BACKGROUNDIncreasing prevalence of Staphylococcus aureus (S. aureus), particularly methicillin-resistant S. aureus (MRSA) has been reported in China. In this study, we investigated the drug resistance characteristic, genetic background, and molecular epidemiological characteristic of S. aureus in Changsha.

METHODSBetween January 2006 and December 2008, 293 clinical isolates of S. aureus were collected from 11 hospitals in Changsha and identified by the Vitek-2 system. All the isolates were verified as MRSA by PCR amplification of both femA and mecA genes. K-B disk method was used to test drug sensitivity of S. aureus to antibiotics. Pulsed-field gel electrophoresis (PFGE) was performed for genotypic and homologous analysis of 115 isolates randomly selected from the original 293 clinical S. aureus isolates.

RESULTSS. aureus was highly resistant to penicillin, ampicillin, erythromycin, and clindamycin with resistant rates of 96.6%, 96.6%, 77.1%, and 67.2% respectively. All the isolates were susceptible to tecoplanin, vancomycin, and linezolid. MRSA accounted for 64.8% (190/293) of all the S. aureus strains. The 115 S. aureus isolates were clustered into 39 PFGE types by PFGE typing, with 13 predominant patterns (designated types A to M) accounting for 89 isolates. The most prevalent PFGE type was type A (n = 56, 48.7%) and 100.0% of type A strains were MRSA. PFGE type A included 13 subtypes, and the most prevalent subtype was subtype A1 (46.4%, 26/56). Strains with PFGE type A were isolated from eight hospitals (8/11), and both subtypes A1 and A4 strains were isolated in a university hospital.

CONCLUSIONSClinical isolates of S. aureus in Changsha were resistant to multiple traditional antibiotics. There was an outbreak of PFGE type A MRSA in this area and the A1 subtype was the predominant epidemic clone. Dissemination of the same clone was an important reason for the wide spread of MRSA.

Ampicillin ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; China ; Clindamycin ; pharmacology ; Electrophoresis, Gel, Pulsed-Field ; Erythromycin ; pharmacology ; Humans ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; genetics ; Microbial Sensitivity Tests ; Penicillins ; pharmacology ; Staphylococcus aureus ; drug effects ; genetics ; Vancomycin ; metabolism

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

BACKGROUND: The development of new drugs or alternative therapies effective against methicillin-resistant Staphylococcus aureus (MRSA) is of great importance, and various natural anti-MRSA products are good candidates for combination therapies. We evaluated the antibacterial activities of a Phellinus baumii ethyl acetate extract (PBEAE) and its synergistic effects with beta-lactams against MRSA. METHODS: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the PBEAE. The PBEAE synergistic effects were determined by evaluating the MICs of anti-staphylococcal antibiotic mixtures, with or without PBEAE. Anti-MRSA synergistic bactericidal effects of the PBEAE and beta-lactams were assessed by time-killing assay. An ELISA was used to determine the effect of the PBEAE on penicillin binding protein (PBP)2a production. RESULTS: The MICs and MBCs of PBEAE against MRSA were 256-512 and 1,024-2,048 microg/mL, respectively. The PBEAE significantly reduced MICs of all beta-lactams tested, including oxacillin, cefazolin, cefepime, and penicillin. However, the PBEAE had little or no effect on the activity of non-beta-lactams. Time-killing assays showed that the synergistic effects of two beta-lactams (oxacillin and cefazolin) with the PBEAE were bactericidal in nature (Deltalog10 colony forming unit/mL at 24 hr: 2.34-2.87 and 2.10-3.04, respectively). The PBEAE induced a dose-dependent decrease in PBP2a production by MRSA, suggesting that the inhibition of PBP2a production was a major synergistic mechanism between the beta-lactams and the PBEAE. CONCLUSIONS: PBEAE can enhance the efficacy of beta-lactams for combined therapy in patients infected with MRSA.
Acetates/chemistry ; Agaricales/*chemistry/metabolism ; Anti-Infective Agents/chemistry/*pharmacology ; Drug Synergism ; Enzyme-Linked Immunosorbent Assay ; Methicillin-Resistant Staphylococcus aureus/*drug effects/metabolism ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins/analysis/metabolism ; Plant Extracts/chemistry/*pharmacology ; beta-Lactams/*pharmacology

AIM: Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium that causes both hospital- and community-acquired infections, and for which single-drug treatments are becoming less efficient. Rhizoma coptidis has been used for more than two thousand years in China to treat diarrhea, fever, and jaundice. In this study, the anti-MRSA activity of Rhizoma coptidis is examined and its effective components sought. METHODS: The mecA and norA genes were determined by PCR amplification and sequencing. Drug susceptibility of Staphylococcus aureus ATCC43300 was performed using the VITEK2 compact system. The chemical fingerprint of Rhizoma coptidis was investigated using HPLC and preparative liquid chromatography, and the anti-MRSA activity was determined using an improved broth microdilution method. RESULTS: The drug susceptibility test revealed that the penicillin-binding protein phenotype of the strain changed in comparison to penicillin-sensitive Staphylococcus aureus. Ten batches of Rhizoma coptidis showed anti-MRSA activity on the norA-negative Staphylococcus aureus strain, as well as the strain that contained a norA gene. The spectrum-effect relationship revealed that the berberine alkaloids were the effective components, within which berberine, coptisine, palmatine, epiberberine, and jatrorrhizine were the major components. CONCLUSION This study lays a foundation for in vivo studies of Rhizoma coptidis and for the development of multi-component drugs.
Anti-Bacterial Agents ; chemistry ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; China ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; genetics ; growth & development ; metabolism ; Microbial Sensitivity Tests ; methods ; Ranunculaceae ; chemistry ; Rhizome ; chemistry

OBJECTIVE: To investigate the mechanisms by which MecA gene expression leads to β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), and to study the resistance mechanism of MRSA at the molecular level. METHODS: A variety of molecular biological techniques were employed, including screening MRSA using cefoxitin paper disk method, extraction of MRSA mRNA, reverse transcription into cDNA, real-time fluorescence PCR for quantitation of MecA gene expression, and agar dilution method for assessment of minimum inhibitory concentrations in MRSA treated with cefoxitin, oxacillin, vancomycin, or linezolid. RESULTS: According to the level of resistance of MRSA to cefoxitin, 40 MRSA strains were divided into a low resistance group (n=12), a middle resistance group (n=15), and a high resistance group (n=13). The expression level of the MecA gene in the low resistance group, the middle resistance group, and the high resistance group was 58.87±30.30, 363.37±200.05, and 1257.72±446.63, respectively. MRSA resistance to cefoxitin and oxacillin was 100%; MRSA resistance to vancomycin or linezolid could not be detected. For all 40 MRSA strains the MIC90 for vancomycin was 2.0 μg/mL. CONCLUSION MecA gene expression levels may correlate with the MRSA level of resistance to cefoxitin within a certain range of concentration.
Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Cefoxitin ; pharmacology ; Drug Resistance, Multiple, Bacterial ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; genetics ; metabolism ; Microbial Sensitivity Tests ; methods ; Oxacillin ; pharmacology ; Penicillin-Binding Proteins

The sequences of the ccrAB genes from bovine-, canine- and chicken-originating methicillin-resistant Staphylococcus (S.) epidermidis (MRSE) and bovine methicillin-resistant Staphylococcus (S.) aureus (MRSA) were compared to investigate the frequency of intra-species horizontal transfer of the staphylococcal cassette chromosome mec (SCCmec) complex. Nineteen MRSE strains were isolated from bovine milk, chickens, and dogs, and their genetic characteristics were investigated by multilocus sequence typing and SCCmec typing. Among the animal MRSE strains, the most frequent SCCmec type was type IV, which consisted of the type B mec complex and ccrAB type 2. The ccrA2 and ccrB2 genes were sequenced from the bovine, chicken and canine MRSE strains and compared with those of the bovine MRSA strains. The sequences generally clustered as MRSA and MRSE groups, regardless of the animal source. Additionally, no bovine MRSE sequence was associated with the bovine MRSA groups. Although most of the bovine MRSE and MRSA isolates possessed SCCmec type IV sequences, our results suggest that the intra-species gene transfer of the SCCmec complex between bovine S. aureus and bovine S. epidermidis strains is not a frequent event.
Animals ; Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/*genetics/metabolism ; Bacterial Typing Techniques/veterinary ; Cattle ; Cattle Diseases/epidemiology/metabolism ; Chickens ; Dog Diseases/epidemiology/metabolism ; Dogs ; *Drug Resistance, Bacterial ; *Gene Transfer, Horizontal ; Methicillin/*pharmacology ; Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; Milk/microbiology ; Multilocus Sequence Typing/veterinary ; Poultry Diseases/epidemiology/metabolism ; Prevalence ; Republic of Korea/epidemiology ; Staphylococcal Infections/epidemiology/microbiology/*veterinary ; Staphylococcus epidermidis/genetics/isolation & purification

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) are important pathogens causing nosocomial infections in Korean hospitals. This study aimed to investigate the epidemiological and genetic diversity of clinical S. aureus isolates in healthcare settings from 2001 to 2008. METHODS: Samples and data were obtained from 986 individuals as part of the National Antimicrobial Surveillance Project, involving 10 regions nationwide. Molecular typing studies, including multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were performed, and a representative clone of Korean MRSA was classified by combinational grouping using a DiversiLab (DL; bioMérieux, France) repetitive element polymerase chain reaction (rep-PCR) system. RESULTS: Nine Korean MRSA clones (KMRSA-1 to -9) were identified by analysis of genetic backgrounds and molecular characteristics. KMRSA-1 to -3, expressing clonal complex (CC) 5 (carrying SCCmec II), CC8 (carrying SCCmec III), and CC72 (carrying SCCmec IV) were spread nationwide. In contrast, KMRSA-6 was highly prevalent in Gyeongsangnam-do, and KMRSA-4 was highly prevalent in Jeollanam-do and Jeollabuk-do. CONCLUSIONS: Epidemic KMRSA clones were genetically similar to major clones identified from the USA, with the exception of KMRSA-2, which had the SCCmec III type. Our results provide important insights into the distribution and molecular genetics of MRSA strains in Korea and may aid in the monitoring of MRSA spread throughout the country.
Bacterial Proteins/genetics ; DNA, Bacterial/genetics/metabolism ; Electrophoresis, Gel, Pulsed-Field ; Hospitals ; Humans ; Methicillin-Resistant Staphylococcus aureus/*genetics/isolation & purification ; Multilocus Sequence Typing ; Multiplex Polymerase Chain Reaction ; Prevalence ; Republic of Korea/epidemiology ; Staphylococcal Infections/diagnosis/*epidemiology/microbiology

BACKGROUND: We evaluated the performance of the BD MAX StaphSR Assay (SR assay; BD, USA) for direct detection of Staphylococcus aureus and methicillin resistance not only in S. aureus but also in coagulase-negative Staphylococci (CNS) from positive blood cultures. METHODS: From 228 blood culture bottles, 103 S. aureus [45 methicillin-resistant S. aureus (MRSA), 55 methicillin-susceptible S. aureus (MSSA), 3 mixed infections (1 MRSA+Enterococcus faecalis, 1 MSSA+MRCNS, 1 MSSA+MSCNS)], and 125 CNS (102 MRCNS, 23 MSCNS) were identified by Vitek 2. For further analysis, we obtained the cycle threshold (Ct) values from the BD MAX system software to determine an appropriate cutoff value. For discrepancy analysis, conventional mecA/mecC PCR and oxacillin minimum inhibitory concentrations (MICs) were determined. RESULTS: Compared to Vitek 2, the SR assay identified all 103 S. aureus isolates correctly but failed to detect methicillin resistance in three MRSA isolates. All 55 MSSA isolates were correctly identified by the SR assay. In the concordant cases, the highest Ct values for nuc, mecA, and mec right-extremity junction (MREJ) were 25.6, 22, and 22.2, respectively. Therefore, we selected Ct values from 0-27 as a range of positivity, and applying this cutoff, the sensitivity/specificity of the SR assay were 100%/100% for detecting S. aureus, and 97.9%/98.1% and 99.0%/95.8% for detecting methicillin resistance in S. aureus and CNS, respectively. CONCLUSIONS: We propose a Ct cutoff value for nuc/mec assay without considering MREJ because mixed cultures of MSSA and MRCNS were very rare (0.4%) in the positive blood cultures.
Anti-Bacterial Agents/pharmacology ; Bacteremia/diagnosis/microbiology ; Coagulase/metabolism ; Humans ; Methicillin-Resistant Staphylococcus aureus/drug effects/genetics/*isolation & purification ; Microbial Sensitivity Tests ; Oxacillin/pharmacology ; Reagent Kits, Diagnostic ; Staphylococcus/drug effects/enzymology/genetics/isolation & purification ; Staphylococcus aureus/drug effects/genetics/*isolation & purification

Although pandemic community-associated (CA-) methicillin-resistant Staphylococcus aureus (MRSA) ST30 clone has successfully spread into many Asian countries, there has been no case in Korea. We report the first imported case of infection caused by this clone in a Korean traveler returning from the Philippines. A previously healthy 30-yr-old Korean woman developed a buttock carbuncle while traveling in the Philippines. After coming back to Korea, oral cephalosporin was given by a primary physician without any improvement. Abscess was drained and MRSA strain isolated from her carbuncle was molecularly characterized and it was confirmed as ST30-MRSA-IV. She was successfully treated with vancomycin and surgery. Frequent international travel and migration have increased the risk of international spread of CA-MRSA clones. The efforts to understand the changing epidemiology of CA-MRSA should be continued, and we should raise suspicion of CA-MRSA infection in travelers with skin infections returning from CA-MRSA-endemic countries.
Adult ; Bacterial Toxins/metabolism ; Carbuncle/microbiology ; Cephalosporins/therapeutic use ; Community-Acquired Infections/drug therapy/microbiology ; Exotoxins/metabolism ; Female ; Humans ; Leukocidins/metabolism ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus/*isolation & purification ; Philippines ; Republic of Korea ; Staphylococcal Skin Infections/*drug therapy/surgery ; Travel ; Vancomycin/therapeutic use

PURPOSE: Antimicrobial resistance monitoring could be a useful source of information for treating and controlling nosocomial infections. We analyzed antimicrobial resistance data generated by Korean Hospitals and by a commercial laboratory in 2005 and 2007. MATERIALS AND METHODS: Susceptibility data for 2005 and 2007 were collected from 37 and 41 hospitals, respectively, and from one commercial laboratory. Intermediate susceptibility was not included in the calculation of resistance rates. RESULTS: Methicillin-resistant Staphylococcus aureus (MRSA) (64%), third-generation cephalosporin-resistant Klebsiella pneumoniae (29%), fluoroquinolone-resistant Escherichia coli (27%), Pseudomonas aeruginosa (33%), and Acinetobacter spp. (48%), and amikacin-resistant P. aeruginosa (19%) and Acinetobacter spp. (37%) were prevalent in hospitals in 2007. A gradual increase of vancomycin-resistant Enterococcus faecium and imipenem-resistant Acinetobacter spp. was observed. Higher incidences of third-generation cephalosporin-resistant E. coli and K. pneumoniae and imipenem-resistant P. aeruginosa were found in the commercial laboratory than in the hospitals. CONCLUSION: Methicillin-resistant S. aureus, third-generation cephalosporin-resistant K. pneumoniae, and fluoroquinolone-resistant E. coli, P. aeruginosa and Acinetobacter spp. remain prevalent in Korea, while the incidence of vancomycin-resistant E. faecium and imipenem-resistant Acinetobacter spp. has increased gradually. The higher prevalences of third-generation cephalosporin-resistant E. coli and K. pneumoniae, and imipenem-resistant P. aeruginosa in the commercial laboratory are a new concern.
Acinetobacter/*metabolism ; Bacterial Infections/drug therapy/*epidemiology ; Ceftazidime/*pharmacology ; Cross Infection/drug therapy/*epidemiology ; *Drug Resistance, Bacterial ; Escherichia coli/metabolism ; Fluoroquinolones/*pharmacology ; Humans ; Imipenem/*pharmacology ; Klebsiella Infections/*drug therapy ; Klebsiella pneumoniae/*metabolism ; Methicillin-Resistant Staphylococcus aureus/metabolism ; Pseudomonas aeruginosa/metabolism ; Republic of Korea ; Vancomycin/pharmacology