Lawrence Goodridge

Bacteriophages Contribute to the Spread of Antibiotic Resistance Genes among Foodborne Pathogens of the Enterobacteriaceae Family – A Review

Foodborne illnesses continue to have an economic impact on global health care systems. There is a growing concern regarding the increasing frequency of antibiotic resistance in foodborne bacterial pathogens and how such resistance may affect treatment outcomes. In an effort to better understand how to reduce the spread of resistance, many research studies have been conducted regarding the methods by which antibiotic resistance genes are mobilized and spread between bacteria. Transduction by bacteriophages (phages) is one of many horizontal gene transfer mechanisms, and recent findings have shown phage-mediated transduction to be a significant contributor to dissemination of antibiotic resistance genes. Here, we review the viability of transduction as a contributing factor to the dissemination of antibiotic resistance genes in foodborne pathogens of the Enterobacteriaceae family, including non-typhoidal Salmonella and Shiga toxin-producing Escherichia coli, as well as environmental factors that increase transduction of antibiotic resistance genes.

A Syst-OMICS Approach to Ensuring Food Safety and Reducing the Economic Burden of Salmonellosis

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.

Concentration of enteric viruses from tap water using an anion exchange resin-based method

Detecting low concentrations of enteric viruses in water is needed for public health-related monitoring and control purposes. Thus, there is a need for sensitive, rapid and cost effective enteric viral concentration methods compatible with downstream molecular detection. Here, a virus concentration method based on adsorption of the virus to an anion exchange resin and direct isolation of nucleic acids is presented. Ten liter samples of tap water spiked with different concentrations (10-10,000 TCID50/10 L) of human adenovirus 40 (HAdV-40), hepatitis A virus (HAV) or rotavirus (RV) were concentrated and detected by real time PCR or real time RT-PCR. This method improved viral detection compared to direct testing of spiked water samples where the ΔCt was 12.1 for AdV-40 and 4.3 for HAV. Direct detection of RV in water was only possible for one of the three replicates tested (Ct of 37), but RV detection was improved using the resin method (all replicates tested positive with an average Ct of 30, n=3). The limit of detection of the method was 10 TCID50/10 L for HAdV-40 and HAV, and 100 TCID50/10 L of water for RV. These results compare favorably with detection limits reported for more expensive and laborious methods.

Evaluation of an anion exchange resin-based method for concentration of F-RNA coliphages (enteric virus indicators) from water samples

Enteric viral contaminants in water represent a public health concern, thus methods for detecting these viruses or their indicator microorganisms are needed. Because enteric viruses and their viral indicators are often found at low concentrations in water, their detection requires upfront concentration methods. In this study, a strong basic anion exchange resin was evaluated as an adsorbent material for the concentration of F-RNA coliphages (MS2, Qβ, GA, and HB-P22). These coliphages are recognized as enteric virus surrogates and fecal indicator organisms. Following adsorption of the coliphages from 50ml water samples, direct RNA isolation and real time RT-PCR detection were performed. In water samples containing 10(5)pfu/ml of the F-RNA coliphages, the anion exchange resin (IRA-900) adsorbed over 96.7% of the coliphages present, improving real time RT-PCR detection by 5-7 cycles compared to direct testing. F-RNA coliphage RNA recovery using the integrated method ranged from 12.6% to 77.1%. Resin-based concentration of samples with low levels of the F-RNA coliphages allowed for 10(0)pfu/ml (MS2 and Qβ) and 10(-1)pfu/ml (GA and HB-P22) to be detected. The resin-based method offers considerable advantages in cost, speed, simplicity and field adaptability.

Prophage Integrase Typing Is a Useful Indicator of Genomic Diversity in Salmonella enterica

Salmonella enterica is a bacterial species that is a major cause of illness in humans and food-producing animals. S. enterica exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars that have been identified. The development of methods to assess genome diversity in S. enterica will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate S. enterica genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of S. enterica were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated Salmonella serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49 S. enterica genomes (79 integrase genes in the food-associated Salmonella isolates, 50 integrase genes in S. Enteritidis, and 18 integrase genes in S. Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49 S. enterica genomes (44 integrase genes in the food-associated Salmonella isolates, 21 integrase genes in S. Enteritidis, and 9 integrase genes in S. Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of S. Enteritidis and S. Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23 different integrase genes within the food-associated isolates, but only identified four different phages and integrase genes within clonal isolates of S. Enteritidis and S. Heidelberg. These results demonstrate the potential usefulness of PCR based detection of prophage integrase genes as a rapid indicator of genome diversity in S. enterica.

Characterization of Four Novel Bacteriophages Isolated from British Columbia for Control of Non-typhoidal Salmonella in Vitro and on Sprouting Alfalfa Seeds

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.

Salmonella enterica Prophage Sequence Profiles Reflect Genome Diversity and Can Be Used for High Discrimination Subtyping

Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of Salmonella responsible for disease outbreaks is crucial to minimize infections and eliminate ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP) typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated Salmonella strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in Salmonella. In this study, the prophage sequence diversity in different Salmonella serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of S. enterica representing 151 Salmonella serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in S. enterica genomes. Prophage sequences were highly variable among S. enterica serovars with a median ± interquartile range (IQR) of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of S. Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating S. enterica subtypes during foodborne outbreaks.

Field-based evaluation of a male-specific (F+) RNA coliphage concentration method

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Therefore, sensitive, reliable and easy to use methods for the concentration, detection and quantification of microorganisms associated with the safety and quality of water are needed. In this study, we performed a field evaluation of an anion exchange resin-based method to concentrate male-specific (F+) RNA coliphages (FRNA), fecal indicator organisms, from diverse environmental waters that were suspected to be contaminated with feces. In this system, FRNA coliphages are adsorbed to anion exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase (RT)-PCR detection. Matrix-dependent inhibition of this method was evaluated using known quantities of spiked FRNA coliphages belonging to four genogroups (GI, GII, GII and GIV). RT-PCR-based detection was successful in 97%, 72%, 85% and 98% of the samples spiked (106 pfu/l) with GI, GII, GIII and GIV, respectively. Differential FRNA coliphage genogroup detection was linked to inhibitors that altered RT-PCR assay efficiency. No association between inhibition and the physicochemical properties of the water samples was apparent. Additionally, the anion exchange resin method facilitated detection of naturally present FRNA coliphages in 40 of 65 environmental water samples (61.5%), demonstrating the viability of this system to concentrate FRNA coliphages from water.

Impact of the choice of reference genome on the ability of the core genome SNV methodology to distinguish strains of Salmonella enterica serovar Heidelberg

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. The core genome single nucleotide variant pipeline (cgSNV) is one of several whole genome based sequence typing methods used for the laboratory investigation of foodborne pathogens. SNV detection using this method requires a reference genome. The purpose of this study was to investigate the impact of the choice of the reference genome on the cgSNV-informed phylogenetic clustering and inferred isolate relationships. We found that using a draft or closed genome of S. Heidelberg as reference did not impact the ability of the cgSNV methodology to differentiate among 145 S. Heidelberg isolates involved in foodborne outbreaks. We also found that using a distantly related genome such as S. Dublin as choice of reference led to a loss in resolution since some sporadic isolates were found to cluster together with outbreak isolates. In addition, the genetic distances between outbreak isolates as well as between outbreak and sporadic isolates were overall reduced when S. Dublin was used as the reference genome as opposed to S. Heidelberg.

For the Safety of Fresh Produce: Regulatory Considerations for Canada on the Use of Whole Genome Sequencing to Subtype Salmonella

Salmonella is one of the oldest bacteria known to man, yet it is also one of the most prevalent when it comes to foodborne-related diseases and outbreaks. Naturally present in the environment and difficult to treat on fresh produce, Salmonella represents an important food safety challenge. Emerging technologies such as whole genome sequencing (WGS) and next generation sequencing (NGS) now offer promising applications within the realm of food safety that can significantly change the way routine testing, inspections and disease surveillance are done. They offer potential avenues that may foster more sustainable agricultural and environmental practices to detect and reduce the presence of Salmonella. Strategies are being developed to better cluster, integrate and share genomic data to facilitate the development of diagnostic tests and control methods, as well as generate robust evidence to better inform future policy and regulatory decision-making. Using the approaches developed by the Salmonella Syst-OMICS consortium, a large-scale Canadian-based genomic project, this paper discusses the policy and regulatory considerations for the applications of WGS and NGS technologies in the development of testing and biocontrol tools for food safety. The paper presents an overview of the current regulatory framework for the approval of testing methodologies for Salmonella. It discusses considerations related to (1) the development of a new test for Salmonella, (2) the potential establishment of a Salmonella risk virulence classification scheme, and (3) the development of a biocontrol method to reduce the presence of Salmonella on fresh produce.