High genotypic and phenotypic similarity among Shiga toxin-producing Escherichia coli O111 environmental and outbreak strains.

Escherichia coli serogroup O111 is among the six most commonly reported non-O157:H7 Shiga toxin-producing E. coli (STEC), which are emerging as important foodborne pathogens. We have assembled a collection of environmental and clinical strains of E. coli O111 from diverse sources and investigated various genotypic and phenotypic characteristics of these strains to gain a better understanding of the epidemiology and biology of this serogroup. Sixty-three percent of the strains (24/38) were of H-type 8, which dominated the environmental- and outbreak-strains group, whereas the sporadic-case strains were more heterogeneous in H-type. All of the environmental and outbreak strains harbored the Shiga toxin 1 gene (stx1), eae, and ehx, and a subset of these also carried the Shiga toxin 2 gene (stx2). Only 9 of 16 sporadic-case strains produced stx1 and/or stx2, and these were mostly of H-type 8 and 10. Pulsed-field gel electrophoresis analysis revealed a cluster of environmental, outbreak, and sporadic illness strains with high phylogenetic similarity. Strains in this pulsogroup were all of the H8 type and STEC pathotype, and carried eae and ehx. Smaller clusters of highly similar STEC O111 strains included outbreak and sporadic illness strains isolated during different time periods or from different geographical locations. A distinct aggregative behavior was observed in the cultures of all environmental and outbreak STEC O111 strains, but not in those of sporadic-case strains. Among environmental and outbreaks strains, aggregation was positively correlated with production of curli fimbriae and RpoS function, and negatively with cellulose synthesis, while the nonaggregative behavior of sporadic-case strains correlated (positively) only with cellulose production. Our results indicate that STEC O111 strains sharing high genotypic similarity and important phenotypic traits with STEC O111 outbreak strains are present in the agricultural environment and may contribute to the burden of foodborne disease.

Distinct acid resistance and survival fitness displayed by Curli variants of enterohemorrhagic Escherichia coli O157:H7.

Curli are adhesive fimbriae of Enterobacteriaceae and are involved in surface attachment, cell aggregation, and biofilm formation. Here, we report that both inter- and intrastrain variations in curli production are widespread in enterohemorrhagic Escherichia coli O157:H7. The relative proportions of curli-producing variants (C(+)) and curli-deficient variants (C(-)) in an E. coli O157:H7 cell population varied depending on the growth conditions. In variants derived from the 2006 U.S. spinach outbreak strains, the shift between the C(+) and C(-) subpopulations occurred mostly in response to starvation and was unidirectional from C(-) to C(+); in variants derived from the 1993 hamburger outbreak strains, the shift occurred primarily in response to oxygen depletion and was bidirectional. Furthermore, curli variants derived from the same strain displayed marked differences in survival fitness: C(+) variants grew to higher concentrations in nutrient-limited conditions than C(-) variants, whereas C(-) variants were significantly more acid resistant than C(+) variants. This difference in acid resistance does not appear to be linked to the curli fimbriae per se, since a csgA deletion mutant in either a C(+) or a C(-) variant exhibited an acid resistance similar to that of its parental strain. Our data suggest that natural curli variants of E. coli O157:H7 carry several distinct physiological properties that are important for their environmental survival. Maintenance of curli variants in an E. coli O157:H7 population may provide a survival strategy in which C(+) variants are selected in a nutrient-limited environment, whereas C(-) variants are selected in an acidic environment, such as the stomach of an animal host, including that of a human.

Analysis of fenbendazole residues in bovine milk by ELISA.

Fenbendazole residues in bovine milk were analyzed by ELISAs using two monoclonal antibodies. One monoclonal antibody (MAb 587) bound the major benzimidazole anthelmintic drugs, including fenbendazole, oxfendazole, and fenbendazole sulfone. The other (MAb 591) was more specific for fenbendazole, with 13% cross-reactivity with the sulfone and no significant binding to the sulfoxide metabolite. The limit of detection of the ELISA method in the milk matrix was 7 ppb for MAb 587 and 3 ppb for MAb 591. Fenbendazole was administered in feed, drench, and paste form to three groups of dairy cattle. Milk was collected immediately before dosing and then every 12 h for 5 days. The ELISA indicated that residue levels varied widely among individual cows in each group. Fenbendazole levels peaked at approximately 12-24 h and declined rapidly thereafter. Metabolites were detected at much higher levels than the parent compound, peaked at approximately 24-36 h, and declined gradually. Residue levels were undetectable by 72 h. The ELISA data correlated well with the total residues determined by chromatographic analysis, but the use of the two separate ELISAs did not afford an advantage over ELISA with the single, broadly reactive MAb 587. The ELISA method could be used to flag high-residue samples in on-site monitoring of fenbendazole in milk and is a potential tool for studying drug pharmacokinetics.