Biofilm formation of Staphylococcus aureus dairy isolates representing different genotypes.

The objective of this study was to compare the biofilm-forming capabilities of different genotypes of Staphylococcus aureus dairy isolates from Switzerland and northern Italy, including Staph. aureus genotype B (GTB) and methicillin-resistant Staph.aureus (MRSA). We hypothesized that biofilm formation might be more pronounced in the contagious GTB isolates compared with other genotypes affecting individual animals. Twenty-four dairy isolates, including 9 MRSA, were further characterized by genotyping by using ribosomal spacer PCR, spa typing, biofilm formation under static and dynamic conditions, and scanning electron microscopy. The GTB isolates (n = 6) were more able to form biofilms than other genotypes at 37°C and at 20°C after 48 and 72 h of incubation in the static assay using polystyrene microtiter plates. This result was supported by scanning electron micrographs showing a GTB isolate producing strong biofilm with extracellular matrix in contrast to a genotype C isolate. Furthermore, none of the MRSA isolates formed strong biofilms in the static assay. However, some MRSA produced low or moderate amounts of biofilm depending on the applied conditions. Under dynamic conditions, a much more diverse situation was observed. The ability of GTB isolates to be strong biofilm formers was not observed in all cases, emphasizing the importance of growth conditions for the expression of biofilm-related genes. No specific genotype, spa type, or MRSA isolate could be categorized significantly into one level of biofilm formation. Nineteen percent of isolates behaved similarly under static and dynamic conditions. The results of this study expand our knowledge of different dairy-related Staph. aureus subtypes and indicate the benefit of genotyping when biofilms are studied.

Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese.

The objective of this study was to characterize Staphylococcus aureus isolates from Swiss raw milk cheeses that had been found to be contaminated with coagulase-positive staphylococci and to estimate the frequency of the various genotypes, in particular the mastitis-associated Staph. aureus genotype B (GTB). The isolates were also tested for staphylococcal enterotoxin (SE) genes and other virulence factors. From 623 coagulase-positive staphylococci isolated from 78 contaminated raw milk cheeses, 609 were found to be Staphylococcus aureus. Genotyping of all Staph. aureus isolates was performed by PCR amplification of the 16S-23S rRNA intergenic spacer region, as this method was used previously to differentiate between mastitis subtypes associated with their clinical outcome. In total, 20 different genotypes were obtained and the 5 most frequently occurring genotypes were distributed in 6.4% or more of the samples. The enterotoxin-producing Staph. aureus GTB, known for its high contagiousness and increased pathogenicity in Swiss mastitis herds, was found to be the most abundant subtype at the sample level (71.8%) as well as among the isolates (62.0%). A subset of 107 isolates of the different genotypes were analyzed for the presence of SE genes and revealed 9 different SE gene patterns, with sed being most frequently detected and 26% being PCR-negative for SE genes. Almost all isolates of the major contaminant GTB contained the SE gene pattern sed, sej, ser, with half of them additionally carrying sea. Production of SE in vitro was consistent with the SE genes detected in most of the cases; however, some isolated GTB did not produce SEA. Staphylococcus aureus Protein A (spa) typing revealed 30 different subtypes and most GTB isolates belonged to the bovine spa type t2953; GTB/t2953 was linked among other subtypes to SE production in cheese and staphylococcal intoxication cases. Furthermore, 1 of the 623 isolates was a methicillin-resistant Staph. aureus, which was an seh-carrying Staph. aureus spa type tbl 0635 (non-GTB). We conclude that control and reduction of enterotoxigenic Staph. aureus GTB in dairy herds in Switzerland will not only prevent economic losses at the farm level but also improve the safety of raw milk cheeses; distribution of methicillin-resistant Staph. aureus via raw milk cheese is of less concern.

Short communication: heat resistance of Escherichia coli strains in raw milk at different subpasteurization conditions.

A commonly applied treatment of raw milk to reduce bacterial loads is the short-time application of heat at subpasteurization levels under continuous flow, generally referred to as thermization, because this method retains some of the beneficial properties of raw milk. In a previous study, Escherichia coli strains exhibiting increased thermotolerance were found, demanding investigations into their ability to survive thermization. Nine E. coli strains, including 4 Shiga toxin-producing E. coli (STEC) strains, were investigated for their reduction during a thermization treatment in raw milk using a pilot-plant pasteurizer to reflect typically applied commercial conditions. Six of the 9 E. coli strains, including the 4 STEC strains, were similarly inactivated at 60, 62.5, and 65°C, whereas increased thermotolerance was observed for 3 E. coli strains. All strains were reduced to <2 log10 at 60 and 62.5°C within 25s. At 65°C, 6 of 9 E. coli strains were reduced by at least 5 log10 after 25s, whereas at 67.5°C, such a reduction was observed for 8 strains. A much higher thermotolerance was found for E. coli strain FAM21805. For some E. coli strains, time-temperature combinations above 65°C were required to obtain a substantial reduction during a thermization treatment.

Bovine Staphylococcus aureus: diagnostic properties of specific media.

As accurate discrimination between Staphylococcus (S.) aureus and NSA (non-S. aureus staphylococci) involved in bovine mastitis is essential in terms of clinical prognosis and outcome, the aim of this study was to reevaluate the classical bacteriological procedures to identify these agents. Various media and the coagulase tube test were investigated using 116 strains of S. aureus and 115 of NSA, all isolated from cows with spontaneous intramammary infections (IMI). Furthermore, 25 NSA reference strains were analyzed. The study demonstrated that a few media were appropriate for differentiating S. aureus from NSA, provided that the staphylococci were isolated from bovine IMI. Evaluation of hemolysis further revealed that double or incomplete hemolysis are specific for S. aureus and are, therefore, a decisive diagnostic criterion. For strains showing complete hemolysis, maximal discrimination between S. aureus and NSA was observed by subculturing them on CHROMagar Staph. aureus.

Fate of Shiga toxin-producing and generic Escherichia coli during production and ripening of semihard raw milk cheese.

The fate of 5 different Escherichia coli strains, including 3 Shiga toxin-producing E. coli (STEC) strains, was analyzed during the production and ripening of semihard raw milk cheese. The strains, which were previously isolated from raw milk cheese, were spiked into raw milk before cheese production at 2 different levels (approximately 10(1) and 10(3) cfu/mL, respectively). Two cheese types were produced, which differed in cooking temperatures (40 and 46°C). The cheeses were sampled during manufacture and the 16-wk ripening period. An increase in E. coli counts of approximately 3.5 log(10) cfu/g occurred from raw milk to fresh cheese at d 1, which was attributed to a concentration effect during cheese production and growth of the strains. During ripening over 16 wk, a slow, continuous decrease was observed for all strains. However, significant differences were found between the E. coli strains at the applied spiking levels, whereas the inactivation was similar in the 2 different cheese types. The 2 generic E. coli strains survived at higher counts than did the 3 STEC strains. Nevertheless, only 1 of the 3 STEC strains showed significantly weaker survival at both spiking levels and in both cheese types. Six of 16 cheeses made from raw milk at a low spiking level contained more than 10 cfu/g of STEC at the end of the 16-wk ripening process. After enrichment, STEC were detected in almost all cheeses at both spiking levels. Particularly because of the low infectious dose of highly pathogenic STEC, even low colony counts in raw milk cheese are a matter of concern.

Bovine mastitis: the diagnostic properties of a PCR-based assay to monitor the Staphylococcus aureus genotype B status of a herd, using bulk tank milk.

Staphylococcus aureus genotype B (GTB) is a contagious mastitis pathogen in cattle, occurring in up to 87% of individuals. Because treatment is generally insufficient, culling is often required, leading to large economic loss in the Swiss dairy industry. As the detection of this pathogen in bulk tank milk (BTM) would greatly facilitate its control, a novel real-time quantitative PCR-based assay for BTM has previously been developed and is now being evaluated for its diagnostic properties at the herd level. Herds were initially classified as to their Staph. aureus GTB status by a reference method. Using BTM and herd pools of single-quarter and 4-quarter milk, the herds were then grouped by the novel assay, and the resulting classifications were compared. A total of 54 dairy herds were evaluated. Using the reference method, 21 herds were found to be GTB positive, whereas 33 were found to be negative. Considering the novel assay using both herd pools, all herds were grouped correctly, resulting in maximal diagnostic sensitivities (100%) and specificities (100%). For BTM samples, diagnostic sensitivities and specificities were 90 and 100%, respectively. Two herds were false negative in BTM, because cows with clinical signs of mastitis were not milked into the tank. Besides its excellent diagnostic properties, the assay is characterized by its low detection level, high efficiency, and its suitability for automation. Using the novel knowledge and assay, eradication of Staph. aureus GTB from a dairy herd may be considered as a realistic goal.

The sulfur-regulated arylsulfatase gene cluster of Pseudomonas aeruginosa, a new member of the cys regulon.

A gene cluster upstream of the arylsulfatase gene (atsA) in Pseudomonas aeruginosa was characterized and found to encode a putative ABC-type transporter, AtsRBC. Mutants with insertions in the atsR or atsB gene were unable to grow with hexyl-, octyl-, or nitrocatecholsulfate, although they grew normally with other sulfur sources, such as sulfate, methionine, and aliphatic sulfonates. AtsRBC therefore constitutes a general sulfate ester transport system, and desulfurization of aromatic and medium-chain-length aliphatic sulfate esters occurs in the cytoplasm. Expression of the atsR and atsBCA genes was repressed during growth with sulfate, cysteine, or thiocyanate. No expression of these genes was observed in the cysB mutant PAO-CB, and the ats genes therefore constitute an extension of the cys regulon in this species.

Posttranslational formation of formylglycine in prokaryotic sulfatases by modification of either cysteine or serine.

Eukaryotic sulfatases carry an alpha-formylglycine residue that is essential for activity and is located within the catalytic site. This formylglycine is generated by posttranslational modification of a conserved cysteine residue. The arylsulfatase gene of Pseudomonas aeruginosa also encodes a cysteine at the critical position. This protein could be expressed in active form in a sulfatase-deficient strain of P. aeruginosa, thereby restoring growth on aromatic sulfates as sole sulfur source, and in Escherichia coli. Analysis of the mature protein expressed in E. coli revealed the presence of formylglycine at the expected position, showing that the cysteine is also converted to formylglycine in a prokaryotic sulfatase. Substituting the relevant cysteine by a serine codon in the P. aeruginosa gene led to expression of inactive sulfatase protein, lacking the formylglycine. The machinery catalyzing the modification of the Pseudomonas sulfatase in E. coli therefore resembles the eukaryotic machinery, accepting cysteine but not serine as a modification substrate. By contrast, in the arylsulfatase of Klebsiella pneumoniae a formylglycine is found generated by modification of a serine residue. The expression of both the Klebsiella and the Pseudomonas sulfatases as active enzymes in E. coli suggests that two modification systems are present, or that a common modification system is modulated by a cofactor.