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.

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 intramammary 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 vancomycin-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.

ObjectiveStaphylococcus 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.ResultsFrom 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.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3449-8) contains supplementary material, which is available to authorized users.