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

Background: Differences between Escherichia coli O157 (O157) strains are well-established with some of these strains being associated with major outbreaks in the US. EDL933 is one such O157 strain that caused a multistate outbreak in 1982 and has since been used as a prototype in various O157-related experiments. Objective: As O157 can readily acquire genetic mutations, we sought to determine if the genetic and phenotypic profiles of EDL933 strains from different sources would be consistent. Methods: We evaluated wild-type O157 strains stocked as EDL933 from three different laboratories, in the strain typing Polymorphic Amplified Typing Sequence (PATS) and the bovine rectal-anal junction squamous epithelial (RSE) cell- and HEp-2 cell- adherence assays. In addition, we also verified if Shiga toxins (Stx), the Locus of Enterocyte Effacement (LEE) or curli fimbriae contributed to the adherence phenotypes observed using mutant and wild-type EDL933 isolates. Results: Our results showed differences in PATS profiles and RSE cell-adherence phenotype, with no influence from the Stx or LEE genes, between EDL933 from different sources. Interestingly, the EDL933 strain that demonstrated the most contrasting diffuse adherence phenotype on RSE cells, EDL933-T, had decreased curli production that may have contributed to this phenotype. Conclusion: Our observations suggest that a comprehensive characterization of bacterial isolates, even if assigned to the same strain type prior to use in experiments, is warranted to ensure consistency and reproducibility of results.

Manipulating interactions between T4 phage long tail fibers and Escherichia coli receptors

Bacteriophages are the most abundant and diverse biological entities on Earth. Phages exhibit strict host specificity that is largely conferred by adsorption. However, the mechanism underlying this phage–host specificity remains poorly understood. In this study, we examined the interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the long tail fibers of bacteriophage T4. T4 phage uses OmpC of the K12 strain, but not of the O157 strain, for adsorption, even though OmpC from the two E. coli strains share 94% homology. We identified amino acids P177 and F182 in Loop 4 of the K12 OmpC as essential for T4 phage adsorption in the copresence of Loop 1 and Loop 5. Analyses of phage mutants capable of adsorbing to OmpC mutants demonstrated that amino acids at positions 937 and 942 of the gp37 protein, which is present in the digital tip (DT) region of the T4 long tail fibers, play an important role in adsorption. Furthermore, we created a T4 phage mutant library with artificial modifications in the DT region and isolated and characterized multiple phage mutants capable of adsorbing to OmpC of the O157 strain or lipopolysaccharide of the K12 strain. These results shed light on the mechanism underlying the phage–host specificity mediated by gp37 and OmpC and may be useful in the development of phage therapy via artificial modifications of the DT region of T4 phage. IMPORTANCE Understanding the host specificity of phages will lead to the development of phage therapy. The interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the gp37 protein composing the digital tip (DT) region of the long tail fibers of bacteriophage T4 largely determines its host specificity. Here, we elucidated the amino acid residues important for the interaction between gp37 and OmpC. This result suggests that the shapes of both proteins at the binding interface play important roles in their interactions, which is likely mediated by multiple residues of both binding partners. Additionally, we successfully isolated multiple phage mutants capable of adsorbing to a variety of E. coli receptors using a mutant T4 phage library with artificial modifications in the DT region, providing a foundation for the alteration of the host specificity.

Mechanisms involved in the adaptation of Escherichia coli O157:H7 to the host intestinal microenvironment

Host adaptation of pathogens may increase intra- and interspecies transmission. We showed previously that the passage of a clinically isolated enterohemorrhagic Escherichia coli (EHEC) O157 strain (125/99) through the gastrointestinal tract of mice increases its pathogenicity in the same host. In this work, we aimed to elucidate the underlying mechanism(s) involved in the patho-adaptation of the stool-recovered (125RR) strain. We assessed the global transcription profile by microarray and found almost 100 differentially expressed genes in 125RR strain compared with 125/99 strain. We detected an overexpression of Type Three Secretion System (TTSS) proteins at the mRNA and protein levels and demonstrated increased adhesion to epithelial cell lines for the 125RR strain. Additional key attributes of the 125RR strain were: increased motility on semisolid agar, which correlated with an increased fliC mRNA level; reduced Stx2 production at the mRNA and protein levels; increased survival at pH 2.5, as determined by acid resistance assays. We tested whether the overexpression of the LEE-encoded regulator (ler) in trans in the 125/99 strain could recreate the increased pathogenicity observed in the 125RR strain. As anticipated ler overexpression led to increased expression of TTSS proteins and bacterial adhesion to epithelial cells in vitro but also increased mortality and intestinal colonization in vivo. We conclude that this host-adaptation process required changes in several mechanisms that improved EHEC O157 fitness in the new host. The research highlights some of the bacterial mechanisms required for horizontal transmission of these zoonotic pathogens between their animal and human populations.

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

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.

Integrated veterinary-medical activity on characterization of shigatoxin producing Escherichia coli

Background Shiga toxin-producing Escherichia coli (STEC) are an important public health concern as they cause severe conditions (bloody diarrhea and hemolytic uremic syndrome - HUS) and have epidemic potential. Cattle are the main reservoir for the highly virulent STEC O157 strain, and other HUS-associated non-O157 strains. This study aimed to characterize strains from animals and humans and to compare their molecular profiles. Animal isolates were tested for the presence of virulence factors correlated to their pathogenic potential. Methods 74 STEC human isolates from 43 clinical cases (sporadic or epidemic) and 270 animal isolates from feces from 62 cattle farms were collected. Epidemiological investigation to collect environmental and suspected food samples was carried out for 22 cases. All isolates were typed with PFGE and their serotype was defined by Real-Time PCR. Animal isolates were also tested for the presence of subAB, saa, tia, cfk, adfO, hlyA, efaI1-lifA5’3’, and toxB virulence genes. Results O157 was detected in 44% of human cases, other relevant serogroups O26, O111, O103, O145 in 21, 9, 5 and 5% of cases, respectively. The source of infection was identified in one case (cheese contaminated by a O157 strain). Among animal isolates 2, 0.7, 0.7 and 0.3% were identified as O157, O11, O113, and O145, respectively. PFGE highlighted a high heterogeneicity among animal strains, however no pulsotype common to cattle and clinical isolates was found. adfO, cfk, efaI1-lifA5’3’ and toxB were found significantly correlated to eae (intimin). Conclusions Human and cattle strains were not correlated, however STEC diversity in cattle was very high and included some strains potentially pathogenic to humans. For this reason, upholding an integrated surveillance is very important. Key messages Serotypes relevant to human health were found in cattle in a small but not negligible frequence. No direct correlation was found between animal and clinical isolates.

Characterization and Antimicrobial-Resistance Profile of Escherichia coli O157 and O157: H7 Isolated from Modified Atmosphere Packaged Meat Samples

Shiga-like toxin producing Escherichia coli is still an important public issue which causes extremely dangerous health problems. This study was planned in order to examine the inhibitory effect of Modified Atmosphere Packaging application on E. coli O157 and O157: H7. The purposes of the present study were to detect E. coli O157 and O157: H7 strains from ground and cubed beef. A total of 100 MAP cattle meat products (50 minced meat, 50 meat cubes) were collected from the markets and butchers in Samsun province between May and October 2013. According to results, 1(1/50-2%) E. coli O157 and 1(1/50-2%) E. coli O157: H7 strains isolated from 50 ground beef samples, while 1 (1/50-2%) E. coli O157 strain was identified from 50 cubed beef samples. It was determined that E. coli O157 isolate obtained from the MAP ground beef carried stx1, stx2 genes; E. coli O157: H7 isolate carried stx1, stx2, eaeA and hylA genes while E. coli O157 isolate obtained from the MAP cubed meat only carried the stx2 gene. In antibiogram test, both E. coli O157 isolates were resistant to streptomycin and one E. coli O157: H7 isolate was resistant to streptomycin, cephalothin and tetracycline. As a consequence; in order to protect public health, products should be kept in proper hygienic and technical conditions during sale and storage and use of uncontrolled antibiotics should be avoided.

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

ABSTRACT One of the first identified and best-studied toxin-antitoxin (TA) systems in Escherichia coli is the F-plasmid-based CcdAB system. This system is involved in plasmid maintenance through postsegregational killing. More recently, ccdAB homologs have been found on the chromosome, including in pathogenic strains of E. coli and other bacteria. However, the functional role of chromosomal ccdAB genes, if any, has remained unclear. We show that both the native ccd operon of the E. coli O157 strain (ccd O157) and the ccd operon from the F plasmid (ccd F), when inserted on the E. coli chromosome, lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters, with the O157 operon showing higher protection. While the plasmid-encoded CcdB toxin is a potent gyrase inhibitor and leads to bacterial cell death even under fully repressed conditions, the chromosomally encoded toxin leads to growth inhibition, except at high expression levels, where some cell death is seen. This was further confirmed by transiently activating the chromosomal ccd operon through overexpression of an active-site inactive mutant of F-plasmid-encoded CcdB. Both the ccd F and ccd O157 operons may share common mechanisms for activation under stress conditions, eventually leading to multidrug-tolerant persister cells. This study clearly demonstrates an important role for chromosomal ccd systems in bacterial persistence. IMPORTANCE A large number of free-living and pathogenic bacteria are known to harbor multiple toxin-antitoxin systems, on plasmids as well as on chromosomes. The F-plasmid CcdAB system has been extensively studied and is known to be involved in plasmid maintenance. However, little is known about the function of its chromosomal counterpart, found in several pathogenic E. coli strains. We show that the native chromosomal ccd operon of the E. coli O157 strain is involved in drug tolerance and confers protection from cell death under multiple antibiotic stress conditions. This has implications for generation of potential therapeutics that target these TA systems and has clinical significance because the presence of persisters in an antibiotic-treated population can lead to resuscitation of chronic infection and may contribute to failure of antibiotic treatment.

High-Quality Genome Sequence of an Escherichia coli O157 Strain Carrying an mcr-1 Resistance Gene Isolated from a Patient in the United States

ABSTRACT Enterobacteriaceae carrying plasmid-mediated colistin resistance have been found around the world. We report here the high-quality whole-genome sequence of an Escherichia coli O157:H48 isolate (2016C-3936C1) from Connecticut that carried the mcr-1 resistance gene on an IncX4-type plasmid.

Curli Temper Adherence of Escherichia coli O157:H7 to Squamous Epithelial Cells from the Bovine Recto-Anal Junction in a Strain-Dependent Manner

ABSTRACT Our recent studies have shown that intimin and the locus of enterocyte effacement-encoded proteins do not play a role in Escherichia coli O157:H7 (O157) adherence to the bovine recto-anal junction squamous epithelial (RSE) cells. To define factors that play a contributory role, we investigated the role of curli, fimbrial adhesins commonly implicated in adherence to various fomites and plant and human epithelial cells, in O157 adherence to RSE cells. Specifically, we examined (i) wild-type strains of O157; (ii) curli variants of O157 strains; (iii) isogenic curli deletion mutants of O157; and (iv) adherence inhibition of O157 using anti-curlin sera. Results of these experiments conducted under stringent conditions suggest that curli do not solely contribute to O157 adherence to RSE cells and in fact demonstrate a modulating effect on O157 adherence to RSE cells in contrast to HEp-2 cells (human epidermoid carcinoma of the larynx cells with HeLa contamination). The absence of curli and presence of blocking anti-curli antibodies enhanced O157-RSE cell interactions among some strains, thus alluding to a spatial, tempering effect of curli on O157 adherence to RSE cells when present. At the same time, the presence or absence of curli did not alter RSE cell adherence patterns of another O157 strain. These observations are at variance with the reported role of curli in O157 adherence to human cell lines such as HEp-2 and need to be factored in when developing anti-adherence modalities for preharvest control of O157 in cattle. IMPORTANCE This study demonstrated that O157 strains interact with epithelial cells in a host-specific manner. The fimbriae/adhesins that are significant for adherence to human cell lines may not have a role or may have a modulating role in O157 adherence to bovine cells. Targeting such adhesins may not prevent O157 attachment to bovine cells but instead may result in improved adherence. Hence, conducting host-specific evaluations is critical when selecting targets for O157 control strategies.

Transcriptomic Analysis of Shiga-Toxigenic Bacteriophage Carriage Reveals a Profound Regulatory Effect on Acid Resistance in Escherichia coli

ABSTRACTShiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on theEscherichia colihost. Here we use next-generation sequencing to compare the transcriptomes ofE. colistrains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on theE. colihost. Known phage regulators were overexpressed inE. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of anE. coliO157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage andE. coliacid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology ofE. coli.