Mastitis is a common and costly disease on dairy farms, commonly caused by Staphylococcus spp. though the various species are associated with different clinical outcomes. In the current study, we performed genomic analyses to determine the prevalence of adhesion, biofilm, and related regulatory genes in 478 staphylococcal species isolated from clinical and subclinical mastitis cases deposited in public databases. The most prevalent adhesin genes (ebpS, atl, pls, sasH and sasF) were found in both clinical and subclinical isolates. However, the ebpS gene was absent in subclinical isolates of Staphylococcus arlettae, S. succinus, S. sciuri, S. equorun, S. galinarum, and S. saprophyticus. In contrast, the coa, eap, emp, efb, and vWbp genes were present more frequently in clinical (vs. subclincal) mastitis isolates and were highly correlated with the presence of the biofim operon (icaABCD) and its transcriptional regulator, icaR. Co-phylogenetic analyses suggested that many of these adhesins, biofilm, and associated regulatory genes could have been horizontally disseminated between clinical and subclinical isolates. Our results further suggest that several adhesins, biofilm, and related regulatory genes, which have been overlooked in previous studies, may be of use for virulence profiling of mastitis-related Staphylococcus strains or as potential targets for vaccine development.

The aim of this study was to determinate whether coagulase-negative staphylococci (CNS) from buffalo milk or the milking environment possess virulence factors that are associated with intramanimary infections or antimicrobial resistance. Milk samples (n = 320) from 80 lactating buffalo were evaluated for clinical and subclinical mastitis by physical examination, the strip cup test, California Mastitis Test (CMT), and somatic cell count (SCC) over a 4-mo period. In addition, swabs were obtained from the hands of consenting milkers (16), liners (64), and from the mouths (15) and nostrils (15) of buffalo calves. No clinical cases of mastitis were observed; however, CMT together with SCC results indicated that 8 animals had subclinical mastitis. Eighty-four CNS isolates were identified by MALDI-TOF MS and cydB real-time PCR (qPCR) and then evaluated by qPCR for presence of the eta, etb, sea, sec, cna, seb, sei, seq, sem, seg, see, and tst toxin genes, adhesion- and biofilm-associated genes (eno, ebps, fib, fnbA, coa), and the methicillin resistance gene (mecA). Resistance to antibiotics commonly used for mastitis treatment in Brazil was determined using the Kirby-Bauer test. Two strains were positive for the see and eta toxin genes; and mecA (1), eno (27), ebps (10), fnbA (10), and coa (5) genes were also detected. A notable number of isolates were resistant to erythromycin (30), penicillin (26), and cotrimoxazole (18); importantly, 10 vancornycin-resistant isolates were also detected. A smaller number of isolates were resistant to rifampicin (8), oxacillin (7), clindamycin (5), cefepime (4), tetracycline (3), ciprofloxacin (2), and chloramphenicol (1), and none were resistant to gentamicin or ciprofloxacin. Isolates with resistance to 2 (13 isolates), 3 (3), 4 (3), 5 (1), and 6 (1) antibiotics were detected. Taken together, our findings suggest that CNS isolates may not be a significant cause of clinical or even subclinical mastitis in buffaloes, but they may be a reservoir of virulence and antibiotic resistance genes.

Staphylococcus aureus is a commonly reported cause of buffalo mastitis. However, its prevalence may be overestimated. The aim of this study was to compare S. aureus identification by conventional phenotypic and genotypic assays versus Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and novel real-time quantitative PCR tests for the cytochrome oxidase subunit D II (cydB) and staphylocoagulase (coa) genes. From 408 samples obtained from buffalo milk/milking environment, 32 putative S. aureus strains were identified based on characteristic growth on Baird Parker agar, positive catalase reaction, ability to clot rabbit plasma, and positive Sa442 PCR assay. However, in further testing, only 10 of these strains were positive in latex agglutination tests and by MALDI-TOF MS, only eight of the 32 strains were S. aureus while the rest were S. chromogenes (19), S. agnetis (3), S. cohnii (1), or S. xylosus (1). All eight strains identified as S. aureus by MALDI-TOF analysis and confirmed by 16S RNA gene sequencing were positive in a S. aureus-specific cydB PCR test. As well, 7/8 S. aureus strains were PCR positive in a real-time coa PCR test as were 2/69 S. chromogenes and the lone S. xylosus strain tested.

The consumption of raw milk cheese has been growing worldwide with S. aureus and E. coli been the leading agents in food poisoning. The present work aims to evaluate the microbiological quality of raw milk cheeses commercialized in Brazil, regarding microbiology safety and enterotoxin gene presence. Forty-three raw milk cheeses samples from five different suppliers were analyzed. Counting and identification of S. aureus, E. coli and Salmonella spp were performed according to BAM from the FDA. Further S. aureus identification was performed by the cydB and Salmonella spp by the invA gene. S. aureus toxin genes (sea, seb, sec, see, ses, seh and sei) and E. coli gene LT, STa, Stb, stx1, stx2, eae, rmpA, wabG, mrkD, kfu, mcgA, fimH and uge were analysed. From the 43 samples analyzed, 18 presented S. aureus with two isolates positive for the tst gene, two for the sec gene, two for the seh gene and four for the sei gene. Thirty-five E. coli and seven. Salmonella spp isolates were obtained. E. coli isolates harbored sta and stx2 genes. The results revealed that raw milk cheeses sold can cause harm consumer's health and highlights the importance of adoption good hygienic-sanitary practices and consumers awareness.

The aim of this study was to evaluate somatic cell count (SCC), prevalence and etiology of mastitis in a dairy buffalo herd from Analândia, São Paulo State, Brazil, in the dry and rainy seasons. Additionally, antimicrobial susceptibility profile of microorganisms isolated from milk samples was also evaluated. 1,042 milk samples from female Murrah buffaloes in a dairy farm located in Analândia, São Paulo State, Brazil, collected between May 2011 and November 2012 were analyzed. After the mammary gland physical examination, strip cup test and California Mastitis Test (CMT) were performed. Afterwards, 50mL of milk samples from each mammary quarter were collected aseptically for SCC in automatic equipment and microbiological examination. The antimicrobial sensitivity profile to ampicillin, cefoperazone, ceftiofur, enrofloxacin, gentamicin, neomycin, oxacillin, penicillin, and sulfamethoxazole/trimethoprim was evaluated by disk diffusion method. The monthly average temperature and pluviometric index were obtained from "Centro Integrado de Informações Agrometeorológicas" (CIIAGRO) of "Instituto Agronômico de Campinas" (IAC). Milk samples with positive results in the microbiological test showed average SCC of 137,720 cells/mL in the dry period and 190,309 cells/mL in the rainy period. Although a higher number of isolated microorganisms was observed in buffalo milk samples during the rainy period (69/600) compared to the dry period (50/442), the season had no significant effect on the frequency of isolation of microorganisms. The main genera of microorganisms isolated were coagulase-negative Staphylococcus (38.4%), Streptococcus agalactiae(28.8%), and Bacillus spp. (7.56%) during the dry season and Corynebacterium sp. (23.5%), Streptococcus spp. (32.3%), and Streptococcus agalactiae (9.24%) during the rainy period. Multidrug resistance was observed in 30.1% of the isolated microorganisms.