Siyun Wang

Genotypes Associated with Listeria monocytogenes Isolates Displaying Impaired or Enhanced Tolerances to Cold, Salt, Acid, or Desiccation Stress

The human pathogen Listeria monocytogenes is a large concern in the food industry where its continuous detection in food products has caused a string of recalls in North America and Europe. Most recognized for its ability to grow in foods during refrigerated storage, L. monocytogenes can also tolerate several other food-related stresses with some strains possessing higher levels of tolerances than others. The objective of this study was to use a combination of phenotypic analyses and whole genome sequencing to elucidate potential relationships between L. monocytogenes genotypes and food-related stress tolerance phenotypes. To accomplish this, 166 L. monocytogenes isolates were sequenced and evaluated for their ability to grow in cold (4°C), salt (6% NaCl, 25°C), and acid (pH 5, 25°C) stress conditions as well as survive desiccation (33% RH, 20°C). The results revealed that the stress tolerance of L. monocytogenes is associated with serotype, clonal complex (CC), full length inlA profiles, and the presence of a plasmid which was identified in 55% of isolates. Isolates with full length inlA exhibited significantly (p < 0.001) enhanced cold tolerance relative to those harboring a premature stop codon (PMSC) in this gene. Similarly, isolates possessing a plasmid demonstrated significantly (p = 0.013) enhanced acid tolerance. We also identified nine new L. monocytogenes sequence types, a new inlA PMSC, and several connections between CCs and the presence/absence or variations of specific genetic elements. A whole genome single-nucleotide-variants phylogeny revealed sporadic distribution of tolerant isolates and closely related sensitive and tolerant isolates, highlighting that minor genetic differences can influence the stress tolerance of L. monocytogenes. Specifically, a number of cold and desiccation sensitive isolates contained PMSCs in σB regulator genes (rsbS, rsbU, rsbV). Collectively, the results suggest that knowing the sequence type of an isolate in addition to screening for the presence of full-length inlA and a plasmid, could help food processors and food agency investigators determine why certain isolates might be persisting in a food processing environment. Additionally, increased sequencing of L. monocytogenes isolates in combination with stress tolerance profiling, will enhance the ability to identify genetic elements associated with higher risk strains.

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.

Effect of curing method and freeze-thawing on subsequent growth of Listeria monocytogenes on cold-smoked salmon.

The presence of the foodborne pathogen Listeria monocytogenes on cold-smoked salmon is a major concern for the seafood industry. Understanding processing and postprocessing handling factors that affect the ability of this pathogen to grow on cold-smoked salmon is critical for developing effective control strategies. In this study, we investigated the effect of curing method and freeze-thawing of cold-smoked salmon on (i) physicochemical properties and (ii) subsequent growth of genetically diverse strains of L. monocytogenes (inoculated after freeze-thawing) and endogenous lactic acid bacteria. The majority of the measured physicochemical properties were unaffected by freezing and thawing. Overall, wet-cured cold-smoked salmon had higher pH, water activity, and moisture, as well as lower fat, water-phase salt, and phenolic content compared with dry-cured cold-smoked salmon. The curing method and freeze-thawing did not affect growth of endogenous lactic acid bacteria. Freeze-thawing cold-smoked salmon prior to inoculation led to pronounced growth of L. monocytogenes at 7°C. The increase in cell density between days 0 and 30 was significantly (P = 0.0078) greater for cold-smoked salmon that was frozen and thawed prior to inoculation compared with nonfrozen cold-smoked salmon. On dry-cured, freeze-thawed cold-smoked salmon, L. monocytogenes had a lag phase ranging from 3.7 ± 0.1 to 11.2 ± 1.4 days compared with salmon that was wet cured and freeze-thawed, on which L. monocytogenes began to grow within 24 h. Variation in growth among L. monocytogenes strains was also observed, indicating the significance of assessing multiple strains. Further efforts to understand the impact of processing and postprocessing handling steps of cold-smoked salmon on the growth of genetically diverse L. monocytogenes will contribute to improved challenge study designs and data. This, in turn, will likely lead to more reliable and unbiased risk assessments and control measures.

Assessment criteria and approaches for rapid detection methods to be used in the food industry.

The number of commercially available kits and methods for rapid detection of foodborne pathogens continues to increase at a considerable pace, and the diversity of methods and assay formats is reaching a point where it is very difficult even for experts to weigh the advantages and disadvantages of different methods and to decide which methods to choose for a certain testing need. Although a number of documents outline quantitative criteria that can be used to evaluate different detection methods (e.g., exclusivity and inclusivity), a diversity of criteria is typically used by industry to select specific methods that are used for pathogen detection. This article is intended to provide an overall outline of criteria that the food industry can use to evaluate new rapid detection methods, with a specific focus on nucleic acid-based detection methods.

A Novel gene detection method based on period-3 property

Processing of biomolecular sequences using communication theory techniques provides powerful approaches for solving highly relevant problems in bioinformatics by properly mapping character strings into numerical sequences. We provide an optimized procedure for predicting protein-coding regions in DNA sequences based on the period-3 property of coding region. We present a digital correlating and filtering approach in the process of predicting these regions, and find out their locations by using the magnitude of the output sequence. These approaches result in improved computational techniques for the solution of useful problems in genomic information science and technology.

Occurrence, characterization, and potential predictors of verotoxigenic Escherichia coli, Listeria monocytogenes, and Salmonella in surface water used for produce irrigation in the Lower Mainland of British Columbia, Canada

Produce has become a major source of foodborne illness, and may become contaminated through surface water irrigation. The objectives of this study were to (i) determine the frequency of verotoxigenic E. coli (VTEC), Listeria monocytogenes, and Salmonella in surface waters used for irrigation in the Lower Mainland of British Columbia, (ii) assess the suitability of fecal coliforms and generic E. coli as hygiene indicators, and (iii) investigate the correlations of environmental factors with pathogen occurrence. Water samples were collected semi-monthly for 18 months from seven irrigation ditches across the Serpentine and Sumas watersheds. VTEC colonies on water filters were detected using a verotoxin colony immunoblot, and the presence of virulence genes vt1 and vt2 was ascertained via multiplex PCR. Detection of L. monocytogenes and Salmonella was completed using standard, Health Canada Compendium of Analytical Methods. Fecal coliforms and generic E. coli were enumerated by 3M™ Petrifilm™ and filtration methods, and meteorological and geographic data were collected from government records. VTEC, L. monocytogenes, and Salmonella were detected in 4.93%, 10.3%, and 2.69% of 223 samples, respectively. L. monocytogenes occurrence was greatest in the Serpentine watershed (χ2; p < 0.05), and was most common during the winter and fall (Fisher exact test; p < 0.05). Site dependence of VTEC and Salmonella occurrence was observed within watersheds (Fisher’s exact test; p < 0.10). Pathogen occurrence correlated with fecal coliform counts (r = 0.448), while VTEC occurrence also correlated with precipitation over the five days before sampling (r = 0.239). The density of upstream livestock correlated with VTEC (rs = 0.812), and L. monocytogenes (rs = 0.841) detection. These data show that foodborne pathogens are present in the waters used for irrigation in the Lower Mainland of British Columbia, but their frequency may depend on spatial and temporal factors.

Strand specific RNA-sequencing and membrane lipid profiling reveals growth phase-dependent cold stress response mechanisms in Listeria monocytogenes

The human pathogen Listeria monocytogenes continues to pose a challenge in the food industry, where it is known to contaminate ready-to-eat foods and grow during refrigerated storage. Increased knowledge of the cold-stress response of this pathogen will enhance the ability to control it in the food-supply-chain. This study utilized strand-specific RNA sequencing and whole cell fatty acid (FA) profiling to characterize the bacterium’s cold stress response. RNA and FAs were extracted from a cold-tolerant strain at five time points between early lag phase and late stationary-phase, both at 4°C and 20°C. Overall, more genes (1.3×) were suppressed than induced at 4°C. Late stationary-phase cells exhibited the greatest number (n = 1,431) and magnitude (>1,000-fold) of differentially expressed genes (>2-fold, p<0.05) in response to cold. A core set of 22 genes was upregulated at all growth phases, including nine genes required for branched-chain fatty acid (BCFA) synthesis, the osmolyte transporter genes opuCBCD, and the internalin A and D genes. Genes suppressed at 4°C were largely associated with cobalamin (B12) biosynthesis or the production/export of cell wall components. Antisense transcription accounted for up to 1.6% of total mapped reads with higher levels (2.5×) observed at 4°C than 20°C. The greatest number of upregulated antisense transcripts at 4°C occurred in early lag phase, however, at both temperatures, antisense expression levels were highest in late stationary-phase cells. Cold-induced FA membrane changes included a 15% increase in the proportion of BCFAs and a 15% transient increase in unsaturated FAs between lag and exponential phase. These increases probably reduced the membrane phase transition temperature until optimal levels of BCFAs could be produced. Collectively, this research provides new information regarding cold-induced membrane composition changes in L. monocytogenes, the growth-phase dependency of its cold-stress regulon, and the active roles of antisense transcripts in regulating its cold stress response.

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.

Evidence to indicate that maillard reaction products can provide selective antimicrobial activity

The Maillard reaction (MR) is a non-enzymatic browning reaction that occurs between carbonyl and amino groups present in foods that are processed or cooked at high temperatures; the result being a vast number of reaction products, termed Maillard reaction products (MRPs). Whereas initial stages of the reaction produce small molecular weight, colorless products; some being volatile and contributing to odor, extending the reaction with prolonged exposure to heat will result in the MR reaching final stages and generating high molecular weight products that contribute to color and flavor, important sensory attributes. Many specific MRPs also impart a range of biological properties; the most recently noted being antioxidant capacity and to a comparatively lesser extent antimicrobial activity. The potential antimicrobial activity of MRPs appears to be dependent on the strain of microorganism and internal/external factors that influence the physicochemical reactions occurring between microbe and the MRP, albeit, the mechanism(s) offered by MRPs is somewhat unclear. The objective of this paper is to summarize the evidence that MRPs evoke antimicrobial activity when certain conditions exist in foods that facilitate an inhibitory potential. We review the current knowledge of factors that influence the inhibitory effect of MRPs on microorganism growth and proliferation, proposed mechanisms of antimicrobial properties of MRPs, and discussion of future relevant applications.

Stress survival islet 1 contributes to serotype-specific differences in biofilm formation in Listeria monocytogenes

Listeria monocytogenes has a significant impact on the food industry by forming biofilms on food‐processing equipment. Tandem analysis of whole‐genome sequencing data with biofilm data from 166 environmental and food‐related L. monocytogenes isolates has revealed serotypic and genetic factors that strongly correlate with adherence and biofilm formation, such as lineage, plasmid harbourage, a three‐codon deletion in inlA and the presence of the stress survival islet 1 (SSI‐1). Strains from serotype 1/2b, the majority of which contained SSI‐1, formed the strongest biofilms, while serotype 4b strains, the majority of which did not contain SSI‐1, formed the weakest biofilms. When serotype 1/2a was separated by its SSI‐1 genotype, SSI‐1‐positive 1/2a strains demonstrated significantly higher capacity for biofilm formation after 3 days of growth at 30°C (P < 0·0001). Together, these findings indicate that SSI‐1 may contribute to serotype‐associated differences in the biofilm‐forming capacity in L. monocytogenes.