The contamination of food processing equipment by pathogenic bacteria is a serious threat to food safety and public health. Much attention has recently been focussed on Listeriae because of their ability to both grow over a wide temperature range and to adhere to a wide range of materials. However, whilst many previous studies have studied attachment under static or low Reynolds number (Re) flow, no published work exists on the effects of flow conditions at industrially relevant Re on the phenomenon of attachment. This is examined here using non-pathogenic Listeria innocua—frequently claimed to be a surrogate for the pathogen L. monocytogenes—in relation to stainless steel. This material was chosen because it is widely used in the food industry.

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It was shown that Listeria monocytogenes cells grown in a defined minimal, MCDB202, showed enhanced extracellular polymeric substances production compared to BHI. On the other hand, it was reported that in L. monocytogenes luxS mutant, AI-2 reduction and biofilm enhancement were seen. It is hypotheses that there could be a linkage between the AI-2 signaling system and the EPS formation. The expression of EPS could be induced by the reduction in AI-2. The main aim of the research is to study this EPS formation in minimal media, how is it linked to AI-2 production, the function of the EPS as well as to figure out the linkage between EPS formation with cap genes found in Listeria genome. It was shown that MCDB202 have caused an increase in surface hydrophobicity of the cells. However, cells grown in the defined media did not induced better attachment and biofilm formation towards hydrophobic surfaces. And cells grown in MCDB202 were shown less capable to infect eukaryotic cells in the cell invasion assay. On the other hand, AI-2 production was shown to be relative lower in Listeria cell grown in minimal media MCDB202) than rich media (BHI). Bioinformatics study has shown that only capA homologues, but no capBCDE homologues, were found in Listeria genome. However, the bioinformatics works have shown that the capA homologues are unlikely to be contributing the EPS seen produced in Listeria monocytogenes. This was further supported in the expression assay that the two genes were not highly expressed in MCDB media.

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L’objectif de ces travaux a été d’étudier le rôle des exopolysaccharides dans la formation des biofilms de Listeria monocytogenes et dans leur résistance face à des procédures de nettoyage rencontrées en entreprise dans des circuits fermés. Nous avons montré que l’exopolysaccharide majeur présent dans la matrice extracellulaire des biofilms de L. monocytogenes était de l’acide téichoïque identique à l’acide téichoïque pariétal. Nous avons identifié que sur 93 souches de sérotype 1/2a étudiées la moitié présentaient une mutation sur le gène lmo2550 ce qui pouvait entraîner la non-ramification de l’acide téichoïque avec le résidu N-Acétylglucosamine (GlcNAc). Des mutants de la souche de référence EGD-e inactivés pour les gènes lmo2549 ou lmo2550 intervenant dans la ramification de l’acide teichoïque par le GlcNAc ainsi que des mutants inactivés pour les gènes lmo2537 ou tagO1tagO2 intervenant dans la voie de biosynthèse des acides téichoïques ont été étudiés. Cela nous a permis de montrer que l’absence de GlcNAc sur les acides téichoïques avait modifié les propriétés de surface de L. monocytogenes, diminué l’adhésion à l’acier inoxydable et modifié l’architecture des biofilms de 48h. Par ailleurs, ils étaient plus sensibles à une procédure de nettoyage en circuit fermé avec un détachement plus important, une modification de l’architecture des biofilms et la présence uniquement de cellules viables non cultivables et mortes après passage d’un flux de soude. Le but de ces travaux était de mieux comprendre le rôle de la matrice extracellulaire dans la formation des biofilms de L. monocytogenes pour ensuite trouver le meilleur moyen de l’éradiquer dans l’environnement industriel. / The aim of this work was to study the role of exopolysaccharides in the formation of Listeria monocytogenes biofilm and their resistance to cleaning procedure in industry. We showed that the major exopolysaccharide present in the extracellular matrix of L. monocytogenes biofilm was teichoic acid, which was identical at the structure to the parietal teichoic acid. We identified that 50% of 93 strains of 1/2a serotype studied had a mutation in the lmo2550 gene that could lead to non-branching of the teichoic acid with the N-Acetylglucosamine residue (GlcNAc). We therefore examined mutants of EGD-e reference strain which had inactivated lmo2549 or lmo2550 or lmo2537 or tagO1tagO2 genes allowing highlighting the absence of the GlcNAc residue on teichoic acids. The mutation of these genes had changed the L. monocytogenes surface properties, decreased the adhesion to stainless steel and modified the architecture of 48h-biofilms. Furthermore, they were more susceptible to a circuit cleaning procedure with an important detachment, a change in the architecture of the biofilm and the presence of non-cultivable but viable cells and dead cells after passage of the caustic soda flow versus the wild-type EGD-e strain. The aim of this work was to better understand the role of the extracellular matrix in the formation of L. monocytogenes biofilm and then find the procedure to eradicate it in the industrial environment.

Listeria monocytogenes is a common food borne pathogen which is an important contaminant found in various food factory environments, because of its ability to survive in a wide range of environmental conditions, and to grow at refrigeration temperatures. L. monocytogenes has caused both occasional outbreaks and sporadic cases of food-borne illness characterised by high mortality rates. In the UK and other European countries, there has been a conspicuous rise in the number of reported cases of “Listeriosis” recently. Hence, development of efficient and rapid methods for detection of this microorganism in various foods is of great significance for the food industry; and is needed to ensure the safety of foods that are considered at high risk of contamination. Conventional bacteriological methods (e.g. ISO 11290-1/A1) for the detection and quantification of L. monocytogenes are laborious and time consuming. Therefore, development of a rapid and reliable test capable of detecting very low numbers of the organism in ready-to-eat products is required. To address this, a phage amplification assay has been developed as a rapid method for the detection of L. monocytogenes using the broad host range phage A511. Successful development of the assay required identification of a virucide that could achieve inactivation of the phage without affecting the viability of the target cell to be detected. Several different substances were evaluated as potential virucides, and among the tested materials, tea infusions were found to be the most effective virucidal agent for this experiment. The efficacy of the new assay was tested using Stilton cheese, as a representative high risk dairy product, and a method was developed to use centrifugation to concentrate bacterial cells present in samples of half-Fraser broth enrichments. The cells were detected by using the new phage amplification assay and this combination of techniques was shown to be able to detect low numbers of cells in shorter times than can be achieved using conventional culture methods. An additional molecular identification step was also developed so that the identity of the cells detected could be confirmed using a multiplex PCR which targeted conserved regions of the Listeria 16S rDNA genes. In this assay, two amplified DNA fragments were generated confirming the presence of Listeria genus (400 bp band) and also L. monocytogenes species (287 bp band). An advantage of this combined phage-PCR method its ability to detect only viable cells in food samples. The combined assay was then tested on a wide range of spiked food samples, including Camembert cheese, pasteurised milk, minced meat, turkey meat and smoked salmon. The obtained results showed that the limit of detection was as low as 20 (± 5) cfu per 25 g, and duration needed for the detection and molecular conformation of speciation was 2 days (44 h), compared to 5 days using conventional culture methods. The combined phage-PCR assay was able to achieve a sensitive and specific identification of viable L. monocytogenes present in foods within 48 h, and therefore would allow for rapid screening of food products prior to release from the factory.

The aim of this project was to examine whether exposure to environmental and physiological stress conditions could affect some functional properties for the selection of probiotic microorganisms. The study was focused on two commercial strains of Bifidobacterium animalis ssp. lactis and two non-commercial Bifidobacterium strains, namely B. breve NCTC 11815 and B. longum NCTC 11818. The effects of exposure to acid, bile, osmotic and oxidative stresses on their antimicrobial activity, biofilm formation capacity and antibiotic susceptibility profiles were assessed. The conditions to generate acid stress in the organisms were chosen as pH 3 for one hour, for both B. animalis ssp. lactis strains, and pH 4 for one hour, for B. breve and B. longum. Conditions for bile stress were 1% (w/v) bile for one hour, for both B. animalis ssp. lactis strains and B. breve, and 0.5% (w/v) bile for one hour, for B. longum. Osmotic stress conditions were 3% (w/v) NaCl for one hour, for both B. animalis ssp. lactis strains and B. breve, and 2% (w/v) NaCl for one hour, for B. longum. Oxidative stress was generated for all organisms by shaking at 200 rpm for two hours. The antimicrobial activities of all four bifidobacteria against pathogenic bacteria, namely Escherichia coli NCTC 12900, Salmonella enterica ser. Typhimurium DT124 and S. enterica ser. Enteritidis PT4, were maintained after exposure to each stress, although there appeared to be lower inhibition after exposure to stress. This varied with strain and type of stress. The antibiotic susceptibility profiles of all four bifidobacteria for five antibiotics, namely tetracycline, erythromycin, ampicillin, chloramphenicol and vancomycin, were unchanged after exposure to each stress. The expression of tetracycline resistance gene tet(W) in one of the B. animalis ssp. lactis strains, designated as strain C, was significantly higher (P ≤ 0.05) after exposure to acid, bile and osmotic stresses, although this did not translate to higher resistance of B. animalis ssp. lactis (C) to tetracycline. Effects of each stress on biofilm formation in the four bifidobacteria varied with the strain. In general, more positive effects of exposure to stress were observed in both B. animalis ssp. lactis strains, while more negative effects of exposure to stress were shown by B. breve and B. longum. The expression of exopolysaccharide-synthesis gene gtf01207 in B. animalis ssp. lactis (C) was significantly higher after exposure to osmotic stress, although it also appeared to be higher after exposure to acid and bile stresses. Studying the effects of exposure to stress on in vitro probiotic selection properties could give a better reflection of what applies in vivo, since microorganisms for probiotic use would be inevitably exposed to stresses. This could give a more accurate insight on the potential to provide health benefit. The results of this study may justify the commercial use of the B. animalis ssp. lactis strains.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a wide ecological niche and causes a number of diseases in human and animals. It invades mammalian host cells and escapes from the vacuoles prior to replication in the host cell cytoplasm and infecting adjacent cells via actin-based mobility. Phosphoinositide (PIP) metabolism is essential to mammalian cells in signal transduction, actin remodelling, endosome dynamics and membrane trafficking. Modulation of host PIP metabolism by bacteria PIP phosphatases is important for pathogenicity and virulence of many human pathogens. In this study the function of two L. monocytogenes tyrosine and inositol phosphatases LipA and LipB were studied in vitro. The lipA and lipB deletion mutants generated in EGDe and InlA strains were not affected in invasion but were attenuated in intracellular growth in Caco-2 and Hela M cell lines but not in mouse macrophages. Deletion of lipA or lipB did not affect the actin polymerisation but caused reduced plaque number in the plaque assay. The turnover of five PIPs in Hela M cells during L. monocytogenes infection were studied by expression of fluorescent protein tagged domains that specifically recognizes individual PIPs. L. monocytognenes did not affect the metabolism of PI4P, PI(4,5)P2, PI(3,4,5)P3 but co-localised with PI3P at 1.5 hr post-infection and with PI(3,4)P2 at 6 hr to 24 hr post-infection. The PI(3,4)P2 effector protein lamellipodin was discovered to be recruited to actin-associated L. monocytogenes at 4 hr to 24 hr post-infection in Hela M cells. This discovery leads to the hypothesis of a novel mechanism of lamellipodin-dependant cell-to-cell spread. The lipA mutant was found to be attenuated in PI(3,4)P2 recruitment and therefore hypothesized to participate in the proposed lamellipodin pathway by converting PI(3,5)P2 into PI5P, leading to the activation of PI3K and subsequent production of PI(3,4)P2. LipB showed partial localisation at the Golgi complex when over-expressed in Hela M cells, and it was assumed to act mainly as a protein-tyrosine phosphatase. In summary, this study provides some evidence on L. monocytogenes modulating host PIP metabolism by the production of inositol phosphatases. It gives us a better understanding on the intracellular growth of this pathogenic bacterium, and on the interaction between host and parasite.

Les moisissures sont responsables de contaminations sur les produits de BVP et induisent des pertes économiques conséquentes. Dans ce contexte, les cultures bioprotectrices représentent un intérêt croissant comme alternative aux conservateurs chimiques. L’objectif de la première partie de cette étude a été d’évaluer in vitro et in situ l’activité antifongique de bactéries lactiques et propioniques contre cinq moisissures contaminants isolées de produits de BVP. Les bactéries les plus actives pendant les tests in vitro ont été testées in situ par pulvérisation de surface. Sur le milieu WFH, les isolats bactériens les plus actifs correspondent aux espèces Lactobacillus plantarum, reuteri et au groupe buchneri. Les souches plus actives après ces tests ont été testées par inclusion dans la recette du pain au lait et différentes souches ont montré un effet retard en particulier la souche Leuconostoccitreum qui semble retarder la croissance de Penicillium corylophilum après 10 jours. Dans la deuxième partie de cette étude, les surnageants de cultures actifs sont analysés pour identifier les composés antifongiques grâce à différentes traitements et différentes méthode telle que l’HPLC et la spectrométrie de masse. Les résultats suggèrent que les acides organiques jouent un rôle prépondérant dans l’activité antifongique et ont montré que les composés antifongiques retrouvés correspondaient aux acides lactique, acétique et propionique, à l’éthanol et au peroxyde d’hydrogène, ainsi que d’autres composés mais à plus faible échelle. Sur ces résultats, différentes combinaisons des composés identifiés ont été testées pour leur effet sur la germination et la croissance radiale de P. corylophilum et E. repens. Certaines de ces combinaisons ont montré les mêmes effets que le surnageant actif ce qui confirme l’implication des molécules identifiées dans l’activité. Les résultats suggèrent que l’acide acétique est responsable de la totalité de l’activité antifongique observée sur P. corylophilum et qu’il joue un rôle important dans l’inhibition de E. repens. La souche bactérienne sélectionnée pourrait représenter une possibilité de culture bioprotectrice pour les produits de BVP. / Molds are responsible for the spoilage of bakery products and thus, cause substantial economic losses. In this context, bioprotective cultures represent a growing interest as an alternative to chemical preservatives. The aims of the first part of this study was to evaluate the in vitro and in situ antifungal activity of lactic acid bacteria (LAB) and propionibacteria against five moulds species isolated from bakery products. The most inhibitorybacteria found during the in vitro test were evaluated in situ after surface spraying. In WFH medium, the most active LAB isolates belonged to the Lactobacillus plantarum, reuteri and buchneri groups. The most active strains were added directly during “pains au lait” preparation and differents strains present delayed effect in particular a strain of Leuconostoc citreum which seems to delay the growth of Penicillium corylophilum after 10 days. In the second part, supernatants were analyzed to identified and quantified antifungal compounds by different treatments and different methods like HPLC, mass spectrometry. The results suggested that organic acids played the most important role in the antifungal activity and show that the main antifungal compounds corresponded to lactic, acetic and propionic acids, ethanol and hydrogen peroxide, as well as other compounds present at low levels. Based on these results, various combinations of the identified compounds were used to evaluate their effect on spore germination and fungal growth of P. corylophilum and E. repens. Some combinations presented the same activity than the bacterial culture supernatant thus confirming the involvement of the molecules in the antifungal activity. The results suggested that acetic acid was responsible of the entire antifungal activity against Penicillium corylophilum and played an important role in Eurotium repens inhibition. The selected bacteria provide a future prospect for use as bioprotective cultures on bakery products.

S. thermophilus est une bactérie lactique largement utilisé dans l'industrie laitière et, comme toute bactérie, doit s'adapter à des environnements variés tels que le lait, le yaourt et même le tractus digestif, après que le produit ait été ingéré. Les systèmes à deux composants (TCS) constituent un des mécanismes essentiels qu'utilisent les bactéries pour percevoir et s'adapter à des changements environnementaux. D'un point de vue structural, les TCS sont constitués de deux composants: un « senseur » ou protéine histidine kinase (HK) qui s'auto-phosphoryle en réponse à un stimulus puis transfère son groupement phosphate au « response regulator » (RR), le deuxième composant. Celui-ci se comporte alors le plus souvent comme un régulateur transcriptionnel permettant une réponse physiologique adaptée. Afin de mieux comprendre ces phénomènes de régulation impliqués dans la réponse aux changements environnementaux, nous avons étudié la contribution de chacun des 8 TCS de Streptococcus thermophilus LMD-9 à son adaptation dans le lait. Ainsi, des études transcriptionnelles effectuées sur des cultures en lait montrent que tous les RR sont exprimés, à des niveaux et profils d'expression différents. Nous avons noté en coculture avec Lactobacillus bulgaricus, le partenaire de Streptococcus thermophilus dans le yaourt, une induction de l'expression de 4 RR qui atteint, pour rr02 et rr09, un facteur 6. Nous avons construit par ailleurs des mutants négatifs pour 7 des 8 RR de S. thermophilus et montré l'essentialité de RR05, un orthologue de YycF chez B. subtilis ou de or de WalR chez S. aureus. Pour les 7 autres mutants RR, l'absence d'un seul gène rr n'impacte pas suffisamment la croissance du streptocoque en lait. Enfin, la détermination du régulon du TCS06 par des études post-génomiques a permis de montrer que ce système est impliqué dans la résistance à la bacitracine en modulant entre autres la voie de biosynthèse du polysaccharide à rhamnose (RGP). / The lactic acid bacterium Streptococcus thermophilus is widely used in the dairy industry and, as a food bacterium, has to cope with changing environments such as milk, yogurt as well as the digestive tract, after the product has been ingested. Two-component systems (TCS), typically composed of a sensor kinase (HK) that detects a stimulus and of a response regulator (RR) which acts as a transcriptional regulator, are among the most prevalent means for bacteria to adapt to changing environments via fine-tune gene expression. To get a more comprehensive view of the role of all two-component systems in S. thermophilus physiology, we have investigated the contribution of each S. thermophilus LMD-9 TCS to its fitness and adaptation to milk. Transcriptomic studies (RT-qPCR) and construction of negative mutants of the rr genes were performed for LMD-9 S. thermophilus strain. We have shown that all LMD-9 response regulators were expressed in milk, at different levels and with different profiles of expression during growth. In mixed culture with Lactobacillus bulgaricus, the S. thermophilus partner in yoghurt, the expression of four LMD-9 rr increased; for two of them, rr02 and rr09, the increase reached a factor 6. These results indicate that Lb. bulgaricus induces regulatory changes in S. thermophilus and that S. thermophilus is able to adapt to these changes by probable fine tuning regulations. We constructed negative mutants for 7 out of 8 LMD-9 RRs and we showed that RR05 -an ortholog of B. subtilis YycF or S. aureus WalR- was essential for the optimum growth of S. thermophilus. For the 7 other RR, the absence of a single rr gene was not sufficient to notably impact the growth of LMD-9 in milk. The determination of the TCS06 regulon by post-genomics shows that TCS06 is involved in bacitracin resistance through the modulation of the rhamnose polysaccharide pathway.