Detection and characterization of Bifidobacterium crudilactis and B. mongoliense able to grow during the manufacturing process of French raw milk cheeses.

BACKGROUND: The study of a production chain of raw milk cheeses (St Marcellin, Vercors area, France) led to the isolation of two Bifidobacterium populations: B. crudilactis and B. mongoliense, that were able to grow along the production chain. The aims of this study were to further detect and characterize these bacteria along the process and evaluate the ability of some strains to survive or grow in adverse conditions. RESULTS: Using PCR coupled with restriction fragment length polymorphism, B. crudilactis and B. mongoliense were detected in respectively 77% and 30% of St Marcellin cheeses from production chain after 21 days of ripening. They were present in more than half of all analyzed retail cheeses with counts going from 1.6 to 5 log cfu g-1 for B. crudilactis and 1.4 to 7 log cfu g-1 for B. mongoliense. Bifidobacterium mongoliense was sensitive to pH 2, with an observed decrease of at least 3 log for both studied strains (FR49/f/2 and FR41/2) after 1 h incubation. At pH 3, no significant decrease was observed. Good survival was observed for the same strains in presence of pancreatic juice with a decrease of less than one log. Survival of strain FR49/f/2 was better than FR41/2 with a decrease of 3 logarithms (in presence of 1% bile salts) and almost 2 logarithms (in presence of 0.5% bile salts). The genotypic analyses using total DNA-DNA hybridization, GC% content, 16S rRNA gene sequencing and multilocus sequencing analysis (MLSA) confirmed the classification of Bifidobacterium. crudilactis and B. mongoliense into two different clusters well separated from other bifidobacteria clusters. CONCLUSIONS: According to the observed characteristics such as survival in adverse conditions and their ability to grow under 12 °C during the manufacturing process of the cheeses, which has never been described for bifidobacteria and which is a very interesting technological asset, these B. crudilactis and B. mongoliense strains should be further investigated for a potential use in new food or in food supplements.

Bifidobacterium pseudolongum are efficient indicators of animal fecal contamination in raw milk cheese industry.

BACKGROUND: The contamination of raw milk cheeses (St-Marcellin and Brie) from two plants in France was studied at several steps of production (raw milk, after addition of rennet - St-Marcellin - or after second maturation - Brie -, after removal from the mold and during ripening) using bifidobacteria as indicators of fecal contamination. RESULTS: Bifidobacterium semi-quantitative counts were compared using PCR-RFLP and real-time PCR. B. pseudolongum were detected in 77% (PCR-RFLP; 1.75 to 2.29 log cfu ml(-1)) and 68% (real-time PCR; 2.19 to 2.73 log cfu ml(-1)) of St-Marcellin samples and in 87% (PCR-RFLP; 1.17 to 2.40 log cfu ml(-1)) of Brie cheeses samples. Mean counts of B. pseudolongum remained stable along both processes. Two other populations of bifidobacteria were detected during the ripening stage of St-Marcellin, respectively in 61% and 18% of the samples (PCR-RFLP). The presence of these populations explains the increase in total bifidobacteria observed during ripening. Further characterization of these populations is currently under process. Forty-eight percents (St-Marcellin) and 70% (Brie) of the samples were B. pseudolongum positive/E. coli negative while only 10% (St-Marcellin) and 3% (Brie) were B. pseudolongum negative/E. coli positive. CONCLUSIONS: The increase of total bifidobacteria during ripening in Marcellin's process does not allow their use as fecal indicator. The presence of B. pseudolongum along the processes defined a contamination from animal origin since this species is predominant in cow dung and has never been isolated in human feces. B. pseudolongum was more sensitive as an indicator than E. coli along the two different cheese processes. B. pseudolongum should be used as fecal indicator rather than E. coli to assess the quality of raw milk and raw milk cheeses.

Bacteroides sp. strain D8, the first cholesterol-reducing bacterium isolated from human feces.

The microbial community in the human colon contains bacteria that reduce cholesterol to coprostanol, but the species responsible for this conversion are still unknown. We describe here the first isolation and characterization of a cholesterol-reducing bacterium of human intestinal origin. Strain D8 was isolated from a 10(-8) dilution of a fresh stool sample provided by a senior male volunteer with a high capacity to reduce luminal cholesterol to coprostanol. Cholesterol-to-coprostanol conversion by strain D8 started on the third day, while cells were in stationary phase, and was almost complete after 7 days. Intermediate products (4-cholesten-3-one and coprostanone) were occasionally observed, suggesting an indirect pathway for cholesterol-to-coprostanol conversion. Resting-cell assays showed that strain D8 could reduce 1.5 mumol of cholesterol/mg bacterial protein/h. Strain D8 was a gram-negative, non-spore-forming, rod-shaped organism identified as a member of the genus Bacteroides closely related to Bacteroides vulgatus, based on its morphological and biochemical characteristics. The 16S rRNA gene sequence of strain D8 was most similar (>99.5%) to those of two isolates of the recently described species Bacteroides dorei. Phylogenetic tree construction confirmed that Bacteroides sp. strain D8 clustered within an independent clade together with these B. dorei strains. Nevertheless, no cholesterol-reducing activity could be detected in cultures of the B. dorei type strain. Based on Bacteroides group-specific PCR-temporal temperature gradient gel electrophoresis, there was no correlation between the presence of a band comigrating with the band of Bacteroides sp. strain D8 and cholesterol conversion in 11 human fecal samples, indicating that this strain is unlikely to be mainly responsible for cholesterol conversion in the human population.

Clostridium saccharogumia sp. nov. and Lactonifactor longoviformis gen. nov., sp. nov., two novel human faecal bacteria involved in the conversion of the dietary phytoestrogen secoisolariciresinol diglucoside.

Two anaerobic bacteria involved in the conversion of the plant lignan secoisolariciresinol diglucoside were isolated from faeces of a healthy male adult. The first isolate, strain SDG-Mt85-3Db, was a mesophilic strictly anaerobic Gram-positive helically coiled rod. Based on 16S r RNA gene sequence analysis, its nearest relatives were Clostridium cocleatum (96.7% similarity) and Clostridium ramosum (96.6%). In contrast to these species, the isolate was devoid of alpha-galactosidase and -glucosidase and did not grow on maltose, melibiose, raffinose, rhamnose and trehalose. The hypothesis that strain SDG-Mt85-3Db represents a new bacterial species of the Clostridium cluster XVIII was confirmed by DNA-DNA hybridisation experiments. The G+C content of DNA of strain SDG-Mt85-3Db (30.7+/-0.8 mol%) was comparable with that of Clostridium butyricum, the type species of the genus Clostridium. The name Clostridium saccharogumia is proposed for strain SDG-Mt85-3Db (=DSM 17460T=CCUG 51486T). The second isolate, strain ED-Mt61/PYG-s6, was a mesophilic strictly anaerobic Gram-positive regular rod. Based on 16S rRNA gene sequence analysis, its nearest relatives were Clostridium amygdalinum (93.3%), Clostridium saccharolyticum (93.1%) and Ruminococcus productus (93.0%). The isolate differed from these species in its ability to dehydrogenate enterodiol. It also possessed alpha-arabinosidase and -galactosidase and had a higher G+C content of DNA (48.0 mol%). According to these findings, it is proposed to create a novel genus, Lactonifactor, and a novel species, Lactonifactor longoviformis, to accommodate strain ED-Mt61/PYG-s6. The type strain is DSM 17459T (=CCUG 51487T).

Bifidobacterial lipoglycan as a new cause for false-positive platelia Aspergillus enzyme-linked immunosorbent assay reactivity.

We previously hypothesized that a lipoglycan of Bifidobacterium bifidum subsp. pennsylvanicum cross-reacts with the Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) based on the presence of galactofuranosyl epitopes in the cell wall (M. A. S. H. Mennink-Kersten, R. R. Klont, A. Warris, H. J. M. Op den Camp, and P. E. Verweij, Lancet 363:325-327, 2004). We tested this hypothesis by testing bacterial suspensions of different bifidobacterial species and other gram-positive and -negative bacteria with the PA ELISA, which is used to detect circulating galactomannan for the serodiagnosis of invasive aspergillosis. Furthermore, neonatal fecal samples were enumerated for bifidobacteria by fluorescence in situ hybridization (FISH) and tested for PA ELISA reactivity. All bifidobacteria, except B. infantis and B. adolescentis, showed reactivity 6- to 600-fold higher compared to the controls (i.e., Micrococcus luteus and Propionibacterium freudenreichii, which contain a cell wall lipomannan). Eggerthella lenta showed a 25-fold-higher reactivity. ELISA reactivity was clearly shown to be associated with bacterial lipoglycans containing a beta-1,5-galactofuranosyl chain. All neonatal feces showed PA ELISA reactivity and associated numbers of bifidobacteria. Since high concentrations of bifidobacteria are present in the human gut, these bacteria or excreted lipoglycan may cause false serum PA ELISA reactivity in selected patient groups, especially neonates.

Altered ability of Bacillus cereus spores to grow under unfavorable conditions (presence of nisin, low temperature, acidic pH, presence of NaCl) following heat treatment during sporulation.

The effect of thermal treatment on the heat resistance of Bacillus cereus spores and their ability to germinate and grow under more or less adverse conditions during sporulation was investigated. Spores produced by sporulating cells subjected to a mild heat treatment (at a temperature 15 degrees C higher than the growth temperature) were more resistant to heat than were spores produced by untreated cells. Spore germination and growth (the lag time, the maximal growth rate, and the occurrence of a decrease in population) may be greatly affected by adverse environmental conditions brought about by the addition of nisin, low temperatures, acidic pHs, and, to a lesser extent, the addition of NaCl. Furthermore, heat treatments applied to sporulating cells or to mature spores induced a modification of the lag time (interaction of both treatments). Therefore, mild heat treatments applied during sporulation may affect the heat resistance of spores and the ability of these spores to germinate under adverse conditions and may thus increase the risk associated with the presence of spores in lightly processed foods.

Injury and Lethality of Heat Treatment of Bacillus cereus Spores Suspended in Buffer and in Poultry Meat.

The effect was studied of supplementing the recovery medium with lysozyme, glucose, NaCl, and MgSO4 on the apparent heat resistance of four strains of Bacillus cereus . The composition of an optimal medium (allowing the germination and growth of all surviving spores) and of a selective medium (inhibiting injured spores) was then determined: the optimal medium consisted of nutrient agar supplemented with 50 × 10-6 g of lysozyme and 0.5 g of MgSO4 per liter. The selective medium had the same composition but was supplemented with 15 g of NaCl per liter. The injury and lethality were analyzed of heat treatment of spores of four Bacillus cereus strains in phosphate buffer and in mechanically separated poultry meat. The four strains tested exhibited great differences in injury and death rates, and considerable differences could be observed in survivor counts when spores were suspended in buffer or in meat. Moreover, variability in behavior when the strains were heat treated in buffer could not be extrapolated to the same strains suspended in food. These data showed that it is necessary to be very careful when evaluating potential hazards from B. cereus spores in various foods; heating medium and strain selection must be considered to evaluate the efficiency of heat treatments.