Molecular epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) isolated from milk of cows with subclinical mastitis.
Bovine mastitis has been a concern for dairy herd for decades. The adaptation capacity of one of the main species responsible for this disease, Staphylococcus aureus (S. aureus), plays a pivotal role in this issue. The aim of this study was to establish a molecular and phenotypic profile of 285 S. aureus strains isolated from milk of subclinical mastitis cows from 18 different farms in São Paulo State using spa typing, multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), agr cluster (I, II, III and IV) typing, PCR for genes including enterotoxins (sea, seb, sec, sed, see, seg, seh, sei), toxic shock syndrome toxin (tsst-1), and Panton-Valentine leucocidin (pvl), as well as in vitro resistance assays for 12 antibiotics. The results showed a wide variety of strains with a high toxigenic potential; concomitantly, sec, seg and seh were prevalent. In addition, we observed a predominance of the spa types t605 (ST 126, CC126) and t127 (ST1, CC1) and the unusual presence of t321 causing bovine mastitis, which has been previously reported only in swine. The most frequent ST were ST126 (70.5%) and ST1 (10.5%). Regarding PFGE, we observed four major groups and six profile patterns. The highest resistance was observed for streptomycin (9.5%), followed by tetracycline (3.5%), clindamycin (9.3%), and erythromycin (2.8%). The tsst-1 gene was detected in 36.8% of isolates and pvl was not observed. One hundred and thirty-six (47.7%) isolates possessed agr type II, followed by types III (20%) and I (8.1%), with type IV not being detected. We observed that the same spa type could result in different PFGE profiles, so the exclusive use of spa type sequences can lead to incorrect interpretations regarding the spread of clones in an epidemiological context.
Bonsaglia ECR
，Silva NCC
，Rossi BF
，Camargo CH
，Dantas STA
，Langoni H
，Guimarães FF
，Lima FS
，Fitzgerald JR
，Fernandes A Júnior
，Rall VLM
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Molecular characterization and clonal diversity of methicillin-susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil.
Mastitis is an important disease for the dairy industry worldwide, causing economic losses and reducing milk quality and production. Staphylococcus aureus is a worldwide agent of this intramammary infection, which also causes foodborne diseases. The objective of this study was to determine the frequency of methicillin-susceptible Staphylococcus aureus (MSSA) isolates in milk of mastitis cows in Brazil and to analyze the genetic lineages and the content of antimicrobial resistance genes and virulence factors among these isolates. Fifty-six MSSA isolates were recovered from 1,484 milk samples (positive for the California mastitis test) of 518 cows from 11 different farms in Brazil (representing 51% of total Staph. aureus obtained), and they were further characterized. Methicillin-susceptible Staphylococcus aureus were isolated from 3.7% of California mastitis test-positive tested milk samples and from 6.2% of tested mastitic cows. Methicillin-susceptible Staphylococcus aureus isolates were characterized by spa typing, agr typing, and multilocus sequence typing, and resistance and virulence traits were investigated by PCR. Seven spa types were identified among MSSA (% of isolates): t127 (44.6), t605 (37.5), t002, t1784, t2066 (1.8), and 2 new ones: t10856 (10.7) and t10852 (1.8). Five distinct sequence types (ST) were detected (% of isolates): ST1 (46.4), ST126 (37.5), ST133 (10.7), ST5 (3.6), and a novel ST registered as ST2493 (1.8). Resistances were detected for streptomycin, chloramphenicol, and tetracycline. One strain contained the chloramphenicol resistance gene (fexA; included within transposon Tn558) and 3 strains contained the tetracycline resistance gene [tet(K)]. Methicillin-susceptible Staphylococcus aureus strains were susceptible to most of the antibiotics studied and lacked the virulence genes of Panton-Valentine leukocidin (lukF/S-PV), toxic shock syndrome toxin 1 (tst), exfoliative toxin A (eta), and exfoliative toxin B (etb), as well as the genes of the immune evasion cluster. Methicillin-susceptible Staphylococcus aureus isolates were detected in a relatively low proportion of cows with mastitis (6.2%) and recovered isolates presented high diversity of genetic lineages, with CC1 and CC126 the predominant clonal complexes, and CC133 also being detected. Larger epidemiological studies with molecular characterization of isolates are required to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis.
Silva NCC
，Guimarães FF
，Manzi MP
，Budri PE
，Gómez-Sanz E
，Benito D
，Langoni H
，Rall VLM
，Torres C
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Genotyping of long term persistent Staphylococcus aureus in bovine subclinical mastitis.
Bovine mastitis affects dairy cattle worldwide and Staphylococcus aureus is one of the most common microorganisms involved in subclinical and chronic disease. Superantigens, such as enterotoxins contribute to S. aureus persistence and pathogenicity in this disease. Subclinical and chronic mastitis cases were diagnosed and S. aureus isolates from sub-clinical cases were investigated for carriage of virulence and antibiotic resistance genes that may contribute to long-term carriage and infection. Over a 12-month period, 116 S. aureus strains were recovered from 68 cows with subclinical mastitis. Classical enterotoxin genes (sea-see) were detected in 24.1% of isolates, and pvl and tsst-1 were identified in 3.4% and 46.6% the isolates, respectively. 18.1% that were persistent isolates were identified and characterized by pulsed field gel electrophoresis (PFGE), MLST, spa typing. Four isolates were methicillin-resistant S. aureus (MRSA) and belonged to SCCmec type I. Molecular typing showed that the agrI group was the most frequent, and a rare isolate was positive for both agrI and agrIII groups. Molecular characterization revealed the persistence of the spa type t10856 (ST133, clonal complex CC133, agr I), in a single animal for nine months and the persistence t605 (ST126, CC126) colonizing four animals for four months. These strains have been described recently in other herds in the same region, indicating their transmissibility and clonal expansion. We conclude that animals with subclinical mastitis are an important and somewhat overlooked reservoir for transmission within and between herds, and may carry virulence and antibiotic resistance genes contributing to persistent colonization, hinder the control of mastitis and may cause risks to the public health.
Rossi BF
，Bonsaglia ECR
，Castilho IG
，Dantas STA
，Salina A
，Langoni H
，Pantoja JCF
，Budri PE
，Fitzgerald-Hughes D
，Júnior AF
，Rall VLM
... -
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Prevalence and characterization of Staphylococcus aureus isolates from subclinical bovine mastitis in southern Xinjiang, China.
Staphylococcus aureus is one of the major pathogens causing mastitis in dairy herds. The colonization of dairy cows and subsequent contamination of raw milk by S. aureus, especially strains exhibiting multidrug resistance and biofilm-forming and toxin-producing abilities, remains an important issue for both dairy farmers and public health. In this study, we investigated the prevalence, antimicrobial susceptibility, biofilm formation, and genetic diversity of S. aureus from subclinical bovine mastitis in dairy farms located in southern Xinjiang, China. Sixty-five isolates from 84 subclinical mastitic milk samples were identified as S. aureus. The resistance rates to penicillin, erythromycin, clindamycin, tetracycline, gentamicin, linezolid, rifampicin, quinupudin-dafupudin, ciprofloxacin, norfloxacin, and chloramphenicol were 58.5, 44.6, 40.0, 18.5, 12.3, 10.8, 9.2, 6.2, 4.6, 4.6, and 1.5%, respectively. All isolates were susceptible to cefoxitin, sulfamethoxazole-trimethoprim, and vancomycin. Isolates from farm A showed a significantly higher resistance rate to tetracycline (16.9%) than those from farm B (1.5%). The most frequently detected virulence factors were hla (96.9%, 63/65) and hlb (100.0%, 65/65). The percentage rates of the staphylococcal enterotoxin genes sea, sec, sed, seg, seh, sei, and sej in S. aureus isolates were 4.6, 33.8, 27.7, 3.1, 41.5, 41.5, and 7.7%, respectively. The percentage rate of the sec gene in isolates from farm B (30.8%) was significantly higher than that of farm A (3.1%). The percentage rates of the tsst and pvl genes in S. aureus isolates were 26.2 and 40.0%. The percentage rate of the pvl gene in isolates from farm B (32.3%) was significantly higher than that of farm A (7.7%). The adhesion molecules fnbA, fnbB, clfA, clfB, and cna were detected in 21 (32.3%), 23 (35.4%), 65 (100.0%), 65 (100.0%), and 65 (100.0%) isolates, respectively. The percentage rates of the icaA, sarA, tcaR, ccp, luxS, and sigB genes in S. aureus isolates were 69.2, 100.0, 86.2, 95.4, 84.6, and 100.0%, respectively. The fnbB and icaA genes were more frequently detected in isolates from farm A (29.2 and 40.0%, respectively) than those from farm B (6.2 and 29.2%, respectively). The luxS gene was more often found in isolates from farm B (50.8%) than those from farm A (33.8%). Using the microplate method, 61.5, 26.2, and 10.8% of the isolates showed weak, moderate, and strong biofilm-forming abilities, respectively. Different clonal complex (CC) and spa-types were identified, including CC81, CC398, CC88, CC5405, and CC5406. Importantly, in this study we report for the first time 41 new sequence types (ST) among 44 distinct ST. These results indicated high genetic diversity of S. aureus involved in subclinical bovine mastitis in southern Xinjiang, China. The results also showed that S. aureus from subclinical bovine mastitis cases in southern Xinjiang, China, were mainly resistant to β-lactams, erythromycin, and clindamycin. Also, biofilm- and adhesion-related genes, which are increasingly known as important virulence factors in the pathogenesis of S. aureus infections, were detected at a high rate. This study could help identify predominant clones and provide surveillance measures to decrease or eliminate S. aureus contamination in raw milk of dairy cows with subclinical mastitis.
Ren Q
，Liao G
，Wu Z
，Lv J
，Chen W
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