Alfalfa sprouts have been linked to numerous North American outbreaks of Salmonella in recent years. Conventionally, treatments involving chlorine, heat, and irradiation are used for alfalfa seed sanitation. However, such treatments may be highly variable in their efficacy for pathogen control and/or detrimental to sprout quality, therefore negatively perceived by consumers advocating for natural alternatives. The usage of bacteriophages for pathogen control in sprouts has been previously explored, although with conflicting and inconsistent results. Lytic phages, viral predators of bacteria, represent an attractive approach as they provide several advantages compared to conventional treatments, such as their high specificity for bacterial targets and their ubiquity in nature. In this study, four Salmonella phages were isolated from British Columbia, Canada and characterized with respect to host range, burst size, latent period, and environmental stability to assess their potential to control Salmonella. Phage isolate SI1 showed the greatest host range, highest burst size and shortest latent period, greatest stability across all pH and temperatures and was the most effective in control of S. Enteritidis in vitro. Therefore, SI1 was chosen for treatment of sprouting alfalfa seeds artificially contaminated with S. Enteritidis with a multiplicity of infection (MOI) of ∼110 PFU/CFU. A significant (p < 0.05) reduction of 38.3 ± 3.0% of viable Salmonella cells was observed following two h of phage treatment. On days two to six of the sprouting process, reductions of Salmonella were also observed, but were not significant compared to the control (p > 0.05). It was further demonstrated that the sprout yield was not significantly (p > 0.05) affected by phage treatment. These results highlight the potential of phages recovered from the British Columbia environment for use as biocontrol agents against Salmonella, although differing efficacies in vitro was observed. Moreover, the effectiveness of SI1 to significantly (p < 0.05) control Salmonella on sprouting alfalfa seeds on day 1 of treatment was demonstrated. Although promising, future work should aim to optimize this treatment to achieve more effective, and longer lasting, biocontrol of Salmonella in sprouting alfalfa seeds.

Phages infecting members of the opportunistic human pathogen, Salmonella enterica, are widespread in natural environments and offer a potential source of agents that could be used for controlling populations of this bacterium; yet, relatively little is known about these phages. Here we describe the isolation and characterization of 45 phages of Salmonella enterica from disparate geographic locations within British Columbia, Canada. Host-range profiling revealed host-specific patterns of susceptibility and resistance, with several phages identified that have a broad-host range (i.e., able to lyse >40% of bacterial hosts tested). One phage in particular, SE13, is able to lyse 51 out of the 61 Salmonella strains tested. Comparative genomic analyses also revealed an abundance of sequence diversity in the sequenced phages. Alignment of the genomes grouped the phages into 12 clusters with three singletons. Phages within certain clusters exhibited extraordinarily high genome homology (>98% nucleotide identity), yet between clusters, genomes exhibited a span of diversity (<50% nucleotide identity). Alignment of the major capsid protein also supported the clustering pattern observed with alignment of the whole genomes. We further observed associations between genomic relatedness and the site of isolation, as well as genetic elements related to DNA metabolism and host virulence. Our data support the knowledge framework for phage diversity and phage–host interactions that are required for developing phage-based applications for various sectors, including biocontrol, detection and typing.

The Salmonella Syst-OMICS consortium is sequencing 4,500 Salmonella genomes and building an analysis pipeline for the study of Salmonella genome evolution, antibiotic resistance and virulence genes. Metadata, including phenotypic as well as genomic data, for isolates of the collection are provided through the Salmonella Foodborne Syst-OMICS database (SalFoS), at https://salfos.ibis.ulaval.ca/. Here, we present our strategy and the analysis of the first 3,377 genomes. Our data will be used to draw potential links between strains found in fresh produce, humans, animals and the environment. The ultimate goals are to understand how Salmonella evolves over time, improve the accuracy of diagnostic methods, develop control methods in the field, and identify prognostic markers for evidence-based decisions in epidemiology and surveillance.

Prophages have long been regarded as an important contributor to the evolution of Salmonella and Verotoxin-producing E. coli (VTEC), members of the Enterobacteriaceae that cause millions of cases of foodborne illness in North America. In S. Typhimurium, prophages provide many of the genes required for invasion; similarly, in VTEC, the Verotoxin-encoding genes are located in cryptic prophages. The ability of prophages to quickly acquire and lose genes have driven their rapid evolution, leading to highly diversified populations of phages that can infect distantly-related bacterial hosts. To defend against foreign genetic materials (i.e., phages), bacteria have evolved Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immunity, consisting of variable spacer regions that match short nucleic acid sequences of invaders previously encountered. The number of spacer regions varies widely amongst Enterobacteriaceae, and there is currently no clear consensus if the accumulation of spacers is linked to genomic prophage abundance. Given the immense prophage diversity and contribution to bacterial host phenotypes, we analyzed the prophage sequences within 118 strains of Salmonella and VTEC, 117 of which are of agricultural origin. Overall, 130 unique prophage sequences were identified and they were found to be remarkably diverse with &lt;50% nucleotide similarity, particularly with the Gifsy-1 group which was identified in several Salmonella serovars and interestingly, a strain of VTEC. Additionally, we identified a novel plasmid-like phage that carried antibiotic resistance and bacteriocin resistance genes. The strains analyzed carried at least six distinct spacers which did not possess homology to prophages identified in the same genome. In fact, only a fraction of all identified spacers (14%) possessed significant homology to known prophages. Regression models did not discern a correlation between spacer and prophage abundance in our strains, although the relatively high number of spacers in our strains (an average of 27 in Salmonella and 19 in VTEC) suggest that high rates of infection may occur in agricultural niches and be a contributing driver in bacterial evolution. Cumulatively, these results shed insight into prophage diversity of Salmonella and VTEC, which will have further implications when informing development of phage therapies against these foodborne pathogens.

Salmonella enterica ( S. enterica ) is a causative agent of multiple outbreaks of foodborne illness associated with fresh produce, including pre-cut melon and leafy vegetables. Current industrial antimicrobial interventions have been shown to reduce microbial populations by <90%. Consequently, bacteriophages have been suggested as an alternative to chemical sanitizers. Seven S. enterica strains from four serovars (10 5 CFU/mL) were separately inoculated onto excised pieces of Romaine lettuce leaf and cantaloupe flesh treated with a five-strain bacteriophage cocktail 24 h before S. enterica inoculation. S. enterica , total aerobic populations and water activity were measured immediately after inoculation and after 1 and 2 days of incubation at 8 °C. The efficacy of the bacteriophage cocktail varied between strains. Populations of S. enterica Enteritidis strain S3, S. Javiana S203, S. Javiana S200 were reduced by > 3 log CFU/g and S. Newport S2 by 1 log CFU/g on both lettuce and cantaloupe tissues at all sampling times. In contrast, populations of strains S. Thompson S193 and S194 were reduced by 2 log CFU/g on day 0 on lettuce, but were not significantly different (P > 0.05) from the controls thereafter, S. Newport S195 populations were reduced on lettuce by 1 log CFU/g on day 0 and no reductions were found on cantaloupe tissue. Both aerobic populations and water activity were higher on cantaloupe than on lettuce. The water activity of lettuce decreased significantly (P < 0.05) from 0.845 ± 0.027 on day 0–0.494 ± 0.022 on day 1, but that of cantaloupe remained between 0.977 and 0.993 from day 0–2. The results of this study showed that bacteriophages can reduce S. enterica populations on lettuce and cantaloupe tissues but that the magnitude of the effect was strain-dependent.