Emmanuel Coton

Biogenic amines in fermented foods.

Food-fermenting lactic acid bacteria (LAB) are generally considered to be non-toxic and non-pathogenic. Some species of LAB, however, can produce biogenic amines (BAs). BAs are organic, basic, nitrogenous compounds, mainly formed through decarboxylation of amino acids. BAs are present in a wide range of foods, including dairy products, and can occasionally accumulate in high concentrations. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding BA content in many fermented products, it is generally assumed that they should not be allowed to accumulate. The ability of microorganisms to decarboxylate amino acids is highly variable, often being strain specific, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the likelihood that foods contain BA and to prevent their accumulation in food products. Moreover, improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods.

Low occurrence of safety hazards in coagulase negative staphylococci isolated from fermented foodstuffs

Some coagulase negative staphylococci (CNS) species play an important role in the fermentation of meat and milk products and are considered as food-grade. However, the increasing clinical significance of CNS and the presence of undesirable and unsafe properties in CNS question their presence or use in food. Our goal was to assess the safety of CNS by developing a diagnostic microarray targeting 268 genes corresponding to safety hazards in a food context i.e. toxins (especially enterotoxins) and determinants of antibiotic resistance and biogenic amine production. Target genes were selected among staphylococci and Gram-positive species that may be in contact with CNS in foodstuffs. The diagnostic microarray was used to screen 129 strains belonging to the 2 dominant species isolated from foodstuffs (S. equorum and S. xylosus) and the 2 main species isolated both in foodstuffs and clinical samples (S. epidermidis and S. saprophyticus). Microarray data were further completed by antibiograms and measurement of biogenic amine production. Safety hazards associated with CNS were mostly limited to the presence of antibiotic resistance. Seventy-one percent of the strains possessed at least one gene encoding antibiotic resistance, while only one strain carried an enterotoxin gene. Most strains did not carry any genes encoding staphylococcal toxins (68%), non-staphylococcal toxins (95%) or decarboxylases involved in biogenic amine production (78%). Food safety hazards were more pronounced in S. epidermidis than in the three other species regardless the food or clinical origin of the strains. Seventy-six percent of the strains carrying genes encoding staphylococcal toxin and 69% of strains carrying 5 or more antibiotic determinants belonged to S. epidermidis species. The dominant antibiotic resistance targeted erythromycin, tetracycline and penicillin and were generally traced back to the presence of tetK and blaZ in the two latest cases. Six percent of the food-related strains produced significant amounts of biogenic amines in vitro without any of the corresponding genes detected, reflecting a lack of knowledge on genetic determinants of such production in staphylococci. This work gives a first picture of safety hazards within four species of CNS frequently isolated from food or clinical environment.

Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses.

The goal of this study was to identify at the species level a large collection of Gram-negative dairy bacteria isolated from milks or semi-hard and soft, smear-ripened cheeses (cheese core or surface samples) from different regions of France. The isolates were then assessed for two risk factors, antibiotic resistance and volatile and non-volatile biogenic amine production in vitro. In total, 173 Gram-negative isolates were identified by rrs and/or rpoB gene sequencing. A large biodiversity was observed with nearly half of all Gram-negative isolates belonging to the Enterobacteriaceae family. Overall, 26 different genera represented by 68 species including potential new species were identified among the studied Gram-negative isolates for both surface and milk or cheese core samples. The most frequently isolated genera corresponded to Pseudomonas, Proteus, Psychrobacter, Halomonas and Serratia and represented almost 54% of the dairy collection. After Pseudomonas, Chryseobacterium, Enterobacter and Stenotrophomonas were the most frequently isolated genera found in cheese core and milk samples while Proteus, Psychrobacter, Halomonas and Serratia were the most frequently isolated genera among surface samples. Antibiotic resistance profiles indicated that resistances to the aminosid, imipemen and quinolon were relatively low while more than half of all tested isolates were resistant to antibiotics belonging to the monobactam, cephem, fosfomycin, colistin, phenicol, sulfamid and some from the penam families. Thirty-six% of isolates were negative for in vitro biogenic amine production. Among biogenic amine-producers, cadaverine was the most frequently produced followed by isoamylamine, histamine and putrescine. Only low levels (<75 mg/l) of tyramine were detected in vitro.

Filamentous Fungi and Mycotoxins in Cheese: A Review

Important fungi growing on cheese include Penicillium, Aspergillus, Cladosporium, Geotrichum, Mucor, and Trichoderma. For some cheeses, such as Camembert, Roquefort, molds are intentionally added. However, some contaminating or technological fungal species have the potential to produce undesirable metabolites such as mycotoxins. The most hazardous mycotoxins found in cheese, ochratoxin A and aflatoxin M1, are produced by unwanted fungal species either via direct cheese contamination or indirect milk contamination (animal feed contamination), respectively. To date, no human food poisoning cases have been associated with contaminated cheese consumption. However, although some studies state that cheese is an unfavorable matrix for mycotoxin production; these metabolites are actually detected in cheeses at various concentrations. In this context, questions can be raised concerning mycotoxin production in cheese, the biotic and abiotic factors influencing their production, mycotoxin relative toxicity as well as the methods used for detection and quantification. This review emphasizes future challenges that need to be addressed by the scientific community, fungal culture manufacturers, and artisanal and industrial cheese producers.

Evidence of horizontal transfer as origin of strain to strain variation of the tyramine production trait in Lactobacillus brevis.

Lactobacillus brevis strains with the ability to decarboxylate tyrosine to tyramine have been described and the involvement of several genes constituting a tyrdc operon at the chromosomal level has been demonstrated in this species. In this study, the existence of Lb. brevis strains unable to form tyramine was observed. In order to evaluate if the tyramine-producing ability was strain-dependent or if it could be correlated to the existence of a new species or subspecies, different isolates were analysed. Analysis by M13-RAPD and sequencing of 16S rDNA, 16S-23S ISR and house-keeping gene recA confirmed that all the isolates belonged to the Lb. brevis species. Analysis of the TyrDC pathway encoding operon region in representative strains indicated the existence of a polymorphism. The genetic differences observed showed that the tyrosine decarboxylating ability is not a Lb. brevis species trait but that it is strain-dependent within this species and suggest that the genes encoding the tyramine-producing pathway constitute a genomic island.

Evidence of Distinct Populations and Specific Subpopulations within the Species Oenococcus oeni

ABSTRACT Among the lactic acid bacteria (LAB) present in the oenological microbial ecosystem, Oenococcus oeni, an acidophilic lactic acid bacterium, is essential during winemaking. It outclasses all other bacterial species during malolactic fermentation (MLF). Oenological performances, such as malic acid degradation rate and sensorial impact, vary significantly according to the strain. The genetic diversity of the O. oeni species was evaluated using a multilocus sequence typing (MLST) scheme. Seven housekeeping genes were sequenced for a collection of 258 strains that had been isolated all over the world (particularly Burgundy, Champagne, and Aquitaine, France, Chile, South Africa, and Italy) and in several wine types (red wines, white wines, and champagne) and cider. The allelic diversity was high, with an average of 20.7 alleles per locus, many of them being rare alleles. The collection comprised 127 sequence types, suggesting an important genotypic diversity. The neighbor-joining phylogenetic tree constructed from the concatenated sequence of the seven housekeeping genes showed two major phylogenetic groups, named A and B. One unique strain isolated from cider composed a third group, rooting the phylogenetic tree. However, all other strains isolated from cider were in group B. Eight phylogenetic subgroups were statistically differentiated and could be delineated by the analysis of only 32 mutations instead of the 600 mutations observed in the concatenated sequence of the seven housekeeping genes. Interestingly, in group A, several phylogenetic subgroups were composed mostly of strains coming from a precise geographic origin. Three subgroups were identified, composed of strains from Chile, South Africa, and eastern France.

Ecological and aromatic impact of two Gram-negative bacteria (Psychrobacter celer and Hafnia alvei) inoculated as part of the whole microbial community of an experimental smear soft cheese.

The impact of the growth of two Gram-negative bacteria, Psychrobacter celer and Hafnia alvei, inoculated at 10(2) and 10(6) cfu/g, on the dynamics of a multispecies community as well as on volatile aroma compound production during cheese ripening was investigated. Results showed that P. celer was able to successfully implant itself in cheese, regardless of its inoculation level. However, when it was inoculated at a high level, the bacterial biodiversity was drastically lowered from day 25 to the end of ripening. Overall, the presence of P. celer led to the higher production of volatile aroma compounds such as aldehydes, ketones and sulfur compounds. Regardless of its inoculation level, H. alvei barely affected the growth of the bacterial community and was subdominant at the end of ripening. It influenced total volatile aroma compound production with volatile sulfur compounds being the most abundant. Overall, these two bacteria were able to implant themselves in a cheese community and significantly contributed to the aromatic properties of the cheese. Their role in flavoring and their interactions with the technological microorganisms must be considered during cheese ripening and should be further investigated.

Origin of the Putrescine-Producing Ability of the Coagulase-Negative Bacterium Staphylococcus epidermidis 2015B

ABSTRACT A multiplex PCR method, aimed at the detection of genes associated with biogenic amine production, identified the odc gene encoding ornithine decarboxylase in 1 of 15 strains of Staphylococcus epidermidis. The ability of the positive strain, S. epidermidis 2015B, to produce putrescine in vitro was demonstrated by high-performance liquid chromatography (HPLC). In this strain, the odc gene was detected on plasmid DNA, suggesting that the ability to form putrescine is carried by a mobile element, which explains the fact that the trait is strain dependent within the S. epidermidis species. A 6,292-bp nucleotide sequence harboring the putative odc gene was determined. S. epidermidis ornithine decarboxylase (ODC) showed 60 to 65% sequence identity with known ODCs of Gram-positive as well as Gram-negative bacteria. Downstream of the odc gene, a gene encoding a putative amino acid transporter was found that shared 59% sequence identity with the ornithine/putrescine exchanger (PotE) of Escherichia coli. Cloning and expression of the potE gene of S. epidermis 2015B in Lactococcus lactis demonstrated that the gene product transported ornithine and putrescine into the cells and efficiently exchanged putrescine for ornithine. Analysis of the flanking regions showed high identity levels with different S. epidermidis plasmid sequences, which would confirm the plasmidic location of the odc operon. It follows that the odc and potE gene pair encodes a putrescine-producing pathway in S. epidermis 2015B that was acquired through horizontal gene transfer.

Induction of sexual reproduction and genetic diversity in the cheese fungus Penicillium roqueforti

The emblematic fungus Penicillium roqueforti is used throughout the world as a starter culture in the production of blue‐veined cheeses. Like other industrial filamentous fungi, P. roqueforti was thought to lack a sexual cycle. However, an ability to induce recombination is of great economic and fundamental importance, as it would make it possible to transform and improve industrial strains, promoting the creation of novel phenotypes and eliminating the deleterious mutations that accumulate during clonal propagation. We report here, for the first time, the induction of the sexual structures of P. roqueforti — ascogonia, cleistothecia and ascospores. The progeny of the sexual cycle displayed clear evidence of recombination. We also used the recently published genome sequence for this species to develop microsatellite markers for investigating the footprints of recombination and population structure in a large collection of isolates from around the world and from different environments. Indeed, P. roqueforti also occurs in silage, wood and human‐related environments other than cheese. We found tremendous genetic diversity within P. roqueforti, even within cheese strains and identified six highly differentiated clusters that probably predate the use of this species for cheese production. Screening for phenotypic and metabolic differences between these populations could guide future development strategies.

Genotypic characterization of Enterobacter sakazakii isolates by PFGE, BOX‐PCR and sequencing of the fliC gene

Aims:  Enterobacter sakazakii is an emerging food‐borne pathogen that can cause rare but severe forms of neonatal meningitis, bacteraemia and necrotizing enterocolitis. A rapid typing method at the strain level is needed to determine the monoclonality or polyclonality of the isolates during outbreaks.