Supershed Escherichia coli O157:H7 Has Potential for Increased Persistence on the Rectoanal Junction Squamous Epithelial Cells and Antibiotic Resistance

Abstract

Supershedding cattle shed Escherichia coli O157:H7 (O157) at ≥ 104 colony-forming units/g feces. We recently demonstrated that a supershed O157 (SS-O157) strain, SS-17, hyperadheres to the rectoanal junction (RAJ) squamous epithelial (RSE) cells which may contribute to SS-O157 persistence at this site in greater numbers, thereby increasing the fecal O157 load characterizing the supershedding phenomenon. In order to verify if this would be the signature adherence profile of any SS-O157, we tested additional SS-O157 isolates (n = 101; each from a different animal) in the RSE cell adherence assay. Similar to SS-17, all 101 SS-O157 exhibited aggregative adherence on RSE cells, with 56% attaching strongly (>10 bacteria/cell; hyperadherent) and 44% attaching moderately (1–10 bacteria/cells). Strain typing using Polymorphic Amplified Typing Sequences (PATS) analysis assigned the 101 SS-O157 into 5 major clades but not to any predominant genotype. Interestingly, 69% of SS-O157 isolates were identical to human O157 outbreak strains based on pulsed field gel electrophoresis profiles (CDC PulseNet Database), grouped into two clades by PATS distinguishing them from remaining SS-O157, and were hyperadherent on RSE cells. A subset of SS-O157 isolates (n = 53) representing different PATS and RSE cell adherence profiles were analyzed for antibiotic resistance (AR). Several SS-O157 (30/53) showed resistance to sulfisoxazole, and one isolate was resistant to both sulfisoxazole and tetracycline. Minimum inhibitory concentration (MIC) tests confirmed some of the resistance observed using the Kirby–Bauer disk diffusion test. Each SS-O157 isolate carried at least 10 genes associated with AR. However, genes directly associated with AR were rarely amplified: aac (3)-IV in 2 isolates, sul2 in 3 isolates, and tetB in one isolate. The integrase gene, int, linked with integron-based AR acquisition/transmission, was identified in 92% of SS-O157 isolates. Our results indicate that SS-O157 isolates could potentially persist longer at the bovine RAJ but exhibit limited resistance towards clinical antibiotics.

EDL933 Strains of Escherichia coli O157 can Demonstrate Genetic Diversity and Differential Adherence to Bovine Recto-Anal Junction Squamous Epithelial Cells

“…Assays were performed with two different cell types using previously standardized protocols [21, 45 -47]. As per the protocols, all bacterial strains were cultured overnight in Dulbecco's modified Eagle's medium with low glucose [DMEM-LG (Invitrogen, Carlsbad, CA)] at 37°C without aeration, washed, and re-suspended in DMEM with no glucose (DMEM-NG; Invitrogen) [20,47,48]. Obtained from Dr. Saul Tzipori, Tufts University, North Grafton, MA [39,40].…”

“…Adherence patterns on RSE cells were qualitatively recorded as diffuse, aggregative, or nonadherent, and quantitatively as the percentages of RSE cells with or without adhering bacteria [21,46,47]; adherence was recorded as strongly adherent when more than 50% of RSE cells had 10 adherent bacteria, moderately adherent when 50% or less of the RSE cells had 1 to 10 adherent bacteria, and nonadherent when less than 50% of the RSE cells had only 1 to 5 adherent bacteria. RSE cells with no added bacteria were subjected to the assay procedure and used as negative controls to confirm the absence of pre-existing O157 bacteria.…”

“…Adherence patterns displayed by bacterial strains on HEp-2 cells (human epidermoid carcinoma of the larynx cells with HeLa contamination) (ATCC ® CCL-23™) were determined using the same growth conditions as those used for the RSE adherence assay and as described previously [21,47,48]. The HEp-2 adherence assay was performed twice, with four technical replicates per bacterial strain each time.…”

“…The HEp-2 adherence assay was performed twice, with four technical replicates per bacterial strain each time. Slides were stained with the FITC-labelled goat anti-O157 antibody (KPL) targeting the O157 antigen and the Alexa Fluor 594-labelled Phalloidin (Thermo Scientific) targeting the HEp-2 cell actin filaments [20,21,47]. The adherence was qualitatively and quantitatively evaluated as described for the RSE adherence assay.…”

“…Colony lysates, prepared from each bacterial strain cultured on Luria Bertani agar plates, were each tested in triplicate to confirm the PATS profiles as described previously [49 -52]. Briefly, primer pairs targeting the 8 polymorphic XbaI-, 7 polymorphic AvrII-restriction enzyme sites, and the four virulence genes encoding Shiga toxin 1 and 2 (Stx1 and Stx2), Intimin-γ (eae), and hemolysin-A (hlyA) were used to generate amplicons from the colony lysates in a hot start, touchdown PCR reaction [47,49,50,52,53]. PCR reactions amplifying the AvrII-restriction enzyme site were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA), and digested with the AvrII restriction enzyme (New England Biolabs, Beverly, MA) to confirm the presence of the restriction site.…”