Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopic Observation

International audienceFoodborne pathogens are responsible of foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This extracellular matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can enter a viable but nonculturable (VBNC) state. VBNC cells are characterized by a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples, and thus poses a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method with a combination of propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain

Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopic Observation

International audienceFoodborne pathogens are responsible of foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This extracellular matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can enter a viable but nonculturable (VBNC) state. VBNC cells are characterized by a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples, and thus poses a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method with a combination of propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain

Phenotypic and genotypic characterization of H2S-positive and H2S-negative strains of Shewanella baltica isolated from spoiled whiting ( Merlangius merlangus )

International audienceFour strains were isolated from a spoiled whiting (Merlangius merlangus). All of them were able to grow aerobically from 4 to 30°C and also able to develop anaerobically in the presence of trimethylamine N-Oxide (TMAO) at 25°C. Biochemical characterization did not allow identification of the strains species but showed that one of the four strains was unable to produce H2 S. Two strains synthetized an ornithine decarboxylase being potential putrescine producers. Results of carbon source use highlighted that the four strains were able to use citrate and d-sucrose and one strain was not able to use l-arabinose. Genotypic characterization of the strains thanks to 16S rRNA and gyrB partial gene sequencing led to their identification as members of Shewanella baltica species. These observations suggest that H2 S production may not be the most appropriate screening parameter for Shewanella species and further to monitor the development of spoilage flora

Variable adhesion of Listeria monocytogenes isolates from food-processing facilities and clinical cases to inert surfaces.

One hundred one strains of Listeria monocytogenes isolated from seafood and cheese industry samples and from patients with listeriosis were assessed using a microtiter plate method for adhesion to polystyrene and stainless steel surfaces. The adhesion rate for these strains ranged from 3.10 to 35.29% with an inoculum of 8 x 10(8) cells per well. A strong correlation was found between adhesion to polystyrene and stainless steel microtiter plates, indicating that the intrinsic ability of L. monocytogenes to adhere to inert surfaces is stronger than the influence of the surface's physicochemical properties. The clinical strains were less adherent to inert surfaces than were the industrial strains. By integrating other factors such as location of the industrial strains, contamination type of the clinical strains, serotype, and pulsotype into the analysis, some weak but significant differences were noted. For the industrial isolates, the number of cells attached to both surfaces differed significantly depending on whether they were isolated from food or food-processing environments in the seafood and cheese industry. For clinical isolates, sporadic strains exhibited greater adhesion to polystyrene than did epidemic strains. Strains belonging to the pulsed-field gel electrophoretype clusters A and M (lineages II and I, respectively) were less able to adhere to polystyrene and stainless steel than were strains in the more common clusters

Teichoic acid is the major polysaccharide present in theListeria monocytogenesbiofilm matrix

International audienceThe aim of this study was to characterize the Listeria monocytogenes biofilm and particularly the nature of the carbohydrates in the biofilm extracellular matrix and culture supernatant versus to cell wall carbohydrates. Listeria monocytogenes serotype 1/2a and 4b strains were able to form complex biofilms embedded in an extracellular matrix. The soluble carbohydrates from biofilm extracellular matrix and culture supernatant were identified as teichoic acids, structurally identical to cell wall teichoic acids. In addition, the DSS 1130 BFA2 strain had a serotype 1/2a teichoic acid lacking N-acetyl glucosamine glycosylation due to a mutation in the lmo2550 gene. Consequently, we hypothesized that the extracellular teichoic acids in L. monocytogenes biofilms have the same origin as cell wall teichoic acid

<em>Vibrio</em> species involved in seafood-borne outbreaks (<em>vibrio cholerae, v-parahaemolyticus and v-vulnificus</em>): Review of microbiological versus recent molecular detection methods in seafood products

International audienceSeafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872-1:2007 and TS 21872-2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood

Dynamics of mobile genetic elements of Listeria monocytogenes persisting in ready-to-eat seafood processing plants in France

Background: Listeria monocytogenes Clonal Complexes (CCs) have been epidemiologically associated with foods, especially ready-to-eat (RTE) products for which the most likely source of contamination depends on the occurrence of persisting clones in food-processing environments (FPEs). As the ability of L. monocytogenes to adapt to environmental stressors met in the food chain challenges the efforts to its eradication from FPEs, the threat of persistent strains to the food industry and public health authorities continues to rise. In this study, 94 food and FPEs L. monocytogenes isolates, representing persistent subtypes contaminating three French seafood facilities over 2–6 years, were whole-genome sequenced to characterize their genetic diversity and determine the biomarkers associated with long-term survival in FPEs.Results: Food and FPEs isolates belonged to five CCs, comprising long-term intra- and inter-plant persisting clones. Mobile genetic elements (MGEs) such as plasmids, prophages and transposons were highly conserved within CCs, some of which harboured genes for resistance to chemical compounds and biocides used in the processing plants. Some of these genes were found in a 90.8 kbp plasmid, predicted to be” mobilizable”, identical in isolates from CC204 and CC155, and highly similar to an 81.6 kbp plasmid from isolates belonging to CC7. These similarities suggest horizontal transfer between isolates, accompanied by deletion and homologous recombination in isolates from CC7. Prophage profiles characterized persistent clonal strains and several prophage-loci were plant-associated. Notably, a persistent clone from CC101 harboured a novel 31.5 kbp genomic island that we named Listeria genomic island 3 (LGI3), composed by plant-associated loci and chromosomally integrating cadmium-resistance determinants cadA1C.Conclusions: Genome-wide analysis indicated that inter- and intra-plant persisting clones harbour conserved MGEs, likely acquired in FPEs and maintained by selective pressures. The presence of closely related plasmids in L. monocytogenes CCs supports the hypothesis of horizontal gene transfer conferring enhanced survival to FPE-associated stressors, especially in hard-to-clean harbourage sites. Investigating the MGEs evolutionary and transmission dynamics provides additional resolution to trace-back potentially persistent clones. The biomarkers herein discovered provide new tools for better designing effective strategies for the removal or reduction of resident L. monocytogenes in FPEs to prevent contamination of RTE seafood

Adhesion to stainless steel surfaces and detection of viable but non cultivable cells of Vibrio parahaemolyticus and Vibrio cholerae isolated from shrimps in seafood processing environments: Stayin' alive?

International audienceA single strain of Vibrio parahaemolyticus and a single strain of Vibrio cholerae were isolated from shrimps sourced from seafood processing plants. The isolated strains were evaluated for their ability to adhere to stainless steel surfaces, under conditions encountered in seafood industries. In this way, the impact of different environmental factors such as temperature (8 degrees C or 37 degrees C) and culture media (tryptic soy broth (TSB) 2% NaCl, artificial sea water (ASW) or industrial brine) were studied. The viability of these strains was analyzed by three different methods: the enumeration of cultivable bacteria on agar media, and two PCR methods, i.e. a qPCR and a PMA-qPCR method in order to distinguish between viable and dead cells. The proportion of viable but non-cultivable (VBNC) cells was evaluated by comparing the results of propidium monoazide real-time PCR (PMA-qPCR) with the enumeration results. Cultivable bacteria were not detected in brine, regardless of the conditions tested. However, V. cholerae in the VBNC state was detected in brine at 8 degrees C until 48 h of incubation. V. cholerae exhibited higher viability at 8 degrees C compared to 37 degrees C. V. cholerae also exhibited higher viability compared to V. parahaemolyticus. In contrast, for V. parahaemolyticus, temperature and media (except industrial brine) had little influence on cell viability and adhesion behavior

The absence of N-acetylglucosamine in wall teichoic acids of Listeria monocytogenes modifies biofilm architecture and tolerance to rinsing and cleaning procedures

The wall teichoic acid (WTA) is the major carbohydrate found within the extracellular matrix of the Listeria monocytogenes biofilm. We first addressed the frequency of spontaneous mutations in two genes (lmo2549 and lmo2550) responsible for the GlcNAcylation in 93 serotype 1/2a strains that were mainly isolated from seafood industries. We studied the impact of mutations in lmo2549 or lmo2550 genes on biofilm formation by using one mutant carrying a natural mutation inactivating the lmo2550 gene (DSS 1130 BFA2 strain) and two EGD-e mutants that lack respective genes by in-frame deletion of lmo2549 or lmo2550 using splicing-by-overlap-extension PCR, followed by allelic exchange mutagenesis. The lmo2550 gene mutation, occurring in around 50% isolates, caused a decrease in bacterial adhesion to stainless steel compared to wild-type EGD-e strain during the adhesion step. On the other hand, bacterial population weren’t significantly different after 24h-biofilm formation. The biofilm architecture was different between the wild-type strain and the two mutants inactivated for lmo2549 or lmo2550 genes respectively with the presence of bacterial micro-colonies for mutants which were not observed in the wild-type EGD-e strain biofilm. These differences might account for the stronger hydrophilic surface exhibited by the mutant cells. Upon a water flow or to a cleaning procedure at a shear stress of 0.16 Pa, the mutant biofilms showed the higher detachment rate compared to wild-type strain. Meanwhile, an increase in the amount of residual viable but non-culturable population on stainless steel was recorded in two mutants. Our data suggests that the GlcNAc residue of WTA played a role in adhesion and biofilm formation of Listeria monocyctogenes.ISSN:1932-620