Safety assessment of Gram-negative bacteria associated with traditional French cheeses.

Twenty Gram-negative bacterial (GNB) strains were selected based on the biodiversity previously observed in French traditional cheeses and their safety was assessed considering various safety criteria. For the majority of tested GNB strains, only gastric stress at pH 2 (vs pH 4) resulted in low survival and no regrowth after an additional simulated gastro-intestinal stress. Presence of milk was shown to be rarely protective. The majority of strains was resistant to human serum and had a low level of adherence to Caco-2 cells. When tested for virulence in Galleria mellonella larvae, GNB strains had LD 50 values similar to that of safe controls. However, four strains, Hafnia paralvei 920, Proteus sp. (close to P. hauseri) UCMA 3780, Providencia heimbachae GR4, and Morganella morganii 3A2A were highly toxic to the larvae, which suggests the presence of potential virulent factors in these strains. Noteworthy, to our knowledge, no foodborne intoxication or outbreak has been reported so far for any of the GNB belonging to the genera/species associated with the tested strains. The role of multiple dynamic interactions between cheese microbiota and GIT barriers could be key factors explaining safe consumption of the corresponding cheeses.

From undefined red smear cheese consortia to minimal model communities both exhibiting similar anti-listerial activity on a cheese-like matrix.

Starting from one undefined cheese smear consortium exhibiting anti-listerial activity (signal) at 15 °C, 50 yeasts and 39 bacteria were identified by partial rDNA sequencing. Construction of microbial communities was done either by addition or by erosion approach with the aim to obtain minimal communities having similar signal to that of the initial smear. The signal of these microbial communities was monitored in cheese microcosm for 14 days under ripening conditions. In the addition scheme, strains having significant signals were mixed step by step. Five-member communities, obtained by addition of a Gram negative bacterium to two yeasts and two Gram positive bacteria, enhanced the signal dramatically contrary to six-member communities including two Gram negative bacteria. In the erosion approach, a progressive reduction of 89 initial strains was performed. While intermediate communities (89, 44 and 22 members) exhibited a lower signal than initial smear consortium, eleven- and six-member communities gave a signal almost as efficient. It was noteworthy that the final minimal model communities obtained by erosion and addition approaches both had anti-listerial activity while consisting of different strains. In conclusion, some minimal model communities can have higher anti-listerial effectiveness than individual strains or the initial 89 micro-organisms from smear. Thus, microbial interactions are involved in the production and modulation of anti-listerial signals in cheese surface communities.

Draft Genome Sequence of Enterococcus mundtii QAUEM2808, Isolated from Dahi, a Fermented Milk Product.

Enterococcus mundtii QAUEM2808 has been isolated from dahi, an indigenous fermented milk product of Pakistan. Here, we report the draft genome sequence for this strain, which consists of 160 contigs corresponding to 2,957,514 bp and a G+C content of 38.5%.

Monitoring the microbiology of high quality milk by monthly sampling over 2 years.

The concentrations of seven types of microorganism in the milk produced by farms using high quality milking procedures were monitored monthly. The most commonly encountered bacterium was Pseudomonas, whose concentration varied greatly between samples, but lactococci, lactobacilli and yeasts were also present at more stable concentrations. Staphylococcus aureus and beta-glucuronidase-positive Escherichia coli were occasionally detected. Listeria monocytogenes was found in the samples from only one farm, while Yersinia enterocolitica was never detected. There were seasonal variations in the concentrations of lactobacilli and yeasts over the 2 years monitored. The changes in certain bacteria (Lactococcus, Lactobacillus) on any given farm were very similar from one year to the next. The microbiological characteristics of the milk from the farms examined seemed to be fairly constant. The profiles of some bacteria used for cheesemaking were relatively stable over time. It thus seems that the milk provided by different producers could be typed.

Microbiological composition of raw milk from selected farms in the Camembert region of Normandy.

Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters. Total bacterial counts and somatic cell counts were measured. Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens, yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas, coliforms, Escherichia coli, enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted. Pseudomonas (2000 cfu ml-1), lactococci (760 cfu ml-1) and Micrococcaceae (720 cfu ml-1) were the most numerous groups. Lactic acid bacteria were detected in all samples. Coliforms were present in most samples, but 84% of samples had counts < 100 cfu ml-1. Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml-1. About 80% of supplies had < or = 10 E. coli cfu ml-1 and all samples had < or = 1 Cl. perfringens cfu ml-1. Two of the tested milks were positive for salmonellas (2.9%), four were positive for Listeria monocytogenes (5.8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1.4%).

Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates.

A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L. lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency.

An ecological study of lactococci isolated from raw milk in the camembert cheese registered designation of origin area.

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of "Camembert de Normandie" cheese.

Characterization of lactococci isolated from milk produced in the Camembert region of Normandy.

Thirty-eight Lactococcus strains, isolated from raw milk produced in two dairy areas in Normandy, were identified at the phenotypic level. Only Lactococcus lactis strains with the lactis phenotype were found in the milk samples. Most strains fermented lactose (97%) and showed proteinase activity (76%). Isolates were characterized by RAPD technique and rRNA gene restriction analysis. More L. lactis strains with the lactis genotype were found in the first area, while L. lactis strains with the cremoris genotype predominated in the second area. RAPD was more efficient than rRNA gene restriction analysis in differentiating between strains with the subsp. lactis genotype. For L. lactis with the subsp. cremoris genotype, the second method gave a better result but there was poor discrimination between strains. Plasmid profiles were determined. Patterns ranged in size from 1.3 to 16.5 kbp, and 29 different profiles were found. Six groups of strains were determined, five of which were specific for the area of origin. It is suggested that the region of manufacture could influence organoleptic properties of cheeses because of different Lactococcus strains in the raw milk used for cheese making.

Genetic diversity among Geotrichum candidum strains from various substrates studied using RAM and RAPD-PCR.

AIMS: Assessment of genetic diversity within the species Geotrichum candidum and development of tools to trace the strains that play an important role in the agro food industry. METHODS AND RESULTS: RAM-PCR and RAPD-PCR techniques were assessed for their ability to discriminate 57 strains of various morphotypes, substrates and geographical origin. The techniques were complementary and, when combined, allowed us to discriminate isolates. Moreover, we established a link between a taxon and its occupation of an ecological niche, which should not be confused with the substrate of isolation. CONCLUSIONS: We observed a high degree of diversity, which could be linked to the variety of the ecological niches chosen and to the high degree of morphological polymorphism encountered within the species. SIGNIFICANCE AND IMPACT OF THE STUDY: Used in combination, RAM-PCR and RAPD-PCR permit traceability and monitoring systems for G. candidum strains during food processing.

Flavour sulphides are produced from methionine by two different pathways by Geotrichum candidum.

We have investigated the capacities of Geotrichum candidum strains to produce sulphides from methionine. This attribute is very important in cheese technology because of the flavouring potential of sulphur compounds. A spectrophotometric procedure using 5,5'-dithiobis(2-nitrobenzoic acid) to determine sulphides was tested on a collection of G. candidum strains, and confirmed by gas chromatography-mass spectrometry. The strains were distinguished on the basis of their ability to produce methanethiol. Gas chromatography-mass spectrometry also made it possible to identify other sulphides, such as dimethyl disulphide, dimethyl trisulphide and dimethyl sulphide. Using sonicated cells, the specific production of these four sulphides was studied in presence of L-[S-methyl-2H]methionine. Both methanethiol and dimethyl sulphide were produced from methionine, but two different pathways were used by G. candidum.