bExcision and integration of staphylococcal cassette chromosome mec (SCCmec) are mediated by cassette chromosome recombinases (Ccr), which play a crucial role in the worldwide spread of methicillin resistance in staphylococci. We report a novel ccr gene, ccrC2, in the SCCmec of a Staphylococcus aureus isolate, BA01611, which showed 62.6% to 69.4% sequence identities to all published ccrC1 sequences. A further survey found that the ccrC2 gene was mainly located among coagulase-negative staphylococci (CoNS) and could be found in staphylococcal isolates from China, the United States, France, and Germany. The ccr gene complex harboring the ccrC2 gene was designated a type 9 complex, and the SCCmec of BA01611 was considered a novel type and was designated type XII (9C2). This novel SCCmec element in BA01611 was flanked by a pseudo-SCC element (⌿SCC BA01611 ) carrying a truncated ccrA1 gene. Both individual SCC elements and a composite SCC were excised from the chromosome based on detection of extrachromosomal circular intermediates. We advocate inclusion of the ccrC2 gene and type 9 ccr gene complex during revision of the SCCmec typing method.

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.

In this study, we employed directed evolution and site-directed mutagenesis to screen thermostable mutants of a family 11 xylanase from Neocallimastix patriciarum, and found that the thermostability and specific activity are both enhanced when mutations (G201C and C60A) take place in the interior hydrophobic region of the enzyme. Far-ultraviolet circular dichroism analysis showed that the melting temperatures (T(m)) of the G201C and C60A-G201C mutants are higher than that of the wild type by about 10 and 12 degrees C, respectively. At 72 degrees C, their specific activities are about 4 and 6 times as that of the wild type, respectively. Homology modeling and site-directed mutagenesis demonstrated that the enhanced thermostability of the G201C and C60A-G201C mutants may be mainly attributed to a potential stronger hydrophobic interaction between the two well-packed cysteines at sites 50 and 201, rather than the disulfide bond formation which was ruled out by thiol titration with dithionitrobenzoic acid (DTNB). And the strength of such interaction depends on the packing of the side-chain and hydrophobicity of residues at these two sites. This suggests that cysteine could stabilize a protein not only by forming a disulfide bond, but also by the strong hydrophobicity itself.

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.