Effect of severe weather events on the shedding of Shiga toxigenic Escherichia coli in slaughter cattle and phenotype of serogroup O157 isolates.

High-event periods (HEPs) occur sporadically when beef carcasses and meat have episodes of acute contamination with Shiga toxin-producing Escherichia coli (STEC). In this study, severe weather events were investigated as catalysts for HEPs based on PCR and isolate prevalence of seven E. coli serogroups in slaughter cattle feces. Winter ambient temperatures with daily means 10.5oC warmer or 12.3°C colder than seasonal norms (-10.4°C) most altered STEC shedding. Fecal samples yielded increased proportions (P < 0.05) of O26 and O157 isolates during winter warm periods, and reduced (P < 0.05) O45 isolates during cold periods compared to samplings during seasonal norms. Based on changing PCR prevalence and isolates collected, O157 was the serogroup most responsive to severe weather events. Consequently, O157 isolates (n = 219) were evaluated for heat resistance, biofilm-forming potential and virulence gene subtypes. Two isolates had heat-resistant phenotypes with thermal death time at 60°C (D60) > 10 min and one also had strong biofilm-forming potential. However, this isolate lacked eae and stx genes. Severe weather can influence STEC shedding, particularly of O157, and could possibly trigger HEPs. However, our data suggest that it is unlikely for isolates to carry virulence genes and possess phenotypes capable of evading post-harvest microbiological interventions.

Endemic bacteriophages: a cautionary tale for evaluation of bacteriophage therapy and other interventions for infection control in animals.

BACKGROUND: One of the most effective targets for control of zoonotic foodborne pathogens in the farm to fork continuum is their elimination in food animals destined for market. Phage therapy for Escherichia coli O157:H7 in ruminants, the main animal reservoir of this pathogen, is a popular research topic. Since phages active against this pathogen may be endemic in host animals and their environment, they may emerge during trials of phage therapy or other interventions, rendering interpretation of trials problematic. METHODS: During separate phage therapy trials, sheep and cattle inoculated with 109 to 1010 CFU of E. coli O157:H7 soon began shedding phages dissimilar in plaque morphology to the administered therapeutic phages. None of the former was previously identified in the animals or in their environment. The dissimilar "rogue" phage was isolated and characterized by host range, ultrastructure, and genomic and proteomic analyses. RESULTS: The "rogue" phage (Phage vB_EcoS_Rogue1) is distinctly different from the administered therapeutic Myoviridae phages, being a member of the Siphoviridae (head: 53 nm; striated tail: 152x8 nm). It has a 45.8 kb genome which is most closely related to coliphage JK06, a member of the "T1-like viruses" isolated in Israel. Detailed bioinformatic analysis reveals that the tail of these phages is related to the tail genes of coliphage lambda. The presence of "rogue" phages resulting from natural enrichments can pose problems in the interpretation of phage therapeutic studies. Similarly, evaluation of any interventions for foodborne or other bacterial pathogens in animals may be compromised unless tests for such phages are included to identify their presence and potential impact.

Oral and rectal administration of bacteriophages for control of Escherichia coli O157:H7 in feedlot cattle.

This study compared oral and rectal administration of O157-specific bacteriophages for mitigating the fecal shedding of Escherichia coli O157 by experimentally inoculated steers. Fecal shedding of nalidixic acid-resistant (Nal(R)) E. coli O157:H7 was monitored over 83 days after oral (ORL; 3.3 x 10(11) PFU), rectal (REC; 1.5 x 10(11) PFU), both oral and rectal (O+R; 4.8 x 10(11) PFU), or no (CON; control) treatment with a four-strain O157-specific bacteriophage cocktail in multiple doses. Bacteriophages were enumerated by plaque assay, and NalR E. coli O157:H7 by direct plating on sorbitol MacConkey agar supplemented with cefixime, potassium tellurite, and nalidixic acid. Orally treated steers produced the fewest Nal(R) E. coli O157:H7 culture-positive samples (P < 0.06) compared with REC and O+R steers, but this number was only nominally lower (P = 0.26) than that for the CON steers. The overall mean shedding level (log CFU per gram of feces) was higher for REC steers (P < 0.10) than for steers of the other treatment groups. Despite the shedding of higher mean bacteriophage levels (log PFU per gram of feces) by ORL and O+R than by CON and REC steers, there was no difference (P > 0.05) in the number of E. coli O157-positive samples among treatments. Bacteriophage was isolated from CON steers, indicating that these steers acquired the bacteriophage from the environment and shed the phage at a level similar to that of REC steers (P = 0.39). Continuous bacteriophage therapy may be an efficacious method for mitigating shedding of E. coli O157:H7 in cattle, providing that the host bacterium does not develop resistance. This therapy may be especially advantageous if nontreated cattle can acquire this biocontrol agent from the feedlot environment.

Survival of Escherichia coli O157:H7 in feces from corn- and barley-fed steers.

The survival of Escherichia coli O157:H7 in feces from steers fed corn (CO) or barley (BA) was evaluated at -10, +4 and +22 degrees C. Fecal pats were inoculated with a four-strain mixture of nalidixic-acid resistant E. coli O157:H7 at two levels: 10(3) CFU g(-1) (low, L) and 105 CFU g(-1) (high, H). At -10 degrees C, duration of survival of E. coli O157:H7 was reduced (p < 0.05) in CO-L (35 days) compared to BA-L (49 days), likely due to the effects of fecal volatile fatty acids in combination with a fecal pH of <6.5. At 4 degrees C, E. coli O157:H7 was detected in BA-H, CO-H, CO-L and BA-L for 77, 77, 56 and 63 days, respectively, with no difference (p > 0.05) observed in the duration of survival or rate of decline of E. coli O157:H7 among treatments. Survival of E. coli O157:H7 was twice as likely (p < 0.05) at 22 degrees C than at 4 degrees C and -10 degrees C. While pH and dry matter content increased, and volatile fatty acid concentrations decreased over 84 days at all three temperatures, these changes were most pronounced at 22 degrees C. Survival of E. coli O157:H7 for extended periods of time in feces from both corn- and barley-fed animals was demonstrated, thus fecal material may serve as a vector for the transmission of the organism. The greater survival of E. coli O157:H7 at 22 degrees C suggests that temperature may play a role in the seasonality of transmission and prevalence of this bacterium in feedlot cattle. The reported greater prevalence of E. coli O157:H7 in cattle fed barley as compared to those fed corn does not appear to be related to elevated risk of transmission arising from differential survival of the bacterium in feces.