Simona Gazzola

Gene transfer of vancomycin and tetracycline resistances among Enterococcus faecalis during cheese and sausage fermentations.

This study assessed the frequency of transfer of two mobile genetic elements coding for virulence determinants and antibiotic resistance factors, into food associated enterococci during fermentation processes. First, the transfer of the pheromone-inducible pCF10 plasmid, carrying tetracycline resistance and aggregation substance (AS) as virulence factor, between clinical and food strains of Enterococcus faecalis, was investigated in models of cheese and fermented sausage. The experiments demonstrated that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from E. faecalis OG1rf cells to food strain E. faecalis BF3098c and that the plasmid subsequently persisted in these environments. Very high frequency of transfer was observed in sausage (10(-3)/recipient) if compared to cheese (10(-6)) and plate mating (10(-4)). Transconjugants were subsequently verified by PCR. The second transmissible element was the plasmid harbouring the vancomycin resistance (VanA phenotype) from E. faecalis A256. The transfer of this antibiotic resistance to a food strain of E. faecalis was studied in vitro and in food models. Although the transfer of vancomycin resistance was achieved in all the environments, the highest conjugation frequencies were observed during the ripening of fermented sausages, reaching 10(-3) transconjugants/recipient cell. PCR confirmed the transfer of the VanA genotype into a food associated Enterococcus strain. This study showed that even in the absence of selective pressure, mobile genetic elements carrying antibiotic resistance and virulence determinants can be transferred at high frequency to food associated enterococci during cheese and sausage fermentation.

Contribution of enterococci to the spread of antibiotic resistance in the production chain of swine meat commodities.

Thirty-six samples, including fecal specimens, dry feedstuffs, raw and processed pork meat products, and dry fermented sausages, were collected from two production chains of swine meat commodities and analyzed for the presence of 11 antibiotic resistance (AR) genes. Specific PCR assays carried out on DNA extracted directly from the samples revealed a high incidence of the genes tet(K) (80.5%), ermB (66.7%), and tet(M) (66.7%). Feces and feedstuffs gave the largest number of positive amplifications. To elucidate the contribution of enterococci to the occurrence and spread of AR, 146 resistant enterococci were isolated, and their identity, genetic fingerprints, and AR gene profiles were determined by means of molecular techniques. Enterococcus faecalis and Enterococcus faecium were the predominant isolated species (43.8 and 38.4%, respectively); Other Enterococcus species identified were E. durans (8.9%), E. hirae (2.7%), E. gallinarum (2.1%), E. mundtii (2.1%), and E. casseliflavus (2.1%). A number of isolates displayed a complex AR gene profile comprising up to four different resistance determinants. The genes tet(M) and ermB were highly diffused, being present in 86.9 and 84.9%, respectively, of the isolates. The application of amplified fragment length polymorphism fingerprinting was particularly valuable to monitor the resistant enterococcal isolates along the production chain and to individuate steps in which contamination might occur. In fact, isolates of E. faecalis and E. faecium showing the same amplified fragment length polymorphism profile and AR gene pattern were detected in samples taken at different steps of the food chain suggesting three cases of bacterial clonal spread.

Vancomycin heteroresistance and biofilm formation in Staphylococcus epidermidis from food

Staphylococcus epidermidis CNBL 7032 is a heteroresistant strain, with subpopulations resistant to vancomycin concentrations up to 32 mg l (-1), which was isolated from cured pork meat. The mechanisms of glycopeptide resistance in this strain were investigated in this study. S. epidermidis CNBL 7032 does not harbour enterococcal transmissible vancomycin-resistance genes. Transmission electron microscopy revealed that resistant subpopulations have a thicker cell wall, and that the increase in cell wall thickness is proportional to vancomycin concentration in the growth medium. Scanning electron microscopy showed that S. epidermidis CNBL 7032 forms a biofilm-like structure when grown in the presence of vancomycin. This food isolate harbours the gene atlE, coding for an autolysin with an adhesive function, which is involved in the first phase of biofilm formation. This study has demonstrated an interaction between atlE expression, biofilm formation and glycopeptide antibiotic resistance; transcription analysis demonstrated that the expression of atlE increased proportionally with the vancomycin concentration in the culture. Insertional inactivation of atlE confirmed the role of the AtlE autolysin in biofilm and vancomycin resistance.

A new anti-infective strategy to reduce adhesion-mediated virulence in Staphylococcus aureus affecting surface proteins.

Staphylococcus aureus is a flexible microbial pathogen frequently isolated from community-acquired and nosocomial infections. The use of indwelling medical devices is associated with a significant risk of infection by this bacterium which possesses a variety of virulence factors, including many toxins, and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The present study evaluates the anti-infective properties of serratiopeptidase, a secreted protein of Serratia marcescens, in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. SPEP seems to exert its action by modulating specific proteins. Proteomic studies performed on surface proteins extracted from SPEP-treated S. aureus cultures revealed that a number of proteins are affected by the treatment. Among these we found the adhesin/autolysin Atl, FnBP-A, SecA1, Sbi, EF-Tu, EF-G, and alpha-enolase. EF-Tu, EF-G and alpha-enolase are known to perform a variety of functions, depending on their cytoplasmic or surface localization. All these factors can facilitate bacterial colonization, persistence and invasion of host tissues. Our results suggest that SPEP could be developed as a potential “anti-infective agent” capable to hinder the entry of S. aureus into human tissues, and also impair the ability of this pathogen to form biofilm on prostheses, catheters and medical devices.

Draft Genome Sequence of Clostridium tyrobutyricum Strain UC7086, Isolated from Grana Padano Cheese with Late-Blowing Defect

ABSTRACT Clostridium tyrobutyricum is considered the main agent of late-blowing defect in the production of hard cheese. Here, we described the draft genome sequences and annotation of C. tyrobutyricum strain UC7086, which was isolated from Grana Padano cheese with blowing defect, and C. tyrobutyricum DSM 2637 type strain in a comparative study.

Population structure and safety aspects of Enterococcus strains isolated from artisanal dry fermented sausages produced in Argentina

Enterococci population from Argentinean artisanal dry fermented sausage was identified and their safety aspects were evaluated. Species‐specific PCR was used to distinguish between Enterococcus faecium (56%) and Enterococcus faecalis (17%). Other isolates (27%) were identified as Enterococcus durans, Enterococcus casseliflavus and Enterococcus mundtii by using 16S RNA gene sequence. RAPD analyses showed different biotypes for Ent. faecium and Ent. faecalis species. Low incidence of antibiotic resistance and high virulence traits in Ent. casseliflavus and Ent. faecalis were found; the majority of the Ent. faecium strains were shown to be free of virulence factors. The absence of virulence/resistance traits and the anti‐Listeria activity of Ent. faecium isolates may be exploited to enhance natural preservation thereby guaranteeing organoleptic/safety characteristics of artisanal fermented sausages.

Transcriptome analysis of Bacillus thuringiensis spore life, germination and cell outgrowth in a vegetable-based food model

Toxigenic species belonging to Bacillus cereus sensu lato, including Bacillus thuringiensis, cause foodborne outbreaks thanks to their capacity to survive as spores and to grow in food matrixes. The goal of this work was to assess by means of a genome-wide transcriptional assay, in the food isolate B. thuringiensis UC10070, the gene expression behind the process of spore germination and consequent outgrowth in a vegetable-based food model. Scanning electron microscopy and Energy Dispersive X-ray microanalysis were applied to select the key steps of B. thuringiensis UC10070 cell cycle to be analyzed with DNA-microarrays. At only 40 min from heat activation, germination started rapidly and in less than two hours spores transformed in active growing cells. A total of 1646 genes were found to be differentially expressed and modulated during the entire B. cereus life cycle in the food model, with most of the significant genes belonging to transport, transcriptional regulation and protein synthesis, cell wall and motility and DNA repair groups. Gene expression studies revealed that toxin-coding genes nheC, cytK and hblC were found to be expressed in vegetative cells growing in the food model.

Biofilm Formation on Stainless Steel by Streptococcus thermophilus UC8547 in Milk Environments Is Mediated by the Proteinase PrtS

ABSTRACT In Streptococcus thermophilus, gene transfer events and loss of ancestral traits over the years contribute to its high level of adaptation to milk environments. Biofilm formation capacity, a phenotype that is lost in the majority of strains, plays a role in persistence in dairy environments, such as milk pasteurization and cheese manufacturing plants. To investigate this property, we have studied S. thermophilus UC8547, a fast-acidifying dairy starter culture selected for its high capacity to form biofilm on stainless steel under environmental conditions resembling the dairy environment. Using a dynamic flow cell apparatus, it was shown that S. thermophilus UC8547 biofilm formation on stainless steel depends on the presence of milk proteins. From this strain, which harbors the prtS gene for the cell wall protease and shows an aggregative phenotype, spontaneous mutants with impaired biofilm capacity can be isolated at high frequency. These mutants lack the PrtS expendable island, as confirmed by comparison of the genome sequence of UC8547Δ3 with that of the parent strain. The prtS island excision occurs between two 26-bp direct repeats located in the two copies of the ISSth1 flanking this genomic island. The central role of PrtS was confirmed by analyzing the derivative strain UC8547Δ16, whose prtS gene was interrupted by an insertional mutation, thereby making it incapable of biofilm formation. PrtS, acting as a binding substance between the milk proteins adhered to stainless steel and S. thermophilus cell envelopes, mediates biofilm formation in dairy environments. This feature provides S. thermophilus with an ecological benefit for its survival and persistence in this environment. IMPORTANCE The increased persistence of S. thermophilus biofilm has consequences in the dairy environment: if, on the one hand, the release of this microorganism from biofilm can promote the fermentation of artisanal cheeses, under industrial conditions it may lead to undesirable contamination of dairy products. The study of the molecular mechanism driving S. thermophilus biofilm formation provides increased knowledge on how an ancestral trait affects relevant phenotypes, such as persistence in the environment and efficiency of growth in milk. This study provides insight into the genetic factors affecting biofilm formation at dairy plants.

Genome Sequence of Lactococcus lactis subsp. cremoris Mast36, a Strain Isolated from Bovine Mastitis

ABSTRACT The genome sequence of Lactococcus lactis subsp. cremoris Mast36, isolated from bovine mastitis, is reported here. This strain was shown to be able to grow in milk and still possess genes of vegetable origin. The genome also contains a cluster of genes associated with pathogenicity.

Draft Genome Sequence of Vancomycin-Heteroresistant Staphylococcus epidermidis Strain UC7032, Isolated from Food.

ABSTRACT Staphylococcus epidermidis strain UC7032 was isolated from ready-to-eat cured meat and is heteroresistant to glycopeptide antibiotics. The draft whole-genome analysis revealed that this strain shows common characteristics typical of strains that are involved in nosocomial infections.