Gram-Positive Anaerobic Cocci: Clinical Relevance, Changed Taxonomy, Identification and Antibiotic Resistance

Gram-positive anaerobic cocci: clinical relevance, changed taxonomy, identification and antibiotic resistance

Introduction Gram-positive anaerobic cocci Peptostreptococcus sp. (formerly)

New species and taxonomy

Identification © by author

Antibiotic resistance ESCMID Online Lecture Library Virulence F. magna

GPAC in general

Clinical relevance: ± 30% of all anaerobes recovered from human clinical specimens

Most GPAC: - mixed infections - susceptible for antibiotics usually used to treat anaerobic infections © by author

Metronidazole-resistance: ESCMID- strict anaerobic Online cocci are Lecturegenerally sensitive Library - micro-aerophilic cocci are generally resistent

Introduction

Gram-positive anaerobic cocci (GPAC):

Peptococcus P.niger Seldomly isolated from clinical material

Ruminococcus Faecel microbiota Seldomly ©isolated by fromauthor clinical material Insufficient culture methods?

ESCMIDCoprococcus Online Lecture Library Isolated from faecal samples

Gram positive anaerobic cocci, D.A. Murdoch Clinical Microbiology reviews, 1998

Peptostreptococcus

The genus Peptostreptococcus contained 13 species.

Clinically most relevant (literature): P. anaerobius P. asaccharolyticus P. magnus P. micros © by author ESCMID Online Lecture Library New species, changed taxonomy (in chronological order)

In 1997 3 new species were added to the genus Peptostreptococcus

P. harei P. ivorii P. octavius

Clinical relevance unknown© by author ESCMID Online Lecture Library Description of Three New Species of the Genus Peptostreptococcus from Human Clinical Specimens: P. harei sp. nov., P. ivorii sp. nov., and P. octavius sp. nov., D. A. Murdoch et al., Int. J. Syst. Bact. 1997 New taxonomy

Reclassification of Peptostreptococcus magnus (Prevot 1933) Holdeman and Moore 1972 as Finegoldia magna comb. nov. and Peptostreptococcus micros (Prevot 1933) Smith 1957 as Micromonas micros comb. nov. Murdoch et al., Anaerobe,5, 1999

Proposal of the genera© Anaerococcus by author gen. nov., Peptoniphilus gen. nov. and Gallicola gen. nov. for members of the genus Peptostreptococcus EzakiESCMID et al., Int J syst Online Evol Microbiol, Lecture 51, 2001 Library

Anaerococcus spp.

Anaerobic cocci: anaerobic cocci Finegoldia magna MicromonasParvimonas micramicros Gallicola barnesea Helcococcus kunzii Peptoniphilus spp.

P. anaerobius A rollercoaster of new species (1)

Species name year origin Peptostreptococcus stomatis 2006 Human oral cavity Peptoniphilus allenii 2006 Human clinical specimen Anaerococcus murdochii 2007 Human clinical specimen Peptoniphilus gorbachii 2007 Human clinical specimen Peptoniphilus olsenii 2007 Human clinical specimen Murdochiella asaccharolytica 2010 Human clinical specimen Peptostreptococcus russellii 2011 Swine manure Peptoniphilus methioninivorax© by 2011 author Retail ground beef Peptoniphilus koenoeniae 2011 Human clinical specimen ESCMIDPeptoniphilus duerdenii Online 2011 Lecture Human clinical Library specimen A rollercoaster of new species (2)

Species name year origin Peptostreptococcus canis 2012 Oral dog Anaerococcus obesiensis 2012 Human faeces Anaerococcus senegalensis 2012 Human faeces Peptoniphilus coxii 2012 Human clinical specimen

Peptoniphilus tyrelliae (senegalensis) 2012 Human clinical specimen Peptoniphilus grossensis 2012 Human faeces Peptoniphilus timonensis© by2012 author Human faeces Anaerococcus pacaensis 2013 Human clinical specimen ESCMIDPeptoniphilus obesi Online 2013Lecture Human faeces Library A rollercoaster of new species (3)

Species name year origin Peptoniphilus rhinitidis 2014 Human clinical specimen Peptoniphilus mikwangii 2014 Human clinical specimen Anaerococcus degenerii 2015 Human clinical specimen Anaerococcus nagyae 2016 Human clinical specimen Anaerococcus urinomassiliensis 2016 Human clinical specimen Anaerococcus rubiinfantis 2016 Human faeces Peptoniphilus catoniae © by 2016author Human faeces

Peptostreptococcus 4 species ESCMID Online LectureAnaerococcus Library 14 species Peptoniphilus 19 species Phenotypic identification

Enzymatic tests derived from the API 32A (BioMerieux) Table Wadsworth manual

Reliable identification: - F. magna - P. micra

P. harei – P. asaccharolyticus similar biochemical features

The addition of new species made© available by schemes author for the identification of GPAC less useful

ESCMID Online Lecture Library Identification of GPAC by MALDI-TOF MS Bruker system

• 54% of GPAC encountered in human clinical specimens is identified by MALDI- TOF MS (estimated)

• On target extraction with 70% formic acid is recommended

• A. vaginalis is misidentifed as A. hydrogenalis

• P. harei is correctly identified © by author • New species are not represented in de MALDI-TOF MS database ESCMID Online Lecture Library The optimization and validation of the Biotyper MALDI-TOF MS database for the identification of gram positive anaerobic cocci

A.C.M Veloo1, E.D. de Vries 1, H. Jean-Pierre3,4, U.S. Justesen5, T. Morris6, E. Urban7, I. Wybo8, A.J. van Winkelhoff 1,2, on behalf of the ENRIA workgroup Clin Microbiol Infect 2016; doi: 10.1016/j.cmi.2016.06.016

Added to database: Main Spectral Profiles of 108 clinical GPAC isolates, representing 17 different species

Validation database: © by author 140 clinical GPAC isolates ESCMID Online Lecture Library Dendrogram of MSPs created of strains belonging to the genera

Peptoniphilus and PeptostreptococcusMSP Dendrogram Peptoniphilus lacrimalis ENR0509 Peptoniphilus lacrimalis ENR0094 PeptoniphilusPeptoniphilus lacrimalis lacrimalis ENR0095 Peptoniphilus lacrimalis ENR0093 Peptoniphilus lacrimalis 0061 Peptoniphilus koenoeniae ENR0624 PeptoniphilusPeptoniphilus koenoeneniae koenoeniae CCUG 56067T Peptoniphilus duerdenii ENR0656 Peptoniphilus duerdenii ENR0664 2016 Peptoniphilus duerdenii ENR0650 2016 PeptoniphilusPeptoniphilus duerdenii duerdenii ENR0448 Peptoniphilus duerdenii ENR0415 Peptoniphilus duerdenii CCUG 56055T Peptoniphilus olsenii ENR0511 2016 Peptoniphilus olsenii ENR0510 2016 PeptoniphilusPeptoniphilus olsenii olseniiENR0449 2016 Peptoniphilus olsenii CCUG 53342T PeptoniphilusPeptoniphilus timonensis timonensis ENR0668 Peptoniphilus sp. ENR0666 PeptoniphilusPeptoniphilus sp. ENR0670 rhinitidis Peptoniphilus sp. ENR0665 Peptoniphilus harei ENR0002 2016 Peptoniphilus harei DSM 10021 DSM Peptoniphilus harei DSM 10020T DSM Peptostreptococcus harei ENR0001 Peptoniphilus indolicus ENR0402 PeptoniphilusPeptoniphilus harei ENR0424 harei Peptoniphilus harei ENR0423 Peptoniphilus harei ENR0003 Peptoniphilus harei ENR0564 Peptoniphilus harei 08_570 IBS Peptoniphilus harei 06_622 IBS PeptoniphilusPeptoniphilus tyrelliae tyrelliae CCUG 59621T Peptoniphilus gorbachii VA22133_12 ERL Peptoniphilus gorbachii ENR0602C Peptoniphilus gorbachii ENR0089 PeptoniphilusPeptoniphilus gorbachii gorbachii ENR0090 Peptoniphilus gorbachii ENR0091 2016 Peptoniphilus gorbachii ENR0088 Peptoniphilus grossensis ENR0663 Peptoniphilus grossensis ENR0662 2016 Peptoniphilus grossensis ENR0451 PeptoniphilusPeptoniphilus grossensis grossensis ENR0450 Peptoniphilus grossensis ENR0616 GPAC018B ENR0092 Peptoniphilus coxii ENR0102 Peptoniphilus coxii ENR0101 Peptoniphilus ivorii ENR0287 © by author PeptoniphilusPeptoniphilus coxii ENR0358 coxii Peptoniphilus coxii ENR0297 Peptoniphilus coxii 0118 Peptoniphilus coxii ENR0620 Peptoniphilus coxii 0113 Peptoniphilus ivorii ENR0177 Peptoniphilus ivorii ENR0486 PeptoniphilusPeptoniphilus ivorii ENR0296 ivorii Peptoniphilus ivorii DSM 10022T DSM Peptoniphilus indolicus DSM 20464T DSM PeptoniphilusPeptoniphilus indolicus indolicus 455 RLT Peptoniphilus asaccharolyticus DSM 20463T DSM PeptostreptococcusPeptoniphilus anaerobiusasaccharolyticus DSM 2949T DSM ESCMID Online Lecture LibraryPeptostreptococcus anaerobius ENR0006 Peptostreptococcus anaerobius ENR0005 Peptostreptococcus anaerobius ENR0004 PeptostreptococcusPeptostreptococcus anaerobius anaerobius HU33518_2 PNU Peptostreptococcus anaerobius HU28371_2 PNU Peptostreptococcus anaerobius DSM 20357 DSM Peptostreptococcus canis ENR0657 PeptostreptococcusPeptostreptococcus canis ENR0655 canis Peptostreptococcus stomatis ENR0097 Peptostreptococcus stomatis ENR0038 Peptostreptococcus stomatis 0112 PeptostreptococcusPeptostreptococcus stomatis stomatis ENR0526 Peptostreptococcus stomatis ENR0525 1000 900 800 700 600 500 400 300 200 100 0 Distance Level Dendrogram of MPSs created of strains belonging to the genera Anaerococcus, Murdochiella, Peptococcus, Finegoldia and Parvimonas MSP Dendrogram Murdochiella asaccharolytica 0066 MurdochiellaMurdochiella asaccharolytica asaccharolytica 0064 Murdochia asaccharolytica ENR0024 Anaerococcus tetradius ENR0441 Anaerococcus tetradius ENR0016 Anaerococcus tetradius ENR0326 AnaerococcusAnaerococcus tetradius tetradius DSM 2951T DSM Anaerococcus tetradius ENR0017n Anaerococcus tetradius 123 RLT Anaerococcus lactolyticus ENR0475 AnaerococcusAnaerococcus lactolyticus lactolyticus ENR0480 Anaerococcus lactolyticus ENR0440 Anaerococcus lactolyticus ENR0010x Anaerococcus lactolyticus DSM 7456T DSM Anaerococcus murdochii ENR0012 Anaerococcus murdochii ENR0011 Anaerococcus murdochii DSM 21462T DSM Anaerococcus murdochii ENR0134n AnaerococcusAnaerococcus murdochii murdochii ENR0025 Anaerococcus murdochii ENR0014x Anaerococcus murdochii ENR0622 Anaerococcus murdochii ENR0015 Anaerococcus murdochii ENR0013 nieuw AnaerococcusAnaerococcus gpac104 degenerii ENR0041 Anaerococcus degenerii ENR0615 Finegoldia magna DSM 20472 DSM Finegoldia magna 110706_D1 LUMC Finegoldia magna HU30296_1 PNU Finegoldia magna HU27914 PNU Finegoldia magna HU29644_1 PNU FinegoldiaFinegoldia magna magna DSM 20470T DSM Finegoldia magna HU29642_2 PNU Finegoldia magna DSM 20362 DSM Finegoldia magna DSM 20369 DSM Finegoldia magna 07_937 N5 IBS Finegoldia magna 06_469 IBS Parvimonas micra DSM 20468T DSM Parvimonas micra P6flue_re1AN USH Parvimonas micra P10bue_li5AN USH Parvimonas micra P10bue_li2AN USH ParvimonasParvimonas micra P10flue_li2ANmicra USH Parvimonas micra P10bue_li10AN USH Parvimonas micra CCM 3776 CCM Anaerococcus provenciensis ENR0675 nieuw AnaerococcusAnaerococcus provenciensis provenciensis ENR0667n Anaerococcus octavius DSM 11663T DSM AnaerococcusAnaerococcus octavius octavius DSM 10024 DSM Anaerococcus nagyiae ENR0686 AnaerococcusAnaerococcus nagyiae nagyae ENR0669 Anaerococcus nagyiae ENR0652 Peptococcus niger enr0661 e © by author Peptococcus niger ENR0353 Peptococcus niger enr0645 e PeptococcusPeptococcus niger ENR0087niger Peptococcus niger ENR0086 Peptococcus niger 458 RLT Anaerococcus prevotii ENR0535 Anaerococcus prevotii ENR0527 AnaerococcusAnaerococcus prevotii prevotii DSM 20548T DSM Anaerococcus prevotii DSM 20473 DSM Anaerococcus hydrogenalis DSM 7454T DSM AnaerococcusAnaerococcus hydrogenalis hydrogenalis 41 RLT Anaerococcus hydrogenalis 110706_C2 LUMC AnaerococcusAnaerococcus obesiensis obesiensis ENR0357 ESCMID Online Lecture LibraryAnaerococcus Anaerococcus senegalensis senegalensis ENR0455 Anaerococcus vaginalis DSM 7457T DSM Anaerococcus vaginalis ENR0135 Anaerococcus vaginalis ENR007 Anaerococcus vaginalis ENR0023 Anaerococcus vaginalis ENR0037 Anaerococcus vaginalis ENR0022 AnaerococcusAnaerococcus vaginalis vaginalis ENR0020 Anaerococcus vaginalis ENR0009 Anaerococcus vaginalis ENR0021 Anaerococcus vaginalis ENR0019 Anaerococcus vaginalis ENR0040 Anaerococcus vaginalis ENR0018 Anaerococcus vaginalis ENR0008 1000 900 800 700 600 500 400 300 200 100 0 Distance Level Results validation

Bruker database + ENRIA GPAC species ID Bruker database database no. of strains of which the log score ≥2 score >1.7 - <2 score ≤1.7 score ≥2 score >1.7 - <2 score ≤1.7 score increased P. harei (n=28) 19 8 1 28 0 0 26 F. magna (n=25) 16 8 1 16 8 1 0 P. micra (n=27) 25 2 0 25 2 0 0 A. vaginalis (n=8) 0 2 6 8 0 0 8 A. murdochii (n=5) 4 1 0 5 0 0 1 A. hydrogenalis (n=2) 0 0 2 0 2e 0 2 A. obesiensis (n=2) 0 0 2 0 0 2 0 P. anaerobius (n=8) 7 1 0 8 0 0 8 P. stomatis (n=2) 0 0 2 1 1 0 2 M. asaccharolytica (n=3) 0 0 3 3 0 0 3 P. coxii (n=6) 0 0 6 5 1 0 6 P. lacrimalis (n=3) 0 0 3 3 0 0 3 P. grossensis (n=2) 0 ©1 by 1author 1 1 0 2 P. lactolyticus (n=2) 1 0 1 2 0 0 1 P. gorbachii (n=2) 1 0 1 2 0 0 2 P. duerdenii (n=2) 0 0 2 2 0 0 2 differentESCMID GPAC species (n=4) 0Online 0 Lecture4 4 0Library 0 4 GPAC (9) 2 0 7 2 1 6 2

Total (n) 75 23 42 115 16 9 72 % of all strains 53.6% 16.4% 30.0% 82.1% 11.4% 6.4% 51.4% Resistance GPAC in the Netherlands 2002-2004

Antimicrobial susceptibility of clinically relevant gram-positive anaerobic cocci, collected in a 3-year period in The Netherlands Veloo et al., Antimicrob Agent Chemother, 2011;55:1199-1203 24 64 24 90 MIC © by author ESCMID Online Lecture Library Resistance GPAC in The Netherlands 2011-2013

Antibiotic susceptibility profiles of anaerobic bacteria in the Netherlands Veloo ACM, van Winkelhoff AJ Anaerobe 2015; 31:19-24

2011-2013 MIC90 >256

8 7 6 5 4 3 F. magna 2 P. micra 1 0 P. harei 2002-2004 MIC90

8 © by author7 6 5 4 3 F. magna 2 ESCMID Online Lecture1 Library P. micra 0 P. harei Evaluation of antibiotic susceptibility of gram-positive anaerobic cocci isolated from cancer patients of the N. N. Blokhin Russian cancer research center Shilnikova et al. J Pathogens 2015; article ID 648134

81 GPAC strains collected from 2004-2014

Variable resistance of the different species for:

- penicillin G - amoxicillin/clavulanic acid - metronidazole - ciprofloxacin - levofloxacin

Two metronidazole resistant ©P. micra by strains author

One multi-drug resistant (penG, cipro, levo, amoxi/clav, metro) F. magna strain ESCMID Online Lecture Library Antibiotic susceptibilities of gram-positive anaerobic cocci: results of a sentinel study in England and Wales Brazier JS et al. J Antimicrob Chemother 2003; 52:224-228

113 GPAC strains collected in 2002

Antibiotic % resistance

PenicillinG 7.1 Tetracycline 41.6 Erythromycin 27.4 Cefoxitin 0 Clindamycin 7.1 Chloramphenicol 0 Imipenem 0 Amoxicillin/clavulanic© by acidauthor 3.5 Piperacillin/tazobactam 0 Metronidazole 0 ESCMID Online Lecture Library F. magna

Infections in which anaerobic bacteria play a role:

30 % GPAC 30 % F. magna

Can be isolated in pure culture. Only Bacteroides fragilis is more often encountered in pure culture.

Not present in the oral microbiota Pathogenesis F. magna

Virulence factor Function

Collagenase breakdown collagen PAB binding to human serum albumin Protein L Immunoglobulin (Ig)-binding protein release© by of author “de novo-synthesized mediators” SufAESCMID Online degradation Lecture of fibrinogen Library degradation of antibacterial peptides release of FAF from bacterial cell wall Pathogenesis F. magna

Virulence factor Function

FAF mediation of bacterial aggregation binding with BM40, present on the skin blocking of an antibacterial peptide inactivation© by author of MIG/CXCL9 (=antibacterial peptide)

ESCMID Online Lecture Library Virulence and “site of infection”

Correlation between collagenase production and site of infection Krepel et al.(1991, 1992): F. magna (n) - 222 intra-abdominal samples 11 - 58 non-puerperal breast abscesses 21 - 56 diabetic foot infections 18

ESCMIDF. magna is the Online most common Lecture anaerobe isolatedLibrary from non- puerperal breast abscesses

Virulence and “site of infection”

Correlation between protein L producing F. magna strains and bacterial vaginosis Kastern et al. Infect immun 58: 1217, 1990

Total: 30 F. magna strains

- 19 from healthy© by women author - 4 from healthy men ESCMID - 7 from Online women with Lecture bacterial vaginosis Library 4 strains express protein L Virulence and “site of infection”

de Château et al. J Biol chem 271: 26609, 1996 Expression of protein L and PAB in 48 F. magna strains

- 30 strains from suppurative infections

1 strain protein L, 16 strains PAB

- 10 commensal strains ESCMID No expression Online of proteinLecture L or PAB Library

Protein L and PAB were not expressed at the same time. Message