Short communication: Detection of antibiotic resistance, mecA, and virulence genes in coagulase-negative Staphylococcus spp. from buffalo milk and the milking environment.

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.

Pizauro LJL ，de Almeida CC ，Soltes GA ，Slavic D ，de Ávila FA ，Zafalon LF ，MacInnes JI ... -  《-》

Detection and characterization of methicillin-resistant and susceptible coagulase-negative staphylococci in milk from cows with clinical mastitis in Tunisia.

This study investigated prevalence of methicillin-resistant (MR) and methicillin-susceptible (MS) coagulase-negative staphylococci (CNS) and the implicated mechanisms of resistance and virulence in milk of mastitis cows. In addition, the presence of SCCmec type was analyzed in MR Staphylococcus epidermidis (MRSE). Three hundred milk samples from cows with clinical mastitis were obtained from 30 dairy farms in different regions of Tunisia. Sixty-eight of the 300 tested samples contained CNS strains. Various CNS species were identified, with Staphylococcus xylosus being the most frequently found (40%) followed by Staphylococcus warneri (12%). The mecA gene was present in 14 of 20 MR-CNS isolates. All of them were lacking the mecC gene. The SCCmecIVa was identified in four MRSE isolates. Most of CNS isolates showed penicillin resistance (70.6%) and 58.3% of them carried the blaZ gene. MR-CNS isolates (n = 20) showed resistance to erythromycin, tetracycline and trimethoprim-sulfametoxazole harboring different resistance genes such us erm(B), erm(T), erm(C), mph(C) or msr(A), tet(K) and dfr(A). However, a lower percentage of resistance was observed among 48 MS-CNS isolates: erythromycin (8.3%), tetracycline (6.2%), streptomycin (6.2%), clindamycin (6.2%), and trimethoprim-sulfametoxazole (2%). The Inu(B) gene was detected in one Staphylococcus xylosus strain that showed clindamycin resistance. The virulence gene tsst-1 was observed in one MR-CNS strain. Coagulase-negative staphylococci containing a diversity of antimicrobial resistance genes are frequently detected in milk of mastitis cows. This fact emphasizes the importance of identifying CNS when an intramammary infection is present because of the potential risk of lateral transfer of resistant genes among staphylococcal species and other pathogenic bacteria.

Klibi A ，Maaroufi A ，Torres C ，Jouini A ... -  《-》

The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd.

Staphylococci are the leading pathogens of bovine mastitis which is difficult to control. However, the published data on the prevalence of staphylococcal species, virulence and antibiotic resistance genes in bovine mastitis from China are limited. In this study, 104 out of 209 subclinical mastitis milk samples from a single Chinese dairy herd were cultured-positive for staphylococci (49.8%), which were further identified as coagulase-positive staphylococci (CPS) or coagulase-negative staphylococci (CNS). According to the partial tuf and/or 16S rRNA gene sequence, the 28 CPS isolates were confirmed to be Staphylococcus aureus (26.9%), and 76 CNS isolates were assigned to 13 different species (73.1%) with Staphylococcus arlettae, Staphylococcus sciuri, Staphylococcus xylosus and Staphylococcus chromogenes as the dominant species. In the 28 S. aureus isolates, the most prevalent general virulence genes were coa, Ig and eno (100%), followed by hla (96.4%), hlb (92.9%), fib (92.9%), clfA (89.3%), clfB (85.7%) and nuc (85.7%). Both exotoxin and biofilm-associated genes were significantly less prevalent than the previously reported. Although 19 different virulence gene patterns were found, only one was dominant (32.1%). The prevalence of blaZ (82.1%) or mecA gene (35.7%) was much higher than the previously reported. In the 76 CNS isolates, the virulence genes were significantly less prevalent than that in the S. aureus isolates. Among the 4 main CNS species, S. chromogenes (n = 12) was the only species with high percentage (75%) of blaZ gene, while S. sciuri (n = 12) was the only species with the high percentage (66.7%) of mecA gene. The most of antibiotic resistance genes were present as multi-resistance genes, and the antibiotic resistances were attributed by different resistance genes between resistant S. aureus and CNS isolates. These data suggest that the prevalence of staphylococcal species, virulence and antibiotic resistance in the mastitis milk from the Chinese dairy herd are different from the previously reported, and that the herd- or farm-based diagnosis of staphylococcal bovine mastitis is required.

Xu J ，Tan X ，Zhang X ，Xia X ，Sun H ... -  《-》

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 ... -  《-》

Microarray-based detection of resistance genes in coagulase-negative staphylococci isolated from cattle and buffalo with mastitis in Egypt.

El-Ashker M ，Gwida M ，Monecke S ，Ehricht R ，Elsayed M ，El-Gohary F ，Reißig A ，Müller E ，Paul A ，Igbinosa EO ，Beshiru A ，Maurischat S ... -  《-》