Is the persistence of Listeria monocytogenes in food processing facilities and its resistance to pathogen intervention linked to its phylogeny?

The foodborne pathogen Listeria monocytogenes is differentiated into four distinct lineages which differ in their virulence. It remains unknown, however, whether the four lineages also differ with respect to their ability to persist in food processing facilities, their resistance to high pressure, a preservation method that is used commercially for Listeria control on ready-to-eat meats, and their ability to form biofilms. This study aimed to determine differences in the pressure resistance and biofilm formation of 59 isolates of L. monocytogenes representing lineages I and II. Furthermore, the genetic similarity of 9 isolates of L. monocytogenes that were obtained from a meat processing facility over a period of 1 year and of 20 isolates of L. monocytogenes from food processing facilities was analyzed to assess whether the ability of the lineages of L. monocytogenes to persist in these facilities differs. Analysis of 386 genomes with respect to the source of isolation revealed that genomes of lineage II are over-represented in meat isolates when compared with clinical isolates. Of the 38 strains of Lm. monocytogenes that persisted in food processing facilities (this study or published studies), 31 were assigned to lineage II. Isolates of lineage I were more resistant to treatments at 400 to 600 MPa. The thickness of biofilms did not differ between lineages. In conclusion, strains of lineage II are more likely to persist in food processing facilities while strains of lineage I are more resistant to high pressure.IMPORTANCEListeria monocytogenes substantially contributes to the mortality of foodborne disease in developed countries. The virulence of strains of four lineages of L. monocytogenes differs, indicating that risks associated with the presence of L. monocytogenes are lineage specific. Our study extends the current knowledge by documentation that the lineage-level phylogeny of L. monocytogenes plays a role in the source of isolation, in the persistence in food processing facilities, and in the resistance to pathogen intervention technologies. In short, the control of risks associated with the presence of L. monocytogenes in food is also lineage specific. Understanding the route of contamination L. monocytogenes is an important factor to consider when designing improved control measures.

Intrinsically disordered proteins and liquid-liquid phase separation in SARS-CoV-2 interactomes.

This paper discusses the properties of proteins and their relations in the interactomes of the selected subsets of SARS-CoV-2 proteome-the membrane protein, nonstructural proteins, and, finally, full proteome. Protein disorder according to several measures, liquid-liquid phase separation probabilities, and protein node degrees in the interaction networks were singled out as the features of interest. Additionally, viral interactomes were combined with the interactome of human lung tissue so as to examine if the new connections in the resulting viral-host interactome are linked to protein disorder. Correlation analysis shows that there is no clear relationship between raw features of interest, whereas there is a positive correlation between the protein disorder and its neighborhood mean disorder. There are also indications that highly connected viral hubs tend to be on average more ordered than proteins with a small number of connections. This is in contrast to previous similar studies conducted on eukaryotic interactomes and possibly raises new questions in research on viral interactomes.

Combined Infection Control Interventions Protect Essential Food Workers from Occupational Exposures to SARS-CoV-2 in the Agricultural Environment.

Essential food workers experience elevated risks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection due to prolonged occupational exposures in food production and processing areas, shared transportation (car or bus), and employer-provided shared housing. Our goal was to quantify the daily cumulative risk of SARS-CoV-2 infection for healthy susceptible produce workers and to evaluate the relative reduction in risk attributable to food industry interventions and vaccination. We simulated daily SARS-CoV-2 exposures of indoor and outdoor produce workers through six linked quantitative microbial risk assessment (QMRA) model scenarios. For each scenario, the infectious viral dose emitted by a symptomatic worker was calculated across aerosol, droplet, and fomite-mediated transmission pathways. Standard industry interventions (2-m physical distancing, handwashing, surface disinfection, universal masking, ventilation) were simulated to assess relative risk reductions from baseline risk (no interventions, 1-m distance). Implementation of industry interventions reduced an indoor worker's relative infection risk by 98.0% (0.020; 95% uncertainty interval [UI], 0.005 to 0.104) from baseline risk (1.00; 95% UI, 0.995 to 1.00) and an outdoor worker's relative infection risk by 94.5% (0.027; 95% UI, 0.013 to 0.055) from baseline risk (0.487; 95% UI, 0.257 to 0.825). Integrating these interventions with two-dose mRNA vaccinations (86 to 99% efficacy), representing a worker's protective immunity to infection, reduced the relative infection risk from baseline for indoor workers by 99.9% (0.001; 95% UI, 0.0002 to 0.005) and outdoor workers by 99.6% (0.002; 95% UI, 0.0003 to 0.005). Consistent implementation of combined industry interventions, paired with vaccination, effectively mitigates the elevated risks from occupationally acquired SARS-CoV-2 infection faced by produce workers. IMPORTANCE This is the first study to estimate the daily risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection across a variety of indoor and outdoor environmental settings relevant to food workers (e.g., shared transportation [car or bus], enclosed produce processing facility and accompanying breakroom, outdoor produce harvesting field, shared housing facility) through a linked quantitative microbial risk assessment framework. Our model has demonstrated that the elevated daily SARS-CoV-2 infection risk experienced by indoor and outdoor produce workers can be reduced below 1% when vaccinations (optimal vaccine efficacy, 86 to 99%) are implemented with recommended infection control strategies (e.g., handwashing, surface disinfection, universal masking, physical distancing, and increased ventilation). Our novel findings provide scenario-specific infection risk estimates that can be utilized by food industry managers to target high-risk scenarios with effective infection mitigation strategies, which was informed through more realistic and context-driven modeling estimates of the infection risk faced by essential food workers daily. Bundled interventions, particularly if they include vaccination, yield significant reductions (>99%) in daily SARS-CoV-2 infection risk for essential food workers in enclosed and open-air environments.

Probing antimicrobial resistance and sanitizer tolerance themes and their implications for the food industry through the Listeria monocytogenes lens.

The development of antibiotic resistance is a serious public health crisis, reducing our ability to effectively combat infectious bacterial diseases. The parallel study of reduced susceptibility to sanitizers is growing, particularly for environmental foodborne pathogens, such as Listeria monocytogenes. As regulations demand a seek-and-destroy approach for L. monocytogenes, understanding sanitizer efficacy and its uses are critical for the food industry. Studies have reported the ability of L. monocytogenes to survive in sanitizer concentrations 10-1000 times lower than the manufacturer-recommended concentration (MRC). Notably, data show that at MRC and when applied according to the label instructions, sanitizers remain largely effective. Studies also report that variables such as the presence of organic material, application time/temperature, and bacterial attachment to surfaces can impact sanitizer effectiveness. Due to the lack of standardization in the methodology and definitions of sanitizer resistance, tolerance, and susceptibility, different messages are conveyed in different studies. In this review, we examine the diversity of definitions, terminology, and methodologies used in studies examining L. monocytogenes resistance and susceptibility to antimicrobials. Research available to date fails to demonstrate "resistance" of L. monocytogenes to recommended sanitizer treatments as prescribed by the label. As such, sanitizer tolerance would be a more accurate description of L. monocytogenes response to low sanitizer concentrations (i.e., sub-MRC). Conservative use of word "resistance" will reduce confusion and allow for concise messaging as sanitizer research findings are communicated to industry and regulators.

Relative performance of commercial citric acid and quaternary ammonium sanitizers against Listeria monocytogenes under conditions relevant to food industry.

Growth of Listeria monocytogenes in cold temperatures coupled with its tolerance of antimicrobials can promote its survival and persistence in food processing environments. The food industry relies heavily on cleaning and sanitation to control L. monocytogenes; therefore, it is important to understand the environmental context (i.e., temperature) on the efficacy of antimicrobials used in food industry. The minimum bactericidal concentrations (MBCs) of an "eco-friendly" citric acid-based (CAB) sanitizer and a conventional quaternary ammonium compound (CQAC) sanitizer were determined against 14 L. monocytogenes isolates at 4-30 °C. A subset of isolates (n = 3) was also exposed to sub-lethal concentrations of sanitizers to assess differences in growth behavior. CAB and CQAC were effective at manufacturer recommended concentrations in liquid assays. The MBC of CAB was significantly lower at 4 °C compared to 23 °C (p < 0.05), whereas the MBC of CQAC was unchanged between 4 °C and 23 °C. Manufacturers' recommendations for dose and duration of CAB and CQAC were unable to consistently achieve a >5-log reduction of L. monocytogenes attached to surfaces. Findings from this study demonstrate the importance of sanitizer evaluation under conditions representative of their use in the food industry.

Prevalence of Listeria spp. in produce handling and processing facilities in the Pacific Northwest.

Listeria monocytogenes is a significant concern for the produce industry; however, there is limited information to support the practical decision-making to mitigate this risk. This study investigated the prevalence of Listeria spp. and L. monocytogenes in seven produce handling and processing (PHP) facilities in the Pacific Northwest. PHP facilities were defined as facilities that receive raw agricultural commodities and further handle, pack, wash, or process prior to distribution into the retail sector. Environmental swabs (n = 50/facility) were collected in high-risk areas (e.g., near raw product entry points) from seven PHP facilities over two visits. Listeria spp. were isolated using modified ISO 11290-1 method and speciated with Microgen® Listeria-ID. Listeria spp., including L. monocytogenes, were found in 5/7 PHP. Prevalence of Listeria spp. ranged from 2% to 26% in these five facilities. Drains, entry areas, and portable equipment consistently tested positive for Listeria spp. during active production. Two additional sampling rounds (n = 50/round) were conducted in the highest prevalence facility (Facility #1). Overall, Listeria spp. were detected in 44/150 (29.3%) swabs collected from Facility #1. This study demonstrated the high prevalence of Listeria spp. near raw product entry points across PHP facilities.

Tailoring Atomoxetine Release Rate from DLP 3D-Printed Tablets Using Artificial Neural Networks: Influence of Tablet Thickness and Drug Loading.

Various three-dimensional printing (3DP) technologies have been investigated so far in relation to their potential to produce customizable medicines and medical devices. The aim of this study was to examine the possibility of tailoring drug release rates from immediate to prolonged release by varying the tablet thickness and the drug loading, as well as to develop artificial neural network (ANN) predictive models for atomoxetine (ATH) release rate from DLP 3D-printed tablets. Photoreactive mixtures were comprised of poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) 400 in a constant ratio of 3:1, water, photoinitiator and ATH as a model drug whose content was varied from 5% to 20% (w/w). Designed 3D models of cylindrical shape tablets were of constant diameter, but different thickness. A series of tablets with doses ranging from 2.06 mg to 37.48 mg, exhibiting immediate- and modified-release profiles were successfully fabricated, confirming the potential of this technology in manufacturing dosage forms on demand, with the possibility to adjust the dose and release behavior by varying drug loading and dimensions of tablets. DSC (differential scanning calorimetry), XRPD (X-ray powder diffraction) and microscopic analysis showed that ATH remained in a crystalline form in tablets, while FTIR spectroscopy confirmed that no interactions occurred between ATH and polymers.

Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy.

De novo heterozygous mutations in STXBP1/Munc18-1 cause early infantile epileptic encephalopathies (EIEE4, OMIM #612164) characterized by infantile epilepsy, developmental delay, intellectual disability, and can include autistic features. We characterized the cellular deficits for an allelic series of seven STXBP1 mutations and developed four mouse models that recapitulate the abnormal EEG activity and cognitive aspects of human STXBP1-encephalopathy. Disease-causing STXBP1 variants supported synaptic transmission to a variable extent on a null background, but had no effect when overexpressed on a heterozygous background. All disease variants had severely decreased protein levels. Together, these cellular studies suggest that impaired protein stability and STXBP1 haploinsufficiency explain STXBP1-encephalopathy and that, therefore, Stxbp1+/- mice provide a valid mouse model. Simultaneous video and EEG recordings revealed that Stxbp1+/- mice with different genomic backgrounds recapitulate the seizure/spasm phenotype observed in humans, characterized by myoclonic jerks and spike-wave discharges that were suppressed by the antiepileptic drug levetiracetam. Mice heterozygous for Stxbp1 in GABAergic neurons only, showed impaired viability, 50% died within 2-3 weeks, and the rest showed stronger epileptic activity. c-Fos staining implicated neocortical areas, but not other brain regions, as the seizure foci. Stxbp1+/- mice showed impaired cognitive performance, hyperactivity and anxiety-like behaviour, without altered social behaviour. Taken together, these data demonstrate the construct, face and predictive validity of Stxbp1+/- mice and point to protein instability, haploinsufficiency and imbalanced excitation in neocortex, as the underlying mechanism of STXBP1-encephalopathy. The mouse models reported here are valid models for development of therapeutic interventions targeting STXBP1-encephalopathy.

Structural disorder of plasmid-encoded proteins in Bacteria and Archaea.

BACKGROUND: In the last decade and a half it has been firmly established that a large number of proteins do not adopt a well-defined (ordered) structure under physiological conditions. Such intrinsically disordered proteins (IDPs) and intrinsically disordered (protein) regions (IDRs) are involved in essential cell processes through two basic mechanisms: the entropic chain mechanism which is responsible for rapid fluctuations among many alternative conformations, and molecular recognition via short recognition elements that bind to other molecules. IDPs possess a high adaptive potential and there is special interest in investigating their involvement in organism evolution. RESULTS: We analyzed 2554 Bacterial and 139 Archaeal proteomes, with a total of 8,455,194 proteins for disorder content and its implications for adaptation of organisms, using three disorder predictors and three measures. Along with other findings, we revealed that for all three predictors and all three measures (1) Bacteria exhibit significantly more disorder than Archaea; (2) plasmid-encoded proteins contain considerably more IDRs than proteins encoded on chromosomes (or whole genomes) in both prokaryote superkingdoms; (3) plasmid proteins are significantly more disordered than chromosomal proteins only in the group of proteins with no COG category assigned; (4) antitoxin proteins in comparison to other proteins, are the most disordered (almost double) in both Bacterial and Archaeal proteomes; (5) plasmidal proteins are more disordered than chromosomal proteins in Bacterial antitoxins and toxin-unclassified proteins, but have almost the same disorder content in toxin proteins. CONCLUSION: Our results suggest that while disorder content depends on genome and proteome characteristics, it is more influenced by functional engagements than by gene location (on chromosome or plasmid).

Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE.

A novel genomic island (LGI1) was discovered in Listeria monocytogenes isolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenes emrE [emrELm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion of lmo1851 had no effect on the L. monocytogenes stress response, and deletion of sel1 did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion of emrE resulted in increased susceptibility to QACs (P < 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 µg/ml), 14/16 LGI1 genes were induced, and lmo1861 (putative repressor gene) was constitutively expressed at 4 °C, 37 °C, and 52 °C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 µg/ml), upregulation of emrE (49.6-fold), lmo1851 (2.3-fold), lmo1861 (82.4-fold), and sigB (4.1-fold) occurred. Reserpine visibly suppressed the growth of the ΔemrELm strain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance of L. monocytogenes to QACs via emrELm. Since QACs are commonly used in the food industry, there is a concern that L. monocytogenes strains possessing emrE will have an increased ability to survive this stress and thus to persist in food processing environments.

Antihyperalgesic/antinociceptive effects of ceftriaxone and its synergistic interactions with different analgesics in inflammatory pain in rodents.

BACKGROUND: The ß-lactam antibiotic ceftriaxone stimulates glutamate transporter GLT-1 expression and is effective in neuropathic and visceral pain models. This study examined the effects of ceftriaxone and its interactions with different analgesics (ibuprofen, celecoxib, paracetamol, and levetiracetam) in somatic and visceral pain models in rodents. METHODS: The effects of ceftriaxone (intraperitoneally/intraplantarly), analgesics (orally), and their combinations were examined in the carrageenan-induced paw inflammatory hyperalgesia model in rats (n = 6-12) and in the acetic acid-induced writhing test in mice (n = 6-10). The type of interaction between ceftriaxone and analgesics was determined by isobolographic analysis. RESULTS: Pretreatment with intraperitoneally administered ceftriaxone (10-200 mg/kg per day) for 7 days produced a significant dose-dependent antihyperalgesia in the somatic inflammatory model. Acute administration of ceftriaxone, via either intraperitoneal (10-200 mg/kg) or intraplantar (0.05-0.2 mg per paw) routes, produced a significant and dose-dependent but less efficacious antihyperalgesia. In the visceral pain model, significant dose-dependent antinociception of ceftriaxone (25-200 mg/kg per day) was observed only after the 7-day pretreatment. Isobolographic analysis in the inflammatory hyperalgesia model revealed approximately 10-fold reduction of doses of both drugs in all examined combinations. In the visceral nociception model, more than 7- and 17-fold reduction of doses of both drugs was observed in combinations of ceftriaxone with ibuprofen/paracetamol and celecoxib/levetiracetam, respectively. CONCLUSIONS: Ceftriaxone exerts antihyperalgesia/antinociception in both somatic and visceral inflammatory pain. Its efficacy is higher after a 7-day pretreatment than after acute administration. The two-drug combinations of ceftriaxone and the nonsteroidal analgesics/levetiracetam have synergistic interactions in both pain models. These results suggest that ceftriaxone, particularly in combinations with ibuprofen, celecoxib, paracetamol, or levetiracetam, may provide useful approach to the clinical treatment of inflammation-related pain.

Cold growth behaviour and genetic comparison of Canadian and Swiss Listeria monocytogenes strains associated with the food supply chain and human listeriosis cases.

Sixty-two strains of Listeria monocytogenes isolated in Canada and Switzerland were investigated. Comparison based on molecular genotypes confirmed that strains in these two countries are genetically diverse. Interestingly strains from both countries displayed similar range of cold growth phenotypic profiles. Based on cold growth lag phase duration periods displayed in BHI at 4 °C, the strains were similarly divided into groups of fast, intermediate and slow cold adaptors. Overall Swiss strains had faster exponential cold growth rates compared to Canadian strains. However gene expression analysis revealed no significant differences between fast and slow cold adapting strains in the ability to induce nine cold adaptation genes (lmo0501, cspA, cspD, gbuA, lmo0688, pgpH, sigB, sigH and sigL) in response to cold stress exposure. Neither was the presence of Stress survival islet 1 (SSI-1) analysed by PCR associated with enhanced cold adaptation. Phylogeny based on the sigL gene subdivided strains from these two countries into two major and one minor cluster. Fast cold adaptors were more frequently in one of the major clusters (cluster A), whereas slow cold adaptors were mainly in the other (cluster B). Genetic differences between these two major clusters are associated with various amino acid substitutions in the predicted SigL proteins. Compared to the EGDe type strain and most slow cold adaptors, most fast cold adaptors exhibited five identical amino acid substitutions (M90L, S203A/S203T, S304N, S315N, and I383T) in their SigL proteins. We hypothesize that these amino acid changes might be associated with SigL protein structural and functional changes that may promote differences in cold growth behaviour between L. monocytogenes strains.

E-commerce readiness and training needs of small-scale dairy processors in Serbia: Understanding barriers and knowledge gaps.

The objective of this study was to identify the requirements needed for selling dairy products through e-commerce, as well as current gaps and challenges that exist for small scale dairy processors (SSDPs), and need to be addressed in order to comply with those requirements. A mixed method research design was used for training needs assessment. Qualitative (in-depth interview with 7 online platform representatives (OPRs)) and quantitative approach (survey questionnaire with 58 SSDPs) were conducted. Interview transcripts were coded and codes were grouped into seven themes. Hierarchical cluster analysis was applied to 146 answers from 58 SSDPs. They were divided into 4 clusters. Mean sums of responses between clusters were compared by Mann-Whitney U test. OPRs suggested that SSDPs should be provided with tools and resources to help them achieve food safety and quality targets, as well as practical knowledge and skills. They reported that it is crucial to find a solution for the cold chain transportation, for maintaining consistent product quality. Survey results showed that SSDPs use kitchen equipment (79.3%) and kitchen cleaning products (81.0%) for dairy processing. In total, 43.1% process raw milk and only 24.1% have product label on the package. Only members of cluster 3 and 4 sell their products online (73.7% and 90.0%, respectively), mostly using their own social media platforms (57.9% and 60.0%, respectively), transporting products to end buyers by themselves in hand refrigerators (47.4% and 70.0%, respectively). By analyzing the differences among clusters of SSDPs, trainings can be tailored to the characteristics and knowledge gaps of each group.

Microbiological Assessment of Dairy Products Produced by Small-Scale Dairy Producers in Serbia.

The microbiological quality of dairy products from small-scale producers in Serbia was analysed. A total of 302 dairy products [raw (n = 111) and pasteurized milk cheeses (n = 79) and kajmak (n = 112)], were collected and tested for the presence of pathogens, Listeria monocytogenes and Salmonella spp., and enumerated for Coagulase-positive staphylococci (CPS), Escherichia coli, and yeasts and moulds. None of the samples tested positive for Salmonella spp., while L. monocytogenes was recovered from one raw milk cheese and five kajmak samples. Raw milk cheese and kajmak also had higher levels of indicator microorganisms, namely E. coli and yeast and moulds. Molecular serotyping grouped L. monocytogenes isolates into serogroups 1 (1/2a and 3a) and 3 (1/2b, 3b, and 7). When exposed to eight antibiotics, L. monocytogenes isolates were mostly sensitive, with the exception of oxacillin and reduced susceptibility to clindamycin, penicillin G, and trimethoprim/sulfamethoxazole, emphasizing the importance of continuous surveillance for antimicrobial resistance. Samples that tested positive for Listeria spp. also had higher loads of indicator microorganisms, namely E. coli and yeast and moulds, suggesting lapses in hygiene practices during production. Collectively, these data emphasize the need for improved food safety and hygiene practices among small-scale dairy producers. This is crucial to reduce the microbial contamination and improve both the quality and safety of dairy products in the Serbian market.

Genomic characterization of Listeria monocytogenes recovered from dairy facilities in British Columbia, Canada from 2007 to 2017.

Listeria monocytogenes is a foodborne pathogen of concern in dairy processing facilities, with the potential to cause human illness and trigger regulatory actions if found in the product. Monitoring for Listeria spp. through environmental sampling is recommended to prevent establishment of these microorganisms in dairy processing environments, thereby reducing the risk of product contamination. To inform on L. monocytogenes diversity and transmission, we analyzed genome sequences of L. monocytogenes strains (n = 88) obtained through the British Columbia Dairy Inspection Program. Strains were recovered from five different dairy processing facilities over a 10 year period (2007-2017). Analysis of whole genome sequences (WGS) grouped the isolates into nine sequence types and 11 cgMLST types (CT). The majority of isolates (93%) belonged to lineage II. Within each CT, single nucleotide polymorphism (SNP) differences ranged from 0 to 237 between isolates. A highly similar (0-16 SNPs) cluster of over 60 isolates, collected over 9 years within one facility (#71), was identified suggesting a possible persistent population. Analyses of genome content revealed a low frequency of genes associated with stress tolerance, with the exception of widely disseminated cadmium resistance genes cadA1 and cadA2. The distribution of virulence genes and mutations within internalin genes varied across the isolates and facilities. Further studies are needed to elucidate their phenotypic effect on pathogenicity and stress response. These findings demonstrate the diversity of L. monocytogenes isolates across dairy facilities in the same region. Findings also showed the utility of using WGS to discern potential persistence events within a single facility over time.

Examination of food chain-derived Listeria monocytogenes strains of different serotypes reveals considerable diversity in inlA genotypes, mutability, and adaptation to cold temperatures.

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.

Antimicrobial resistance and co-selection phenomenon in Listeria spp. recovered from food and food production environments.

Antimicrobial resistance (AMR), co-selection phenomenon, and the relationship between reduced susceptibility (RSC) to ciprofloxacin (CIP) and resistance to other antimicrobials in Listeria spp. (n = 103) recovered from food processing environments (FPE) and food were investigated. Resistance of Listeria monocytogenes and other listeriae, respectively, to cefoxitin (FOX; 98% vs. 88%), CIP (7% vs. 4%), clindamycin (CLI; 33% vs. 59%) and tetracycline (6% vs. 8%) was observed, as was RSC to CIP (67% vs. 57%) and CLI (65% vs. 41%). L. monocytogenes also possessed RSC to linezolid (LZD; 6%), rifampicin (2%) and streptomycin (6%), with other listeriae displaying RSC to chloramphenicol (4%). L. monocytogenes serotype 1/2a (90%) isolates were more frequently resistant or possessed RSC to CIP compared to serotype 4b (55%) (p = 0.015). When eight strains were experimentally adapted to high concentrations of CIP, co-selection occurred as MICs to benzalkonium chloride (BAC) increased (n = 5), gentamicin MICs remained the same (n = 6) or increased 2-fold (n = 2), and led to RSC to LZD (n = 1) and resistance to CLI (n = 8). Overall, levels of resistance/RSC to CIP in food chain isolates, particularly 1/2a, are concerning. Further, reduced sensitivity to disparate antimicrobials following CIP exposure highlights the need for increased knowledge of co-selection phenomenon linked with antimicrobial agents.

Before and After: Evaluation of Microbial and Organic Loads in Produce Handling and Packing Operations with Diverse Cleaning and Sanitizing Procedures.

Inadequate cleaning and/or sanitation (C/S) of food contact surfaces (FCSs) has been frequently reported during Produce Safety Rule inspections; however, limited data are available evaluating the effectiveness of C/S processes in produce operations. Different C/S practices were evaluated in four fresh produce operations for their efficacy in reducing microbial and organic loads on various FCSs. Microbial (aerobic plate counts; APC) and organic (ATP) loads were quantified during production, after cleaning, and after sanitizing, if applicable. Operations included: a berry packinghouse (BerryPK; wet cleaning), a blueberry harvest contractor (BerryHC; cleaning + sanitizing, C+S), and two mixed vegetable packinghouses (MixedV1; C+S, and MixedV2; rinsing + sanitizing, R+S). Following wet cleaning, significant reductions in APCs (p < 0.05) were seen on high-density polyethylene (HDPE) storage trays (n = 50) in BerryPK (3.1 ± 0.9 to 2.5 ± 0.7 log CFU/100 cm2). In BerryHC, a greater reduction in APCs was seen on HDPE harvest buckets (n = 25) following C+S (3.8 ± 0.5 to 1.1 ± 0.4 log CFU/100 cm2), compared to wet cleaning only in BerryPK. Stainless steel and conveyor belt FCSs (n = 16) in MixedV1 were sampled, and a significant reduction in APCs (p < 0.05) was observed when comparing in-use (4.8 ± 1.3 log CFU/100 cm2) to post-C+S (3.9 ± 0.7 log CFU/100 cm2). When similar FCSs (n = 17) were sampled in MixedV2, R+S also led to significant reduction in APCs (3.3 ± 0.6 to 1.9 ± 0.6 log CFU/100 cm2) (p < 0.05). ATP testing in fresh produce settings yielded inconsistent results, with no correlation between organic and bacterial loads detected during production (R2 = 0.00) across four operations, and weak correlations observed after cleaning (R2 = 0.18) and after sanitation (R2 = 0.33). The results from this study provide the foundational basis for future research on practical and effective C/S methods tailored to the produce industry.

Occurrence and characterization of Listeria spp. in ready-to-eat retail foods from Vancouver, British Columbia.

The occurrence of Listeria spp. and Listeria monocytogenes in retail RTE meat and fish products in Vancouver, British Columbia (B.C.) was investigated. To assess potential consumer health risk, recovered L. monocytogenes isolates were subjected to genotypic and phenotypic characterization. Conventional methods were used to recover Listeria spp. from deli meat (n = 40) and fish (n = 40) samples collected from 17 stores. Listeria spp. were recovered only from fish samples (20%); 5% harboured Listeria innocua, 5% had L. monocytogenes and 10% contained Listeria welshimeri. L. monocytogenes isolates serotyped as 1/2a and 1/2b, possessed dissimilar PFGE patterns, and had full-length InlA. Three 1/2a clonal isolates encoded the 50 kb genomic island, LGI1. Antimicrobial resistance (AMR) profiling showed all Listeria spp. possessed resistance to cefoxitin and nalidixic acid. L. monocytogenes were resistant to clindamycin, two were resistant to streptomycin, and one to amikacin. Reduced susceptibility to ciprofloxacin was seen in all L. monocytogenes, L. innocua and three L. welshimeri isolates. Reduced susceptibility to amikacin and chloramphenicol was also observed in one L. monocytogenes and three L. welshimeri isolates, respectively. Recovery of L. monocytogenes in fish samples possessing AMR, full-length InlA, LGI1, and serotypes frequently associated with listeriosis suggest B.C. consumers are exposed to high-risk strains.