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 ... -  《-》

Molecular epidemiology and characterization of antimicrobial-resistant Staphylococcus haemolyticus strains isolated from dairy cattle milk in Northwest, China.

Non-aureus Staphylococcus (NAS) species are currently the most commonly identified microbial agents causing sub-clinical infections of the udder and are also deemed as opportunistic pathogens of clinical mastitis in dairy cattle. More than 10 NAS species have been identified and studied but little is known about S. haemolyticus in accordance with dairy mastitis. The present study focused on the molecular epidemiology and genotypic characterization of S. haemolyticus isolated from dairy cattle milk in Northwest, China. In this study, a total of 356 milk samples were collected from large dairy farms in three provinces in Northwest, China. The bacterial isolation and presumptive identification were done by microbiological and biochemical methods following the molecular confirmation by 16S rRNA gene sequencing. The antimicrobial susceptibility testing (AST) was done by Kirby-Bauer disk diffusion assay and antibiotic-resistance genes (ARGs) were identified by PCR. The phylogenetic grouping and sequence typing was done by Pulsed Field Gel Electrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST) respectively. In total, 39/356 (11.0%) were identified as positive for S. haemolyticus. The overall prevalence of other Staphylococcus species was noted to be 39.6% (141/356), while the species distribution was as follows: S. aureus 14.9%, S. sciuri 10.4%, S. saprophyticus 7.6%, S. chromogenes 4.2%, S. simulans 1.4%, and S. epidermidis 1.1%. The antimicrobial susceptibility of 39 S. haemolyticus strains exhibited higher resistance to erythromycin (92.3%) followed by trimethoprim-sulfamethoxazole (51.3%), ciprofloxacin (43.6%), florfenicol (30.8%), cefoxitin (28.2%), and gentamicin (23.1%). All of the S. haemolyticus strains were susceptible to tetracycline, vancomycin, and linezolid. The overall percentage of multi-drug resistant (MDR) S. haemolyticus strains was noted to be 46.15% (18/39). Among ARGs, mphC was identified as predominant (82.05%), followed by ermB (33.33%), floR (30.77%), gyrA (30.77%), sul1 (28.21%), ermA (23.08%), aadD (12.82%), grlA (12.82%), aacA-aphD (10.26%), sul2 (10.26%), dfrA (7.69%), and dfrG (5.13%). The PFGE categorized 39 S. haemolyticus strains into A-H phylogenetic groups while the MLST categorized strains into eight STs with ST8 being the most predominant while other STs identified were ST3, ST11, ST22, ST32, ST19, ST16, and ST7. These findings provided new insights into our understanding of the epidemiology and genetic characteristics of S. haemolyticus in dairy farms to inform interventions limiting the spread of AMR in dairy production.

Shoaib M ，Xu J ，Meng X ，Wu Z ，Hou X ，He Z ，Shang R ，Zhang H ，Pu W ... -  《Frontiers in Cellular and Infection Microbiology》

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 ... -  《-》

Antimicrobial resistance profiles of 5 common bovine mastitis pathogens in large Chinese dairy herds.

The prevalence of antimicrobial resistance (AMR) is increasing in human and animal pathogens, becoming a concern worldwide. However, prevalence and characteristics of AMR of bovine mastitis pathogens in large Chinese dairy herds are still unclear. Therefore, our objective was to determine the AMR profile of bacteria isolated from clinical mastitis in large (>500 cows) Chinese dairy herds. A total of 541 isolates of the 5 most common species, Staphylococcus aureus (n = 103), non-aureus staphylococci (NAS; n = 107), Streptococcus species (n = 101), Klebsiella species (n = 130), and Escherichia coli (n = 100), isolated from bovine clinical mastitis on 45 dairy farms located in 10 provinces of China were included. Presence of AMR was determined by minimum inhibitory concentrations using the microdilution method. Prevalence of multidrug resistance (resistance to >2 antimicrobials) was 27% (148/541). A very wide distribution of minimum inhibitory concentrations was screened in all isolates, including Staph. aureus isolates, which were resistant to penicillin (66%). In addition, NAS (30%) were more resistant than Staph. aureus to oxacillin (84%), penicillin (62%), tetracycline (34%), and clindamycin (33%). Prevalence of resistance to tetracycline was high (59%) in Streptococcus spp. Additionally, prevalence of resistance of both E. coli and Klebsiella spp. was high to amoxicillin/clavulanate potassium (81 and 38%, respectively), followed by tetracycline (only Klebsiella spp. 32%). A high proportion (27%) of isolates were multidrug resistant; the most frequent combinations were clindamycin-cefalexin-tetracycline or enrofloxacin-cefalexin-penicillin patterns for Staph. aureus; enrofloxacin-oxacillin-penicillin-tetracycline patterns for NAS; clindamycin-enrofloxacin-tetracycline patterns for Streptococcus spp.; amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for Klebsiella spp.; and amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for E. coli. Resistance for 4 kinds of antimicrobials highly critical for human medicine, including daptomycin, vancomycin, imipenem, and polymyxin B, ranged from 0 to 24%. In conclusion, prevalence of AMR in mastitis pathogens was high on large Chinese dairy farms, potentially jeopardizing both antimicrobial efficacy and public health. Results of this study highlighted the need for improvements in antimicrobial stewardship and infection control programs in large Chinese dairy farms to reduce emergence of AMR.

Cheng J ，Qu W ，Barkema HW ，Nobrega DB ，Gao J ，Liu G ，De Buck J ，Kastelic JP ，Sun H ，Han B ... -  《-》

Prevalence of non-aureus staphylococci species causing intramammary infections in Canadian dairy herds.

Non-aureus staphylococci (NAS), the microorganisms most frequently isolated from bovine milk worldwide, are a heterogeneous group of numerous species. To establish their importance as a group, the distribution of individual species needs to be determined. In the present study, NAS intramammary infection (IMI) was defined as a milk sample containing ≥1,000 cfu/mL in pure or mixed culture that was obtained from a cohort of cows assembled by the Canadian Bovine Mastitis Research Network. Overall, 6,213 (6.3%) of 98,233 quarter-milk samples from 5,149 cows and 20,305 udder quarters were associated with an NAS IMI. Of the 6,213 phenotypically identified NAS isolates, 5,509 (89%) were stored by the Canadian Bovine Mastitis Research Network Mastitis Pathogen Collection and characterized using partial sequencing of the rpoB housekeeping gene, confirming 5,434 isolates as NAS. Prevalence of each NAS species IMI was estimated using Bayesian models, with presence of a specific NAS species as the outcome. Overall quarter-level NAS IMI prevalence was 26%. The most prevalent species causing IMI were Staphylococcus chromogenes (13%), Staphylococcus simulans (4%), Staphylococcus haemolyticus (3%), Staphylococcus xylosus (2%), and Staphylococcus epidermidis (1%). The prevalence of NAS IMI as a group was highest in first-parity heifers and was evenly distributed throughout cows in parities ≥2. The IMI prevalence of some species such as S. chromogenes, S. simulans, and S. epidermidis differed among parities. Overall prevalence of NAS IMI was 35% at calving, decreased over the next 10 d, and then gradually increased until the end of lactation. The prevalence of S. chromogenes, Staphylococcus gallinarum, Staphylococcus cohnii, and Staphylococcus capitis was highest at calving, whereas the prevalence of S. chromogenes, S. haemolyticus, S. xylosus, and S. cohnii increased during lactation. Although the overall prevalence of NAS IMI was similar across barn types, the prevalence of S. simulans, S. xylosus, S. cohnii, Staphylococcus saprophyticus, S. capitis, and Staphylococcus arlettae IMI was higher in tiestall barns; the prevalence of S. epidermidis IMI was lowest; and the prevalence of S. chromogenes and Staphylococcus sciuri IMI was highest in bedded-pack barns. Staphylococcus simulans, S. epidermidis, S. xylosus, and S. cohnii IMI were more prevalent in herds with intermediate to high bulk milk somatic cell count (BMSCC) and S. haemolyticus IMI was more prevalent in herds with high BMSCC, whereas other common NAS species IMI were equally prevalent in all 3 BMSCC categories. Distribution of NAS species IMI differed among the 4 regions of Canada. In conclusion, distribution differed considerably among NAS species IMI; therefore, accurate identification (species level) is essential for studying NAS epidemiology.

Condas LAZ ，De Buck J ，Nobrega DB ，Carson DA ，Naushad S ，De Vliegher S ，Zadoks RN ，Middleton JR ，Dufour S ，Kastelic JP ，Barkema HW ... -  《-》