Michel Hébraud

Listeria monocytogenes LO28: Surface Physicochemical Properties and Ability To Form Biofilms at Different Temperatures and Growth Phases

ABSTRACT The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20°C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37°C. At 8°C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.

Attachment and biofilm formation by foodborne bacteria in meat processing environments: Causes, implications, role of bacterial interactions and control by alternative novel methods

Attachment of potential spoilage and pathogenic bacteria to food contact surfaces and the subsequent biofilm formation represent serious challenges to the meat industry, since these may lead to cross-contamination of the products, resulting in lowered-shelf life and transmission of diseases. In meat processing environments, microorganisms are sometimes associated to surfaces in complex multispecies communities, while bacterial interactions have been shown to play a key role in cell attachment and detachment from biofilms, as well as in the resistance of biofilm community members against antimicrobial treatments. Disinfection of food contact surfaces in such environments is a challenging task, aggravated by the great antimicrobial resistance of biofilm associated bacteria. In recent years, several alternative novel methods, such as essential oils and bacteriophages, have been successfully tested as an alternative means for the disinfection of microbial-contaminated food contact surfaces. In this review, all these aspects of biofilm formation in meat processing environments are discussed from a microbial meat-quality and safety perspective.

Effect of osmotic, alkaline, acid or thermal stresses on the growth and inhibition of Listeria monocytogenes

Five strains of Listeria monocytogenes (a, b, c, d and e) isolated from industrial plants have been subjected to different osmotic, alkaline, acid or thermal stresses. The effects of these treatments on lag‐phase (L) and growth rate (μ) of cells in mid‐log phase have been followed using an automated optical density monitoring system. Increasing the osmotic pressure by the addition of different amounts of NaCl increased the lag phase and decreased the growth rate. The same phenomena were observed after decreasing the pH of the medium to 5·8, 5·6 or 5·4 by addition of acetic, lactic or hydrochloric acids. The inhibitory effect was: acetic acid > lactic acid > hydrochloric acid. The addition of NaOH to attain pH values of 9·5, 10·0, 10·5 or 11·0 in the medium produced a dramatic increase of the lag phase at pH 10·5 and 11. Growth rates were also decreased while the maximal population increased with high pH values. These effects varied according to strains. Strains d and e were the most resistant to acidic and alkaline stresses, and e was the most affected by the addition of NaCl. A cold shock of 30 min at 0 °C had limited effects on growth parameters. On the other hand, hyperthermal shocks (55 or 63 °C, 30 min) led to similar increased lag phases and to significant increases of the maximal population in all five strains.

Molecular biology of surface colonization by Listeria monocytogenes: an additional facet of an opportunistic Gram‐positive foodborne pathogen

The opportunistic and facultative intracellular pathogenic bacterium Listeria monocytogenes causes a rare but severe foodborne disease called listeriosis, the outcome of which can be fatal. The infection cycle and key virulence factors are now well characterized in this species. Nonetheless, this knowledge has not prevented the re-emergence of listeriosis, as recently reported in several European countries. Listeria monocytogenes is particularly problematic in the food industry since it can survive and multiply under conditions frequently used for food preservation. Moreover, this foodborne pathogen also forms biofilms, which increase its persistence and resistance in industrial production lines, leading to contamination of food products. Significant differences have been reported regarding the ability of different isolates to form biofilms, but no clear correlation can be established with serovars or lineages. The architecture of listerial biofilms varies greatly from one strain to another as it ranges from bacterial monolayers to the most recently described network of knitted chains. While the role of polysaccharides as part of the extracellular matrix contributing to listerial biofilm formation remains elusive, the importance of eDNA has been demonstrated. The involvement of flagella in biofilm formation has also been pointed out, but their exact role in the process remains to be clarified because of conflicting results. Two cell-cell communication systems LuxS and Agr have been shown to take part in the regulation of biofilm formation. Several additional molecular determinants have been identified by functional genetic analyses, such as the (p)ppGpp synthetase RelA and more recently BapL. Future directions and questions about the molecular mechanisms of biofilm formation in L. monocytogenes are further discussed, such as correlation between clonal complexes as revealed by MLST and biofilm formation, the swarming over swimming regulation hypothesis regarding the role of the flagella, and the involvement of microbial surface components recognizing adhesive matrix molecules in the colonization of abiotic and biotic surfaces.

Antimicrobial effects of sanitizers against planktonic and sessile Listeria monocytogenes cells according to the growth phase

This study was designed to investigate the individual or combined effects of sanitizers on survival of planktonic or sessile Listeria monocytogenes cells at different phase of growth. The sanitizers tested included: (i) acetic acid (pH 5.0), (ii) NaOH (pH 12.0), (iii) 10% Na2SO4, (iv) 10% Na2SO4 and acetic acid (pH 5.0), (v) 10% Na2SO4 and NaOH (pH 12.0), (vi) a quaternary ammonium (20 ppm) and (vii) glyceryl monolaurate (75 ppm). Results revealed a great efficacy of alkaline treatments on both sessile and planktonic cells with a slightly higher resistance of 6 h biofilms. Quaternary ammonium appeared very effective in killing more than 98% of cells, but a resistance of 7 days biofilm was observed. Other sanitizers did not succeed in inhibiting totally the pathogen but acted in a similar way on both sessile and planktonic cells. Renewing the medium or not do not seem to be the major cause of a resistance emergence.

Critical review on biofilm methods

Abstract Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

Intra- and inter-species interactions within biofilms of important foodborne bacterial pathogens.

A community-based sessile life style is the normal mode of growth and survival for many bacterial species. Under such conditions, cell-to-cell interactions are inevitable and ultimately lead to the establishment of dense, complex and highly structured biofilm populations encapsulated in a self-produced extracellular matrix and capable of coordinated and collective behavior. Remarkably, in food processing environments, a variety of different bacteria may attach to surfaces, survive, grow, and form biofilms. Salmonella enterica, Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus are important bacterial pathogens commonly implicated in outbreaks of foodborne diseases, while all are known to be able to create biofilms on both abiotic and biotic surfaces. Particularly challenging is the attempt to understand the complexity of inter-bacterial interactions that can be encountered in such unwanted consortia, such as competitive and cooperative ones, together with their impact on the final outcome of these communities (e.g., maturation, physiology, antimicrobial resistance, virulence, dispersal). In this review, up-to-date data on both the intra- and inter-species interactions encountered in biofilms of these pathogens are presented. A better understanding of these interactions, both at molecular and biophysical levels, could lead to novel intervention strategies for controlling pathogenic biofilm formation in food processing environments and thus improve food safety.

Characterization of Glycoside and Polysaccharide Hydrolases Secreted by the Rumen Anaerobic Fungi Neocallimastix frontalis, Sphaeromonas communis and Piromonas communis

SUMMARY: The rumen anaerobic fungi Neocallimastix frontalis, Sphaeromonas communis and Piromonas communis were grown in the presence of cellulosic substrate (sisal fibres) and the properties of the glycosidases and polysaccharide-degrading enzymes produced by the organisms were studied. β-D-Glucosidase (EC 3.2.1.21), β-D-fucosidase (EC 3.2.1.38), β-D-galactosidase (EC 3.2.1.23), β-1,3-glucanase (EC 3.2.1.6), β-1,4-glucanase (EC 3.2.1.4) and β-xylanase (EC 3.2.1.8) had pH optima of 6.0 and temperature optima under the conditions of assay of 50-55 °C, whereas for β-xylosidase (EC 3.2.1.37) the optima were 6.5 and 39 °C respectively. The apparent K m values of individual enzymes secreted by the three fungi were similar. The results show that S. communis, P. communis and N. frontalis produce the same range of enzymes, with similar properties, able to degrade cellulose and hemicellulose.

Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions

Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.

Insight into the core and variant exoproteomes of Listeria monocytogenes species by comparative subproteomic analysis

While Listeria monocytogenes is responsible for listeriosis, it is also a saprophytic species with exceptional survival aptitudes. Secreted proteins are one of the main tools used by bacteria to interact with their environment. In order to take into account the biodiversity of L. monocytogenes species, exoproteomic analysis was carried out on 12 representative strains. Following 2‐DE and MALDI‐TOF MS, a total of 151 spots were identified and corresponded to 60 non‐orthologous proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach predicting final subcellular localization was carried out. Fifty‐two out of the 60 proteins identified (86.7%) were indeed predicted as localized in the extracellular milieu (gene ontology (GO): 0005576). Most of them (65.4%) were actually predicted as secreted via the Sec translocon. Comparative analysis allowed for proteins found in all or only in a subset of L. monocytogenes strains to be defined. While the core exoproteome included most proteins related to bacterial virulence, cell wall biogenesis, as well as proteins secreted by unknown pathways, a slight variation in the protein members of these categories were observed and constituted the variant exoproteome. This investigation resulted in the first definition of the core and variant exoproteomes of L. monocytogenes where corollaries on bacterial physiology are further discussed.