Identification and molecular phylogeny of coagulase-negative staphylococci isolates from Minas Frescal cheese in southeastern Brazil: Superantigenic toxin production and antibiotic resistance.

Minas Frescal is a typical Brazilian fresh cheese and one of the most popular dairy products in the country. This white soft, semiskimmed, nonripened cheese with high moisture content is obtained by enzymatic coagulation of cow milk using calf rennet or coagulants, usually in industrial dairy plants, but is also manufactured in small farms. Contamination of Minas Frescal by several staphylococci has been frequently reported. Coagulase-negative staphylococci (CNS) strains are maybe the most harmful, as they are able to produce heat-stable enterotoxins with super antigenic activities in food matrices, especially in dairy products such as soft cheeses. The aim of the present study was to investigate the presence of CNS strains in Minas Frescal marketed in southeastern Brazil concerning the risk of staphylococci food poisoning by the consumption of improperly manufactured cheese and the possibility of these food matrices being a reservoir of staphylococcal resistance to antimicrobials. Ten distinct CNS strains were found in 6 cheeses from distinct brands. The most frequent species were Staphylococcus saprophyticus (40%), Staphylococcus xylosus (30%), Staphylococcus sciuri (20%), and Staphylococcus piscifermentans (10%). Three strains were identified to the Staphylococcus genera. Three major species groups composed of 3 refined clusters were grouped by phylogenetic analyses with similarities over to 90%. All CNS strains carried multiple enterotoxin genes, with high incidence of sea and seb (90 and 70%, respectively), followed by sec/see, seh/sei, and sed with intermediate incidence (60, 50, and 40%, respectively), and, finally, seg/selk/selq/selr and selu with the lowest incidence (20 and 10%, respectively). Real-time reverse transcription PCR and ELISA assays confirmed the enteroxigenic character of the CNS strains, which expressed and produced the enterotoxins in vitro. The CNS strains showed multiresistance to antimicrobial agents such as ß-lactams, vancomycin, and linezolid, which have therapeutic importance in both human and veterinarian medicines. The risk of staphylococci food poisoning by the consumption of improperly manufactured Minas Frescal was emphasized, in addition to the possibility of these food matrices being a reservoir for antibiotic resistance. More effective control measures concerning the presence and typing of staphylococci in raw milk and dairy derivatives should be included to prevent the spread of pathogenic strains.

Purification and characterization of the chaperone-like Hsp26 from Saccharomyces cerevisiae.

sHsps are ubiquitous ATP-independent molecular chaperones, which efficiently prevent the unspecific aggregation of non-native proteins. Here, we described the purification of the small heat shock protein Hsp26 from a Saccharomyces cerevisiae strain harboring a multicopy plasmid carrying HSP26 gene under the control of its native promoter. A 26 kDa protein was purified to apparent homogeneity with a recovery of 74% by a very reproducible three steps procedure consisting of ethanol precipitation, sucrose gradient ultracentrifugation, and heat inactivation of residual contaminants. The purified polypeptide was unequivocally identified as Hsp26 using a specific Hsp26 polyclonal antibody as a probe. The analysis of the purified protein by electron microscopy revealed near spherical particles with a diameter of 12.0 nm (n=57, standard deviation +/-1.6 nm), displaying a dispersion in size ranging from 9.2 to 16.1 nm, identical to Methanococcus jannaschii Hsp16.5 and in the range of the size estimated for yeast Hsp26, in a previous report. Purified yeast Hsp26 was able to suppress 72% of the heat-induced aggregation of citrate synthase at a ratio of 1:1 (Hsp26 24-mer complex to citrate synthase dimer), and 86% of the heat-induced aggregation of lysozyme at a molar ratio of 1:16 (Hsp26 24-mer complex to lysozyme monomer). In conclusion, the Hsp26 protein purified as described here has structure and activity similar to the previously described preparations. As advantages, this new protocol is very reproducible and requires simple apparatuses which are found in all standard biochemistry laboratories.