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Enteroinvasive Escherichia coli (EIEC) is a unique pathovar that has a pathogenic mechanism nearly indistinguishable from that of Shigella species. In contrast to isolates of the four Shigella species, which are widespread and can be frequent causes of human illness, EIEC causes far fewer reported illnesses each year. In this study, we analyzed the genome sequences of 20 EIEC isolates, including 14 first described in this study. Phylogenomic analysis of the EIEC genomes demonstrated that 17 of the isolates are present in three distinct lineages that contained only EIEC genomes, compared to reference genomes from each of the E. coli pathovars and Shigella species. Comparative genomic analysis identified genes that were unique to each of the three identified EIEC lineages. While many of the EIEC lineage-specific genes have unknown functions, those with predicted functions included a colicin and putative proteins involved in transcriptional regulation or carbohydrate metabolism. In silico detection of the Shigella virulence plasmid (pINV), which is essential for the invasion of host cells, demonstrated that a form of pINV was present in nearly all EIEC genomes, but the Mxi-Spa-Ipa region of the plasmid that encodes the invasion-associated proteins was absent from several of the EIEC isolates. The comparative genomic findings in this study support the hypothesis that multiple EIEC lineages have evolved independently from multiple distinct lineages of E. coli via the acquisition of the Shigella virulence plasmid and, in some cases, the Shigella pathogenicity islands.

ITALIC! Shigella boydiiis one of the four ITALIC! Shigellaspecies that causes disease worldwide; however, there are few published studies that examine the genomic variation of this species. This study compares genomes of 72 total isolates; 28 ITALIC! S. boydiifrom Bangladesh and The Gambia that were recently isolated as part of the Global Enteric Multicenter Study (GEMS), 14 historical ITALIC! S. boydiigenomes in the public domain and 30 ITALIC! Escherichia coliand ITALIC! Shigellareference genomes that represent the genomic diversity of these pathogens. This comparative analysis of these 72 genomes identified that the ITALIC! S. boydiiisolates separate into three phylogenomic clades, each with specific gene content. Each of the clades contains ITALIC! S. boydiiisolates from geographic and temporally distant sources, indicating that the ITALIC! S. boydiiisolates from the GEMS are representative of ITALIC! S. boydii.This study describes the genome sequences of a collection of novel ITALIC! S. boydiiisolates and provides insight into the diversity of this species in comparison to the ITALIC! E. coliand other ITALIC! Shigellaspecies.

An increase in the frequency of seafood-borne gastroenteritis in humans and Vibrio-related disease of fish and invertebrates has generated interest in the ecology of disease-causing Vibrios and the mechanisms driving their evolution. Genome sequencing studies have indicated a substantial contribution of horizontal gene transfer (HGT) to the evolution of Vibrios. Of particular interest is the contribution of HGT to the evolution of Vibrios pathogens and the adaptation of disease-causing Vibrios for survival in diverse environments. In this review, we discuss the diversity and distribution of mobile genetic elements (MGEs) isolated from Vibrios and the contribution of these elements to the expansion of the ecological and pathogenic niches of the host strain. Much of the research on Vibrio MGEs has focused on understanding phages and plasmids and we will primarily discuss the evolution of these elements and also briefly highlight the other diverse elements characterized from Vibrios, which includes genomic islands and conjugative elements.

Inactivation of mismatch repair (MMR) has been shown to increase the accumulation of spontaneous mutations and frequency of recombination for diverse pathogenic bacteria. Currently, little is known regarding the role of mutator phenotypes for the diversification of natural populations of opportunistic human pathogens in marine environments. In this study, a higher frequency of mutators was detected among V. parahaemolyticus strains obtained from environmental sources compared with clinical sources. Inactivation of the MMR gene mutS caused increased antibiotic resistance and phase variation resulting in translucent colony morphologies. Increased nucleotide diversity in mutS and rpoB alleles from mutator compared with wild-type strains indicated a significant contribution of the mutator phenotype to the evolution of select genes. The results of this study indicate that the inactivation of MMR in V. parahaemolyticus leads to increased genetic and phenotypic diversity. This study is the first to report a higher frequency of natural mutators among Vibrio environmental strains and to provide evidence that inactivation of MMR increases the diversity of V. parahaemolyticus.

Enteropathogenic Escherichia coli (EPEC) are diarrhoeagenic E. coli, and are a significant cause of gastrointestinal illness among young children in developing countries. Typical EPEC are identified by the presence of the bundle-forming pilus encoded by a virulence plasmid, which has been linked to an increased severity of illness, while atypical EPEC lack this feature. Comparative genomics of 70 total EPEC from lethal (LI), non-lethal symptomatic (NSI) or asymptomatic (AI) cases of diarrhoeal illness in children enrolled in the Global Enteric Multicenter Study was used to investigate the genomic differences in EPEC isolates obtained from individuals with various clinical outcomes. A comparison of the genomes of isolates from different clinical outcomes identified genes that were significantly more prevalent in EPEC isolates of symptomatic and lethal outcomes than in EPEC isolates of asymptomatic outcomes. These EPEC isolates exhibited previously unappreciated phylogenomic diversity and combinations of virulence factors. These comparative results highlight the diversity of the pathogen, as well as the complexity of the EPEC virulence factor repertoire. Comparative genomics of 70 lethal, non-lethal symptomatic and asymptomatic enteropathogenic Escherichia coli (EPEC) isolates identifies the virulence-associated genes that are significantly more prevalent in symptomatic and lethal infections.

A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli., A novel isolate of Escherichia coli with features of extraintestinal and enteropathogenic strains was isolated from the blood of a man with multiorgan dysfunction following 3 weeks of diarrhea., Graphical Abstract Figure. A novel isolate of Escherichia coli with features of extraintestinal and enteropathogenic strains was isolated from the blood of a man with multiorgan dysfunction following 3 weeks of diarrhea.

This article examines the role of genomics in the understanding and identification of O157:H7 enterohemorrhagic Escherichia coli (EHEC). We highlight the development of novel molecular typing systems that are based on the genomic sequence that has been generated for this pathotype. The genomic comparisons of EHEC to other E. coli strains highlight the close relatedness of the O157 and O55 isolates and also identify other non-O157 clades of isolates that appear to have a different genomic history. Analysis within the EHEC isolates must be completed on a fine scale using whole-genome sequence-based approaches to assess both the conserved and lateral acquired gene content. The plethora of genomic data for EHEC isolates has provided the ability to examine this pathotype in detail, which has provided opportunities for novel surveillance, detection, and diagnostics.

O157:H7 is the most common enterohemorrhagic Escherichia coli (EHEC) serotype in North America, and it has been the principal causative agent of numerous food-poisoning outbreaks worldwide ( 1 , 2 ). Initially E. coli O157:H7 was recognized as a human pathogen in 1982 when it was isolated from 47 persons in two states who had developed bloody diarrhea after consuming hamburgers contaminated with this organism ( 3 ). Since then, E. coli O157:H7 has emerged as a major enteric pathogen, capable of causing localized infections and large outbreaks of gastrointestinal disease ( 4 ). Data accumulated from 1982 to 1996 showed that approximately two-thirds of the 3,000 cases of E. coli infections from 139 recognized outbreaks were associated with the ingestion of contaminated food products, whereas 22% of the reported cases were from direct person-to-person transmission and 10% were from drinking water ( 5 ). Surveillance data have demonstrated a high prevalence of E. coli O157:H7 among cattle and their environment, but a relatively low incidence of human infection. This supports the potential hypothesis that a subset of E. coli O157:H7 harbored in cattle may be responsible for the majority of human disease ( 6 ). To minimize or eradicate adverse effects on public health, the E. coli O157:H7 lineage has been the focus of numerous epidemiological, microbiological, genomic, forensic, and diagnostic studies. Overall, it is estimated that E. coli O157:H7 alone causes more than 76,000 infections and 61 deaths in humans due to severe complications annually in the United States ( 7 ). Symptoms include a range of gastrointestinal morbidities, such as severe abdominal cramping accompanied with little or no associated fever and a watery diarrhea that leads to severe bloody diarrhea ( 8 ). Although many infected individuals remain asymptomatic, approximately 15 to 20% of people infected with EHEC present severe enough symptoms to require hospitalization. In such severe cases, patients display renal dysfunction known as hemolytic-uremic syndrome (HUS), hemorrhagic colitis, and central nervous system failure with potentially lethal outcomes ( 9 – 11 ).

Vibrio parahaemolyticus is an aquatic halophilic bacterium that occupies estuarine and coastal marine environments, and is a leading cause of seafood-borne food poisoning cases. To investigate the environmental reservoir and potential gene flow that occurs among V. parahaemolyticus isolates, the virulence-associated gene content and genome diversity of a collection of 133 V. parahaemolyticus isolates were analyzed. Phylogenetic analysis of housekeeping genes, and pulsed-field gel electrophoresis, demonstrated that there is genetic similarity among V. parahaemolyticus clinical and environmental isolates. Whole-genome sequencing and comparative analysis of six representative V. parahaemolyticus isolates was used to identify genes that are unique to the clinical and environmental isolates examined. Comparative genomics demonstrated an O3:K6 environmental isolate, AF91, which was cultured from sediment collected in Florida in 2006, has significant genomic similarity to the post-1995 O3:K6 isolates. However, AF91 lacks the majority of the virulence-associated genes and genomic islands associated with these highly virulent post-1995 O3:K6 genomes. These findings demonstrate that although they do not contain most of the known virulence-associated regions, some V. parahaemolyticus environmental isolates exhibit significant genetic similarity to clinical isolates. This highlights the dynamic nature of the V. parahaemolyticus genome allowing them to transition between aquatic and host-pathogen states.

The whole-genome sequence of a carbapenem-resistant Klebsiella pneumoniae strain, PittNDM01, which coproduces NDM-1 and OXA-232 carbapenemases, was determined in this study. The use of single-molecule, real-time (SMRT) sequencing provided a closed genome in a single sequencing run. K. pneumoniae PittNDM01 has a single chromosome of 5,348,284 bp and four plasmids: pPKPN1 (283,371 bp), pPKPN2 (103,694 bp), pPKPN3 (70,814 bp), and pPKPN4 (6,141 bp). The contents of the chromosome were similar to that of the K. pneumoniae reference genome strain MGH 78578, with the exception of a large inversion spanning 23.3% of the chromosome. In contrast, three of the four plasmids are unique. The plasmid pPKPN1, an IncHI1B-like plasmid, carries the bla NDM-1 , armA , and qnrB1 genes, along with tellurium and mercury resistance operons. bla NDM-1 is carried on a unique structure in which Tn 125 is further bracketed by IS 26 downstream of a class 1 integron. The IncFIA-like plasmid pPKPN3 also carries an array of resistance elements, including bla CTX-M-15 and a mercury resistance operon. The ColE-type plasmid pPKPN4 carrying bla OXA-232 is identical to a plasmid previously reported from France. SMRT sequencing was useful in resolving the complex bacterial genomic structures in the de novo assemblies.

We report here the draft genome sequences of nine enteropathogenic Escherichia coli (EPEC) strains isolated from children in Kenya who died during hospitalization with diarrhea. Each of the isolates possess the EPEC adherence factor (EAF) plasmid encoding the bundle-forming pilus, which is characteristic of EPEC. These isolates represent diverse serogroups and EPEC phylogenomic lineages.

A Vibrio parahaemolyticus strain isolated from the rhizosphere of the ecosystem dominant estuarine grass, Spartina alterniflora , was characterized and shown to carry nifH , the gene encoding the nitrogenase iron protein, and to fix N 2 . Nitrogen fixation may contribute substantially to the adaptability, niche breadth, and ecological significance of V. parahaemolyticus .

First identified in 1982 as a human pathogen, enterohemorrhagic Escherichia coli of the O157:H7 serotype is a major cause of food-borne acquired human infections. Here, we report the genome sequence of the first known strain of this serotype isolated in the United States.

We report the draft genome sequences of three enteropathogenic Escherichia coli (EPEC) isolates that display the O157 serogroup but do not have the Shiga toxin genes ( stx ), which are characteristic of O157 enterohemorrhagic E. coli (EHEC). E. coli strain RN587/1 has the O157:H8 serotype and possesses the EAF plasmid characteristic of typical EPEC (J. B. Kaper, J. P. Nataro, and H. L. Mobley, Nat. Rev. Microbiol. 2:123–140, 2004). The other two isolates, strains C844-97 and C639-08, are both O157:H45 and possess the locus of enterocyte effacement (LEE) pathogenicity island; however, they do not contain the EAF plasmid or the stx -carrying phage.

The attaching and effacing Escherichia coli (AEEC) are characterized by the presence of a type III secretion system encoded by the locus of enterocyte effacement (LEE). Enterohemorrhagic E. coli (EHEC) are often identified as isolates that are LEE+ and carry the Shiga toxin ( stx )-encoding phage, which are labeled Shiga toxin-producing E. coli ; whereas enteropathogenic E. coli (EPEC) are LEE+ and often carry the EPEC adherence factor plasmid-encoded bundle-forming pilus ( bfp ) genes. All other LEE+ /bfp−/stx− isolates have been historically designated atypical EPEC. These groups have been defined based on the presence or absence of a limited number of virulence factors, many of which are encoded on mobile elements. This study describes the comparative analysis of the genomes of 114 LEE+ E. coli isolates. Based on a whole-genome phylogeny and analysis of type III secretion system effectors, the AEEC are divided into five distinct genomic lineages. The LEE+ /stx+/bfp− genomes were primarily divided into two genomic lineages, the O157/O55 EHEC1 and non-O157 EHEC2. The LEE +/bfp+/stx− AEEC isolates sequenced in this study separated into the EPEC1, EPEC2, and EPEC4 genomic lineages. A multiplex PCR assay for identification of each of these AEEC genomic lineages was developed. Of the 114 AEEC genomes analyzed, 31 LEE + isolates were not in any of the known AEEC lineages and thus represent unclassified AEEC that in most cases are more similar to other E. coli pathovars than to text modification AEEC. Our findings demonstrate evolutionary relationships among diverse AEEC pathogens and the utility of phylogenomics for lineage-specific identification of AEEC clinical isolates.

This study reports the release of draft genome sequences of five isolates of uropathogenic Escherichia coli (UPEC), isolated from patients suffering from uncomplicated cystitis in 2012 in Ann Arbor, Michigan. Phylogenetic analyses revealed that these strains belonged to E. coli phylogroups B2 and D and are closely related to known UPEC strains. Comparative genomic analysis revealed that more conserved proteins were shared between these recent isolates and UPEC strains causing cystitis than those causing pyelonephritis. Additional genomic comparisons identified that three isolates encode a type III secretion system (T3SS) and a putative T3SS effector gene cluster along with an invasin-like outer membrane protein. The presence of T3SS genes is a rare occurrence among UPEC strains. These genomes further substantiate the heterogeneity of the gene pool of UPEC and provide a foundation for comparative genomic studies using recent clinical isolates.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries. EPEC strain E2348/69 is used worldwide as a prototype to study EPEC genetics and disease. However, isolates of E2348/69 differ phenotypically, reflecting a history of in vitro selection. To identify the genomic and phenotypic changes in the prototype strain, we sequenced the genome of the nalidixic acid-resistant (Nal r ) E2348/69 clone. We also sequenced a recent nleF mutant derived by one-step PCR mutagenesis from the Nal r strain. The sequencing results revealed no unintended changes between the mutant and the parent strain. However, loss of the pE2348-2 plasmid and 3 nonsynonymous mutations were found in comparison to the published streptomycin-resistant (Str r ) E2348/69 reference genome. One mutation is a conservative amino acid substitution in ftsK . Another, in gyrA , is a mutation known to result in resistance to nalidixic acid. The third mutation converts a stop codon to a tryptophan, predicted to result in the fusion of hflD , the lysogenization regulator, to purB . The purB gene encodes an adenylosuccinate lyase involved in purine biosynthesis. The Nal r clone has a lower growth rate than the Str r isolate when cultured in minimal media, a difference which is corrected upon addition of adenine or by genetic complementation with purB . Addition of adenine or genetic complementation also restored the invasion efficiency of the Nal r clone. This report reconciles longstanding inconsistencies in phenotypic properties of an archetypal strain and provides both reassurance and cautions regarding intentional and unintentional evolution in vitro .

Klebsiella oxytoca strain 11492-1 was isolated from a perianal swab culture from a patient at the University of Maryland Medical Center in 2005. The K. oxytoca 11492-1 draft genome contains multiple antibiotic resistance genes, including a FOX-5 AmpC β-lactamase encoded on a large IncA/C plasmid.

We report the draft genome sequences of the collection referred to as the Escherichia coli DECA collection, which was assembled to contain representative isolates of the 15 most common diarrheagenic clones in humans ( http://shigatox.net/new/ ). These genomes represent a valuable resource to the community of researchers who examine these enteric pathogens.

An increase in the frequency of seafood-borne gastroenteritis in humans and Vibrio-related disease of fish and invertebrates has generated interest in the ecology of disease-causing Vibrios and the mechanisms driving their evolution. Genome sequencing studies have indicated a substantial contribution of horizontal gene transfer (HGT) to the evolution of Vibrios. Of particular interest is the contribution of HGT to the evolution of Vibrios pathogens and the adaptation of disease-causing Vibrios for survival in diverse environments. In this review, we discuss the diversity and distribution of mobile genetic elements (MGEs) isolated from Vibrios and the contribution of these elements to the expansion of the ecological and pathogenic niches of the host strain. Much of the research on Vibrio MGEs has focused on understanding phages and plasmids and we will primarily discuss the evolution of these elements and also briefly highlight the other diverse elements characterized from Vibrios, which includes genomic islands and conjugative elements.

Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus -related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus . We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus , V. harveyi , and V. campbellii . The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus .

The pool of mobile genetic elements (MGE) in microbial communities consists of viruses, plasmids, and associated elements (insertion sequences, transposons, and integrons) that are either self-transmissible or use mobile plasmids and viruses as vehicles for their dissemination. This mobilome facilitates the horizontal transfer of genes that promote the evolution and adaptation of microbial communities. Efforts to characterize MGEs from microbial populations resident in a variety of ecological habitats have revealed a surprisingly novel and seemingly untapped biodiversity. To better understand the impact of horizontal gene transfer (HGT), as well as the agents that promote HGT in marine ecosystems and to determine whether or not environmental parameters can effect the composition and structure of the mobilome in marine microbial communities, information on the distribution, diversity, and ecological traits of the marine mobilome is presented. In this chapter we discuss recent insights gained from different methodological approaches used to characterize the biodiversity and ecology of MGE in marine environments and their contributions to HGT. In addition, we present case studies that highlight specific HGT examples in coastal, open-ocean, and deep-sea marine ecosystems.

The pool of mobile genetic elements (MGE) in microbial communities consists of viruses, plasmids, and associated elements (insertion sequences, transposons, and integrons) that are either self-transmissible or use mobile plasmids and viruses as vehicles for their dissemination. This mobilome facilitates the horizontal transfer of genes that promote the evolution and adaptation of microbial communities. Efforts to characterize MGEs from microbial populations resident in a variety of ecological habitats have revealed a surprisingly novel and seemingly untapped biodiversity. To better understand the impact of horizontal gene transfer (HGT), as well as the agents that promote HGT in marine ecosystems and to determine whether or not environmental parameters can effect the composition and structure of the mobilome in marine microbial communities, information on the distribution, diversity, and ecological traits of the marine mobilome is presented. In this chapter we discuss recent insights gained from different methodological approaches used to characterize the biodiversity and ecology of MGE in marine environments and their contributions to HGT. In addition, we present case studies that highlight specific HGT examples in coastal, open-ocean, and deep-sea marine ecosystems.

The horizontal transfer of genes by mobile genetic elements such as plasmids and phages can accelerate genome diversification of Vibrio spp., affecting their physiology, pathogenicity, and ecological character. In this study, sequence analysis of three plasmids from Vibrio spp. previously isolated from salt marsh sediment revealed the remarkable diversity of these elements. Plasmids p0908 (81.4 kb), p23023 (52.5 kb), and p09022 (31.0 kb) had a predicted 99, 64, and 32 protein-coding sequences and G+C contents of 49.2%, 44.7%, and 42.4%, respectively. A phylogenetic tree based on concatenation of the host 16S rRNA and rpoA nucleotide sequences indicated p23023 and p09022 were isolated from strains most closely related to V. mediterranei and V. campbellii , respectively, while the host of p0908 forms a clade with V. fluvialis and V. furnissii . Many predicted proteins had amino acid identities to proteins of previously characterized phages and plasmids (24 to 94%). Predicted proteins with similarity to chromosomally encoded proteins included RecA, a nucleoid-associated protein (NdpA), a type IV helicase (UvrD), and multiple hypothetical proteins. Plasmid p0908 had striking similarity to enterobacteria phage P1, sharing genetic organization and amino acid identity for 23 predicted proteins. This study provides evidence of genetic exchange between Vibrio plasmids, phages, and chromosomes among diverse Vibrio spp.

Escherichia coli of the O157 serogroup are comprised of a diverse collection of more than 100 O157:non-H7 serotypes that are found in the environment, animal reservoir and infected patients and some have been linked to severe outbreaks of human disease. Among these, the enteropathogenic E. coli O157:non-H7 serotypes carry virulence factors that are hallmarks of enterohemorrhagic E. coli, such as causing attaching and effacing lesions during human gastrointestinal tract infections. Given the shared virulence gene pool between O157:H7 and O157:non-H7 serotypes, our objective was to examine the prevalence of virulence traits of O157:non-H7 serotypes within and across their H-serotype and when compared to other E. coli pathovars. We sequenced six O157:non-H7 genomes complemented by four genomes from public repositories in an effort to determine their virulence state and genetic relatedness to the highly pathogenic enterohemorrhagic O157:H7 lineage and its ancestral O55:H7 serotype. Whole-genome-based phylogenomic analysis and molecular typing is indicative of a non-monophyletic origin of the heterogeneous O157:non-H7 serotypes that are only distantly related to the O157:H7 serotype. The availability of multiple genomes enables robust phylogenomic placement of these strains into their evolutionary context, and the assessment of the pathogenic potential of the O157:non-H7 strains in causing human disease.