Joseph E. LeClerc

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.

Quality Sample Collection, Handling, and Preservation for an Effective Microbial Forensics Program.

Science can be part of an effective investigative response to a bioterrorism event or a biocrime by providing capabilities to analyze biological and associated signatures in collected evidence. Microbial forensics, a discipline comprised of several scientific fields, is dedicated to the analysis of evidence from such criminal acts to help determine the responsible party and to exonerate the innocent (6). A partnership among a number of government agencies, academia, and the private sector has been formed to better respond to and deter potential perpetrators of bioterrorism or biocrimes. This partnership leverages our national scientific and analytical capabilities to support activities of law enforcement agencies. The Department of Homeland Security (DHS), whose mission is, in part, to respond to and to prevent acts of terrorism against the United States, has established the National Bioforensics Analysis Center (NBFAC) (4, 6). The NBFAC, in partnership with the Federal Bureau of Investigation (FBI), (i) provides a state-of-the-art central laboratory for analysis of microbial forensic evidence and (ii) serves as a nexus for integrating the national resources to increase the effectiveness of law enforcement in obtaining the highest level of attribution possible in criminal cases where the weapon is a biological agent. One approach used by the NBFAC to establish a sound foundation, to foster communication, and to facilitate integration across government and other agencies is to promote independent meetings, which address specific needs and provide a forum for input from the broader scientific community, on the best scientific practices in microbial forensics (5). As part of this ongoing effort, a series of meetings sponsored by DHS were held at the Banbury Center of the Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, to address specific issues for the enhancement of microbial forensic capability. One such meeting, held on 16 to 19 October 2005, focused on the collection, handling, and storage of samples. These issues had been identified at previous meetings (5, 6) as some of the most critical issues confronting a crime scene investigation and subsequent analysis of evidence. The participants represented diverse scientific entities within academia, the private sector, the national laboratories, and several federal agencies (Central Intelligence Agency, Centers for Disease Control and Prevention, DHS, FBI, Food and Drug Administration, and U.S. Department of Agriculture), some of which have been involved in evidence collection for purposes related to forensics, public health, or plant and animal health. The collection and preservation of microbial forensic evidence are paramount to efficient and successful investigation and attribution. If evidence (when available) is not collected, degrades, or is contaminated during collection, handling, transport, or storage, the downstream characterization and attribution analyses may be compromised. Retrieving sufficient quantities and maintaining the integrity of the evidence increase the chances of being able to characterize the material to obtain the highest level of attribution possible. This paper presents issues related to the practices of sample collection, handling, transportation, and storage and includes recommendations for future directions for the field of microbial forensics and people participating in it. The recommendations apply to the NBFAC, as well as to other facilities and practitioners.

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

BackgroundThe 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.ResultsFrom 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.ConclusionsThese 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 Analysis of Immune Response against Vibrio cholerae Hemolysin in Caenorhabditis elegans

Vibrio cholerae cytolysin (VCC) is among the accessory V. cholerae virulence factors that may contribute to disease pathogenesis in humans. VCC, encoded by hlyA gene, belongs to the most common class of bacterial toxins, known as pore-forming toxins (PFTs). V. cholerae infects and kills Caenorhabditis elegans via cholerae toxin independent manner. VCC is required for the lethality, growth retardation and intestinal cell vacuolation during the infection. However, little is known about the host gene expression responses against VCC. To address this question we performed a microarray study in C. elegans exposed to V. cholerae strains with intact and deleted hlyA genes. Many of the VCC regulated genes identified, including C-type lectins, Prion-like (glutamine [Q]/asparagine [N]-rich)-domain containing genes, genes regulated by insulin/IGF-1-mediated signaling (IIS) pathway, were previously reported as mediators of innate immune response against other bacteria in C. elegans. Protective function of the subset of the genes up-regulated by VCC was confirmed using RNAi. By means of a machine learning algorithm called FastMEDUSA, we identified several putative VCC induced immune regulatory transcriptional factors and transcription factor binding motifs. Our results suggest that VCC is a major virulence factor, which induces a wide variety of immune response- related genes during V. cholerae infection in C. elegans.

Genome Signatures of Escherichia coli O157:H7 Isolates from the Bovine Host Reservoir

ABSTRACT 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.

Reticulate Evolution Among the Group I Salmonellae: An Ongoing Role for Horizontal Gene Transfer

Salmonella enterica is responsible for 1.4 million cases of foodborne salmonellosis in the United States annually making it the number one causative agent of bacterial foodborne illnesses (CDC, 2007). Infection can occur after eating undercooked meat, poultry and eggs that have been contaminated with Salmonella (CDC, 2007). In recent years several outbreaks have occurred in the United States that were associated with Salmonella contamination of produce, the most recent being a S. enterica Saintpaul outbreak associated with tomatoes, jalapeno and serrano peppers that sickened over 1400 individuals (CDC, 2008). The movement of several serovars of Salmonella into previously naive niches (i.e., producegrowing environs) suggests that the pathogen is readily adapting to new environments. An understanding of the reticulate evolutionary mechanisms that underpin the acquisition and composition of the requisite genetic and phenotypic features of Salmonella is essential to more accurate risk assessment of this pathogen (Hohmann, 2001).