Genomic Epidemiology and Characterization of Neonatal Meningitis Escherichia Coli and Their Virulence Plasmids Bryon Alex Nicholson Iowa State University

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2015 Genomic epidemiology and characterization of neonatal meningitis Escherichia coli and their virulence plasmids Bryon Alex Nicholson Iowa State University

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Genomic epidemiology and characterization of Neonatal Meningitis Escherichia coli and their virulence plasmids

Bryon Alex Nicholson

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Microbiology

Program of Study Committee: Ganwu Li, Co-Major Professor Lisa K Nolan, Co-Major Professor Qijing Zhang F. Chris Minion Gwyn Beattie

Iowa State University

Ames, Iowa

2015

TABLE OF CONTENTS

NOMENCLATURE ...... iii ACKNOWLEDGMENTS ...... iv ABSTRACT ...... v

CHAPTER 1 INTRODUCTION ...... 1 General biology of Escherichia coli ...... 1 Genetic diversity of E. coli ...... 3 Intestinal pathogenic E. coli ...... 7 Extra intestinal pathogenic E. coli ...... 7 Virulence plasmids of extra intestinal pathogenic E. coli ...... 18 Neonatal bacterial meningitis ...... 22 Pathogenesis of neonatal meningitis E. coli ...... 24 Role of adhesion and invasion in Neonatal Meningitis E. coli ...... 26 Immunomodulation by Neonatal Meningitis E. coli ...... 28 Aims and significance of this research ...... 31 Organization of this dissertation ...... 33 References ...... 33

CHAPTER 2 EPIDEMIOLOGICAL ANALYSIS OF NEONATAL MENINGITIS ESCHERICHIA COLI : GENETIC DISTRIBUTION, VARIANCE, AND RELATION TO OTHER EXTRA-INTESTINAL PATHOGENIC E. COLI ...... 47 Introduction ...... 48 Materials and Methods ...... 51 Results ...... 58 Discussion ...... 73 Acknowledgements ...... 84 References ...... 84

CHAPTER 3 LARGE VIRULENCE PLASMIDS IN NEONATAL MENINGITIS ESCHERICHIA COLI : PHYLOGENETIC RELATIONSHIPS AND CORE AND PAN-GENOME ...... 92 Abstract ...... 93 Importance ...... 94 Introduction ...... 94 Materials and Methods ...... 96 Results ...... 101 Discussion ...... 112 Acknowledgements ...... 117 References ...... 117

iii

CHAPTER 4 COMPLETE GENOME SEQUENCE OF THE NEONATAL MENINGITIS ESCHERICHIA COLI STRAIN, NMEC O18 ...... 121 Abstract ...... 122 Genome Announcement...... 122 References ...... 124

CHAPTER 5 COMPLETE GENOME SEQUENCE OF THE AVIAN PATHOGENIC ESCHERICHIA COLI STRAIN, APEC O18 ...... 125 Abstract ...... 126 Genome Announcement...... 126 References ...... 127

CHAPTER 6 CONCLUSION AND DISCUSSION ...... 128

NOMENCLATURE

ETEC Enterotoxigenic E. coli

EPEC Enteropathogenic E. coli

EHEC Enterohaemorrhagic E. coli

EAEC Enteroaggregative E. coli

DAEC Diffusely Adherent E. coli

EIEC Enteroinvasive E. coli

AIEC Adherent Invasive E. coli

ExPEC Extra intestinal Pathogenic E. coli

NMEC Neonatal Meningitis E. coli

APEC Avian Pathogenic E. coli

UPEC Urinary Pathogenic E. coli

NBM Neonatal Bacterial Meningitis

BBB Blood-Brain Barrier

CNS Central Nervous System

CSF Cerebrospinal Fluid

FimH Fimbrial protein H

OmpA Outer Membrane Protein A

NF-κB Nuclear Factor Kappa-light-chain-enhancer of activated B cells

HBMEC Human Brain Microvascular Endothelial Cells

PMN Polymorphonuclear

ACKNOWLEDGMENTS

First and foremost, I would like to thank my major professors, Dr. Ganwu Li and

Dr. Lisa K. Nolan, for allowing me the opportunity to work and learn with them. A special thanks to Yvonne Wannemuehler, who provided me with my initial laboratory training and constant support throughout my research. I would also like to Dr. Catherine

Logue for her tireless efforts in teaching me experimental methods and providing editorial input, and Dr. Nicolle Barbieri for generously assisting me in performing cell culture and animal assays and being a constant source for thought-provoking discussion and ideas.

I would also like to thank my committee members: Drs. Chris Minion, Qijing

Zhang, and Gwyn Beattie for contributing their time, knowledge, expertise, and support.

Finally, I would like to thank my wife, Jennifer, my parents, and the rest of my friends and family for their encouragement and support.

ABSTRACT

Neonatal meningitis Eschericha coli (NMEC), a sub-pathotype of extra intestinal

E. coli (ExPEC) , is one of the most common causative agents of neonatal bacterial

meningitis (NBM). NBM is a devastating disease affecting neonates less than four weeks

old, causing acute onset of sepsis and invasion of the central nervous system. Without

treatment, NBM is almost certainly fatal, but early intervention and empiric treatment, as

well as new broad spectrum antibiotics, have reduced the mortality rates from 32% in

1975 to as low as 8.5% in the United States.

Recent studies have greatly improved knowledge of NMEC pathogenic

mechanisms by characterizing multiple NMEC virulence factors; however, nearly all

experiments were performed using a single NMEC strain, RS218, and its derivative

mutants. Broader studies examining larger populations of NMEC have begun to show

that some of the virulence factors critical for NMEC RS218 pathogenesis are uncommon

in other NMEC, while other virulence factors identified using NMEC RS218 are present

in multiple E. coli sub-pathotypes and even non-pathogenic E. coli. Such findings beg

the questions, what genes define the NMEC sub-pathotype? How diverse are members of

the NMEC sub-pathotype? And can another NMEC strain, other than NMEC RS218,

serve as a more representative prototype of the NMEC group?

Additionally, other studies have found that some Avian Pathogenic E. coli

(APEC) strains can cause NBM in the rat model of human disease, while some NMEC

are able to cause disease in the day-old chick model of avian colibacillosis. Such

findings suggest that NBM and avian colibacillosis, caused by ExPEC, could be zoonotic vii

diseases, a contention that requires further study for both the benefit of human and animal

health.

In sum, these studies challenge current definitions of ExPEC sub-pathotypes.

This dissertation focused on on generating high quality genomic and plasmid sequences

for a random population of NMEC and analyzed these data in silico to better define the

NMEC sub-pathotype.

Initial characterization began with sequence and assembly of a population of 48

NMEC strains from the United States and Europe, searching for novel virulence and resistance factors. As part of this comparative study of NMEC strains, a polymorphic analysis of ubiquitous E. coli genes, thought to contribute to the pathogenesis of ExPEC diseases, but also present in non-pathogenic E. coli was undertaken. Analysis of the results showed that genes that are strongly linked to NMEC virulence in the literature, such as cnf1, sfaS, sfaC, and traJ , were found in less than 50% of the NMEC surveyed, while the K1 capsule, aslA, chuA , and papX were found in more than 75% of the strains.

Analysis of the sequences of the ubiquitously occurring and well-known genes, fimH and ompA , demonstrated that both harbored polymorphisms in sites important to protein activity and substrate binding. When these NMEC polymorphisms were compared to those in other ExPEC sub-pathotypes (namely, uropathogenic E. coli and APEC), distinct polymorphic patterns were discerned that differentiated members of the three ExPEC sub-pathotypes. This finding is quite intriguing, as these genes encode a number of key processes in ExPEC pathogenesis, suggesting that they could explain tissue tropism, host specificity and/or capability of a strain to cause a specific syndrome. Future study will need to be done to explore these issues. viii

In addition to comparative analysis of NMEC chromosomes, the large putative virulence plasmids of several NMEC isolates were sequenced and compared to define the core and pan-plasmidome of NMEC. These sequences were also scanned for novel virulence and resistance genes, and the sequences compared to one another and to plasmids sequences of other ExPEC sub-pathotypes. Results show NMEC plasmids sequenced in this dissertation were genetically similar to plasmids of APEC strains.

Neither core plasmid polymorphism phylogenies nor differences in plasmid-borne genes could distinguish APEC plasmids from NMEC plasmids.

Finally, high quality complete genomic sequences for NMEC O18 and APEC

O18 strains with zoonotic potential were generated. NMEC O18 is a highly virulent

NMEC strain able to cause similar disease in the rat model of human neonatal meningitis.

Interestingly, it lacks cnf1 and traJ , both thought to be crucial for NMEC pathogenesis.

APEC O18 is highly virulent in chick embryos and day old chicks but is also highly

virulent in the rat model of human meningitis. This paper reports the first sequence of an

APEC O18 strain causing neonatal meningitis at the same level as NMEC O18 and

RS218. 1

CHAPTER 1. INTRODUCTION

Escherichia coli is a diverse and prolific species of bacteria commonly found in the environment and inhabiting the gastrointestinal tract of mammals (1). While most E. coli are harmless, some possess the potential to cause disease in humans and animals. To distinguish between different disease causing isolates, pathogenic E. coli are commonly grouped into pathotypes, or groups of isolates that cause the same or similar disease. One group of these pathotypes, Extra intestinal Pathogenic E. coli (ExPEC), are able to cause a range of diseases in different hosts, including colibacillosis in poultry (2), urinary tract infections, neonatal meningitis and sepsis in humans and other animals such as neonatal pigs and dogs (3-5). Collectively, these diseases are a major burden to the healthcare costs annually (6). Urinary tract infection (UTI) symptoms caused approximately 10.5 million doctor visits, and approximately 2.5 million emergency room visits, in the year

2007 alone (7). After including costs for lost work productivity, the estimated costs of

UTI infections in the United States is estimated at 3.5 billion dollars per year as of 2007

(2, 6-8).

General Biology of E. coli

Escherichia coli are Gram-negative, facultative anaerobic, rod-shaped bacteria belonging to the family Enterobacteriaceae of the Proteobacteria phylum (1).

Escherichia. coli are non-spore forming and may be motile through possession of flagella

(9), occurring in a peritrichous arrangement, or more rarely through twitching motility mediated by pili (10). Growth temperature ranges and optimums vary based on each individual strain; however, optimal growth occurs at 37°C (1) with the typical strain 2 showing at least minimal sustained growth between 7°C and 42°C (11, 12). Classically,

E. coli are identified by their ability to ferment lactose, produce β-glucosidase, and

convert tryptophan to indole, while not possessing cytochrome C oxidase, creating an

oxidase test negative phenotype (1, 13, 14).

Escherichia coli possess a wide range of metabolic genes and commonly use a diverse set of carbon sources including D-galactose and glycerol (1), as well as some amino such as α-ketoglutarate acid, pyruvate, or oxaloacetate (1). Escherichia coli

generally can metabolize amino acids and grow in minimal media containing as few

chemicals as glucose, ammonium chloride, magnesium sulfate, and calcium chloride

(15); however, some strains may be natural or engineered auxotrophs (16, 17).

Engineered E. coli auxotrophs are often used for research, including metabolic

engineering (18), unknown gene function research (19), or specific amino acid synthases

to select for a specific mutation or maintenance of genetic components such as a

recombinant plasmid (17, 19).

A major agent of genetic diversity in E. coli is horizontal gene transfer of mobile

genetic elements, most notably, plasmids (20), and less commonly through phage

transduction of genes (21). Plasmids, as well as bacteriouphage islands, are a common

feature of many E. coli (21); both often contain critical components for E. coli

pathogenesis (20, 22). In general, E. coli are not competent under laboratory conditions

(23), and are unable to maintain imported extracellular DNA due to nucleases such as

those encoded by recBCD that degrade linear DNA strands; however, degraded

nucleotides can be used for cellular processes (24). Given that E. coli does possess genes 3 to import linear DNA, some have suggested E. coli may be naturally competent in conditions that are currently unknown (25-28).

Genetic Diversity of E. coli

E. coli , including, Shigella spp., is an exceptionally diverse genus of bacteria and shows genetic heterogeneity, with genomes ranging in size from 4.56 Mbp to 5.7 Mbp, a

20% difference in size (29). This size difference hints of the diversity of E. coli . In the

1960s, genomic relatedness was determined using DNA-DNA hybridization, with a cutoff of 70% being set to delineate species (30). Since that time, other methods, such as restriction length polymorphism analysis and pulse field gel electrophoresis analysis have been used to determine genetic relatedness (31), but perhaps, the most definitive means to assess E. coli strains’ relatedness is the use of full genome sequence comparisons (32-

34).

Initial genomic comparisons were carried out for the first publicly available E. coli sequences, pathogenic E. coli strains EDL933 (EHEC) released in 2001, CFT073

(UPEC), released in 2002, and the laboratory adapted strain MG1655 released in 1997

(35). These results showed a 39.2% overlap in protein coding genes, giving the first indications that E. coli were a much more diverse species than previously thought, and well beyond the theoretical 70% cutoff value (36). Further, it was found upon comparison of fragmented sequences of UPEC strains J96 and 536 that the evolution of

UPEC had occurred independently from different lineages (36).

A later study examining 61 sequences of non-pathogenic and pathogenic E. coli of various pathotypes revealed that the maximum common E. coli genome was 4 approximately 1 Mbp (~20%), less than the difference in genome size between some E. coli strains (21). Further, this study showed a pan-genome of 16,000 gene families, while the typical E. coli genome only contains 5,000 genes. Given the large diversity among E. coli strains and the recognition that virulence in E. coli had evolved independently multiple times, scientists in E. coli genomics cast doubt on the idea that single or small groups of representative isolates could adequately represent the species or pathotype (29).

E. coli can be classified in several ways, e.g., by assignment to serotype or serogroup, pathotype, multilocus sequence type and phylogenetic group (29, 32, 37-40).

Originally, serotype was based on recognition of antigen of the lipopolysaccharide of the outer membrane, K capsular antigen and flagellar, and/or H antigen by certain antisera

(41); whereas, serogroup was based on the recognition of O antigen alone (1). Recently, however, a shift is occurring away from more time-consuming and difficult serology methods to faster and sometimes cheaper DNA-based or computational methods (42).

Serotyping of E. coli strains is performed by agglutination assay of specific rabbit antisera in response to multiple antigens on the bacterial cell surface: the O antigen,

(n~173) consisting of the oligosaccharide portion of the exterior lipopolysaccharides coating the bacterial surface LPS; the K antigen, (n~60) consisting of the acidic capsular polysaccharide layer; and the H antigen, (~53) characterizing the flagella (1, 37, 41). The combination of O, K and H typing is commonly known as a serotype, while the O antigen type itself is referred to as the serogroup (41). This method historically has suffered many difficulties, as cross reactions between O types were common, as well as cross reactions between O and K antigens (1, 41), in part due to some capsular chains being bound to the cell membrane (41). Even though cross reactions exist, serotyping has 5 historically been used in E. coli since 1943 when it was described by Kauffmann (43),

and has provided typing based on surface factors involved in virulence processes such as

attachment and immune recognition. Serotyping is still a common and important method

of discriminating among E. coli strains .

Though a large number of serotypes exist, fewer serotypes or serogroups are commonly observed in infections, and can be associated with specific pathotypes. The most well-known example being enterohemorrhagic E. coli strain serotype O157:H7, though serogroups O26, O103, O111, and O145 currently comprise the majority of

EHEC strains (44). This pattern holds true to varying degrees for all pathogenic E. coli , with example pathotypes containing common serogroups including enteroaggregative E. coli (O3, O15, O44, O86, O119, and O125) (1), UPEC (O1, O2, O4, O6, O7, O8, O16,

O18, O25, and O75) (45), NMEC (O1, O6, O7, O8, O18, and O83) (46), and APEC (O1,

O2, O25, O78 and O119) (47).

The phylogenetic type of an E. coli strain was initially based on multilocus enzyme electrophoresis (MLEE) profiles of 10 genes, with each disease or pathotype using individual numbering systems for enzyme mobility; which were eventually combined into a single system in 1997 (31). Studies by Whittam with MLEE were the first to statistically link frequencies of 16 possible observed allele combinations to four major alleles, A, B, C, and D, (48), with occasional subvariations including E, B1, B2,

B3, and A1 (49, 50). These groups were refined to A, B1, B2 and D following inclusion of more and diverse samples (51). These alleles have since been termed Phylogenetic groups, and PCR amplification methods eventually replaced MLEE due to PCR needing fewer control strains, and being overall simpler and faster (39). Today, seven major 6 phylogenetic groups exist (A, B1, B2, C, D, E, F), and PCR is a primary method for phylogenetic typing, correctly assigning 99.5% of strains to the proper phylogenetic group (40). Finally multilocus sequence type (MLST) methods specific to E. coli with publicly accessible databases were first published in 2005 (52). Currently two E. coli

MLST databases are widely used, and may also be used to determine phylogenetic typing

(40, 52, 53). The recently formed phylogenetic group C further separates A and B1

strains, while group E enhances resolution between phylogenetic group B1 and group D.

Phylogenetic group F represents a sister lineage to B2 strains.

Such groupings give real insight into the structural make-up of different E. coli types and their evolutionary background. Typically, enterohemorrhagic E. coli belong to phylogenetic groups B1 and E (54), while human ExPEC belong to phylogenetic group

B2, and to a lesser extent, D (55, 56). APEC were previously classified as primarily phylogenetic groups A and D (~30%) with some belonging to groups B1 and B2 (~15%)

(56); however, this classification is not correct. Original study of E. coli strains using

MLEE and the original 4 phylogenetic group PCR were based only on a small collection of environmental and non-pathogenic isolates combined with isolates from human UTI and meningitis (39). Excluding E. coli causing animal disease, such as APEC, biased the phylogenetic typing scheme, and thus many APEC were improperly classified as groups

A and D. Reclassification of APEC using the revised 7 major group method (40) resulted in massive APEC phylogenetic group rearrangements, with phylogenetic groups A and D shrinking to 11% and 5% incidence respectively. Phylogenetic group C became the most commonly occurring group among APEC (27%), while the incidence of groups B1 and 7

B2 stayed consistent with group F joining at 20%. Less than 1% of strains grouped as untypeable (unpublished data).

Intestinal Pathogenic E. coli

Intestinal pathogenic E. coli, also known as diarrheagenic E. coli, are a subset of pathogenic E. coli causing disease localized to the mammalian intestines. Currently, intestinal pathogenic E. coli are categorized into six major pathotypes. Each uses unique mechanisms to cause disease in the gastrointestinal tract of humans and some other animals, but utilize common host factors to cause disease. Many of these pathogenic strategies, such as cellular adhesion and invasion, are shared between intestinal pathogenic E. coli , but the mechanisms used to accomplish these goals differ and are not covered in this literature review.

Extraintestinal Pathogenic E. coli

Intestinal pathogenic E. coli are a diverse set of organisms containing functionally distinct mechanisms enabling the organisms to cause disease. These distinct mechanisms, though hybrid strains do exist, allow classification into functional groups and types of intestinal pathogenesis. In the case of ExPEC, heterogeneous distribution of virulence genes between pathotypes is common, making distinctions between ExPEC pathotypes more problematic and more reliant on the syndrome, tissue and host from which the strain was isolated (46, 57-59). There are, however, some trends in the presence of virulence genes by an ExPEC sub-pathotypes, likely emphasizing the important of certain traits in causing certain syndromes. Notably, many ExPEC pathogens possess a limited number of serotypes, and commonly produce sialic acid type

K1 capsules. 8

Uropathogenic E. coli (UPEC)

UPEC are the most common causative agent of urinary tract infections throughout

the world, accounting for an estimated 80% of all urinary tract infections (6, 60). Urinary

tract infections predominantly affect women with as many as 40% of women

experiencing a urinary tract infection at some point in their lives (6, 7). Once a urinary

tract infection occurs in an individual, the person is more likely to become infected again,

with a quarter of patients experiencing a second infection less than a year after clearance

of the first infection (61). In general, UPEC infections are not life threatening; however,

UPEC infection can ascend through the urinary tract to the kidneys, resulting in

pyelonephritis, or into the bloodstream causing urosepsis (62). The latter two infections

are quite serious, as pyelonephritis may cause permanent kidney scarring and loss of

function or renal failure (60, 63), while the mortality rate from urosepsis ranges from 20

to 40% and is likely higher in older patients, patients with diabetes, or other

complications involving the urinary tract (64).

It is thought that UPEC originate from the colon, contaminate the perineum and

enter the urethra to cause infection (60). From there, UPEC ascend through the urethra to

enter the bladder, where they cause cystitis (60). While in the bladder, attachment to host

cells, followed by cellular invasion, is critical for UPEC virulence (65). Strong

attachment to host cells is especially critical to avoiding washout due to large volume

discharges (66). For these reasons some UPEC strains possess more than 10 fimbrial

operons to facilitate adherence, including P, type 1, F1C, S, Dr, and M (67). Certain

UPEC fimbriae undergo phase variation in response to environmental cues (68). Phase

variation within populations is also hypothesized to increase the probability of adherence 9 to different host tissues (68). Studies have shown that while many fimbriae exist in

UPEC, type 1 fimbriae are largely responsible for adherence to uroepithelial cells, with the fimbrial tip adhesin, FimH, playing a critical role in UPEC colonization (66, 67, 69).

FimH adhesin largely mediates mannose recognition, including recognition of mannose- containing glycoproteins on the surface of host cells (70). This variability in FimH facilitates binding to a wide range of host receptors and substrates, and may contribute to a wider host range (71). Interestingly, though fimH is a critical virulence gene for UPEC, it is found widely among pathogenic and non-pathogenic strains of E. coli alike (71), begging the question, are there differences in the structure of fimH in pathogenic and non-pathogenic E. coli enabling the pathogens to adhere to a broad spectrum of host receptors, potentially increasing host range as well as possibly reducing immunogenic residues? Recently, further analysis of fimH variants from urinary tract infections showed amino acid residues critical for mannose binding were conserved, but variable amino acid sequences were found in other positions. Analysis of these polymorphisms showed different fimH alleles possess significantly different levels of binding and adherence when assayed in the same genetic background, and indicated specific polymorphisms common for UPEC strains may increase binding fitness in the urogenital environment (69).

Toxins are also important virulence factors in UPEC pathogenesis and include those encoded by hlyA , vat/sat , and cnf1 (65). UPEC, distinct from some intestinal pathogenic E. coli , do not commonly possess a functional type III secretion system.

Therefore, UPEC toxins are either excreted through type I or IV secretion systems or encode their own transport (65). HlyA is an α-hemolytic protein toxin, which causes cell 10 lysis at high concentrations, while at lower concentration its effects on host cells are more subtle, causing activation of MAP kinase, calcium oscillation signals, histone modification, and inactivation of the Akt kinase (65, 72). Vat, also known as Sat (73), is a serine protease autotransporter protein toxin shown to cause elongation of urinary epithelial cells and loosening of cell junctions, which is thought to increase cellular invasion or entry into the bloodstream (73). Cnf1 is a dermonecrotic toxin, so named for its first recognized phenotypes (74). Cnf1 binds to epithelial cell laminin receptors and heparin sulfate proteoglycan co-receptors, where it is endocytosed and transported to the endosomal compartment (75). Once in the endosomal compartment, the toxin inserts into the endosomal membrane, and the active toxin is released into the cell cytoplasm, where it causes constitutive activation of Rho GTP-binding proteins (76), inducing stress fiber formation, which plays a critical role in multiple cellular adhesion processes (cell adhesion, barrier formation, tissue structure) and cell shape (77).

Evasion of host immune responses is another critical component for UPEC pathogenesis in the urinary tract. Immune evasion is in many cases mediated by adhesins and toxins. Examples of this include FimH-mediated adherence and invasion of bladder epithelial cells (78) or HlyA-deactivation of Akt kinase, causing reduced vesicle trafficking, inflammation signaling, and decreased leukocyte phagocytosis and chemotaxis (65) (79). Genes more specific for immune invasion include O antigen LPS genes and LPS recycling genes, which function to reduce pathogen-associated molecular patterns (80), biofilm synthesis and export by capsular genes such as kpsT and kpsM (81).

The outer membrane protein OmpA is also thought to increase serum resistance. Studies examining UPEC strains and NMEC strains have shown OmpA facilitates binding to 11 macrophages as well as invasion of macrophages (82), binds complement protein C4bp to facilitate degradation of complement proteins C3b and C4b (83), and prevents transcription of pro-inflammatory cytokines and chemokines by preventing phosphorylation and degradation of inhibitor κB proteins (84).

Nutrient acquisition genes comprise another group of important UPEC virulence

genes. In the extraintestinal environment, key nutrients for growth are often sparse,

making nutrient acquisition a critical need (85-87). Indeed, many host mechanisms exist

to reduce the bioavailability of key nutrients such as iron, and iron sequestration is a

common defense mechanism for host-pathogen resistance (86). While most bacteria

contain mechanisms to acquire iron from the environment (88), UPEC possesses multiple

systems, commonly encoding the siderophore systems for enterobactin, salmochelin,

aerobactin, and yersiniabactin (65). In addition, UPEC commonly possess the sit metal

transport operon and the chu operon, which encodes utilization of heme or hemoglobin

(65), as opposed to intestinal or non-pathogenic E. coli , which generally encode fewer

metal acquisition operons (89). Despite its high affinity for iron, enterobactin is bound

and inactivated by the human protein lipocalin 2, while salmochelin (encoded by the iro

operon) has been shown to function in the presence of lipocalin 2 and may enhance in-

vivo iron acquisition (90). UPEC’s possession of multiple uptake systems likely

enhances fitness in many environments, including the urinary tract, and offers functional

redundancy to promote survival in inhospitable environments (87, 91, 92).

Similar to iron acquisition, UPEC strains frequently carry other metabolic genes hypothesized to confer a selective advantage for survival, including D-serine deaminase, tetrathionate reductase, urease, and α-ketoglutarate import and utilization enzymes (19, 12

93, 94). These genes are often found on genomic islands, which have been horizontally transferred from other pathogenic species, such as Salmonella enterica subsp. enterica serotype Typhimurium (tetrathionate reductase) or Helicobacter pylori (Urease). D- serine deaminase and its associated permease and regulatory protein are encoded by the operon dsdCXA . This operon allows for the import and utilization of D-serine as a sole carbon and nitrogen source converting by D-serine to pyruvate and ammonia. While D- serine is found in nature, it is much less common than the L-amino acid enantiomers (95).

In humans, it is found only in brain, plasma, and urine, which are locations associated with ExPEC infection, and UPEC has been shown to alter gene regulation in response to

D-serine exposure (96). Tetrathionate reductase, encoded by the gene cluster ttrABCRS , encodes a two component regulatory system (94) as well the ttr gene cluster is shown to facilitate anaerobic B 12 -dependent utilization of tetrathionate to thiosulfate, which can

further be reduced to H 2S in Salmonella ; however, genes responsible for the further

reduction to H 2S are not generally present in E. coli . This absence is a trait commonly used in metabolic identification of E. coli (1). More recent studies of tetrathionate in S.

Typhimurium have shown that tetrathionate mediated resistance to NADPH oxidase and

nitric oxide radicals, both of which are commonly used by the host to inhibit or eliminate

bacterial infections (97).

Urease has been previously reported to be produced by some pathogenic E. coli

strains such as EHEC (98), but often the urease-producing operon ureDABCEFG is

incomplete or non-functional. Some UPEC strains, containing the Helicobacter genomic

island, can produce urease under anaerobic, nitrogen-limited conditions, generating an

alternative nitrogen source, and promoting inflammation of the bladder epithelium (19). 13

Finally, an α-ketoglutarate utilization system has been shown to be present in 70% of a

sample of UPEC (19). This system encodes a two-component regulatory system that

responds to α-ketoglutarate and regulates expression of 8 genes (19). The 5-carbon compound α-ketoglutarate is a citric acid cycle intermediate and is used as an organic anion in the concentration gradients of the kidney’s proximal tubules for antiport of organic anions (99). Chlamydia trachomatis , another intracellular uropathogenic organism, also uses α-ketoglutarate for metabolism (100). This organism actually lacks

the citric acid cycle enzymes citrate synthase, aconitase, and isocitrate dehydrogenase

which are necessary to generate α-ketoglutarate (100). These genes when taken together

show specific adaption to the urinary tract environment, and with multiple mechanisms of

nutrient acquisition by these organisms.

In conclusion UPEC strains are heterogeneous and diverse, possessing a multitude

of virulence factors and other characteristics that contribute to uropathogenesis. While

intestinal pathogenic E. coli often possess well-defined virulence genes encoding well-

characterized mechanisms for virulence, UPEC strains possess a large variety of

virulence genes. Many of these virulence genes encode functionally similar or redundant

proteins, while other genes encode virulence factors essential or significantly contributing

to pathogenesis. These are shared with non-pathogenic E. coli and are present in many E.

coli sub-pathotypes. These indicate the population of UPEC likely possesses a large

collection of virulence factors not all of which are necessary for pathogenicity, and

distinct combination of virulence genes are likely responsible for variable severity in

UPEC infections (29).

Avian Pathogenic E. coli

Avian pathogenic E. coli (APEC) is a common sub-pathotype of ExPEC and one of the leading causes of mortality and morbidity in the poultry industry for broiler and layer chickens, turkeys, ducks and other production birds worldwide (101, 102). APEC cause colibacillosis, which is an umbrella term encompassing a wide range of systemic and localized infections (101). Some of the most severe infections for the health of the bird involve septicemia, while other infections like cellulitis or Swollen Head are more localized (103). Regardless, these conditions are responsible for major losses to the poultry industries due to mortality, morbidity, loss of production and condemnations

(104).

APEC is thought to enter the bird in different ways such as via inhalation of contaminated dust, through abraded skin, via cracks in embryonating eggs, and by retrograde movement of bacteria from the cloaca (2). Colibacillosis is likely to be both a primary and secondary disease depending on the strain of APEC involved in infection

(101). Some APEC are very well equipped pathogens while others are likely relatively avirulent, but because of predisposing infections, are able to cause disease (101). Such predisposing conditions can involve high ammonia and dust levels or previous infection with pathogens such as Mycoplasma spp. or Newcastle virus (105). These stressors are thought to weaken the respiratory immune response and/or damage tracheal cilia, enhancing APEC’s ability to colonize the respiratory tract (2, 105).

When APEC use a respiratory route in infecting birds, the organisms are inhaled, and colonize the upper and lower respiratory tract to cause various respiratory conditions such as airsacculitis. From there, APEC can gain entry to submucosal tissue, where they 15 cross to the bloodstream by unknown mechanisms. Once in the bloodstream, APEC cause septicemia, leading to development of pericarditis and perihepatitis. As a result,

APEC may be isolated from the brain, spleen, and bone of infected birds, as well as in blood, urine and synovial fluid (106).

APEC’s diversity is also seen in its possession of known and putative virulence genes, with APEC varying greatly in their virulence genotypes (102, 107, 108). APEC harbor a wide range of potential virulence factors, but their role of these factors in pathogenesis is unknown. Similar to UPEC, APEC often demonstrate redundancy of virulence genes performing critical functions during pathogenesis (87, 109, 110). APEC strains frequently possess large ColBM and ColV plasmids which carry pathogenicity islands that contain many virulence genes known to contribute to APEC pathogenesis

(102, 111-115). These plasmids serve as one of the most distinguishing characteristics separating APEC strains from fecal E. coli strains and are present in over 90% of APEC strains. As in UPEC, virulence factors of APEC can be broadly categorized as adhesins, toxins, and metabolic genes.

Adhesins represent one of the better-defined APEC virulence factors, although much is still unknown. The fimbrial proteins Stg, P, Yqi, type I, Curli, E. coli common pilus, as well as the temperature sensitive hemagglutinin protein Tsh, have all been implicated as important APEC virulence factors, with one study finding the Tsh protein as well as the Yqi and Stg pili are necessary for APEC colonization of the trachea and lung specifically (116). The other adhesion factors have been shown to reduce virulence in strains, their specific functions are yet unknown (117). The adhesion and invasion gene, ibeA, has also been implicated as an APEC virulence gene, functioning to resist 16 oxidative stress (118), but again, this gene is only found in 14% of APEC strains

(unpublished data).

According to genotypic studies of APEC strains, toxins are uncommon in APEC strains, with the only confirmed toxin being the vacuolating autotransporter toxin Vat also found in UPEC (47, 73). The vat gene’s prevalence in APEC strains is approximately 33% (102) and was found to be an unreliable target for APEC identification. While this gene is uncommon, another study has found that in APEC strain Ec222 the vat gene is necessary for pathogenesis (119); however given the absence of this gene in a large majority of APEC strains indicates this toxin is not a requisite virulence factor. Further, the lack of novel toxin discovery through sequencing of multiple diverse APEC, and the high incidence of virulence plasmids not containing toxins, suggests toxins may not generally be involved in APEC pathogenesis.

APEC, like other ExPEC, possess unique metabolic genes to help survive in the extra intestinal environment. Iron acquisition genes are exceptionally prevalent in APEC, with aerobactin, salmochelin, and enterobactin commonly found in APEC strains. The salmochelin and aerobactin siderophores are commonly found on the aforementioned virulence plasmids. APEC also commonly possess the putative metal transport gene cluster sitABC (96%). Further studies show deletion of the phosphate-specific phosphate transport system operon pstCAB gene causes a reduction in virulence in APEC (120); however, this study indicated that the pst operon may serve as a primary phosphate

sensor, and mutation of the pst operon causes constitutive expression of the pho operon.

The pho operon is a two-component regulatory operon known to regulate 39 genes, and

potentially regulates an additional 98 genes. Though this gene deletion causes 17 attenuation, interference with one or more regulatory systems precludes correlating the attenuation with a specific gene. Another study found expression of nirC from strain

SCI-07 in vivo was significantly upregulated compared to growth in regular serum and hypothesized the gene may play a role in downregulating macrophage production of nitric oxide. Further analysis showed the gene played a highly nuanced role in virulence causing increased expression of the nitric oxide resistance genes hmp and ytfE , but

overall decreasing motility important for colisepticemia, in the wild type strain. Net

effects of nirC deletion delayed infection onset, but increased duration of infection, lesion

score, and increased bacterial counts in organs and macrophage.

Overall, the specific mechanisms of APEC pathogenesis are poorly understood,

and efforts to determine pathogenicity are hampered by strains possessing diverse genetic

content. Some characterization has been performed to test specific toxins and adhesins

and metabolic genes, but the majority of this work has been done for genes that are not

pervasive in APEC strains. Currently, common and confirmed APEC virulence factors

include only the ColV/ColBM plasmids that encode siderophores, a putative adhesin gene

tsh , and a putative serum resistance gene iss . Much study is still needed to determine

specific pathogenic mechanisms for APEC.

Neonatal Meningitis causing E. coli

Neonatal meningitis E. coli (NMEC) is the final major sub-pathotype of extra

intestinal E. coli and is primary focus of this dissertation . NMEC is included here for

completion. A detailed analysis of NMEC pathogenesis begins on page 24.

Virulence Plasmids of Extra-Intestinal Pathogenic E. coli

Plasmids are extrachromosomal self-replicating genetic elements most often transferrable, and are categorized either by their incompatibility/replicon type or, for E. coli plasmids, by which colicins are produced (20). Plasmids are often a source of genetic diversity in microbial populations, granting enhanced fitness under specific environmental conditions by providing accessory metabolic, virulence, and/or resistance genes. Virulence plasmids are known to play a significant role in the pathogenesis of many E. coli including ETEC, EAEC, EIEC, EHEC, EPEC, and ExPEC (20) and in general code for toxins, adhesins, and/or other virulence enhancing factors. Among

ExPEC pathotypes, virulence plasmids are defining characteristics of APEC and NMEC, generally 90% or greater prevalence has been reported (112). Virulence plasmids are less common in UPEC, with indications they may be present in approximately 30% of UPEC strains (121). Three major types of plasmids are found in ExPEC: colicin V producing

(ColV), colicin B and M producing (ColBM), and Vir plasmids, all of which are in the

RepFIB/FIIA incompatibility groups (20), and generally carry virulence genes. This section will detail the proposed virulence phenotypes of ColV, ColBM, and Vir plasmids, highlighting that although these virulence phenotypes and genes are common, they are not shared by all plasmids.

Colicin V and Colicin BM Virulence Plasmids

ColV virulence plasmids have been associated with ExPEC virulence since 1962, when an F-type plasmid producing colicin V was found to be transferrable (122). Later studies suggested colicin V may be a virulence factor (123); however transposon mutagenesis showed that other plasmid genes, and not colicin, were likely responsible for 19 enhanced virulence (124). More contemporary studies show ColV plasmids not only enhance virulence but can cause pathogenic gain of function in previously non- pathogenic strains (114).

The colicin plasmids, named for their antimicrobial phenotypes (125),

characteristically possess genes to produce colicin, a colicin immunity protein conferring

immunity to carried colicin, and cell lysis proteins or other export systems (125). Colicin

proteins are categorized by the colicin’s outer membrane surface receptor and the inner

membrane receptor (126), and function as antimicrobial peptides affecting closely related

bacterial species (125). While colicins are diverse and effective antimicrobials,

transcription and translation of lysis protein-encoding genes in the operon is lethal.

Transcription and translation of the colicin operon causes a buildup of colicin and lysis

proteins in the cytoplasm. Lysis proteins are transported to the outer membrane via type

II secretion (125). A critical concentration of lysis protein in the outer membrane causes

OmpLA activation and expression of proteases to lyse the cell (125). Once released,

colicin B binds to the enterobactin receptor protein FepA and is transported to the

periplasm (126), where it forms a pore in the target cell disrupting membrane stability

and ion gradients causing cell death (126). Colicin M instead binds to outer membrane

ferrichrome receptor FhuA, and once in the cytoplasm colicin M degrades peptidoglycan

precursors preventing peptidoglycan synthesis and eventually causing cell death (127).

Colicin V is unique in that it utilizes an ABC transporter operon, cvaABCI, instead of

lysis proteins allowing non-lethal colicin release (128). Colicin V enters host cells via

interaction with the outer membrane protein Cir (129). Once inside the periplasm,

Colicin V interacts with inner membrane protein SdaC forming a pore and destroying 20 membrane potential (129). It has been shown that colicins themselves provide little benefit to strains during pathogenesis, instead they likely assist plasmid-containing strains to compete in the intestinal tract or other environments, while other genes contribute to virulence (124).

Siderophores and metal acquisition genes are common in ColV plasmids, usually encoding both the aerobactin and salmochelin siderophores, as well as the sitABCD operon. Aerobactin and salmochelin, as previously described, are functional siderophore systems in the presence of lipocalin 2 in human serum, and are upregulated in human and avian serum. When the aerobactin and salmochelin operons are deleted, a corresponding reduction in virulence has been noted in avian models (87). The sit operon encodes divalent metal ion transporters, and are similar to chromosomally-encoded sit genes located in some Shigella species (130). In vitro studies have shown the sitABCD operon cloned into siderophore deficient strains can orchestrate the uptake and utilization of iron in the presence of iron chelator 2,2’-dipryidyl, by non-specific transport that instead favors manganese binding (131). Furthermore, the sitABCD gene cassette was shown to increase resistance to oxidative stress in the presence of hydrogen peroxide (131).

Transport systems are also commonly found in ColV plasmids. As one may expect, colicin export genes cvaABC are encoded by these colicin producing plasmids, as well as etsABC , encoding a putative iron transport system; however no functional characterization of these genes has occurred (109).

Finally, outer membrane proteins and toxins are regularly encoded on ColV-type

plasmids. The membrane proteins OmpTp and Iss are routinely found on plasmids, as 21 well as the temperature-sensitive hemagglutinin autotransporter Tsh. The ompTp gene

encodes an outer membrane protein homologous to ompT in the omptin family of outer membrane proteases. OmpT was suggested to play a role in activating other secreted proteins (132) while in vitro study has shown OmpT can degrade antimicrobial peptides

present in human urine (133). The observant reader may have noted, OmpT and OmpTp

are highly homologous but distinct proteins, with one present on the ColV and ColBM

virulence plasmids, while the other is chromosomally encoded. Overall these genes are

highly homologous, but changes in ompT orthologs are known to affect substrate

specificity. The exact property of the OmpTp protease have yet to be determined. The

Iss protein(s) are closely related to chromosomally-encoded lambda phage gene bor ,

encoding an outer membrane protein. Three allelic variants of iss exist, as well as bor ,

and while bor is only found chromosomally, iss can be plasmid or chromosomally

located, and their confusion has hampered studies to this day (134). The Iss protein is a

dominant feature of APEC, and studies utilizing Iss as a vaccine target show Iss may

provide protection against heterogeneous APEC strains (135, 136). While these results

are promising, surprisingly little research exists regarding the specific mechanism by

which the iss gene facilitates enhanced serum survival (137). The tsh gene of ColV

plasmids encodes a temperature-sensitive hemagglutinin gene autotransporter, able to

bind to red blood cells, purified hemoglobin, and fibronectin and collagen IV, while also

having protease function with the ability to cleave casein, though an in vivo target that

has not identified (138). ColBM plasmids are similar in nature to the ColV plasmids

previously discussed; however, ColBM plasmids have been discovered that are missing

typical regions found on ColV plasmids, and instead contain insertions of antimicrobial 22 resistance genes, and may be a potential source of virulence and resistance plasmid hybrids (113).

Vir Plasmids

Vir plasmids were discovered in 1974 and belong to the replicon types

RepFIB/FIIA. Vir plasmids are found in E. coli associated with healthy bovine as well as in intestinal and extra intestinal disease of humans and animals, but are much rarer than

ColV and ColBM plasmids (139). Overall three genes are characteristic of Vir plasmids:

CNF-2, type III cytolethal distending toxin (CDT), and P17b fimbriae with a fourth putative fimbrial gene cluster tibAC . CNF-2 protein is thought to function similarly to chromosomally encoded CNF-1, and is commonly found in E. coli isolated from healthy cattle. The type III cytolethal toxin is encoded by three genes, two of which facilitate host cell delivery (140). Once inside the host cell, CDT functions as a DNase, causing double-stranded DNA breaks (140, 141). The F17 fimbria encoded by NTEC plasmids is a variant of a chromosomally encoded E. coli common fimbriae. Recent studies have shown F17 fimbrial variants exist which are associated with septic disease in lambs and calves, but distinct from F17a, F17c, and F17d variants associated with bovine ETEC, bovine diarrhea/sepsis, and human UPEC respectively (142).

Neonatal Bacterial Meningitis

Neonatal bacterial meningitis (NBM) is an insidious disease affecting 1 in every

1,000 children during the first 4 weeks of life. Without treatment, NBM is normally fatal. Clinically NBM is diagnosed by bacterial presence in the blood, and presence of bacteria, antigens, or pleocytosis in the cerebrospinal fluid (CSF) (143). NBM onset is sudden and characterized by rapid progression of symptoms, faster than culture results 23 can be obtained. Current medical literature suggests empiric treatment with penicillin and cefotaxime/aminoglycosides modified if necessary according to local prevalence of meningitis causing organisms and antimicrobial resistance. After obtaining positive culture identification and sensitivity reports, treatment protocols are optimized to the specific organism and its antimicrobial susceptibilities (144).

Bacterial meningitis presenting in the first two days to week of life is indicative of vertical transmission from the mother, termed early-onset, while infection after the first week up to 4 weeks suggests acquired infection, termed late-onset (145). Infections may also be categorized by the gestational age of the child. Term birth indicates a gestation age > 37 weeks, late preterm gestation age is between 33 and 37 weeks inclusive, and preterm gestational age is < 33 weeks (8).

A major French observational study over a seven year period from 2001 through

2007 in 114 pediatric wards throughout France is currently the best source of NBM data for incidence, disease onset data, and cultured causative organisms. This study found children born at term comprised the majority of observed infections (n=423, 78%), with late-preterm following (16%), and the lowest incidence of infection in pre-term birth

(6%); however term births account for 90% of births, while late-preterm account for approximately 8%, and preterm 2%, indicating infants born before a GA of 37 weeks are more susceptible to NBM (8, 146). The major causative organisms of NBM in order of incidence have been identified as Group B streptococci (~60%)(GBS) and E. coli (~30%) with isolated incidence of infection seen by other Gram-negative bacilli, other Gram- positive cocci, Neisseria meningitidis (~5%), and Listeria monocytogenes (~3%) (147).

Group B streptococci were causative agents in both early-onset and late-onset NBM, but 24

E. coli prevalence rises from 18% to 33% from early-onset to late-onset, with a corresponding drop in GBS incidence. Overall mortality was 13% with higher mortality rates observed in pre-term infants (26%) compared to full term infants (10%), while low- weight infants regardless of term have higher mortality rates (25%) than appropriate birthweight infants (12%) (8).

Follow-up studies on nine-year-old survivors of NBM reveals the harsh effects of

NBM on surviving neonates: neurological sequelae. NBM survivors were compared to non-NBM case children with the same birthdate and sex from the same doctor. Mortality before the age of 9 occurred in 3% of NBM cases while 20% of NBM survivors presented with moderate or severe neurological sequelae, with a higher incidence in lower weight and gestational neonates at time of infection. No corresponding control children presented with these sequelae. Severe impairments included cerebral palsy with loss of function or several functions such as, significant learning disabilities, and mental retardation determined by an IQ < 55 (100 indicating normal). Moderate impairments were defined by mild cerebral palsy, learning impairment determined by IQ between 55-

69 inclusive, partial paralysis, and hearing loss.

Pathogenesis of Neonatal Meningitis causing E. coli

The pathogenic process of NMEC has been almost entirely determined through study of the NMEC strain RS218. Strain RS218 is a highly pathogenic strain isolated from a human case of neonatal meningitis, and this strain or its derivatives are used solely in most experiments characterizing NMEC (46, 82, 84, 148-167). 25

Current models of NMEC pathogenesis derived from study of strain RS218 indicate infection begins through perinatal exposure of NMEC to the digestive tract (4,

158). Once exposed, NMEC are thought to travel the digestive system to the intestinal tract, where they attach to enterocytes and are transcytosed into the bloodstream (158).

In the bloodstream, NMEC utilizes multiple mechanisms to evade the innate immune system. NMEC strains generally produce capsular glycoproteins mimicking human glycoproteins (K1) (148), as well as O-acetyltransferase via the neuO gene, providing variation of the O antigen (168). In addition to mimicry, NMEC are resistant to killing by the classical complement system (83). While in the bloodstream, NMEC also invade and replicate in macrophages (82, 166), evading the immune system while simultaneously disrupting pro-inflammatory cytokine production, further obfuscating infection (84).

Proliferation of the bacteria in the bloodstream and macrophage quickly overwhelms the immune response and causes acute bacteremia (4). Bacteremia exposes high numbers of bacteria to the blood-brain barrier (BBB), a tight junction of microvascular endothelial cells separating the central nervous system (CNS) from the blood stream (153). NMEC strains are thought to adhere to the cells of the BBB, and through the actions of adhesins (169), toxins (152, 153, 164), and invasion factors (151), invade human brain microvascular endothelial cells (HBMEC) and astrocytes to transverse the BBB into the CNS, or to cross the BBB inside macrophages, utilizing a

Trojan horse mechanism to enter the CNS (163, 170). In either case, NMEC in the CNS further invade astrocytes resulting in meningitis. 26

During the process of pathogenesis NMEC must adhere to multiple cell types, survive the host immune response, invade immune and epithelial cells, and cross the

BBB. Current knowledge of these processes and their mechanisms are described in the following sections.

Role of adhesion and invasion in neonatal meningitis E. coli

Adhesion is an important factor for bacterial pathogens (71, 142, 171), and

NMEC is no different. NMEC pathogenesis involves adhesion and invasion of three major cell types, Enterocytes, Monocytes, and HBMEC.

Enterocyte Adhesion and Invasion

The first step in the current model of NMEC pathogenesis necessitates transit

from the intestines to the circulatory system. As a naturally enteric organism, E. coli

often express fimbriae and pili for adhesion to intestinal epithelial cells (71). Pathogenic

bacteria may also carry additional proteins specific for binding to proteoglycans

expressed on host cells (4). In ETEC, the Tia protein is one example, while NMEC

possess the hek gene. The hek gene facilitates binding to all proteoglycans, with

preference for heparin-containing proteoglycans (158), and is sufficient to cause in vitro

invasion of enterocytes when cloned into a non-pathogenic K-12 strain, but is less

invasive than wild-type RS218 (158). The genes ompA as well as ibeA are also

implicated in enterocyte binding and invasion, but their roles are not yet defined (158).

Monocyte Adhesion and Invasion

NMEC strains bind to monocytes and macrophage cells both in vitro and in vivo

(82) . In the case of strain RS218, studies in macrophage-depleted mice have shown the 27 absence of macrophage fully attenuates RS218 (166) though studies on NMEC 58 in this dissertation show little to no invasion of macrophage in vitro (unpublished data). The

major binding protein showing contribution to macrophage adherence is the outer

membrane protein OmpA (82). The OmpA protein is known to bind receptor CD64A,

and is necessary for RS218 binding and subsequent invasion of the macrophage cell line

RAW 264.7 (160). The CD64A protein is an Fc-gamma receptor expressed by all

macrophages that binds monomeric IgG antibodies, and in conjunction with gamma-

chain binding, facilitates macrophage killing (160). When OmpA is bound to the CD64A

protein, significantly less gamma chain association to CD64A is seen in comparison to

OmpA negative mutants, demonstrating OmpA can block CD64A-mediated macrophage

binding to inhibit a lethal macrophage phenotype. Further OmpA possess a higher

affinity for the CD64A binding site than IgG, and can supplant bound IgG, causing

further resisting host immunity (160).

Blood-Brain Barrier Adhesion and Invasion

Not all neonatal pathogens or pathogenic E. coli strains are capable of causing meningitis, which is defined by bacterial invasion of the CNS (172). To transverse the blood-brain barrier, NMEC utilize type 1 fimbriae and OmpA proteins as the major adherence factors. The FimH tip adhesion of type 1 fimbriae interacts with host cell

CD48; the binding phenotype is lost when antibodies to either FimH or CD48 are added in vitro (155). CD48 is thought to mediate adhesion and interactions between human epithelial and immune cells, and may be involved in macrophage transcytosis through the

BBB (173). 28

The OmpA protein, as previously implicated with enterocytes and macrophages, also contributes to BBB binding. OmpA interacts with N-acetylglucosamine regions of glycoproteins on the BBB, specifically glycoprotein 96, also present on colonocytes (156,

157, 162, 174, 175), and is necessary for HBMEC binding and invasion, but may not be sufficient itself (167). This glycoprotein specificity is not surprising as many other pathogens including Clostridium difficile, Neisseria gonorrheae, Staphylococcus aureus, and adherent-invasive E. coli are known to interact with gp96 (167, 176-180).

OmpA and FimH by themselves may not be sufficient for invasion of the BBB, as other factors are thought to assist in the process (148, 151, 152, 161, 164). Some of the most common and best studied factors include: IbeA, Cnf1, and the K1 capsule, which are all though to contribute to invasion of the blood brain barrier. The ibeA gene encodes

an invasion factor uncommon in all ExPEC, but associated with NMEC and found in

approximately half of strains (181). The IbeA protein binds vimentin (170), an

intermediate filament protein normally expressed in host cell cytoplasm, but may

circumstantially express on the cell surface under certain conditions. IbeA was shown as

sufficient to cause low-level invasion of HBMEC in vitro (182). While another study has

shown IbeA binding of vimentin greatly increases PNM transversal of the BBB, perhaps

suggesting a Trojan horse mechanism for CNF invasion facilitated by IbeA (163).

Though aslA is known to contribute to invasion of HBMEC, the specific mechanisms by

which aslA increases invasion are currently unknown (150).

Immunomodulation by neonatal meningitis E. coli

During infection, NMEC are exposed to factors of the neonatal immune system.

Though not fully mature, the immune system still presents significant challenges which 29 bacteria must overcome. NMEC use the properties of mimicry, complement resistance, macrophage invasion, and cytokine modulation to avoid the killing effects of the immune system.

Antiphagocytic Properties and Mimicry

The K1 capsule is the major agent of host mimicry in meningitis E. coli . The K1

capsule is composed of poly-α-2,8-N-acetylneuraminic acid, a sialic acid (183, 184).

Sialic acid and its structural derivatives are common on the surface of mammalian and

avian cells, and function as self-recognition molecules for many cellular processes (185).

Mimicry of the sialic acid residues obscures NMEC from the immune system, and

downregulates complement deposition on the cell surface (186), as well as downregulates

macrophage phagocytosis of cells (184).

Complement resistance

Complement resistance in NMEC is mediated by multiple factors. The K1 capsule interferes with complement factor C3 binding to the cell surface (186). In addition to the capsule, the NMEC outer membrane protein OmpA also interferes with the complement system. OmpA binds complement binding protein 4 (C4bp), which acts as an inhibitor of C3b (187). C3b is a required component for both the classical and the mannose-binding lectin pathway of complement killing (188). Studies of neonatal cord blood have shown decreased concentrations of alternative complement factors, especially factors P and B (189). These factors are essential for activation of the alternative pathway of complement (188). Adults possessing genetic mutations causing deficiency or inactivation of complement factor P show increased susceptibility to bacterial infections, especially acute meningeal infections such as Neisseria meningitidis (189). 30

Macrophage invasion

Macrophages present another facet of NMEC immune invasion. NMEC have been shown to adhere to, and invade, macrophage cells. Replication of type strain RS218 in macrophage requires expression of the OmpA protein (82). NMEC invade macrophage, form vacuoles and resist macrophage killing. While inside these vacuoles

NMEC continue to replicate relatively unhindered. Eventually prolific bacterial replication damages and kills the infected macrophage, which bursts and rereleases

NMEC into the bloodstream (82).

Cytokine Modulation

While inside monocytes, disruption of cytokine production occurs by preventing the inhibitor kappa B protein from phosphorylation, preventing transcription of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) (84), which normally acts in macrophage as a transcriptional regulator activating pro-inflammatory cytokines such as interleukin 1 and tumor necrosis factor α (190). Simultaneously, NMEC induce

production of Bcl XL , causing downregulation of cellular apoptosis (154). The

combination of multiple complement resistance factors, resistance to macrophage killing,

and inhibition of pro-inflammatory cytokine production allows NMEC to effectively

evade early immune response to infection and multiply undetected, which causes

bacteremia that overwhelms the host immune system. The high level bacteremia

facilitates NMEC exposure to the blood brain barrier, where NMEC eventually invade

and cause meningitis (4).

Aims and Significance of this Research

A considerable body of research has been established on NMEC pathogenesis and mechanisms of specific virulence genes during the course of infection. Almost all of this research has been performed using the strain RS218, or its derivatives (82-84, 148-150,

152-167, 174, 191). Given the diverse nature of E. coli , researchers already doubt the ability of single isolate-based studies to represent an E. coli pathotype (29). More troubling, the strain RS218 contains uncommon virulence factors, such as the cnf1 gene present in less than 5% of NMEC strains isolated from human cases (181). The cnf1 gene is widely considered a critical virulence component for BBB invasion, and is found more commonly in UPEC strains (27.5%) (Unpublished data). Finally, a diverse group of NMEC serotypes, phylogenetic groups, and genotypes have been observed in NMEC strains, indicating understanding of NMEC pathogenesis is likely too narrow and does not accurately reflect the entire NMEC sub-pathotype.

Previous studies in population genetics of ExPEC frequently reveal heterogeneous distributions of virulence factors in ExPEC, with no genes solely associated with specific sub-pathotypes, as well as similar prevalence of known virulence factors (181). Attempts to characterize the ExPEC pathotypes have met limited success, and generally distinguish

ExPEC sub-pathotypes from fecal E. coli , but cannot distinguish between all ExPEC sub- pathotypes (46, 58, 110). The current classification system for ExPEC is solely based on disease caused by the isolated E. coli , but studies have shown some ExPEC can cause diseases of multiple sub-pathotypes (58). Select APEC strains cause high mortality meningitis in the neonatal model, while some NMEC strains also cause colibacillosis in day-old chicks (58); unfortunately no studies have examined UPEC strains in the avian or 32 meningitis models. Due to these genomic similarities, and examples of virulence in multiple disease models and hosts, studies now suggest APEC, NMEC, and UPEC are potentially zoonotic pathogens (58, 59, 110, 134, 181, 192, 193).

With these factors in mind, this dissertation sought to use a whole-genome epidemiological approach to define a sample population of NMEC strains. We sequenced 49 randomly selected NMEC genomes isolated from clinical cases of neonatal meningitis from the United States and the Netherlands, and compared incidence and polymorphisms of virulence genes. Our ultimate goal was to determine whether a larger sample of NMEC strains coincided with the current model of NMEC pathogenesis.

Further, we sought to better define the characteristic RepFIIA/FIB plasmids of NMEC strains, and if and how NMEC plasmids differ from one another and from other ExPEC sub-pathotype plasmids. Finally, we provided whole-genomic sequence of strains APEC

O18 and NMEC O18, which were both previously characterized and shown to cause disease in both the neonatal meningitis and colibacilosis models.

This research characterized the broadest collection of NMEC strains to date. The work is significant due to use of genomic population-based knowledge to NMEC study, and challenges, deeply entrenched concepts of NMEC research based on a single strain and thus do not account for the overall diversity within population of NMEC. Finally this research significantly increased the amount of genomic data from NMEC publicly available, and contributed the first sequenced ExPEC strains confirmed to cause disease in both human and avian disease models.

Organization of this Dissertation

This dissertation is organized in journal format with 6 chapters. Chapter 1 contains a literature review of Escherichia coli . Chapter 2 addresses chromosomal components of NMEC virulence as well as the distribution and variance of these factors within a sample population of sequenced NMEC strains. Chapter 3 follows the chromosomal study with a genomic survey of NMEC virulence plasmids, including their common and accessory genome, effects on NMEC virulence, and phenotypes conferred.

Chapter 4 details the complete genome announcement of NMEC strain O18, possessing the ability to cause mortality in chick embryos and in the rat model of meningitis.

Chapter 5 contains the genome announcement of APEC strain APEC O18, which shares the O18 serotype with NMEC O18 and causing similar mortality rates to NMEC O18 in both chick embryos and neonatal rats. Chapter 6 concludes with a summary of the dissertation.

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CHAPTER 2. EPIDEMIOLOGICAL ANALYSIS OF MENINGITIS

ESCHERICHIA COLI: GENETIC DISTRIBUTION, VARIANCE, AND

RELATION TO OTHER EXTRA-INTESTINAL PATHOGENIC E. COLI

A paper to be submitted to BMC Genomics

Bryon A. Nicholsona, Nicolle Barbieria, Yvonne Wannemuehlera, Ganwu Lib, Catherine

M. Loguea, Lisa K. Nolana

aDepartment of Veterinary Microbiology and Preventive Medicine, College of Veterinary

Medicine, Iowa State University, Ames, Iowa, USA

bDepartment of Veterinary Diagnostic and Production Animal Medicine, College of

Veterinary Medicine, 1802 University Blvd., Iowa State University, Ames, Iowa 50011,

USA

Introduction

Disease-causing Escherichia coli strains can be grouped into two broad categories based on the tissues in which they cause disease. They are, intestinal pathogenic E. coli and extraintestinal pathogenic E. coli (ExPEC) (1). The ExPEC pathotype encompasses several sub-pathotypes including uropathogenic E. coli (UPEC) (1), septicemia-causing

E. coli (2), neonatal meningitis E. coli (NMEC) (2) and avian pathogenic E. coli (APEC)

(3). Among these, the NMEC sub-pathotype is one of the least studied despite the fact that NMEC are one of the most common causative agents of neonatal bacterial meningitis

(4).

Neonatal bacterial meningitis is a severe and rapidly progressing infectious disease of children during the first four weeks of life (5). During the course of disease the host generally develops profound bacteremia which leads to invasion and inflammation of the meninges (4). Observed hospital mortality rates due to bacterial meningitis in neonates range from in 28% to 40% regardless of etiology (4). However, if left untreated, this disease is usually fatal with survivors suffering severe lifelong neurologic sequelae (6, 7).

NMEC pathogenesis includes initial attachment and invasion of enterocytes in the small intestine. Attachment of NMEC to enterocytes is mediated by the fimbrial tip adhesin FimH, of the E. coli type 1 pilus (8, 9). Following attachment, the protein Hek interacts with host proteoglycans to facilitate transcytosis of the enterocytes where it gains access to the bloodstream. Following entry into the bloodstream, NMEC must survive the bloodstream. Bloodstream survival is facilitated by is mediated by K1 49 capsule (10) and OmpA (11-13). OmpA, a bacterial outer membrane protein coded by the ompA gene, is a multifunctional protein that interrupts activation of the complement cascade via the classical pathway through binding of the C4b-binding protein (13).

OmpA further facilitates bloodstream survival by modulating cytokine release following macrophage invasion (11, 14). The resulting bacteremia brings NMEC in contact with the blood-brain barrier (BBB) (2). Transcytosis of the BBB, frequently mimicked in vitro using human brain microvascular cell lines (HBMEC), is a complex process and is thought to involve the protein products of fimH, ompA, cnf1, and ibeA. The FimH protein mediates binding of NMEC to the human brain microvascular endothelial cells and is hypothesized as necessary for HBMEC invasion, recognizing host protein CD48. FimH binding to CD48 causes host cell calcium release and is thought to activate RhoA to induce cytoskeletal rearrangement (15). The protein OmpA is shown to facilitate binding to HBMEC to the stress protein Ecgp (16-19). OmpA also induces phosphorylation of signal transducer and activator of transcription 3 protein (STAT3), activating RhoA and

Rac1 to induce cytoskeletal rearrangements (19), but full downstream effects are not known. These studies clearly suggest OmpA contributes to NMEC virulence but it is still unclear if they are necessary, as ompA-negative strain RS218 mutants still invade

HBMEC in vitro (20). The protein IbeA binds to brain microvascular epithelial cell surface protein vimentin where it induces PMN transmigration into the cerebrospinal fluid (21-23) leading to “Trojan horse” invasion; however, the IbeA protein in other studies was shown to be sufficient to cause bacterial invasion of HBMEC without PMN interaction (24). 50

Much of what we know about NMEC pathogenesis is based on study of a single

NMEC strain, RS218, a member of the O18:K1:H7 serotype (8-11, 14, 15, 17, 23, 25-

29). This organism was the first NMEC characterized and has served as a useful model of the NMEC sub-pathotype for some time, with this strain and its derivatives being the focus of most NMEC research to date. However, recent studies have underscored the challenges in generalizing what we know of this strain to the entire NMEC sub-pathotype

(30, 31). For instance, recent population-based studies have shown that some virulence factors important to NMEC RS218’s pathogenesis, such as cnf1, are absent in the majority of NMEC strains (30, 31). Similarly, multiple strains deviating from the

O18:K1 serotype, which is thought to typify the NMEC, have also been reported (32, 33).

Finally, the virulence factors thought to be essential to NMEC pathogenesis, ompA and fimH, are ubiquitous in E. coli (34-41), are a known virulence factors of UPEC, enterotoxigenic E. coli, and also a hypothesized virulence factor of adherent-invasive E. coli further complicating the relationship between sub-pathotypes and virulence factors

(42, 43).

Recently, more broad-based studies have identified a substantial degree of overlap between NMEC and APEC in their pathogenic mechanisms and abilities to cause disease in models of human and avian disease. This has led to the hypothesis that NMEC, which cause human meningitis, might be derived from Avian Pathogenic E. coli (APEC), an important and widespread pathogen of poultry (44) and contaminant of retail poultry meat (45).

Currently, sequence data on NMEC strains are limited. The first publicly available NMEC-sequenced strain was IHE3034 published in 2010 (46). Strain IHE3034 51 has lost a large virulence plasmid, which have been found in other strains to contribute to virulence (28, 32). NMEC strain CE10 was the second publically available sequenced

NMEC genome (33). CE10 was sequenced due to the novelty of containing a type III secretion system and atypical O7 serogroup (33). NMEC strain RS218 was publically released as whole genome shotgun sequences in August of 2015, and is described as the prototypical Escherichia coli K1 strain causing meningitis (47). While study of these genomes has greatly enhanced knowledge of meningitis caused by E. coli, population studies have shown they may not fully account for the diversity of NMEC strains, or represent typical NMEC strains (30, 31, 45, 48).

Here, we seek to broaden the community’s access to genomic data from a diverse group of NMEC, define the NMEC core and accessory genomes, and ascertain if these differ greatly from what is known about the APEC core and accessory genomes. In addition, we will attempt to discern through phylogenomic analysis the relationship of

NMEC to that of other ExPEC.

Materials and Methods

Bacterial Strains and Selection. From a group of 91 E. coli strains isolated from cases of human neonatal bacterial meningitis in the United States and the Netherlands, 48 isolates were selected via random number generation for whole genome sequencing.

These NMEC had been previously serogrouped at the E. coli reference center

(Pennsylvania State University), genotyped for virulence and resistance genes by PCR

(49), and assigned to phylogenetic groups (50) (Table 1).

Table 1. Strains sequenced and used in this study and their general properties.

Strain Serotype Capsule Phylogroup Cluster () Hemolysis Source Distribution NMEC 2 18 1 B2 8 γ This study O-Type Phylogroup Cluster () NMEC 3 18ac 1 B2 8 γ This study 1 5 A 3 1 6 NMEC 6 6 1 B2 4 γ This study 2 1 B1 2 2 3 NMEC 7 - 1 B2 8 β This study 6 2 B2 43 3 1 NMEC 8 83 1 B2 8 γ This study 7 5 C 3 4 5 NMEC 11 - 1 B2 8 γ This study 9 1 D 1 5 1 NMEC 14 7 80 C 2 γ This study 12 3 E 1 6 6 NMEC 15 18 1 B2 8 γ This study 14 2 F 5 7 5 NMEC 16 83 1 B2 8 γ This study 16 1 8 23 NMEC 18 18 1 B2 8 γ This study 18 9 9 1 NMEC 19 - 1 B2 8 γ This study 18ac 1 NMEC 20 7 - D 1 γ This study 21 2 NMEC 22 - 1 B2 8 γ This study 23 1 NMEC 23 7 1 F 1 γ This study 25 1 NMEC 26 21 - B2 6 β This study 45 2 NMEC 27 12 - A 1 γ This study 78 1 NMEC 28 18 - B2 8 γ This study 83 6 NMEC 29 135 1 B2 7 γ This study 135 1 NMEC 30 18 1 B2 8 γ This study 166 1 NMEC 34 - 1 F 9 γ This study auto 2 NMEC 35 166 1 B2 4 γ This study - 10 NMEC 36 14 1 B2 7 γ This study NMEC 37 14 1 B2 8 γ This study NMEC 38 18 1 B2 8 γ This study NMEC 40 83 1 B2 5 β This study NMEC 42 auto - B2 8 γ This study NMEC 43 - - B2 8 γ This study NMEC 45 23 1 B1 3 β This study NMEC 49 auto 1 B2 8 γ This study NMEC 52 25 10 B2 4 γ This study NMEC 55 9 36 C 2 γ This study NMEC 57 83 1 B2 8 γ This study NMEC 58 18 1 B2 8 γ This study NMEC 59 1 1 B2 6 β This study NMEC 60 1 1 B2 8 γ This study NMEC 65 83 1 B2 8 γ This study NMEC 67 83 1 B2 8 γ This study NMEC 70 - 1 B2 7 γ This study NMEC 71 21 unknown B2 6 γ This study NMEC 72 - unknown B2 6 β This study NMEC 73 - unknown B1 2 γ This study NMEC 74 - unknown E 1 γ This study NMEC 81 12 unknown B2 6 β This study NMEC 82 1 unknown B2 7 γ This study NMEC 83 1 unknown F 4 γ This study NMEC 84 16 unknown B2 4 γ This study NMEC 86 12 unknown A 1 γ This study NMEC 89 45 unknown B2 7 γ This study NMEC 91 7 unknown F 1 γ This study RS218 18 1 B2 6 β CP007149 S88 45 1 B2 8 + CU928161.2 CE10 7 1 D (F*) unknown unknown NC_017646 UTI89 18 1 B2 - + NC_007946 CFT073 6 2 B2 - + AE014075 APEC O1 1 1 B2 - γ NC_008563 APEC O78 78 unknown C - γ NC_020163 IMT5155 2 1 B2 - unknown NZ_CP005930 DH1 85 12 C - γ NC_017625 W3110 - (16) 12 A - γ AP009048 Strain Origin Netherlands US France Germany Unknown 53

DNA Isolation and Library Preparation. Bacterial strains for sequencing were removed from -80°C glycerol stock, struck to MacConkey agar plates and incubated overnight at 37°C. Isolated single colonies were picked and inoculated into 2ml of Luria

Bertani broth (Becton Dickinson Franklin Lakes, NJ) and incubated at 37°C with shaking for 18 hours. Bacterial cells were pelleted by centrifugation at 7000xg for 10 minutes and resuspended in buffer containing 0.25mg/ml RNaseA and 2µg/ml Proteinase K.

Cells were lysed by heating at 80°C for 90 minutes. DNA was harvested from the lysate using ChargeSwitch gDNA mini bacteria kit (Life Technologies Carlsbad, CA), and the

DNA yields were quantified using a Qubit fluorometer dsDNA HS kit (Life Technologies

Carlsbad, CA). Genomic libraries were prepared using the Nextera XT kit (Illumina San

Diego, California) for paired end barcoded samples. Genomic library DNA was quantified by qPCR using KAPA library quantification kits (Kapa Biosystems

Wilmington, MA), and normalized to equivalent amplifying concentrations.

Genome Sequencing and Assembly. Genomic libraries were sequenced on an

Illumina MiSeq Desktop Sequencer using Miseq Reagent Kit v2 for 500 cycles (paired- end 250) at the USDA National Animal Disease Center (Ames, Iowa). Two runs, each with 24 indexed libraries were sequenced. Resultant reads were de-multiplexed, and resultant fastq files were assessed using the FastQC program to assess reads for quality, duplications, and per base sequence content. Read positions with an average Phred quality scores below 18, duplications, or average read position base content skewed 10% or more from overall average were trimmed.

De-multiplexed reads were assembled using the SPAdes Genome Assembler version 3.5.0 for Linux (51) using assembly options for paired-end reads and Burrows 54

Wheeler Aligner mismatch correction. Post-assembly quality assessment was performed using the software Quast (52) to determine contig sizes, N50, and assembly size.

Sequencing Treatment and Statistics. Following post-sequencing quality trimming and adapter removal, the mean read length for sequenced genomes was 220 base pairs per end. The mean number of non-scaffolded contigs, greater than 1,000 base pairs, was 122. The mean genome size of sequenced strains was 5,006,856 base pairs.

Percentage GC averaged 50.60%. Individual genome assembly statistics are described in

Table 2.

Whole Genome Alignment and Phylogeny. NMEC genome assemblies from this study were compared to previously sequenced NMEC genomes (n = 4), genomic sequences from other ExPEC (n = 5) and laboratory strains (n = 2). Previously sequenced strains used in this study included NMEC strains CE10, RS218, IHE3034, and

S88; APEC strains IMT5155, APEC O78, and APEC O1; UPEC strains UTI89 and

CFT073; and the laboratory strains, E. coli DH1 and W3110. Perl scripts were used to remove assembled contigs less than 800 base pairs in size or having less than 8-fold coverage. Harvest genomics tools version 1.1.2 (53) were used to align and visualize the core and pan genome of these strains using Muscle. Single Nucleotide Polymorphisms

(SNPs) from aligned core genomic regions were removed and concatenated. Whole genome SNPs were used to generate a maximum likelihood phylogenetic tree using

FastTree2 (54).

Table 2. Sequencing and Assembly Statistics.

Strain Contigs Large Contigs Largest Contig (bp) Total Length (Mb) GC% N50 NMEC 2 239 137 239,391 5.21 50.64 81594 NMEC 3 252 95 452,385 5.28 50.68 196320 NMEC 6 567 347 102,147 4.96 50.94 24868 NMEC 7 311 197 171,252 5.20 50.63 58893 NMEC 8 160 52 531,258 4.92 50.55 177127 NMEC 11 305 193 250,823 5.21 50.6 57634 NMEC 14 244 137 284,584 5.05 50.66 117543 NMEC 15 247 105 418,432 5.23 50.56 154846 NMEC 16 137 64 516,108 4.93 50.56 171565 NMEC 18 171 62 703,355 5.08 50.57 322711 NMEC 19 147 76 401,764 4.91 50.61 139554 NMEC 20 178 76 457,407 5.01 50.62 172171 NMEC 22 168 73 812,195 5.22 50.71 196545 NMEC 23 450 216 292,119 5.26 50.53 74570 NMEC 26 199 81 612,860 5.05 50.56 173783 NMEC 27 352 192 266,060 5.05 50.68 89766 NMEC 28 194 92 432,717 5.18 50.7 170238 NMEC 29 210 77 421,341 5.21 50.66 266902 NMEC 30 282 96 440,598 5.22 50.66 191667 NMEC 34 421 233 247,335 5.14 50.58 44186 NMEC 35 410 171 231,118 5.13 50.6 84412 NMEC 36 216 100 451,924 5.16 50.52 186035 NMEC 37 287 108 552,239 5.17 50.55 173395 NMEC 38 270 129 434,159 5.22 50.56 114966 NMEC 40 127 67 433,073 5.08 50.41 168383 NMEC 42 194 94 349,405 5.12 50.36 80856 NMEC 43 179 90 702,431 5.17 50.57 173442 NMEC 45 180 199 283,798 4.99 50.65 92111 NMEC 49 133 62 397,745 4.95 50.51 182434 NMEC 52 117 52 546,582 5.03 50.62 190962 NMEC 55 181 105 364,933 5.10 50.73 149429 NMEC 57 157 68 455,812 4.93 50.55 169504 NMEC 59 163 83 578,758 4.98 50.55 213151 NMEC 60 114 55 681,191 5.27 50.6 195201 NMEC 65 117 55 516,850 4.94 50.58 211094 NMEC 67 110 54 658,344 4.93 50.57 231496 NMEC 70 239 90 419,133 5.16 50.59 203905 NMEC 71 125 57 444,515 4.88 50.6 253886 NMEC 72 169 76 444,419 5.03 50.53 175273 NMEC 73 177 96 219,994 4.74 50.82 123827 NMEC 74 425 230 214,651 5.42 50.54 63216 NMEC 81 237 98 368,051 5.13 50.34 156132 NMEC 82 216 85 453,216 5.16 50.52 203913 NMEC 83 407 235 214,378 5.23 50.6 47944 NMEC 84 85 34 585,631 4.84 50.53 343102 NMEC 86 960 504 73,433 5.14 51.15 18407 NMEC 89 247 97 577,928 5.13 50.65 203906 NMEC 91 284 157 285,197 5.26 50.51 112369 56

Virulence Genotyping. To assess the virulome of NMEC, all sequenced NMEC, selected ExPEC, and laboratory E. coli strains were assessed for their content of virulence genes using using MvirDB available through the Lawrence Livermore National

Laboratory (55) using an all-versus-all nucleotide BLAST (56). A Perl script was used to remove low quality blast hits, removing hits where the subject sequence was less than

95% of the query sequence’s length, below 95% sequence identity, or expected value was above 1.0 e-20. Resultant BLAST hits were examined manually to remove duplicate gene entries.

Antimicrobial Resistance Genotyping. Antimicrobial resistance profiles for sequenced genomes were determined by comparison to the Comprehensive Antibiotic

Resistance Database (CARD) (57). Nucleotide sequences for antimicrobial resistance were compared to a database of assembled NMEC sequences using default parameters.

A Perl script was used to filter the resultant gene list. Hits where the subject sequence was less than 95% of the query length, 95% sequence identity, or expected value above

1.0 e-20 were removed from the analysis.

Antimicrobial Susceptibility Phenotyping. Antimicrobial susceptibility of the

NMEC isolates used in this study was determined using the broth microdilution method and the National Antimicrobial Resistance Monitoring Scheme (NARMS) (58)

(Sensititre, Trek Diagnostics, Cleveland, OH) according to the Clinical Laboratory

Standards Institute. NMEC strains were tested for succeptibility to the following antimicrobials: amikacin (0.5 - 64 μg/ml), ampicillin (1 - 32 μg/ml), amoxicillin/clavulanic acid (1/0.5 - 32/16 μg/ml), ceftriaxone (0.25 - 64 μg/ml), chloramphenicol (2 - 32 μg/ml), ciprofloxacin (0.015 - 4 μg/ml), 57 trimethoprim/sulfamethoxazole (0.12/2.38 - 4/76 μg/ml), cefoxitin (0.5 - 32 μg/ml), gentamicin (0.25 - 16 μg/ml), kanamycin (8 - 64 μg/ml), nalidixic acid (0.5 - 32 μg/ml), sulfisoxazole (15-256 μg/ml), streptomycin (32 - 64 μg/ml), tetracycline (4 - 32 μg/ml), and ceftiofur (0.12 - 8 μg/ml). Resistance and susceptibility cutoffs were determined using CLSI interpretive criteria as follows: Amikacin (≥ 64 μg/ml), ampicillin (≥ 32

μg/ml), amoxicillin/clavulanic acid (≥ 32/16 μg/ml), ceftriaxone (≥ 4 μg/ml), chloramphenicol (≥ 32 μg/ml), ciprofloxacin (≥ 4 μg/ml), trimethoprim/ sulfamethoxazole (≥ 4/76 μg/ml), cefoxitin (≥ 32 μg/ml), gentamicin (≥ 16 μg/ml), kanamycin (≥ 64 μg/ml), nalidixic acid (≥ 32 μg/ml), sulfisoxazole (≥ 512 μg/ml), streptomycin (≥ 64 μg/ml), tetracycline (≥ 16 μg/ml), and ceftiofur (≥ 8 μg/ml).

ompA and fimH polymorphism analyses. Nucleotide sequences for the ExPEC virulence genes fimH and ompA were extracted from the genome of NMEC strain S88 and queried against a BLAST database of assembled genomes. A BLAST hit cutoff, excluding hits with an overlap length and total identities of less than 90%, was used to remove erroneous fragments from the analysis. Sequences were concatenated and aligned using ClustalOmega using default parameters (59). The resulting alignment was used to generate a phylogeny using FastTree2, options for GTR+CAT model and gamma distributed rate variation. The nucleotide sequences were then translated into protein sequences and aligned to their known structures to compare amino acid changes within protein active sites.

Results

NMEC and ExPEC Core Genome Phylogenies Show Distinct NMEC Lineages.

A total of 48 NMEC strains, isolated from different patients with neonatal bacterial meningitis in the United States and the Netherlands representing phylogenetic groups A, B1, B2, C, D, E, and F were selected for genome sequencing. Strains sequenced were also highly divergent in terms of serogroup, but a significant number of

O18, O83 and untypeable serogroups were found among the sequenced group. To determine the relatedness of NMEC strains to each other and other E. coli and to elucidate their evolutionary history, SNPs from strains sequenced in this study and all previous publically available NMEC genomes were combined with representative strains from other ExPEC sub-pathotypes APEC and UPEC as well as non-pathogenic laboratory strains W3110 and DH1, (Figures 1 and 2). The core shared genome of ExPEC and laboratory strains in this study was 2.2Mbp. The SNP-based phylogenies revealed large evolutionary differences among all of the NMEC strains examined, but close relationships were observed between many of the NMEC based on where they clustered.

Lab strains, E. coli W3110 and DH1, formed a new distinct node when added. The three

APEC strains examined, APEC O1 and O78, did not form a single node but clustered within nodes containing NMEC strains. Uropathogenic Escherichia coli (UPEC) strains

UTI89 and CFT073 clustered into separate nodes. These distinct clustered nodes and sister nodes contained NMEC strains and indicate, on a whole genomic level, a closer relationship of these APEC and UPEC strains to NMEC than each other.

Segregation of taxa within the SNP phylogeny suggests strong association between strains based on serogroup. The predominant NMEC serogroup, O18, were all 59 of the B2 phylogenetic group and clustered into closely related sister nodes, along with

APEC O18, CFT073, and UTI89. Similarly, five of the six O83 strains, all assigned to phylogenetic group B2, clustered together in closely related sister nodes distinct from the

O18 serogroups. Serogroups O21 and O45 were also closely clustered in the SNP phylogeny, but the limited number of sequenced samples limits inference of the similarities of O21 and O45 strain. Serogroups O1, O6, O7, O12, O14 and untypeable strains, regardless of sub-pathotype, did not form independent nodes, and were interspersed throughout the phylogeny. Strain phylogenetic type also showed an apparent correlation with the SNP derived phylogenetic landscape. As evolutionary distance from laboratory strains increased in the SNP phylogeny, the strains of the same clermont phylogenetic type clustered into nodes or sister nodes following a progression (A-(C or

B1) EDFB2) similar to previous study (50), and the predominant strains in the latter portion, B2, D, and F, coincides with studies implicating these strains more commonly in human disease (60). Correlations of genome size between phylogenetic branches or serogroup were not performed, as the presence of plasmid DNA in the strains sequenced in this study as well as the incomplete assembly of the sequenced reduces the value of such comparisons. The phylogeny was also assessed for the relationship between country of isolation and phylogenetic relationship. There was no readily apparent pattern of segregation by country of origin in the phylogeny.

Figure 1. Whole genome maximum likelihood hylogeny of previously sequenced, and NMEC strains sequenced in this study, through single nucleotide variant concatenation and alignment. Whole genomes or assembled contigs aligned using the Muscle algorithm in Harvest Genomic Tools Parsnp version 1.2. The phylogeny was generating using FastTree2 and visualized using FigTree v1.4.2. Colored taxa represent Clermont expanded phylogenetic groups: Black A, Maroon C, Orange, B1, Gold E, Cyan D, Green F, and Red B2. 1

Figure 2. Whole genome maximum likelihood phylogeny of previously ExPEC strains and NMEC strains in this study. Single nucleotide polymorphism variants concatenation and alignment. Whole genomes or assembled contigs aligned using the Muscle algorithm in Harvest Genomic Tools Parsnp version 1.2. The phylogeny was generating using FastTree2 and visualized using FigTree v1.4.2. Each strain’s O-serotype follows the taxa label. Colored taxa represent Clermont expanded phylogenetic groups: Dark Blue A, Maroon C, Orange, B1, Gold E, Cyan D, Green F, and Red B2 62

Virulence factor database screening.

Assembled NMEC sequences as well as additional ExPEC and laboratory E. coli strains were assessed using the Lawrence Livermore National Laboratory LLNL virulence database and nucleotide BLAST for putative virulence factors. Initial results identified 4,875 potential virulence genes among all of the genomes examined. After removing entries with identical distribution, gene length, and gene names in addition to manual inspection, the potential gene pool was reduced to 1,231 (61, 62). Among them, only 299 genes were present in greater than 50% of ExPEC strains, and 208 genes including ompA are also present in E. coli K12 strains like W3110 and DH11.

The prevalence of genes previously identified as contributors to NMEC pathogenesis including the genes encoding K1 capsular synthesis, kpsDMT (63, 64) arylsulfatase aslA (26), invasion protein ibeA (24, 65, 66), the S fimbrial switch protein sfaC, (66, 67), fimbrial adhesin fimH (15, 68) and outer membrane ompA (11, 69, 70) was determined in all ExPEC and laboratory strains in this study. Additional virulence factors of ExPEC that have not been associated with NMEC before were also found in this pool, including the fimbrial and motility, regulating genes papX and prsX (71), the serine protease autotransporter protein vat (61, 62), and the hemin receptor protein chuA

(72). Additionally 24 genes related to iron acquisition including the yersiniabactin, enterobactin, and aerobactin operons, as well as the gene encoding the iron sensing protein fhuF (73) and metabolic genes likely to enhance siderophore production, were identified. 63

Genes putatively associated with two multi-drug efflux pumps, three ABC transporter systems, two non-ribosomal peptide synthesis systems, and a putative transcriptional regulator were also found in ExPEC strains but not laboratory strains. The remaining genes of the pool were associated with phage proteins, mobile genetic elements, and insertion sequences. The full list of virulence genes, percentage incidence, presumed function, and individual strain incidence are reported in Table S1.

fimH Polymorphism Analysis Reveals Novel Polymorphic Patterns.

FimH has been previously correlated with NMEC and UPEC pathogenesis (15,

43, 68, 74). Although fimH was found to be ubiquitous among the assayed strains whether from ExPEC or laboratory strains, previous studies showed that adhesion FimH was subjected to positive selection and certain site mutations have been associated with increased in vivo fitness. The sequences of fimH showed 95.9% nucleotide identity in the gene. Conversion to amino acid sequence showed polymorphisms in 24 of the 300 residues, corresponding to 8% of the protein in positions. Amino acid residues comprising the mannose binding pocket, or mannose chaperoning residues (positions 1,

13, 46-48, 52, 54, 133, 135, 140, and 142) contained no amino acid polymorphisms as found in previous studies (43, 74). In total, 32 polymorphic amino acid sites were found among all strains, while 10 sites showed increased incidence of polymorphisms (Figure

3). UPEC FimH sites previously defined as under positive selection and enhancing uroepithelial cell binding (V27, S62, and V163) were examined (74). Sequenced NMEC strains were compared with previously sequenced APEC strains including χ7122 (75)

(n=4), sequenced APEC fimH genes (n=23) (76) as well as sequenced UPEC genomes and the Broad Institute’s Escherichia coli as a human pathogen project (n=143). 65

Table S1. Listing of all virulence genes detected in NMEC strains sequenced. Grid association of virulence genes incidence and the respective genomes. Red indicates virulence gene positive strains, while black indicates negative strains.

Gene Putative Function NM2 NM3 NM6 NM7 NM8 NM11 NM14 NM15 NM16 NM18 NM19 NM20 NM22 NM23 NM26 NM27 NM28 NM29 NM30 NM34 NM35 NM36 NM37 NM38 NM40 NM42 NM43 NM45 NM49 NM52 NM55 NM57 NM58 NM59 NM60 NM65 NM67 NM70 NM71 NM72 NM73 NM74 NM81 NM82 NM83 NM84 NM86 NM89 NM91 RS218 S88 IHE3034 CE10 W3110 mdtN Multidrug resistance protein MdtN 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 0 gspL Putative type II secretion system protein L 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 entE 2,3-dihydroxybenzoate-AMP ligase 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 fyuA Pesticin Receptor 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 kpsD Polysialic acid transport protein KpsD 1 1 1 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 0 ybtE Salicyl-AMP ligase 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 ybtT Yersiniabactin biosynthetic protein YbtT 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 ybtU Yersiniabactin biosynthetic protein YbtU 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 chuA Outer membrane heme/hemoglobin receptor chuA 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 0 chuT putative periplasmic binding protein 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 0 hemF/chuW Oxygen-dependent coproporphyrinogen-III oxidase 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 0 ybtA Yersiniabactin transcriptional regulator, YbtA 0 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 ybtQ Inner membrane ABC-transporter YbtQ 0 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 ybtX Yersiniabactin-iron transporter permease YbtX 0 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 aslA Arylsulfatase 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 chuS putative heme/hemoglobin transport protein 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 0 chuX Putative heme utilization carrier protein ChuX 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 ybtP Lipoprotein inner membrane ABC-transporter 0 1 0 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 irp2 Yersiniabactin non-ribosomal peptide synthase 0 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 1 1 1 1 1 1 0 irp9 salicylate synthase Irp9 0 1 0 0 1 1 1 1 1 1 1 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 irp1 HMWP1 nonribosomal peptide/polyketide synthase 0 1 0 1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 0 1 0 1 1 1 1 1 1 0 yeeS UPF0758 protein YeeS 1 1 0 1 1 0 1 0 1 0 1 1 0 1 1 1 0 1 1 0 1 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 fepG Ferric enterobactin transport system permease protein FepG 1 1 0 1 1 1 0 1 1 1 1 0 1 1 1 0 1 1 1 0 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 0 1 1 0 1 0 1 1 1 1 1 1 0 fhuF Ferric iron reductase protein FhuF 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1 1 0 1 1 0 1 1 0 0 1 1 0 1 1 0 kspM Polysialic acid transport protein KpsM 1 1 0 1 1 1 0 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 0 1 1 1 1 1 1 1 0 0 0 0 1 1 1 1 0 1 1 1 1 1 1 0 iroD Ferric enterochelin esterase 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 1 0 1 0 1 0 1 0 0 iroE IroE, putative hydrolase 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 1 0 1 0 1 0 1 0 0 papX/prsX HTH-type transcriptional regulator PapX 1 1 1 0 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 0 0 1 1 0 0 1 1 1 1 1 1 1 0 1 0 0 1 1 1 0 0 1 1 1 1 1 1 0 yeeP 50S ribosome-binding GTPase family protein 1 0 1 1 1 1 0 1 1 1 1 0 1 1 1 0 1 0 1 1 0 0 1 1 1 1 1 1 1 0 1 1 1 0 1 1 1 1 1 1 0 1 0 0 1 0 0 1 1 1 1 1 1 0 iroC ABC transporter protein IroC 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 0 1 0 1 0 1 0 1 0 0 iucB Aerobactin biosynthesis protein IucB 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 0 1 1 0 1 1 1 1 1 0 1 0 1 1 1 1 0 1 1 1 0 0 0 1 0 iucC Aerobactin biosynthesis protein IucC 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 0 1 1 0 1 1 1 1 1 0 1 0 1 1 1 1 0 1 1 1 0 0 0 1 0 iucD Aerobactin biosynthesis protein IucD 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 0 1 1 0 1 1 1 1 1 0 1 0 1 1 1 1 0 1 1 1 0 0 0 1 0 kpsT Polysialic acid transport ATP-binding protein KpsT 1 1 0 1 1 1 0 1 1 1 1 0 1 1 0 0 1 1 1 1 1 1 1 1 0 1 1 0 1 0 0 1 1 1 1 1 1 1 0 0 0 0 1 1 1 1 0 1 1 1 1 1 1 0 emrE Multidrug transporter EmrE 1 1 0 1 0 1 0 1 0 1 0 0 1 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 0 0 1 1 1 0 0 1 1 1 0 1 1 1 0 1 0 1 0 1 1 1 0 0

iroB Glucosyltransferase 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 0 1 1 0 0 0 1 0 1 0 1 0 1 0 1 0 1 0 0 64 rusA Holliday junction nuclease RusA 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 1 0 1 1 1 1 0 0 0 0 1 0 1 1 1 0 0 1 1 1 0 0 0 1 0 1 0 1 1 1 1 1 1 1 1 0 - putative MarR-family transcriptional regulator 1 1 0 0 1 1 0 1 1 1 1 0 1 0 1 0 1 1 1 0 0 1 1 1 1 1 1 0 1 0 0 1 1 1 1 1 1 1 1 1 0 0 1 1 0 1 0 1 0 0 1 1 0 0

fepA Ferrienterobactin receptor 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 0 0 1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 0 1 1 0 0 0 1 0 1 0 1 0 1 0 1 0 1 0 0 gspH putative type II secretion protein 1 1 0 1 0 1 1 1 0 1 0 1 1 0 1 0 1 1 1 0 0 1 1 1 1 0 0 1 0 0 0 0 1 1 1 0 0 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 0 0 - putative hemin receptor 1 1 0 1 1 1 0 1 1 1 1 0 1 1 0 1 1 0 1 1 1 0 1 1 0 1 1 0 1 0 0 1 1 1 0 1 1 0 0 0 0 1 1 0 1 0 0 0 1 0 0 1 0 0 c3581 hypothetical protein c3581, Escherichia coli CFT073 0 1 0 1 1 0 1 0 1 0 1 0 0 0 1 0 0 1 0 1 0 1 1 1 0 0 0 1 0 0 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 0 0 0 parB ParB partioning protein 1 1 0 1 1 1 0 1 1 0 1 0 0 1 1 1 0 0 1 1 1 0 0 1 0 0 0 0 1 0 1 1 0 0 0 1 1 1 1 1 1 1 1 1 0 0 1 0 1 0 0 1 1 0 ibeA invasion protein IbeA 1 1 1 1 1 1 0 1 1 1 1 0 1 0 0 0 1 0 1 0 1 0 1 1 1 1 1 0 1 1 0 1 1 0 0 1 1 0 0 0 0 0 1 0 0 1 0 0 0 1 0 1 0 0 mchE microcin H47 secretion protein 1 1 0 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 1 1 1 0 0 1 1 1 0 0 1 1 0 0 1 1 1 1 0 0 0 1 0 1 0 1 0 1 0 0 0 0 0 0 sfaC Putative F1C and S fimbrial switch regulatory protein SfaC 1 1 0 1 1 1 0 1 1 1 1 0 1 0 0 1 1 0 1 1 1 0 1 1 1 0 0 1 1 0 0 1 1 1 0 1 1 0 0 0 0 0 0 0 1 0 0 0 1 1 0 1 1 0 vat Vacuolating autotransporter toxin 1 0 1 0 0 1 0 1 0 1 0 0 1 0 0 0 1 1 1 0 1 1 0 0 1 1 1 0 1 0 0 0 1 1 1 1 0 1 0 0 0 0 1 1 0 1 0 1 0 1 1 1 0 0 fliC Flagellin 1 1 0 1 0 1 0 1 0 1 0 0 1 0 0 0 1 1 1 1 1 1 1 1 0 1 1 0 0 0 0 0 1 1 1 0 0 1 0 0 0 0 0 1 1 0 0 1 0 1 1 1 0 0 - hypothetical protein c5153 0 1 0 1 1 0 1 0 1 0 1 0 0 0 1 1 0 1 1 0 1 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1 1 1 1 1 1 1 1 1 0 - hypothetical protein 1 1 1 1 1 1 0 1 1 1 1 0 1 0 1 0 1 1 1 0 1 1 1 1 1 1 1 0 1 0 0 1 1 1 1 1 1 1 1 1 0 0 1 1 0 1 0 1 0 1 1 1 0 0 - hypothetical protein 0 0 0 1 1 0 1 1 1 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 1 0 1 1 1 1 1 0 - hypothetical protein c3187 0 0 0 0 1 0 0 0 1 0 1 1 0 0 0 0 0 1 0 0 1 1 0 1 0 1 1 0 1 0 1 1 1 0 1 1 1 0 0 0 0 1 0 1 1 1 0 1 1 1 1 1 1 0 - hypothetical protein 1 1 0 1 1 1 0 1 1 1 1 0 1 1 0 1 1 0 1 1 1 0 1 1 0 1 1 0 1 0 0 1 1 1 0 1 1 0 0 0 0 0 1 0 1 0 0 0 0 1 0 1 0 0 - hypothetical protein 1 1 0 1 1 1 0 1 1 1 1 0 1 0 0 0 1 1 1 0 0 1 1 1 0 0 0 0 1 0 0 1 1 1 1 1 1 1 0 0 0 0 1 0 0 0 0 1 0 1 1 1 0 0 Z Putative tail component of prophage CP-933O 1 0 1 0 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 1 0 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 O partial O replication protein for prophage CP-933H 1 1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 1 1 1 0 1 1 1 1 0 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 1 1 1 0 0 ECs1109 putative head decoration protein 0 1 1 0 1 1 0 1 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 0 1 0 0 1 1 1 1 1 1 0 1 1 0 0 1 0 0 1 1 1 1 1 1 1 1 0 - putative minor tail protein 1 1 0 1 1 1 0 1 1 1 1 0 1 0 1 1 1 1 1 0 1 1 1 1 0 0 0 0 1 0 0 1 1 1 1 1 1 1 1 1 0 0 1 0 1 0 1 1 1 1 1 1 1 0 - putative tail component of prophage 0 1 1 0 1 1 1 1 1 1 1 0 1 1 0 0 1 1 1 1 0 1 1 1 0 0 0 0 1 1 0 1 1 1 1 1 1 1 0 0 1 1 1 1 1 0 0 0 1 0 1 1 1 0 - putative minor tail protein 1 1 0 1 1 1 0 1 1 1 1 0 1 1 1 0 1 1 1 1 1 1 1 1 0 0 0 0 1 0 0 1 1 1 1 1 1 1 1 1 0 0 0 0 1 0 0 1 1 1 1 1 1 0 - putative head-tail joining protein of prophage 1 1 0 1 1 1 1 1 1 0 1 0 0 1 1 1 0 0 1 1 1 0 1 1 1 0 0 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 0 1 1 0 - putative head completion protein 1 1 1 0 1 0 0 0 1 1 1 0 1 1 1 1 1 1 1 1 0 0 0 0 0 1 1 0 1 0 0 1 1 1 0 1 1 1 1 1 0 0 1 1 0 1 0 0 1 1 1 1 1 0 - putative tail component of prophage 1 1 1 1 1 1 0 1 0 1 0 0 1 1 0 0 1 1 1 1 1 1 1 1 1 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0 1 0 1 1 1 0 1 1 1 1 1 1 0 - putative head-tail joining protein of prophage 1 1 0 1 1 1 1 1 1 0 1 0 0 1 1 1 0 0 1 0 1 0 1 1 1 0 0 0 1 0 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 0 1 1 0 - putative DNA packaging protein of prophage 1 1 0 1 0 1 1 1 1 0 1 0 0 0 1 1 0 0 1 1 1 0 1 1 1 0 0 1 1 1 1 1 0 0 0 1 1 1 1 1 1 0 1 1 1 0 1 0 1 0 0 1 1 0 - hypothetical protein Z1653 1 1 1 1 0 1 0 1 0 1 0 0 1 1 0 0 1 1 1 0 1 1 1 1 0 1 1 0 0 1 0 0 1 1 1 0 0 1 0 0 0 1 0 1 0 1 0 1 1 1 1 1 1 0 - putative tail component of prophage 0 1 1 1 1 0 1 0 1 0 1 0 0 0 1 1 0 0 1 0 1 0 0 0 0 0 0 0 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 0 0 0 1 1 1 0 - putative head-DNA stabilization protein of prophage 1 1 0 1 1 1 1 1 1 0 1 0 0 1 1 1 0 0 1 1 1 0 1 1 1 0 0 0 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 0 1 0 0 - putative capsid protein of prophage 1 1 0 1 1 1 0 1 1 0 1 0 0 0 1 1 0 0 1 0 1 0 1 1 1 0 0 0 1 0 1 1 0 0 0 1 1 1 1 1 1 1 1 1 0 0 1 0 1 0 0 1 1 0 - putative tail component of prophage 0 1 1 1 1 1 0 1 1 1 1 0 1 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 1 0 1 1 1 1 1 1 1 1 0 0 1 1 1 0 0 0 0 0 0 1 0 1 0 0 - putative minor tail protein 1 1 0 1 1 1 0 1 1 1 1 0 1 0 0 0 1 1 1 0 1 1 1 1 0 0 0 0 1 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 0 1 1 1 0 0 int bacteriophage integrase 0 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 1 1 0 IS629 IS629 ORF1 0 1 0 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 1 1 0 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 0 1 1 1 1 0 1 0 insA DNA-binding protein InsA 1 1 0 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 1 1 0 1 1 1 1 1 1 1 0 1 1 0 1 1 1 0 0 0 1 1 1 0 0 1 0 insA truncated InsA protein 1 1 1 1 1 1 0 1 1 1 1 0 1 0 1 0 1 1 1 0 1 1 1 1 1 1 1 0 1 0 0 1 1 1 1 1 1 1 1 1 0 0 1 1 0 1 0 1 0 1 1 1 0 0 insB truncated InsB protein 1 1 1 1 1 1 0 1 1 1 1 0 1 0 1 0 1 1 1 0 1 1 1 1 1 1 1 0 1 0 0 1 1 1 1 1 1 1 1 1 0 0 1 1 0 1 0 1 0 1 1 1 0 0 tnp IS629 ORF1 IS3 family transposase fragment 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 0 1 1 0 1 1 1 1 1 0 1 0 1 1 1 1 0 1 1 1 0 0 0 1 0 IS629 transposase IS629 1 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 1 1 1 0 0 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 0 1 0 1 1 1 0 0 1 1 1 0 1 0 1 0 IS629 hypothetical protein c3596 0 1 0 1 1 1 1 0 0 1 0 1 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 1 1 0 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 0 1 1 0 1 0 1 0 insE putative transposase 0 1 0 1 1 1 0 0 0 1 0 1 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 1 1 0 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 0 1 1 0 1 0 1 0 - putative transposase 0 1 0 1 1 1 0 0 1 1 1 0 1 1 0 1 1 1 1 0 0 1 0 1 1 0 0 0 1 0 0 1 0 1 1 1 1 1 0 1 0 1 1 1 0 0 1 1 1 1 1 0 1 0 IS629 transposase IS629 1 1 0 1 1 1 0 1 1 1 1 0 1 1 0 0 1 1 1 0 0 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 0 0 0 1 1 1 0 0 1 1 1 0 1 0 1 0 - putative transposase 0 1 0 1 1 1 0 1 1 1 1 0 1 1 0 0 1 1 1 0 0 1 0 1 1 0 0 0 1 0 0 1 0 1 1 1 1 1 0 1 0 1 1 1 0 0 1 1 0 1 1 0 1 0 - hypothetical transposase 0 1 0 1 1 1 0 0 0 1 0 1 1 0 1 0 1 1 1 0 1 1 0 1 1 0 0 1 1 0 1 1 0 1 1 1 1 0 1 1 0 1 1 1 0 0 0 1 1 1 0 0 1 0 IS629 IS629 ORF2 1 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 1 1 1 0 0 1 1 1 1 0 0 0 1 0 1 1 1 0 1 1 1 1 0 0 0 1 0 1 0 0 1 1 1 0 0 0 1 0 IS629 transposase IS629 1 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 1 1 1 0 0 1 1 1 1 0 0 0 1 0 1 1 1 0 1 1 1 0 0 0 0 1 0 1 0 0 1 1 1 0 0 0 1 0 IS629 IS629 ORF2 1 1 0 1 1 1 1 1 1 1 1 0 1 1 0 0 1 1 1 0 1 1 0 1 1 0 0 0 1 0 0 1 0 0 1 1 1 0 0 0 0 1 0 1 0 0 1 1 0 0 0 0 1 0 orfA ORF A protein 1 1 1 1 0 1 1 1 0 1 0 0 1 1 0 0 1 0 1 1 0 0 1 1 0 1 1 1 0 1 0 0 1 0 1 0 0 1 0 0 1 0 1 0 1 1 0 0 1 0 0 1 1 0 65

Figure 3. Amino acid alignment of variable amino acid residues in FimH. Alignment of the 10 most variable amino acid residues among all ExPEC in this study. Fisher’s exact test to compare amino frequencies among pathotypes. *** p< 0.0001: ** p< 0.005: * p< 0.01

Using this three-polymorphism system, 89% of NMEC, 55% of UPEC, and 96% of

APEC were classified as A27 S62 V163. The remaining 44% of UPEC strains possessed

27V 62S 163V polymorphism patterns, as seen in K12 E. coli strains (Table 3). This

same set of genomes were compared to adherent-invasive E. coli in 10 mutational

hotspots (43). The resulting distribution shows separation beginnings of separation

between APEC, NMEC, and UPEC strains, but the large number residues examined

creates numerous polymorphic patterns present in only one or two strains (Table 4).

Total NMEC UPEC APEC Table 3. FimH alleles based on positively ASV 154 47 81 26 selected UPEC residues and incidence among ASA 1 0 1 0 CFT073 pathotypes. AAV 3 2 1 0 UTI89 Table 4. FimH alleles based on adherent- VAA 0 0 0 0

Table 4. FimH alleles based on adherent- AAA 0 0 0 0 VSV 73 4 65 1 K12 Total 231 53 148 27

Total NMEC UPEC APEC ASV 66.7% 88.7% 54.7% 96.3% ASA 0.4% 0.0% 0.7% 0.0% CFT073 AAV 1.3% 3.8% 0.7% 0.0% UTI89 VAA 0.0% 0.0% 0.0% 0.0% AAA 0.0% 0.0% 0.0% 0.0% VSV 31.6% 7.5% 43.9% 3.7% K12

Table 4. FimH alleles based on adherent-invasive E. coli

Total NMEC UPEC APEC Total NMEC UPEC APEC TVGNGATVRY 78 4 65 1 K12 TVGNGATVRY 28.5% 7.5% 43.9% 3.7% K12 TAGNGATVRY 64 5 31 20 TAGNGATVRY 23.4% 9.4% 20.9% 74.1% NAGNGATVRY 40 5 25 0 NAGNGATVRY 14.6% 9.4% 16.9% 0.0% TAGSGATVRY 26 16 3 5 UTI89 TAGSGATVRY 9.5% 30.2% 2.0% 18.5% UTI89 TAGNGATVHY 19 3 15 1 TAGNGATVHY 6.9% 5.7% 10.1% 3.7% TACNGATVRY 7 3 3 0 TACNGATVRY 2.6% 5.7% 2.0% 0.0% TAGSGVTVSY 6 6 0 0 TAGSGVTVSY 2.2% 11.3% 0.0% 0.0% TASSGATVRY 4 3 1 0 TASSGATVRY 1.5% 5.7% 0.7% 0.0% TAGNGATVHH 3 0 0 0 TAGNGATVHH 1.1% 0.0% 0.0% 0.0% TAGNGATVRF 3 2 0 0 TAGNGATVRF 1.1% 3.8% 0.0% 0.0% TASNGATVRY 3 0 3 0 TASNGATVRY 1.1% 0.0% 2.0% 0.0% TADNGATVRY 2 1 1 0 TADNGATVRY 0.7% 1.9% 0.7% 0.0% TAGNAATVRY 2 0 0 0 TAGNAATVRY 0.7% 0.0% 0.0% 0.0% TAGNWATVRY 2 0 0 0 TAGNWATVRY 0.7% 0.0% 0.0% 0.0% TAGSGATARY 2 0 1 0 CFT073 TAGSGATARY 0.7% 0.0% 0.7% 0.0% CFT073 NAGNGATVRF 1 1 0 0 NAGNGATVRF 0.4% 1.9% 0.0% 0.0% PAGNGATVRY 1 1 0 0 PAGNGATVRY 0.4% 1.9% 0.0% 0.0% PAGSGVTVSY 1 1 0 0 PAGSGVTVSY 0.4% 1.9% 0.0% 0.0% SAGSGATVRY 1 1 0 0 SAGSGATVRY 0.4% 1.9% 0.0% 0.0% TAASGTTVRY 1 0 0 0 TAASGTTVRY 0.4% 0.0% 0.0% 0.0% TAGNGATVCC 1 0 0 0 TAGNGATVCC 0.4% 0.0% 0.0% 0.0% TAGNGATVSY 1 0 0 0 TAGNGATVSY 0.4% 0.0% 0.0% 0.0% TAGSEATVRY 1 0 0 0 TAGSEATVRY 0.4% 0.0% 0.0% 0.0% TAGSGAAVRY 1 0 0 0 TAGSGAAVRY 0.4% 0.0% 0.0% 0.0% TAGSGAPVRY 1 0 0 0 TAGSGAPVRY 0.4% 0.0% 0.0% 0.0% TAGSGVTVRY 1 1 0 0 TAGSGVTVRY 0.4% 1.9% 0.0% 0.0% TAGSRATVRY 1 0 0 0 TAGSRATVRY 0.4% 0.0% 0.0% 0.0% TAGSWATVRY 1 0 0 0 TAGSWATVRY 0.4% 0.0% 0.0% 0.0% Sum 274 53 148 27

Identification of the most common amino acid residues at each polymorphic site and its incidence was performed for all strains, and results were grouped by sub- pathotype (Table 5). The resulting analysis showed four sites (positions 27, 70, 78 and

119) with significantly different prevalence of mutations between at least two sub- pathotypes. Concatenation of these polymorphic sites resulted in 5 distinct FimH polymorphic patterns, with the most prevalent patterns being: ASNA in NMEC (52.8%),

VSNA in UPEC (53.9%), and ANSA in APEC (66.7%), all of which were significantly more prevalent in their respective sub-pathotype at P < 0.01 as determined by Fisher’s exact test.

Table 5. FimH allele analysis based on residue variance in positions 27, 70, 78, and 119

2 Total NMEC UPEC APEC X VNSA 78 4 65 1 K12 P< 0.0001 ASNA 47 28 5 5 CFT073 UTI89 P< 0.01 ANNA 12 4 0 3 P< 0.05 ANSA 97 12 53 18 ANSV 39 5 24 0 Sum 273 53 147 27

Total NMEC UPEC APEC VNSA 28.6% 7.5% 44.2% 3.7% K12 ASNA 17.2% 52.8% 3.4% 18.5% CFT073 UTI89 ANNA 4.4% 7.5% 0.0% 11.1% ANSA 35.5% 22.6% 36.1% 66.7% ANSV 14.3% 9.4% 16.3% 0.0%

Sequenced ompA of NMEC display 7 unique patterns of polymorphisms, while OmpA of

UPEC is relatively conserved.

The sequences of the outer membrane protein gene ompA were extracted from the assembled genomic sequences of NMEC, ExPEC, and the laboratory strains. The extracted 1056 bp nucleotide sequences contained nucleotide polymorphisms or gaps in

91 positions (8.6%). Transmembrane loops and periplasmic turns were identified in all

ExPEC and laboratory sequences used in this study. Resulting concatenation of protein polymorphic sites resulted in 7 distinct OmpA polymorphic alleles differing at 16 residues (Figure 4). Some OmpA alleles differ by as little as one amino acid, while other alleles differ in all 16 amino acid positions. The most frequently occurring polymorphic sites in NMEC when compared to UPEC were in loop positions 1-3. These polymorphisms frequently altered hydrophobicity or charge of the extracellular loop residues. Loop 3 polymorphisms often included a 5 amino acid insertion, increasing the 65

Extracellular Loop L1 L2 L3 L4 C-terminal AA Position 46 87 88 89 114 129 131 132 133 134 135 136 137 138 140 177 187 201 229 277 MG1655 N S V E I S V - - - - - Y G N H M A N G NMEC N S V E V S V - - - - - Y G N H M A T A 0.566038 0.641509 0.641509 0.641509 0.679245 0.622642 0.566038 0.622642 0.622642 0.622642 0.622642 0.622642 0.566038 0.622642 0.641509 0.962264 0.943396 0.962264 0.867925 0.811321 UPEC N S V E V S V - - - - - Y G N H M A T G 0.992248 0.992248 0.992248 0.976744 0.72093 0.992248 0.976744 0.992248 0.992248 0.992248 0.992248 0.992248 0.976744 0.992248 0.992248 1 1 0.992248 0.728682 0.534884

Figure 4. Amino acid alignment of variable amino acid residues of OmpA.

69 loop size to 9 amino acids. Polymorphisms of UPEC were primarily found in the C terminal end of the protein, and were not contained in the extracellular loops used for

OmpA typing in this study. UPEC most often possessed the OmpA allele 1 from this study, with only 5 strains (3.4%) possessing a different polymorphic pattern, and are strikingly different from NMEC, where 48.1% of strains carried a different allele. When examining the polymorphisms in the C-terminal protein portion, polymorphisms occurred more frequently in UPEC strains (54.3%) versus NMEC strains (24.5%), but little no studies have been performed regarding the function of this region in NMEC or UPEC virulence.

Antibiotic Resistance Genotyping.

Assembled NMEC strains (48), previously assembled NMEC strains (5) as well as UPEC (2) and APEC (3) genomes were assessed using BLAST and the CARD antimicrobial resistance database to identify antimicrobial resistance genes. The resultant analysis identified 481 possible antimicrobial resistance-associated genes amongst all of the assembled NMEC isolates. Scripts were used to assess the pool of 481 candidate genes for duplications. A majority of duplicated or repeated resistance genes resulted from polymorphisms occurring within similar resistance genes, notably blaTEM, encoding resistance to extended spectrum beta-lactamases; sul1, encoding resistance to sulfonamides; and aadA conferring resistance to aminoglycosides. Such duplicates were removed from further consideration. The resulting ‘de-duplicated’ resistance gene pool contained 22 genes encoding specific antibiotic resistances, while 45 putative non- specific genes were identified, encoding efflux systems or genetic regulatory elements

(Table 6). Resulting resistance profiles revealed the most frequent antimicrobial 1

Table 6. Heatmap listing of all antimicrobial resistance genes. Grid map of all antimicrobial resistance genes associated with NMEC genomes. Red indicates positive resistance genotype; black indicates susceptible genotype.

Gene Antibiotic NM3 NM11 NM14 NM15 NM19 NM20 NM22 NM23 NM26 NM27 NM28 NM34 NM38 NM40 NM49 NM55 NM57 NM67 NM70 NM74 NM86 NM89 NM91 bacA Bacitracin 0 0 1 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 0 0 1 0 1 strA Streptomycin 1 0 0 0 1 0 0 0 0 0 1 0 0 1 1 0 1 1 0 1 1 1 0 sul2 Sulfonamides 1 0 0 0 1 0 0 0 0 0 1 0 1 1 1 0 1 1 0 1 0 1 0 VC Aminoglycoside 1 0 0 0 1 0 0 0 0 0 1 0 0 1 1 0 1 1 0 1 1 1 0 aadA Streptomycin, Spectomycin 1 1 0 1 0 1 1 0 1 1 0 0 0 1 0 1 0 0 1 0 0 0 0 strB Streptomycin 0 0 0 0 1 0 0 0 0 0 1 0 0 1 1 0 1 1 0 1 1 1 0 TEM-1 Beta lactam 1 0 0 0 0 1 0 0 1 0 1 1 0 1 0 1 0 0 1 0 1 1 0 aadA15 Streptomycin, Spectomycin 1 1 0 1 0 1 1 0 1 0 0 0 0 1 0 1 0 0 1 0 0 0 0 aadA22 Streptomycin, Spectomycin 1 1 0 1 0 1 1 0 0 1 0 0 0 1 0 1 0 0 1 0 0 0 0 sulI Sulfonamides 0 1 0 1 0 1 1 0 1 0 0 0 0 1 0 1 0 0 1 0 0 0 0 aadA3 Aminoglycoside 0 1 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 aadA39 Streptomycin 0 1 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

70 dfrA1 Trimethoprim 1 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 0 0 0 0 0 CatA1 Chloramphenicol 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 aadA16 Streptomycin, Spectomycin 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 aphA1 Kanamycin, Amikacin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 mphA Macrolide 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 mphB Macrolide 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 sat Streptothricin 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 tetA Tetracycline 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 tetD Tetracycline 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 dfrA12 Trimethoprim 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 71

resistances were for aminoglycoside antimicrobials, with strains often possessing multiple aminoglycoside adenylyltransferases and phosphotransferase. Profiles for sulfonamide resistance were similar, with a number of strains possessing plasmid borne sul2 (22%) or integron based sul1 (16%) (77), with a single strain, NMEC 40, possessing copies of both genes. Lower frequencies of occurrence were observed for genes encoding resistance to phenicols and tetracyclines (<5%). Resulting non-specific resistances were encoded by regulatory proteins, such as HNS and emrAB or non-specific efflux pumps.

To verify the genotyping of the NMEC strains, we performed phenotypic testing on the NMEC strains sequenced in this study. Totaled, 27 of the sequenced NMEC strains harbored 65 resistance phenotypes, compared with the genotypic analysis predicting 113 putative resistance genes conferring resistance in 22 sequenced genomes.

Five of the sequenced genomes, NMEC 58, 59 and 82-84, showed phenotypic antimicrobial resistance, but the genotypic analysis failed to identify genes known to grant resistance (Table 7).

Inconsistencies between observed genotype and phenotype occurred in 19 genomes.

Genotypic testing predicted aminoglycoside resistance in six NMEC strains and sulfonamide resistance in one separate strain where none was observed phenotypically. In addition, genotyping failed to identify tetracycline resistance in seven strains, aminoglycoside resistance in four strains, and beta-lactamase resistance to ampicillin in 2 72

Table 7. NARMS antimicrobial resistance patterns for sequenced neonatal bacterial meningitis strains and comparison with observed antibiotic genotypes. Comparison of observed phenotypic antimicrobial resistance by broth microdilution and predicted antimicrobial resistance genotypes. Alpha error corresponds to a genotypically positive, but phenotypically negative result (false positive) while beta error refers to a genotypically negative phenotypically positive result (false negative).

Strain NARMS Resistance Phenotype Putative Resistance Genes ε NMEC 3 Aminoglycoside VC α Ampicillin blaTEM Streptomycin strA Sulfisoxazole sul Co-Trimoxazole dfrA NMEC 11 Streptomycin aadA α Sulfisoxazole sul NMEC 15 Streptomycin aadA α Sulfisoxazole sul NMEC 19 Aminoglycoside VC α Streptomycin strA strB Sulfisoxazole sul NMEC 20 Ampicillin blaTEM Streptomycin aadA α Sulfisoxazole sul Tetracycline - β NMEC 22 Streptomycin aadA α Sulfisoxazole sul Tetracycline - NMEC 26 Ampicillin blaTEM Kanamycin - β Macrolide mphA Streptomycin aadA α Sulfisoxazole sul Tetracycline - β Co-Trimoxazole dfrA NMEC 27 Bacitracin bacA Streptomycin aadA Streptothrycin sat Co-Trimoxazole dfrA α NMEC 28 Aminoglycoside VC α Ampicillin blaTEM Streptomycin strA strB Sulfisoxazole sul Tetracycline - β NMEC 34 Ampicillin blaTEM NMEC 38 Sulfisoxazole sul α NMEC 40 Aminoglycoside VC α Ampicillin blaTEM Chloramphenicol catA1 Macrolide mphB Streptomycin strA strB aadA Sulfisoxazole sul Tetracycline - β Co-Trimoxazole dfrA NMEC 45 Aminoglycoside VC α Bacitracin bacA Streptomycin strA strB Sulfisoxazole sul Tetracycline - β NMEC 49 Aminoglycoside VC α Streptomycin strA strB Sulfisoxazole sul NMEC 55 Ampicillin blaTEM Bacitracin bacA Streptomycin aadA Sulfisoxazole sul Tetracycline - β Co-Trimoxazole dfrA NMEC 57 Aminoglycoside VC α Streptomycin strA strB Sulfisoxazole sul NMEC 58 Co-Amoxiclav - β Ampicillin - β NMEC 59 Co-Amoxiclav - β Ampicillin - β NMEC 67 Aminoglycoside VC α Streptomycin strA strB Sulfisoxazole sul NMEC 70 Ampicillin blaTEM Chloramphenicol CatA1 Streptomycin aadA Sulfisoxazole sul NMEC 74 Aminoglycoside VC Kanamycin aphA1 Streptomycin strA strB Sulfisoxazole sul Tetracycline tetA tetD NMEC 82 Streptomycin - β NMEC 83 Streptomycin - β NMEC 84 Streptomycin - β NMEC 86 Aminoglycoside VC α Ampicillin blaTEM Bacitracin bacA Streptomycin strA strB Tetracycline - β NMEC 89 Aminoglycoside VC α Ampicillin blaTEM Streptomycin strA strB Sulfisoxazole sul Co-Trimoxazole NMEC 91 Bacitracin bacA Streptomycin - β 73 strains and amoxicillin/clavulanic acid in two other strains that was detected phenotypically (Table 8). Resistance gene occurrence without the corresponding phenotype can be explained by mutations rendering the gene ineffective, non-expression of the antimicrobial resistance gene in the assay, or a resistance gene conferring resistance to a specific antimicrobial not tested. Strains possessing an antimicrobial resistance phenotype without a corresponding genotype could occur for multiple reasons, such as a novel resistance gene not facilitating resistance or activity of efflux systems not currently characterized to export antimicrobials or general export systems. Finally a resistance gene could be present in these strains, but located in an unassembled chromosomal or plasmid region.

Table 8. Overall incidence of genotypic and phenotypic antimicrobial resistance

Phenotype Genotype Antibiotic Phenotype Total Percent Antibiotic Genotype Total Percent Antimicrobial Amikacin 0 0% aadA 9 18% Streptomycin Co-Amoxiclav 2 4% Spectomycin Ampicillin 12 24% aphA1 1 2% Kanamycin Cefoxitin 0 0% bacA 5 10% Bacitracin Ceftiofur 0 0% blaTEM 10 20% β-lactam Ceftriaxone 0 0% catA1 2 4% Chloramphenicol Chloramphenicol 2 4% dfrA 5 10% Co-Trimoxazole Ciprofloxacin 0 0% mphA 1 2% Erythromycin Gentamicin 0 0% mphB 1 2% Macrolide Kanamycin 2 4% sat 1 2% Streptothrycin Nalidixic Acid 0 0% strA 1 2% Streptomycin Streptomycin 16 33% strAB 10 20% Streptomycin Sulfisoxazole 17 35% sul 18 37% Sulfonamide Tetracycline 9 18% tetAD 1 2% Tetracycline Co-Trimoxazole 5 10% VC 11 22% Aminoglycoside

Discussion

Current knowledge on the pathogenesis of NMEC reflects decades of research on the phenotypic and genetic properties of a select group of NMEC strains, namely those sharing the O18:K1:H7 serotype and in the B2 phylogenetic group. In particular, much of what we know is owed to intensive study of a single strain of NMEC, NMEC RS218

(20, 24, 26-29, 69, 78). While a great deal has been discovered studying NMEC RS218, it 74 has become clear that it does not represent all NMEC strains (30, 31). Compounding this narrow focus is the paucity of diverse NMEC genomic sequences available in the public domain, as such sequences are often critical reagents supporting state-of-the-art approaches to pathogenesis studies. A number of NMEC described in the literature are assigned to different serotypes and phylogenetic groups than RS218 (30, 32, 33), lack K1 capsule, and harbor diverse sets of virulence genes, including several genes thought to be critical to NMEC pathogenesis, such as ibeA, gimB, cnf1, and aslA (22, 23, 26, 78).

Currently, sequenced NMEC include NMEC RS218 and IHE 3034, both of the O18:K1 serotype (46). Though there have been efforts to expand the sequenced NMEC strains to better reflect this diversity (NMEC CE10 (33) and NMEC S88 (32)), relatively few pathogenesis studies have been carried out using these strains. As part of our purpose here, we seek to ascertain the diversity of NMEC, generate new genomic sequences to support community-wide study of diverse NMEC determine the prevalence of known essential virulence mechanisms, and determine if whole genome and polymorphic methods can distinguish between ExPEC sub-pathotypes.

To fulfill this purpose, we sequenced 48 randomly selected NMEC strains that were isolated from human infants with meningitis in the United States and Europe, and used a genomic epidemiological approach to better characterize the NMEC sub- pathotype. In this manner we further enhanced the body of genomic data available for researchers to study in meningitis-causing E. coli, gain greater understanding of the disease as a whole, and glean insight into NMEC’s relationship to other ExPEC.

Incidence of NMEC virulence factors in sequenced strains.

Numerous studies have characterized NMEC virulence factors (2). These include cnf1, ompA, fimH, ibeA, aslA, and K1 capsular synthesis genes, all of which have been shown to contribute to NMEC pathogenesis (8, 39, 68, 78-81). For instance, OmpA changes the secretion pattern of cytokines in macrophage and dendritic cells, facilitates binding to macrophage and HBMEC, and prevents classical complement activation by promoting degradation of complement factors C3b and C4b (13). The K1 capsule contributes to invasion and resistance to the innate immune response (13). All of these genes are described as significant contributors to virulence or are considered to be essential for NMEC pathogenesis. Thus, it would seem that these genes would occur among all or most NMEC and, perhaps, few or no laboratory strains of E. coli. This was not the case.

cnf1 was found in only 3 NMEC strains sequenced in this study (NMECs 40, 42,

45) as well as RS218, displaying a prevalence similar to previous studies (30, 31).

Current literature suggests cnf1 facilitates invasion of the blood-brain barrier by myosin rearrangement facilitated by RhoGTPases (2, 78, 82, 83), but the lack of cnf1 in the majority of strains sequenced in this study suggests that alternate mechanisms of BBB transversal are more commonly utilized. Additionally the S-fimbrial gene, sfaS, which encodes an adhesin shown previously to adhere to brain glycolipids and reduce phagocytosis (25, 84), was present in only 39.6% of sequenced NMEC strains. ibeA, thought to be critical in invasion of the blood brain barrier (22, 23) was found in only 27

NMEC isolates sequenced in this study (54.7%) as well as IHE3034 and RS218. The prevalence of aslA, previously described as enhancing HBMEC invasion as well as being 76 necessary for invading HBMEC in vitro and in vivo (20, 26), and K1 capsule, which is thought to be critical for NMEC’s survival during HBMEC invasion (80), was high among sequenced NMEC was much higher (86.8% and 80% of all sequenced NMEC strains respectively); however, aslA was also found in APEC O1 and APEC IMT5155, and UPEC CFT073 and UPEC UTI89, suggesting it may play a role in pathogenicity or virulence of multiple ExPEC sub-pathotypes, not just NMEC. Similarly, K1 capsule occurs commonly in many ExPEC as fecal flora of healthy individuals (85). K1 capsule consists of a linear polymer of sialic acid, which mimics the polysialic acid located on human cells, and its poor immunogenicity likely functions to shield NMEC from the immune system (85). In addition to these properties, K1 capsule confer resistance to bacteriophage infection (86), resistance to desiccation (87), and survival in serum (87,

88).

This study did not identify new virulence factors in NMEC strains, and confirmed lower prevalence of the virulence genes cnf1, ibeA, and sfaS than found in other studies

(30). Virulence factors most common in the NMEC population were the K1 capsule, the chuSTX heme utilization genes, and the putative arylsulfatase gene aslA. The K1 capsule is well described in NMEC (10, 85), but studies of chuSTX and aslA in NMEC are limited. Sulfatases are known for involvement in cell regulation, development, and signaling (89). The AslA protein may modulate these cellular process to enhance NMEC fitness. Similarly chu genes are known for heme utilization and are largely thought to contribute to iron acquisition (72, 90), but a recent study shows chu system expression modulates macrophage production of IL-12 and reduces host inflammatory response (91). 77

This suggests the chu system’s role in NMEC pathogenesis may be more nuanced and may explain the high prevalence of this gene in the NMEC population.

The low incidence of virulence genes and factors currently thought critical for the pathogenesis of NMEC, combined with some of their functional redundancy, suggests current knowledge of NMEC pathogenesis may be too reliant on study of few strains, and that pathogenesis is not fully understood. The redundancy observed in virulence functions within NMEC strains indicates NMEC may utilize a “grab bag” approach, requiring combinations of essential genes. This is supported by difficulties in studies attempting to define the NMEC sub-pathotype based on virulence gene prevalence. One such study defining characteristics between NMEC and human fecal E. coli most of the defining characteristics separating NMEC from fecal E. coli were plasmid borne virulence genes such as hlyF ompTp and iss, commonly found in APEC. A more recent study indicates 6 properties: kpsII (APEC 25%, UPEC 77.5%, NMEC 84.6%), K1 capsule, neuC, iucC, sitA (AP 89.6%, UP 77%, NM 94.5%, and vat (APEC 33.6%, UPEC

63.5%, NMEC 74.7%) can be used to define the NMEC sub-pathotype. While these genes occur less frequently in other ExPEC sub-pathotypes, the gene prevalence differences between ExPEC sub-pathotypes are not significant (30). Similarly, the genes iucC, sitA, and vat are found on large transmissible virulence plasmids. Previous studies into these virulence plasmids have shown that they enhance the virulence of meningitis- causing strains, but are not solely responsible for pathogenesis (28). Previous studies have cited the zoonotic potential of APEC strains and indicated some APEC may be able to cause disease in multiple host species and tissues due to genetic similarity (45, 92-94).

Taken together with studies underscoring continued difficulties defining distinct ExPEC 78 sub-pathotypes (30, 31), the close evolutionary relationship of ExPEC sub-pathotypes, and studies showing sub-pathotypes causing disease in multiple ExPEC models (95), it is likely ExPEC are zoonotic pathogens. When combined, these data show factors that enhance and contribute to pathogenicity, but further study regarding combinations of virulence factors necessary and sufficient to cause disease among the entire ExPEC sub- pathotype are necessary.

Polymorphism analysis of the fimH gene in comparison to UPEC and APEC.

Previous studies of FimH, the tip adhesin of the Type 1 pilus, in UPEC (74) have shown that polymorphisms in amino acid residues outside of the mannose-binding pocket are correlated with different attachment affinities to bladder and kidney cells (74). This study identified amino acid residues 27, 62, and 163 as mutational hotspots in fimH that impact bacterial persistence in the mouse model of chronic cystitis, identifying three pathogenic polymorphic patterns (74). Based on these observations, the authors constructed a typing scheme. Using this typing scheme, we found 47 of the 53 NMEC strains in this study (88.6%) possessed the A27/S62/V163 allele: NMEC IHE3034 and

S88 possessed the A27/A62/V163 allele similar to UPEC UTI89: and four NMEC possessed the VSV allele seen in laboratory E. coli strains DH1 and W3110. This result is not unexpected, as this typing scheme was originally developed to differentiate fecal E. coli from UPEC, and not distinguish between ExPEC sub-pathotypes (74). According to work performed in UPEC strains (74), the A27/S62/V163 allele, which we found most commonly among NMEC, confers the ability to adhere to bladder epithelial cells but not as strongly as allele AAV (74). Further analysis of FimH proteins obtained from the

Broad Institute’s E. coli as a human pathogen project show less variation at these 79 positively selected sites. No strains from this collection possessed the enhanced bladder epithelial binding polymorphisms ASA or AAV found in strains CFT073 and UTI89 respectively (74). Instead the preponderance of UPEC strains (54.7%) showed the same

ASV allele dominantly found in NMEC strains (88.7%), as well as a 43.9% minority possessing the VSV allele possessed by K12 E. coli. These two alleles accounted for all

UPEC strains in the study except CFT073 and UTI89. This pattern indicated that polymorphisms in positions 27, 62 and 163 may not be the best discriminators of sub- pathotypes in this study. Analysis of all strains in this study, organized by sub-pathotype, instead diverged most frequently in positions 27, 70, 78, and to a lesser degree position

119 creating 5 distinct alleles. Fisher’s exact test showed the amino acids were significantly different between APEC, UPEC, and NMEC sub-pathotypes at position 70 and 78 (P< 0.01). In position 27 NMEC and APEC significantly differed from UPEC but not from each other (p< 0.0001). Finally position 119 showed a significant difference between APEC and UPEC only (p< 0.01). The VSNA allele was more common in

UPEC strains (P< 0.0001), while NMEC possessed the ASNA allele significantly more often than other sub-pathotypes (P< 0.005), and finally APEC possessed the ANSA allele more often than both NMEC and UPEC (P< 0.01). When taken together these data associate specific FimH polymorphic patterns with different ExPEC sub-pathotypes, and indicate the fimH gene may be under differing selection pressure in each of the sub- pathotypes.

Multiple Distinct OmpA alleles are distributed across NMEC strains but not UPEC strains.

OmpA is an important and multifunctional outer membrane protein in E. coli.

OmpA has four outer membrane loops, 8 transmembrane beta strands, and three periplasmic turns (96). Many of the residues in this protein are highly conserved; however, polymorphisms within the OmpA protein’s four outer membrane loops have been previously reported (97). In addition to its role as a phage receptor, OmpA has been shown to contribute to many cellular processes including: adhesion, cell invasion, biofilm formation, ion transport, immune recognition, and immune evasion (41). Given its importance in many processes in the pathogenesis of NMEC, we sought to further examine ompA gene for any polymorphisms that might impact NMEC virulence. Strains containing the OmpA type 1 allele in this study were by far the most common, found in

51.9% of NMEC and 96.1% of UPEC strains. The next most common allele was

OmpA5, present in 32.7% of NMEC strains, while absent from UPEC strains. Indeed the only other alleles in UPEC strains were OmpA type 2, which differs very little from type

1, and single isolate types 9 and 10. This stark difference between UPEC and NMEC strains strongly suggests that OmpA of NMEC strains is currently is undergoing selection pressure. The OmpA protein in NMEC contributes to innate immune complement resistance, macrophage invasion, macrophage survival, alteration of macrophage cytokine expression, and binding to HBMEC, and suggests these polymorphisms may enhance NMEC fittness.

Current study of OmpA in NMEC strains is limited to strain RS218, carrying the

OmpA type 1 allele (14, 98), and two studies of OmpA loop mutants in RS218 (14, 98). 81

The studies of OmpA loop mutations either deleted entire outer membrane loops (14), or mutated 3 to 4 consecutive outer membrane loop amino acids to alanine (98). These studies showed loops 1 and 2 contributed to serum survival (14), and deletion or poly- alanine replacements in loops 1, 2, and 3 significantly affected strain RS218’s ability to invade HBMEC (14, 98).

Revisiting studies describing the multiple functions of OmpA and its diverse functions during NMEC pathogenesis is likely warranted. OmpA alleles 4 to 7 all contain multiple polymorphisms in loops 1-3, many of which alter fundamental amino acid properties such as polarity and charge, and were already shown to have significant effects on NMEC HBMEC invasion (14, 98).

NMEC harbor UPEC-like resistance patterns.

Antimicrobial resistance gene screening was performed on isolates in this study.

Overall, 47% of meningitis-causing E. coli strains in this study were genotype positive for at least one antimicrobial resistance gene. The most common resistance genes were in the sulI family (37%), conferring resistance to sulfonamides; the blaTEM family

(20%), conferring resistance to beta-lactamases; and the strAB genes (20%), conferring resistance to streptomycin (99). Antimicrobial phenotype testing was previously performed on the 48 strains sequenced in this study and NMEC 58. The resultant phenotypes were generally consistent with predicted genotypic resistance genes, except in the case of few specific antimicrobial agents. Four strains, NMEC 82, 83, 84, and 91, were phenotypically resistant to streptomycin but harbored no gene known to encode streptomycin resistance. Similarly, the tetracycline-resistance phenotype was found in seven strains, but no corresponding resistance gene was found for either tetracycline 82 degradation or ribosomal protection, nor tetracycline-resistant ribosomal sequences.

While no specific resistance gene was found in corresponding strains, many also possessed uncharacterized putative ABC transporter systems, as well as characterized efflux systems that could account for antimicrobial resistance. Similarly, specific resistance genes encoded on transmissible R type plasmids or in chromosomal integrons can be difficult to assemble in whole genomic sequencing due to high repetitive sequence content and multiple phage insertions, which could have caused removal from this analysis (100). Finally, these phenotypic resistances could be caused by a novel antimicrobial resistance gene, or an antimicrobial resistance gene not entered into the database.

The overall incidence antimicrobial resistance for antimicrobial agents in the

NARMS testing scheme was nearly identical to the resistance pattern seen in UPEC

[unpublished data], and significantly less frequent compared to antimicrobial resistance phenotypes tested in APEC [unpublished data]. The lower occurrence of resistance genes in NMEC in comparison to APEC is likely due to differing antimicrobial exposure, as

APEC strains are known to gain resistance genes after antimicrobial introduction in production facilities (101, 102). Comparison of NMEC and UPEC strains shows remarkable similarity in resistance profiles, especially regarding sulfonamides, ampicillin, and streptomycin. Sulfonamides, particularly trimethoprime/sulfamethoxazole, are currently advised for treating cystitis in the United

States and is a second line treatment for pyelonephritis (103). High resistance levels in

UPEC is in agreement with these data, but NMEC strains phenotypically resistant to sulfisoxazole and cotrimoxazole (trimethoprim/sulfamethoxazole) is aberrant. Though 83 sulfisoxazole and cotrimoxazole both reach high plasma concentrations and can penetrate the BBB, use in neonates and children below the age of 2 months is contraindicated.

Studies performed in 1956 and 1959 (104, 105) showed sulfisoxazole caused increased levels of plasma bilirubin, possibly causing kernicterus and death. These studies resulted in contraindication in both neonates and during pregnancy. NMEC in this study were most commonly resistant to sulfisoxazole, with cotrimoxazole resistance being the fourth most prevalent. The sulI gene encoding dihydropteroate synthase is present in all sulfisoxazole resistant strains, and is commonly encoded on resistance plasmids. This indicates likely acquisition of resistance plasmids from UPEC strains, or could be an indication of UPEC strains as a common ancestor for some NMEC.

Summary

The population of NMEC strains sequenced in this study showed a heterogeneous mixture of virulence genes, and often lacked virulence factors thought critical to meningitis pathogenesis. Further phenotypic analysis of the common NMEC virulence genes fimH and ompA revealed a high level of polymorphisms present in both proteins in sites that phenotypically alter their function. This indicates that current knowledge of

NMEC pathogenesis, which is largely based on analysis of one allele of fimH and ompA, may not accurately describe pathogenesis of all NMEC. Comparison of polymorphic sites in populations of NMEC, UPEC, and APEC showed some polymorphic patterns are significantly associated with specific ExPEC sub-pathotypes. Comparison of phenotypic and genotypic antimicrobial resistance predictions in NMEC strains were largely in agreement, though genotypic prediction of tetracycline and tetracycline resistance was highly inaccurate. Finally, the overall antimicrobial resistance patterns of UPEC and 84

NMEC were highly similar, with predominant resistance to sulfonamides in NMEC likely indicating resistance plasmid transfer from, or recent common ancestry to, UPEC.

Acknowledgements

We would like to thank Brett J. Kail for his assistance with Perl programming and debugging.

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CHAPTER 3. LARGE VIRULENCE PLASMIDS IN NEONATAL MENINGITIS

ESCHERICHIA COLI : PHYLOGENETIC RELATIONSHIPS AND CORE AND

ACCESSORY GENOME

A paper submitted to the journal PLOS ONE

Bryon A. Nicholson a, Aaron C. West b, Paul Mangiamele a, Nicolle Barbieri a,

Yvonne Wannemuehler a, Lisa K. Nolan a, Catherine M. Logue a, Ganwu Li c, d

aDepartment of Veterinary Microbiology and Preventive Medicine, College of

Veterinary Medicine, Iowa State University, Ames, Iowa, USA

bDepartment of Chemistry, College of Liberal Arts and Sciences, Iowa State

University, Ames, Iowa, USA

cDepartment of Veterinary Diagnostic and Production Animal Medicine, College

of Veterinary Medicine, 1802 University Blvd., Iowa State University, Ames, Iowa

50011, USA

dDepartment of Veterinary Preventive Medicine, College of Veterinary Medicine,

Nanjing Agricultural University, Nanjing, Jiangsu 210095, P. R. China

Abstract

Neonatal Meningitis Escherichia coli (NMEC) is one of the most common causes

of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or

neurologic deficits in survivors. Large plasmids have been shown experimentally to

increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9

virulence plasmids with various genetic profiles were isolated from NMEC and

sequenced to identify the core and pan plasmid genes of NMEC and create an expanded

plasmid phylogeny. Results showed NMEC virulence plasmids carry a strongly

conserved core of genes with predicted functions in five distinct categories including:

virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The

major functions of virulence-associated and plasmid core genes serve to increase in vivo

fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate

NMEC’s survival in the host’s low iron environment, and systems to enhance bacterial

resistance to host innate immunity. Phylogenetic analysis based on these core plasmid

genes showed that at least two lineages of NMEC virulence plasmids could be discerned.

Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could

not be differentiated based solely on the genes of the core plasmid genome.

Keywords: Avian Pathogenic E. coli (APEC), Extra intestinal pathogenic E. coli (ExPEC), Neonatal Meningitis E. coli (NMEC), Virulence plasmid, Virulence,

Evolution

Importance

Neonatal meningitis-associated Escherichia coli (NMEC) as well as some other types of extra intestinal pathogenic E. coli (ExPEC) are largely defined by the large plasmid; however relatively few NMEC plasmids have been sequenced and relatively few studies have examined their effects on pathogenicity. In this study we sequenced 9 plasmids from cases of neonatal bacterial meningitis to determine the core set of genes, and to determine if an evolutionary relationship between ExPEC plasmids exists. Our results show the core plasmid genome of NMEC strains to include genes related to virulence and metabolism as well as a high degree of relatedness to other large ExPEC plasmids.

Introduction

Escherichia coli is one of the world’s most well-studied organisms. Ubiquitous in

nature and prevalent in biotechnology, it is also one of the most commonly isolated

pathogens from human disease. Though some strains are specialized to cause disease

inside the intestinal tract, others are equipped to cause extraintestinal disease (1, 2).

These extraintestinal pathogenic E. coli (ExPEC) include neonatal meningitis E. coli

(NMEC), one of the predominant agents of neonatal bacterial meningitis (3). Neonatal

bacterial meningitis has a mortality rate between 15 to 40% (4) with 30% of survivors

showing serious neurological sequelae including profound intellectual disability,

epilepsy, or deafness (3). Reviews suggest that NMEC are acquired by neonates from

their mothers perinatally (4, 5). From the initial site of colonization, NMEC transcytose

the gastrointestinal tract into the bloodstream, and from there, they traverse the blood

brain barrier (BBB) (the tight barrier formed by human brain microvascular endothelial 95

cells (HBMEC)) into the central nervous system (CNS). Since successful CNS invasion

requires a high bacteremia, NMEC must survive in the bloodstream, a trait that is

mediated by two bacterial components, an anti-phagocytic capsule and outer membrane

protein A (OmpA), which has an anti-complement role. In addition, NMEC’s replication

in immune cells may play a role in achieving the requisite bacteremia. Also, NMEC’s

traversal of HBMEC is preceded by its attachment to the BBB via Type 1 pili and OmpA,

with invasion itself being mediated by Ibe proteins, FimH (tip adhesin of Type 1 pili),

OmpA, and Cytotoxic Necrotizing Factor 1 (CNF1). K1 capsule may also contribute to

invasion by preventing lysosomal fusion, allowing delivery of live bacteria into the CNS

(4, 5).

Plasmids also seem to contribute to NMEC virulence. A recent study by

Kariyawasam and colleagues indicates that a large virulence plasmid increases the

virulence of NMEC RS218 in vivo (6), while previous studies have shown that the

acquisition of large virulence plasmids is one of the primary sources of genetic variance

and virulence genes in different E. coli pathotypes (7). The majority of these plasmids has been identified as IncF - type plasmids, encoding pathogenicity islands, metabolic genes, antimicrobial resistance genes, and hypothetical genes. Acquisition of these plasmids may provide an advantage for survival during infection and in suboptimal environments, as evidenced by multiple studies showing that these plasmids confer a fitness advantage to NMEC during in host tissues (6, 8, 9). Further, plasmids are associated with an increase in NMEC virulence in animal models (6, 8, 9), and cause cross-species and cross pathogenic group gain of virulence function (10). 96

Here, we seek to better understand the evolution of NMEC’s virulence plasmids and ascertain their similarity to other ExPEC plasmids. While plasmids from NMEC strains S88 (O45:K1), S286 (O78:K1), RS218 (O18:K1) and CE10 (O7:K1) have been sequenced and are available in the public domain, it is our contention that the full diversity of NMEC plasmid sequences is not well documented. We base this hypothesis on the variability found among NMEC in their possession of plasmid-associated genes

(11). We further contend that this knowledge gap could inhibit future study to identify rational strategies designed to control NMEC-caused disease. We will address some of these shortcomings by sequencing a collection of diverse NMEC plasmids, identify orthologous genes shared between NMEC virulence plasmids in an effort to identify their core and pan plasmid genomes, ascertain the phylogenetic relationships between NMEC plasmids and determine their similarities to plasmids occurring in other ExPEC .

Materials and Methods

Bacterial Strains.

A total of 11 E. coli isolates from cases of human neonatal meningitis were used as a source of plasmids in this study (Table 1). These isolates are part of a larger collection, containing 91 neonatal meningitis E. coli (NMEC) isolated from the

Netherlands and United States. The bacterial strains were serogrouped at the E. coli

Reference Center (Pennsylvania State University), genotyped for chromosomal and plasmid virulence genes and plasmid replicon types, and assigned to phylogenetic groups as previously described (11-13).

Virulence Gene Amplification.

As described in prior work, PCR was performed to determine the presence of

chromosomal and plasmid-derived virulence genes (11, 14). Briefly, all strains were

removed from freezer stock and struck to MacConkey agar plates. Isolates were then

transferred to LB broth and grown at 37°C overnight. Bacterial DNA was harvested

using the boil preparation method previously described (15). For PCR analysis, isolates

were tested for 205 genes associated with virulence and their allelic variants,

antimicrobial resistance, plasmid replicons, and pathogenicity islands. Primers were

obtained from Integrated DNA Technologies (Coralville, Iowa). Replicon typing was

carried out using the methodology described by Johnson (7) using multiplex PCR.

Phylogrouping was carried out using the revised method of Clermont, assigning strains to

8 phylogenetic groups (16).

Selection of strains for sequencing

Strains for sequencing were selected based initially on the presence or absence of nine known plasmid virulence genes ( ompTp, hlyF, cvaC, etsA, iutA, iroN, tsh, iss, and sitC ). To be considered virulence plasmids in this analysis, plasmids must contain at least two known plasmid virulence genes. Candidate plasmid-containing strains were further refined by the number of virulence genes detected by PCR, attempting to select for plasmids containing different combinations of virulence genes, as well as selecting plasmid containing strains originating from different serogroups, phylogenetic groups, and capsule types in order to capture the most possible depth.

Plasmid Isolation.

The selected strains were grown overnight in 5ml of Luria Bertani (LB) broth, the

bacterial cells pelleted by centrifugation, and their plasmid DNA isolated using Qiagen

midi plasmid prep kits according to manufacturer’s specifications (Venlo, Netherlands).

The plasmid DNA pellet was re-suspended in Qiagen buffer EB. Plasmid DNA

concentration was measured using a NanoDrop 1000 (Thermo Scientific, Wilmington,

DE).

Plasmid separation was achieved using pulse field gel electrophoresis (PFGE) as described by Tivendale et al (6). PFGE was carried out using a CHEF Mapper XA system (BioRad Hercules, California). Plasmids were separated in 1.0% w/v Seakem

Gold (Lonza, Bessel, Switzerland) agarose in a 0.5X TBE buffer (Invitrogen Carlsbad,

CA). Size controls used were pAPEC-O1-ColBM, pAPEC-O2-ColV, and pAPEC χ7112.

Electrophoresis was carried out at 6v/cm with an angle of 120° for 20h with an initial

switch time of 1s and a final switch time of 20s (17). Following electrophoresis, the gels

were stained in distilled water containing 0.5mg/ml of ethidium bromide and de-stained

in water as necessary. All plasmids were visualized using UV transillumination.

Individual plasmid bands were identified and excised using a clean straight razor and

placed in 1.5 ml tubes. The plasmids were dissolved in Qiagen buffer QG in an

incubator-shaker at 37°C for 30 minutes to remove them from the agarose. The

dissolved gel fragments were subjected to sonication to fragment DNA below the

maximum threshold for Qiagen column purification using a Diagenode Bioruptor

Standard (Diagenode Seraing, Belgium) at high power using 30 second on/off cycles for

5 minutes. The samples were kept on ice before, during, and post sonication. Post 99 sonication, DNA samples were purified as per the Qiagen protocol for PCR purification using manufacturer’s specifications and eluted from purification columns using 11ul of

Qiagen buffer EB. After purification, plasmids were quantified using the Q-bit 2.0

Fluorometer using the q-bit HS assay kit (Life Technologies Carlsbad, CA) according to manufacturer’s specifications.

Plasmid Sequencing.

Paired-end plasmid DNA samples were prepared for sequencing by the Dana

Farber/Harvard Cancer Center DNA Resource Core using the Nextera DNA sample preparation kit (Illumina San Diego, CA) according to manufacturer’s specifications for indexed multiplexing using 50ng of input DNA. Input DNA was fragmented and tagged with the transposon, adding adapter sequences and allowing for amplification.

Tagmented DNA was cleaned to remove any leftover transposon fragments. Purified

DNA was then PCR amplified for 8 cycles and purified of enzymes and DNTPs. Library adapter ligation and amplification were tested by qPCR, and libraries were pooled.

Library fragments in the size range of 500 to 600bp were selected using a Pippin Prep from Sage Science (Beverly, MA) according to manufacturer’s specifications.

Sequencing of the tagged plasmids was carried out using an Illumina Mi-Seq machine

(Illumina San Diego, CA) using a single lane. Output files ranged in size from 1 to 5 GB and were quality trimmed based on the FASTq PHRED quality scores, cutting off at Q25 and imported into the assembly software. Velvet Optimizer 2.1.4 was used to determine optimal k-mer length and coverage cutoff. Plasmid assembly was achieved using Velvet short read assembler (version 1.2.03; European Bioinformatics Institute) for final assembly (18). In general, most plasmids assembled into one to three large contigs with 100 few small contigs or contaminating sequences. Plasmid sequences requiring closure were closed using the previously sequenced pS88 as a backbone against which to assemble small contigs remaining from the Velvet assembly.

Plasmid annotation was carried out for NMEC strains sequenced in this study and elsewhere using the Prokka automated annotation analysis pipeline in order to ensure consistency in our data. The plasmids were annotated using the “-usegenus and –strain” options with a custom database generated from all APEC and NMEC plasmids in the

NCBI database.

Plasmid Core and Accessory Genome Analysis.

The plasmid genomes of the fully sequenced NMEC plasmids from this study and those available online (11) were entered into an NCBI database, where an all-versus-all blast was performed to ascertain orthologous gene clusters (COGs). Genes showing

>90% sequence identity, e-values of lower than 1e-10, and overlap percentages of >90% were determined to be orthologous. Orthologous genes were then sorted into strongly connected components using the algorithm of Tarjan in Perl (19). Strongly connected components where an orthologous gene was present in each plasmid were grouped as members of a core gene set, while genes present in at least one genome were said to be members of the accessory genome.

Plasmid Phylogenetic Analysis.

Phylogenetic analysis was performed for the core genome of the NMEC and two

APEC plasmids in order to compare different plasmids from different ExPEC sub- 101 pathotypes. Core genome nucleotide cDNA sequences were extracted from files, and concatenated to form a mega alignment using custom Perl scripts. ClustalOmega (20) was used to align the nucleotide sequences and using default nucleotide settings and interleaved PHYLIP output. The PHYLIP alignment was then used in seqboot from the

PHYLIP software package to create 100 bootstrapped replacements in the tree, using the

–m weights option. This set of 100 weights was then used in the dnaml program from the

PHYLIP software package to create a bootstrapped maximum likelihood tree using the parameters T=2.5, S, m,1. The bootstrapped trees were then condensed using the consense program from the PHYLIP software package to give a consensus tree with bootstrap values using default parameters. This tree was then reanalyzed in dnaml using the options –u, –t 2.5, to redraw branch lengths assuming the phylogeny was represented

by the bootstrap inferred tree.

Plasmid sequencing data accession numbers. Plasmid sequences were

deposited in GenBank under numbers SAMN03580091 (pNMEC-14-ColV),

SAMN03580092 (pNMEC-15-ColV), SAMN03580093 (pNMEC-16-ColV),

SAMN03580094 (pNMEC-19-ColV), SAMN03580095 (pNMEC-26-ColV),

SAMN03580096 (pNMEC 36-ColV), SAMN03580097 (pNMEC 38-ColV),

SAMN03580098 (pNMEC-49), and SAMN03580099 (pNMEC-58-ColV).

Results

Strain Selection, Purification and Sequence Assembly.

A total of 11 plasmids was sequenced from nine NMEC strains, each isolated

from different patients. Ten of eleven plasmid-containing strains were of the phylogroup 102

B2, and the dominant serogroup was O18:K1 (Table 1). The source strains of these plasmids were previously assigned to clusters as described elsewhere (11) and were chosen for this study based on the diversity of the host strain serogroup, phylogroup, different virulence gene combinations, and cluster analysis.

Multiple plasmids were recovered from some of the NMEC strains: NMEC 15 and NMEC 36 both carried smaller cryptic plasmids. NMEC 15 contained a 114.75kb cryptic plasmid containing hypothetical and phage genes, while NMEC 36 carried a smaller 41.394 kb plasmid.

Table 1: Serogroups, phylogroups and genome statistics of selected sequenced NMEC strains used in the study.

Plasmid Phylogroup O:K Cluster [1] Size (bp) GC% ORFs Source pNM14 C 7:80 2 126,885 49.4 136 This Study pNM15 B2 18:1 8 148,173 51.3 157 This Study pNM16 B2 83:1 8 130,692 49.3 136 This Study pNM19 B2 -:1 8 133,585 48.8 142 This Study pNM26 B2 21:- 6 152,078 49.4 168 This Study pNM36 B2 14:1 7 129,262 49.2 131 This Study pNM38 B2 18:1 8 128,645 49.7 136 This Study pNM49 B2 auto:1 8 121,081 49.6 119 This Study pNM58 B2 18ac:1 8 157,260 49.7 157 This Study pS88 B2 45:1 6 133,853 49.3 157 NC_011747.1 pS286 C 78:- - 97,818 48.6 104 HF922624.1 pAPEC-O1-ColBM B2 1:1 - 174,241 49.6 199 DQ381420.1 pAPEC-O2-ColV B2 2:1 - 184,501 49.2 203 AY545598.5 pAPEC-O103-ColBM B1 103:- - 124,705 50.8 126 NC_011964 p1ColV5155 B2 2:1 - 194,170 49.4 199 CP005930 pVM01 - -:28 - 151,002 49.5 150 NC_010409 pAPEC-1 B1 78:- - 103,275 45.1 164 CP000836 pAPEC-O78-ColV - 78:- - 144859 50.4 158 NZ_CP010316

These plasmids contained a large number of hypothetical genes, as well as phage-related genes. Since we wished to focus our study on the plasmids in NMEC that serve a role in virulence and that are considered to be one of the defining traits of the NMEC sub- 103 pathotype, all plasmids to be sequenced had to contain at least two plasmid-borne virulence genes from a list of nine commonly associated with large ExPEC virulence plasmdis ( ompTp , hlyF , cvaC , etsA, iutA , iroN , tsh , iss, sitC ). Imposition of this selection criterion ensured that only plasmids containing some virulence associated genes would be included, and that cryptic or other plasmids were excluded.

Nine of the 11 plasmids sequenced in this study passed quality control and selection criteria following annotation. Two plasmids from strains NMEC 49 and NMEC

84 had a lower depth of coverage, and contained segments of contaminating DNA, resulting in poor assembly and their subsequent exclusion from analysis beyond assembly

(Table 1). The plasmids pNM15c and pNM36c both contained fewer than three of the virulence-associated genes. Further inspection of these plasmids showed that the majority of the plasmid genome consisted of hypothetical genes, phage genes, and genes of unknown function, and included no known virulence genes, and thus were also excluded from the analysis [Data not shown]. The resulting nine sequenced plasmids were combined with publically available NMEC plasmid sequences meeting the defined criteria described above, pS88 and pS286.

NMEC Virulence Plasmid Accessory Genome.

The virulence plasmid “accessory genome” of these 11 NMEC plasmids is defined as any COGs or single genes not found in the core genome, but present in at least one plasmid. COGs from the accessory genome identified 123 putative genes present in at least two genomes, and 197 putative genes unique to only one plasmid. Each gene’s description was derived from the consensus of the genes’ annotations. The accessory genome consisted of 142 genes appearing in at least two plasmids (Figure 1) and 92 non- 104 orthologous genes appearing in only one plasmid. A heat map of the 142 genes present in two or more genomes is found in Figure S1.

The plasmid accessory genome contained a large number of hypothetical genes, genes of unknown function, phage genes, and insertion sequences. Other notable genes in the accessory genome included tsh the temperature sensitive hemagglutinin, and a

homologue of the outer membrane protease ompT , though all plasmids carried another

distinct copy of the ompT protease.

Figure 1. Genes of the NMEC plasmid accessory genome present in two or more plasmids organized into 8 functional categories based on protein prediction.

105

NMEC Plasmid Core Genome.

To ascertain the range of extant genes in the plasmids of NMEC strains, all sequenced and annotated NMEC virulence plasmids passing selection criteria and two previously sequenced plasmids pS88 (21) and pS286 (22) were concatenated into a cDNA BLAST (22) database. An “all versus all” blast analysis was performed to assign genes to clusters of orthologous groups (COGs), or groups of genes derived from a common ancestor. COGs in which an ortholog from each plasmid was found were considered to be “core plasmid” genes and were further subjected to protein function analysis using the CombFunc pipeline (16). Genes in the core genome were then categorized into eight functional categories based on known function or results from the combFunc pipeline (Table 2). A core genome of 46 genes was predicted (see Figure 2).

The core genome genes can be divided into five categories by putative functions: virulence associated; metabolic and signaling; unknown; plasmid stability and transfer; and mobile elements (Table 2).

The virulence-associated genes of the NMEC plasmid core included all genes of the aerobactin ( iutA/iucABCD ), sit iron/manganese transporter (sitABCD ), and salmochelin ( iroBCDEN) operons. All three of these operons encode high-affinity iron- transport systems that are used by bacteria to obtain iron in low iron conditions such as those they encounter in host fluids and tissues. These operons have previously been reported in virulence plasmids of uropathogenic Escherichia coli (UPEC), APEC, and

NMEC with high frequency and have been associated with ExPEC virulence. Another gene found in the core genome of NMEC large virulence plasmids is iss . This gene encodes a protein linked with increased serum survival in human E. coli isolates. 106

Numerous studies have documented its strong alignment with virulent but not avirulent

E. coli strains (23). In addition, the genes ompT

Figure 2. Visualization of NMEC plasmid core genome sites. Genes within the plasmid core genome are listed in the orange core genome section, while linear representations of the chromosomes of the NMEC plasmids are arranged in the circle. Lines are drawn to the position of the core plasmid genes in their location within the plasmid to show core gene clustering.

107

Table 2: Core plasmid genome of sequenced NMEC plasmids and their functions.

Gene Name Description Function hlyF Hemolysin ompT Outer membrane protease Iss Increased serum survival protein iroB putative glucosyltransferase iroC ATP binding cassette ABC transport homolog iroD putative ferric enterochelin esterase iroE putative hydrolase iroN outer membrane receptor fepA iucA aerobactin siderophore biosynthesis protein Virulence iucB N(6)-hydroxylysine acetylase iucC aerobactin siderophore biosynthesis protein iucD L-lysine 6 monooxigenase iutA ferric aerobactin receptor precursor sitA iron/manganese transport, periplasmic-binding protein sitB iron/manganese transport protein ATP-binding component sitC Iron/manganese transport inner membrane component iron/manganese transport protein, inner membrane sitD component putative cobalamin synthase Putative Cobalamin synthase Metabolic ssbL DHAP synthase for shikimate pathway and truncated enolase putative enolase fragment Signaling crcB camphor resistance protein repA RepFIB replication protein repA1 Plasmid replication protein repA2 Plasmid replication Protein Plasmid Stability traA conjugal transfer pilin subunit and traE conjugal transfer pilus assembly protein Transfer traL conjugal transfer pilus assembly protein yacB putative plasmid stabalization system protein finO conjugal transfer fertility inhibition protein hypothetical mobile element putative mobile element protein/integrase hypothetical phage protein hypothetical phage protein hypothetical transposase hypothetical transposase orfA transposase from plasmid origin Mobile hypothetical protein DNA Binding Function Elements hypothetical protein DNA polymerase hypothetical protein Helicase activity Int Hypothetical integrase protein etsA putative type I secretion membrane-fusion protein etsB putative type I secretion ATP binding protein etsC putative type I secretion outer membrane protein shiF putative MFS family membrane transport protein hypothetical protein Unknown protein Unknown hypothetical protein Unknown protein yubQ putative transglycolylase hypothetical siderophore Putative Siderophore mig-14 putative Mig-14 protein

108 and hlyF are also found in the core genome of APEC’s large virulence plasmids. ompT is predicted to encode a 42-kDa pro-protein, which is processed in the membrane to a 40k-

Da mature form. Mature Iss functions as a narrowly specific outer membrane endoprotease (22) that 1) cleaves paired basic residues and is involved in membrane protein turnover, 2) can degrade interferon-gamma in-vitro (24), and 3) cleaves the human defensin LL-37 (25). The hlyF gene is predicted to encode a putative hemolysin gene; however, the exact function of this gene is unknown.

Metabolic genes of the NMEC plasmid core genome consists of three genes, ssbL , eno and a cobalamin synthesis gene. ssbL is a truncated enolase gene and a putative cobalamin (coenzyme B12) synthase gene. ssbL encodes a phospho-2-dehydro-3-

deoxyheptonate aldolase or DHAP synthase. It is the first enzyme in a series of

metabolic reactions known as the shikimate pathway, leading to the production of

chorismate, which is responsible for the biosynthesis of the amino acids phenylalanine,

tyrosine, and tryptophan and catecholate/phenolate siderophores. This gene co-locates

with the iro operon in ExPEC plasmids (26). The putative enolase gene found in the

plasmid core genome is very small, comprising 33% of the eno gene from S88 at the C-

terminus at 51% identity. The eno gene catalyzes conversion of 2-phosphoglycerate to

phosphoenolpyruvate (PEP) (27). PEP is another reactant in the shikimate pathway for

aromatic amino acid synthesis (28). Finally, a putative cobalamin synthesis gene was

also found. This putative protein contains two domains homologous to cobalamin

synthesis protein p47 metal binding motifs and may act as a chaperone in metal binding.

Previous studies of APEC virulence have shown several vitamin synthesis pathways are

crucial for APEC’s in vivo survival (29). 109

Nine genes comprise the third major gene group of the NMEC plasmid core genome. These genes are responsible for plasmid transfer and stability and include replication genes ( repA , repA1 and repA2 ), as well as finO , encoding an anti-sense RNA molecule inhibiting traJ (30) and the supercoiling and camphor-resistance gene crcB .

The replication genes are responsible for control of plasmid copy number in cells and

prevent replication by binding to plasmid sites, thereby inhibiting DNA replication. This

inhibition can be reversed by high dnaG expression (31-33). Notably absent from this

list are many of the tra genes from the type IV secretion system that responsible for

plasmid conjugal transfer. They do not show up in the core because they are not present

in the plasmid pS286. However, a case can be made for their inclusion since they occur

in all others suggesting that pS286 is atypical, incomplete or aberrant. Blast sequence

comparison between crcB found in this study and all other E. coli found that the crcB

genes of NMEC plasmids and UPEC strains UCI57, UCI58, KTE76, KTE102, KTE240,

and E. coli 908585 are distinct from the crcB gene found in other E. coli , showing only

53% amino acid identity. Such differences suggest the possibilities of different binding

ligands or function. Upon initial discovery, CrcB was indicated in plasmid supercoiling

and resistance to camphor when overexpressed (34). More recent studies show CrcB to

be a putative membrane protein containing a metabolite-binding RNA structure, indicated

to be fluoride in these studies, which can alter downstream gene expression (35).

The NMEC plasmid core also includes mobile genetic elements and phage

insertions. Mobile element genes consists of phage genes, as well as transposable

elements and integration proteins. These elements recognize specific DNA sequences in 110 the plasmid, and can cause DNA transposition for incorporating virulence genes into pathogenicity islands.

The NMEC plasmid core also includes genes of unknown function. The etsABC

operon encodes a putative ABC transport system. It is predicted that etsA encodes a putative membrane fusion protein, etsB encodes ATP-binding/permease protein, and etsC

encodes an efflux protein. These genes are strongly associated with NMEC pathogenicity

islands (11). The shiF gene is a putative member of the COG0477 permease family. The

shiF gene homolog lies upstream of aerobactin synthesis genes; no evidence that its

function is related to this aerobactin system has been found in E. coli. The gene yubQ

encodes an X-polypeptide. It is a protein homologous to lytic transglycosylases (LT) of

family four, thought to originate from phage (36). This class of proteins in general is

thought to act on the cell wall, degrading peptidoglycan into metabolites which can be

recycled in the cytoplasm. Most E. coli contain approximately seven distinct LT proteins

with unknown functional differences. LT proteins are essential genes in E. coli , deletion of all LT genes results in a lethal phenotype. There are two hypothetical genes of unknown function that encode no known protein domains and the combFunc pipeline was unable to predict any functions of these proteins.

Phylogenetic Analysis.

The NMEC core plasmid genome was used to develop an ExPEC plasmid

phylogeny. To relate the evolution of these plasmids to other E. coli plasmids, three

APEC plasmids, pAPEC-O1-ColBM, pAPEC-O2-ColV and pAPEC-O103 were included

in the analysis (Figure 3). These plasmids meet the same three criteria for inclusion as 111

Figure 3. Phylogeny of core NMEC plasmid genes and APEC plasmid genes. Alignment was based on concatenation of core genes shared between APEC and NMEC plasmids. Genes were identified using an all-versus-all nucleotide blast using the blastn algorithm using default parameters. Blast matches sorted into strongly connected components using Tarjan’s algorithm. Nucleotide sequences from strongly connected groups were extracted in a randomized order and concatenated. Bootstrap analysis was performed using Phylip seqboot algorithm creating 10 sets of 10 weights using default parameters, which were then used in 10 separate runs of dnaml using the parameters T 2.5, S, M, 1. Phylip consense was used to condense all output trees into a consensus bootstrap analysis using default parameters described above and were included in the analysis to better understand differences between plasmids from different ExPEC sub-pathotypes. However, UPEC plasmids were not included in this analysis as no publically available sequenced UPEC plasmids met inclusion criteria. The APEC/NMEC core plasmid genome consisted of 15 genes

(21,496bp), containing the iro and iut operons as well as replication proteins repA and repB , which was used to generate a plasmid phylogeny. Additionally, one outlier was observed in this analysis: pAPEC-O103, which is a hybrid plasmid containing a pathogenicity island and multi-drug resistance island and lacking the complete tra operon. As a result of this analysis, it appears that there are at least two lineages of 112

NMEC virulence plasmids. In addition, as a result of the comparative analysis, it appears that APEC and NMEC plasmids cannot be differentiated solely based on the presence or absence of genes in a core genome of plasmid.

Discussion

The contributions made to ExPEC pathogenesis by large virulence plasmids has

received increased scrutiny in recent years. In part, this scrutiny is due to creation of the

reagents needed for their study, such as plasmid genomic sequences (6, 9, 21, 26), and

dissemination of new plasmid isolation protocols that enabled harvesting of large,

naturally occurring plasmids from wild-type bacterial strains (37). Previous to that the

presence of large ExPEC plasmids was likely overlooked (14). Here, in an effort to

define the core and accessory genome of NMEC plasmids, nine new plasmids isolated

from NMEC were sequenced. Plasmids were selected so as to enrich the diversity of

NMEC plasmid sequences available in the public domain and in order to describe the

broadest core and accessory genome. Despite this emphasis on diversity, clusters 5 and 9

contained only a single isolate, while clusters 2 and 3 primarily consisted of human fecal

E. coli biasing the strain selection towards groups 6-8, which were determined to be the

most pathogenic and most common clusters for NMEC in the analysis (11). Efforts were

made to sequence strains from different serogroups where multiple options existed,

attempting to capture the dominant O-types of O18, O83, and O7, while still sequencing

representatives from less common serogroups, such as O14, O21, as well as untypeable

NMEC. Selecting for strains outside of the predominant B2 phylogroup is difficult, as

our collection of 94 NMEC isolates contains predominantly B2 strains (78%), with the

next most common phylogenetic group being F, representing 6.8% of the isolates in the 113 full 91 sample collection. Finally, K type was used if all other parameters from the analysis were the same, which included one strain of the K80 type in the analysis, as well as multiple untypeable capsules. The previously published NMEC plasmids pRS218 and pCE10 were excluded from the analysis due to the absence of all virulence genes used to define a virulence plasmid in this study. Further analysis of strain CE10 showed the iutABCD and sitABCDC genes are present in the chromosome, while RS218 did not possess any of these virulence genes on the plasmid or the chromosome.

Assembled plasmids varied largely in terms of size, but surprisingly had similar

GC content and numbers of open reading frames, indicating that a large proportion of these plasmids may not encode functional proteins. This seems to be borne out by the significant number of hypothetical genes in the accessory genome of the NMEC plasmids. It is unclear whether this result suggests that some plasmids are accumulating genetic information that is not used, or whether they have not yet shed excess genetic information that may not be functional or whether these hypothetical or pseudo genes have an unknown function. Classical genes related to antibiotic resistance were not found in any of these plasmids, with only one plasmid harboring a single putative antibiotic resistance gene. The lack of resistance genes is corroborated by the lack of multiple drug resistant strains in our NMEC collection (38), as well as those available publically for other NMEC strains. Furthermore a complete and intact transfer region was not found on all of the NMEC plasmids, suggesting these plasmids lost the transmissible capability during the evolution course.

The core genome of the NMEC plasmids contains 46 genes, most of which encode components of iron uptake systems. Iron uptake systems are necessary for the 114 full virulence of septicemia-associated E. coli (6, 11, 21), uropathogenic E. coli (39) , and avian pathogenic E. coli (12, 14, 40, 41). Although it has not yet been reported, this study suggests that iron acquisition is important for NMEC virulence since human serum and cerebrospinal fluid (CSF) are also iron-deficient environments. Indeed, the presence of iron acquisition genes in NMEC isolates has been well established (6, 11) and NMEC,

UPEC, and APEC share a similar content of iron uptake systems that are not found in non-virulent fecal E. coli strains from both human and animal sources (11). Moreover, unpublished data from our group has found that the expression of several iron uptake systems was significantly upregulated when NMEC were cultured in human serum and

CSF compared to strains cultured in LB (Unpublished data). The data presented here indicate that three iron uptake systems, the ferric aerobactin system ( iutA/iucABCD ), the salmochelin siderophore system ( iroBCDEN) , and SitABCD system, have been

associated with ExPEC virulence, and are encoded by core the genome of NMEC large

virulence plasmids. Interestingly, the metabolism gene ( ssbL ) found in the core genome of NMEC virulence plasmids appears to be co-located with these iron genes. This ssbL

gene encodes a putative DHAP synthase which may boost production of aromatic amino

acids via the shikimate pathway by increasing concentrations of available precursor

molecules. Aromatic amino acids are necessary for large amount production of

enterobactin, salmochelin SX, and yersiniabactin (26). The finding of ssbL gene in the

core genome of the NMEC virulence plasmid appears to reinforce the importance of the

iron uptake system for NMEC virulence.

Based on the findings published thus far, NMEC’s ability to cause meningitis

requires at least two major steps and one of them is intravascular multiplication leading to 115 high-level bacteremia. Thus, NMEC must be able to resist or avoid the detrimental impact of the host complement, antimicrobial peptides, polymorphonuclear leukocytes, and other specific and innate immunity factors. In the core genome of NMEC virulence plasmids, the iss gene was found, which is homologous to the bor gene of bacteriophage

λ. The Iss protein increases E. coli ’s serum survival likely by restricting C3 deposition on the bacterial surface (23) and blockage of the membrane attack complex (42). Another core genome gene of the NMEC virulence plasmid involved in resistance to innate immunity is ompT . The ompT gene encodes an outer membrane protease (22) that can degrade interferon-gamma. More recent studies on ompT in enteropathogenic E. coli

(EPEC) and enterohaemorrhagic E. coli (EHEC) showed ompT cleaves the human defensin LL-37 (25), a pore forming antimicrobial peptide expressed by neutrophils, epithelial cells, and bone marrow that serves both antimicrobial and immunomodulatory functions. The ompT gene in the core genome of NMEC virulence plasmids shares approximately 74% of amino acid identity to those encoded by EPEC and EHEC. Thus, it could be inferred that OmpT can degrade interferon and human antimicrobial peptide increasing NMEC’s resistance to human innate immunity, thus contributing to NMEC’s pathogenesis. In addition, several other accessory genome genes of NMEC virulence plasmids were found to contribute to NMEC’s resistance to host immunity. The gene tsh , which has been previously reported to be an important virulence factor for closely related

APEC strains and is present in 11 to 50 percent of NMEC samples, encodes a protein able to cleave leukocyte glycoproteins, impair chemotaxis and transmigration, and activate leukocytes’ programmed cell death (43). 116

The homolog of CrcB was shown to be involved in the chromosome condensation

and plasmid supercoiling, causing a secondary effect of resistance to camphor, which

decreases chromosome condensation (34). Overexpression of the gene increases

chromosomal condensation and can correct nucleoid morphology defects in some

mutants, and may help these large plasmids partition into daughter cells. As we know the

E. coli chromosome, like its eukaryotic counterparts, must be condensed approximately

2000-fold to fit inside an E. coli cell. Given the large size of the virulence plasmids for

NMEC, it is likely that the CrcB gene carried on the plasmid is necessary to fit the plasmid into the cells, and may increase the likelihood of the plasmid successfully passing to daughter cells post replication.

In conclusion, this is the first study to define the core and pan NMEC plasmid genome, and perform a phylogenetic analysis of NMEC large virulence plasmids. The role of the core genome of NMEC plasmids is likely to increase the bacteria’s in vivo

fitness by enhanced uptake of iron in a deficient environment, increase resistance to host

innate immunity, as well as maintain the plasmid in the bacterial population with systems

for conjugation and plasmid stability. Phylogenetic analysis of the NMEC plasmids

shows that at least two lineages can be discerned and they are nearly indistinguishable

from those of the APEC plasmid. We hope this and future studies lead to a better

understanding of the molecular mechanisms by which NMEC large plasmids contribute

to virulence and their evolution.

117

Acknowledgements

We thankfully acknowledge Kwang S. Kim (Johns Hopkins University), Chitrita

DebRoy ( E. coli reference center Pennsylvania State University) and Lodewijk Spanjaard

(Netherlands Reference Laboratory for Bacterial Meningitis) for their gift of NMEC strains. Additionally we would like to thank Brett J. Kail his assistance with Perl programming.

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121

CHAPTER 4. COMPLETE GENOME SEQUENCE OF THE NEONATAL

MENINGITIS-CAUSING ESCHERICHIA COLI STRAIN NMEC O18

An article for submission to the journal Genome Announcement

Bryon A. Nicholson a, Yvonne M. Wannemuehler a, Catherine M. Logue a, Ganwu Li b, and

Lisa K. Nolan a

aDepartment of Veterinary Microbiology and Preventive Medicine, College of Veterinary

Medicine, VMRI 2, Iowa State University, Ames, Iowa, USA

bDepartment of Veterinary Diagnostic and Production Animal Medicine, College of

Veterinary Medicine, 1802 University Blvd., Iowa State University, Ames, Iowa, USA; 122

Abstract

Neonatal meningitis E. coli (NMEC) is a common agent of neonatal bacterial meningitis, causing high neonatal mortality and neurologic sequelae. Here, we present the complete genome sequence of NMEC O18 (also known as NMEC 58), a highly virulent O18ac:K1 serotype and B2 phylogenetic group strain of NMEC from the ST416 sequence type. NMEC O18 has been shown to cause 91% mortality in the rat model of human neonatal meningitis.

Genome Announcement

Neonatal bacterial meningitis (NBM) is a devastating disease of newborns. NBM occurs during the first four weeks of life (1, 2). NMEC are the second leading cause of

NBM (1), and predominantly infect newborns after four days of life (2), but can cause disease in newborns less than one day of age (2). NBM rapidly progresses in the infant with empiric treatment often performed before a definitive diagnosis of meningitis is reached (3). Reported mortality rates vary from 10% to 50%, based on study location, gestational age, and weight of the neonate (2, 4). Survivors of NBM often suffer severe neurological sequelae that may include seizures, hearing loss, cerebral palsy, and delayed development, occurring in 12% to 44% of survivors (5).

NMEC O18 has been characterized in the rat model of meningitis. In neonatal rats, it caused 91% mortality at 24 hours post infection, with mortality observed as early as 14 hours (6). NMEC O18 shares the same serogroup and phylogenetic group as the prototypic NMEC strain, NMEC RS218. Unlike NMEC RS218, NMEC O18 contains additional genes encoding aerobactin synthesis as well as the outer membrane proteins 123

OmpTp, HlyF, and Iss but lacks the gene encoding cytotoxic necrotizing factor 1 found in

NMEC RS218 (7, 8). Similar to RS218, NMEC O18 contains a large virulence plasmid carrying genes encoding multiple siderophores and other virulence factors. NMEC O18’s high degree of virulence, despite deviating from the well-described pattern of virulence factors found in NMEC RS218, makes NMEC O18 an interesting candidate for future studies into NMEC pathogenesis.

NMEC O18 is a member of the O18:K1 serotype, the B2 phylogenetic group and the ST 416 sequence type (6). NMEC O18 was isolated from the cerebrospinal fluid of an infant with neonatal bacterial meningitis in the Netherlands (6). NMEC O18 genomic

DNA was subjected to genomic sequencing using Roche/454 FLX genome and Illumina

Hi-Seq 2000 sequencers, followed by hybrid assembly. The following datasets were used in the final assembly: (i) GS-FLX, with 456,508 paired reads and 213,580 shotgun reads totaling 128 Mb (~26-fold coverage) and (ii) Illumina 100 bp paired-end library with

15,458,728 reads totaling 1,236 Mb (~247-fold coverage). Both 454-read sets were assembled de novo using Newbler 2.6 (Roche), and Illumina reads were assembled separately with Velvet 1.1 (8). The genome was closed using 454 scaffolds against

NMEC strain S88 sequence for velvet assembly. Whole-genome optical mapping

(OpGen, Gaithersburg, MD) was used to validate scaffolds and contig order. The assembly was confirmed using PCR and Sanger sequencing and validated by consistency of paired-end evidence from 454 and Illumina reads.

The assembled genome consists of a single chromosome (5,002,781bp; 50.75%

GC content) and a single plasmid (153,378bp; 48.49% GC content). The chromosome contains 4,659 protein-encoding genes and 90 tRNA-genes. 124

Acknowledgement

This work was supported by grant USDA-NIFA award 0826675 and grant NIH award

007134-00001.

References

1. Heath PT, Nik Yusoff NK, Baker CJ. 2003. Neonatal meningitis. Arch Dis Child Fetal Neonatal Ed 88: F173-178. 2. Gaschignard J, Levy C, Romain O, Cohen R, Bingen E, Aujard Y, Boileau P. 2011. Neonatal Bacterial Meningitis: 444 Cases in 7 Years. Pediatr Infect Dis J 30: 212-217. 3. Sivanandan S, Soraisham AS, Swarnam K. 2011. Choice and duration of antimicrobial therapy for neonatal sepsis and meningitis. Int J Pediatr 2011: 712150. 4. Weston EJ, Pondo T, Lewis MM, Martell-Cleary P, Morin C, Jewell B, Daily P, Apostol M, Petit S, Farley M, Lynfield R, Reingold A, Hansen NI, Stoll BJ, Shane AL, Zell E, Schrag SJ. 2011. The burden of invasive early-onset neonatal sepsis in the United States, 2005-2008. Pediatr Infect Dis J 30: 937-941. 5. Stevens JP, Eames M, Kent A, Halket S, Holt D, Harvey D. 2003. Long term outcome of neonatal meningitis. Arch Dis Child Fetal Neonatal Ed 88: F179-184. 6. Tivendale KA, Logue CM, Kariyawasam S, Jordan D, Hussein A, Li G, Wannemuehler Y, Nolan LK. 2010. Avian-pathogenic Escherichia coli strains are similar to neonatal meningitis E. coli strains and are able to cause meningitis in the rat model of human disease. Infect Immun 78: 3412-3419. 7. Khan NA, Wang Y, Kim KJ, Chung JW, Wass CA, Kim KS. 2002. Cytotoxic necrotizing factor-1 contributes to Escherichia coli K1 invasion of the central nervous system. J Biol Chem 277: 15607-15612. 8. Wang MH, Kim KS. 2013. Cytotoxic necrotizing factor 1 contributes to Escherichia coli meningitis. Toxins (Basel) 5: 2270-2280.

125

CHAPTER 5. COMPLETE GENOME SEQUENCE OF THE AVIAN

PATHOGENIC ESCHERICHIA COLI STRAIN APEC O18

An article for submission to the journal Genome Announcements

Bryon A. Nicholson a, Yvonne M. Wannemuehler a, Catherine M. Logue a, Ganwu Li b, and

Lisa K. Nolan a

aDepartment of Veterinary Microbiology and Preventive Medicine, College of Veterinary

Medicine, VMRI 2, Iowa State University, Ames, Iowa, USA

bDepartment of Veterinary Diagnostic and Production Animal Medicine, College of

Veterinary Medicine, 1802 University Blvd., Iowa State University, Ames, Iowa, USA; 126

Abstract

Extra intestinal pathogenic Escherichia coli (ExPEC) are responsible for some of the most widespread and consequential diseases of human and animal hosts. Genetic similarities between human and avian ExPEC strains plus similarities in the mechanisms by which they cause disease have led to suspicion that some ExPEC are zoonotic pathogens. Especially notable in this regard are Neonatal Meningitis E. coli (NMEC) and

Avian Pathogenic E. coli (APEC). Here, we document the first fully closed genomic sequence of an Avian Pathogenic E. coli strain of serogroup O18. APEC O18 shows potential as a zoonotic pathogenic capable of causing neonatal meningitis in human newborns.

Genome Announcement

APEC O18 is a serotype O18 sequence type ST95 strain isolated from the pericardial and lung tissue of a case of colibacillosis in Nebraska. APEC O18, previously labeled APEC 380, has been characterized in the chicken model of colibacillosis, where it caused 80% mortality at 24 hours and in the rat model of neonatal meningitis, where it causes 75% mortality at 24 hours (1).

Genomic sequencing was performed using complementary sequencing technologies, combining results obtained from a Roche/454 FLX genome sequencer (GS) instrument and an Illumina Hi-Seq 2000. The following datasets were used in the final assembly: (i) GS-FLX, with 152,602 paired reads and 423,865 shotgun reads totaling 155

Mb (~31-fold coverage) (ii) Illumina 100 bp paired-end library with 11,193,121 reads totaling 839Mb (~168-fold coverage). Both 454-read sets were assembled de novo using 127

Newbler 2.6 (Roche), and Illumina reads were assembled separately with Velvet 1.1 (2).

The genome was closed using 454 assemblies as a “reference” sequence with the

Illumina data used to add depth, correct errors, and close gaps. Whole-genome optical mapping (OpGen, Gaithersburg, MD) was used to validate scaffolds and contig order.

The assembly was confirmed using PCR and Sanger sequencing and validated by consistency of paired-end evidence from 454 and Illumina reads.

The assembled genome consists of a single chromosome (5,006,568 bp; 50.70%

GC content) containing 4,674 protein-encoding genes and 85 tRNA-carrying genes. The

APEC O18 is larger than previously sequenced strains APEC O78 and APEC χ7122 and shares similarities to strain APEC O1, a strain which does not cause disease in the meningitis model. Genomic comparisons of this isolate with other sequenced ExPEC genomes is ongoing.

Acknowledgement

This work was supported by grant USDA-NIFA award 0826675

References

1. Tivendale KA, Logue CM, Kariyawasam S, Jordan D, Hussein A, Li G, Wannemuehler Y, Nolan LK. 2010. Avian-pathogenic Escherichia coli strains are similar to neonatal meningitis E. coli strains and are able to cause meningitis in the rat model of human disease. Infect Immun 78: 3412-3419. 2. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18: 821-829.

128

CHAPTER 6. CONCLUSION AND DISCUSSION

Pathogenic E. coli are an exceptionally diverse group of organisms highly adaptable to a wide range of environments, hosts, and host sites. Pathogenic E. coli thrive in these environments due to their large and variable genetic content, encoding adhesins, invasion factors, toxins, nutrient acquisition, and protection systems. Often these pathogenic systems are redundantly encoded, such as in the case of iron acquisition, possibly to enhance fitness in differing environments, or for reasons currently unknown.

This diversity and functional redundancy is especially prevalent in extra intestinal strains, leading scientists to suggest ExPEC strains cannot be characterized by single representative isolates as this cannot encompass their breadth of diversity. Nearly all studies into the functionality of NMEC are based on strain RS218 or mutants thereof.

While NMEC typically possess a serotype and capsule similar to RS218, these traits are by no means universal. Similarly virulence gene content varies widely between strains of

NMEC. This focus on RS218 has created a narrow view of NMEC pathogens. When this narrow view is examined with genomic epidemiology of a larger set of NMEC strains, serious gaps in knowledge of pathogenesis become apparent. While all the research of strain RS218 has given insight into the virulence mechanisms of NMEC, factors such as K1 capsule, S fimbriae, Cnf1, and IbeA are not universally present in

NMEC. Perhaps the most suggestive data supporting NMEC diversity is NMEC strain

NMEC O18, a highly pathogenic strain of NMEC that does not invade macrophages as other reported strains of NMEC, but is still highly pathogenic. The differences in genotypes and phenotypes between NMEC strains are highly suggestive that more study 129 is needed regarding the current model of NMEC pathogenesis and that multiple routes of pathogenesis exist in NMEC. Regardless, analysis and characterization of differing

NMEC strains should be a high priority.

Further care must be taken when examining virulence factors such as fimH and ompA , genes common to nearly all E. coli and different E. coli pathotypes where polymorphic sites are known to greatly influence phenotypes. This research has shown that in NMEC, multiple and very distinct alleles of both FimH and OmpA exist in

NMEC. While these alleles have not been tested in NMEC, their study in adherent- invasive E. coli and UPEC shows polymorphisms correlated with altered phenotypes in binding and receptor specificity.

Plasmids are another area of pathogenesis requiring more study. Our studies ascertained little to no differences in virulence in strain NMEC 58 when the large RepFIB virulence plasmid was removed [Unpublished data], indicating that in NMEC 58, the virulence plasmid may not be necessary to cause meningitis, NMEC strain RS218 when tested without its virulence plasmid showed significant attenuation, even though strain

RS218 and NMEC 58 share extremely recent common ancestry.

In conclusion NMEC are important pathogens of humans, and while some information is known about their pathogenicity, further characterization of NMEC should include more strain diversity to broaden our understanding of the pathogen and the disease or diseases they cause.