76 genome sequence. This strain was originally isolated from a patient in Norway in In chapter 5 we describe the distribution of fHbp, NadA and NHBA variants of meningococcal 1976 and is widely used in molecular genetics studies, including those related to serogroup B isolates collected in the Netherlands over a period of 50 years. This collection of isolates vaccine development. We compared ~11,500 nucleotides of the obtained genome sequence covers a longer time period than any other collection available worldwide. Two of three with closely related strain MC58,20 and found zero mismatches. This indicates a very high investigated antigens were present in all isolates. Most variants were observed only once, quality of our whole-genome sequencing strategies used in chapters 6, 7 and 8. The suggesting that these are likely less fit and thereby excluded from the population by availability of the H44/76 genome helps future research that involves this laboratory strain. selection. The annual incidence of the fHbp, NadA and NHBA variants included in the 4CMenB vaccine, ranged from 0 to 20%, 0 to 50% and 5 to 40%, respectively. The varying In chapter 7, the last meningococcal virulence factor we investigated was lipopolysaccharide incidence over time of these variants could be of concern, as the effectiveness of the vaccine (LPS), a major component of the meningococcal outer membrane. The hallmark of could change as a result. The 4CMenB variants persisted during thirty, thirty and fifty years, meningococcal disease is the excessive stimulation of the immune system by LPS. Thus far, respectively. These results may be indicative for long-term broad coverage of 4CMenB, as no naturally occurring LPS-deficient meningococcal isolate has been known to cause clinical the antigens it targets have tended to persist. Moreover, the NHBA variant included in the disease. Our research group previously described that 12% of meningococcal isolates causing 18 vaccine could also provide cross protection to other NHBA variants , but cross protection of meningitis induce a reduced inflammatory response. All but one of these isolates had a 11 the included fHbp variant is expected to be poor. To predict the 4CMenB vaccine mutation in lpxL1, one of the 9 genes involved in lipid A biosynthesis, resulting in an altered 9 effectiveness, the meningococcal antigen typing system (MATS), which combines PorA LPS structure. In chapter 7 we report on the isolate that caused the reduced inflammatory genotyping with an enzyme-linked immunosorbent assay [ELISA] to quantify the expression response, but had no mutations in lpxL1. The infected patient presented with isolated 19 and the cross-reactivity of antigenic variants, can be used. Moreover, further studies of thunderclap headache, a very uncommon clinical presentation of meningococcal meningococcal population structure and evolution are essential in predicting vaccine meningitis.21 It appeared that the causative isolate lacked LPS completely. Whole-genome coverage. sequencing revealed a mutation located in lpxH, which encodes an enzyme in the lipid A

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biosynthesis pathway, explaining its LPS-deficiency. The absence of LPS could have caused and accessory genomes, this chapter has provided insight in the extensive pneumococcal the inflammatory reaction to develop less effectively, providing an explanation for the genetic diversity in relation to clinical outcome. delayed development of symptoms such as fever and neck stiffness, which did not occur Future recommendations until 12 hours after admission. Since 2002, routine vaccination with a single dose of conjugated meningococcal C vaccine at Part II: Streptococcus pneumoniae (pneumococcus) the age of 14 months and a catch-up campaign has almost eradicated meningococcal In this part of the thesis we focus on the pneumococcus, the most common causative serogroup C disease in the Netherlands and other European countries, leaving serogroup B pathogen of bacterial meningitis in Western countries. Few studies have investigated the the most common cause of meningococcal meningitis.27,28 Whether serogroup B clonal role of bacterial factors in invasive pneumococcal disease, including meningitis. Host factors complexes are associated with more severe disease remains to be determined, but we did have shown to contribute to susceptibility22 and outcome.23 Pneumococcal capsular not observe such association in our 1998-2002 cohort. The results from a new prospective polysaccharides have been associated with disease severity and are used for phenotypical cohort study including roughly 85% of patients with community acquired bacterial meningitis characterization.24 The pneumococcal capsular phenotype has been linked to genotype but from 2006-2014 in the Netherlands, and the association of meningococcal and the mechanisms of virulence have not been fully explored.25 In chapter 8 we identified pneumococcal sequence types with patient characteristics are forthcoming. In this cohort, pneumococcal arginine biosynthesis genes to be associated with outcome in patients with complete genomes from more than 1000 causative isolates, regardless of the species, are pneumococcal meningitis, using a clinical phenotype-based approach combined with sequenced and will become publicly available. To expand our investigations on the bacterial whole-genome sequencing. Arginine is a precursor in the biosynthesis of association of bacterial virulence factors with outcome, these bacterial genomes should be polyamines, which have been implicated in oxidative stress responses and protection against analyzed using the same strategy that we used in chapter 8: First, in a hypothesis-free free radicals.26 We found that a wild-type pneumococcal strain harboring these genes approach, all bacterial genomes sequences from patients with favorable outcome should be showed increased growth in human blood and cerebrospinal fluid, compared with an compared with all bacterial genomes sequences from patients with unfavorable outcome. arginine biosynthesis knockout strain. Moreover, the wild-type strain was more resistant to This can be done on a gene content level, as we have done in chapter 8, by analyzing

H2O2 than the knockout strain. In mouse models of meningitis and pneumonia we showed variable sequence elements and on a single nucleotide polymorphism (SNP)-based level. that the knockout strain was attenuated in growth and/or cleared from lung, blood and Ultimately, associations between host DNA polymorphisms and bacterial sequence variation cerebrospinal fluid. can be assessed. To be able to compare these amounts of highly diverse bacterial genome sequences, we should collaborate with bioinformatics laboratories closely. Our data indicated that pneumococcal arginine biosynthesis genes contribute to virulence by influencing bacterial growth. Presence of arginine biosynthesis genes was strongly When the above strategy has resulted in the discovery of new putative bacterial virulence associated with pneumococcal serotype 7F. In fact, 85% of serotype 7F strains contained the factors, the phenotype of wild-type and isogenic knockouts can be investigated for example arginine biosynthesis genes. This serotype was the most common pneumococcal serotype in by determining immune responses in whole-blood stimulation essays (as we have used as a a Dutch cohort study of pneumococcal meningitis patients from 2006-2014.27 The method in our investigations on TPS systems in chapter 4). Multiplex ELISA assays can be association between arginine biosynthesis genes and clinical outcome remained robust in a used to provide a read-out of the immune response. Finally, our zebrafish or pneumococcal multivariate analysis including pneumococcal serotype. By analyzing the pneumococcal core meningitis animal models can be used for in vivo experiments.

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H2O2 than the knockout strain. In mouse models of meningitis and pneumonia we showed variable sequence elements and on a single nucleotide polymorphism (SNP)-based level. that the knockout strain was attenuated in growth and/or cleared from lung, blood and Ultimately, associations between host DNA polymorphisms and bacterial sequence variation cerebrospinal fluid. can be assessed. To be able to compare these amounts of highly diverse bacterial genome sequences, we should collaborate with bioinformatics laboratories closely. Our data indicated that pneumococcal arginine biosynthesis genes contribute to virulence by influencing bacterial growth. Presence of arginine biosynthesis genes was strongly When the above strategy has resulted in the discovery of new putative bacterial virulence associated with pneumococcal serotype 7F. In fact, 85% of serotype 7F strains contained the factors, the phenotype of wild-type and isogenic knockouts can be investigated for example 9 arginine biosynthesis genes. This serotype was the most common pneumococcal serotype in by determining immune responses in whole-blood stimulation essays (as we have used as a a Dutch cohort study of pneumococcal meningitis patients from 2006-2014.27 The method in our investigations on TPS systems in chapter 4). Multiplex ELISA assays can be association between arginine biosynthesis genes and clinical outcome remained robust in a used to provide a read-out of the immune response. Finally, our zebrafish or pneumococcal multivariate analysis including pneumococcal serotype. By analyzing the pneumococcal core meningitis animal models can be used for in vivo experiments.

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Future bacterial genetic epidemiological surveillance studies remain necessary, because community acquired bacterial meningitis and collect the clinical phenotypes and the DNA of meningococcal and pneumococcal populations are capable of rapidly changing their genetic causative bacteria, patients and controls. content. We have observed this phenomenon when we analyzed the meningococcal TPS system distribution in the 1998-2002 and 2006-2014 cohort studies (chapter 4). We observed, for example, that the proportion of isolates without TPS systems, increased from 2% in the 1998-2002 cohort to 16% in the 2006-2014 cohort. Together with our investigation on the pneumococcal arginine biosynthesis gene distribution in (parts of) these cohorts (chapter 8), these studies suggest that the genetic content of genomic islands, which are involved in exchange of genetic content within one species and between species, is clinically relevant.

This thesis, in which more than half of our analyses are based on newly sequenced bacterial whole genome sequences, serves as a proof of principle that bacterial whole-genome sequencing can give answers to research questions that previously remained unanswered. Since bacterial whole-genome sequencing is becoming more readily available, it is very likely that in the forthcoming years, whole genome sequences of thousands of pathogens will be analyzed. The focus of investigators will probably switch from obtaining bacterial genomes to being able to analyze these genomes more extensively. In this perspective, bioinformatics specialists play an important role and will be needed far more often.

This thesis has contributed to a better understanding of the role of bacterial genetics in the clinical course and outcome of bacterial meningitis. We have only begun to understand the contribution of bacterial genetic factors to pathogenesis. From a clinical perspective, in 10 years it is not unlikely that the complete genome of the causative agent from a patient with bacterial meningitis will be routinely sequenced during admission to provide answers to clinical questions about treatment options and prognosis. In the acute phase of treatment of infectious diseases antibiotics will probably remain the cornerstone of treatment (and in the case of bacterial meningitis immunosuppressive drugs such as adjunctive dexamethasone). It is not likely that better treatment options will become available in the acute setting in the foreseeable future. Therefore, to further decrease the disease burden of bacterial meningitis, research shoulf focus on vaccination strategies. Finally, multicenter prospective association studies, such as the MeninGene study, should continue to include patients with

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This thesis has contributed to a better understanding of the role of bacterial genetics in the clinical course and outcome of bacterial meningitis. We have only begun to understand the contribution of bacterial genetic factors to pathogenesis. From a clinical perspective, in 10 years it is not unlikely that the complete genome of the causative agent from a patient with bacterial meningitis will be routinely sequenced during admission to provide answers to clinical questions about treatment options and prognosis. In the acute phase of treatment of infectious diseases antibiotics will probably remain the cornerstone of treatment (and in the case of bacterial meningitis immunosuppressive drugs such as adjunctive dexamethasone). It 9 is not likely that better treatment options will become available in the acute setting in the foreseeable future. Therefore, to further decrease the disease burden of bacterial meningitis, research shoulf focus on vaccination strategies. Finally, multicenter prospective association studies, such as the MeninGene study, should continue to include patients with

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REFERENCES 15. Pizza M, Scarlato V, Masignani V, et al. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 2000;287:1816- 1. Madico G, Welsch JA, Lewis LA, et al. The meningococcal vaccine candidate 1820. GNA1870 binds the complement regulatory protein factor H and enhances serum resistance. J Immunol 2006;177:501-510. 16. Rappuoli R. Reverse vaccinology, a genome-based approach to vaccine development. Vaccine 2001;19:2688-2691. 2. Schneider MC, Prosser BE, Caesar JJ, et al. Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates. Nature 2009;458:890-893. 17. Read RC, Baxter D, Chadwick DR, et al. Effect of a quadrivalent meningococcal ACWY glycoconjugate or a serogroup B meningococcal vaccine on meningococcal carriage: 3. Masignani V, Comanducci M, Giuliani MM, et al. Vaccination against Neisseria an observer-blind, phase 3 randomised clinical trial. Lancet 2014;384:2123-2131. meningitidis using three variants of the lipoprotein GNA1870. J Exp Med 2003;197:789-799. 18. Giuliani MM, Adu-Bobie J, Comanducci M, et al. A universal vaccine for serogroup B meningococcus. Proc Natl Acad Sci U S A 2006;103:10834-10839. 4. Lemee L, Hong E, Etienne M, et al. Genetic diversity and levels of expression of factor H binding protein among carriage isolates of Neisseria meningitidis. PLoS One 19. Donnelly J, Medini D, Boccadifuoco G, et al. Qualitative and quantitative assessment 2014;9:e107240. of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines. Proc Natl Acad Sci U S A 2010;107:19490-19495. 5. Dunphy KY, Beernink PT, Brogioni B, Granoff DM. Effect of Factor H-Binding Protein Sequence Variation on Factor H Binding and Survival of Neisseria meningitidis in 20. Tettelin H, Saunders NJ, Heidelberg J, et al. Complete genome sequence of Neisseria Human Blood. Infect Immun 2011;79:353-359. meningitidis serogroup B strain MC58. Science 2000;287:1809-1815.

6. Kelly A, Jacobsson S, Hussain S, Olcen P, Molling P. Gene variability and degree of 21. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial expression of vaccine candidate factor H binding protein in clinical isolates of meningitis in adults. N Engl J Med 2006;354:44-53. Neisseria meningitidis. APMIS 2013;121:56-63. 22. Brouwer MC, de Gans J, Heckenberg SG, Zwinderman AH, van der Poll T, van de 7. van Ulsen P, Rutten L, Feller M, Tommassen J, van der Ende A. Two-partner Beek D. Host genetic susceptibility to pneumococcal and meningococcal disease: a secretion systems of Neisseria meningitidis associated with invasive clonal systematic review and meta-analysis. Lancet Infect Dis 2009;9:31-44. complexes. Infect Immun 2008;76:4649-4658. 23. Woehrl B, Brouwer MC, Murr C, et al. Complement component 5 contributes to poor 8. Tala A, Progida C, De SM, et al. The HrpB-HrpA two-partner secretion system is disease outcome in humans and mice with pneumococcal meningitis. J Clin Invest essential for intracellular survival of Neisseria meningitidis. Cell Microbiol 2011;121:3943-3953. 2008;10:2461-2482. 24. Brueggemann AB, Griffiths DT, Meats E, Peto T, Crook DW, Spratt BG. Clonal 9. van Ulsen P, Tommassen J. Protein secretion and secreted proteins in pathogenic relationships between invasive and carriage Streptococcus pneumoniae and Neisseriaceae. FEMS Microbiol Rev 2006;30:292-319. serotype- and clone-specific differences in invasive disease potential. J Infect Dis 2003;187:1424-1432. 10. Schmitt C, Turner D, Boesl M, Abele M, Frosch M, Kurzai O. A functional two-partner secretion system contributes to adhesion of Neisseria meningitidis to epithelial cells. 25. Enright MC, Spratt BG. A multilocus sequence typing scheme for Streptococcus J Bacteriol 2007;189:7968-7976. pneumoniae: identification of clones associated with serious invasive disease. Microbiology 1998;144 ( Pt 11):3049-3060. 11. Andrews SM, Pollard AJ. A vaccine against serogroup B Neisseria meningitidis: dealing with uncertainty. Lancet Infect Dis 2014;14:426-434. 26. Ha HC, Sirisoma NS, Kuppusamy P, Zweier JL, Woster PM, Casero RA, Jr. The natural polyamine spermine functions directly as a free radical scavenger. Proc Natl Acad Sci 12. McIntyre PB, O'Brien KL, Greenwood B, van de Beek D. Effect of vaccines on U S A 1998;95:11140-11145. bacterial meningitis worldwide. Lancet 2012;380:1703-1711. 27. Bijlsma MW, Brouwer MC, Kasanmoentalib ES, et al. Community-acquired bacterial 13. Holst J, Martin D, Arnold R, et al. Properties and clinical performance of vaccines meningitis in adults in the Netherlands, 2006-14: a prospective cohort study. Lancet containing outer membrane vesicles from Neisseria meningitidis. Vaccine 2009;27 Infect Dis 2015. Suppl 2:B3-12.

14. Tettelin H, Nelson KE, Paulsen IT, et al. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 2001;293:498-506.

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6. Kelly A, Jacobsson S, Hussain S, Olcen P, Molling P. Gene variability and degree of 21. van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial expression of vaccine candidate factor H binding protein in clinical isolates of meningitis in adults. N Engl J Med 2006;354:44-53. Neisseria meningitidis. APMIS 2013;121:56-63. 22. Brouwer MC, de Gans J, Heckenberg SG, Zwinderman AH, van der Poll T, van de 7. van Ulsen P, Rutten L, Feller M, Tommassen J, van der Ende A. Two-partner Beek D. Host genetic susceptibility to pneumococcal and meningococcal disease: a secretion systems of Neisseria meningitidis associated with invasive clonal systematic review and meta-analysis. Lancet Infect Dis 2009;9:31-44. complexes. Infect Immun 2008;76:4649-4658. 23. Woehrl B, Brouwer MC, Murr C, et al. Complement component 5 contributes to poor 8. Tala A, Progida C, De SM, et al. The HrpB-HrpA two-partner secretion system is disease outcome in humans and mice with pneumococcal meningitis. J Clin Invest essential for intracellular survival of Neisseria meningitidis. Cell Microbiol 2011;121:3943-3953. 2008;10:2461-2482. 24. Brueggemann AB, Griffiths DT, Meats E, Peto T, Crook DW, Spratt BG. Clonal 9. van Ulsen P, Tommassen J. Protein secretion and secreted proteins in pathogenic relationships between invasive and carriage Streptococcus pneumoniae and Neisseriaceae. FEMS Microbiol Rev 2006;30:292-319. serotype- and clone-specific differences in invasive disease potential. J Infect Dis 2003;187:1424-1432. 10. Schmitt C, Turner D, Boesl M, Abele M, Frosch M, Kurzai O. A functional two-partner secretion system contributes to adhesion of Neisseria meningitidis to epithelial cells. 25. Enright MC, Spratt BG. A multilocus sequence typing scheme for Streptococcus J Bacteriol 2007;189:7968-7976. pneumoniae: identification of clones associated with serious invasive disease. Microbiology 1998;144 ( Pt 11):3049-3060. 11. Andrews SM, Pollard AJ. A vaccine against serogroup B Neisseria meningitidis: dealing with uncertainty. Lancet Infect Dis 2014;14:426-434. 26. Ha HC, Sirisoma NS, Kuppusamy P, Zweier JL, Woster PM, Casero RA, Jr. The natural polyamine spermine functions directly as a free radical scavenger. Proc Natl Acad Sci 9 12. McIntyre PB, O'Brien KL, Greenwood B, van de Beek D. Effect of vaccines on U S A 1998;95:11140-11145. bacterial meningitis worldwide. Lancet 2012;380:1703-1711. 27. Bijlsma MW, Brouwer MC, Kasanmoentalib ES, et al. Community-acquired bacterial 13. Holst J, Martin D, Arnold R, et al. Properties and clinical performance of vaccines meningitis in adults in the Netherlands, 2006-14: a prospective cohort study. Lancet containing outer membrane vesicles from Neisseria meningitidis. Vaccine 2009;27 Infect Dis 2015. Suppl 2:B3-12.

14. Tettelin H, Nelson KE, Paulsen IT, et al. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 2001;293:498-506.

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28. Trotter CL, Ramsay ME. Vaccination against meningococcal disease in Europe: review and recommendations for the use of conjugate vaccines. FEMS Microbiol Rev 2007;31:101-107.

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28. Trotter CL, Ramsay ME. Vaccination against meningococcal disease in Europe: review and recommendations for the use of conjugate vaccines. FEMS Microbiol Rev 2007;31:101-107.

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vormen een “sequence type” en als deze onderling erg op elkaar lijken, kunnen ze worden met factor H. Patiënten die geïnfecteerd waren met meningokokken met de fHbpD184 variant gegroepeerd in clonal complexes (cc’s). Hyper-invasieve clonal complexes (gedefinieerd als (een zuur aminozuur is op positie 184 in het eiwit vervangen door een basisch aminozuur) cc8, cc11, cc32, cc41/44 en cc269) zijn typen meningokokken die vaker invasieve ziekte hadden een hogere kans op het ontwikkelen van sepsis tijdens hun opname, wat resulteerde veroorzaken dan andere clonal complexes. In onze cohort studie was infectie met in een slechtere uitkomst bij deze patiënten. Van deze patiënten was 67% geïnfecteerd door

meningokokken van cc11 (allen serogroep C) gerelateerd aan een slechte uitkomst. De een meningokok die behoorde tot cc11. Het vervangen van H184 door D184 in de fHbp-fH meest voorkomende serogroep B cc’s (cc41/44 [60%], cc41 [24%] en cc269 [8%]) waren niet model structuur suggereert een sterkere binding tussen fHbp en fH. Theoretisch zou het

geassocieerd met de ernst van de ziekte. hebben van fHbpD184 kunnen lijden tot een betere bescherming van de meningokok in de bloedsomloop, wat zou overeenkomen met het ernstiger ziektebeloop van patiënten die Dit onderzoek ondersteunt de theorie dat meningokokken genen invloed hebben op ziekte geïnfecteerd zijn met dit type meningokokken. Deze theorie is niet in overeenstemming met uitkomst. Omdat de genen die bij MLST worden onderzocht, niet virulente huishoud genen een eerder onderzoek dat suggereerde dat ernstiger ziek zijn wordt veroorzaakt door zijn, is het waarschijnlijk dat andere genetische factoren binnen bepaalde clonal complexes meningokokken die minder fHbp tot expressie brengen of een verminderde fHbp de associatie met ziekte uitkomst veroorzaken. Vaak is geprobeerd om virulentiefactoren bindingscapaciteit hebben. Onze bindingsexperimenten lieten geen significante verschillen van meningokokken in vitro te onderzoeken, maar de klinische relevantie is veelal zien in bindingsaffiniteit tussen fH en verschillende fHbp184 varianten. Dit zou kunnen onduidelijk. Idealiter zouden dergelijke factoren in vivo in een meningokokken meningitis worden veroorzaakt door een lage sensitiviteit van onze test of door een nog onverklaarde diermodel worden onderzocht. Doordat de mens de enige gastheer is van de meningokok is

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Het doel van dit proefschrift is om meer inzicht te verkrijgen in de associatie tussen tot dusver de ontwikkeling van dergelijke modellen onmogelijk gebleken. Daarom hebben bacteriële genetica en klinische kenmerken van patiënten met bacteriële meningitis. we in de volgende hoofdstukken in deel 1 van dit proefschrift geprobeerd om bekende Hieronder worden de belangrijkste resultaten van dit proefschrift samengevat en de virulentiefactoren van meningokokken in verband te brengen met klinische uitkomst. bevindingen in een breder perspectief geplaatst. Als laatste worden er suggesties gegeven Factor H bindend eiwit (fHbp) is een virulentiefactor van meningokokken die we hebben voor toekomstig onderzoek. onderzocht in hoofdstuk 3. Dit eiwit bindt het menselijke complement onderdeel factor H Deel 1: Neisseria meningitidis (meningokok) (fH), waardoor complement activatie wordt geremd en complement gemedieerde lysis wordt verminderd. De laatste 10 jaar is er veel interesse geweest in fHbp: het is een Onderzoek naar virulentie factoren van meningokokken in klinische cohort studies belangrijk onderdeel in 2 aankomende vaccins. Relatief grote hoeveelheden fHbp is In hoofdstuk 2 van dit proefschrift beschrijven we een Nederlands prospectief cohort van gevonden in hypervirulente meningokokken stammen. Daarnaast laten invasieve stammen patiënten met meningokokken meningitis gedurende de periode 1998-2002, tezamen met een hogere expressie van fHbp zien dan dragerstammen. Daarnaast is fHbp noodzakelijk het bacteriële genotype. Dertig van de 258 patiënten (12%) had een slechte uitkomst en de voor de overleving van meningokokken in menselijk bloed. Onderzoek naar fHbp varianten is mortaliteit was 7%. N.meningitidis kan in serogroepen worden verdeeld op basis van de van belang met betrekking tot vaccin ontwikkeling, omdat de inclusie van de juiste varianten immunologische reactie van de polysachariden in het kapsel van de bacterie. De proportie kan resulteren in een meer effectief vaccin. Het is nog onduidelijk wat de relatie is tussen serogroep B meningokokken was 68% en de proportie serogroep C was 31%. De verschillende fHbp eiwitten en ziekte uitkomst. Daarom hebben we gekeken naar de meningokokken werden getypeerd door middel van de multilocus sequence typing (MLST) distributie van de fHbp eiwitten in meningokokken isolaten van bacteriële meningitis techniek, waarbij delen van het bacteriële DNA worden gesequenced, waarna allelnummers patiënten in ons cohort van 1998-2002 en hebben de fHbp eiwit typen gecorreleerd aan de aan 7 verschillende “huishoud” genen worden toebedeeld. De combinatie van de allelen klinische uitkomst. In het fHbp-fH complex hebben 17 aminozuren van fHbp een interactie vormen een “sequence type” en als deze onderling erg op elkaar lijken, kunnen ze worden met factor H. Patiënten die geïnfecteerd waren met meningokokken met de fHbpD184 variant gegroepeerd in clonal complexes (cc’s). Hyper-invasieve clonal complexes (gedefinieerd als (een zuur aminozuur is op positie 184 in het eiwit vervangen door een basisch aminozuur) cc8, cc11, cc32, cc41/44 en cc269) zijn typen meningokokken die vaker invasieve ziekte hadden een hogere kans op het ontwikkelen van sepsis tijdens hun opname, wat resulteerde veroorzaken dan andere clonal complexes. In onze cohort studie was infectie met in een slechtere uitkomst bij deze patiënten. Van deze patiënten was 67% geïnfecteerd door meningokokken van cc11 (allen serogroep C) gerelateerd aan een slechte uitkomst. De een meningokok die behoorde tot cc11. Het vervangen van H184 door D184 in de fHbp-fH meest voorkomende serogroep B cc’s (cc41/44 [60%], cc41 [24%] en cc269 [8%]) waren niet model structuur suggereert een sterkere binding tussen fHbp en fH. Theoretisch zou het geassocieerd met de ernst van de ziekte. hebben van fHbpD184 kunnen lijden tot een betere bescherming van de meningokok in de bloedsomloop, wat zou overeenkomen met het ernstiger ziektebeloop van patiënten die Dit onderzoek ondersteunt de theorie dat meningokokken genen invloed hebben op ziekte geïnfecteerd zijn met dit type meningokokken. Deze theorie is niet in overeenstemming met uitkomst. Omdat de genen die bij MLST worden onderzocht, niet virulente huishoud genen een eerder onderzoek dat suggereerde dat ernstiger ziek zijn wordt veroorzaakt door zijn, is het waarschijnlijk dat andere genetische factoren binnen bepaalde clonal complexes meningokokken die minder fHbp tot expressie brengen of een verminderde fHbp de associatie met ziekte uitkomst veroorzaken. Vaak is geprobeerd om virulentiefactoren bindingscapaciteit hebben. Onze bindingsexperimenten lieten geen significante verschillen van meningokokken in vitro te onderzoeken, maar de klinische relevantie is veelal zien in bindingsaffiniteit tussen fH en verschillende fHbp184 varianten. Dit zou kunnen onduidelijk. Idealiter zouden dergelijke factoren in vivo in een meningokokken meningitis worden veroorzaakt door een lage sensitiviteit van onze test of door een nog onverklaarde diermodel worden onderzocht. Doordat de mens de enige gastheer is van de meningokok is

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veranderde functie van fHbp die wordt veroorzaakt door aanpassing van het eiwit op deze waren onze bacteriële knock-out studies, waarbij de gedeelde genetische achtergrond een locatie. ideale omstandigheid is om een virulentiefactor te onderzoeken, niet in staat om een verklaring te bieden voor de gevonden associaties. Zoals te zien is in figuur 1 is er een In hoofdstuk 4 onderzochten we two-partner secretion (TPS) systemen. Deze eiwit systemen aanzienlijke overlap tussen de isolaten die het fHbpD184 eiwit type hebben, isolaten die geen exporteren grote TpsA eiwitten naar het celoppervlak en naar het extracellulaire milieu. immunogeen TPS systeem hebben, en isolaten die tot cc11 behoren. Mogelijk zijn onze Meningokokken kunnen 3 verschillende typen TPS systemen hebben, waarbij systeem type 2 gevonden associaties met ziekte uitkomst het resultaat van een andere genetische factor die en 3, met elk een ander TpsA type, kunnen worden gedetecteerd door het menselijk is gekoppeld is aan de onderzochte factoren. Dit zou suggereren dat er andere nog immuunsysteem. TPS systemen van meningokokken zijn betrokken bij de intracellulaire onbekende virulentie factoren aanwezig zijn in deze stammen. Echter, een nationale overleving en adhesie aan andere cellen. We bekeken de distributie van TPS systemen in meningokokken serogroep C vaccinatie campagne in 2002 heeft meningitis, veroorzaakt meningokokken in onze 1998-2002 en 2006-2014 prospectieve cohort studies van patiënten door meningokokken van cc11, bijna helemaal uitgeroeid waarna de mortaliteit en slechte met bacteriële meningitis (totaal 369 meningokokken isolaten onderzocht) met behulp van uitkomst ongeveer hetzelfde zijn gebleven. Daarom is het onwaarschijnlijk dat mogelijk nog PCR en door de hele bacteriële genomen te sequencen (whole-genome sequencing). TPS onbekende virulentiefactoren enkel in cc11 aanwezig zijn. Samengevat geven deze systeem 1 was aanwezig in 91% van de isolaten en TPS systeem 2 en/of 3 was aanwezig in hoofdstukken meer inzicht in de relevantie van virulentiefactoren van meningokokken. 67% van de isolaten. De distributie van TPS systemen was erg gerelateerd aan het type Daarnaast dragen deze hoofdstukken bij aan de discussie omtrent de hyper-invasiviteit van clonal complex. Van de stammen die alleen TPS1 bevatten, was 82% een serogroep C stam sommige clonal complexes. en 68% behoorde tot het hyper-invasieve cc11. Daarna onderzochten we de relatie tussen infectie met stammen die een immunogeen TPS systeem hadden en de ernst van de ziekte. Patiënten die geïnfecteerd waren door meningokokken met een immunogeen TPS systeem, presenteerden zich met minder ernstige ziekte en hadden een betere ziekte uitkomst vergeleken met patiënten die geïnfecteerd waren door meningokokken zonder een immunogeen TPS systeem. Een mogelijke verklaring voor deze bevinding is dat isolaten die enkel TPS systeem 1 bevatten mogelijk een overlevingsvoordeel hebben doordat ze het immuunsysteem kunnen omzeilen, waardoor ze dan een ernstiger ziekte beloop veroorzaken. Om deze hypothese te testen, hebben we wild-type stammen, tps knock-out stammen en gecomplementeerde knock-out stammen met elkaar vergeleken. In experimenten waarbij deze bacteriën hebben laten groeien in menselijk, vonden we echter geen verschil in cytokine respons tussen TPS systeem 2 en 3 knock-out stammen en wild- type stammen die alle TPS systemen hadden.

Onze resultaten van hoofdstukken 2, 3 en 4 wat betreft de klinische associaties met clonal complexes, fHbp type en TPS systeem distributie, laten een duidelijke relatie zien tussen bacteriële genetische factoren en ziekte uitkomst. Helaas waren we niet in staat om een causaal verband aan te tonen tussen deze virulentiefactoren en klinische uitkomst. Ook

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Figuur 1. Venn diagram van genetische factoren van meningokokken die geassocieerd zijn met ziekte tegen een beperkt aantal typen meningokokken bescherming bieden. Daarom is het nodig uitkomst. om andere methoden te gebruiken om vaccins tegen serogroep B meningokokken te kunnen maken. Omdat bacteriën een wisselende genetische inhoud hebben, is het van belang welke vaccinatie targets worden gebruikt in vaccins. Dit kan namelijk effect hebben op de effectiviteit van een vaccin. Recent is het 4CMenB (Bexsero® - Novartis) vaccine ontwikkeld wat nu beschikbaar is in Europa, Canada en Australië. Dit vaccin met meerdere componenten kan het meningokokken dragerschap reduceren met 18%. Het vaccin bevat buitenmembraan onderdelen van meningokokken en daarnaast 3 antigeen varianten: fHbp variant 1.1, Neisserial Heparin-Binding Antigen (NHBA) variant 2 en Neisseria adhesion A (NadA) variant 3.8.

In hoofdstuk 5 beschrijven we de distributie van de fHbp, NadA en NHBA varianten binnen meningokokken die in Nederland gedurende 50 jaar zijn verzameld. Deze collectie beslaat wereldwijd de langste periode die tot nu toe is beschreven. Twee van de drie antigenen waren aanwezig in alle isolaten. De meeste antigeen varianten werden slechts eenmalig gevonden, wat suggereert dat deze niet tot een evolutionair voordeel kunnen lijden, In ons cohort van 254 isolaten van patiënten met meningokokken meningitis is de overlap te zien van isolaten waardoor ze door selectie uit de meningokokken populatie verdwijnen. De jaarlijkse die behoren tot cc11, isolaten die het fHbpD184 polymorfisme hebben, en isolaten die geen immunogeen TPS systeem hebben (totaal aantal: 102). incidentie van de fHbp, NadA en NHBA varianten die in het 4CMenB vaccin zitten, varieerden respectievelijk van 0 tot 20%, 0 tot 50% en van 5 tot 40%. De variërende incidentie kan van belang zijn omdat dit de effectiviteit kan beïnvloeden. De 4CMenB varianten waren Vaccin onderzoek aanwezig gedurende respectievelijk dertig, dertig en vijftig jaar, wat zou kunnen suggereren Polysacharide vaccins en conjugaat vaccins (waarbij het polysacharide is gekoppeld aan een dat het 4CMenB vaccin gedurende langere tijd effectief kan zijn. Wat een ander positief dragereiwit) kunnen bescherming geven tegen serogroep A, C, W en Y meningokokken. effect zou kunnen zijn, is dat het waarschijnlijk is dat de NHBA variant die in het vaccin zit, Serogroep C conjugaat vaccins zijn erg effectief gebleken en hebben bijna serogroep C kruisprotectie kan geven tegen andere varianten. Daarentegen is er minder te verwachten meninogkokkken ziekte uitgeroeid in landen waar dit vaccin is opgenomen in de standaard van mogelijke kruisprotectie van de geïncludeerde fHbp variant. Om de effectiviteit van het vaccinatieschema’s. Daarentegen is het ontwikkelen van vaccins tegen serogroep B 4CMenB vaccin te kunnen voorspellen zou het meningococcal antigen typing system (MATS) meningokokken erg lastig. Het polysacharide kapsel van serogroep B meningokokken kan kunnen worden gebruikt. Dit systeem combineert PorA genotypering met een enzyme- geen immuunreactie opwekken omdat dit qua structuur erg lijkt op onderdelen van linked immunosorbent assay [ELISA] waarmee de expressie en kruisreactiviteit van de menselijke cellen. Hierdoor wordt het polysacharide van de meningokok niet herkend, antigeen varianten kan worden bepaald. Daarnaast blijft het van belang om onderzoek te waardoor er geen beschermende antilichamen kunnen worden aangemaakt. Daarentegen, blijven doen naar de evolutie en populatiestructuur van meningokokken om de vaccin vaccins gebaseerd op blaasjes die bestaan uit onderdelen van meningokokken membranen effectiviteit te kunnen voorspellen. hebben serogroep B epidemieën weten in te dammen. Helaas kunnen deze vaccins maar

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Onderzoek naar complete bacteriële genomen geheel geen LPS kon produceren. De analyse van het hele genoom van dit isolaat liet een mutatie in lpxH zien. Dit gen codeert ook voor een enzym in de lipid A biosynthese, wat de Het onderzoek in het veld van de bacteriële genetica is hard aan het veranderen, waarbij afwezigheid van LPS verklaarde. De patiënt kreeg erg laat in het ziekte beloop symptomen: PCR-gebaseerde sequencing methoden worden vervangen door “next-generation” pas 12 uur na opname kreeg hij koorts en klachten van nekstijfheid. Dit zou kunnen worden technieken die vele malen meer effectief zijn. In 2016 kost het minder dan €50 om een verklaard doordat de LPS-loze stam ervoor heeft gezorgd dat de immuunreactie minder compleet bacterieel genoom te sequencen, afhankelijk van de sequencing techniek . Deze efficiënt op gang is gekomen. nieuwe technieken zijn inmiddels wereldwijd beschikbaar gekomen. In hoofdstukken 4, 6, 7 en 8 gebruiken we deze technieken als een hulpmiddel bij ons onderzoek naar bacteriële Deel 2: Streptococcus pneumoniae (pneumokok) virulentiefactoren. De genoomsequenties die we hebben geanalyseerd in hoofdstukken 6,7 In dit gedeelte van het proefschrift focussen we ons op de pneumokok. Dit is de meest en 8 zijn in het Academisch Medisch Centrum (AMC) gesequenced met de shotgun 454 voorkomende verwekker van bacteriële meningitis in de Westerse wereld. De rol van Titanium (Roche) pyrosequencing techniek, waarna de genomen verder digitaal werden pneumokokken factoren op invasieve ziekte zoals meningitis is nog onvoldoende opgebouwd met behulp van een in het AMC ontwikkelde methode. onderzocht. Gastheer-gerelateerde factoren dragen bij aan de mate waarin de gastheer In 2010 waren er nog maar 7 complete genomen van N. meningitidis gesequenced, waarvan ontvankelijk is voor de ziekte en tevens ook aan de ziekte uitkomst. Het polysacharide kapsel er 1 van serogroup B is. In hoofdstuk 6 beschrijven we de genoom sequentie van de N. van de pneumokok is geassocieerd met uitkomst en wordt tevens gebruikt voor het meningitidis serogroup B H44/76 stam. Dit isolaat, verkregen in 1976, is afkomstig van een karakteriseren van de bacterie. Het fenotype van het kapsel is gerelateerd aan het genotype, Noorse patiënt. Het wordt veel gebruikt in genetisch onderzoek, waaronder ook serogroep B maar de virulentiemechanismen van de pneumokok zijn nog onvoldoende uitgezocht. In vaccinatie ontwikkel studies. We vergeleken ~11,500 nucleotiden van het gesequencede hoofdstuk 8 ontdekken we dat de arginine biosynthese genen van pneumokokken zijn genoom met de MC58 stam die er erg op lijkt en vonden geen verschil. Dit laat zien dat de geassocieerd met uitkomst van patiënten met pneumokokken meningitis. Hiervoor technieken die we hebben gebruikt om complete genomen te verkrijgen in hoofdstukken 6, gebruikten we een onderzoeksopzet waarbij we uitgingen van het klinisch fenotype, en 7 en 8 kwalitatief in orde zijn. combineerden dit met het sequencen van hele bacteriële genomen. Arginine is een precursor bij de biosynthese van polyaminen. Deze stoffen zijn betrokken bij de oxidatieve In hoofdstuk 7 onderzochten we als laatste de virulentiefactor lipopolysacharide (LPS), wat stress respons en bij de bescherming tegen vrije radicalen. We vonden dat een wild-type een belangrijk onderdeel van de buitenmembraan van de meningokok is. Hét kenmerk van pneumokok die deze genen bij zich droeg een versterkte groei liet zien in bloed en meningokokken ziekte is de excessieve stimulatie van het immuunsysteem door LPS. Tot nu hersenvocht, vergeleken met een knock-out stam die deze genen niet had. Daarnaast was de toe is er nog nooit een klinische meningokokkenstam beschreven die geen LPS maakt. Onze wild-type stam resistenter tegen H2O2 dan de knock-out stam. Daarnaast lieten we zien dat onderzoeksgroep heeft eerder beschreven dat 12% van de meningokokken meningitis in modellen voor meningitis en longontsteking de knock-out minder goed groeide in longen, isolaten een verminderde immuunreactie teweeg bracht. Op één na hadden al deze bloed en hersenvocht, vergeleken met het wild-type. stammen een mutatie in lpxL1, 1 van de 9 genen betrokken bij de lipid A biosynthese, wat resulteerde in een veranderde LPS structuur. In hoofdstuk 7 beschrijven we de stam die wél Onze resultaten lieten zien dat de arginine biosynthese genen van pneumokokken bijdragen een verminderde immuunreactie laat zien, maar geen mutaties in lpxL1 had. De patiënt die aan de virulentie van de bacterie omdat ze de groei van de bacterie bevorderen. De door deze stam was geïnfecteerd presenteerde zich met donderslag hoofdpijn, wat een erg aanwezigheid van deze genen was sterk geassocieerd met het 7F pneumokokken serotype. ongewone presentatie van meningokokken meningitis is. Het bleek dat deze stam in het Zelfs 85% van alle serotype 7F stammen droeg deze genen bij zich. Dit serotype is het meest

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voorkomende serotype in de Nederlandse cohort studie van pneumokokken meningitis worden gedaan volgens de methode die we hebben gebruikt in hoofdstuk 4, waarbij de patiënten van 2006 tot 2014. De associatie tussen arginine biosynthese genen en uitkomst immuunreactie van verschillende stammen werd gemeten in menselijk bloed. Multiplex was nog steeds significant na correctie in een multivariate analyse waarbij het serotype van ELISA assays zouden kunnen worden gebruikt om de immuunreactie te meten. Daarna de pneumokok werd meegenomen. Door het analyseren van de pneumokokken genomen in zouden potentiele bacteriële virulentiefactoren verder kunnen worden onderzocht in ons dit hoofdstuk werd meer inzicht verkregen in de genetische diversiteit van de pneumokok zebravis model of meningokokken meningitis diermodel. met betrekking tot ziekte uitkomst. Het is noodzakelijk om onderzoek te blijven doen naar de epidemiologie van meningokokken Toekomstperspectieven en pneumokokken, omdat de genetische eigenschappen van deze bacteriën razendsnel kunnen veranderen. Dit fenomeen hebben we gezien toen we de TPS systeem distributie Door een routine vaccinatie campagne met een geconjugeerd meningokokken serogroep C analyseerden in de cohort studies van 1998-2002 en 2006-2014 (hoofdstuk 4). Hierin zagen vaccin bij kinderen van 14 maanden oud, is meningokokken serogroep C ziekte bijna geheel we dat de proportie van patiënten zonder een TPS systeem steeg van 2% in 1998-2002 tot verdwenen in Nederland en de rest van Europa. Serogroep B meningokokken zijn nu de 16% in 2006-2014. De arginine biosynthese genen (hoofdstuk 8), en de TPS systeem genen meest voorkomende verwekker van meningokokken meningitis. Of clonal complexes binnen liggen op genomische eilanden in het DNA van de bacterie. Deze eilanden zijn betrokken bij deze serogroep geassocieerd zullen zijn met ziekte uitkomst moet nog worden bezien: dit het uitwisselen van DNA tussen bacteriën onderling. Onze studies laten zien dat de hebben we niet kunnen aantonen in onze cohort studie van 1998-2002. Analyses van de genetische inhoud van deze eilanden klinisch relevant is. nieuwe cohort studie van patiënten met bacteriële meningitis tussen 2006 en 2014, worden de komende jaren verwacht. In dit cohort 2014 worden de complete genomen van meer dan In de helft van de hoofdstukken van dit proefschrift analyseren we nieuw gesequencede hele 1000 meningitis verwekkers gesequenced. De verkregen genomen zijn voor iedereen bacteriële genomen. Hierbij laten we zien dat deze techniek ons kan helpen om antwoord toegankelijk die hier onderzoek naar wil doen. Om meer inzicht te krijgen in de associatie kan geven op vooralsnog onbeantwoorde vragen. Nu het sequencen van hele bacteriële tussen bacteriële virulentiefactoren en uitkomst, zou kunnen worden begonnen met de genomen steeds toegankelijker wordt, zal het aantal bacteriële genomen dat de komende aanpak die we in hoofdstuk 8 hebben gebruikt: als eerste kunnen alle bacteriële genomen jaren wordt gesequenced, alleen maar toenemen. Hierbij zal de nadruk bij dergelijk van patiënten met een goede uitkomst worden vergeleken met de genomen van de bacterieel genetisch onderzoek moeten komen te liggen op het effectief kunnen analyseren bacteriën van patiënten met een slechte uitkomst. Deze analyse kan op meerdere niveaus van deze grote hoeveelheden data. Hierbij zal de bio-informatica een belangrijke rol gaan worden gedaan: op gen-inhoud niveau zoals we hebben gedaan in hoofdstuk 8, op het spelen. niveau van variabele sequentie lengte en op single nucleotide polymorphism (SNP) niveau. De inhoud van dit proefschrift heeft bijgedragen aan de kennis over de rol van bacteriële Uiteindelijk zou het zeer interessant zijn om een verband te kunnen leggen tussen genetica bij het ziekte beloop en de uitkomst van bacteriële meningitis. Op dit moment polymorfismen in het DNA van de gastheer en het DNA van de pathogeen. Om dergelijke staan we pas aan het begin van de gedachtevorming omtrent de invloed van bacteriële ingewikkelde genetische analyses te kunnen uitvoeren, is een nauwe samenwerking met bio- genetische factoren op de pathogenese van bacteriële meningitis. Over 10 jaar is het niet informatica afdelingen noodzakelijk. onwaarschijnlijk dat gedurende de ziekenhuisopname van een patiënt met bacteriële Zodra bovenstaande strategie heeft geleid tot de ontdekking van mogelijke bacteriële meningitis, het genoom van de verwekker routinematig wordt gesequenced. Mogelijk kan er virulentiefactoren, zou als eerste stap het fenotype van een wild-type stam kunnen worden dan op basis van deze informatie uitspraak worden gedaan over de beste behandelmethode vergeleken met het fenotype van een isogene knock-out stam. Dit kan dan bijvoorbeeld en de prognose van de ziekte. In de acute fase van de behandeling van een infectieziekte

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zullen antibiotica (en in het geval van bacteriële meningitis de toevoeging van immunosuppressiva) de hoeksteen van de behandeling blijven vormen. Het is onwaarschijnlijk dat dit in de nabije toekomst snel zal veranderen. Daarom is het belangrijk dat toekomstig onderzoek zich blijft focussen op vaccinatie strategieën. Tenslotte is het van belang dat multicenter prospectieve associatiestudies zoals de MeninGene studie, doorgaan met het includeren van bacteriële meningitis patiënten. Hierbij dienen dan patiënten karakteristieken, en het DNA van de ziekteverwekker, gastheer en gezonde controles te worden verzameld.

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Dankwoord

En dan het hoofdstuk wat in proefschriften over het algemeen goed wordt gelezen… Het dankwoord! Dit proefschrift is het resultaat van diverse samenwerkingen en ik wil in dit hoofdstuk de mensen bedanken die hebben bijgedragen aan het onderzoek.

Diederik, mijn promotor, ik had al een artikel met je geschreven voordat ik je ooit had ontmoet! Ik waardeer onze discussies over het onderzoek en ook het feit dat je geïnteresseerd bent in je promovendi buiten de muren van het AMC. Daarnaast weet je binnen onze onderzoeksgroep altijd een topsfeer neer te zetten en je zorgt dat we naar fantastische congressen kunnen gaan. Bij jou smaakt onderzoek doen altijd naar meer.

Arie, mijn copromotor, door jou is 15 jaar geleden mijn interesse in de medische microbiologie gewekt. We kennen elkaar al een lange tijd! Jij weet elke keer weer een draai te geven aan alle, soms wat onverwachte, bevindingen van onze experimenten. Je staat altijd klaar voor overleg en ik wil je bijzonder bedanken voor onze samenwerking.

Members of the thesis defence committee, thank you for taking the time to read my thesis. Stephen, I am deeply honored that you have joined the committee. I would like to thank you for your efforts and I hope you will be able to attend in June!

Mijn paranimfen, Take en Daan, wat fantastisch dat jullie me bijstaan! Take, ik ken je al sinds de E-tjes en we zijn inmiddels allebei een mooi leven aan het opbouwen. Vrienden, sport, wonen: je bent er altijd onderdeel van geweest. Nu dus ook een beetje bij werk! Daan, op dag 1 samen in het AMC begonnen wat de start is geweest van een mooie vriendschap. Het is altijd fantastisch om met jou te keuvelen over de zin en de onzin van het leven.

Robert, toen ik begon met mijn promotietraject was jij er ook net op L1. Het is/was geweldig om met jou te sparren over Next Gen Sequencing en zowel binnen als buiten het AMC hebben we veel meegemaakt. Gelukkig heb ik je binnen de ring kunnen houden!

Alle co-auteurs van de artikelen, met name Afshin, Madelijn en Peter. Dank voor de samenwerking!

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Dankwoord

Alle co-auteurs van de artikelen, met name Afshin, Madelijn en Peter. Dank voor de samenwerking!

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Matthijs: zonder jou geen meningitis onderzoek. Je bent voor iedereen altijd bereikbaar voor Mijn (magische) vrienden: Casper, Friso, Jeroen, Jurriaan, Karsten & Wienke, Martijn, Ron overleg. Bedankt voor jouw medewerking en visie op sommige van mijn stukken. (thanx for proofreading!), Take & Eline en mijn andere vrienden. Bedankt voor jullie vriendschap! Merche, met jou kan ik altijd goed van gedachte wisselen en onderzoeksopzetten uitwerken. We hebben een fantastisch knutsel half jaar gehad toen we aan het sequencen waren! Mijn schoonfamilie, ook altijd geïnteresseerd in het wel en wee in het AMC. Ik ben blij met jullie! Barbara, het komt al langs in de discussie van mijn proefschrift: ik bewonder hoe jij als bio- informaticus zeer grote hoeveelheden data analyseert. Zonder jou geen bacteriële Mijn ouders, Rob en Loura: het kroost is goed terecht gekomen en daar hebben jullie voor genoomanalyse! Bedankt! gezorgd! Bedankt voor alle liefde, betrokkenheid en steun die jullie mij geven. Mijn zusje Hélène en natuurlijk Herman en Lotte: we hebben het altijd gezellig samen en bedankt voor Medewerkers van het Referentielaboratorium voor Bacteriële meningitis: in het bijzonder al jullie interesse! Virma, Agaath en Wendy. Heel veel dank voor jullie hulp en begeleiding rondom het bacteriële typeerwerk en bedankt voor de gezelligheid op het Reflab! Marjolein, wat ben ik blij dat wij elkaar hebben gevonden. Jij vult me aan en brengt lol en liefde in mijn leven. Ik vind het telkens weer een eer om jouw partner te mogen zijn. Wat Alle laboranten bij de medische microbiologie en met name Ewout, Leonie, Kim en Sandra. Ik superleuk dat we binnen vijf dagen van elkaar ons proefschrift verdedigen (ladies first)! waardeer jullie motivatie en bedankt voor het lab werk wat zeer veel heeft bijgedragen aan diverse stukken in dit proefschrift.

Alle medewerkers van het laboratorium voor genoomanalyse en het sequence lab: Marja, Olaf, Ted, Ferry en Frank: het waren mooie tijden toen de eerste 454 sequencer in het AMC werd geplaatst. Ik kan me nog goed herinneren hoe eufoor ik was bij de eerste succesvolle sequence run! Heel veel dank voor de intensieve samenwerking en de gezelligheid.

Mensen met wie ik heb samengewerkt bij het CEMM op K2: Regina, Daniëlle, Linda en Alex. Dank voor jullie input bij onder andere de ELISA’s!

Alle (ex-) AIOS Neurologie: ook al is de samenstelling van de groep dynamisch, de sfeer is constant. En die is top!

Meningitis mede-onderzoekers: Sebastiaan, Barry, Kirsten, Madelijn, Merijn, Joost, Inge, Philip, Soemirien, Merel, Anne en Daan. Wat een gezelligheid en korte lijntjes voor overleg hadden en hebben we op H2. Alle meningitis congressen met jullie waren priceless!

Squashers: mens sana in corpore sano. Bedankt voor het gehak en plezier op de baan!

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Mensen met wie ik heb samengewerkt bij het CEMM op K2: Regina, Daniëlle, Linda en Alex. Dank voor jullie input bij onder andere de ELISA’s!

Alle (ex-) AIOS Neurologie: ook al is de samenstelling van de groep dynamisch, de sfeer is constant. En die is top!

Squashers: mens sana in corpore sano. Bedankt voor het gehak en plezier op de baan!

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List of abbreviations HIV human immunodeficiency virus IAS standardized index of association 4CMenB multicomponent meningococcal serogroup B vaccine (Bexsero®) IgG immunoglobulin G ABC ATP-binding cassette IL-6 interleukin 6 AMC Academic medical center IMDM Iscove's modified Dulbecco's medium Arg arginine IPTG Isopropyl β-D-1-thiogalactopyranoside Asp aspartic acid IQR interquartile range ATP adenosine triphosphate IS insertion sequence BLAST basic local alignment search tool K lysine BLAT BLAST-like alignment tool ka association rate constant BP blood pressure kan kanamycin cc clonal complex Kd thermodynamic dissociation constant CFU colony-forming unit kd dissociation rate constant CI confidence interval lac lactose CRP C-reactive protein LPS lipopolysaccharide CSF cerebrospinal fluid Lys lysine CT computed tomography MATS meningococcal antigen typing system δ* dinucleotide relative abundance Mb megabases DNA deoxyribonucleic acid MenB meningococcal serogroup B E glutamic acid MID multiplex identifier ELISA enzyme-linked immunosorbent assay MLEE multilocus enzyme electrophoresis ery erythromycin MLST multilocus sequence typing ESR erythrocyte sedimentation rate MM6 mono mac 6 FCS fetal calf serum NadA Neisseria adhesin A fH factor H ND not determined fHbp factor H binding protein NHBA Neisserial heparin-binding antigen GCS Glasgow coma scale NO nitric oxide Gly glycine NRLBM Netherlands reference laboratory for bacterial meningitis GNA genome-derived neisserial antigen ns non-significant GOS Glasgow outcome scale NWO Nederlandse organisatie voor wetenschappelijk onderzoek H histidine OD optical density His histidine OMV outer membrane vesicle

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OR odds ratio ORF open reading frame PAGE polyacrylamide gel electrophoresis PBS phosphate-buffered saline PCR polymerase chain reaction Phe phenlyalanine PorA porine A rLP2086 Meningococcal serogroup B bivalent vaccine (Trumenba®) rRNA ribosomal ribonucleic acid RT reverse transcription SD standard deviation SDS sodium dodecyl sulfate SNP single nucleotide polymorphism ST sequence type T threonine Taq Thermus aquaticus THY Todd-Hewitt broth supplemented with yeast TLR toll-like receptor TMB tetramethylbenzidine TPS two-partner secretion tRNA transfer ribonucleic acid TSA trypticase soya agar UDP uridine diphosphate Val valine WBC white blood cells WBCC white blood cell count WGS whole-genome sequencing

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Jan de Gans, Department of Neurology, Center of Infection and Immunity Amsterdam Contributing authors and affiliations (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

Frank Baas, Department of Genome Analysis, Academic Medical Center, University of Madelijn Geldhoff, Department of Neurology, Center of Infection and Immunity Amsterdam Amsterdam, Amsterdam, the Netherlands (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

Stefania Bambini, Novartis Vaccines and Diagnostics, Siena, Italy Sebastiaan G.B. Heckenberg, Department of Neurology, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Diederik van de Beek, Department of Neurology, Center of Infection and Immunity Netherlands Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Robert A.G. Huis in ’t Veld, Department of Medical Microbiology, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Stephen D. Bentley, Wellcome Trust Sanger Institute, Hinxton, United Kingdom Amsterdam, the Netherlands Sandra Bovenkerk, Department of Medical Microbiology, Center of Infection and Immunity Marja E. Jakobs, Department of Genome Analysis, Academic Medical Center, University of Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Amsterdam, Amsterdam, the Netherlands Netherlands Antoine H.C. van Kampen, Bioinformatics Laboratory, Department of Clinical Epidemiology, Matthijs C. Brouwer, Department of Neurology, Center of Infection and Immunity Biostatistics and Bioinformatics, Academic Medical Center, and Biosystems Data Analysis, Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Swammerdam Institute for Life Science, University of Amsterdam, Amsterdam, the Netherlands Netherlands Sara Comandi, Novartis Vaccines and Diagnostics, Siena, Italy Wendy Keijzers, Department of Medical Microbiology, Center of Infection and Immunity Maurizio Comanducci, Novartis Vaccines and Diagnostics, Siena, Italy Amsterdam (CINIMA) and Netherlands Reference Laboratory for Bacterial Meningitis, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Lisa De Tora, Novartis Vaccines and Diagnostics, Siena, Italy Peter van der Ley, Institute for Translational Vaccinology (InTraVacc), Bilthoven, the Arie van der Ende, Department of Medical Microbiology, Center of Infection and Immunity Netherlands Amsterdam (CINIMA) and Netherlands Reference Laboratory for Bacterial Meningitis, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Alessandro Muzzi, Novartis Vaccines and Diagnostics, Siena, Italy

Rachel Exley, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Yvonne Pannekoek, Department of Medical Microbiology, Center of Infection and Immunity Kingdom Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Floris Fransen, Institute for Translational Vaccinology (InTraVacc), Bilthoven, the Netherlands and Department of Infectious Diseases and Immunology, Utrecht University, the Netherlands Mariagrazia Pizza, Novartis Vaccines and Diagnostics, Siena, Italy

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Tom van der Poll, Center for Experimental and Molecular Medicine (CEMM) and Division of Infectious Diseases, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

Sadeeq ur Rahman, Section of Molecular Microbiology, Department of Molecular Cell Biology, Vrije Universiteit, Amsterdam, the Netherlands

Rino Rappuoli, Novartis Vaccines and Diagnostics, Siena, Italy

Barbara D.C. van Schaik, Bioinformatics Laboratory, Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, Amsterdam, the Netherlands

Kim Schipper, Department of Medical Microbiology, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

Lodewijk Spanjaard, Netherlands Reference Laboratory for Bacterial Meningitis, Department of Medical Microbiology, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

Peter van Ulsen, Section of Molecular Microbiology, Department of Molecular Cell Biology, Vrije Universiteit, Amsterdam, the Netherlands

Mercedes Valls Seron, Department of Neurology, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

Stijn van der Veen, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

Martijn Weisfelt, Department of Neurology, Kennemer Gasthuis, Haarlem, the Netherlands

Afshin Zariri, Institute for Translational Vaccinology (InTraVacc), Bilthoven, the Netherlands and Department of Infectious Diseases and Immunology, Utrecht University, the Netherlands

Aeilko H. Zwinderman, Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, Amsterdam, the Netherlands

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