Anti-Bacterial Agents ; pharmacology ; Humans ; Methicillin ; pharmacology ; Methicillin Resistance ; genetics ; Sequence Homology ; Staphylococcus aureus ; drug effects ; genetics

OBJECTIVETo investigate the genotypic diversity of Methicillin-resistant Staphylococcus aureus (MRSA) isolated from pigs and retail foods from different geographical areas in China and further to study the routes and rates of transmission of this pathogen from animals to food.

METHODSSeventy-one MRSA isolates were obtained from pigs and retail foods and then characterized by multi-locus sequencing typing (MLST), spa typing, multiple-locus variable number of tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing.

RESULTSAll isolated MRSA exhibited multi-drug resistance (MDR). Greater diversity was found in food-associated MRSA (7 STs, 8 spa types, and 10 MLVA patterns) compared to pig-associated MRSA (3 STs, 1 spa type, and 6 MLVA patterns). PFGE patterns were more diverse for pig-associated MRSA than those of food-associated isolates (40 vs. 11 pulse types). Among the pig-associated isolates, CC9-ST9-t899-MC2236 was the most prevalent clone (96.4%), and CC9-ST9-t437-MC621 (20.0%) was the predominant clone among the food-associated isolates. The CC9-ST9 isolates showed significantly higher antimicrobial resistance than other clones. Interestingly, CC398-ST398-t034 clone was identified from both pig- and food-associated isolates. Of note, some community- and hospital-associated MRSA strains (t030, t172, t1244, and t4549) were also identified as food-associated isolates.

CONCLUSIONCC9-ST9-t899-MC2236-MDR was the most predominant clone in pigs, but significant genetic diversity was observed in food-associated MRSA. Our results demonstrate the great need for improved surveillance of MRSA in livestock and food and effective prevention strategies to limit MDR-MRSA infections in China.

Animals ; Anti-Bacterial Agents ; pharmacology ; China ; Food Microbiology ; Humans ; Methicillin ; pharmacology ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; genetics ; isolation & purification ; Nose ; microbiology ; Swine ; microbiology

Anthracosis ; complications ; Bacterial Proteins ; genetics ; Humans ; Methicillin Resistance ; genetics ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; genetics ; Penicillin-Binding Proteins ; Respiratory Tract Infections ; complications ; microbiology ; Sputum ; microbiology

Evolution, Molecular ; Methicillin Resistance ; Staphylococcus aureus ; classification ; drug effects ; genetics ; isolation & purification

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method.
Aminoglycosides/*pharmacology ; DNA/chemistry ; Drug Resistance, Bacterial/*genetics ; Human ; Methicillin/*pharmacology ; Polymerase Chain Reaction/*methods ; Species Specificity ; Staphylococcus aureus/*genetics

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method.
Aminoglycosides/*pharmacology ; DNA/chemistry ; Drug Resistance, Bacterial/*genetics ; Human ; Methicillin/*pharmacology ; Polymerase Chain Reaction/*methods ; Species Specificity ; Staphylococcus aureus/*genetics

Emergence and spread of low-level mupirocin resistance in staphylococci have been increasingly reported in recent years. The aim of this study was to characterize missense mutations within the chromosomal isoleucyl-tRNA synthetase gene (ileS) among clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) with low-level mupirocin resistance. A total of 20 isolates of MRSA with low-level mupirocin resistance (minimal inhibitory concentration, 16-64 microgram/mL) were collected from 79 patients in intensive care units for six months. The isolates were analyzed for isoleucyl-tRNA synthetase (IleS) mutations that might affect the binding of mupirocin to the three-dimensional structure of the S. aureus IleS enzyme. All isolates with low-level mupirocin resistance contained the known V588F mutation affecting the Rossman fold, and some of them additionally had previously unidentified mutations such as P187F, K226T, F227L, Q612H, or V767D. Interestingly, Q612H was a novel mutation that was involved in stabilizing the conformation of the catalytic loop containing the KMSKS motif. In conclusion, this study confirms that molecular heterogeneity in ileS gene is common among clinical MRSA isolates with low-level mupirocin resistance, and further study on clinical mutants is needed to understand the structural basis of low-level mupirocin resistance.
Staphylococcus aureus/drug effects/*genetics ; *Mutation, Missense ; Mupirocin/*pharmacology ; Methicillin Resistance ; Isoleucine-tRNA Ligase/*genetics ; Intensive Care Units ; Humans ; Electrophoresis, Gel, Pulsed-Field ; Drug Resistance, Bacterial

OBJECTIVE: To detect the active efflux gene qac gene in methicillin-resistant Staphylococcus aureus (MRSA) by hem-nested polymerase chain reaction (PCR) and to learn the carrier condition of qac gene. METHODS: The active efflux gene qacA/B and qacB of 80 strains MRSA isolated from clinical specimens from Aug 2006 to March 2008 were amplified in vitro by hem-nested PCR with the primers designed by computers based on qac information of Genbank, and the PCR fragments were sequenced and analyzed. RESULTS: We detected qacA/B in 19 out of the 80 MRSA strains (23.75%) and qacB in 18 out of the 80 MRSA strains (22.5%). Compared with sequences of qacA (NO.X56628) and qacB(NO.AF535087) in the Genbank, 98% and 97% were identical, respectively. CONCLUSION The active efflux gene qac gene in MRSA is detected by hem-nested PCR. The proportion of qac gene positive strains is high in clinical practice, which is related to its multi-antibiotic resistance.
Bacterial Proteins ; analysis ; genetics ; Base Sequence ; Drug Resistance, Multiple ; genetics ; Membrane Transport Proteins ; analysis ; genetics ; Methicillin-Resistant Staphylococcus aureus ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods

OBJECTIVETo clone, express and identify the mecA fragment which encoded penicillin binding protein 2a (PBP2a) from methicillin-resistant staphylococcus aureus (MRSA) isolated from patients by gene recombination method.

METHODSAccording to the sequence of mecA gene recorded in GenBank, the primer of mecA fragment which encoded amino acids 25 - 668 of PBP2a was designed. Then the mecA fragment was amplified by PCR and cloned into pQE30 plasmid. After being identified by enzyme digestion and sequencing, the recombinant plasmid was transferred into E. coli M15 [pREP4], and then its expression was induced by 1 mmol/L Isopropy-beta-D-Thiogalactoside (IPTG). The expression product was analyzed by SDS-PAGE, protein sequencing and mass spectroscopy.

RESULTSThe recombinant pQE30- mecA had been successfully constructed. The result of sequencing showed that the mecA fragment had 1932 bases, including 9 bases undergoing mutation. After being induced for 6 hours by IPTG, the soluble protein in M15 (pQE30- mecA), with a relative molecular weight of 74 x 10(3), was found by SDS-PAGE. The soluble protein had been confirmed to be PBP2a after identification.

CONCLUSIONThe soluble PBP2a of MRSA isolated from patients is expressed successfully by gene recombinant technology.

Base Sequence ; Cloning, Molecular ; Gene Expression ; Humans ; Methicillin Resistance ; genetics ; Methicillin-Resistant Staphylococcus aureus ; genetics ; isolation & purification ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins ; genetics ; metabolism ; Peptide Synthases ; genetics ; metabolism ; Plasmids

OBJECTIVETo investigate the spread and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) at hospital.

METHODSTotally 110 strains of Staphylococcus aureus (SA) were isolated from the clinical samples of patients in 4 hospitals and 30 strains of SA were isolated from the hospital environment and personnel in Xiangya Hospital. MRSA was detected using oxacillin disk diffusion test, cefoxitin disk diffusion test and MecA, FemA gene PCR assay. Beta-lactamase was detected using nitrocephin sticks. The antimicrobial susceptibility of MRSA was tested by K-B disk diffusion test.

RESULTSAmong the 140 strains, 89 were MRSA, accounting for 63.57% of the total SA. The isolation rates of MRSA in clinical strains and environment strains were 64.55% and 60.00% (P > 0.05). All MRSA strains were sensitive to vancomycin and linezolid, 87 MRSA strains (97.75%) were sensitive to teicoplanin, most of which, however, were resistant to other antibiotics. Among the 89 strains, 85 MRSA strains (95.51% ) expressed beta-lactamase.

CONCLUSIONSMRSA is highly prevalent in hospitals. Most MRSA strains are multi-drug resistant, but are still sensitive to vancomycin, linezolid, and teicoplanin.

Anti-Bacterial Agents ; pharmacology ; Cross Infection ; microbiology ; Drug Resistance, Multiple, Bacterial ; Hospitalization ; Humans ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; genetics ; isolation & purification ; Microbial Sensitivity Tests ; Staphylococcal Infections ; microbiology