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L’effet de la température et de la composition du milieu en nutriments sur les propriétés des spores (résistance et germination) de B. cereus a été largement étudié contrairement à l'effet de l'anaérobiose. Or, les cellules végétatives de B. cereus peuvent se retrouver dans une grande variété de milieux naturels avec un faible niveau d'oxygène (intestin, sol, lignes de traitement des aliments…) où la sporulation peut avoir lieu. Les spores produites dans ces conditions anaérobies pourraient donc avoir des propriétés particulières. Dans ce travail, un panel de 18 souches de B. cereus appartenant aux groupes phylogénétiques de II à VII a été étudié pour sa capacité à sporuler en anaérobiose dans un milieu de sporulation approprié que nous avons développé (MODS). En anaérobiose, la capacité de sporulation a été plus faible et plus hétérogène qu’en aérobiose. La souche AH187 a produit le niveau de spores le plus important en anaérobiose, elle a donc été choisie pour étudier les propriétés de ces spores. Les spores produites en anaérobiose étaient plus résistantes à la chaleur humide entre 90°C et 100°C, à 1M de NaOH, 1M d'acide nitreux et à la lumière pulsée. Aucune différence dans la résistance à 5 % de peroxyde d'hydrogène ou à 0.25 mM de formaldéhyde, ni aux UV-C, n'a été observée entre les deux conditions. En présence de L- alanine, les spores produites en anaérobiose germaient plus efficacement que celles produites en aérobiose tandis qu’aucune différence dans la germination n’a été observée en présence d'inosine. Aucune différence dans la taille des spores produites dans les deux conditions n’a été observée par microscopie électronique à transmission. Toutefois, les spores obtenues dans des conditions anaérobies avaient un exosporium endommagé ou dans certains cas un exosporium complètement détaché, contrairement aux spores produites dans des conditions aérobies. Afin de comprendre les différences dans la capacité de sporulation de B.cereus entre les 2 conditions, des PCR en temps réel (RT-PCR) ont été utilisées pour étudier l'expression des gènes de l'initiation de la sporulation spo0A, spo0B, spo0F, KinA et kinB. Les cinétiques d'expressions des gènes spo0A, spo0B, spo0F et KinA avaient la même tendance. Ils étaient caractérisés par une expression plus élevée en anaérobiose par rapport à l’aérobiose au début et à la fin de la phase exponentielle de croissance. En outre, l'expression du gène kinB était caractérisée par une augmentation en anaérobiose par rapport à l’aérobiose pour atteindre un pic entre 4 h (milieu de phase exponentielle) et 6 h (début de phase stationnaire) de croissance. Les gènes spo0A, spo0B, spo0F, KinA et kinB sont exprimés de manière différentielle entre l’aérobiose et l’anaérobiose. Ces données pourraient aider à comprendre la différence de capacité de sporulation de B. cereus entre la condition aérobie et anaérobie, The effect of temperature and nutrient composition of the medium on B. cereus spore properties (resistance and germination) has been extensively studied unlike to the effect of anaerobiosis. Nevertheless, B. cereus vegetative cells can be found in a large variety of natural environments with low oxygen level (intestine, soil, food processing line) where sporulation take place. Spores produced in these anaerobic environments could have particular properties. In this work, a panel of B. cereus strains belonging to phylogenetic groups II to VII was studied for their capacity to sporulate in anaerobiosis in an appropriate sporulation medium we developed (MODS). In anaerobiosis, sporulation ability was lower and more heterogeneous than in aerobiosis. The B. cereus AH187 strain produced the highest level of spores in anaerobiosis, it was therefore chosen to study spore properties. Spores produced in anaerobiosis were more resistant to wet heat from 90°C to 100 °C, 1M NaOH, 1M nitrous acid and pulsed light. No difference in resistance to 5 % hydrogen peroxide or 0.25 mM formaldehyde or UV-C was observed between these two conditions. In the presence of L-alanine, spores produced in anaerobiosis germinated more efficiently than spore produced in aerobiosis. No difference in germination was observed with inosine. No difference in the spores size produced in the two conditions was observed by transmission electron microscopy. However, spores obtained under anaerobic conditions had a damaged exosporium, or in some cases a completely detached exosporium, unlike spores produced under aerobic conditions. To understand differences in sporulation ability between both conditions, Real-time reverse transcription-PCR was used to study the expression the expression of sporulation initiation genes spo0A, spo0B, spo0F, kinA and kinB. The kinetics of gene expression spo0A, spo0B, spo0F and kinA had the same trend. They were characterized by a higher expression in anaerobiosis compared to aerobiosis at the beginning and the end of exponential growth phase. Furthermore, kinB gene expression was characterized by an increase in anaerobiosis compared to aerobiosis to achieve a peak between 4 (middle exponential phase) and 6 (early stationary phase) hours of growth. The spo0A, spo0B, spo0F, kinA and kinB genes are differentially expressed between aerobiosis and anaerobiosis. These data may help to understand the difference in B. cereus sporulation capacity between aerobic and anaerobic condition

International audience; This study was performed to evaluate the effect of anaerobiosis on the formation of Bacillus cereus spores and their resulting properties. For this purpose, an appropriate sporulation medium was developed (MODs). Sporulation of 18 strains from different phylogenetic groups of B. cereus was studied in MODs medium in aerobiosis and anaerobiosis. In anaerobiosis, sporulation ability was weaker and more heterogeneous than in aerobiosis. Among tested strains, B. cereus AH187 produced the highest level of spores in anaerobiosis. This strain was therefore chosen to study spore properties. Spores produced in anaerobiosis were more resistant to wet heat at 90 °C, 92.5 °C, 95 °C, 97.5 °C and 100 °C. For example, D90 were 21,09 1.70 and 81.87 2.00 for aerobiosis and anaerobiosis conditions, respectively. Spores produced in anaerobiosis have a z-value of 7.70 °C compared with 10.52 °C for spores produced in aerobiosis. Spores produced in anaerobiosis were also more resistant to 1 M NaOH, 1 M nitrous acid and pulsed light at fluences of 0.34 J cm-2 and 0.49 J cm-2. No difference in resistance to UV-C, 5% hydrogen peroxide or 0.25 mM formaldehyde was observed between these two conditions. In the presence of L-alanine, spores produced in anaerobiosis germinated more efficiently than spore produced in aerobiosis. No difference in germination was observed with inosine as inducer. No difference in the size of spores produced in the different conditions was observed by transmission electron microscopy. However, spores obtained under anaerobic conditions had a damaged exosporium, or in some cases a completely detached exosporium, unlike spores produced under aerobic conditions. This study shows that few spores are formed under anaerobic condition; nevertheless, this condition has an impact on the spore properties of B. cereus AH 187 strain. Spores obtained under anaerobic condition were more resistant to heat and to some chemical compounds. This is an important feature, considering the risk associated with the presence of this pathogen in thermally processed and packaged food in absence of oxygen.

The aim of this study was to explore diversity and dynamic of indigenous LAB strains associated with a long ripened hard cheese produced from raw milk and undefined natural starter such as PDO Grana Padano cheese. Samples of milk, curd, natural whey culture and cheeses (2nd, 6th, 9th and 13th months of ripening) were collected from 6 cheese factories in northern Italy. DNA was extracted from each sample and from 194 LAB isolates. tRNA(Ala)-23S rDNA-RFLP was applied to identify isolates. Strain diversity was assessed by (GTG)5 rep-PCR and RAPD(P1)-PCR. Finally, culture-independent LH-PCR (V1-V2 16S-rDNA), was considered to explore structure and dynamic of the microbiota. Grana Padano LAB were represented mainly by Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus helveticus and Pediococcus acidilactici, while the structure and dynamic of microbiota at different localities was specific. The strength of this work is to have focused the study on isolates coming from more than one cheese factories rather than a high number of isolates from one unique production. We provided a valuable insight into inter and intraspecies diversity of typical LAB strains during ripening of traditional PDO Grana Padano, contributing to the understanding of specific microbial ecosystem of this cheese.

Pigs are important reservoirs for Arcobacter. Since 1978, Arcobacter species have been associated with reproduction disorders, but excretion by clinically healthy pigs has been frequently reported as well. Information on Arcobacter colonization of the porcine gastrointestinal tract is lacking. In the present study, gastrointestinal tracts of 12 pigs were collected, and the content and mucus of eight sections were examined. Arcobacters were enumerated and isolated by a selective quantitative and qualitative method, respectively, and identified by multiplex-polymerase chain reaction (PCR). Their genetic diversity was examined by enterobacterial repetitive intergenic consensus PCR and pulsed-field gel electrophoresis. Arcobacter species were isolated from at least two gastrointestinal sections of all pigs in levels up to 10(5) colony-forming units (CFU) g(-1) in content and 10(4) CFU g(-1) in mucus. Characterization of the isolates revealed a high degree of genotypic diversity. In general, the highest counts, and greatest species and strain diversity was obtained from the large intestine, and especially from the rectum. Though Arcobacter strains were mostly detected in one gastrointestinal section, several unique strains were also recovered from the content and/or mucus of various gastrointestinal sections of individual pigs. In the gastrointestinal tract, Arcobacter is present with species distributions, numbers, and strain heterogeneity comparable to those reported on porcine carcasses post slaughter, thus confirming the potential route of transmission to carcasses by fecal contamination during processing.

The mumps virus (MuV) molecular evolution is characterized by the co-circulation of numerous distinct strains. Standardized phylogenetic analyses based on the nucleotide sequences of the SH gene are important for mumps surveillance, but lack the information regarding antigenic properties. So far, the location of antigenic epitopes has been determined for two MuV proteins, the hemagglutinin-neuraminidase (HN) and the nucleocapsid (N) protein. We performed multiple sequence comparisons of putative HN and N protein sequences in order to describe their diversity and plasticity, and to determine the level of similarity between vaccine and wild-type strains. The results of full-length HN or N protein phylogeny showed that MuV strains form a number of differing clades which are in concordance with grouping obtained by standard MuV genotyping. When vaccine strains are compared to all wild-type strains, the highest mean percentage of amino acid differences in both HN and N protein analysis was found for Jeryl Lynn 5 and Jeryl Lynn 2 strains while the lowest value was obtained for Leningrad-3 and L-Zagreb strains. When only 3 antigenic regions of the HN protein, comprising 45 amino acids in total, were investigated, the diversity is considerably diminished: 51.5% of all putative HN proteins show identical sequences (including those of vaccine strains L-Zagreb, Leningrad-3, Hoshino and Urabe). Another 26.5% proteins (including Miyahara vaccine strain) differ in only one amino acid, while the others differ in two to five amino acids from the most common sequence. Jeryl Lynn 2 and Jeryl Lynn 5 strains differ in four amino acids each. N protein antigenic sites have been mapped within its hypervariable C-terminus. Our results indicate that there might be genotype-specific amino acids residing in this antigenic region. The results of our study present the background information for investigations of MuV heterogeneity and antigenic diversity.

Aims: Our aim was to assess the diversity of the nutrient germination response of Bacillus cereus spores. Methods and Results: B. cereus spore germination was monitored by decrease in optical density using a Bioscreen C analyser in response to the major germinant substances inosine and l-alanine. Spores of a set of 12 strains taken to illustrate the diversity of the B. cereus group showed ranging germination capacities. Two strains never germinated in the presence of l-alanine, at any of the germinant concentrations tested. Both the extent and rate of spore germination were affected by low pH and high NaCl concentration, but differently according to the strain. Conclusions: A broad diversity was observed in nutrient-triggered spore germination among the members of the B. cereus group. Spore germination of some strains occurred at low concentrations of inosine or l-alanine, suggesting high receptor sensitivity to germinants. The activity of these receptors was also affected by pH or high NaCl concentration. Significance and Impact of the Study: The greater ability of some strains to germinate in response to l-alanine and inosine is one criterion among others for B. cereus strain selection in food processing or storage studies, before confirmation in complex food or laboratory media. The diversity in response to germinants found among the B. cereus strains suggests a differential expression and (or) absence of some germination genes involved in the response, mainly to l-alanine.

The study comprises an analysis of the yeast microbiota that participated in the spontaneous fermentation of crushed Wegierka Zwykla plum fruit, which is the raw material for slivovitz production in the mountain region in the south of Poland. Saccharomyces cerevisiae yeast strains were differentiated by means of the killer sensitivity analysis related to a killer reference panel of 9 well-known killer yeast strains. The first phase of the fermentation was dominated by the representatives of Kloeckera apiculata and Candida pulcherrima species, which reached their maximum concentration (1.4·106 CFU/mL) after 48 h of the process. Almost all yeasts isolated during the following days were classified as S. cerevisiae and the killer sensitivity analysis revealed a high population diversity of this species and the presence of 14 different strains that changed quantitatively and qualitatively throughout the fermentation period.