Co-Occurrence of L. monocytogenes with Other Bacterial Genera and Bacterial Diversity on Cleaned Conveyor Surfaces in a Swine Slaughterhouse.

Bacterial pathogens, such as Listeria monocytogenes, can show resistance to disinfection and persistence on working surfaces, permitting them to survive and contaminate food products. Persistence-a complex phenomenon involving interactions between many bacteria within a biofilm-is modulated by in situ characteristics. This study aimed to describe, in silico, the microbiota identified in a swine slaughterhouse after sanitation procedures to better understand the presence of L. monocytogenes on these surfaces. Molecular tools for characterization of microbial communities were used to assess the relative contribution of different bacteria resulting from this phenomenon, and the 16S rRNA sequencing method was used on samples from meat conveyor belt surfaces collected on four sampling visits to study the co-occurrence between L. monocytogenes and other bacteria. From the background microbiota, a total of six genera were found to be negatively correlated with Listeria spp., suggesting Listeria growth inhibition, competition, or at least an absence of shared habitats. Based on these results, a complete scenario of interactions of Listeria with components of background microbiota was established. This work contributes to identifying avenues that could prevent the growth and persistence of L. monocytogenes on food-processing surfaces.

Counteracting Bacterial Motility: A Promising Strategy to Narrow Listeria monocytogenes Biofilm in Food Processing Industry.

Listeria monocytogenes (L. monocytogenes) is often associated with processed food as it can form biofilms that represent a source of contamination at all stages of the manufacturing chain. The control and prevention of biofilms in food-processing plants are of utmost importance. This study explores the efficacy of prospect molecules for counteracting bacterial mechanisms leading to biofilm formation. The compounds included the phytomolecule tomatidine, zinc chloride (ZnCl2), ethylenediaminetetraacetic acid (EDTA), and a more complexed mixture of bacterial compounds from coagulase-negative staphylococci (CNS exoproducts). Significant inhibition of L. monocytogenes biofilm formation was evidenced using a microfluidic system and confocal microscopic analyses (p < 0.001). Active molecules were effective at an early stage of biofilm development (≥50% of inhibition) but failed to disperse mature biofilms of L. monocytogenes. According to our findings, prevention of surface attachment was associated with a disruption of bacterial motility. Indeed, agar cell motility assays demonstrated the effectiveness of these molecules. Overall, results highlighted the critical role of motility in biofilm formation and allow to consider flagellum-mediated motility as a promising molecular target in control strategies against L. monocytogenes in food processing environments.

Distribution, diversity and persistence of Listeria monocytogenes in swine slaughterhouses and their association with food and human listeriosis strains.

Listeria monocytogenes is the etiological agent of listeriosis, a major foodborne disease and an important public health concern. Contamination of meat with L. monocytogenes occurs frequently at the slaughterhouse. Our aims were; 1) to investigate the distribution of L. monocytogenes in the processing areas of four swine slaughterhouses; 2) to describe the diversity of L. monocytogenes strains by pulsed-field gel electrophoresis; 3) to identify persistent L. monocytogenes strains and describe their distribution; 4) to investigate the associations between persistence of strains and their following characteristics: detection in food isolates, detection in human clinical isolates, and the presence of benzalkonium chloride (BAC) resistance genes. Various operation areas within the four swine slaughterhouses were sampled on four occasions. A total of 2496 samples were analyzed, and L. monocytogenes was successfully isolated from 243 samples. The proportion of positive samples ranged from 32 to 58% in each slaughterhouse and from 24 to 68% in each operation area. Fifty-eight different pulsotypes were identified and eight pulsotypes, present in samples collected during 4 visits, were considered persistent. The persistent pulsotypes were significantly more likely to be detected in food (P < 0.01, exact χ²) and human clinical cases (P < 0.01, exact χ²), respectively. Among pulsotypes harboring the BAC bcrABC resistance cassette or the emrE multidrug transporter gene, 42.8% were persistent compared to 4.5% for pulsotypes without these resistance genes (P < 0.01, exact χ²). Our study highlights the importance of persistent L. monocytogenes strains in the environmental contamination of slaughterhouses, which may lead to repeated contamination of meat products. It also shows that the presence of disinfectants resistance genes is an important contributing factor.

Genomic characterization of Listeria monocytogenes isolates reveals that their persistence in a pig slaughterhouse is linked to the presence of benzalkonium chloride resistance genes.

BACKGROUND: The aim of this study was to characterize the genomes of 30 Listeria monocytogenes isolates collected at a pig slaughterhouse to determine the molecular basis for their persistence. RESULTS: Comparison of the 30 L. monocytogenes genomes showed that successive isolates (i.e., persistent types) recovered from thew sampling site could be linked on the basis of single nucleotide variants confined to prophage regions. In addition, our study revealed the presence among these strains of the bcrABC cassette which is known to produce efflux pump-mediated benzalkonium chloride resistance, and which may account for the persistence of these isolates in the slaughterhouse environment. The presence of the bcrABC cassette was confirmed by WGS and PCR and the resistance phenotype was determined by measuring minimum inhibitory concentrations. Furthermore, the BC-resistant strains were found to produce lower amounts of biofilm in the presence of sublethal concentrations of BC. CONCLUSIONS: High resolution SNP-based typing and determination of the bcrABC cassette may provide a means of distinguishing between resident and sporadic L. monocytogenes isolates, and this in turn will support better management of this pathogen in the food industry.

Characterisation of InlA truncation in Listeria monocytogenes isolates from farm animals and human cases in the province of Quebec.

The introduction of Listeria monocytogenes into the food production chain is a concern, with numerous grouped cases of listeriosis associated with milk-derived or pork-derived products have been documented. Management of this zoonotic pathogen considers all strains as an equal risk. Recently, a new perspective for characterisation of strain virulence was introduced with the discovery of the unaltered sequence of InlA as a determinant of strain virulence; this has also been reported as an infrequent finding among so-called environmental strains, that is, strains isolated from food or from surfaces in food industries. The aim of this study was to differentiate L monocytogenes strains isolated from animal cases versus those from human cases and to differentiate clinical strains from environmental ones using a Caenorhabditis elegans virulence testing model. In Quebec in 2013/2014, the surveillance of L monocytogenes clinical isolates registered a total of 20 strains of animal origin and 16 pulsed-field gel electrophoresis types isolated from human cases. The mixed PCR multiplex agglutination protocol used for geno-serotyping clearly discriminated genogroup IVB strains from bovine and human origins. The presence of a premature stop codon single nucleotide polymorphism in the inlA gene sequence in clinical strains and the identical behaviour of particular strains in the C elegans model are discussed in this paper from the perspective of industrial management of L monocytogenes risk.

Impact of Nutrient Restriction on the Structure of Listeria monocytogenes Biofilm Grown in a Microfluidic System.

Biofilm formation by the pathogen Listeria monocytogenes is a major concern in food industries. The aim of this work was to elucidate the effect of nutrient limitation on both biofilm architecture and on the viability of the bacteria in microfluidic growth conditions. Biofilm formation by two L. monocytogenes strains was performed in a rich medium (BHI) and in a 10-fold diluted BHI (BHI/10) at 30°C for 24 h by using both static conditions and the microfluidic system Bioflux. In dynamic conditions, biofilms grown in rich and poor medium showed significant differences as well in structure and in the resulting biovolume. In BHI/10, biofilm was organized in a knitted network where cells formed long chains, whereas in the rich medium, the observed structure was homogeneous cellular multilayers. Biofilm biovolume production in BHI/10 was significantly higher than in BHI in these dynamic conditions. Interestingly, biovolume of dead cells in biofilms formed under limited nutrient conditions (BHI/10) was significantly higher than in biofilms formed in the BHI medium. In the other hand, in static conditions, biofilm is organized in a multilayer cells and dispersed cells in a rich medium BHI and poor medium BHI/10 respectively. There was significantly more biomass in the rich medium compared to BHI/10 but no difference was noted in the dead/damaged subpopulation showing how L. monocytogenes biofilm could be affected by the growth conditions. This work demonstrated that nutrient concentration affects biofilm structure and the proportion of dead cells in biofilms under microfluidic condition. Our study also showed that limited nutrients play an important role in the structural stability of L. monocytogenes biofilm by enhancing cell death and liberating extracellular DNA.

Single cell synchrotron FT-IR microspectroscopy reveals a link between neutral lipid and storage carbohydrate fluxes in S. cerevisiae.

In most organisms, storage lipids are packaged into specialized structures called lipid droplets. These contain a core of neutral lipids surrounded by a monolayer of phospholipids, and various proteins which vary depending on the species. Hydrophobic structural proteins stabilize the interface between the lipid core and aqueous cellular environment (perilipin family of proteins, apolipoproteins, oleosins). We developed a genetic approach using heterologous expression in Saccharomyces cerevisiae of the Arabidopsis thaliana lipid droplet oleosin and caleosin proteins AtOle1 and AtClo1. These transformed yeasts overaccumulate lipid droplets, leading to a specific increase in storage lipids. The phenotype of these cells was explored using synchrotron FT-IR microspectroscopy to investigate the dynamics of lipid storage and cellular carbon fluxes reflected as changes in spectral fingerprints. Multivariate statistical analysis of the data showed a clear effect on storage carbohydrates and more specifically, a decrease in glycogen in our modified strains. These observations were confirmed by biochemical quantification of the storage carbohydrates glycogen and trehalose. Our results demonstrate that neutral lipid and storage carbohydrate fluxes are tightly connected and co-regulated.