Genomic elements located in the accessory repertoire drive the adaptation to biocides in Listeria monocytogenes strains from different ecological niches.

In response to the massive use of biocides for controlling Listeria monocytogenes (hereafter Lm) contaminations along the food chain, strains showing biocide tolerance emerged. Here, accessory genomic elements were associated with biocide tolerance through pangenome-wide associations performed on 197 Lm strains from different lineages, ecological, geographical and temporal origins. Mobile elements, including prophage-related loci, the Tn6188_qacH transposon and pLMST6_emrC plasmid, were widespread across lineage I and II food strains and associated with tolerance to benzalkonium-chloride (BC), a quaternary ammonium compound (QAC) widely used in food processing. The pLMST6_emrC was also associated with tolerance to another QAC, the didecyldimethylammonium-chloride, displaying a pleiotropic effect. While no associations were detected for chemically reactive biocides (alcohols and chlorines), genes encoding for cell-surface proteins were associated with BC or polymeric biguanide tolerance. The latter was restricted to lineage I strains from animal and the environment. In conclusion, different genetic markers, with polygenic nature or not, appear to have driven the Lm adaptation to biocide, especially in food strains but also from animal and the environment. These markers could aid to monitor and predict the spread of biocide tolerant Lm genotypes across different ecological niches, finally reducing the risk of such strains in food industrial settings.

Impact of temperature and oxygen on the fate of Bacillus weihenstephanensis in a food-based medium.

The capacity of the Bacillus weihenstephanensis KBAB4 strain, a psychrotolerant species of the B. cereus sensu lato group, to multiply in carrot broth at 8 °C and 30 °C, in presence or absence of oxygen was determined. In aerobic carrot broth tyndallized in presence of oxygen, at both temperatures, the population of vegetative cells of B. weihenstephanensis inoculated at a level of 103 or 106 CFU/ml dropped immediately. After 16 h at 30 °C, B. weihenstephanensis reached around 103 CFU/ml, indicating that some vegetative cells had survived and multiplied, with lipid inclusions accumulated in cells, indicating possible stressing conditions. At 8 °C, no multiplication of B. weihenstephanensis was observed during 3 days to at least 12 days, depending of carrot broth batches. In anaerobic carrot broth tyndallized without oxygen, the vegetative cells of B. weihenstephanensis were not killed upon inoculation and multiplied in the broth at both 30 °C and 8 °C. Comparison with results from previous studies shows that B. weihenstephanensis behaves differently in carrot broth and in laboratory media at 8 °C with regards to presence or absence of oxygen.

Heat-resistance of psychrotolerant Bacillus cereus vegetative cells.

Spores of psychrotolerant strains of the foodborne pathogen Bacillus cereus can multiply during storage of cooked or pasteurized, refrigerated foods and can represent a risk if these cells are not eliminated during reheating of food product before consumption. We determined the heat-resistance of psychrotolerant B. cereus vegetative cells at different heating temperatures in laboratory medium and compared it with that of thermotolerant B. cereus vegetative cells. The z values, based on times for a 3 log10 reduction, of the vegetative cells of the three psychrotolerant phylogenetic groups of B. cereus varied between 3.02 °C and 4.84 °C. The temperature at which a 3 log10 reduction was achieved in 10 min varied between 47.6 °C and 49.2 °C for psychrotolerant vegetative cells and it was around 54.8 °C for thermotolerant vegetative cells. Moreover, 0.4 min at 60 °C would be sufficient for a 6 log10 CFU/ml reduction of the most heat resistant psychrotolerant B. cereus vegetative cells. These data clearly showed that psychrotolerant B. cereus vegetative cells can be rapidly eliminated by a mild heat treatment such as food reheating.

Cereulide production by Bacillus weihenstephanensis strains during growth at different pH values and temperatures.

Besides Bacillus cereus, some strains of the psychrotolerant, potentially foodborne pathogen Bacillus weihenstephanensis can produce the emetic toxine (cereulide). This toxin is a heat- and acid-stable cyclic dodecadepsipeptide that causes food intoxication with vomiting. However, some severe clinical cases with lethal outcomes have been described. If cereulide can be produced during refrigerated storage, it will not be inactivated by reheating food, representing an important risk of food intoxication for consumers. In this paper, we determined the capacity of the B. weihenstephanensis strains BtB2-4 and MC67 to grow and produce cereulide on agar media at temperatures from 8 °C to 25 °C and at a pH from 5.4 to 7.0. At 8 °C, strain BtB2-4 produced quantifiable amounts of cereulide, whereas the limit of detection was reached for strain MC67. For BtB2-4, cereulide production increased 5-fold between 8 °C and 10-15 °C and by more than 100-fold between 15 °C and 25 °C. At temperatures of 10 °C and higher, cereulide concentrations were within the range of those reported by previous works in foods implicated in emetic poisoning. At 25 °C, decreasing the pH to 5.4 reduced cereulide production by strain BtB2-4 by at least 20-fold.

Combined effect of anaerobiosis, low pH and cold temperatures on the growth capacities of psychrotrophic Bacillus cereus.

Psychrotrophic strains of the foodborne pathogen Bacillus cereus can multiply during the refrigerated storage of food products. The aim of this study was to determine the impact of anaerobiosis on the growth of two psychrotrophic B. cereus strains exposed to acidic pH at a cold temperature in a laboratory medium. At 10 °C, growth occurred at pH values equal to or higher than 5.7 during anaerobiosis, whereas aerobic growth was observed from pH 5.4. Growth rates during aerobiosis were similar at pH 5.4 and pH 7. No growth was observed for the two tested strains at 8 °C without oxygen regardless of the pH; however, both strains grew at this temperature from pH 5.4 in the presence of oxygen. These pH growth limits in aerobiosis are consistent with those reported for different strains and different foods or media, but no other studies have described anaerobic growth at acidic pH values. The maximal B. cereus concentration was approximately 6.0 log10 CFU/ml for cultures in the absence of oxygen and approximately 8.0 log10 CFU/ml for cultures in the presence of oxygen. In conclusion, we found that the combination of anaerobiosis, pH < 5.7 at 10 °C, or anaerobiosis and temperatures ≤8 °C prevent psychrotrophic B. cereus growth.

Exposure to Quaternary Ammonium Compounds Selects Resistance to Ciprofloxacin in Listeria monocytogenes.

In this contribution, the antimicrobial susceptibility toward 11 antibiotics and four biocides of a panel of 205 Listeria monocytogenes (Lm) strains isolated from different ecological niches (i.e., food, animals and natural environment) was evaluated. The impact of exposure to biocides on the antibiotic susceptibilities of Lm was also investigated. Lm strains isolated from food exhibited overall a lower susceptibility (higher minimal inhibitory concentrations, MIC) for ammonium quaternary compounds (QACs) and peracetic acid (PAC) than strains isolated from animals and natural environments. Conversely, the ecological origins of Lm strains did not significantly affect their susceptibilities towards antibiotics. Interestingly, repeated exposure to QACs recurrently led to a decrease in susceptibility toward ciprofloxacin (CIP), a fluoroquinolone antibiotic, largely used in human medicine. Moreover, these lower levels of susceptibility to CIP remained stable in most Lm strains even after subcultures without biocide selection pressure, suggesting an adaptation involving modifications at the genetic level. Results underlined the ability of Lm to adapt to biocides, especially QACs, and the potential link between this adaptation and the selection of resistance toward critical antibiotics such as ciprofloxacin. These data support a potential role of the extensive use of QACs from "farm to fork" in the selection of biocide and antibiotic resistance in pathogenic bacteria such as Lm.

Membrane Proteocomplexome of Campylobacter jejuni Using 2-D Blue Native/SDS-PAGE Combined to Bioinformatics Analysis.

Campylobacter is the leading cause of the human bacterial foodborne infections in the developed countries. The perception cues from biotic or abiotic environments by the bacteria are often related to bacterial surface and membrane proteins that mediate the cellular response for the adaptation of Campylobacter jejuni to the environment. These proteins function rarely as a unique entity, they are often organized in functional complexes. In C. jejuni, these complexes are not fully identified and some of them remain unknown. To identify putative functional multi-subunit entities at the membrane subproteome level of C. jejuni, a holistic non a priori method was addressed using two-dimensional blue native/Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in strain C. jejuni 81-176. Couples of acrylamide gradient/migration-time, membrane detergent concentration and hand-made strips were optimized to obtain reproducible extraction and separation of intact membrane protein complexes (MPCs). The MPCs were subsequently denatured using SDS-PAGE and each spot from each MPCs was identified by mass spectrometry. Altogether, 21 MPCs could be detected including multi homo-oligomeric and multi hetero-oligomeric complexes distributed in both inner and outer membranes. The function, the conservation and the regulation of the MPCs across C. jejuni strains were inspected by functional and genomic comparison analyses. In this study, relatedness between subunits of two efflux pumps, CmeABC and MacABputC was observed. In addition, a consensus sequence CosR-binding box in promoter regions of MacABputC was present in C. jejuni but not in Campylobacter coli. The MPCs identified in C. jejuni 81-176 membrane are involved in protein folding, molecule trafficking, oxidative phosphorylation, membrane structuration, peptidoglycan biosynthesis, motility and chemotaxis, stress signaling, efflux pumps and virulence.