Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains producing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of pig postweaning diarrhea (PWD). We recently identified neutralizing epitopes of fimbriae K88 and F18, heat-labile toxin (LT), heat-stable toxins type I (STa) and type II (STb), and Shiga toxin 2e (Stx2e). In this study, we explored a novel epitope- and structure-based vaccinology platform, multiepitope fusion antigen (MEFA), for PWD vaccine development. By using an epitope substitution LT toxoid, which lacks enterotoxicity but retains immunogenicity, as the backbone to present neutralizing epitopes of two ETEC fimbriae and four toxins, we generated PWD fimbria-toxin MEFA to mimic epitope native antigenicity. We then examined MEFA protein immunogenicity and evaluated MEFA application in PWD vaccine development. Mice subcutaneously immunized with PWD MEFA protein developed strong IgG responses to K88, F18, LT, and STb and moderate responses to the toxins Stx2e and STa. Importantly, MEFA-induced antibodies inhibited adherence of K88 or F18 fimbrial bacteria to pig intestinal cells and also neutralized LT, STa, STb, and Stx2e toxicity. These results indicated that PWD fimbria-toxin MEFA induced neutralizing antibodies against an unprecedent two fimbriae and four toxins and strongly suggested a potential application of this MEFA protein in developing a broadly protective PWD vaccine.IMPORTANCE ETEC-associated postweaning diarrhea (PWD) causes significant economic losses to swine producers worldwide. Currently, there is no effective prevention against PWD. A vaccine that blocks ETEC fimbriae (K88 and F18) from attaching to host receptors and prevents enterotoxins from stimulating water hypersecretion in pig small intestinal epithelial cells can effectively protect against PWD and significantly improves pig health and well-being. The fimbria-toxin MEFA generated from this study induced neutralizing antibodies against both ETEC fimbriae and all four ETEC toxins, suggesting a great potential of this fimbria-toxin MEFA in PWD vaccine development and further supporting the general application of this novel MEFA vaccinology platform for multivalent vaccine development.

Mapping the Neutralizing Epitopes of Enterotoxigenic Escherichia coli K88 (F4) Fimbrial Adhesin and Major Subunit FaeG.

Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Galactooligosaccharide supplementation provides protection against Citrobacter rodentium-induced colitis without limiting pathogen burden.

Many enteric pathogens, including Salmonella and enteropathogenic and enterohemorrhagic Escherichia coli, express adhesins that recognize and bind to carbohydrate moieties expressed on epithelial cells. An attractive strategy for inhibiting bacterial adherence employs molecules that mimic these epithelial binding sites. Prebiotic oligosaccharides are non-digestible, fermentable fibres capable of modulating the gut microbiota. Moreover, they may act as molecular decoys that competitively inhibit adherence of pathogens to host cells. In particular, galactooligosaccharides (GOS) and other prebiotic fibres have been shown to inhibit pathogen adherence to epithelial cells in vitro. In the present study, we determined the ability of prophylactic GOS administration to reduce enteric pathogen adherence both in vitro and in vivo as well as protect against intestinal inflammation. GOS supplementation significantly reduced the adherence of the epithelial-adherent murine bacterial pathogen Citrobacter rodentium in a dose-dependent manner to the surface of epithelial cells in vitro. A 1- to 2-log reduction in bacterial adherence was observed at the lowest and highest doses tested, respectively. However, mouse studies revealed that treatment with GOS neither reduced the adherence of C. rodentium to the distal colon nor decreased its dissemination to systemic organs. Despite the absence of adherence inhibition, colonic disease scores for GOS-treated, C. rodentium-infected mice were significantly lower than those of untreated C. rodentium-infected animals (P=0.028). Together, these data suggest that GOS has a direct protective effect in ameliorating disease severity following C. rodentium infection through an anti-adherence-independent mechanism.

Tellurite Resistance in Shiga Toxin-Producing Escherichia coli.

Potassium tellurite (K2TeO3) is an effective selective agent for O157:H7 Shiga toxin-producing Escherichia coli (STEC), whereas tellurite resistance in non-O157 STEC is variable with information on O45 minimal. High-level K2TeO3 resistance in STEC is attributable to the ter gene cluster with terD an indicator of the cluster's presence. Polymerase chain reactions for terD and K2TeO3 minimum inhibitory concentration (MIC) determinations in broth cultures were conducted on 70 STEC and 40 non-STEC control organisms. Sixty-six STEC strains (94.3%) were terD+ compared to 28 control organisms (70.0%; P < 0.001). The prevalence of terD in O103 STEC strains was 70%, whereas in all other serogroups it was ≥ 90%. The K2TeO3 geometric mean MIC ranking for STEC serogroups from highest to lowest was O111 > O26 > O145 > O157 > O103 > O121 = O45. The K2TeO3 geometric mean MIC was significantly higher in terD+ than in terD- STEC, but not in terD+ versus terD- control strains. Resistance to K2TeO3 (MIC ≥ 25 mg/L) was exhibited by 65/66 terD+ and 0/4 terD- STEC strains, compared to 12/28 terD+ and 8/12 terD- control strains. These results confirm previous studies showing the significantly higher prevalence of the ter gene cluster in STEC strains, and the relationship between these genes and K2TeO3 resistance in STEC and especially intimin (eae)-positive STEC, in contrast to non-STEC organisms. O45 and O121 STEC, although frequently terD positive, on average had significantly lower levels of K2TeO3 resistance than O26, O111, and O145 STEC.

Culture-Based Quantification with Molecular Characterization of Non-O157 and O157 Enterohemorrhagic Escherichia coli Isolates from Rectoanal Mucosal Swabs of Feedlot Cattle.

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens carried in the intestinal tracts of ruminants and shed in the feces. High concentrations (≥104 colony-forming units [CFU]/g) of EHEC in cattle feces are associated with contamination of hides, and subsequently, carcasses and beef. Several studies using agar media have quantified O157 but few have quantified non-O157 EHEC in samples from cattle. Thus, the objective of this study was to determine the concentration of O157 and non-O157 EHEC in cattle, and to characterize the associated EHEC isolates for their virulence potential. Two hundred feedlot steers were sampled by rectoanal mucosal swab (RAMS) every 35 days over four sampling periods, and a spiral plating method using modified Possé differential agar was used to quantify EHEC organisms in these samples. Bacterial colonies from agar plates were tested by multiplex PCR for Shiga toxin and intimin genes (stx and eae, respectively), and confirmed EHEC isolates (i.e., positive for both stx and eae) were serotyped and characterized for virulence genes using a microarray. Organisms detected in this study included O26, O101, O103, O109, O121, O145, O157, and O177 EHEC, with all except O121 quantifiable and measuring within a range from 9.0 × 102 to 3.0 × 105 CFU/g of RAMS sample. Organisms of the same EHEC serogroup were not detected in quantifiable concentrations from a single animal more than once. EHEC organisms most commonly detected at quantifiable levels were O26, O157, and O177. Interestingly, O26 EHEC isolates tested negative for stx1 but positive for stx2a. High concentrations of EHEC were detected in 11 (5.5%) of the steers at least once over the sampling period. These results indicate that in addition to O157, non-O157 EHEC are transiently present in high concentrations in the rectoanal mucosal region of cattle.

Comparison of Enrichment Broths for Supporting Growth of Shiga Toxin-Producing Escherichia coli.

Detection of Shiga toxin-producing Escherichia coli (STEC) in complex sample matrices remains challenging. In an attempt to improve detection, nonselective and selective enrichment broths were compared as follows: (1) trypticase soy broth (TSB) was compared with TSB plus novobiocin, vancomycin, rifampicin, bile salts, and potassium tellurite (TSB-NVRBT) for supporting growth of STEC in pure culture; (2) E. coli broth (EC), TSB, and TSB plus bile salts (mTSB) were compared for enrichment of STEC O26, O45, O103, O104, O111, O121, O145, and O157 (STEC-8) in inoculated cattle fecal samples; (3) EC, TSB, and mTSB were compared for the detection of STEC-8 in inoculated cattle fecal samples. Fecal samples were inoculated with wild-type STEC-8 or nalidixic acid- or rifampicin-resistant derivatives of the same strains at 100, 1000, or 10,000 colony-forming units per gram (CFU/g) of feces. In pure culture, the mean STEC CFU/mL following enrichment in TSB was 1.17 log10 greater than that in TSB-NVRBT (P < 0.05). In inoculated fecal samples, EC enrichment yielded growth of STEC-8 (6.42 log10 CFU/g) that was significantly greater than in TSB (6.23 log10 CFU/g; P < 0.05), and numerically but not significantly greater than in mTSB (6.37 log10 CFU/g; P = 0.60). Wild-type STEC strains were detected in 43.8 % (21/48) of the samples enriched in EC and mTSB compared to 27.1 % (13/48) of the samples enriched in TSB (P = 0.15). Overall, STEC grew significantly better when enriched in EC compared to TSB. Modification of TSB by the addition of bile salts improved the growth and detection of STEC compared to TSB alone.

Prevalence of Enterohemorrhagic Escherichia coli O26, O45, O103, O111, O121, O145, and O157 on Hides and Preintervention Carcass Surfaces of Feedlot Cattle at Harvest.

Cattle hides are a main source of enterohemorrhagic Escherichia coli (EHEC) contamination of beef carcasses. The objectives of this study were to (1) determine the prevalence of "top 6" non-O157 plus O157:H7 EHEC (EHEC-7) on feedlot cattle hides and their matched preintervention carcasses; (2) assess the agreement among detection methods for these matrices; and (3) conduct a molecular risk assessment of EHEC-7 isolates. Samples from 576 feedlot cattle were obtained at a commercial harvest facility and tested for EHEC-7 by a culture-based method and the polymerase chain reaction/mass spectrometry-based NeoSEEK(™) STEC Detection and Identification test (NS). Prevalence data were analyzed with generalized linear mixed models. The cumulative prevalence of EHEC-7 in hide samples as detected by NS was 80.7%, with a distribution of 49.9%, O145; 37.1%, O45; 12.5%, O103; 11.0%, O157; 2.2%, O111; 2.0%, O121; and 0.2%, O26. In contrast, the cumulative prevalence of EHEC-7 in hide samples by culture was 1.2%, with a distribution of 0.6%, O157; 0.4%, O26; 0.2%, O145; and 0%, O45, O103, O111, and O121. The cumulative prevalence of EHEC-7 on matched preintervention carcasses as detected by NS was 6.0%, with a distribution of 2.8%, O157; 1.6%, O145; 1.2%, O103; 1.1%, O45; 0.2%, O26; and 0.0%, O111 and O121. Although the culture-based method detected fewer positive hide samples than NS, it detected EHEC in five hide samples that tested negative for the respective organism by NS. McNemar's chi-square tests indicated significant (p<0.05) disagreement between methods. All EHEC-7 isolates recovered from hides were seropathotype A or B, with compatible virulence gene content. This study indicates that "top 6" and O157:H7 EHEC are present on hides, and to a lesser extent, preintervention carcasses of feedlot cattle at harvest. However, continued improvement in non-O157 detection methods is needed for accurate estimation of prevalence, given the discordant results across protocols.

Assessment of heterogeneity of efficacy of a three-dose regimen of a type III secreted protein vaccine for reducing STEC O157 in feces of feedlot cattle.

Preharvest control of Escherichia coli O157:H7 (STEC O157) may prevent human illness by reducing the presence of STEC O157 throughout the beef production chain. Immunization of cattle with a type III secreted protein vaccine inhibits colonization of cattle with STEC O157 and reduces the probability of fecal shedding and hide contamination. Our objectives were to perform a meta-analysis to estimate efficacy of a three-dose regimen of TTSP vaccine at reducing the presence of STEC O157 in the feces of feedlot cattle and to test factors that might modify vaccine efficacy. Pen-level data (n=184 pens, 1462 cattle) from four randomized controlled vaccine trials conducted from 2002 to 2008 at the University of Nebraska-Lincoln were analyzed. Factors explaining a culture-positive fecal sample were tested in generalized estimating equations logistic regression and log-binomial models. An autoregressive correlation structure was defined to account for clustering of repeated test-periods within block. Clustering or potential confounding by study was accounted for by treating study as a fixed effect. STEC O157 was detected from 661 of 5451 postvaccination fecal samples. The probability to detect STEC O157 postvaccination was 8.4% and 15.8% in vaccinated and unvaccinated cattle, respectively. Interactions between vaccination and (1) study; (2) prevalence of control pens within each time-place cluster; and (3) days from vaccination were not significant or fit poorly with observed data. Adjusting for study, cattle in pens receiving three doses of vaccine were less likely to shed STEC O157 (odds ratio=0.46, p<0.0001). Model-adjusted vaccine efficacy was 48% (95% confidence interval: 0.37-0.57). We concluded that a three-dose regimen type III secreted protein vaccine was efficacious at reducing the probability of detecting STEC O157 in the feces of cattle and that vaccine efficacy was not modified by study or level of prevalence observed in control pens.

Effect of potassium tellurite concentration in a chromogenic agar medium on isolation of tellurite-resistant "Top Seven" Shiga toxin-producing Escherichia coli from ground beef.

The effect of potassium tellurite concentration in a chromogenic agar medium on the detection of tellurite-resistant "top seven" Shiga toxin-producing Escherichia coli (STEC) in beef was evaluated. Samples of ground beef were inoculated with tellurite-resistant STEC O26, O45, O103, O111, O121, O145, or O157 strains at geometric mean (±standard error of the mean) levels of 0, 49 (±1), 490 (±1), or 4900 (±1) CFU/10 g and enriched 1:10 (90 mL) in EC broth (40°C for 6 h). Following enrichment, aliquots of broth culture were treated by immunomagnetic separation with one of three pools of beads against STEC serogroups; pool I: O26, O45, and O121; pool II: O103, O111, and O145; and pool III: O157. After immunomagnetic separation, 50 µL of washed bead suspensions in buffered peptone water was spiral plated onto a modified Possé medium containing 0.5, 1.0, or 1.5 mg/L potassium tellurite, and incubated at 37°C for 18 h. Up to four isolated colonies were picked from each spiral plate based on expected colony phenotypes for STEC, and isolate identity was confirmed with an 11-plex PCR assay targeting the O serogroups and virulence genes. Overall, across all inoculum levels and strains, modified Possé media containing 0.5, 1.0, or 1.5 mg/L potassium tellurite each had a positive predictive value of 100%, and medium containing 0.5 mg/L potassium tellurite had numerically the highest sensitivity (100%) and negative predictive value (100%), which was significantly different from 1.5 mg/L (92.9% and 40.0%, respectively; P < 0.05). Similarly, there was an inverse relationship between potassium tellurite concentration and analytical specificity (number of colonies tested that were STEC-positive): 0.5 (1463 of 1482; 98.7%), 1.0 (1356 of 1411; 96.1%), and 1.5 mg/L (1187 of 1278; 92.9%; P < 0.05). These results suggest that 0.5 mg/L gives better performance than 1.0 or 1.5 mg/L of potassium tellurite in Possé medium for isolation of tellurite-resistant "top seven" STEC from ground beef.

Association of ISVsa3 with Multidrug Resistance in Salmonella enterica Isolates from Cattle (Bos taurus).

Salmonella enterica is, globally, an important cause of human illness with beef being a significant attributable source. In the human patient, systemic Salmonella infection requires antibiotic therapy, and when strains are multidrug resistant (MDR), no effective treatment may be available. MDR in bacteria is often associated with the presence of mobile genetic elements (MGE) that mediate horizontal spread of antimicrobial resistance (AMR) genes. In this study, we sought to determine the potential relationship of MDR in bovine Salmonella isolates with MGE. The present study involved 111 bovine Salmonella isolates obtained collectively from specimens derived from healthy cattle or their environments at Midwestern U.S. feedyards (2000-2001, n = 19), or specimens from sick cattle submitted to the Nebraska Veterinary Diagnostic Center (2010-2020, n = 92). Phenotypically, 33/111 isolates (29.7%) were MDR (resistant to ≥3 drug classes). Based on whole-genome sequencing (WGS; n = 41) and PCR (n = 111), a MDR phenotype was strongly associated (OR = 186; p < 0.0001) with carriage of ISVsa3, an IS91-like Family transposase. In all 41 isolates analyzed by WGS ((31 MDR and 10 non-MDR (resistant to 0-2 antibiotic classes)), MDR genes were associated with carriage of ISVsa3, most often on an IncC type plasmid carrying blaCMY-2. The typical arrangement was floR, tet(A), aph(6)-Id, aph(3″)-Ib, and sul2 flanked by ISVsa3. These results suggest that AMR genes in MDR S. enterica isolates of cattle are frequently associated with ISVsa3 and carried on IncC plasmids. Further research is needed to better understand the role of ISVsa3 in dissemination of MDR Salmonella strains.

Evidence for a Causal Role for Escherichia coli Strains Identified as Adherent-Invasive (AIEC) in Intestinal Inflammation.

Enrichment of adherent-invasive Escherichia coli (AIEC) has been consistently detected in subsets of inflammatory bowel disease (IBD) patients. Although some AIEC strains cause colitis in animal models, these studies did not systematically compare AIEC with non-AIEC strains, and causal links between AIEC and disease are still disputed. Specifically, it remains unclear whether AIEC shows enhanced pathogenicity compared to that of commensal E. coli found in the same ecological microhabitat and if the in vitro phenotypes used to classify strains as AIEC are pathologically relevant. Here, we utilized in vitro phenotyping and a murine model of intestinal inflammation to systematically compare strains identified as AIEC with those identified as non-AIEC and relate AIEC phenotypes to pathogenicity. Strains identified as AIEC caused, on average, more severe intestinal inflammation. Intracellular survival/replication phenotypes routinely used to classify AIEC positively correlated with disease, while adherence to epithelial cells and tumor necrosis factor alpha production by macrophages did not. This knowledge was then applied to design and test a strategy to prevent inflammation by selecting E. coli strains that adhered to epithelial cells but poorly survived/replicated intracellularly. Two E. coli strains that ameliorated AIEC-mediated disease were subsequently identified. In summary, our results show a relationship between intracellular survival/replication in E. coli and pathology in murine colitis, suggesting that strains possessing these phenotypes might not only become enriched in human IBD but also contribute to disease. We provide new evidence that specific AIEC phenotypes are pathologically relevant and proof of principle that such mechanistic information can be therapeutically exploited to alleviate intestinal inflammation. IMPORTANCE Inflammatory bowel disease (IBD) is associated with an altered gut microbiota composition, including expansion of Proteobacteria. Many species in this phylum are thought to contribute to disease under certain conditions, including adherent-invasive Escherichia coli (AIEC) strains, which are enriched in some patients. However, whether this bloom contributes to disease or is just a response to IBD-associated physiological changes is unknown. Although assigning causality is challenging, appropriate animal models can test the hypothesis that AIEC strains have an enhanced ability to cause colitis in comparison to other gut commensal E. coli strains and to identify bacterial traits contributing to virulence. We observed that AIEC strains are generally more pathogenic than commensal E. coli and that bacterial intracellular survival/replication phenotypes contributed to disease. We also found that E. coli strains lacking primary virulence traits can prevent inflammation. Our findings provide critical information on E. coli pathogenicity that may inform development of IBD diagnostic tools and therapies.

Isolation and characterization of Escherichia albertii in poultry at the pre-harvest level.

Escherichia albertii, often misidentified as Escherichia coli, has become an emerging foodborne human enteric pathogen. However, the prevalence and major animal reservoirs of this significant pathogen are still not clear. Here, we performed comprehensive microbiological, molecular, comparative genomics and animal studies to understand the status and features of E. albertii in the US domestic and food animals. Although no E. albertii was identified in a total of 1,022 diverse E. coli strains isolated from pets and food animals in a retrospective screening, in a pilot study, E. albertii was successfully isolated from a broiler farm (6 out of 20 chickens). The chicken E. albertii isolates showed clonal relationship as indicated by both pulsed-field gel electrophoresis (PFGE) and whole-genome sequence analysis. The isolated chicken E. albertii displayed multidrug resistance; all the resistance determinants including the extended-spectrum beta-lactamase gene, carried by plasmids, could be conjugatively transferred to E. coli, which was further confirmed by S1-PFGE and Southern hybridization. Whole-genome sequence-based phylogenetic analysis showed the chicken E. albertii strains were phylogenetically close to those of human origins. Challenge experiment demonstrated that the E. albertii strains isolated from human and wild bird could successfully colonize in the chicken intestine. Together, this study, for the first time, reported the isolation of E. albertii in poultry at the pre-hrvest level. The findings from multi-tier characterization of the chicken E. albertii strains indicated the importance of chickens as a reservoir for E. albertii. A large scale of E. albertii survey in poultry production at the pre-harvest level is highly warranted in the future.

Recovery Rate of Cells of the Seven Regulated Serogroups of Shiga Toxin-Producing Escherichia coli from Raw Veal Cutlets, Ground Veal, and Ground Beef from Retail Stores in the Mid-Atlantic Region of the United States.

ABSTRACT: A total of 482 veal cutlet, 555 ground veal, and 540 ground beef samples were purchased from retail establishments in the mid-Atlantic region of the United States over a noncontiguous 2-year period between 2014 and 2017. Samples (325 g each) were individually enriched and screened via real-time PCR for all seven regulated serogroups of Shiga toxin-producing Escherichia coli (STEC). Presumptive STEC-positive samples were subjected to serogroup-specific immunomagnetic separation and plated onto selective media. Up to five isolates typical for STEC from each sample were analyzed via multiplex PCR for both the virulence genes (i.e., eae, stx1 and/or stx2, and ehxA) and serogroup-specific gene(s) for the seven regulated STEC serogroups. The recovery rates of non-O157 STEC from veal cutlets (3.94%, 19 of 482 samples) and ground veal (7.03%, 39 of 555 samples) were significantly higher (P < 0.05) than that from ground beef (0.93%, 5 of 540 samples). In contrast, only a single isolate of STEC O157:H7 was recovered; this isolate originated from 1 (0.18%) of 555 samples of ground veal. Recovery rates for STEC were not associated with state, season, packaging type, or store type (P > 0.05) but were associated with brand and fat content (P < 0.05). Pulsed-field subtyping of the 270 viable and confirmed STEC isolates from the 64 total samples testing positive revealed 78 pulsotypes (50 to 80% similarity) belonging to 39 pulsogroups, with ≥90% similarity among pulsotypes within pulsogroups. Multiple isolates from 43 (67.7%) of 64 samples testing positive had an indistinguishable pulsotype. STEC serotypes O26 and O103 were the most prevalent serogroups in beef and veal, respectively. These findings support related findings from regulatory sampling studies over the past decade and confirm that recovery rates for the regulated STEC serogroups are higher for raw veal than for raw beef samples, as was observed in the present study of meat purchased at food retailers in the mid-Atlantic region of the United States.

Determining relationships between the seasonal occurrence of Escherichia coli O157:H7 in live cattle, ground beef, and humans.

The prevalence and concentration of many foodborne pathogens exhibit seasonal patterns at different stages of the farm-to-table continuum. Escherichia coli O157:H7 is one such pathogen. While numerous studies have described the seasonal trend of E. coli O157:H7 in live cattle, ground beef, and human cases, it is difficult to relate the results from these different studies and determine the interrelationships that drive the seasonal pattern of beef-related human illnesses. This study uses a common modeling approach, which facilitates the comparisons across data sets, to relate prevalence in live cattle to raw ground beef and human illness. The results support an intuitive model where a seasonal rise of E. coli O157:H7 in cattle drives increased ground beef prevalence and a corresponding rise in the human case rate. We also demonstrate the use of these models to assess the public health impact of consumer behaviors. We present an example that suggests that the probability of illness, associated with summertime cooking and handling practices, is not substantially higher than the baseline probability associated with more conventional cooking and handling practices during the remainder of the year.

Special Issue: Shiga Toxin-Producing Escherichia coli.

Globally, Shiga toxin-producing Escherichia coli (STEC) is an important cause of diarrheal disease, most notably hemorrhagic colitis, and post-diarrheal sequela, such as hemolytic-uremic syndrome (HUS) [...].

Performance of Chromogenic Agar Media for Isolation of Shiga Toxin-Producing Escherichia coli from Ground Beef.

ABSTRACT: The performance of three chromogenic agar media for detection of the "top seven" Shiga toxin-producing Escherichia coli (STEC) in beef was compared. Samples of retail ground beef were inoculated with STEC O26, O45, O103, O111, O121, O145, or O157 at geometric mean (±standard error of the mean) levels of 0, 48 (±1), 420 (±1), 4,100 (±1), or 45,000 (±1) CFU/10 g and enriched 1:10 (90 mL) in EC broth (40°C for 6 h). Following enrichment, aliquots of broth culture were treated by immunomagnetic separation with one of three pools of beads against the seven STEC serogroups: pool I, O26, O45, and O121; pool II, O103, O111, and O145; and pool III, O157. After immunomagnetic separation, 50 µL of washed bead suspensions in buffered peptone water were spiral plated onto modified Rainbow Agar O157 (mRBA), CHROMagar STEC (CS), or modified Possé differential medium (mPossé2) and incubated at 37°C for 18 h. Up to six isolated colonies were picked from each spiral plate based on expected colony phenotypes for STEC on the respective media, and isolate identity was confirmed with an 11-plex PCR assay targeting the O serogroups and virulence genes. Overall, mRBA had the highest sensitivity (99.2%), correctly detecting a significantly higher proportion of STEC serogroups than either CS (79.4%; P < 0.05) or mPossé2 (91.7%; P < 0.05). mRBA also had the highest negative predictive value (90.0%), correctly identifying a significantly higher proportion of true-negative samples compared with CS (25.7%; P < 0.05) and mPossé2 (46.2%; P < 0.05). However, mRBA also had the lowest analytical specificity of 83.2% (P < 0.05), yielding the lowest proportion of colonies tested that were STEC positive (3,548 of 4,263) compared with 97.7% (3,607 of 3,693) for mPossé2 and 98.0% (2,875 of 2,935) for CS. Reduced specificity results in more work and higher expense due to the increased number of colonies that must be tested. Further improvements in agar culture media for non-O157 STEC isolation are needed.

Correction: Moxley, R.A., et al. Intimate Attachment of Escherichia coli O157:H7 to Urinary Bladder Epithelium in the Gnotobiotic Piglet Model. Microorganisms 2020, 8, 263.

The authors wish to make the following corrections to this paper [...].

Intimate Attachment of Escherichia coli O157:H7 to Urinary Bladder Epithelium in the Gnotobiotic Piglet Model.

Enterohemorrhagic Escherichia coli (EHEC), a pathogenic subset of Shiga toxin-producing E. coli (STEC), is an important cause of hemorrhagic colitis and hemolytic-uremic syndrome (HUS), and a rare cause of urinary tract infections (UTIs) with associated HUS. EHEC strains attach intimately to intestinal epithelium with formation of actin pedestals (attaching-effacing (A/E) lesions); however, the mechanism of EHEC attachment to the uroepithelium is unknown. We conducted a retrospective study on archived urinary bladder specimens from gnotobiotic piglets that naturally developed cystitis associated with EHEC O157:H7 infection following oral inoculation and fecal shedding. Paraffin-embedded bladder tissues from three piglets with cystitis and immunohistochemical evidence of EHEC O157:H7 adherence to the uroepithelium were processed for and examined by transmission electron microscopy. EHEC O157:H7 bacteria were found in one of three piglets, intimately attached to pedestals on the apical surfaces of the superficial urothelium (umbrella cells). Cystitis was significantly associated with the length of survival of the piglets post-inoculation (p = 0.0339; estimated odds ratio = 2.6652). This is the first report of E. coli causing A/E-like lesions in the uroepithelium, and also evidence of the utility of the gnotobiotic piglet as a model for studies of the pathogenesis of EHEC UTIs.

A randomized longitudinal trial to test the effect of regional vaccination within a cattle feedyard on Escherichia coli O157:H7 rectal colonization, fecal shedding, and hide contamination.

We tested the efficacy of vaccinating all cattle within a region of a cattle feedlot using a two-dose regimen of a vaccine against type III secreted proteins of Escherichia coli O157:H7. Cattle (n = 504) were randomly assigned to 63 pens (8 steers/pen) within 3 treatment regions of the feedyard. All pens within each region were assigned: (1) two doses of vaccine (ALLVAC), (2) two doses of adjuvant as placebo (NOVAC), or (3) commingled vaccination (HALFVAC), four of eight cattle in each pen receiving two doses of vaccine, and the others adjuvant. Binary outcomes were (1) fecal shedding of E. coli O157:H7 42, 63, and 84 days postvaccination (dpv), (2) hide contamination 42, 63, and 84 dpv, and at the abattoir 85 dpv, and (3) colonization of the terminal rectal mucosa at the abattoir 85 dpv. For each outcome, multilevel logistic regression tested the effect of regional vaccination (ALLVAC vs. NOVAC), and compared commingled vaccinated versus placebo-treated cattle within HALFVAC pens. For fecal shedding, regional vaccine efficacy of ALLVAC compared to NOVAC pens was 63% (OR = 0.34, p = 0.0009), similar to vaccine efficacy of 52% for vaccinated cattle compared to placebo-treated cattle within HALFVAC pens (OR = 0.48, p = 0.014). For hide contamination, vaccine efficacy was 55% for regional vaccination of cattle in ALLVAC pens compared to NOVAC pens (OR = 0.43, p = 0.014). However, commingling vaccinated and placebo-treated cattle was not protective of hide contamination (OR = 0.67, p = 0.33). Colonization of cattle at the abattoir was not different among vaccinated and placebo-treated cattle (p = 0.63). We concluded that the two-dose vaccine regimen effectively reduced E. coli O157:H7 fecal shedding and hide contamination, and that vaccination of cattle within regions of the feedyard provided greater protection against hide contamination than commingling vaccinates and nonvaccinates.