Associations between Listeria monocytogenes genomic characteristics and adhesion to polystyrene at 8 °C.

Listeria monocytogenes remains a threat to the food system and has led to numerous foodborne outbreaks worldwide. L. monocytogenes can establish itself in food production facilities by adhering to surfaces, resulting in increased resistance to environmental stressors. The aim of this study was to evaluate the adhesion ability of L. monocytogenes at 8 °C and to analyse associations between the observed phenotypes and genetic factors such as internalin A (inlA) genotypes, stress survival islet 1 (SSI-1) genotype, and clonal complex (CC). L. monocytogenes isolates (n = 184) were grown at 8 °C and 100% relative humidity for 15 days. The growth was measured by optical density at 600 nm every 24 h. Adherent cells were stained using crystal violet and quantified spectrophotometrically. Genotyping of inlA and SSI-1, multi-locus sequence typing, and a genome-wide association study (GWAS) were performed to elucidate the phenotype-genotype relationships in L. monocytogenes cold adhesion. Among all inlA genotypes, truncated inlA isolates had the highest mean adhered cells, ABS595nm = 0.30 ± 0.15 (Tukey HSD; P < 0.05), while three-codon deletion inlA isolates had the least mean adhered cells (Tukey HSD; P < 0.05). When SSI-1 was present, more cells adhered; less cells adhered when SSI-1 was absent (Welch's t-test; P < 0.05). Adhesion was associated with clonal complexes which have low clinical frequency, while reduced adhesion was associated with clonal complexes which have high frequency. The results of this study support that premature stop codons in the virulence gene inlA are associated with increased cold adhesion and that an invasion enhancing deletion in inlA is associated with decreased cold adhesion. This study also provides evidence to suggest that there is an evolutionary trade off between virulence and adhesion in L. monocytogenes. These results provide a greater understanding of L. monocytogenes adhesion which will aid in the development of strategies to reduce L. monocytogenes in the food system.

Safety and Quality of Fish and Game Meats Prepared by First Nations Communities in British Columbia, Canada.

ABSTRACT: Canadian First Nations communities rely on traditional preservation methods such as the smoking, drying, and canning of fish and game meats to ensure long-term food security. Unlike commercial food production, there are no recognized official standards for these methods, rendering their efficacy in producing microbiologically safe foods relatively unknown. In this study, 81 fresh or processed fish and game samples obtained from four British Columbia First Nations communities were analyzed for quality indicator microbes, foodborne pathogens, and mineral levels. Aerobic counts, coliforms (CC), Escherichia coli (EC), lactic acid bacteria, Staphylococcus aureus (STA), and yeast and molds (YM) were enumerated using the TEMPO, whereas the presence of E. coli O157:H7, Listeria monocytogenes, and Salmonella were detected using the VIDAS immunoassay system. The opportunistic pathogens Enterococcus faecalis and Enterococcus faecium were additionally detected using culture methods with subsequent PCR confirmation, and minerals (Ca, Cu, K, Mg, Mn, Na, and Zn) were detected using mass spectrometry. With the exception of STA, microbial loads were significantly (P < 0.05) higher in processed fish and meat samples compared with unprocessed samples, and game samples contained higher microbial levels than fish; however, differences were only significant (P < 0.05) for coliforms, E. coli, and STA. E. coli O157:H7 was detected in one smoked moose sample, and E. faecalis and E. faecium were isolated from 21 and 2 samples, respectively. Although smoked samples contained significantly higher Na levels, they were effective in reducing microbial levels. These results indicate that current food preservation methods practiced by British Columbia First Nations communities are infrequently effective at reducing microbial populations, and in many cases, resulted in increased microbial loads. More efforts should be made to improve the dissemination of safe food handling and processing knowledge to ensure long-term food security and well-being.

Comparative Analysis of Listeria monocytogenes Plasmids and Expression Levels of Plasmid-Encoded Genes during Growth under Salt and Acid Stress Conditions.

Listeria monocytogenes strains are known to harbour plasmids that confer resistance to sanitizers, heavy metals, and antibiotics; however, very little research has been conducted into how plasmids may influence L. monocytogenes' ability to tolerate food-related stresses. To investigate this, a library (n = 93) of L. monocytogenes plasmid sequences were compared. Plasmid sequences were divided into two groups (G1 and G2) based on a repA phylogeny. Twenty-six unique plasmid types were observed, with 13 belonging to each of the two repA-based groups. G1 plasmids were significantly (p < 0.05) smaller than G2 plasmids but contained a larger diversity of genes. The most prevalent G1 plasmid (57,083 bp) was observed in 26 strains from both Switzerland and Canada and a variety of serotypes. Quantitative PCR (qPCR) revealed a >2-fold induction of plasmid-contained genes encoding an NADH peroxidase, cadmium ATPase, multicopper oxidase, and a ClpL chaperone protein during growth under salt (6% NaCl) and acid conditions (pH 5) and ProW, an osmolyte transporter, under salt stress conditions. No differences in salt and acid tolerance were observed between plasmid-cured and wildtype strains. This work highlights the abundance of specific plasmid types among food-related L. monocytogenes strains, the unique characteristics of G1 and G2 plasmids, and the possible contributions of plasmids to L. monocytogenes tolerance to food-related stresses.

Characterization of Listeria monocytogenes enhanced cold-tolerance variants isolated during prolonged cold storage.

In this study, we show that growth and prolonged storage of Listeria monocytogenes at 4 °C can promote the selection of variants with enhanced cold and heat tolerance. Enhanced cold-tolerance (ECT) variants (n = 12) were successfully isolated from a strain with impaired cold growth abilities following 84 days of storage at 4 °C in brain heart infusion broth (BHIB). Whole genome sequencing, membrane fatty acid analysis, and stress tolerance profiling were performed on the parent strain and two ECT variants: one displaying regular-sized colonies and the other displaying small colonies when grown at 37 °C on BHI agar. Under cold stress conditions, the parent strain exhibited an impaired ability to produce branched-chain fatty acids which are known to be important for cold adaptation in L.monocytogenes. The ECT variants were able to overcome this limitation, a finding which is hypothesized to be associated with the identification of two independent single-nucleotide polymorphisms in genes encoding subunits of acetyl-coA carboxylase, an enzyme critical for fatty acid biosynthesis. While the ECT phenotype was not found to be associated with improved salt (BHIB + 6% NaCl, 25 °C), acid (BHIB pH 5, 25 °C) or desiccation (33% RH, 20 °C) tolerance, the small-colony variant exhibited significantly (p < 0.05) enhanced heat tolerance at 52 °C in buffered peptone water compared to the parent strain and the other variant. The results from this study demonstrate that the continuous use of refrigeration along the food-supply chain has the potential to select for L.monocytogenes variants with enhanced cold and heat tolerance, highlighting the impact that microbial intervention strategies can have on the evolution of bacterial strains and likewise, food safety.

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.

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.

Genes involved in Listeria monocytogenes biofilm formation at a simulated food processing plant temperature of 15 °C.

Listeria monocytogenes is a pathogenic foodborne bacterium whose persistence in food processing environments is in part attributed to its biofilm formation. Most biofilm studies have been carried out at 30-37 °C rather than at temperatures found in the food processing plants (i.e., 10-20 °C). The objective of the present study was to mine for novel genes that contribute to L. monocytogenes biofilm formation at 15 °C using the random insertional mutagenesis approach. A library of 11,024 L. monocytogenes 568 (serotype 1/2a) Himar1 insertional mutants was created. Mutants with reduced or enhanced biofilm formation at 15 °C were detected in microtiter plate assays with crystal violet and safranin staining. Fourteen mutants expressed enhanced biofilm phenotypes, and harbored transposon insertions in genes encoding cell wall biosynthesis, motility, metabolism, stress response, and cell surface associated proteins. Deficient mutants (n=5) contained interruptions in genes related to peptidoglycan, teichoic acid, or lipoproteins. Enhanced mutants produced significantly (p<0.05) higher cell densities in biofilm formed on stainless steel (SS) coupons at 15 °C (48 h) than deficient mutants, which were also more sensitive to benzalkonium chloride. All biofilm deficient mutants and four enhanced mutants in the microtiter plate assay (flaA, cheR, lmo2563 and lmo2488) formed no biofilm in a peg lid assay (Calgary biofilm device) while insertions in lmo1224 and lmo0543 led to excess biofilm in all assays. Two enhanced biofilm formers were more resistant to enzymatic removal with DNase, proteinase K or pectinase than the parent strain. Scanning electron microscopy of individual biofilms made by five mutants and the parent on SS surfaces showed formation of heterogeneous biofilm with dense zones by immotile mutants, while deficient mutants exhibited sparse growth. In conclusion, interruptions of 9 genes not previously linked to biofilm formation in L. monocytogenes (lmo2572, lmo2488 (uvrA), lmo1224, lmo0434 (inlB), lmo0263 (inlH), lmo0543, lmo0057 (EsaA), lmo2563, lmo0453), caused enhanced biofilm formation in the bacterium at 15 °C. The remaining mutants harbored interruptions in 10 genetic loci previously associated with biofilm formation at higher temperatures, indicating some temperature driven differences in the formation of biofilm by L. monocytogenes.

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis.

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm(2) relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.

Role of initial contamination levels, biofilm maturity and presence of salt and fat on desiccation survival of Listeria monocytogenes on stainless steel surfaces.

This study investigated the effect of initial contamination levels, biofilm maturity and presence of salt and fatty food soils on desiccation survival of Listeria monocytogenes on stainless steel (SS) coupons. L. monocytogenes cultures grown (at 15 °C for 48 h) in Tryptic Soy Broth with 1% glucose (TSB-glu) containing either 0.5 or 5% (w/v) NaCl were re-suspended in TSB-glu containing either 0.5 or 5% NaCl and used to contaminate SS coupons at levels of 3.5, 5.5, and 7.5 log CFU/cm². Desiccation (at 15 °C for 20 days, 43% RH) commenced immediately (non-biofilm) or following biofilm formation (at 15 °C for 48 h, 100% RH). To study the impact of food lipids, non-biofilm L. monocytogenes cells were suspended in TSB-glu containing either canola oil (5-10%) or lard (20-60%) and desiccated as above on SS coupons. Following desiccation for 20 days, survivors decreased by 1.4-3.7 log CFU/cm² for non-biofilm L. monocytogenes cells. The contamination level had no significant (p > 0.05) effect on survival kinetics. SEM micrographs showed mature biofilms on coupons initially contaminated with 5.5 and 7.5 log CFU/cm². Mature biofilm cells were significantly (p < 0.05) more desiccation resistant than cells in immature biofilms formed by the lowest contamination level. Besides biofilm maturity/formation, previous osmoadaptation, exposure to lard (20-60%) or salt (5%) during desiccation significantly (p < 0.05) increased the bacterium's survival. In conclusion, L. monocytogenes desiccation survival can be greatly reduced by preventing presence of mature biofilms and salty or fatty soils on food contact surfaces.