Forming biofilm is a strategy utilized by Shiga toxin-producing Escherichia coli (STEC) to survive and persist in food processing environments. We investigated the biofilm-forming potential of STEC strains from 10 clinically important serogroups on stainless steel at 22 °C or 13 °C after 24, 48, and 72 h of incubation. Results from crystal violet staining, plate counts, and scanning electron microscopy (SEM) identified a single isolate from each of the O113, O145, O91, O157, and O121 serogroups that was capable of forming strong or moderate biofilms on stainless steel at 22 °C. However, the biofilm-forming strength of these five strains was reduced when incubation time progressed. Moreover, we found that these strains formed a dense pellicle at the air-liquid interface on stainless steel, which suggests that oxygen was conducive to biofilm formation. At 13 °C, biofilm formation by these strains decreased (P < 0.05), but gradually increased over time. Overall, STEC biofilm formation was most prominent at 22 °C up to 24 h. The findings in this study identify the environmental conditions that may promote STEC biofilm formation in food processing facilities and suggest that the ability of specific strains to form biofilms contributes to their persistence within these environments.

Escherichia coli are commensal bacteria in the gastrointestinal tract of mammals, but some strains have acquired Shiga-toxins and can cause enterohemorrhagic diarrhoea and kidney failure in humans. Shiga-toxigenic E. coli (STEC) strains such as E. coli O157:H7 and some non-O157 strains also contain other virulence traits, some of which contribute to their ability to form biofilms. This study characterized non-O157 E. coli from South African cattle faecal samples for their virulence potential, antimicrobial resistance (AMR), biofilm-forming ability, and genetic relatedness using culture-based methods, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing (WGS). Of 80 isolates screened, 77.5% (62/80) possessed Shiga-toxins genes. Of 18 antimicrobials tested, phenotypic resistance was detected against seven antimicrobials. Resistance ranged from 1.3% (1/80) for ampicillin-sulbactam to 20% (16/80) for tetracycline. Antimicrobial resistance genes were infrequently detected except for tetA, which was found in 31.3% (25/80) and tetB detected in 11.3% (9/80) of isolates. Eight biofilm-forming associated genes were detected in STEC isolates (n = 62) and two non-STEC strains. Prevalence of biofilm genes ranged from 31.3% (20/64) for ehaAβ passenger to 100% for curli structural subunit (csgA) and curli regulators (csgA and crl). Of the 64 STEC and multi-drug resistant isolates, 70.3% (45/64) and 37.5% (24/64) formed strong biofilms on polystyrene at 22 and 37 °C, respectively. Of 59 isolates screened by PFGE, 37 showed unique patterns and the remaining isolates were grouped into five clusters with a ≥90% relatedness. In silico serotyping following WGS on a subset of 24 non-O157 STEC isolates predicted 20 serotypes comprising three novel serotypes, indicating their diversity as potential pathogens. These findings show that North West South African cattle harbour genetically diverse, virulent, antimicrobial-resistant and biofilm-forming non-O157 E. coli. Biofilm-forming ability may increase the likelihood of persistence of these pathogens in the environment and facilitate their dissemination, increasing the risk of cross contamination or establishment of infections in hosts.

This study was done to examine the rhizobacteria from field grown Genetically Modified (GM)

Non-O157 Shiga toxin-producing Escherichia coli (STEC) E. coli are emerging pathotypes that are frequently associated with diseases in humans around the world. The consequences of these serogroups for public health is a concern given the lack of effective prevention and treatment measures. In this study, ten bacteriophages (phages; SA20RB, SA79RD, SA126VB, SA30RD, SA32RD, SA35RD, SA21RB, SA80RD, SA12KD and SA91KD) isolated from cattle faeces collected in the North-West of South Africa were characterized. Activity of these phages against non-O157 STEC isolates served as hosts for these phages. All of the phages except SA80RD displayed lytic against non-O157 E. coli isolates. Of 22 non-O157 E. coli isolates, 14 were sensitive to 9 of the 10 phages tested. Phage SA35RD was able to lyse 13 isolates representing a diverse group of non-O157 E. coli serotypes including a novel O-antigen Shiga toxigenic (wzx-Onovel5:H19) strain. However, non-O157 E. coli serotypes O76:H34, O99:H9, O129:H23 and O136:H30 were insensitive to all phages. Based on transmission electron microscopy, the non-O157 STEC phages were placed into Myoviridae (n = 5) and Siphoviridae (n = 5). Genome of the phage ranged from 44 to 184.3 kb. All but three phages (SA91KD, SA80RD and SA126VB) were insensitive to EcoRI-HF and HindIII nucleases. This is the first study illustrating that cattle from North-West South Africa harbour phages with lytic potentials that could potentially be exploited for biocontrol against a diverse group of non-O157 STEC isolated from the same region.

This study examined the biofilm-forming ability of six non-O157 Shiga-toxin-producing Escherichia coli (STEC) strains: O116:H21, wzx-Onovel5:H19, O129:H21, O129:H23, O26:H11, and O154:H10 on stainless steel coupons after 24, 48, and 72 h of incubation at 22 °C and after 168 h at 10 °C. The results of crystal violet staining revealed that strains O129:H23 and O154:H10 were able to form biofilms on both the submerged surface and the air–liquid interface of coupons, whereas strains O116:H21, wzx-Onovel5:H19, O129:H21, and O26:H11 formed biofilm only at the air–liquid interface. Viable cell counts and scanning electron microscopy showed that biofilm formation increased (p < 0.05) over time. The biofilm-forming ability of non-O157 STEC was strongest (p < 0.05) at 22 °C after 48 h of incubation. The strongest biofilm former regardless of temperature was O129:H23. Generally, at 10 °C, weak to no biofilm was observed for isolates O154:H10, O116:H21, wzx-Onovel5:H19, O26:H11, and O129:H21 after 168 h. This study found that temperature affected the biofilm-forming ability of non-O157 STEC strains. Overall, our data indicate a high potential for biofilm formation by the isolates at 22 °C, suggesting that non-O157 STEC strains could colonize stainless steel within food-processing facilities. This could serve as a potential source of adulteration and promote the dissemination of these potential pathogens in food.

Shiga toxin (stx) is the principal virulence factor of the foodborne pathogen, Shiga toxin-producing Escherichia coli (STEC) O157:H7 and is associated with various lambdoid bacterio (phages). A comparative genomic analysis was performed on STEC O157 isolates from cattle (n = 125) and clinical (n = 127) samples to characterize virulence genes, stx-phage insertion sites and antimicrobial resistance genes that may segregate strains circulating in the same geographic region. In silico analyses revealed that O157 isolates harboured the toxin subtypes stx1a and stx2a. Most cattle (76.0%) and clinical (76.4%) isolates carried the virulence gene combination of stx1, stx2, eae and hlyA. Characterization of stx1 and stx2-carrying phages in assembled contigs revealed that they were associated with mlrA and wrbA insertion sites, respectively. In cattle isolates, mlrA and wrbA insertion sites were occupied more often (77% and 79% isolates respectively) than in clinical isolates (38% and 1.6% isolates, respectively). Profiling of antimicrobial resistance genes (ARGs) in the assembled contigs revealed that 8.8% of cattle (11/125) and 8.7% of clinical (11/127) isolates harboured ARGs. Eight antimicrobial resistance genes cassettes (ARCs) were identified in 14 isolates (cattle, n = 8 and clinical, n = 6) with streptomycin (aadA1, aadA2, ant(3’’)-Ia and aph(3’’)-Ib) being the most prevalent gene in ARCs. The profound disparity between the cattle and clinical strains in occupancy of the wrbA locus suggests that this trait may serve to differentiate cattle from human clinical STEC O157:H7. These findings are important for stx screening and stx-phage insertion site genotyping as well as monitoring ARGs in isolates from cattle and clinical samples.

Bacteriophages, natural killers of bacteria, and plant secondary metabolites, such as condensed tannins, are potential agents for the control of foodborne pathogens. The first objective of this study evaluated the efficacy of a bacteriophage SA21RB in reducing pre-formed biofilms on stainless-steel produced by two Shiga toxin-producing Escherichia coli (STEC) strains, one from South Africa and the other from Canada. The second objective examined the anti-bacterial and anti-biofilm activity of condensed tannin (CT) from purple prairie clover and phlorotannins (PT) from brown seaweed against these strains. For 24-h-old biofilms, (O113:H21; 6.2 log10 colony-forming units per square centimeter (CFU/cm2) and O154:H10; 5.4 log10 CFU/cm2), 3 h of exposure to phage (1013 plaque-forming units per milliliter (PFU/mL)) reduced (p ≤ 0.05) the number of viable cells attached to stainless-steel coupons by 2.5 and 2.1 log10 CFU/cm2 for O113:H21 and O154:H10, respectively. However, as biofilms matured, the ability of phage to control biofilm formation declined. In biofilms formed for 72 h (O113:H21; 5.4 log10 CFU/cm2 and O154:H10; 7 log10 CFU/cm2), reductions after the same duration of phage treatment were only 0.9 and 1.3 log10 CFU/cm2 for O113:H21 and O154:H10, respectively. Initial screening of CT and PT for anti-bacterial activity by a microplate assay indicated that both STEC strains were less sensitive (p ≤ 0.05) to CT than PT over a concentration range of 25–400 µg/mL. Based on the lower activity of CT (25–400 µg/mL), they were not further examined. Accordingly, PT (50 µg/mL) inhibited (p ≤ 0.05) biofilm formation for up to 24 h of incubation at 22 °C, but this inhibition progressively declined over 72 h for both O154:H10 and O113:H21. Scanning electron microscopy revealed that both SA21RB and PT eliminated 24 h biofilms, but that both strains were able to adhere and form biofilms on stainless-steel coupons at longer incubation times. These findings revealed that phage SA21RB is more effective at disrupting 24 than 72 h biofilms and that PT were able to inhibit biofilm formation of both E. coli O154:H10 and O113:H21 for up to 24 h.

Foodborne illness is exacerbated by novel and emerging pathotypes, persistent contamination, antimicrobial resistance, an ever-changing environment, and the complexity of food production systems. Sporadic and outbreak events of common foodborne pathogens like Shiga toxigenic E. coli (STEC), Salmonella, Campylobacter, and Listeria monocytogenes are increasingly identified. Methods of controlling human infections linked with food products are essential to improve food safety and public health and to avoid economic losses associated with contaminated food product recalls and litigations. Bacteriophages (phages) are an attractive additional weapon in the ongoing search for preventative measures to improve food safety and public health. However, like all other antimicrobial interventions that are being employed in food production systems, phages are not a panacea to all food safety challenges. Therefore, while phage-based biocontrol can be promising in combating foodborne pathogens, their antibacterial spectrum is generally narrower than most antibiotics. The emergence of phage-insensitive single-cell variants and the formulation of effective cocktails are some of the challenges faced by phage-based biocontrol methods. This review examines phage-based applications at critical control points in food production systems with an emphasis on when and where they can be successfully applied at production and processing levels. Shortcomings associated with phage-based control measures are outlined together with strategies that can be applied to improve phage utility for current and future applications in food safety.