Shiga Toxin-encoding Bacteriophage Research Articles

Influence of cheese making process on STEC bacteriophage release.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens implicated in diseases including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). The main virulence factor are Shiga toxins; their production and secretion are by-products of the expression of late genes of prophages upon sub-lethal environmental stimuli exposure. Hence, the lysogenic prophage after a stress switch to lytic cycle spreading the Stx phages. In the present study, 35 STEC were screened for the presence and the ability to release Shiga toxin-encoding bacteriophages. Three bacterial strains showed signals of prophage presence both in plate and in PCR. Subsequently, these bacterial strains were subjected to stressors that simulate cheese manufacturing conditions: NaCl (1, 1.5 and 2% w/v), lactic acid (0.5, 1.5 and 3% v/v), anaerobic growth, pasteurization (72°C for 15 s), UV irradiation. The ability to release prophage was evaluated by Real Time qPCR. Induction of the prophages showed that the addition of NaCl at 1.5 and 2% significantly increased viral release compared to control. Conversely, the addition of lactic acid had a significant repressive effect. The other applied stressors had no significant effect in phage release according to the experimental conditions adopted.

Persistence of a Stx-Encoding Bacteriophage in Minced Meat Investigated by Application of an Improved DNA Extraction Method and Digital Droplet PCR

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens with Shiga toxins as the main virulence factor. Shiga toxins are encoded on Shiga toxin-encoding bacteriophages (Stx phages). Stx phages may exist as free bacteriophages in the environment or in foods or as prophages integrated into the host genome. From a food safety perspective, it is important to have knowledge on the survival and persistence of Stx phages in food products since these may integrate into the bacterial hosts through transduction if conditions are right. Here, we present the results from a study investigating the survival of a Stx phage in minced meat from beef stored at a suboptimal temperature (8°C) for food storage along with modifications and optimizations of the methods applied. Minced meat from beef was inoculated with known levels of a labeled Stx phage prior to storage. Phage filtrates were used for plaque assays and DNA extraction, followed by real-time PCR and digital droplet PCR (ddPCR). The results from the pilot study suggested that the initial DNA extraction protocol was not optimal, and several modifications were tested before a final protocol was defined. The final DNA extraction protocol comprised ultra-centrifugation of the entire phage filtrate for concentrating phages and two times phenol–chloroform extraction. The protocol was used for two spiking experiments. The DNA extraction protocol resulted in flexibility in the amount of DNA available for use in PCR analyses, ultimately increasing the sensitivity of the method used for quantification of phages in a sample. All three quantification methods employed (i.e., plaque assays, real-time PCR, and ddPCR) showed similar trends in the development of the phages during storage, where ddPCR has the benefit of giving absolute quantification of DNA copies in a simple experimental setup. The results indicate that the Stx phages persist and remain infective for at least 20 days under the storage conditions used in the present study. Stx phages in foods might represent a potential risk for humans. Although it can be speculated that transduction may take place at 8°C with subsequent forming of STEC, it can be expected to be a rare event. However, such an event may possibly take place under more optimal conditions, such as an increase in storage temperature of foods or in the gastrointestinal tract of humans.

Structural annotation of the conserved carbohydrate esterase vb_24B_21 from Shiga toxin-encoding bacteriophage Φ24B

Shiga toxin-encoding bacteriophages transfer Shiga toxin genes to Escherichia coli and are responsible for the emergence of pathogenic bacterial strains that cause severe foodborne human diseases. Gene vb_24B_21 is the most highly conserved gene across sequenced Shiga bacteriophages. Protein vb_24B_21 (also termed 933Wp42 and NanS-p) is a carbohydrate esterase with homology to the E. coli chromosomally encoded NanS that deacetylates sialic acid in the intestinal mucus. To assist the functional characterization of vb_24B_21, we have studied its molecular structure by homology modelling its esterase domain and by elucidating the crystal structure of its uncharacterized C-terminal domain at the atomic resolution of 0.97 Å. Our modelling confirms that NanS from the E. coli host is the closest structurally characterized homolog to the esterase domain of vb_24B_21. Like NanS, vb_24B_21 has an atypical active site, comprising a simple catalytic dyad Ser-His and a divergent oxyanion hole. The crystal structure of the C-terminal domain reveals a lectin-like, jelly-roll β-sandwich fold. The domain displays a prominent cleft that bioinformatics analysis predicts to be a carbohydrate binding site without catalytic properties. In summary, our study indicates that vb_24B_21 is a NanS-like atypical esterase that is assisted by a carbohydrate-binding module of yet undetermined binding specificity.

Assessing the genomic relatedness and evolutionary rates of persistent verotoxigenic Escherichia coli serotypes within a closed beef herd in Canada.

Verotoxigenic Escherichia coli (VTEC) are food- and water-borne pathogens associated with both sporadic illness and outbreaks of enteric disease. While it is known that cattle are reservoirs of VTEC, little is known about the genomic variation of VTEC in cattle, and whether the variation in genomes reported for human outbreak strains is consistent with individual animal or group/herd sources of infection. A previous study of VTEC prevalence identified serotypes carried persistently by three consecutive cohorts of heifers within a closed herd of cattle. This present study aimed to: (i) determine whether the genomic relatedness of bovine isolates is similar to that reported for human strains associated with single source outbreaks, (ii) estimate the rates of genome change among dominant serotypes over time within a cattle herd, and (iii) identify genomic features of serotypes associated with persistence in cattle. Illumina MiSeq genome sequencing and genotyping based on allelic and single nucleotide variations were completed, while genome change over time was measured using Bayesian evolutionary analysis sampling trees. The accessory genome, including the non-protein-encoding intergenic regions (IGRs), virulence factors, antimicrobial-resistance genes and plasmid gene content of representative persistent and sporadic cattle strains were compared using Fisher’s exact test corrected for multiple comparisons. Herd strains from serotypes O6:H34 (n=22), O22:H8 (n=30), O108:H8 (n=39), O139:H19 (n=44) and O157:H7 (n=106) were readily distinguishable from epidemiologically unrelated strains of the same serotype using a similarity threshold of 10 or fewer allele differences between adjacent nodes. Temporal-cohort clustering within each serotype was supported by date randomization analysis. Substitutions per site per year were consistent with previously reported values for E. coli ; however, there was low branch support for these values. Acquisition of the phage-encoded Shiga toxin 2 gene in serotype O22:H8 was observed. Pan-genome analyses identified accessory regions that were more prevalent in persistent serotypes (P≤0.05) than in sporadic serotypes. These results suggest that VTEC serotypes from a specific cattle population are highly clonal with a similar level of relatedness as human single-source outbreak-associated strains, but changes in the genome occur gradually over time. Additionally, elements in the accessory genomes may provide a selective advantage for persistence of VTEC within cattle herds.

Comparison of Shiga toxin-encoding bacteriophages in highly pathogenic strains of Shiga toxin-producing Escherichia coli O157:H7 in the UK.

Over the last 35 years in the UK, the burden of Shiga toxin-producing Escherichia coli (STEC) O157:H7 infection has, during different periods of time, been associated with five different sub-lineages (1983–1995, Ia, I/IIa and I/IIb; 1996–2014, Ic; and 2015–2018, IIb). The acquisition of a stx2a-encoding bacteriophage by these five sub-lineages appears to have coincided with their respective emergences. The Oxford Nanopore Technologies (ONT) system was used to sequence, characterize and compare the stx-encoding prophages harboured by each sub-lineage to investigate the integration of this key virulence factor. The stx2a-encoding prophages from each of the lineages causing clinical disease in the UK were all different, including the two UK sub-lineages (Ia and I/IIa) circulating concurrently and causing severe disease in the early 1980s. Comparisons between the stx2a-encoding prophage in sub-lineages I/IIb and IIb revealed similarity to the prophage commonly found to encode stx2c, and the same site of bacteriophage integration (sbcB) as stx2c-encoding prophage. These data suggest independent acquisition of previously unobserved stx2a-encoding phage is more likely to have contributed to the emergence of STEC O157:H7 sub-lineages in the UK than intra-UK lineage to lineage phage transmission. In contrast, the stx2c-encoding prophage showed a high level of similarity across lineages and time, consistent with the model of stx2c being present in the common ancestor to extant STEC O157:H7 and maintained by vertical inheritance in the majority of the population. Studying the nature of the stx-encoding bacteriophage contributes to our understanding of the emergence of highly pathogenic strains of STEC O157:H7.

Contribution and Interaction of Shiga Toxin Genes to Escherichia coli O157:H7 Virulence.

Escherichia coli O157:H7 is the predominant cause of diarrhea-associated hemolytic uremic syndrome (HUS) worldwide. Its cardinal virulence traits are Shiga toxins, which are encoded by stx genes, the most common of which are stx1a, stx2a, and stx2c. The toxins these genes encode differ in their in vitro and experimental phenotypes, but the human population-level impact of these differences is poorly understood. Using Shiga toxin-encoding bacteriophage insertion typing and real-time polymerase chain reaction, we genotyped isolates from 936 E. coli O157:H7 cases and verified HUS status via chart review. We compared the HUS risk between isolates with stx2a and those with stx2a and another gene and estimated additive interaction of the stx genes. Adjusted for age and symptoms, the HUS incidence of E. coli O157:H7 containing stx2a alone was 4.4% greater (95% confidence interval (CI) −0.3%, 9.1%) than when it occurred with stx1a. When stx1a and stx2a occur together, the risk of HUS was 27.1% lower (95% CI −87.8%, −2.3%) than would be expected if interaction were not present. At the population level, temporal or geographic shifts toward these genotypes should be monitored, and stx genotype may be an important consideration in clinically predicting HUS among E. coli O157:H7 cases.

Cheating, facilitation and cooperation regulate the effectiveness of phage-encoded exotoxins as antipredator molecules.

Temperate phage encoded Shiga toxin (Stx) kills the bacterivorous predator, Tetrahymena thermophila, providing Stx+ Escherichia coli with a survival advantage over Stx− cells. Although bacterial death accompanies Stx release, since bacteria grow clonally the fitness benefits of predator killing accrue to the kin of the sacrificed organism, meaning Stx‐mediated protist killing is a form of self‐destructive cooperation. We show here that the fitness benefits of Stx production are not restricted to the kin of the phage‐encoding bacteria. Instead, nearby “free loading” bacteria, irrespective of their genotype, also reap the benefit of Stx‐mediated predator killing. This finding indicates that the phage‐borne Stx exotoxin behaves as a public good. Stx is encoded by a mobile phage. We find that Stx‐encoding phage can use susceptible bacteria in the population as surrogates to enhance toxin and phage production. Moreover, our findings also demonstrate that engulfment and concentration of Stx‐encoding and susceptible Stx− bacteria in the Tetrahymena phagosome enhances the transfer of Stx‐encoding temperate phage from the host to the susceptible bacteria. This transfer increases the population of cooperating bacteria within the community. Since these bacteria now encode Stx, the predation‐stimulated increase in phage transfer increases the population of toxin encoding bacteria in the environment.

Erratum for Quirós et al., “Improving Detection of Shiga Toxin-Producing Escherichia coli by Molecular Methods by Reducing the Interference of Free Shiga Toxin-Encoding Bacteriophages”

Erratum for Quirós et al., “Improving Detection of Shiga Toxin-Producing Escherichia coli by Molecular Methods by Reducing the Interference of Free Shiga Toxin-Encoding Bacteriophages”

Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic E. coli Infection Using Food-Borne Inoculation.

Upon colonization of the intestinal epithelium, the attaching and effacing (AE) pathogen Enterohemorrhagic Escherichia coli (EHEC) effaces microvilli and forms pedestal-like structures beneath the adherent bacterium. The production of one of its virulence factors, the phage-encoded Shiga toxin (Stx) results in systemic disease, including the development of renal failure. Although EHEC does not productively infect conventional mice, EHEC infection can be modeled in mice utilizing a derivative of the natural murine AE pathogen Citrobacter rodentium (CR). Gavage of mice with CR(ΦStx2dact), a C. rodentium lysogenized by a phage encoding an Stx variant with high potency in mice, features AE lesion formation on intestinal epithelium and Stx-mediated systemic disease, including renal damage. This model is somewhat limited by mouse-to-mouse variation in the course of disease, with the time to severe morbidity (and required euthanasia) varying by as many as 5 days, a feature that limits pathological analysis at defined stages of disease. In the current study, we altered and optimized the preparation, dose, and mode of delivery of CR(ΦStx2dact), using food-borne route of infection to generate highly synchronous disease model. We found that food-borne inoculation of as few as 3 × 104 CR(ΦStx2dact) resulted in productive colonization and severe systemic disease. Upon inoculation of 1 × 108 bacteria, the majority of infected animals suffered weight loss beginning 5 days post-infection and all required euthanasia on day 6 or 7. This enhanced murine model for EHEC infection should facilitate characterization of the pathology associated with specific phases of Stx-mediated disease.

Protozoan Predation of Escherichia coli O157:H7 Is Unaffected by the Carriage of Shiga Toxin-Encoding Bacteriophages.

Escherichia coli O157:H7 is a food-borne bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans. While cattle are a known source of E. coli O157:H7 exposure resulting in human infection, environmental reservoirs may also be important sources of infection for both cattle and humans. Bacteriophage-encoded Shiga toxins (Stx) carried by E. coli O157:H7 may provide a selective advantage for survival of these bacteria in the environment, possibly through their toxic effects on grazing protozoa. To determine Stx effects on protozoan grazing, we co-cultured Paramecium caudatum, a common ciliate protozoon in cattle water sources, with multiple strains of Shiga-toxigenic E. coli O157:H7 and non-Shiga toxigenic cattle commensal E. coli. Over three days at ambient laboratory temperature, P. caudatum consistently reduced both E. coli O157:H7 and non-Shiga toxigenic E. coli populations by 1–3 log cfu. Furthermore, a wild-type strain of Shiga-toxigenic E. coli O157:H7 (EDL933) and isogenic mutants lacking the A subunit of Stx 2a, the entire Stx 2a-encoding bacteriophage, and/or the entire Stx 1-encoding bacteriophage were grazed with similar efficacy by both P. caudatum and Tetrahymena pyriformis (another ciliate protozoon). Therefore, our data provided no evidence of a protective effect of either Stx or the products of other bacteriophage genes on protozoan predation of E. coli. Further research is necessary to determine if the grazing activity of naturally-occurring protozoa in cattle water troughs can serve to decrease cattle exposure to E. coli O157:H7 and other Shiga-toxigenic E. coli.

Highly Virulent Non-O157 Enterohemorrhagic Escherichia coli (EHEC) Serotypes Reflect Similar Phylogenetic Lineages, Providing New Insights into the Evolution of EHEC.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene (stx), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the “big five”), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx-negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC.

BaeSR, involved in envelope stress response, protects against lysogenic conversion by Shiga toxin 2-encoding phages.

Infection and lysogenic conversion with Shiga toxin-encoding bacteriophages (Stx phages) drive the emergence of new Shiga toxin-producing Escherichia coli strains. Phage attachment to the bacterial surface is the first stage of phage infection. Envelope perturbation causes activation of envelope stress responses in bacterial cells. Although many external factors are known to activate envelope stress responses, the role of these responses in the phage-bacterium interaction remains unexplored. Here, we investigate the link between three envelope signaling systems in E. coli (RcsBC, CpxAR, and BaeSR) and Stx2 phage infection by determining the success of bacterial lysogenic conversion. For this purpose, E. coli DH5α wild-type (WT) and mutant strains lacking RcsBC, CpxAR, or BaeSR signaling systems were incubated with a recombinant Stx2 phage (933W). Notably, the number of lysogens obtained with the BaeSR mutant was 5 log10 units higher than with the WT, and the same differences were observed when using 7 different Stx2 phages. To assess whether the membrane receptor used by Stx phages, BamA, was involved in the differences observed, bamA gene expression was monitored by reverse transcription-quantitative PCR (RT-qPCR) in all host strains. A 4-fold-higher bamA expression level was observed in the BaeSR mutant than in the WT strain, suggesting that differential expression of the receptor used by Stx phages accounted for the increase in the number of lysogenization events. Establishing the link between the role of stress responses and phage infection has important implications for understanding the factors affecting lysogenic conversion, which drives the emergence of new pathogenic clones.

Genetic diversity of Shiga toxin-producing Escherichia coli O157 : H7 recovered from human and food sources.

The aim of this study was to identify an epidemiological association between Shiga toxin-producing Escherichia coli O157 : H7 strains associated with human infection and with food sources. Frequency distributions of different genetic markers of E. coli O157 : H7 strains recovered from human and food sources were compared using molecular assays to identify E. coli O157 : H7 genotypes associated with variation in pathogenic potential and host specificity. Genotypic characterization included: lineage-specific polymorphism assay (LSPA-6), clade typing, tir (A255T) polymorphism, Shiga toxin-encoding bacteriophage insertion site analysis and variant analysis of Shiga toxin 2 gene (stx2a and stx2c) and antiterminator Q genes (Q933 and Q21). The intermediate lineage (LI/II) dominated among both food and human strains. Compared to other clades, clades 7 and 8 were more frequent among food and human strains, respectively. The tir (255T) polymorphism occurred more frequently among human strains than food strains. Q21 and Q933 + Q21 were found at significantly higher frequencies among food and human strains, respectively. Moreover, stx2a and stx2a+c were detected at significantly higher frequencies among human strains compared to food strains. Bivariate analysis revealed significant concordance (P<0.05) between the LSPA-6 assay and the other typing methods. Multivariable regression analysis suggested that tir (255T) was the most distinctive genotype that can be used to detect bacterial clones with potential risk for human illness from food sources. This study supported previous reports of the existence of diversity in genetic markers among different isolation sources by including E. coli O157 : H7 strains from both food and human sources. This might enable tracking genotypes with potential risk for human illness from food sources.

Persistence of Infectious Shiga Toxin-Encoding Bacteriophages after Disinfection Treatments

In Shiga toxin-producing Escherichia coli (STEC), induction of Shiga toxin-encoding bacteriophages (Stx phages) causes the release of free phages that can later be found in the environment. The ability of Stx phages to survive different inactivation conditions determines their prevalence in the environment, the risk of stx transduction, and the generation of new STEC strains. We evaluated the infectivity and genomes of two Stx phages (Φ534 and Φ557) under different conditions. Infectious Stx phages were stable at 4, 22, and 37°C and at pH 7 and 9 after 1 month of storage but were completely inactivated at pH 3. Infective Stx phages decreased moderately when treated with UV (2.2-log10 reduction for an estimated UV dose of 178.2 mJ/cm(2)) or after treatment at 60 and 68°C for 60 min (2.2- and 2.5-log10 reductions, respectively) and were highly inactivated (3 log10) by 10 ppm of chlorine in 1 min. Assays in a mesocosm showed lower inactivation of all microorganisms in winter than in summer. The number of Stx phage genomes did not decrease significantly in most cases, and STEC inactivation was higher than phage inactivation under all conditions. Moreover, Stx phages retained the ability to lysogenize E. coli after some of the treatments.

Emerging infectious colitis

The present review will highlight recent advances in the knowledge of emerging pathogens causing infectious colitis and provide a description of the most important food-borne outbreaks. Outbreaks of enteric disease caused by Salmonella spp., Campylobacter spp., and Shiga toxin-producing Escherichia coli (STEC) continue to surprise with new epidemiological findings or changing virulence characteristics. These pathogens evolve to exploit novel opportunities for spread and transmission, such as fresh produce within the food chain, and generate new public health challenges. Organic sprouts were recently considered as the source responsible for a large German disease outbreak comprising 3842 patients. The outbreak strain was identified as an enteroaggregative STEC O104:H4 (EAggC), a rare hybrid pathogen that harbours the phage encoded Shiga toxin gene and antibiotic resistance in an EAggEC background. Clostridium difficile PCR ribotype 078 is emerging across Europe, causing severe disease outside healthcare facilities as well as disease in farm animals, indicating that the species border has been crossed. Although the global impact of cryptosporidiosis is less pronounced, these organisms have been responsible for large outbreaks of infectious diarrhoea, often not reported. Invasive listeriosis is a serious food-borne illness and was found recently in 28 US states affecting 147 patients, associated with eating contaminated cantaloupe. Outbreaks of gastroenteritis caused by Listeria monocytogenes are most likely severely underestimated. Centralized surveillance of food-borne enteropathogens is essential for the early detection of disease outbreaks and for the organization of an immediate and appropriate response. An improved understanding of the pathogenesis, pathology and epidemiology of emerging enteropathogens causing infectious colitis will provide new approaches for disease prevention and control.

The agricultural antibiotic carbadox induces phage-mediated gene transfer in Salmonella

Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the US during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli (STEC) and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness genes in the environment.

Potentially Pathogenic Escherichia coli Can Form a Biofilm under Conditions Relevant to the Food Production Chain

The biofilm-producing abilities of potentially human-pathogenic serotypes of Escherichia coli from the ovine reservoir were studied at different temperatures and on different surfaces. A possible influence of the hydrophobicity of the bacterial cells, as well as the presence of two virulence factors, the Shiga toxin-encoding (Stx) bacteriophage and the eae gene, was also studied. A total of 99 E. coli isolates of serotypes O26:H11, O103:H2, and O103:H25 isolated from sheep feces were included. The results show that isolates of all three E. coli serotypes investigated can produce biofilm on stainless steel, glass, and polystyrene at 12, 20, and 37°C. There was a good general correlation between the results obtained on the different surfaces. E. coli O103:H2 isolates produced much more biofilm than those of the other two serotypes at all three temperatures. In addition, isolates of serotype O26:H11 produced more biofilm than those of O103:H25 at 37°C. The hydrophobicity of the isolates varied between serotypes and was also influenced by temperature. The results strongly indicated that hydrophobicity influenced the attachment of the bacteria rather than their ability to form biofilm once attached. Isolates with the eae gene produced less biofilm at 37°C than isolates without this gene. The presence of a Stx bacteriophage did not influence biofilm production. In conclusion, our results show that potentially human-pathogenic E. coli from the ovine reservoir can form biofilm on various surfaces and at several temperatures relevant for food production and handling.

A prospective case–control and molecular epidemiological study of human cases of Shiga toxin-producing Escherichia coli in New Zealand

BackgroundShiga toxin-producing Escherichia coli (STEC) O157:H7 and related non-O157 STEC strains are enteric pathogens of public health concern worldwide, causing life-threatening diseases. Cattle are considered the principal hosts and have been shown to be a source of infection for both foodborne and environmental outbreaks in humans. The aims of this study were to investigate risk factors associated with sporadic STEC infections in humans in New Zealand and to provide epidemiological information about the source and exposure pathways.MethodsDuring a national prospective case–control study from July 2011 to July 2012, any confirmed case of STEC infection notified to regional public health units, together with a random selection of controls intended to be representative of the national demography, were interviewed for risk factor evaluation. Isolates from each case were genotyped using pulsed-field gel electrophoresis (PFGE) and Shiga toxin-encoding bacteriophage insertion (SBI) typing.ResultsQuestionnaire data from 113 eligible cases and 506 controls were analysed using multivariate logistic regression. Statistically significant animal and environmental risk factors for human STEC infections were identified, notably 'Cattle livestock present in meshblock’ (the smallest geographical unit) (odds ratio 1.89, 95% CI 1.04–3.42), 'Contact with animal manure’ (OR 2.09, 95% CI 1.12–3.90), and 'Contact with recreational waters’ (OR 2.95, 95% CI 1.30–6.70). No food-associated risk factors were identified as sources of STEC infection. E. coli O157:H7 caused 100/113 (88.5%) of clinical STEC infections in this study, and 97/100 isolates were available for molecular analysis. PFGE profiles of isolates revealed three distinctive clusters of genotypes, and these were strongly correlated with SBI type. The variable 'Island of residence’ (North or South Island of New Zealand) was significantly associated with PFGE genotype (p = 0.012).ConclusionsOur findings implicate environmental and animal contact, but not food, as significant exposure pathways for sporadic STEC infections in humans in New Zealand. Risk factors associated with beef and dairy cattle suggest that ruminants are the most important sources of STEC infection. Notably, outbreaks of STEC infections are rare in New Zealand and this further suggests that food is not a significant exposure pathway.

The Trojan Horse of the microbiological arms race: phage‐encoded toxins as a defence against eukaryotic predators

Phage-encoded Shiga toxin (Stx) acts as a bacterial defence against the eukaryotic predator Tetrahymena. To function as an effective bacterial anti-predator defence, Stx must kill a broad spectrum of predators. Consistent with that assertion, we show here that bacterially encoded Stx efficiently kills the bacteriovore Acanthamoeba castellanii in co-culture. We also show that, in addition to Stx, the phage-encoded exotoxin, diphtheria toxin (Dtx) expressed by Corynebacterium diphtheriae also can function as part of an anti-predator strategy; it kills Acanthamoeba in co-culture. Interestingly, only exotoxins produced by bacteria internalized by the Acanthamoeba predator are cytolethal; the presence of purified Dtx or Stx in culture medium has no effect on predator viability. This finding is consistent with our results indicating that intoxication of Acanthamoeba by these exotoxins does not require a receptor. Thus bacteria, in the disguise of a food source, function as a 'Trojan Horse', carrying genes encoding an exotoxin into target organisms. This 'Trojan Horse' mechanism of exotoxin delivery into predator cells allows intoxication of predators that lack a cell surface receptor for the particular toxin, allowing bacteria-bearing exotoxins to kill a broader spectrum of predators, increasing the fitness of the otherwise 'defenceless' prey bacteria.

Lessons Learned From Outbreaks of Shiga Toxin Producing Escherichia coli.

In 2011, a large outbreak caused by a Shiga toxin producing E. coli (STEC) occurred in Northern Germany, with a satellite outbreak in Western France, including the highest number of hemolytic uremic syndrome (HUS) cases ever encountered during a STEC outbreak. The outbreak strain was characterized as an enteroaggregative E. coli of serotype O104:H4 expressing a phage-encoded Shiga toxin 2. The majority of STEC infections and HUS cases were observed in adults, with a preponderance of the female gender. The outbreak imposed huge challenges on clinicians, microbiologists, and epidemiologists but also provided important new insight for the understanding of STEC infection. Thus, novel therapeutic strategies in the treatment of HUS in adults and for decolonization of long-term STEC carriers were evaluated. This review highlights the unusual features of the recent O104:H4 outbreak and focuses on emerging new strategies in diagnostics and treatment of acute STEC-related disease, as well as STEC long-term carriage.