Genomic diversity of 2010 Haitian cholera outbreak strains.

The millions of deaths from cholera during the past 200 y, coupled with the morbidity and mortality of cholera in Haiti since October 2010, are grim reminders that Vibrio cholerae, the etiologic agent of cholera, remains a scourge. We report the isolation of both V. cholerae O1 and non-O1/O139 early in the Haiti cholera epidemic from samples collected from victims in 18 towns across eight Arrondissements of Haiti. The results showed two distinct populations of V. cholerae coexisted in Haiti early in the epidemic. As non-O1/O139 V. cholerae was the sole pathogen isolated from 21% of the clinical specimens, its role in this epidemic, either alone or in concert with V. cholerae O1, cannot be dismissed. A genomic approach was used to examine similarities and differences among the Haitian V. cholerae O1 and V. cholerae non-O1/O139 strains. A total of 47 V. cholerae O1 and 29 V. cholerae non-O1/O139 isolates from patients and the environment were sequenced. Comparative genome analyses of the 76 genomes and eight reference strains of V. cholerae isolated in concurrent epidemics outside Haiti and 27 V. cholerae genomes available in the public database demonstrated substantial diversity of V. cholerae and ongoing flux within its genome.

Genomic anatomy of Escherichia coli O157:H7 outbreaks.

The rapid emergence of Escherichia coli O157:H7 from an unknown strain in 1982 to the dominant hemorrhagic E. coli serotype in the United States and the cause of widespread outbreaks of human food-borne illness highlights a need to evaluate critically the extent to which genomic plasticity of this important enteric pathogen contributes to its pathogenic potential and its evolution as well as its adaptation in different ecological niches. Aimed at a better understanding of the evolution of the E. coli O157:H7 pathogenome, the present study presents the high-quality sequencing and comparative phylogenomic analysis of a comprehensive panel of 25 E. coli O157:H7 strains associated with three nearly simultaneous food-borne outbreaks of human disease in the United States. Here we present a population genetic analysis of more than 200 related strains recovered from patients, contaminated produce, and zoonotic sources. High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. A total of 1,225 SNPs were identified in the present study and are now available for typing of the E. coli O157:H7 lineage. These data should prove useful for the development of a refined phylogenomic framework for forensic, diagnostic, and epidemiological studies to define better risk in response to novel and emerging E. coli O157:H7 resistance and virulence phenotypes.

Comparative genomics of 28 Salmonella enterica isolates: evidence for CRISPR-mediated adaptive sublineage evolution.

Despite extensive surveillance, food-borne Salmonella enterica infections continue to be a significant burden on public health systems worldwide. As the S. enterica species comprises sublineages that differ greatly in antigenic representation, virulence, and antimicrobial resistance phenotypes, a better understanding of the species' evolution is critical for the prediction and prevention of future outbreaks. The roles that virulence and resistance phenotype acquisition, exchange, and loss play in the evolution of S. enterica sublineages, which to a certain extent are represented by serotypes, remains mostly uncharacterized. Here, we compare 17 newly sequenced and phenotypically characterized nontyphoidal S. enterica strains to 11 previously sequenced S. enterica genomes to carry out the most comprehensive comparative analysis of this species so far. These phenotypic and genotypic data comparisons in the phylogenetic species context suggest that the evolution of known S. enterica sublineages is mediated mostly by two mechanisms, (i) the loss of coding sequences with known metabolic functions, which leads to functional reduction, and (ii) the acquisition of horizontally transferred phage and plasmid DNA, which provides virulence and resistance functions and leads to increasing specialization. Matches between S. enterica clustered regularly interspaced short palindromic repeats (CRISPR), part of a defense mechanism against invading plasmid and phage DNA, and plasmid and prophage regions suggest that CRISPR-mediated immunity could control short-term phenotype changes and mediate long-term sublineage evolution. CRISPR analysis could therefore be critical in assessing the evolutionary potential of S. enterica sublineages and aid in the prediction and prevention of future S. enterica outbreaks.

Investigating the global genomic diversity of Escherichia coli using a multi-genome DNA microarray platform with novel gene prediction strategies.

BACKGROUND: The gene content of a diverse group of 183 unique Escherichia coli and Shigella isolates was determined using the Affymetrix GeneChip® E. coli Genome 2.0 Array, originally designed for transcriptome analysis, as a genotyping tool. The probe set design utilized by this array provided the opportunity to determine the gene content of each strain very accurately and reliably. This array constitutes 10,112 independent genes representing four individual E. coli genomes, therefore providing the ability to survey genes of several different pathogen types. The entire ECOR collection, 80 EHEC-like isolates, and a diverse set of isolates from our FDA strain repository were included in our analysis. RESULTS: From this study we were able to define sets of genes that correspond to, and therefore define, the EHEC pathogen type. Furthermore, our sampling of 63 unique strains of O157:H7 showed the ability of this array to discriminate between closely related strains. We found that individual strains of O157:H7 differed, on average, by 197 probe sets. Finally, we describe an analysis method that utilizes the power of the probe sets to determine accurately the presence/absence of each gene represented on this array. CONCLUSIONS: These elements provide insights into understanding the microbial diversity that exists within extant E. coli populations. Moreover, these data demonstrate that this novel microarray-based analysis is a powerful tool in the field of molecular epidemiology and the newly emerging field of microbial forensics.

Genomic characterization of asymptomatic Escherichia coli isolated from the neobladder.

The replacement of the bladder with a neobladder made from ileal tissue is the prescribed treatment in some cases of bladder cancer or trauma. Studies have demonstrated that individuals with an ileal neobladder have recurrent colonization by Escherichia coli and other species that are commonly associated with urinary tract infections; however, pyelonephritis and complicated symptomatic infections with ileal neobladders are relatively rare. This study examines the genomic content of two E. coli isolates from individuals with neobladders using comparative genomic hybridization (CGH) with a pan-E. coli/Shigella microarray. Comparisons of the neobladder genome hybridization patterns with reference genomes demonstrate that the neobladder isolates are more similar to the commensal, laboratory-adapted E. coli and a subset of enteroaggregative E. coli than they are to uropathogenic E. coli isolates. Genes identified by CGH as exclusively present in the neobladder isolates among the 30 examined isolates were primarily from large enteric isolate plasmids. Isolations identified a large plasmid in each isolate, and sequencing confirmed similarity to previously identified plasmids of enteric species. Screening, via PCR, of more than 100 isolates of E. coli from environmental, diarrhoeagenic and urinary tract sources did not identify neobladder-specific genes that were widely distributed in these populations. These results taken together demonstrate that the neobladder isolates, while distinct, are genomically more similar to gastrointestinal or commensal E. coli, suggesting why they can colonize the transplanted intestinal tissue but rarely progress to acute pyelonephritis or more severe disease.

Genome signatures of Escherichia coli O157:H7 isolates from the bovine host reservoir.

Cattle comprise a main reservoir of Shiga toxin-producing Escherichia coli O157:H7 (STEC). The significant differences in host prevalence, transmissibility, and virulence phenotypes among strains from bovine and human sources are of major interest to the public health community and livestock industry. Genomic analysis revealed divergence into three lineages: lineage I and lineage I/II strains are commonly associated with human disease, while lineage II strains are overrepresented in the asymptomatic bovine host reservoir. Growing evidence suggests that genotypic differences between these lineages, such as polymorphisms in Shiga toxin subtypes and synergistically acting virulence factors, are correlated with phenotypic differences in virulence, host ecology, and epidemiology. To assess the genomic plasticity on a genome-wide scale, we have sequenced the whole genome of strain EC869, a bovine-associated E. coli O157:H7 isolate. Comparative phylogenomic analysis of this key isolate enabled us to place accurately bovine lineage II strains within the genetically homogenous E. coli O157:H7 clade. Identification of polymorphic loci that are anchored both in the chromosomal backbone and horizontally acquired regions allowed us to associate bovine genotypes with altered virulence phenotypes and host prevalence. This study catalogued numerous novel lineage II-specific genome signatures, some of which appear to be associated intimately with the altered pathogenic potential and niche adaptation within the bovine rumen. The presented extended list of polymorphic markers is valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies of this emerging human pathogen.

Comparative genomics of the IncA/C multidrug resistance plasmid family.

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

Antimicrobial resistance-conferring plasmids with similarity to virulence plasmids from avian pathogenic Escherichia coli strains in Salmonella enterica serovar Kentucky isolates from poultry.

Salmonella enterica, a leading cause of food-borne gastroenteritis worldwide, may be found in any raw food of animal, vegetable, or fruit origin. Salmonella serovars differ in distribution, virulence, and host specificity. Salmonella enterica serovar Kentucky, though often found in the food supply, is less commonly isolated from ill humans. The multidrug-resistant isolate S. Kentucky CVM29188, isolated from a chicken breast sample in 2003, contains three plasmids (146,811 bp, 101,461 bp, and 46,121 bp), two of which carry resistance determinants (pCVM29188_146 [strAB and tetRA] and pCVM29188_101 [bla(CMY-2) and sugE]). Both resistance plasmids were transferable by conjugation, alone or in combination, to S. Kentucky, Salmonella enterica serovar Newport, and Escherichia coli recipients. pCVM29188_146 shares a highly conserved plasmid backbone of 106 kb (>90% nucleotide identity) with two virulence plasmids from avian pathogenic Escherichia coli strains (pAPEC-O1-ColBM and pAPEC-O2-ColV). Shared avian pathogenic E. coli (APEC) virulence factors include iutA iucABCD, sitABCD, etsABC, iss, and iroBCDEN. PCR analyses of recent (1997 to 2005) S. Kentucky isolates from food animal, retail meat, and human sources revealed that 172 (60%) contained similar APEC-like plasmid backbones. Notably, though rare in human- and cattle-derived isolates, this plasmid backbone was found at a high frequency (50 to 100%) among S. Kentucky isolates from chickens within the same time span. Ninety-four percent of the APEC-positive isolates showed resistance to tetracycline and streptomycin. Together, our findings of a resistance-conferring APEC virulence plasmid in a poultry-derived S. Kentucky isolate and of similar resistance/virulence plasmids in most recent S. Kentucky isolates from chickens and, to lesser degree, from humans and cattle highlight the need for additional research in order to examine the prevalence and spread of combined virulence and resistance plasmids in bacteria in agricultural, environmental, and clinical settings.

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

Altered utilization of N-acetyl-D-galactosamine by Escherichia coli O157:H7 from the 2006 spinach outbreak.

In silico analyses of previously sequenced strains of Escherichia coli O157:H7, EDL933 and Sakai, localized the gene cluster for the utilization of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam). This gene cluster encodes the Aga phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of Aga. As the complete coding sequences for enzyme IIA (EIIA)(Aga/Gam), EIIB(Aga), EIIC(Aga), and EIID(Aga) of the Aga PTS are present, E. coli O157:H7 strains normally are able to utilize Aga as a sole carbon source. The Gam PTS complex, in contrast, lacks EIIC(Gam), and consequently, E. coli O157:H7 strains cannot utilize Gam. Phenotypic analyses of 120 independent isolates of E. coli O157:H7 from our culture collection revealed that the overwhelming majority (118/120) displayed the expected Aga+ Gam- phenotype. Yet, when 194 individual isolates, derived from a 2006 spinach-associated E. coli O157:H7 outbreak, were analyzed, all (194/194) displayed an Aga- Gam- phenotype. Comparison of aga/gam sequences from two spinach isolates with those of EDL933 and Sakai revealed a single nucleotide change (G:C-->A:T) in the agaF gene in the spinach-associated isolates. The base substitution in agaF, which encodes EIIA(Aga/Gam) of the PTS, changes a conserved glycine residue to serine (Gly91Ser). Pyrosequencing of this region showed that all spinach-associated E. coli O157:H7 isolates harbored this same G:C-->A:T substitution. Notably, when agaF+ was cloned into an expression vector and transformed into six spinach isolates, all (6/6) were able to grow on Aga, thus demonstrating that the Gly91Ser substitution underlies the Aga- phenotype in these isolates.