Different distribution of antimicrobial resistance genes and virulence profiles of Staphylococcus aureus strains isolated from clinical mastitis in six countries.
Staphylococcus aureus is recognized worldwide as one of the main contagious mastitis agents in cattle and can express a set of antimicrobial resistance genes and virulence-associated genes that explain the wide range of outcomes of intramammary infections. Staphylococcus aureus strains are heterogeneous: their different resistance and virulence patterns, associated with host-level factors and treatment factors, are related to the severity of infection. The aim of this study was to determine phenotypic antibiotic susceptibility, occurrence of selected antimicrobial resistance genes and other virulence genes in 93 S. aureus strains isolated from clinical mastitis in 6 countries: Argentina, Brazil, Germany, Italy, the United States (New York State), and South Africa. These isolates were tested against a total of 16 drugs (amoxicillin-clavulanate, ampicillin, cefazolin, cefoperazone, cefquinome, enrofloxacin, erythromycin, gentamicin, kanamycin, lincomycin, oxacillin, penicillin, rifampin, spiramycin, sulfamethoxazole/trimethoprim, tylosin) by minimum inhibitory concentration (MIC) assay, and examined for the presence of 6 antibiotic-resistance genes (blaZ, mecA, mecC, ermA, ermB, ermC) and 6 virulence-associated genes (scn, chp, sak, hla, hlb, sea) via PCR analysis. The phenotypic results of this study revealed the presence of 19.4% penicillin-resistant strains, whereas 22.6% of the strains were classified as having resistance (5.4%) or intermediate resistance (17.2%) to erythromycin. Most (96.8%) of the isolates were inhibited by cephalosporins, and all were susceptible to amoxicillin-clavulanate. Two strains (1 from Germany, 1 from Italy) were resistant to oxacillin and were positive for mecA. Among the other antimicrobial resistance genes, the most frequently detected was blaZ (46.2%), and 32.3% of the isolates were positive for erm genes: ermC (21.5%) and ermB (10.8%). The most prevalent virulence gene was hla (100%), followed by hlb (84.9%) and sea (65.6%). These results show a low prevalence of antibiotic multidrug resistance in S. aureus isolates, even if the detection of selected antimicrobial resistance genes did not always correspond with the occurrence of phenotypic antibiotic resistance; the immune evasion cluster gene prevalence was quite low in the samples analyzed.
Monistero V
，Barberio A
，Biscarini F
，Cremonesi P
，Castiglioni B
，Graber HU
，Bottini E
，Ceballos-Marquez A
，Kroemker V
，Petzer IM
，Pollera C
，Santisteban C
，Veiga Dos Santos M
，Bronzo V
，Piccinini R
，Re G
，Cocchi M
，Moroni P
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Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.
Staphylococcus species, categorized into Staphylococcus aureus and non-aureus staphylococci (NAS), are frequent causes of mastitis in dairy cattle around the world. Current treatments using antimicrobials are under increasing scrutiny due to rising prevalence of multi-drug resistance in S. aureus. Objectives of this study were to determine: (1) genetic diversity of Staphylococcus species isolated from clinical mastitis in cows from large Chinese dairy farms; and (2) prevalence and distribution of antimicrobial resistance genes (ARG) in these isolates. Staphylococcus aureus (n = 96) were isolated from 26 herds located in 12 provinces of China, whereas NAS (n = 112) were isolated from 59 herds located in 18 provinces of China. The NAS were identified at the species level using a partial 16S rRNA sequencing method, whereas random amplification of polymorphic DNA (RAPD) PCR was done to determine genetic relationships of isolates. Finally, PCR was used to detect resistance and biofilm formation genes. Staphylococcus chromogenes (33%) was the most common NAS species, followed by Staphylococcus sciuri (17%) and Staphylococcus epidermidis (8%). Staphylococcus aureus was grouped in 12 genotypes, of which 2 types represented 56% of isolates. Staphylococcus chromogenes (n = 37) clustered into 8 RAPD types, with 2 prevalent types containing 73% of isolates. The most prevalent ARG in S. aureus isolates was blaZ (95%), followed by tetM (33%), tetK (31%), ermT (26%), and aacA-aphD (23%). The mecA and vanA were detected in 16 and 4% of isolates, respectively. In NAS, blaZ (100%), mecA (73%), tetK (79%), tetM (96%), mphC (63%), and msrA (54%) were frequently detected. Antimicrobial resistance genes mecA, tetK, tetL, tetM, dfrG, ermB, msrA, mphC, aadD, and aphA3 were more commonly detected in NAS than in S. aureus. Biofilm formation genes (icaA and icaD) were frequently detected in staphylococci isolated from bovine clinical mastitis. The existence of predominant RAPD types in S. aureus and S. chromogenes isolates across Chinese dairy farms indicated that specific genotypes had disseminated within herds and become more udder-adapted. High prevalence of ARG, especially in NAS, highlighted the risk of selection of multi-drug resistant staphylococci with potential as a reservoir of ARG.
Qu Y
，Zhao H
，Nobrega DB
，Cobo ER
，Han B
，Zhao Z
，Li S
，Li M
，Barkema HW
，Gao J
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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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