Pathogenomes of Shiga Toxin Positive and Negative Escherichia coli O157:H7 Strains TT12A and TT12B: Comprehensive Phylogenomic Analysis Using Closed Genomes.

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx- strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx1a- and ΦStx2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains' distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx-) evolutionary paths.

Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) of non-O157:H7 serotypes are responsible for global and widespread human food-borne disease. Among these serogroups, O26, O45, O103, O111, O121, and O145 account for the majority of clinical infections and are colloquially referred to as the "Big Six." The "Big Six" strain panel we sequenced and analyzed in this study are reference type cultures comprised of six strains representing each of the non-O157 STEC serogroups curated and distributed by the American Type Culture Collection (ATCC) as a resource to the research community under panel number ATCC MP-9. The application of long- and short-read hybrid sequencing yielded closed chromosomes and a total of 14 plasmids of diverse functions. Through high-resolution comparative phylogenomics, we cataloged the shared and strain-specific virulence and resistance gene content and established the close relationship of serogroup O26 and O103 strains featuring flagellar H-type 11. Virulence phenotyping revealed statistically significant differences in the Stx-production capabilities that we found to be correlated to the strain's individual stx-status. Among the carried Stx1a, Stx2a, and Stx2d phages, the Stx2a phage is by far the most responsive upon RecA-mediated phage mobilization, and in consequence, stx2a + isolates produced the highest-level of toxin in this panel. The availability of high-quality closed genomes for this "Big Six" reference set, including carried plasmids, along with the recorded genomic virulence profiles and Stx-production phenotypes will provide a valuable foundation to further explore the plasticity in evolutionary trajectories in these emerging non-O157 STEC lineages, which are major culprits of human food-borne disease.

Population structure and genetic diversity of Streptococcus suis isolates obtained from the United States.

Diseases caused by the zoonotic pathogen Streptococcus suis are an extensive economic problem as well as an animal welfare concern for the global swine industry. Previous studies have evaluated the genomic diversity and population structure of S. suis isolates, however, the majority of these studies utilized isolates obtained from countries other than the U.S. This study applied whole genome sequencing and cgMLST-based typing to evaluate the population structure and genetic relatedness among S. suis isolates obtained within the U.S. The established high-resolution phylogenomic framework revealed extensive genomic variation and diversity among the sampled S. suis isolates, with isolates from the U.S. and from countries outside the U.S. found interspersed in the phylogeny. S. suis isolates obtained within the U.S. did not cluster by state or geographic location, however, isolates with similar serotypes, both obtained from within and outside the U.S., generally clustered together. Average nucleotide identity (ANI) values determined for the S. suis genomes were extensively broad, approaching the recommended species demarcation value, and correlated with the phylogenetic group distribution of the cgMLST-based tree. Numerous antimicrobial resistance (AMR) elements were identified among both U.S. and non-U.S. isolates with ble, tetO, and ermB genes identified as the most prevalent. The epf, mrp, and sly genes, historically used as markers for virulence potential, were also observed in the genomes of isolates that grouped together forming a subclade of clonal complex 1 (CC1) isolates. Collectively, the data in this report provides critical information needed to address potential biosurveillance needs and insights into the genetic diversity and population structure of S. suis isolates obtained within the U.S.

Escherichia coli O157:H7 tir 255 T > A allele strains differ in chromosomal and plasmid composition.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains with the T allele in the translocated intimin receptor polymorphism (tir) 255 A > T gene associate with human disease more than strains with an A allele; however, the allele is not thought to be the direct cause of this difference. We sequenced a diverse set of STEC O157:H7 strains (26% A allele, 74% T allele) to identify linked differences that might underlie disease association. The average chromosome and pO157 plasmid size and gene content were significantly greater within the tir 255 A allele strains. Eighteen coding sequences were unique to tir 255 A allele chromosomes, and three were unique to tir 255 T allele chromosomes. There also were non-pO157 plasmids that were unique to each tir 255 allele variant. The overall average number of prophages did not differ between tir 255 allele strains; however, there were different types between the strains. Genomic and mobile element variation linked to the tir 255 polymorphism may account for the increased frequency of the T allele isolates in human disease.

Pathogenome comparison and global phylogeny of Escherichia coli ST1485 strains.

Escherichia coli ST1485 strains belong to the clinically important phylogroup F and have disseminated worldwide in humans, animals, and the environment. Here, we elucidated the pathogenome of a global collection of E. coli ST1485 isolates from diverse sources retrieved from public databases and a high-quality sequenced complete genome of colistin-resistant E. coli strain CFSAN061771 isolated from raw milk cheese which designated as a reference strain. CFSAN061771 belongs to O83:H42-ST1485 pathotype and carries a conjugative ColV plasmid, pCFSAN061771_01, combining extraintestinal virulence genes (ompt, sitA, iroN, etsC, traT, cvaC, hylF, iss, tsh, mchf, iucC, iutA) with a multidrug resistance island (blaTEM-1, aph(6)-Id, aph(3″)-Ib, sul2, dfrA14). Comparative genomic analysis revealed a high frequency of pCFSAN061771_01-like plasmids in E. coli ST1485. A notable evolutionary genetic event in E. coli ST1485 strains is the acquisition of a pCFSAN061771_02-like plasmid, which confers resistance to several antimicrobials, tellurium, and quaternary ammonium compounds. The identical virulence and antibiotic resistance profiles identified in some human and animal strains are worrisome. This is the first study to emphasize the significance of E. coli ST1485 as a global high-risk virulent and multidrug-resistant clone with zoonotic potential.

Genome Sequence Analysis and Characterization of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains Associated With a Laboratory Infection.

A laboratory-acquired E. coli O157:H7 infection with associated severe sequelae including hemolytic uremic syndrome occurred in an individual working in the laboratory with a mixture of nalidixic acid-resistant (NalR) O157:H7 mutant strains in a soil-biochar blend. The patient was hospitalized and treated with an intravenous combination of metronidazole and levofloxacin. The present study investigated the source of this severe laboratory acquired infection and further examined the influence of the antibiotics used during treatment on the expression and production of Shiga toxin. Genomes of two Stx2a-and eae-positive O157:H7 strains isolated from the patient's stool were sequenced along with two pairs of the wt strains and their derived NalR mutants used in the laboratory experiments. High-resolution SNP typing determined the strains' individual genetic relatedness and unambiguously identified the two laboratory-derived NalR mutant strains as the source of the researcher's life-threatening disease, rather than a conceivable ingestion of unrelated O157:H7 isolates circulating at the same time. It was further confirmed that in sublethal doses, the antibiotics increased toxin expression and production. Our results support a simultaneous co-infection with clinical strains in the laboratory, which were the causative agents of previous O157:H7 outbreaks, and further that the administration of antibiotics may have impacted the outcome of the infection.

Pathogenomes and variations in Shiga toxin production among geographically distinct clones of Escherichia coli O113:H21.

Infections with globally disseminated Shiga toxin-producing Escherichia coli (STEC) of the O113:H21 serotype can progress to severe clinical complications, such as hemolytic uremic syndrome (HUS). Two phylogeographically distinct clonal complexes have been established by multi locus sequence typing (MLST). Infections with ST-820 isolates circulating exclusively in Australia have caused severe human disease, such as HUS. Conversely, ST-223 isolates prevalent in the US and outside Australia seem to rarely cause severe human disease but are frequent contaminants. Following a genomic epidemiology approach, we wanted to gain insights into the underlying cause for this disparity. We examined the plasticity in the genome make-up and Shiga toxin production in a collection of 20 ST-820 and ST-223 strains isolated from produce, the bovine reservoir, and clinical cases. STEC are notorious for assembly into fragmented draft sequences when using short-read sequencing technologies due to the extensive and partly homologous phage complement. The application of long-read technology (LRT) sequencing yielded closed reference chromosomes and plasmids for two representative ST-820 and ST-223 strains. The established high-resolution framework, based on whole genome alignments, single nucleotide polymorphism (SNP)-typing and MLST, includes the chromosomes and plasmids of other publicly available O113:H21 sequences and allowed us to refine the phylogeographical boundaries of ST-820 and ST-223 complex isolates and to further identify a historic non-shigatoxigenic strain from Mexico as a quasi-intermediate. Plasmid comparison revealed strong correlations between the strains' featured pO113 plasmid genotypes and chromosomally inferred ST, which suggests coevolution of the chromosome and virulence plasmids. Our pathogenicity assessment revealed statistically significant differences in the Stx2a-production capabilities of ST-820 as compared to ST-223 strains under RecA-induced Stx phage mobilization, a condition that mimics Stx-phage induction. These observations suggest that ST-820 strains may confer an increased pathogenic potential in line with the strain-associated epidemiological metadata. Still, some of the tested ST-223 cultures sourced from contaminated produce or the bovine reservoir also produced Stx at levels comparable to those of ST-820 isolates, which calls for awareness and for continued surveillance of this lineage.

Genetic and Virulence Profiles of Enteroaggregative Escherichia coli (EAEC) Isolated From Deployed Military Personnel (DMP) With Travelers' Diarrhea.

To discern if there was a particular genotype associated with clinical enteroaggregative Escherichia coli (EAEC) strains isolated from deployed military personnel (DMP) with travelers' diarrhea (TD), we characterized a collection of EAEC from DMP deployed to Afghanistan, Djibouti, Kenya, or Honduras. Although we did not identify a specific EAEC genotype associated with TD in DMP, we found that EAEC isolated at the first clinic visit were more likely to encode the dispersin gene aap than EAEC collected at follow-up visits. A majority of the EAEC isolates were typical EAEC that adhered to HEp-2 cells, formed biofilms, and harbored genes for aggregative adherence fimbriae (AAF), AggR, and serine protease autotransporters of Enterobacteriaceae (SPATEs). A separate subset of the EAEC had aggR and genes for SPATEs but encoded a gene highly homologous to that for CS22, a fimbriae more commonly found in enterotoxigenic E. coli. None of these CS22-encoding EAEC formed biofilms in vitro or adhered to HEp-2 cells. Whole genome sequence and single nucleotide polymorphism analyses demonstrated that most of the strains were genetically diverse, but that a few were closely related. Isolation of these related strains occurred within days to more than a year apart, a finding that suggests a persistent source and genomic stability. In an ampicillin-treated mouse model we found that an agg4A+ aar- isolate formed a biofilm in the intestine and caused reduced weight gain in mice, whereas a strain that did not form an in vivo biofilm caused no morbidity. Our diverse strain collection from DMP displays the heterogeneity of EAEC strains isolated from human patients, and our mouse model of infection indicated the genotype agg4A+ aar- and/or capacity to form biofilm in vivo may correlate to disease severity.

Pathogenomes of Atypical Non-shigatoxigenic Escherichia coli NSF/SF O157:H7/NM: Comprehensive Phylogenomic Analysis Using Closed Genomes.

The toxigenic conversion of Escherichia coli strains by Shiga toxin-converting (Stx) bacteriophages were prominent and recurring events in the stepwise evolution of enterohemorrhagic E. coli (EHEC) O157:H7 from an enteropathogenic (EPEC) O55:H7 ancestor. Atypical, attenuated isolates have been described for both non-sorbitol fermenting (NSF) O157:H7 and SF O157:NM serotypes, which are distinguished by the absence of Stx, the characteristic virulence hallmark of Stx-producing E. coli (STEC). Such atypical isolates either never acquired Stx-phages or may have secondarily lost stx during the course of infection, isolation, or routine subculture; the latter are commonly referred to as LST (Lost Shiga Toxin)-isolates. In this study we analyzed the genomes of 15 NSF O157:H7 and SF O157:NM strains from North America, Europe, and Asia that are characterized by the absence of stx, the virulence hallmark of STEC. The individual genomic basis of the Stx (-) phenotype has remained largely undetermined as the majority of STEC genomes in public genome repositories were generated using short read technology and are in draft stage, posing a major obstacle for the high-resolution whole genome sequence typing (WGST). The application of LRT (long-read technology) sequencing provided us with closed genomes, which proved critical to put the atypical non-shigatoxigenic NSF O157:H7 and SF O157:NM strains into the phylogenomic context of the stepwise evolutionary model. Availability of closed chromosomes for representative Stx (-) NSF O157:H7 and SF O157:NM strains allowed to describe the genomic basis and individual evolutionary trajectories underlying the absence of Stx at high accuracy and resolution. The ability of LRT to recover and accurately assemble plasmids revealed a strong correlation between the strains' featured plasmid genotype and chromosomally inferred clade, which suggests the coevolution of the chromosome and accessory plasmids. The identified ancestral traits in the pSFO157 plasmid of NSF O157:H7 strain LSU-61 provided additional evidence for its intermediate status. Taken together, these observations highlight the utility of LRTs for advancing our understanding of EHEC O157:H7/NM pathogenome evolution. Insights into the genomic and phenotypic plasticity of STEC on a lineage- and genome-wide scale are foundational to improve and inform risk assessment, biosurveillance, and prevention strategies.

Comparative Virulence and Genomic Analysis of Streptococcus suis Isolates.

Streptococcus suis is a zoonotic bacterial swine pathogen causing substantial economic and health burdens to the pork industry. Mechanisms used by S. suis to colonize and cause disease remain unknown and vaccines and/or intervention strategies currently do not exist. Studies addressing virulence mechanisms used by S. suis have been complicated because different isolates can cause a spectrum of disease outcomes ranging from lethal systemic disease to asymptomatic carriage. The objectives of this study were to evaluate the virulence capacity of nine United States S. suis isolates following intranasal challenge in swine and then perform comparative genomic analyses to identify genomic attributes associated with swine-virulent phenotypes. No correlation was found between the capacity to cause disease in swine and the functional characteristics of genome size, serotype, sequence type (ST), or in vitro virulence-associated phenotypes. A search for orthologs found in highly virulent isolates and not found in non-virulent isolates revealed numerous predicted protein coding sequences specific to each category. While none of these predicted protein coding sequences have been previously characterized as potential virulence factors, this analysis does provide a reliable one-to-one assignment of specific genes of interest that could prove useful in future allelic replacement and/or functional genomic studies. Collectively, this report provides a framework for future allelic replacement and/or functional genomic studies investigating genetic characteristics underlying the spectrum of disease outcomes caused by S. suis isolates.

Genomic features of colistin resistant Escherichia coli ST69 strain harboring mcr-1 on IncHI2 plasmid from raw milk cheese in Egypt.

There is emerging evidence that food of animal origin may be responsible for the spread of multidrug resistant extraintestinal pathogenic Escherichia coli in the community. Here, we describe the emergence of colistin resistance gene, mcr-1, in a strain belonging to the dominant uropathogenic E. coli ST69 lineage. E. coli strain CFSAN061770 was isolated during monitoring of the popular Egyptian raw milk cheese, karish cheese, for the presence of colistin resistance. The complete genome of E. coli strain CFSAN061770 comprises a chromosome of 5,292,297 bp with a G + C content of 50.6%. Further, three plasmids named pEGY1-MCR-1, pEGY2 and pEGY3 of 228,947 bp, 103,234 bp and 87,012 bp were detected, respectively. Plasmid pEGY1-MCR-1 belongs to the IncHI2 incompatibility group and carries the colistin resistance mcr-1 gene flanked by two ISApl1 elements and forms a composite transposon. It mediates resistance to aminoglycosides (aadA1 and aadA2), phenicol (cmlA1 and floR), sulfonamides (sul3), and tetracycline (tet(A)), and these loci were found clustered in a multidrug resistant region. Plasmid pEGY3 carries a complex multiple resistance locus (CMR) (aph(3')-Ia, strA, strB, sul2, and blaTEM-1) encoding resistance to different classes of antibiotics. Interestingly, the closest plasmids to plasmid pEGY1-MCR-1 detected from the NCBI Blast search belonged to the incompatibility group IncHI2 and were from the Kingdom of Saudi Arabia and Qatar which suggests a dissemination of pEGY1-MCR-1-like plasmids in the Middle East. Most striking, and of great public health concern is that strain CFSAN061770 carries five virulence genes (iss, fimH, iutA, kpsMIII and kpsTIII) which were identified in clinical extraintestinal pathogenic E. coli. Besides that, it carries the astA gene, which codes for the enteroaggregative E. coli heat-stable toxin 1 (EAST1).

Closed Genome Sequence of Escherichia coli K-12 Group Strain C600.

Escherichia coli strain C600 is a prototypical K-12 derived laboratory strain which has been broadly used for molecular microbiology and bacterial physiology studies since its isolation in 1954. Here, we present the closed genome sequence of E. coli strain C600, retrieved from the American Type Culture Collection (ATCC 23724).

Single Nucleotide Polymorphism Analysis Indicates Genetic Distinction and Reduced Diversity of Swine-Associated Methicillin Resistant Staphylococcus aureus (MRSA) ST5 Isolates Compared to Clinical MRSA ST5 Isolates.

Livestock associated methicillin resistant S. aureus (LA-MRSA) are lineages adapted to livestock species. LA-MRSA can be transmitted to humans and public health concerns exist because livestock may be the largest MRSA reservoir outside of hospital settings. Although the predominant European (ST398) and Asian (ST9) lineages of LA-MRSA are considered livestock adapted, North American swine also harbor ST5, a globally disseminated and highly pathogenic lineage. This study applied whole genome sequencing and single nucleotide polymorphism (SNP) typing to compare the population structure and genetic relatedness between swine associated and human clinical MRSA ST5 isolates. The established high-resolution phylogenomic framework revealed that LA-MRSA and human clinical MRSA ST5 are genetically distinct. LA-MRSA isolates were found to be clonal within farms, while greater genome diversity was observed among sampled clinical MRSA ST5. Analysis of the accessory genome demonstrated that LA-MRSA ST5 isolates and clinical MRSA ST5 isolates harbor different AMR genes and virulence factors, consistent with the SNP analysis. Collectively, our data indicate LA-MRSA and clinical MRSA ST5 isolates are distinct and the swine reservoir is likely of minimal significance as a source of clinical MRSA ST5 infections.

Closed Genome and Comparative Phylogenetic Analysis of the Clinical Multidrug Resistant Shigella sonnei Strain 866.

Shigella sonnei is responsible for the majority of shigellosis infections in the US with over 500,000 cases reported annually. Here, we present the complete genome of the clinical multidrug resistant (MDR) strain 866, which is highly susceptible to bacteriophage infections. The strain has a circular chromosome of 4.85 Mb and carries a 113 kb MDR plasmid. This IncB/O/K/Z-type plasmid, termed p866, confers resistance to five different classes of antibiotics including ß-lactamase, sulfonamide, tetracycline, aminoglycoside, and trimethoprim. Comparative analysis of the plasmid architecture and gene inventory revealed that p866 shares its plasmid backbone with previously described IncB/O/K/Z-type Shigella spp. and Escherichia coli plasmids, but is differentiated by the insertion of antibiotic resistance cassettes, which we found associated with mobile genetic elements such as Tn3, Tn7, and Tn10. A whole genome-derived phylogenetic reconstruction showed the evolutionary relationships of S. sonnei strain 866 and the four established Shigella species, highlighting the clonal nature of S. sonnei.

Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A.

Multidrug-resistant Acinetobacter baumannii is among the most common causes of infectious complications associated with combat-related trauma in military personnel serving overseas. However, little is currently known about its pathogenesis. While the gastrointestinal (GI) tract has been found to be a major reservoir for A. baumannii, as well as to potentially contribute to development of multidrug resistance, no studies have addressed the mechanisms involved in gut colonization. In this study, we address this critical gap in knowledge by first assessing the interaction between secretory IgA (SIgA), the principal humoral immune defense on mucosal surfaces, and the A. baumannii clinical isolate Ci79. Surprisingly, SIgA appeared to enhance A. baumannii GI tract colonization, in a process mediated by bacterial thioredoxin A (TrxA), as evidenced by reduction of bacterial attachment in the presence of TrxA inhibitors. Additionally, a trxA targeted deletion mutant (ΔtrxA) showed reduced bacterial burdens within the GI tract 24 h after oral challenge by in vivo live imaging, along with loss of thiol-reductase activity. Surprisingly, not only was GI tract colonization greatly reduced but the associated 50% lethal dose (LD50) of the ΔtrxA mutant was increased nearly 100-fold in an intraperitoneal sepsis model. These data suggest that TrxA not only mediates A. baumannii GI tract colonization but also may contribute to pathogenesis in A. baumannii sepsis following escape from the GI tract under conditions when the intestinal barrier is compromised, as occurs with cases of severe shock and trauma.IMPORTANCEAcinetobacter baumannii is an emerging bacterial pathogen recently classified as a serious threat to U.S. and global health by both the Centers for Disease Control and Prevention and the World Health Organization. It also is one of the leading causes of combat-related infections associated with injured military personnel serving overseas. Little is known regarding mechanisms of gastrointestinal tract colonization despite this site being shown to serve as a reservoir for multidrug-resistant (MDR) A. baumannii isolates. Here, we establish that secretory IgA, the major immunoglobulin of mucosal surfaces, promotes A. baumannii GI tract colonization via bacterial thioredoxin A as evidenced through significant reduction in colonization in IgA-deficient animals. Additionally, bacterial colonization and mortality were significantly reduced in animals challenged with a thioredoxin A-deficient A. baumannii mutant. Combined, these data suggest that thioredoxin A is a novel virulence factor, for which antithioredoxin therapies could be developed, for this important multidrug-resistant pathogen.

Closed Genome Sequence of Vibrio cholerae O1 El Tor Inaba Strain A1552.

Vibrio cholerae is a Gram-negative waterborne human pathogen and the causative agent of cholera. Here, we present the complete genome sequence of the seventh pandemic O1 biovar El Tor Inaba strain A1552 isolated in 1992. This clinical strain has served as an important model strain for studying cholera pathogenicity traits.

Identification of a mouse Lactobacillus johnsonii strain with deconjugase activity against the FXR antagonist T-ß-MCA.

Bile salt hydrolase (BSH) activity against the bile acid tauro-beta-muricholic acid (T-ß-MCA) was recently reported to mediate host bile acid, glucose, and lipid homeostasis via the farnesoid X receptor (FXR) signaling pathway. An earlier study correlated decreased Lactobacillus abundance in the cecum with increased concentrations of intestinal T-ß-MCA, an FXR antagonist. While several studies have characterized BSHs in lactobacilli, deconjugation of T-ß-MCA remains poorly characterized among members of this genus, and therefore it was unclear what strain(s) were responsible for this activity. Here, a strain of L. johnsonii with robust BSH activity against T-ß-MCA in vitro was isolated from the cecum of a C57BL/6J mouse. A screening assay performed on a collection of 14 Lactobacillus strains from nine different species identified BSH substrate specificity for T-ß-MCA only in two of three L. johnsonii strains. Genomic analysis of the two strains with this BSH activity revealed the presence of three bsh genes that are homologous to bsh genes in the previously sequenced human-associated strain L. johnsonii NCC533. Heterologous expression of several bsh genes in E. coli followed by enzymatic assays revealed broad differences in substrate specificity even among closely related bsh homologs, and suggests that the phylogeny of these enzymes does not closely correlate with substrate specificity. Predictive modeling allowed us to propose a potential mechanism driving differences in BSH activity for T-ß-MCA in these homologs. Our data suggests that L. johnsonii regulates T-ß-MCA levels in the mouse intestinal environment, and that this species may play a central role in FXR signaling in the mouse.

Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks.

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.

Genome Sequences of Five Clinical Isolates of Klebsiella pneumoniae.

Klebsiella pneumoniae is a nosocomial pathogen of emerging importance and displays resistance to broad-spectrum antibiotics, such as carbapenems. Here, we report the genome sequences of five clinical K. pneumoniae isolates, four of which are carbapenem resistant. Carbapenem resistance is conferred by hydrolyzing class A ß-lactamases found adjacent to transposases.

Phylogenetic Diversity of Vibrio cholerae Associated with Endemic Cholera in Mexico from 1991 to 2008.

UNLABELLED: An outbreak of cholera occurred in 1991 in Mexico, where it had not been reported for more than a century and is now endemic. Vibrio cholerae O1 prototype El Tor and classical strains coexist with altered El Tor strains (1991 to 1997). Nontoxigenic (CTX(-)) V. cholerae El Tor dominated toxigenic (CTX(+)) strains (2001 to 2003), but V. cholerae CTX(+) variant El Tor was isolated during 2004 to 2008, outcompeting CTX(-) V. cholerae. Genomes of six Mexican V. cholerae O1 strains isolated during 1991 to 2008 were sequenced and compared with both contemporary and archived strains of V. cholerae. Three were CTX(+) El Tor, two were CTX(-) El Tor, and the remaining strain was a CTX(+) classical isolate. Whole-genome sequence analysis showed the six isolates belonged to five distinct phylogenetic clades. One CTX(-) isolate is ancestral to the 6th and 7th pandemic CTX(+) V. cholerae isolates. The other CTX(-) isolate joined with CTX(-) non-O1/O139 isolates from Haiti and seroconverted O1 isolates from Brazil and Amazonia. One CTX(+) isolate was phylogenetically placed with the sixth pandemic classical clade and the V. cholerae O395 classical reference strain. Two CTX(+) El Tor isolates possessing intact Vibrio seventh pandemic island II (VSP-II) are related to hybrid El Tor isolates from Mozambique and Bangladesh. The third CTX(+) El Tor isolate contained West African-South American (WASA) recombination in VSP-II and showed relatedness to isolates from Peru and Brazil. Except for one isolate, all Mexican isolates lack SXT/R391 integrative conjugative elements (ICEs) and sensitivity to selected antibiotics, with one isolate resistant to streptomycin. No isolates were related to contemporary isolates from Asia, Africa, or Haiti, indicating phylogenetic diversity. IMPORTANCE: Sequencing of genomes of V. cholerae is critical if genetic changes occurring over time in the circulating population of an area of endemicity are to be understood. Although cholera outbreaks occurred rarely in Mexico prior to the 1990s, genetically diverse V. cholerae O1 strains were isolated between 1991 and 2008. Despite the lack of strong evidence, the notion that cholera was transmitted from Africa to Latin America has been proposed in the literature. In this study, we have applied whole-genome sequence analysis to a set of 124 V. cholerae strains, including six Mexican isolates, to determine their phylogenetic relationships. Phylogenetic analysis indicated the six V. cholerae O1 isolates belong to five phylogenetic clades: i.e., basal, nontoxigenic, classical, El Tor, and hybrid El Tor. Thus, the results of phylogenetic analysis, coupled with CTXϕ array and antibiotic susceptibility, do not support single-source transmission of cholera to Mexico from African countries. The association of indigenous populations of V. cholerae that has been observed in this study suggests it plays a significant role in the dynamics of cholera in Mexico.