Effectiveness of bacteriophages AKFV33 (Tequintavirus, T5) and AHP24 (Rogunavirus, T1), wV7 (Tequatrovirus, T4), and AHP24S (Vequintavirus, rV5), as well as 11 cocktails of combinations of the four phages, were evaluatedin vitrofor biocontrol of six common phage types ofEscherichia coliO157 (human and bovine origins) at different multiplicities of infection (MOIs; 0.01–1,000), temperatures (37 or 22°C), and exposure times (10–22 h). Phage efficacy against O157 was highest at MOI 1,000 (P< 0.001) and after 14-18 h of exposure at 22°C (P< 0.001). The activity of individual phages against O157 did not predict the activity of a cocktail of these phages even at the same temperature and MOI. Combinations of phages were neutral (no better or worse than the most effective constituent phages acting alone), displayed facilitation (greater efficacy than the most effective constituent phages acting alone), or antagonistic (lower efficacy than the most effective constituent phages acting alone). Across MOIs, temperatures, exposure time, and O157 strains, a cocktail of T1, T4, and rV5 was most effective (P< 0.05) against O157, although T1 and rV5 were less effective (P< 0.001) than other individual phages. T5 was the most effective individual phages (P< 0.05), but was antagonistic to other phages, particularly rV5 and T4 + rV5. Interactions among phages were influenced by phage genera and phage combination, O157 strains, MOIs, incubation temperatures, and times. Based on this study, future development of phage cocktails should, as a minimum, include confirmation of a lack of antagonism among constituent phages and preferably confirmation of facilitation or synergistic effects.

Over a two-year period, Mannheimia haemolytica (MH; n = 113), Pasteurella multocida (PM; n = 47), Histophilus somni (HS; n = 41) and Mycoplasma bovis (MB; n = 227) were isolated from bovine lung tissue at necropsy from cattle raised conventionally (CON, n = 29 feedlots) or without antimicrobials [natural (NAT), n = 2 feedlots]. Excluding MB, isolates were assayed by PCR to detect the presence of 13 antimicrobial resistance (AMR) genes and five core genes associated with integrative and conjugative elements (ICEs). Antimicrobial susceptibility phenotypes and minimum inhibitory concentrations (MICs, µg/mL) were determined for a subset of isolates (MH, n = 104; PM, n = 45; HS, n = 23; and MB, n = 61) using Sensititre analyses. A subset of isolates (n = 21) was also evaluated by whole-genome sequencing (WGS) based on variation in AMR phenotype. All five ICE core genes were detected in PM and HS by PCR, but only 3/5 were present in MH. Presence of mco and tnpA ICE core genes in MH was associated with higher MICs (p < 0.05) for all tetracyclines, and 2/3 of all macrolides, aminoglycosides and fluoroquinolones evaluated. In contrast, association of ICE core genes with MICs was largely restricted to macrolides for PM and to individual tetracyclines and macrolides for HS. For MH, the average number of AMR genes markedly increased (p < 0.05) in year 2 of the study due to the emergence of a strain that was PCR positive for all 13 PCR-tested AMR genes as well as two additional AMR genes (aadA31 and bla_ROB-1) detected by WGS. Conventional management of cattle increased (p < 0.05) MICs of tilmicosin and tulathromycin for MH; neomycin and spectinomycin for PM; and gamithromycin and tulathromycin for MB. The average number of PCR-detected AMR genes in PM was also increased (p < 0.05) in CON mortalities. This study demonstrates increased AMR especially to macrolides by bovine respiratory disease organisms in CON as compared to NAT feedlots and a rapid increase in AMR following dissemination of strain(s) carrying ICE-associated multidrug resistance.

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.

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.

Rapid and accurate diagnosis of bovine respiratory disease (BRD) presents a substantial challenge to the North American cattle industry. Here we utilize recombinase polymerase amplification (RPA), a fast and sensitive isothermal DNA-based technology for the detection of four BRD pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis), genes coding antimicrobial resistance (AMR) and integrative conjugative elements (ICE) which can harbor AMR genes. Eleven RPA assays were designed and validated including: a) one conventional species-specific multiplex assay targeting the 4 BRD pathogens, b) two species-specific real-time multiplex RPA assays targeting M. haemolytica/M. bovis and P. multocida/H. somni, respectively with a novel competitive internal amplification control, c) seven conventional assays targeting AMR genes (tetH, tetR, msrE, mphE, sul2, floR, erm42), and d) one real-time assay targeting ICE. Each real-time RPA assay was tested on 100 deep nasopharyngeal swabs (DNPS) collected from feedlot cattle previously assessed for targets using either culture methods and/or polymerase chain reaction (PCR) verification (TC-PCR). The developed RPA assays enabled sensitive and accurate identification of BRD agents and AMR/ICE genes directly from DNPS, in a shorter period than TC-PCR, showing considerable promise as a tool for point-of-care identification of BRD pathogens and antimicrobial resistance genes.

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 ehaA^β 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.