Mastitis in dairy cattle continues to be an economically important disease. However, control is complicated by a high prevalence of resistance to antibiotics. Phage therapy, therefore, is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a highly virulent phage SPW from wastewater of dairy farm, which possesses a strong lytic capability against mastitis-associated Staphylococcus aureus, the most important pathogen in bovine clinical and subclinical mastitis. The phage SPW produced large, round and clear plaques on bacterial culture plates. TEM showed phage SPW has an icosahedral head 62.5 nm in diameter and long tail of 106 nm, head and tail were held together by a connector of 18 ± 1.5 nm long and can be classified as a member of the Myoviridae family. Restriction analysis indicated that phage SPW was a dsDNA virus with an approximate genome size of 65-69 kb. One-step growth kinetics showed a short latency period of about 10-15 min and a rise period of 50 min and a relatively small burst size was 44 ± 3 phages particles/infected cell. Moreover, adsorption rates were not influenced by calcium ions and phage SPW was relatively stable in a wide range of temperature and pH values, and resistant to chloroform and isopropanol. The optimal multiplicity of infection (MOI) was 0.01. When phage SPW was used to infect five other clinically isolated pathogenic isolates, it showed relatively wide spectrum host range. Phage SPW was capable of eliciting efficient lysis of S. aureus, revealing it potentially as an effective approach to prophylaxis or treatment of S. aureus-associated mastitis in dairy cows.

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) in bovine milk is a major public health concern. The primary purpose of this research was to determine molecular genetic characteristics and antibiotic resistance of staphylococcal isolates recovered from milk of mastitic cows in the Shaanxi Province in Northwestern China. One hundred and thirteen methicillin-susceptible Staphylococcus aureus (MSSA), one mecA-positive and phenotype-positive MRSA, seven mecA- and mecC- negative but phenotype-positive MRSA and two MR-CoNS including one oxacillin-susceptible mecA-positive Staphylococcus haemolyticus (OS-MRSH) and one mecA-positive and methicillin-resistant Staphylococcus epidermidis (MRSE) isolates were recovered from 214 quarter milk samples on 4 dairy farms. All above 123 isolates were subjected to antibiotic resistance profiling. S. aureus isolates were also genotyped using the spa typing and the multilocus sequence typing (MLST). Eight MRSA and 2 MR-CoNS isolates were additionally tested for SCCmec types. Resistance was common among isolates against ampicillin or penicillin (80.5%), kanamycin (68.3%), gentamicin (67.5%), tetracycline (43.9%) and chloramphenicol (30.1%). However, no isolate was resistant to vancomycin or teicoplanin. Twenty, 29 and 58 isolates showed resistance to 1, 2 or more than 2 antibiotics, respectively. The predominant multidrug resistance profile was penicillin/ampicillin/kanamycin/gentamicin/tetracycline (46 isolates). Most S. aureus isolates belonged to spa types t524 (n = 63), t11772 (a new type, n = 31) and t4207 (n = 15). At the same time, MLST types ST71 (n = 67) and ST2738 (a new type, n = 45) were identified as dominant sequence types. The mecA-positive and phenotype-positive MRSA isolate had a composite genotype t524-ST71-SCCmecIVa, while 7 mecA-negative but phenotype-positive MRSA isolates were all t524-ST71. The OS-MRSH isolate contained a type V SCCmec cassette, while the MRSE isolate possessed a non-typeable SCCmec. The spa-MLST types t11772-ST2738 (n = 27), t11807-ST2683 (n = 4) and t11771-ST2738 (n = 3) were newly identified genotypes of S. aureus. These new genotypes and multidrug-resistant staphylococci could pose additional threat to animal and human health.

BackgroundThere are limited data available on macrolide-lincosamide-streptogramin (MLS) resistance of Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CoNS) from bovine milk in China. To address this knowledge gap, MLS resistance was determined in 121 S. aureus and 97 CoNS isolates. Minimum inhibitory concentrations (MICs) of MLS antibiotics were determined by an agar dilution method, while differentiation of MLS phenotypes was performed by a double-disc diffusion test. MLS resistance genotypes were determined by PCR for corresponding resistance genes.ResultsForty (33.1 %) S. aureus and 65 (67.0 %) CoNS were resistant to erythromycin, whereas all 218 isolates were susceptible to quinupristin/dalfopristin. Among 40 erythromycin-resistant (ER-R) S. aureus and 65 ER-R CoNS isolates, 38 S. aureus and 40 CoNS isolates exhibited the inducible MLS (iMLS) resistance phenotype and 2 S. aureus and 20 CoNS isolates expressed the constitutive MLS resistance (cMLS) phenotype. At the same time, 5 CoNS isolates exhibited resistance to erythromycin but susceptibility to clindamycin (the MS phenotype). An inactivating enzyme gene lnu(A), methylase genes erm(C) and erm(B), efflux genes msr(A)/msr(B), a phosphotransferase gene mph(C), an esterase gene ere(A) and the streptogramin resistance determinant vga(A) were detected individually or in combinations. Among them, genes lnu(A), erm(C) and mph(C) predominated. The ereA gene was detected for the first time in staphylococci of bovine milk origin. Resistance genes also existed in erythromycin-susceptible isolates.ConclusionsOur study demonstrated a high level of resistance to MLS antibiotics in staphylococci from bovine mastitic milk, especially with a high rate of the iMLS phenotype in S. aureus isolates. These data suggest that MLS antibiotics should be used judiciously to treat or prevent bovine mastitis caused by staphylococci.

BackgroundIntegrons are mobile DNA elements and they have an important role in acquisition and dissemination of antimicrobial resistance genes. However, there are limited data available on integrons of Staphylococcus aureus (S. aureus) from bovine mastitis, especially from Chinese dairy cows. To address this knowledge gap, bovine mastitis-inducing S. aureus isolates were investigated for the presence of integrons as well as characterization of gene cassettes. Integrons were detected using PCR reactions and then further characterized by a restriction fragment-length polymorphism analysis and amplicon sequencing.ResultsAll 121 S. aureus isolates carried the class 1 integrase gene intI1, with no intI2 and intI3 genes detected. One hundred and three isolates were positive for the presence of 12 resistance genes, either alone or in combination with other gene cassettes. These resistance genes encoded resistance to trimethoprim (dhfrV, dfrA1, dfrA12), aminoglycosides (aadA1, aadA5, aadA4, aadA24, aacA4, aadA2, aadB), chloramphenicol (cmlA6) and quaternary ammonium compound (qacH) and were organized into 11 different gene cassettes arrangements (A-K). The gene cassette arrays dfrA1-aadA1 (D, 44.6%), aadA2 (K, 31.4%), dfrA12-orfX2-aadA2 (G, 27.3%) and aadA1 (A, 25.6%) were most prevalent. Furthermore, 74 isolates contained combinations of 2 to 4 gene cassette arrays. Finally, all of the integron/cassettes-positive isolates were resistant to aminoglycoside antibiotics.ConclusionsThis is the first study on the integrons and gene cassette arrays in S. aureus isolates from milk of mastitic cows from Northwestern China and provide the evidence for class 1 integron as possible antibiotic resistance determinants on dairy farms.

The structure of a composite staphylococcal cassette chromosome (SCC) carried by a methicillin-resistant Staphylococcus haemolyticus (NW19A) isolated from a bovine milk sample was analyzed. The formation of the circular forms of both single SCC elements and composite SCC elements was detected in NW19A. Twenty heavy metal and antibiotic resistance-related genes coexisted in this composite SCC, suggesting that these genes might be coselected under environmental pressure. The mec gene complex in NW19A, designated type C3, is different from classic C1 or C2 gene complexes structurally and likely evolves differently. Furthermore, results from alignment of the SCC composite island of NW19A with 50 related sequences from different staphylococcal strains provided additional evidence to support the notion that coagulase-negative staphylococci (CoNS) are the original host of heavy metal resistance genes among staphylococci. Given that a SCC composite island could transfer freely among different staphylococcal species from different hosts, more attention should be paid to contamination with heavy metals and antibiotics in dairy farming environments, including wastewater, soil, feces, and feed. C oresistance, one type of coselection mechanism, occurs when genes specifying resistant phenotypes are located together on the same mobile genetic element (MGE), such as plasmids, transposons, and staphylococcal cassette chromosome (SCC) elements (1). SCC elements are widely distributed in different staphylococci and highly diverse in their structural organization and genetic contents. SCCmec is a type of SCC element. It carries the mec gene complex and has broad-spectrum beta-lactam resistance encoded by the mecA (or mecC) gene. Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of health care-associated infections and community-associated infections worldwide and can be associated with mastitis in dairy cattle (2, 3). MRSA is generated when methicillin-sensitive Staphylococcus aureus (MSSA) exogenously acquires an SCCmec element, but with an unclear mechanism (4). There is increasing, but largely indirect, evidence suggesting that coagulase-negative staphylococci (CoNS) might act as a source of SCCmec for MRSA. However, very little is known about genetics of SCCmec in CoNS (5).In China, the average usage of veterinary antibiotics has reached approximately 6,000 tons annually (6), and numerous antibiotic-resistant bacteria have appeared (7). Abundant antibiotics are also used in dairy cattle in China because of the high incidence of mastitis and endometritis (8) and may increase the prevalence of antibiotic-resistant bacteria. In order to investigate antibiotic-resistant staphylococci from mastitis milk, in this study, a methicillin-resistant Staphylococcus haemolyticus (MRSH) strain from a milk sample from a Holstein cow was studied for its SCC element in terms of structural organization and genetic contents. Besides a mecA gene, a tetracycline resistance gene and many heavy metal resistance genes were ...