Comparative analysis of superantigen genes in Staphylococcus xylosus and Staphylococcus aureus isolates collected from a single mammary quarter of cows with mastitis.

The purpose of this study was to analyze and compare genes encoding superantigens (SAgs) in Staphylococcus xylosus and Staphylococcus aureus isolates collected simultaneously from milk of the same cows with clinical mastitis. Genes encoding staphylococcal enterotoxins and enterotoxin-like proteins (sea-selu), toxic shock syndrome toxin 1 (tst-1) and exfoliative toxins (eta and etd) were investigated. It was found that among 30 isolates of S. xylosus, 16 (53.3%) harbored from 1 to 10 SAg genes. In total, in 16 SAg positive S. xylosus, 11 different enterotoxin genes were detected: sec, sed, seg, seh, sei, selm, seln, selo, selp, ser, selu and one etd gene encoding exfoliative toxin D. The most prevalent genes were ser, selu, and selo. Among all the positive isolates of S. xylosus, a total of 14 different SAg gene combinations were detected. One combination was repeated in 3 isolates, whereas the rest were detected only once. However, in the case of S. aureus all the 30 isolates harbored the same combination of SAg genes: seg, sei, selm, seln, selo and on the basis of PFGE analysis all belonged to the same clonal type. Also noteworthy was the observation that SAg genes detected in S. aureus have also been found in S. xylosus. The findings of this study further extend previous observations that SAg genes are present not only in S. aureus but also in coagulase-negative staphylococci, including S. xylosus. Therefore, taking into account that the SAg genes are encoded on mobile genetic elements it is possible that these genes can be transferred between different species of coexisting staphylococci.

Superantigen gene profiles, genetic relatedness and biological activity of exosecretions of Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis.

This study evaluated the superantigen gene profiles, genetic relatedness and biological activity of exosecretions of 50 Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis. Genomic relatedness of S. aureus was determined by pulsed field gel electrophoresis analysis of macro-restricted chromosomes. The presence of genes encoding superantigens was confirmed by multiplex PCR. To study the biological activity of S. aureus exosecretions, the supernatants from bacterial liquid cultures were classified into three groups: those with leukotoxin­like properties, those with superantigen­like properties and those with no particular activity on leukocytes cultured in vitro. It was shown that all analyzed bacterial isolates belonged to the same clonal type and harbored the same combination of superantigen genes, namely sed, selj and ser. However, 22% of all isolates produced factors with superantigen­like and 48% of them with leukotoxin­like activities. Finally, although there were no detectable genetic differences between the analyzed bacterial isolates, the virulence factors secreted by them differed considerably.

Influence of S. aureus exosecretions on cytokine profile in bovine leukocyte cultures in vitro.

The aim of the research was to evaluate the in vitro effect of Staphylococcus aureus exosecretions on the expression of genes encoding IL-2 and IL-12 and secretion of IFN-γ and TNF-α, in bovine leukocyte cultures in vitro. The research was based on 30 S. aureus isolates collected from milk samples from cows with clinical mastitis. Supernatants prepared from the bacterial liquid cultures, which were used to treat leukocytes, were divided into three groups: one with superantigen-like properties, one with leukotoxic-like properties and the one without superantigen or leukotoxic-like properties. The MNC, PMN and MIX (consisted of MNC and PMN leukocytes) cultures were grown and treated with the supernatants. The work shows that the effect on the cytokine gene expression and cytokine secretion caused by S. aureus exosecretions is mainly due to the presence of virulence factors connected with superantigen-like activity and less with leukotoxic-like activity whereas exosecretions of other activity are not or only slightly involved in this process.

Identification and methicillin resistance of coagulase-negative staphylococci isolated from nasal cavity of healthy horses.

The aim of this study was an analysis of the staphylococcal flora of the nasal cavity of 42 healthy horses from 4 farms, along with species identification of CoNS isolates and determination of resistance to 18 antimicrobial agents, particularly phenotypic and genotypic methicillin resistance. From the 81 swabs, 87 staphylococci were isolated. All isolates possessed the gap gene but the coa gene was not detected in any of these isolates. Using PCR-RFLP of the gap gene, 82.8% of CoNS were identified: S. equorum (14.9%), S. warneri (14.9%), S. sciuri (12.6%), S. vitulinus (12.6%), S. xylosus (11.5%), S. felis (5.7%), S. haemolyticus (3.4%), S. simulans (3.4%), S. capitis (1.1%), S. chromogenes (1.1%), and S. cohnii subsp. urealyticus (1.1%). To our knowledge, this was the first isolation of S. felis from a horse. The species identity of the remaining Staphylococcus spp. isolates (17.2%) could not be determined from the gap gene PCR-RFLP analysis and 16S rRNA gene sequencing data. Based on 16S-23S intergenic transcribed spacer PCR, 11 different ITS-PCR profiles were identified for the 87 analyzed isolates. Results of API Staph were consistent with molecular identification of 17 (19.5%) isolates. Resistance was detected to only 1 or 2 of the 18 antimicrobial agents tested in the 17.2% CoNS isolates, including 6.9% MRCoNS. The mecA gene was detected in each of the 5 (5.7%) phenotypically cefoxitin-resistant isolates and in 12 (13.8%) isolates susceptible to cefoxitin. In total, from 12 horses (28.6%), 17 (19.5%) MRCoNS were isolated. The highest percentage of MRCoNS was noted among S. sciuri isolates (100%).

The effect of auto-vaccination therapy on the phenotypic variation of one clonal type of Staphylococcus aureus isolated from cows with mastitis.

The aim of this study was to demonstrate the effect of auto-vaccine therapy on selected properties of Staphylococcus aureus strains, isolated from milk of cows with subclinical mastitis. The experiment was based on auto-vaccines which were prepared from S. aureus strains isolated from 16 cows. S. aureus strains isolated from cows on the 7th, 21st and 35th day following auto-vaccination, were analyzed phenotypically and genotypically. The isolated strains represented 17 biotypes all belonging to one clonal type. Increases of new biotypes of S. aureus were detected on the 35th day of therapy. Among 48 re-isolated strains, 18.75% (9/48) revealed single and 12.50% (6/48) multiple phenotypical changes. The present study demonstrated that during auto-vaccine therapy, S. aureus strains can change phenotypically, pointing out the necessity for using precise diagnostic methods, that would make possible a better assessment of the used therapy.

Secretory virulence factors produced by Staphylococcus aureus isolates obtained from mastitic bovine milk--effect on bovine polymorphonuclear neutrophils.

The aim of the research was to test whether exogenic virulence factors secreted by Staphylococcus aureus isolates are involved in mechanisms that allow the bacteria to modulate and evade phagocytosis by bovine polymorphonuclear neutrophils. The research was based on the comparison of the effects of supernatants, prepared from cultures of 30 S. aureus isolates, on the functional properties of bovine neutrophils in vitro. S. aureus isolates were collected from milk samples from cows with clinical mastitis. Supernatants, which were used to treat leukocytes, were prepared from 18 h S. aureus cultures. Exogenic virulence factors secreted by S. aureus isolates significantly influenced the phagocytosis parameters evaluated. Depending on their leukotoxic or superantigenic properties, supernatants could affect the ingestion process, and also showed an influence on the digestion efficiency and phagocytosis carried out by bovine polymorphonuclear neutrophils in vitro.