Genetic and Phenotypic Factors Associated with Persistent Shedding of Shiga Toxin-Producing Escherichia coli by Beef Cattle.

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infections. Cattle are an important STEC reservoir, although little is known about specific pathogen traits that impact persistence in the farm environment. Hence, we sought to evaluate STEC isolates recovered from beef cattle in a single herd in Michigan. To do this, we collected fecal grabs from 26 cattle and resampled 13 of these animals at 3 additional visits over a 3-month period. In all, 66 STEC isolates were recovered for genomics and biofilm quantification using crystal violet assays. The STEC population was diverse, representing seven serotypes, including O157:H7, O26:H11, and O103:H2, which are commonly associated with human infections. Although a core genome analysis of 2,933 genes grouped isolates into clusters based on serogroups, some isolates within each cluster had variable biofilm levels and virulence gene profiles. Most (77.8%; n = 49) isolates harbored stx2a, while 38 (57.5%) isolates formed strong biofilms. Isolates belonging to the predominant serogroup O6 (n = 36; 54.5%) were more likely to form strong biofilms, persistently colonize multiple cattle, and be acquired over time. A high-quality single nucleotide polymorphism (SNP) analysis of 33 O6 isolates detected between 0 and 13 single nucleotide polymorphism (SNP) differences between strains, indicating that highly similar strain types were persisting in this herd. Similar findings were observed for other persistent serogroups, although key genes were found to differ among strong and weak biofilm producers. Together, these data highlight the diversity and persistent nature of some STEC types in this important food animal reservoir.IMPORTANCE Food animal reservoirs contribute to Shiga toxin-producing Escherichia coli (STEC) evolution via the acquisition of horizontally acquired elements like Shiga toxin bacteriophages that enhance pathogenicity. In cattle, persistent fecal shedding of STEC contributes to contamination of beef and dairy products and to crops being exposed to contaminated water systems. Hence, identifying factors important for STEC persistence is critical. This longitudinal study enhances our understanding of the genetic diversity of STEC types circulating in a cattle herd and identifies genotypic and phenotypic traits associated with persistence. Key findings demonstrate that multiple STEC types readily persist in and are transmitted across cattle in a shared environment. These dynamics also enhance the persistence of virulence genes that can be transferred between bacterial hosts, resulting in the emergence of novel STEC strain types. Understanding how pathogens persist and diversify in reservoirs is important for guiding new preharvest prevention strategies aimed at reducing foodborne transmission to humans.

Dissemination of a highly virulent pathogen: tracking the early events that define infection.

The series of events that occurs immediately after pathogen entrance into the body is largely speculative. Key aspects of these events are pathogen dissemination and pathogen interactions with the immune response as the invader moves into deeper tissues. We sought to define major events that occur early during infection of a highly virulent pathogen. To this end, we tracked early dissemination of Yersinia pestis, a highly pathogenic bacterium that causes bubonic plague in mammals. Specifically, we addressed two fundamental questions: (1) do the bacteria encounter barriers in disseminating to draining lymph nodes (LN), and (2) what mechanism does this nonmotile bacterium use to reach the LN compartment, as the prevailing model predicts trafficking in association with host cells. Infection was followed through microscopy imaging in addition to assessing bacterial population dynamics during dissemination from the skin. We found and characterized an unexpected bottleneck that severely restricts bacterial dissemination to LNs. The bacteria that do not pass through this bottleneck are confined to the skin, where large numbers of neutrophils arrive and efficiently control bacterial proliferation. Notably, bottleneck formation is route dependent, as it is abrogated after subcutaneous inoculation. Using a combination of approaches, including microscopy imaging, we tested the prevailing model of bacterial dissemination from the skin into LNs and found no evidence of involvement of migrating phagocytes in dissemination. Thus, early stages of infection are defined by a bottleneck that restricts bacterial dissemination and by neutrophil-dependent control of bacterial proliferation in the skin. Furthermore, and as opposed to current models, our data indicate an intracellular stage is not required by Y. pestis to disseminate from the skin to draining LNs. Because our findings address events that occur during early encounters of pathogen with the immune response, this work can inform efforts to prevent or control infection.

Predicting the public health benefit of vaccinating cattle against Escherichia coli O157.

Identifying the major sources of risk in disease transmission is key to designing effective controls. However, understanding of transmission dynamics across species boundaries is typically poor, making the design and evaluation of controls particularly challenging for zoonotic pathogens. One such global pathogen is Escherichia coli O157, which causes a serious and sometimes fatal gastrointestinal illness. Cattle are the main reservoir for E. coli O157, and vaccines for cattle now exist. However, adoption of vaccines is being delayed by conflicting responsibilities of veterinary and public health agencies, economic drivers, and because clinical trials cannot easily test interventions across species boundaries, lack of information on the public health benefits. Here, we examine transmission risk across the cattle-human species boundary and show three key results. First, supershedding of the pathogen by cattle is associated with the genetic marker stx2. Second, by quantifying the link between shedding density in cattle and human risk, we show that only the relatively rare supershedding events contribute significantly to human risk. Third, we show that this finding has profound consequences for the public health benefits of the cattle vaccine. A naïve evaluation based on efficacy in cattle would suggest a 50% reduction in risk; however, because the vaccine targets the major source of human risk, we predict a reduction in human cases of nearly 85%. By accounting for nonlinearities in transmission across the human-animal interface, we show that adoption of these vaccines by the livestock industry could prevent substantial numbers of human E. coli O157 cases.

New perspectives to the enterotoxigenic E. coli F4 porcine infection model: Susceptibility genotypes in relation to performance, diarrhoea and bacterial shedding.

Enterotoxigenic E. coli (ETEC), causing post-weaning diarrhoea, is a major problem in weaned piglets. Individual animal responses to ETEC infection show high variability in animal experiments. Two studies were designed to optimize the ETEC F4ac infection model in piglets by combining the genotype susceptibility with performance, diarrhoea incidence and bacterial shedding. The studies were performed with respectively 120 and 80 male piglets that were tested for susceptibility or resistance towards ETEC O149:F4ac by a DNA marker based test. Three different genotypes were observed; resistant (RR), susceptible heterozygote (RS) and susceptible homozygote (SS). Piglets, were orally infected with an inoculum suspension (containing 1.5E8 CFU/ml ETEC F4ac) at day 0, 1 and 2 of the study. Performance, diarrhoea incidence and bacterial shedding were followed for 21days. In the first week after challenge a difference in average daily gain was observed between resistant and susceptible piglets in both studies. For the complete study period no significant differences were observed. Diarrhoea incidence was significantly higher in susceptible pigs compared to the resistant pigs in the first week after challenge. Bacterial shedding was much higher in the susceptible pigs and ETEC excretion lasted longer. ETEC was hardly detected in the faecal material of the resistant pigs. In conclusion, susceptible pigs showed higher diarrhoea incidence and higher numbers of faecal ETEC shedding in the first week after challenge compared to resistant pigs. The DNA marker based test can be used to select pigs that are susceptible for ETEC for inclusion in ETEC infection model, resulting in less animals needed to perform infection studies.

Comparative analysis of super-shedder strains of Escherichia coli O157:H7 reveals distinctive genomic features and a strongly aggregative adherent phenotype on bovine rectoanal junction squamous epithelial cells.

Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as "super-shedders" (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

TLR4 single nucleotide polymorphisms (SNPs) associated with Salmonella shedding in pigs.

Toll-like receptor 4 (TLR4) is a key factor in the innate immune recognition of lipopolysaccharide (LPS) from Gram-negative bacteria. Previous studies from our group identified differences in the expression profile of TLR4 and genes affected by the TLR4 signaling pathway among pigs that shed varying levels of Salmonella, a Gram-negative bacterium. Therefore, genetic variation in this gene may be involved with the host's immune response to bacterial infections. The current study screened for single nucleotide polymorphisms (SNPs) in the TLR4 gene and tested their association with Salmonella fecal shedding. Pigs (n = 117) were intranasally challenged at 7 weeks of age with 1 × 10(9) CFU of S. Typhimurium χ4232 and were classified as low or persistent Salmonella shedders based on the levels of Salmonella being excreted in fecal material. Salmonella fecal shedding was determined by quantitative bacteriology on days 2, 7, 14, and 20/21 post exposure, and the cumulative levels of Salmonella were calculated to identify the low (n = 20) and persistent (n = 20) Salmonella shedder pigs. From those 40 animals, the TLR4 region was sequenced, and 18 single nucleotide polymorphisms (SNPs) in TLR4 were identified. Twelve SNPs have been previously described and six are novel SNPs of which five are in the 5' untranslated region and one is in intron 2. Single marker association test identified 13 SNPs associated with the qualitative trait of Salmonella fecal shedding, and seven of those SNPs were also associated with a quantitative measurement of fecal shedding (P < 0.05). Using a stepwise regression process, a haplotype composed of SNPs rs80787918 and rs80907449 (P ≤ 4.0 × 10(-3)) spanning a region of 4.9 Kb was identified, thereby providing additional information of the influence of those SNPs on Salmonella fecal shedding in pigs.