Foi realizado o monitoramento epidemiológico molecular de estirpes de Staphylococcus aureus potencialmente toxigênicas isoladas no processo de produção do queijo Minas frescal em micro-usina do Estado de São Paulo. Para tanto, foram realizadas seis amostragens durante o período de junho de 2008 a julho de 2009, de modo a perfazer um total de 140 amostras. Essas amostras foram colhidas da superfície dos tanques de recepção e estocagem do leite cru, da superfície do tanque de equilíbrio do leite pasteurizado, da rede de abastecimento de água, das tubulações e equipamentos, das mãos do manipulador e de queijos embalados prontos para consumo. As colônias isoladas em Agar Baird-Parker confirmadas como cocos Gram positivos e que mostravam-se positivas às provas de catalase, coagulase e da produção de acetoína, foram submetidas à extração do DNA bacteriano através da utilização do Kit Invitek - Uniscience®. A confirmação molecular da espécie dos isolados e a presença de enterotoxinas SEA, SEB, SEC, SED e da toxina TSST-1 foi realizada a partir da amplificação dos fragmentos de DNA cromossômico específico. Entre as 74 estirpes de estafilococos coagulase positivos isoladas, somente 41 (55.4%) amostras foram confirmadas como sendo Staphylococcus aureus, das quais 25 (61,0%) mostraram-se positivas na pesquisa de toxinas estafilocócicas. A enterotoxina de maior frequência identificada foi a SEA. As estirpes de Staphylococcus aureus toxigênico foram mais isoladas nas mãos do manipulador (16,0%), no leite cru do tanque de recepção (12,0%), no leite pasteurizado para elaboração do queijo (12,0%) e no queijo Minas frescal pronto para consumo (12,0%).

We studied the molecular epidemiology of Staphylococcus aureus strains potentially toxigenic, isolated from the production process of Minas frescal cheese in a small dairy plant in the state of São Paulo. For this, samples were taken during the period from June 2008 to July 2009. Samples were collected from the surface of the receiving and storage tanks of raw milk, the surface of the balance tank of pasteurized milk, the water supply system, the pipes and equipments, the hands of the handler and from the packaged cheese, totaling 140 samples. The colonies isolated on Baird-Parker Agar confirmed as Gram positive and positive for catalase, coagulase and acetoin production, were submitted to extraction of bacterial DNA using the Invitek - Uniscience® kit. Confirmation of the isolated species and enterotoxins SEA, SEB, SEC, SED and TSST-1 toxin was carried out through the amplification of specific fragments of chromosomal DNA. Among the 74 strains of isolated coagulase-positive staphylococci, only 41 (55.4%) strains were confirmed as Staphylococcus aureus, of which 25 (61.0%) were positive to the presence of staphylococcal toxins. The most frequently identified enterotoxin was SEA. The toxigenic strains of Staphylococcus aureus were more frequently isolated from hands of the handler (16.0%), raw milk receiving tank (12.0%), pasteurized milk for cheese making (12.0%) and fresh white cheese ready for consumption (12.0%).

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.

The objective of the present study was to evaluate the occurrence of Staphylococcus aureus in milk produced in 37 farms located in the regions of Ribeirão Preto and São Carlos, state of São Paulo, Brazil. Two-hundred and eight samples of milk from individual cows showing subclinical mastitis, and 37 samples of bulk tank milk were analyzed. S. aureus strains were detected in 18 (7.3%) milk samples: 14 (6.7%) from samples of individual cows, and 4 (10.8%) from bulk tank milk. Two individual milk samples (14.3%) and two bulk milk samples contained enterotoxigenic S. aureus. PFGE analysis revealed the genetic heterogeneity of the strains isolated from raw milk, which presented to 13 S. aureus patterns. Results confirmed the potential transmission of staphylococcal food poisoning to consumers via milk of cows affected by subclinical mastitis, mainly when raw milk is ingested.

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 Estado de São Paulo, Brasil. Os resultados obtidos revelaram quatro diferentes padrões de resistência a antimicrobianos, sendo observada a predominância de resistência à lincomicina entre 19 (24,7%) estirpes de S.aureus. As 58 (75,3%) estirpes restantes foram sensíveis aos 12 antimicrobianos testados. A PCR-ribotipagem revelou a ocorrência de nove padrões genotípicos distintos, além de apresentar uma capacidade discriminatória maior (D = 0,82) que a obtida nos antibiogramas (D = 0,42). Entre as 19 estirpes resistentes aos antimicrobianos, 14 (73,7%) foram agrupadas em três padrões de ribotipagem e, destas, 13 (92,9%) apresentaram resistência à eritromicina e à lincomicina, isoladamente ou em associação. O grande número de ribotipos e de padrões de resistência a antimicrobianos observados nesta propriedade demonstrou que há grande heterogeneidade genética em populações naturais de S. aureus, fato este que deve ser levado em consideração em programas de controle da mastite bovina.

Seventy-seven S. aureus strains, isolated in milk samples obtained from 40 Holstein cows suffering from subclinical mastitis in a dairy herd in the state of São Paulo - Brazil, were undergone to PCR-ribotyping and in vitro susceptibility testing to 12 antimicrobial drugs. PCR-ribotyping revealed nine different patterns and presented a higher discriminatory power (D = 0.82) in comparison with the in vitro antibiotic susceptibility test (D = 0.42). Moreover,, the results showed four antibiotic resistance patterns, with the resistance to lincomycin being the most predominant and comprising 19 (24.7%) S. aureus strains. The other 58 (75.3%) isolates were sensitive to all antibiotics tested. Of the 19 isolates which presented resistance to antibiotics, 14 (73.7%) were grouped in three ribotypes, and 13 (92.9%) of them were resistant to eritromycin and/or lincomycin. The large number of ribotypes and antibiotic resistance patterns found in this single dairy herd showed that one must be careful when considering the results of in vitro susceptibility tests obtained from a few S. aureus strains, because there is a high heterogeneity among naturally occurring populations of S. aureus, which must be taken into consideration in control programs against bovine mastitis.