Biofilm formation is considered to be a selective advantage for Staphylococcus aureus mastitis isolates by facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers. The objective of this study was to determine the sensitivity and specificity of three techniques for the detection of S. aureus biofilm-positive strains. Two phenotypic tests, including growth on microtitre plates and Congo red agar, were compared with a PCR technique using 94 S. aureus strains obtained from cows with subclinical mastitis from two farms in the state of São Paulo. These strains were characterised by in vitro slime production on Congo red agar, biofilm formation on microtitre plates and the presence of the icaA and icaD genes. The results revealed that 85% of the isolates tested produced slime on the Congo red agar, 98.9% of the isolates produced biofilms in vitro by adhering to sterile 96-well “U” bottom polystyrene tissue culture plates, and 95.7% of the isolates carried the icaA and icaD genes. The results of the phenotypic tests for biofilm formation were compared with those of the molecular analysis, and the sensitivity and specificity of the Congo red agar test were 88.9% and 100%, respectively, while those of the microtitre plate test were 100% and 25%, respectively. When the phenotypic methods for the detection of biofilm producers, namely growth on microtitre plates and Congo red agar, were compared, the sensitivity and specificity were 86% and 100%, respectively. Therefore, growth on Congo red agar and the microtitre plate test are methods that could be used to determine whether an isolate has the potential for biofilm production.

Coagulase-negative staphylococci (CNS) are among the main responsible agents for mastitis in sheep. Cure rates can be reduced due to several causes, such as those related to virulence factors presented by microorganisms. This study aims at characterizing the virulence and resistance factors to antimicrobial agents in different CNS species isolated from sheep milk. After collecting milk samples, the samples were analyzed and the CNS species were identified. After identification, the susceptibility-sensitivity profile was examined using the disk diffusion technique for 10 antimicrobial agents. The DNA was extracted to detect the presence of the mecA gene, biofilm (icaADBC, bap, and bhp) and toxin genes (sea, seb, sec, sed, tst, and luk-PV) by PCR. Samples carrying toxin genes had their expression assessed using the reverse-transcription PCR technique. The biofilm production was assessed using the adherence method on a polystyrene plate. One hundred twelve CNS samples were isolated, 53 (47.3%) from animals with subclinical mastitis and 59 (52.7%) from healthy animals. Drugs tested have shown to be efficient for most CNS samples. The largest resistance percentage of CNS was found for the penicillin (17.0%) and tetracycline (10.7%) and 4 samples carried the mecA gene. As for the biofilm genes, the icaADBC operon was found in 10 (8.9%) samples, the bap gene was found in 16 (14.3%), and the bhp gene was found in 3 (2.7%). In addition, 69 (61.6%) samples produced biofilm. The survey of toxin genes has shown that 70 (62.5%) samples showed some toxin-encoding gene. However, none of the samples has expressed any of the genes from those toxins studied.

Avaliou-se a relação custo-benefício do tratamento da mastite subclínica bovina causada por Staphylococcus aureus. Foram selecionados 270 quartos mamários com mastite subclínica e sadios, divididos em quatro grupos de acordo com o estádio de lactação e o tratamento. O grupo 1 foi formado por animais entre 10 e 60 dias da lactação e tratados contra mastites; o grupo 2 incluiu animais entre 61 dias da lactação e dois meses antes da secagem e tratados contra mastite; o grupo 3 foi formado por animais entre 10 e 60 dias da lactação, não tratados contra mastite; e o grupo 4 foi formado por animais entre 61 dias em lactação e dois meses antes da secagem, não tratados. O tratamento foi realizado pela infusão intramamária de 150mg de gentamicina, uma vez ao dia. A reavaliação foi efetuada após 30 dias. Para os cálculos dos custos com o tratamento, foram considerados uma prevalência de S. aureus de 5% e os gastos com medicamento, descarte do leite, antibiograma e mão-de-obra. Observou-se redução de 2% e 14% das receitas nos grupos 1 e 2, respectivamente, quando comparada com as receitas obtidas antes do tratamento, demonstrando ser economicamente inviável o tratamento da mastite subclínica bovina causada por S. aureus, durante a lactação.

Incorrect identification of Staphylococcus spp. can have serious clinical and zoonotic repercussions. Accordingly, the aim of this study was to determine if matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or cydB real- time quantitative PCR (qPCR) could be used to accurately identify coagulase negative Staphylococcus spp. (CoNS) obtained from buffalo milk and milking environment samples. Seventy-five of 84 CoNS isolates could be identified to the species level (score value >1.99) using MALDI-TOF MS. However, as determined by cytochrome d ubiquinol oxidase subunit II (cydB) qPCR and by 16S RNA and cydB gene sequencing, 10S. agnetis strains were wrongly identified as S. hyicus by MALDI-TOF MS. In addition, 9 isolates identified by MALDI-TOF only to the genus level (score values between 1.70 and 1.99) could be identified to species by cydB qPCR. Our findings suggest that MALDI-TOF MS is a reliable method for rapid identification of S. chromogenes and S. epidermidis (species of interest both in human and veterinary medicine) and may be able to correctly identify other Staphylococcus spp. However, at present not all Staphylococcus spp. found in buffalo milk can be accurately identified by MALDI-TOF MS and for these organisms, the cydB qPCR developed in the current study may provide a reliable alternative method for rapid identification of CoNS species.

Staphylococcus aureus is among the main etiologic agents of bovine mastitis. A total of 83 isolates of S. aureus from mammary glands of primiparous heifers were collected in the prepartum, calving and during lactation. For lactating cows, a total of 27 isolates of S. aureus from mammary glands were collected during lactation. The samples were taken in two dairy farms located in Sao Paulo State, Brazil. The highest frequency of S. aureus isolation in heifers was at the end of lactation. Strains were typified through Pulsed-field gel electrophoresis (PFGE) and grouped according to patterns of restriction enzyme SmaI. PFGE generated seven clonal profiles that were grouped into three different lineages, with the LA lineage being predominant and identified in heifers, as well as in the cows from the two regions studied. It was concluded that the cows showed a significant source of dispersion of S. aureus. At the first lactation the heifers were infected by the same clonal profiles of S. aureus which were isolated from multiparous lactating cows. The heifers were infected during milking over the months of lactation.

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Foram estudadas as provas de condutividade elétrica, utilizando-se um medidor manual, e do conteúdo de cloretos do leite como métodos auxiliares para o diagnóstico da mastite subclínica bovina na identificação de quartos mamários doentes em que Staphylococcus aureus e microrganismos do grupo Corynebacterium foram isolados posteriormente. Os exames foram realizados durante o período de dois anos, em animais da raça Holandesa, em propriedade rural produtora de leite do tipo C, onde a ordenha era realizada uma vez ao dia. A sensibilidade das provas de condutividade elétrica e do conteúdo de cloretos do leite originado dos quartos mamários em que foram isolados o Corynebacterium sp (65,3% e 78,3%, respectivamente) foi superior à encontrada para os quartos mamários em que o Staphylococcus aureus foi identificado (55,4% e 68,2%, respectivamente). As eficiências das duas provas diagnósticas foram semelhantes. Foi demonstrada significância estatística nas análises de regressão das duas provas acompanhadas para os quartos mamários sadios e quartos com mastite subclínica por Staphylococcus aureus.

RESUMO Foram submetidas a PCR-Ribotipagem e aos testes de sensibilidade in vitro frente a 12 antimicrobianos 77 estirpes de Staphylococcus aureus isoladas em amostras de leite procedentes de 40 vacas da raça holandesa que apresentaram mastite subclínica, em uma propriedade rural localizada no