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.

Molecular characterization of O157:H7, O26:H11 and O103:H2 Shiga toxin-producing Escherichia coli isolated from dairy products.

Pathogenic Shiga toxin-producing E. coli (STEC) are recognized worldwide as environment and foodborne pathogens which can be transmitted by ingestion of ready-to-eat food such as raw milk-derived products. STEC show a prevalence rate in dairy products of 0.9%, yet comparably few outbreaks have been related to dairy products consumption. In this study, we used rt-qPCR to identify the virulence potential of O157, O26 and O103 STEC strains isolated from raw-milk dairy products by analyzing virulence-related gene frequencies and associations with O-island (OI) 44, OI-48, OI-50, OI-57, OI-71 and OI-122. Results showed that 100% of STEC strains investigated harbored genes associated with EHEC-related virulence profile patterns (eae and stx, with either espK, espV, ureD and/or Z2098). We also found similarities in virulence-related gene content between O157:H7 and O103:H2 dairy and non-dairy STEC strains, especially isolates from human cases. The O26:H11-serotype STEC strains investigated harbor the arcA-allele 2 gene associated with specific genetic markers. These profiles are associated with high-virulence seropathotype-A STEC. However, the low frequency of stx2 gene associated with absence of other virulence genes in dairy isolates of O26:H11 remains a promising avenue of investigation to estimate their real pathogenicity. All O26:H11 attaching-effacing E. coli (AEEC) strains carried CRISPRO26:H11SP_O26_E but not genetic markers espK, espV, ureD and/or Z2098 associated with the emerging potentially high-virulence "new French clone". These strains are potentially as "EHEC-like" strains because they may acquire (or have lost) stx gene. In this study, O157:H7, O103:H2 and O26:H11 STEC strains isolated from dairy products were assigned as potential pathogens. However, research now needs to investigate the impact of dairy product environment and dairy processing on the expression of their pathogenicity.

Invited review: Anti-adhesive properties of bovine oligosaccharides and bovine milk fat globule membrane-associated glycoconjugates against bacterial food enteropathogens.

The prevalence of the main raw milk and raw milk-derived dairy product enteropathogens (Campylobacter, Shiga toxin-producing Escherichia coli, Listeria, and Salmonella) is higher than the number of epidemiological cases related to ingesting these foodstuffs. Bovine milk oligosaccharides and milk fat globule membrane (MFGM)-linked glycoconjugates interact with foodborne enteropathogens to inhibit their adhesion to intestinal cells and tissues. This review examines the main mechanisms and strategies used by enteropathogens to adhere to their target, details the anti-adhesive properties of MFGM against enteropathogens and enterotoxins, assesses the integrity of bacteria-MFGM complexes during dairy product manufacture and digestion, and discusses the potential for using these macromolecules and glycoconjugates in foods for public health.

Short communication: Behavior of different Shiga toxin-producing Escherichia coli serotypes (O26:H11, O103:H2, O145:H28, O157:H7) during the manufacture, ripening, and storage of a white mold cheese.

Ruminants are healthy carriers of Shiga toxin-producing Escherichia coli (STEC). If good hygienic and agricultural practices at the farm level, especially during the milking process, are not adequately followed, milk and dairy products made with raw milk could become contaminated. Sporadic cases and rare food outbreaks have been linked with dairy products. Consequently, understanding STEC behavior in cheeses would help to evaluate risks for human health. The behavior of 4 different STEC strains belonging to the serotypes O26:H11, O103:H2, O145:H28, and O157:H7 were monitored during the manufacture, ripening, and storage of a white mold soft cheese. These strains, originating from dairy products, were inoculated individually in raw milk from cow at 10(2) cfu/mL. During the first 24 to 36h of the manufacturing stage, the STEC level increased by 2 to 3 log10 cfu/g. Over the course of the ripening stage, the concentration of the non-O157 STEC remained relatively constant, whereas a decrease of the E. coli O157:H7 concentration was observed. During the storage stage, the level of the different non-O157 STEC strains decreased slowly in the core and in the rind of cheeses. The non-O157 STEC level reached between 3.1 and 4.1 log10 cfu/g at d 56. Interestingly, the concentration of the E. coli O157:H7 strain decreased dramatically: the strains remained detectable only after enrichment. During ripening and storage, STEC levels were generally higher in rinds than in cheese cores. In contrast to what was seen in cheese cores, the E. coli O157:H7 strain remained enumerable in rinds during these steps. These results highlight that STEC can grow during the manufacture and survive during the ripening and storage of a white mold soft cheese.

Behavior of Escherichia coli O26:H11 in the presence of Hafnia alvei in a model cheese ecosystem.

This study was designed to evaluate the capacity of three Hafnia strains to inhibit the growth of an E. coli strain O26:H11 in an uncooked pressed model cheese, in the presence or absence of a microbial consortium added to mimic a cheese microbial community. Inoculated at 2 log CFU/ml into pasteurized milk without Hafnia, the E. coli O26:H11 strain reached 5 log CFU/g during cheese-making and survived at levels of 4 to 5 log CFU/g beyond 40 days. Inoculated into milk at 6 log CFU/ml, all three tested Hafnia strains (H. alvei B16 and HA, H. paralvei 920) reached values close to 8 log CFU/g and reduced E. coli O26:H11 counts in cheese on day 1 by 0.8 to 1.4 log CFU/g compared to cheeses inoculated with E. coli O26:H11 and the microbial consortium only. The Hafnia strains slightly reduced counts of Enterococcus faecalis (~-0.5 log from day 1) and promoted Lactobacillus plantarum growth (+0.2 to 0.5 log from day 8) in cheese. They produced small amounts of putrescine (~1.3 mmol/kg) and cadaverine (~0.9 mmol/kg) in cheese after 28 days, and did not affect levels of volatile aroma compounds. Further work on H. alvei strain B16 showed that E. coli O26:H11, inoculated at 2 log CFU/ml, was inhibited by H. alvei B16 inoculated at 6 log CFU/ml and not at 4.5 log CFU/ml. The inhibition was associated neither with lower pH values in cheese after 6 or 24h, nor with higher concentrations of lactic acid. Enhanced concentrations of acetic acid on day 1 in cheese inoculated with H. alvei B16 (4 to 11 mmol/kg) could not fully explain the reduction in E. coli O26:H11 growth. A synergistic interaction between H. alvei B16 and the microbial consortium, resulting in an additional 0.7-log reduction in E. coli O26:H11 counts, was observed from day 8 in model cheeses made from pasteurized milk. However, E. coli O26:H11 survived better during ripening in model cheeses made from raw milk than in those made from pasteurized milk, but this was not associated with an increase in pH values. In vitro approaches are required to investigate the mechanisms and causative agents of this interaction. H. alvei B16 appears to be a promising strain for reducing E. coli O26:H11 growth in cheese, as part of a multi-hurdle approach.

Milk fatty acid composition and cheese texture and appearance from cows fed hay or different grazing systems on upland pastures.

The objective of this work was to compare milk fatty acid (FA) profile and texture and appearance of Cantal cheeses obtained from cows grazing 2 different upland grasslands: a highly diversified pasture (74 species) of area 12.5 ha managed under continuous mode (C), and a weakly diversified pasture (31 species) of area 7.7 ha (an old temporary grassland) managed under rotational mode (R). A control group of cows fed a hay-based diet (indoors, I) was used. Three equivalent groups of 12 Montbéliarde cows underwent the 3 treatments from May to September 2008. The cheeses were manufactured during 3 consecutive days in early June, early July, and late August (27 cheeses in all). The texture, appearance, and chemical composition of the cheeses were determined after 12 wk of ripening. Concentrations of total saturated FA and monounsaturated FA were higher and lower, respectively, in I milks compared with pasture milks. The concentrations of trans-11-C18:1 and cis-9-C18:1, and polyunsaturated FA as well as yellowness decreased during the season in C-derived milk but remained constant in R-derived milk, through a combined effect of grass development stage and the cows' grazing selection. The I cheeses were, on average, firmer, less creamy, less elastic, and less yellow than the pasture cheeses. Decreasing and increasing trends in texture firmness during the season were observed for C and R cheeses, respectively. The rind of the pasture-fed cow cheese had fewer, less intensely colored, and less prominent spots than did that of I cheeses. This difference was probably due to greater migration of fat to the rind during pressing because of the lower fat melting point of the pasture-fed cow cheeses, which had higher unsaturated FA content. The greater amounts of fat deposited on the rind of the pasture-fed cow cheeses may have partially inhibited the microbial activity responsible for rind appearance. Our trial underlines the importance of the effects of grazing management associated with vegetation type on milk and cheese characteristics.

Effect of Lactococcus garvieae, Lactococcus lactis and Enterococcus faecalis on the behaviour of Staphylococcus aureus in microfiltered milk.

The effect of four strains of Lactococcus garvieae, three strains of Lactococcus lactis and one strain of Enterococcus faecalis on Staphylococcus aureus SA15 growth in microfiltered milk was evaluated. Lactococcus and Enterococcus strains were co-cultured with S. aureus in microfiltered milk and in medium buffered at pH 6.8. All Lactococcus and Enterococcus strains were able to inhibit S. aureus growth after 6h of incubation. Inhibition by L. lactis and E. faecalis strains could be partially attributed to the decrease in pH below 6.0 as it has been observed in medium buffered at pH 6.8. L. garvieae strains were the most effective to inhibit S. aureus growth without acidification. Inhibition of S. aureus could not be attributed neither to production of lactate, acetate or nor to antistaphylococcal substance. Amino acids competition was not involved in the inhibition by L. garvieae as addition of valine, isoleucine, threonine, methionine and phenylalanine did not suppress the inhibition of S. aureus.

Effect of temperature and pH on growth of Staphylococcus aureus in co-culture with Lactococcus garvieae.

Staphylococcus aureus growth and enterotoxin production in co-culture with Lactococcus garvieae were studied in laboratory medium as a function of incubation temperature and pH values. Doehlert experimental design was used to study the effect of L. garvieae concentration, temperature, and pH on S. aureus growth in laboratory medium. The mathematical model obtained was validated in cheeses. The inhibition of S. aureus growth by L. garvieae was more important during the first 6 hours of incubation, and its effect increased when its concentration increased. After 24 and 48 hours, the effect of L. garvieae decreased, and the growth of S. aureus was positively influenced by higher temperature and pH values. Staphylococcal enterotoxins were detected in only one experimental set after 48 hours of incubation at 30 degrees C at pH 6.8. Our results argue in favor of adding antagonist strain early in the cheese-making process.

Application of Single Strand Conformation Polymorphism --PCR method for distinguishing cheese bacterial communities that inhibit Listeria monocytogenes.

The aim of this study was to compare the microbial communities of different cheeses where Listeria monocytogenes either grew or did not grow. For this purpose, (i) isolates from the most inhibitory cheese ecosystem were identified and their ability to produce anti-Listeria substances was determined, (ii) bacterial communities of cheeses with and without L. monocytogenes growth were compared using the Single Strand Conformation Polymorphism method. The study showed SSCP to be an effective tool for differentiating between the bacterial communities of different cheeses manufactured with the same technology. All the cheeses with the lowest L. monocytogenes counts on day 8 were distinguished by the dominance in their SSCP profiles, after amplification of the V2 region of the 16S rRNA gene, of 3 peaks whose nucleotide sequences comigrated with Enterococcus faecium and Enterococcus saccharominimus, Chryseobacterium sp and Corynebacterium flavescens, Lactococcus garvieae and Lactococcus lactis respectively. However, no anti-Listeria compounds were produced under our experimental conditions. These six bacterial species were inoculated, separately or together, into pasteurised milk and their anti-listerial activity in cheese was evaluated. The area of inhibition between the control and trial curves confirmed that L. monocytogenes is inhibited by E. saccharominimus, C. flavescens, L. lactis, L. garvieae and the mixture of all six bacterial strains. Further studies should be performed to determine the metabolites involved in L. monocytogenes inhibition.

Control of Listeria monocytogenes in raw-milk cheeses.

The development of Listeria monocytogenes in cheeses made with raw-milk originating from six different farms and according to the Saint-Nectaire cheesemaking technology was studied. Milk was inoculated with two strains of L. monocytogenes at 5 to 10 CFU/25 ml. Microbial and chemical analyses were carried out at appropriate intervals during ripening. L. monocytogenes did not grow in the cores of cheeses prepared with milk originating from three farms. That inhibition could be partially attributed to the pH values and L-lactate content. There was no growth in cheeses with pH below 5.2 and lactate content around 14 mg/g. In all cheeses, L. monocytogenes stopped growing in the cores of cheeses after eight days and some other factors may be involved in the inhibition. No relation was found between L. monocytogenes count and other microbial counts. Growth occurred on cheese surfaces between eight and eighteen days, when the pH significantly increased. The lowest L. monocytogenes growth was found on the surface of cheeses with the lowest pH and without any core growth. Further studies will be performed to clarify the involvement of the microbial community in L. monocytogenes inhibition, in particular during the ripening period.

The effect of raw milk microbial flora on the sensory characteristics of Salers-type cheeses.

The sensory characteristics of Salers Protected Denomination of Origin raw-milk cheeses are linked to the biochemical composition of the raw material (milk) and to the resultant microbial community. To evaluate the influence of the microbial community on sensory characteristics, Salers-type cheeses were manufactured with the same pasteurized milk, reinoculated with 3 different microbial communities from 3 different filtrates from microfiltered milks. Each cheese was subjected to microbial counts (on selective media), biochemical tests, and volatile and sensory component analyses at different times of ripening. Adding different microbial communities to specimens of the same (biochemically identical) pasteurized milk lead to different sensory characteristics of the cheeses. Cheeses with fresh cream, hazelnut, and caramel attributes were opposed to those with fermented cream, chemical, and garlic flavors. The aromatic compounds identified (esters, acids, alcohols, and aldehydes) in these cheeses were quite similar. Nevertheless, one milk was distinguished by a higher content of acetoin, and lower 2-butanone and 3-methylpentanone concentrations. Over the production period of 1 mo, the different cheeses were characterized by the same balance of the microbial population assessed by microbial counts on different media. This was associated with the stability of some sensory attributes describing these cheeses. Nevertheless, there was no linear correlation between microbial flora data and sensory characteristics as measured in this study.

Design and application of a Staphylococcus-specific single strand conformation polymorphism-PCR analysis to monitor Staphylococcus populations diversity and dynamics during production of raw milk cheese.

AIM: Development of a nested-PCR single strand conformation polymorphism (SSCP) assay targeting the 16S rRNA genes of the Staphylococcus genus, to monitor staphylococci in cheese. METHODS AND RESULTS: New primer sets to specifically amplify 16S rDNA of staphylococci were designed to be used in a nested-PCR SSCP assay. The method was efficient in discriminating the staphylococcal species most frequently found in cheese. It was validated by monitoring Staphylococcus populations in three productions of raw milk cheese. Analysis of milk samples revealed dominant SSCP peaks corresponding to Staphylococcus aureus, Staphylococcus equorum and Staphylococcus saprophyticus. After 12 h, the S. aureus peak became dominant. CONCLUSIONS: The combination of specific Staphylococcus nested-PCR and SSCP allows rapid and direct monitoring of staphylococci diversity and dynamics in milk and cheese. In the core of the cheeses studied, S. aureus may have ecological advantages against other Staphylococcus populations. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a promising tool to study the ecology of staphylococci in cheeses and in other food samples.

Diversity, dynamics and activity of bacterial populations in 'Registered Designation of Origin' Salers cheese by single-strand conformation polymorphism analysis of 16S rRNA genes.

AIMS: The aim of this work was to measure the dynamic global metabolic activities of the microbial community during ripening of RDO Salers cheese by using a direct molecular approach. METHODS AND RESULTS: A culture-independent approach including PCR, reverse transcriptase PCR (RT-PCR) and single strand conformation polymorphism (SSCP) analysis of 16S rRNA genes was applied on 'Registered Designation of Origin' Salers cheese samples collected in three farms. The evolution of the global structure of the microbial community in terms of structure or global activities was assessed using ecological indices. The diversity of the global population was higher on RNA patterns than on DNA patterns, because of less dominance and greater richness. Comparison of the SSCP patterns derived from RNA and DNA analysis indicated that the dominant population was not necessarily the most active. The metabolic activities of each bacterial group changed significantly during ripening. Besides lactic acid bacteria that were dominant on both DNA and RNA patterns, the dynamics of the presence and activity of microbial groups rarely studied in the core of cheese, such as corynebacteria, or of unidentified peaks were reported. CONCLUSIONS: By using SSCP RNA analysis, we were able to obtain information about the activity of bacterial population in cheese, which varied a lot between cheeses and was changing perpetually during ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: Double DNA-RNA SSCP analysis opens up future prospects in the microbial ecology of cheeses. It will have many applications for controlling of microbial community during cheese processing.

Analysis of biogenic amines in northern and southern European sausages and role of flora in amine production.

The biogenic amine contents, microbial counts and flora producing amines were investigated in four types of fermented sausages. Southern type European sausages (Italian and Belgian) showed higher tyramine and phenylethylamine values than northern type ones (Norwegian and Belgian). The spontaneous non-starter lactic acid bacteria could be responsible for the production of these amines in the Italian products, and the cocci Gram positive in the Belgian South ones. The Norwegian sausages showed the lowest total amine content of those studied. The two Belgian types were characterised by the highest putrescine contents, associated with high counts of Enterococcus. The production of amines in vitro by the starter cultures used in the manufacture of the sausages revealed that none of the Lactobacillus species produced any amines and only Kocuria varians and Staphylococcus carnosus showed phenylethylamine and tryptamine production. High correlations were found between the content of putrescine, histamine and cadaverine.

Use of green fluorescent protein to monitor Lactobacillus sakei in fermented meat products.

Lactobacillus sakei is a lactic acid bacterium naturally found on meat and often used as starter for the production of dry sausages or other fermented meat products. The gene encoding the green fluorescent protein (GFP) was cloned downstream from the constitutive L-lactate dehydrogenase promoter (pldhL) of L. sakei. The pldhL::gfp fusion was introduced in L. sakei either on a replicative plasmid or by double crossover integration into the chromosome, as a single copy. Both constructions were stable. Expression of GFP did not alter growth and was detectable by epifluorescence microscopy allowing the detection and monitoring of the development of GFP+ specific L. sakei strains both under growth laboratory conditions and in dry sausage samples.

Growth and effect of staphylococci and lactic acid bacteria on unsaturated free fatty acids.

The growth and the effects of four species of staphylococci and six lactic acid bacteria (LAB) of the genus Carnobacterium, Lactobacillus and Pediococcus on unsaturated free fatty acids were studied. The strains were grown in complex medium supplemented either with oleic, linoleic or linolenic acids. Growth was followed and oxidation of the substrates measured by TBARS. The strains of Staphylococcus xylosus 873, 16, Staphylococcus warneri 863 and Staphylococcus saprophyticus grew well on all the substrates. Whereas, the growth of the two strains of Staphylococcus carnosus and Staphylococcus xylosus 831 was inhibited in the media with linolenic acid. The addition of manganese to this media allowed good growth of these strains. All the LAB did not grow well in the media with linoleic acid, but their growth was favoured by addition of manganese to the media. Under our conditions, only linoleic and linolenic acids were oxidised. All the strains had no prooxidant activity. All the staphylococci limited oxidation of linoleic acid and had a small effect on linolenic acid. LAB did not limit oxidation of linoleic acid. With manganese in the media: the oxidation of the sterile controls was delayed for 2 days and then increased; strains of S. carnosus and S. xylosus inhibited oxidation of linolenic acid; and Lactobacillus plantarum and Pediococcus pentosaceus limited oxidation of linoleic acid. The two Carnobacterium, whatever the conditions, had no antioxidant properties.

Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci.

The objective of this work was to study the production of catalase and nitrate reductase by staphylococci in order to understand their role in lipid oxidation during sausage manufacturing. Catalase and nitrate reductase were measured in resting cells and supernatants of staphylococci grown in different conditions. All staphylococci (except S. warneri) synthetized nitrate reductase. In static condition, the synthesis was maximal during exponential growth phase, whereas in shaking condition, the synthesis was maximal at the beginning of stationary phase. The production of nitrate reductase was increased in presence of nitrate, this effect was particularly important for the two S. carnosus strains which exhibited the highest activity. For all staphylococci, the production of catalase was maximal at the end of the exponential growth phase. The lowest amount of catalase was produced by S. warneri and the highest by S. carnosus. Only S. xylosus 873 and S. saprophyticus 852 released high amounts of catalase in the supernatant growth. Staphylococci produced higher amounts of catalase in shaking conditions. Addition of nitrate in the growth media favoured the synthesis of catalase, with a pronounced effect for S. carnosus. Nitrate also favoured the release of catalase.

Factors influencing leucine catabolism by a strain of Staphylococcus carnosus.

The metabolism of leucine by resting cells of Staphylococcus carnosus 833 was studied according to three physicochemical factors: preculture condition (defined medium; complex medium), nitrate concentration (0% and 0.03%) and stirring condition (static or shaking). A factorial design was set up to test the effects of these factors, each at two levels. The results showed that resting cells of S. carnosus 833 produced 3-methyl butanal, 3-methyl butanol and 3-methyl butanoic acid from leucine. Whatever the incubation conditions, there was greater quantity of 3-methyl butanoic acid than 3-methyl butanal and 3-methyl butanol. The preculture and incubation conditions influenced the level of production of the 3 metabolites. The highest overall production of the 3 metabolites was observed when cells were incubated without nitrate in the reaction mixture. 3-methylbutanoic acid production was enhanced when S. carnosus 833 was precultivated in complex medium. 3-methylbutanal was only detected when cells were precultivated in defined medium. Stirring condition had no effect on leucine catabolism of S. carnosus 833.

Identification of Staphylococcus carnosus and Staphylococcus warneri isolated from meat by fluorescent in situ hybridization with 16S rRNA-targeted oligonucleotide probes.

16S rRNA targeted oligonucleotide probes were designed by sequence analysis of an rRNA database to discriminate S. carnosus, S. warneri, and S. saprophyticus species. After establishing hybridization conditions by RNA dot blot hybridization with reference species, our probes were shown to be specific. By in situ hybridization only S-S-S.carno-0440-a-A-23 and S-S-S.war-0180-a-A-23 can specifically detect S. carnosus and S. warneri, respectively. The detection of old cells of S. carnosus 833 was more limited by the permeabilisation than by the low rRNA content. One day old cells could be permeabilized with lysostaphin, whereas young cells were permeabilized with lysozyme.

Tyramine production by a strain of Carnobacterium divergens inoculated in meat-fat mixture.

This study determined if a strain of Carnobacterium divergens producing tyramine in laboratory medium is able to produce this amine in meat. Thus the strain was inoculated into a sterile meat-fat mixture of initial pH 5.3 or 4.9 and incubated at 15°C or 25°C. Amine production in these samples was measured at 12 days and compared to that in non-inoculated samples. Non-inoculated samples and those inoculated with C. divergens had the same low content of tryptamine, putrescine, cadaverine, histamine, spermine and spermine. Maximum tyramine and phenylethylamine production was observed in sample of initial pH 5.3 inoculated with C. divergens and incubated at 25°C. The amount of tyramine produced correlated with the growth of the strain, and the amount of lactate and acetate produced.