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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–producing Escherichia coli (STEC) are a leading cause of foodborne illnesses worldwide, with beef and beef products as a common food reservoir. STEC strains may be present in beef-proce...

A previously isolated a bacteriophage, vB_EcoS_AKFV33 of T5virus, demonstrated great potential in biocontrol of Shiga toxigenic Escherichia coli (STEC) O157. This study further evaluated its potential as a biocontrol agent in broth culture against other important non-O157 serogroups of STEC and Salmonella. AKFV33 was capable of lysing isolates of STEC serogroups O26 (n = 1), O145 (n = 1) and Salmonella enterica serovars (n = 6). In a broth culture microplate system, efficacy of AKFV33 for killing STEC O26:H11, O145:NM and Salmonella was improved (P Salmonella in mixtures with enhanced activity (P < 0.05) against O157:H7 and O26:H11, offering great promise for control of multiple zoonotic pathogens at both pre and post-harvest.

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant bacteria, novel antibacterial agents, are urgently needed. A bacteriophage-based biocontrol strategy is the therapeutic use of phages to control bacterial pathogens in agricultural settings. Phage biocontrol is increasingly accepted as a sustainable technology, effective at decontaminating foodborne pathogens. To ensure effective biocontrol outcomes, systematic screening of phage combinations against targeted bacteria under required environmental conditions is crucial. Antibacterial efficacy of phage cocktails may be affected by phage genera and combination, targeted bacterial strains, the multiplicity of infection, temperature, and time. To formulate a phage cocktail with superior efficacy, the proposed method was to systematically evaluate the effectiveness of individual phages and phage cocktails in killing foodborne bacterial pathogens under targeted conditions. Bacterial killing efficacy was monitored by measuring optical density at desired temperatures and durations. Superior phage efficacy was determined by complete inhibition of bacterial growth. The proposed method is a robust, evidence-based approach to facilitate formulating phage cocktails with superior antibacterial efficacy.

Grazing Swan geese (Anser cygnoides) have good meat quality but grow slowly. This study aimed to study whether supplemental feeding could improve growth performance of grazing Swan geese and investigate a suitable dietary metabolizable energy (ME) level of supplemental diet for grazing Swan geese. Naturalized healthy male Swan geese (n = 144, 42 ± 2.0 days and 1.21 ± 0.17 kg) were randomly allocated into 4 groups and grazed on pasture alone (control, CON) or offered supplemental diets with ME of 9.5, 11.5, or 13.5 MJ/kg of DM after grazing. Growth performance and body-size measurements (including bone development) were lower (p &lt, 0.05) in CON versus supplemented geese, as well as slaughter measurements on days 28 and 56. The DM intake linearly decreased (p &lt, 0.01) with increasing dietary ME from day 29 to 56. Slaughter, semi-eviscerated, eviscerated, and thigh muscle yield linearly (p &lt, 0.01) decreased with increasing dietary ME on day 56. Lightness (L*) and yellowness (b*) for breast and thigh muscle on days 28 and 56, and breast muscle shear force on day 56, were lower (p &lt, 0.01) in supplemented versus CON geese. In conclusion, supplemental feeding improved growth performance and carcass characteristics of grazing Swan geese, and supplemental feed with ME of 9.5 MJ/kg of DM could be offered to improve growth and meat quality of grazing Swan geese.

Multidrug-resistant (MDR; resistance to ≥3 antimicrobial classes) members of the Pasteurellaceae family may compromise the efficacy of therapies used to prevent and treat bovine respiratory disease (BRD) in feedlot cattle. This study examined the prevalence of multidrug resistance in strains of Mannheimia haemolytica and Pasteurella multocida collected from BRD cattle mortalities in North America. Isolates of M. haemolytica (n = 147) and P. multocida (n = 70) spanning 69 Alberta feedlots from 2011 to 2016 and two United States feedlots from 2011 to 2012 were examined for antimicrobial resistance (AMR) in association with integrative and conjugative elements (ICEs). Overall, resistance was high in both bacterial species with an increase in the prevalence of MDR isolates between 2011 and 2016. Resistance to >7 antimicrobial drugs occurred in 31% of M. haemolytica and 83% of P. multocida isolates. Resistance to sulfadimethoxine, trimethoprim/sulfamethoxazole, neomycin, clindamycin oxytetracycline, spectinomycin, tylosin, tilmicosin, and tulathromycin was most common. Although >80% of strains harbored three or more ICE-associated genes, only 12% of M. haemolytica and 77% of P. multocida contained all six, reflecting the diversity of ICEs. There was evidence of clonal spread as P. multocida and M. haemolytica isolates with the same pulsed-field gel electrophoresis profile from the United States in 2011 were isolated in Alberta in 2015–2016. This work highlights that MDR strains of Pasteurellaceae containing ICEs are widespread and may be contributing to BRD therapy failure in feedlot cattle. Given the antimicrobial resistance gene profiles identified, these MDR isolates may be selected for by the use of macrolides, tetracyclines, and/or in-feed supplements containing heavy metals.

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 evaluated in vitro for biocontrol of six common phage types of Escherichia coli O157 (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.

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 &le, 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 &le, 0.05) to CT than PT over a concentration range of 25&ndash, 400 &micro, g/mL. Based on the lower activity of CT (25&ndash, g/mL), they were not further examined. Accordingly, PT (50 &micro, g/mL) inhibited (p &le, 0.05) biofilm formation for up to 24 h of incubation at 22 &deg, 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.

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.

Shiga toxigenic Escherichia coli (STEC) can form biofilms and frequently cause serious foodborne illnesses. A strain of STEC O145:H25 (EC19990166) known to be a strong biofilm former was used to evaluate the efficacy of bacteriophage AZO145A against biofilms formed on stainless steel (SS) coupons. Exposure of STEC O145:H25 to phage AZO145A (1010 PFU/mL) for 2 h resulted in a 4.0 log10 reduction (P

Shiga toxin-producing

Methicillin-resistant Staphylococcus aureus (MRSA) has become a worrisome superbug, due to its wide distribution and multidrug resistance. To characterize effects of a newly identified plantaricin GZ1-27 on MRSA, transcriptomic and proteomic profiling of MRSA strain ATCC43300 was performed in response to sub-MIC (16 μg/mL) plantaricin GZ1-27 stress. In total, 1090 differentially expressed genes (padj 0.05) and 418 differentially expressed proteins (fold change 1.2, p 0.05) were identified. Centralized protein expression clusters were predicted in biological functions (biofilm formation, DNA replication and repair, and heat-shock) and metabolic pathways (purine metabolism, amino acid metabolism, and biosynthesis of secondary metabolites). Moreover, a capacity of inhibition MRSA biofilm formation and killing biofilm cells were verified using crystal violet staining, scanning electron microscopy, and confocal laser-scanning microscopy. These findings yielded comprehensive new data regarding responses induced by plantaricin and could inform evidence-based methods to mitigate MRSA biofilm formation.

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&beta, 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 &deg, C, respectively. Of 59 isolates screened by PFGE, 37 showed unique patterns and the remaining isolates were grouped into five clusters with a &ge, 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.

The objectives of this study were to characterize the phenotype and genotype of 36 non-O157 Shiga toxin-producing Escherichia coli (STEC) strains isolated from humans, ovines, or bovines, including the top 6 (O26, O45, O103, O111, O121, and O145) and three other serogroups implicated in serious illness (O91, O113, and O128). Biofilms were formed by all strains with intermediate to strong biofilm producers (n = 24) more common at 22°C than at 37°C (p 0.001) and 48 and 72 h (p 0.001) than 24 h of incubation time. Biofilm-forming potential differed by serogroup and origin with O113 and human strains exhibiting the highest potential (p 0.001). Biofilm-associated genes, csgA/csgD/crl/fimH (100%), flu (94%), rpoS (92%), ehaA(α) (89%), and cah (72%), were most prevalent, while mlrA (22%) and ehaA(β) (14%) were least prevalent, although there was no clear compliment of genes associated with strains exhibiting the greatest biofilm-forming capacity. Among 12 virulence genes screened, iha and ehxA were present in 92% of the strains. The occurrence of stx1 in the top 6 serogroups (8/12, 67%) did not differ (p = 0.8) from other serogroups (17/24, 71%), but stx2 was less likely (confidence interval [CI] = 0.14-1.12; p = 0.04) to be in the former (9/24, 38%) than the latter (9/12, 75%). Excluding serogroups, O91 and O121, at least one strain per serogroup was resistant to between three and six antimicrobials. Streptomycin (31%), sulfisoxazole (31%), and tetracycline (25%) resistance was most common and was 35-50% less likely (p 0.05) in human than animal strains. All non-O157 STEC strains were able to form biofilms on an abiotic surface, with some exhibiting resistance to multiple antimicrobials. Potential as a reservoir of antimicrobial resistance genes may be another hazard of biofilms in food-processing plants. As a result, future strategies to control these pathogens may include measures to prevent biofilms.

Bacteriophages (phages) have been used for biocontrol of Escherichia coli O157 and other pathogenic bacteria in many different matrices and foods, but few studies have included inactivation of residual phages in culture medium before plating and enumeration of surviving host bacteria for the assessment of phage efficacy. This oversight may lead to overestimation of phage efficacy. The ability of virucidal solution containing a mixture of ferrous sulfate [iron(II) sulfate, FeSO4] and tea extract [Fe(II)T] to inactivate residual T5-like, T1-like, T4-like, and rV5-like phages was assessed using E. coli O157 as the host. At concentrations of ≥10 mM FeSO4, all phages were not detected after 20 min in a broth culture model. Compared with the virucidal solution-free samples (1 to 96% recovery), Fe(II)T (10 mM FeSO4 plus 15% tea extract) recovered a greater (P < 0.01) number of E. coli O157 from phage-treated broth culture (97 to 100% recovery) and beef samples (52 to 100% recovery). Moreover, with the addition of Fe(II)T, the number of bacteria surviving after exposure to T5-like or T4-like phages was greater (P < 0.01) than that after exposure to T1-like or rV5-like phages. Consequently, use of a virucide for phage inactivation is recommended to improve the accuracy of evaluations of phage efficacy for biocontrol of E. coli O157.

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, &micro, g/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 &lt, 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 &lt, 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 blaROB-1) detected by WGS. Conventional management of cattle increased (p &lt, 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 &lt, 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.

The purpose of this study was to determine the effectiveness of a commercial Salmonella bacteriophage mixture (SalmoFresh™ 6-phage strains) and to compare its effectiveness with a chlorinated water treatment to reduce Salmonella on produce and seeds at different temperatures and storage times. Two sets of experiments were designed to test phage and chlorinated water effectiveness on produce at 2, 10 and 25 °C at different storage times (1, 24, 48 and 72 h). First, SalmoFresh™ was applied to the surface of lettuce, mung bean sprouts and mung bean seeds that were spot-inoculated with a five Salmonella strain mixture (Newport, Braenderup, Typhimurium, Kentucky, and Heidelberg, 105 CFU/mL) by spraying phages onto lettuce (n = 48 pieces, 3×3 cm2 per treatment) and sprouts (n = 48 pieces per treatment). A second set of experiments (scaled-up) consisted in the application of phages by immersion to Salmonella adulterated lettuce (600 g), 300 g sprouts (300 g) or mung bean seeds (30 g) in a phage cocktail (108 PFU/mL) for 15 min (lettuce and sprouts) or 1 h (seeds). Another group of samples was washed with chlorinated water and yet another group was treated with a combination of chlorinated water followed by phage cocktail. Each experiment was repeated three times by quadruplicates. After the treatments for spot-inoculated and scaled-up experiments, lettuce and sprouts were separated into different lots (10 g/lot) and stored at 2, 10 and 25 °C; Salmonella was enumerated after 1, 24, 48 and 72 h. Adulterated phage-treated seeds were packaged and stored dry at 25 °C. Salmonella was enumerated after 72 h of storage. Groups of phage treated mung bean seeds (720 g) were germinated, and the reduction in Salmonella determined. Results of microplate virulence assays indicated that SalmoFresh™ reduced (P = 0.007) Salmonella by an average of 5.34 logs CFU/mL after 5 h at 25 °C. Spraying SalmoFresh™ onto lettuce and sprouts reduced Salmonella by 0.76 and 0.83 log10 CFU/g, respectively (P

Purple prairie clover (PPC; Dalea purpurea Vent.) containing 84.5 g/kg dry matter (DM) of condensed tannin (CT) was ensiled without (control) or with polyethylene glycol (PEG) for 76 days, followed by 14 days of aerobic exposure. Changes in fermentation characteristics were determined, and the composition of bacterial and fungal communities were assessed using metagenomic sequencing. The addition of polyethylene glycol (PEG) that deactivated CT at ensiling increased ( P < 0.05 to ∼0.001) soluble N, nonprotein N, lactic acid, total volatile fatty acids, ammonia N, deoxynivalenol (DON), and ochratoxin A (OTA) but decreased ( P < 0.001) pH and water-soluble carbohydrates. The concentrations of DON and OTA increased ( P < 0.001) for both silages, with the extent of increase being greater for control than for PEG-treated silage during aerobic exposure. The PEG-treated silage exhibited higher ( P < 0.01 to ∼0.001) copy numbers of total bacteria, Lactobacillus , yeasts, and fungi than the control. The addition of PEG decreased ( P < 0.01) bacterial diversity during both ensiling and aerobic exposure, whereas it increased ( P < 0.05) fungal diversity during aerobic exposure. The addition of PEG at ensiling increased ( P < 0.05) the abundances of Lactobacillus and Pediococcus species but decreased ( P < 0.01) the abundances of Lactococcus and Leuconostoc species. Filamentous fungi were found in the microbiome at ensiling and after aerobic exposure, whereas Bacillus spp. were the dominate bacteria after aerobic exposure. In conclusion, CT decreased protein degradation and improved the aerobic stability of silage. These desirable outcomes likely reflect the ability of PPC CT to inhibit those microorganisms involved in lowering silage quality and in the production of mycotoxins. IMPORTANCE The present study reports the effects of condensed tannins on the complex microbial communities involved in ensiling and aerobic exposure of purple prairie clover. This study documents the ability of condensed tannins to lower mycotoxin production and the associated microbiome. Taxonomic bacterial community profiles were dominated by Lactobacillales after fermentation, with a notable increase in Bacillus spp. as a result of aerobic exposure. It is interesting to observe that condensed tannins decreased bacterial diversity during both ensiling and aerobic exposure but increased fungal diversity during aerobic exposure only. The present study indicates that the effects of condensed tannins on microbial communities lead to reduced lactic acid and total volatile fatty acid production, proteolysis, and mycotoxin concentration in the terminal silage and improved aerobic stability. Condensed tannins could be used as an additive to control unfavorable microbial development and maybe enhanced feed safety.

Efficacy of four bacteriophages (phages) and a cocktail for biocontrol of Escherichia coli O157 was assessed on beef samples stored at 4, 22 and 37 °C. Samples (3 × 3 × 1 cm) were contaminated with E. coli O157 (10(4) CFU/cm(2)) and treated with single phages: T5-like (T5), T1-like (T1), T4-like (T4) and O1-like (O1), or a cocktail at two titers: multiplicity of infection (MOI) = 1000 and MOI = 10. In contrast to previous studies, use of virucidal solution prevented over-estimation of phage efficacy. Irrespective of temperature and MOIs, T5 was most (P 0.001) and O1 least (P 0.05) effective for biocontrol of E. coli O157, with relative efficacy of other phages temperature dependent. At 4 °C, T1 (P 0.05) and cocktail (P 0.001) were more effective than T4. In contrast, T4 was equally (P = 0.08, at 37 °C) or less effective (P = 0.003, at 22 °C) than T5. Phages were more effective (P 0.001) against E. coli O157 at warmer temperatures and high MOI. As the beef supply chain includes hours of storage or transport at temperatures near 4 °C, this study demonstrates phages could significantly reduce E. coli O157 during this period.

Bovine respiratory disease (BRD) is a significant and costly illness in feedlot cattle. Metagenomic analysis was performed on bronchoalveolar lavage samples obtained from 18 feedlot cattle that died of BRD.

Bacteriophages are associated with reduced fecal shedding of Shiga-toxin-producing Escherichia coli O157:H7 (STEC O157:H7) in cattle. Four phages exhibiting activity against 12 of 14 STEC O157:H7 strains, representing 11 common phage types, were isolated. Phages did not lyse non-O157 E. coli, with 11 of the 12 STEC strains exhibiting extreme susceptibility (average multiplicity of infection (MOI) = 0.0003−0.0007). All phages had icosahedral heads with tapered, noncontractile tails, a morphology indicative of T1-like Siphoviridae. Genome size of all phages was ∼44 kb, but EcoRІ or HindIII digestion profiles differed among phages. Based on restriction enzyme digestion profiles, phages AHP24, AHS24, and AHP42 were more related (66.7%−82.4%) to each other than to AKS96, while AHP24 and AHS24, isolated from the same feedlot pen, exhibited the highest identity (88.9%−92.3%). Phages AHP24 and AHS24 exhibited the broadest host range and strongest lytic activity against STEC O157:H7, making them strong candidates for biocontrol of this bacterium in cattle.

Bacteriophages are natural predators of bacteria and may mitigate Escherichia coli O157:H7 in cattle and their environment. As bacteriophages targeted to E. coli O157:H7 (phages) lose activity at low pH, protection from gastric acidity may enhance efficacy of orally administered phages. Polymer encapsulation of four phages, wV8, rV5, wV7, and wV11, and exposure to pH 3.0 for 20 min resulted in an average 13.6% recovery of phages after release from encapsulation at pH 7.2. In contrast, untreated phages under similar conditions had a complete loss of activity. Steers (n = 24) received 1011 CFU of naladixic acid–resistant E. coli O157:H7 on day 0 and were housed in six pens of four steers. Two pens were control (naladixic acid–resistant E. coli O157:H7 only), and the remaining pens received polymer-encapsulated phages (Ephage) on days −1, 1, 3, 6, and 8. Two pens received Ephage orally in gelatin capsules (bolus; 1010 PFU per steer per day), and the remaining two pens received Ephage top-dressed on their fee...

Aims: To evaluate host range and lytic capability of four bacteriophages (rV5, wV7, wV8 and wV11) against Escherichia coli O157:H7 (STEC O157:H7) from cattle and humans. Methods and Results: Four hundred and twenty-two STEC O157:H7 isolates (297 bovine; 125 human) were obtained in Alberta, Canada. The four phages were serially diluted and incubated for 5 h with overnight cultures of STEC O157:H7 to estimate their multiplicity of infection (MOI). All bovine STEC O157:H7 were subjected to pulsed-field gel electrophoresis (PFGE) and phage typing (PT). Phage wV7 lysed all human and bovine isolates irrespective of PFGE genotype or PT phenotype and exhibited the lowest MOI (0·004–0·006, P

This study was conducted to compare fecal grab (FEC) and rectoanal mucosal swab (RAMS) techniques as sampling methods for surveillance of Escherichia coli O157:H7 in conjunction with administration of a mitigation therapy. The study was nested within a larger experiment that investigated bacteriophage as a preharvest strategy for controlling E. coli O157:H7 in feedlot steers. Samples (FEC and RAMS) were collected from 16 of the 32 feedlot steers (control and oral bacteriophage treatment; n = 8) involved in the mitigation study. All steers had been inoculated on day 0 with 10(10) CFU of nalidixic acid-resistant E. coli O157:H7, and samples were collected on 16 occasions over the next 83 days. FEC samples were assessed by direct plating of serial dilutions in PBS, plus a 6-h enrichment and immunomagnetic separation when E. coli O157:H7 concentrations were below limits detectable by direct plating (i.e.

Previously, bacteriophage vB_EcoM_AYO145A, which lyses Shiga toxin-producing Escherichia coli O145:NM, was classified as an O1-like virus of the Myoviridae family. Here, we report the complete genome sequence of this phage and a comparative genomic analysis with other known O1-like phages.

This study aimed to isolate and characterize bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces. Of 37 non-O157 STEC-infecting phages isolated, those targeting O26 (AXO26A, AYO26A, AYO26B), O103 (AXO103A, AYO103A), O111 (AXO111A, AYO111A), O121 (AXO121A, AXO121B), and O145 (AYO145A, AYO145B) were further characterized. Transmission electron microscopy showed that the 11 isolates belonged to 3 families and 6 genera: the families Myoviridae (types rV5, T4, ViI, O1), Siphoviridae (type T5), and Podoviridae (type T7). Genome size of the phages as determined by pulsed-field gel electrophoresis ranged from 38 to 177 kb. Excluding phages AXO26A, AYO103A, AYO145A, and AYO145B, all other phages were capable of lysing more than 1 clinically important strain from serogroups of O26, O91, O103, O111, O113, O121, and O128, but none exhibited infectivity across all serogroups. Moreover, phages AYO26A, AXO121A, and AXO121B were also able to lyse 4 common phage types of STEC O157:H7. Our findings show that a diversity of non-O157 STEC-infecting phages are harbored in bovine feces. Phages AYO26A, AYO26B, AXO103A, AXO111A, AYO111A, AXO121A, and AXO121B exhibited a broad host range against a number of serogroups of STEC and have potential for the biocontrol of STEC in the environment.

The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink both in vitro and in vivo. Five Pseudomonas aeruginosa (P. aeruginosa) strains were isolated from lungs of mink with suspected hemorrhagic pneumonia and their identity was confirmed by morphological observation and 16S rDNA sequence analysis. Compared to P. aeruginosa strains isolated from mink with hemorrhagic pneumonia in 2002, these isolates were more resistant to antibiotics selected. A lytic phage vB_PaeP_PPA-ABTNL (PPA-ABTNL) of the Podoviridae family was isolated from hospital sewage using a P. aeruginosa isolate as host, showing broad host range against P. aeruginosa. A one-step growth curve analysis of PPA-ABTNL revealed eclipse and latent periods of 20 and 35 min, respectively, with a burst size of about 110 PFU per infected cell. Phage PPA-ABTNL significantly reduced the growth of P. aeruginosa isolates in vitro. The genome of PPA-ABTNL was 43,227 bp (62.4% G+C) containing 54 open reading frames and lacked regions encoding known virulence factors, integration-related proteins and antibiotic resistance determinants. Genome architecture analysis showed that PPA-ABTNL belonged to the "phiKMV-like Viruses" group. A repeated dose inhalational toxicity study using PPA-ABTNL crude preparation was conducted in mice and no significantly abnormal histological changes, morbidity or mortality were observed. There was no indication of any potential risk associated with using PPA-ABTNL as a therapeutic agent. The results of a curative treatment experiment demonstrated that atomization by ultrasonic treatment could efficiently deliver phage to the lungs of mink and a dose of 10 multiplicity of infection was optimal for treating mink hemorrhagic pneumonia. Our work demonstrated the potential for phage to fight P. aeruginosa involved in mink lung infections when administered by means of ultrasonic nebulization.

The objectives of this study were to identify endemic bacteriophages (phages) in the feedlot environment and determine relationships of these phages to Escherichia coli O157:H7 from cattle shedding high and low numbers of naturally occurring E. coli O157:H7. Angus crossbred steers were purchased from a southern Alberta (Canada) feedlot where cattle excreting ≥10 4 CFU · g −1 of E. coli O157:H7 in feces at a single time point were identified as supershedders (SS; n = 6), and cattle excreting 4 CFU · g −1 of feces were identified as low shedders (LS; n = 5). Fecal pats or fecal grabs were collected daily from individual cattle for 5 weeks. E. coli O157:H7 in feces was detected by immunomagnetic separation and enumerated by direct plating, and phages were isolated using short- and overnight-enrichment methods. The total prevalence of E. coli O157:H7 isolated from feces was 14.4% and did not differ between LS and SS ( P = 0.972). The total prevalence of phages was higher in the LS group (20.9%) than in the SS group (8.3%; P = 0.01). Based on genome size estimated by pulsed-field gel electrophoresis and morphology determined by transmission electron microscopy, T4- and O1-like phages of Myoviridae and T1-like phage of Siphoviridae were isolated. Compared to T1- and O1-like phages, T4-like phages exhibited a broad host range and strong lytic capability when targeting E. coli O157:H7. Moreover, the T4-like phages were more frequently isolated from feces of LS than SS, suggesting that endemic phages may impact the shedding dynamics of E. coli O157:H7 in cattle.

Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol ofE. colirequire several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intactE. colicells that survive phage exposure.Escherichia coliO157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cellsE. coliO157:H7. Compared to PMA-qPCR, direct plating overestimated (P< 0.01) phage efficacy as cell surface-attached phage particles lysedE. coliO157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol ofE. coliO157:H7.

Despite multiple control measures, Escherichia coli O157:H7 (STEC O157:H7) continues to be responsible for many food borne outbreaks in North America and elsewhere. Bacteriophage therapy may prove useful for controlling this pathogen in the host, their environment and food. Bacteriophage vB_EcoS_AKFV33 (AKFV33), a T5-like phage of Siphoviridae lysed common phage types of STEC O157:H7 and not non-O157 E. coli. Moreover, STEC O157:H7 isolated from the same feedlot pen from which the phage was obtained, were highly susceptible to AKFV33. Adsorption rate constant and burst size were estimated to be 9.31 × 10(-9) ml/min and 350 PFU/infected cell, respectively. The genome of AKVF33 was 108,853 bp (38.95% G+C), containing 160 open reading frames (ORFs), 22 tRNA genes and 32 strong promoters recognized by host RNA polymerase. Of 12 ORFs without homologues to T5-like phages, 7 predicted novel proteins while others exhibited low identity (

This chapter reviews published scientific literature on: (i) the nature of bacterial contamination of the food supply; (ii) the isolation of zoonotic bacteria-specific bacteriophages; and (iii) the use of bacteriophages against foodborne bacterial pathogens, with emphasis on treatment of farm animals (such as poultry, pigs, sheep and cattle), in food processing plants, and in ready-to-eat foods.

The relationship between endemic bacteriophages infecting E. coli O157:H7 (referred to as “phage”) and levels of shedding of E. coli O157:H7 by cattle was investigated in two commercial feedlots in southern Alberta, Canada. Between May and November 2007, 10 pens of cattle were monitored by collection of pooled fecal pats, water with sediment from troughs, manure slurry from the pen floor, and rectal fecal samples from individual animals (20 per pen) at two separate times. Bacteriophages infecting E. coli O157:H7 were detected more frequently ( P < 0.001) after 18 to 20 h enrichment than by initial screening and were recovered in 239 of 855 samples (26.5% of 411 pooled fecal pats, 23.8% of 320 fecal grab samples, 21.8% of 87 water trough samples, and 94.6% of 37 pen floor slurry samples). Overall, prevalence of phage was highest ( P < 0.001) in slurry. Recovery of phage from pooled fecal pats was highest ( P < 0.05) in May. Overall recovery did not differ ( P > 0.10) between fecal grab samples and pooled fecal pats. A higher prevalence of phage in fecal pats or water trough samples was associated ( P < 0.01) with reduced prevalence of E. coli O157:H7 in rectal fecal samples. There was a weak but significant negative correlation between isolation of phage and E. coli O157:H7 in fecal grab samples ( r = −0.11; P < 0.05). These data demonstrate that the prevalence of phage fluctuates in a manner similar to that described for E. coli O157:H7. Phage were more prevalent in manure slurry than other environmental sources. The likelihood of fecal shedding of E. coli O157:H7 was reduced if cattle in the pen harbored phage.

The T1-like bacteriophages vB_EcoS_AHP24, AHS24, AHP42 and AKS96 of the family Siphoviridae were shown to lyse common phage types of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157:H7), but not non-O157 E. coli. All contained circularly permuted genomes of 45.7–46.8 kb (43.8–44 mol% G+C) encoding 74–81 open reading frames and 1 arginyl-tRNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the structural proteins were identical among the four phages. Further proteomic analysis identified seven structural proteins responsible for tail fiber, tail tape measure protein, major capsid, portal protein as well as major and minor tail proteins. Bioinformatic analyses on the proteins revealed that genomes of AHP24, AHS24, AHP42 and AKS96 did not encode for bacterial virulence factors, integration-related proteins or antibiotic resistance determinants. All four phages were highly lytic to STEC O157:H7 with considerable potential as biocontrol agents. Comparative genomic, proteomic and phylogenetic analysis suggested that the four phages along with 17 T1-like phage genomes from database of National Center for Biotechnology Information (NCBI) can be assigned into a proposed subfamily “Tunavirinae” with further classification into five genera, namely “Tlslikevirus” (TLS, FSL SP-126), “Kp36likevirus” (KP36, F20), Tunalikevirus (T1, ADB-2 and Shf1), “Rtplikevirus” (RTP, vB_EcoS_ACG-M12) and “Jk06likevirus” (JK06, vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96, phiJLA23, phiKP26, phiEB49). The fact that the viruses related to JK06 have been isolated independently in Israel (JK06) (GenBank Assession #, NC_007291), Canada (vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96) and Mexico (phiKP26, phiJLA23) (between 2005 and 2011) indicates that these similar phages are widely distributed, and that horizontal gene transfer does not always prevent the characterization of bacteriophage evolution. With this new scheme, any new discovered phages with same type can be more properly identified. Genomic- and proteomic- based taxonomic classification of phages would facilitate better understanding phages diversity and genetic traits involved in phage evolution.

Background Mannheimia haemolytica is a commensal bacterium that resides in the upper respiratory tract of cattle that can play a role in bovine respiratory disease. Prophages are common in the M. haemolytica genome and contribute significantly to host diversity. The objective of this research was to undertake comparative genomic analysis of phages induced from strains of M. haemolytica serotype A1 (535A and 2256A), A2 (587A and 1127A) and A6 (1152A and 3927A). Results Overall, four P2-like (535AP1, 587AP1, 1127AP1 and 2256AP1; genomes: 34.9–35.7 kb; G+C content: 41.5–42.1 %; genes: 51–53 coding sequences, CDSs), four λ-like (535AP2, 587AP2, 1152AP2 and 3927AP1; genomes: 48.6–52.1 kb; 41.1–41.4 % mol G+C; genes: 77–83 CDSs and 2 tRNAs) and one Mu-like (3927AP2; genome: 33.8 kb; 43.1 % mol G+C; encoding 50 CDSs) phages were identified. All P2-like phages are collinear with the temperate phage φMhaA1-PHL101 with 535AP1, 2256AP1 and 1152AP1 being most closely related, followed by 587AP1 and 1127AP1. Lambdoid phages are not collinear with any other known λ-type phages, with 587AP2 being distinct from 535AP2, 3927AP1 and 1152AP2. All λ-like phages contain genes encoding a toxin-antitoxin (TA) system and cell-associated haemolysin XhlA. The Mu-like phage induced from 3927A is closely related to the phage remnant φMhaMu2 from M. haemolytica PHL21, with similar Mu-like phages existing in the genomes of M. haemolytica 535A and 587A. Conclusions This is among the first reports of both λ- and Mu-type phages being induced from M. haemolytica. Compared to phages induced from commensal strains of M. haemolytica serotype A2, those induced from the more virulent A1 and A6 serotypes are more closely related. Moreover, when P2-, λ- and Mu-like phages co-existed in the M. haemolytica genome, only P2- and λ-like phages were detected upon induction, suggesting that Mu-type phages may be more resistant to induction. Toxin-antitoxin gene cassettes in λ-like phages may contribute to their genomic persistence or the establishment of persister subpopulations of M. haemolytica. Further work is required to determine if the cell-associated haemolysin XhlA encoded by λ-like phages contributes to the pathogenicity and ecological fitness of M. haemolytica. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0494-5) contains supplementary material, which is available to authorized users.