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

Coagulase-negative staphylococci (CoNS) are reported to be the leading cause of nosocomial bloodstream infections. Staphylococcus pettenkoferi is a novel member of CoNS that was first isolated from the human blood and bursitis wound in 2002. We have reported cases of 6 S. pettenkoferi strains isolated from blood specimens, including one pathogen and 5 contaminants and catheter colonizers. Brucker Biotyper (Brucker Daltonics, Bremen, Germany) and molecular typing with 16S rRNA gene sequencing confirmed the 6 isolates as S. pettenkoferi. The conventional phenotypic identification of these isolates is not reliable owing to their inconsistent biochemical characteristics. Five of the 6 isolates were found to be resistant to oxacillin, and all isolates showed susceptibility to vancomycin and linezolid. For accurate identification of this novel species, advanced methods by using Brucker Biotyper or molecular methods such as 16S rRNA gene sequencing are required.
Aged ; Aged, 80 and over ; Anti-Bacterial Agents/pharmacology/therapeutic use ; DNA, Bacterial/chemistry/metabolism ; Drug Resistance, Bacterial/drug effects ; Female ; Humans ; Linezolid/pharmacology ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Oxacillin/pharmacology ; Phenotype ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; Sequence Analysis, DNA ; Staphylococcal Infections/drug therapy/*microbiology/pathology ; Staphylococcus/drug effects/*genetics/isolation & purification ; Vancomycin/pharmacology

BACKGROUND: We performed surveillance cultures of the surfaces of X-ray cassettes to assess contamination with methicillin-resistant Staphylococcus aureus (MRSA). METHODS: The surfaces of 37 X-ray cassettes stored in a radiology department were cultured using mannitol salt agar containing 6 microg/mL oxacillin. Suspected methicillin-resistant staphylococcal colonies were isolated and identified by biochemical testing. Pulsed-field gel electrophoresis (PFGE) analysis was performed to determine the clonal relationships of the contaminants. RESULTS: Six X-ray cassettes (16.2%) were contaminated with MRSA. During the isolation procedure, we also detected 19 X-ray cassettes (51.4%) contaminated with methicillin-resistant Staphylococcus haemolyticus (MRSH), identified as yellow colonies resembling MRSA on mannitol salt agar. PFGE analysis of the MRSA and MRSH isolates revealed that most isolates of each organism were identical or closely related to each other, suggesting a common source of contamination. CONCLUSIONS: X-ray cassettes, which are commonly in direct contact with patients, were contaminated with MRSA and MRSH. In hospital environments, contaminated X-ray cassettes may serve as fomites for methicillin-resistant staphylococci.
Anti-Bacterial Agents/pharmacology ; Diagnostic Equipment/*microbiology ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus/drug effects/*isolation & purification ; Microbial Sensitivity Tests ; Oxacillin/pharmacology ; Staphylococcus haemolyticus/drug effects/*isolation & purification

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

BACKGROUND: Few studies have evaluated the performance of the recently introduced MicroScan Synergies plus Positive Combo 3 Panels (SIPC3) (Dade Behring Inc., USA). We evaluated the clinical efficacy of the panels in identification (ID) and antimicrobial susceptibility testing (AST) of Staphylococcusaureus and enterococci. METHODS: To evaluate the panels' accuracy of identification, the results obtained using the test panels were compared with those obtained by using conventional biochemical tests in conjunction with VITEK 2 system (bio-Merieux, USA). In addition, the AST results obtained using the panels were compared with those obtained by performing CLSI broth microdilution. RESULTS: The overall agreement between the approaches for the ID of S. aureus and enterococci was 100% and 96%, respectively. The categorical and essential agreements (CA and EA) for S. aureus were 98%, each. Very major errors (VME), major errors (ME), and minor error (mE) for S. aureus were 0.45%, 0.3%, and 4.2%, respectively. The majority of VMEs were for oxacillin (8.6%), penicillin (2.0%), erythromycin (7.9%), clindamycin (3.8%), and tetracycline (4.1%). For enterococci, the CA, EA, VME, ME, and mE were 88.8%, 93.7%, 4.4%, 0%, and 2.8%, respectively. The 80.5% (29/36) of Enterococcus faecium had concordant ID with the reference. Most of the categorical errors (3 VMEs and 14 mEs) were observed for quinupristin/dalfopristin (Synercid; Catalytica Pharmaceuticals Inc., USA). CONCLUSIONS: The panels compared favorably with conventional methods for the ID and AST of S. aureus. However, we expected a better performance for ID of E. faecium and AST using Synercid.
Anti-Bacterial Agents/*pharmacology ; Clindamycin/pharmacology ; Drug Resistance, Bacterial ; Enterococcus/*drug effects/isolation & purification ; Erythromycin/pharmacology ; Microbial Sensitivity Tests/instrumentation/*methods ; Oxacillin/pharmacology ; Penicillins/pharmacology ; Reagent Kits, Diagnostic ; Staphylococcus aureus/*drug effects/isolation & purification ; Tetracycline/pharmacology

OBJECTIVETo establish a rapid multiplex PCR (MPCR) detection system of oxacillin and erythromycin resistance genes in Staphylococcus aureus (S. aureus) and evaluate the genotype distribution of the genes associated to mecA, ermA and ermC resistance in Guangzhou.

METHODSThe S. aureus strains were identified and susceptibility tests were performed using VITEK-60 or PHOENIX-100 system. The inducible resistance to clindamycin of strains with of erythromycin resistance was conducted using D-test, and the MPCR system of for detecting the antibiotic resistance genes was optimized.

RESULTSThe MPCR assay for detecting the resistance genes was constructed successfully. According to the results of MPCR, the positivity rates for mecA, ermA and ermC genes among the 124 strains of S. aureus isolated from clinical samples were 56.5%, 50% and 33.9%, respectively. Good correlation was observed between the antibiotic resistance phenotypes and the S. aureus genotypes. mecA were detected in all the methicillin-resistant S. aureus strains, and ermA and/or ermC in 97.7% of the S. aureus strains with erythromycin resistance.

CONCLUSIONThis MPCR system allows rapid and reliable analysis of antibiotic resistance genotypes of S. aureus isolated from clinical samples. mecA, ermA, and ermC genes are among the predominant genetic determinants for the resistance to oxacillin and erythromycin in S. aureus isolates in Guangzhou.

Anti-Bacterial Agents ; pharmacology ; Drug Resistance, Multiple, Bacterial ; genetics ; Erythromycin ; pharmacology ; Methicillin-Resistant Staphylococcus aureus ; genetics ; Oxacillin ; pharmacology ; Polymerase Chain Reaction ; methods ; Staphylococcus aureus ; genetics

OBJECTIVETo investigate the distribution and drug resistance spectrum of clinical bacterial and Candida isolates.

METHODSMost of the bacterial isolates were identified using automated BD Phoenix, and a few with K-B method carried out manually. Candida isolates were identified by color-display plate and K-B method.

RESULTSThe most common isolates in the 2478 strains were P. aeruginosa (15.6%), E. coli (11.5%), C. albicans (9.6%), K. pneumoniae (9.3%), S. aureu (8.2%), and S. epidermidis (7.5%). In gram-negative isolates, the antibiotics with the lowest resistance rate were meraopenem (14.4%), cefoperazone/Sulbactam (14.8%), Imipenem (21.9%), piperacillin/tazobactam (27.4%), ceftazidime (30.0%), amikacin (31.1%), and cefepime (33.1%). The detection rate of E.coli and K. pneumoniae isolates producing extended spectrum beta-lactamase (ESBLs) were 47.4% and 37.3% respectively. In gram-positive isolates, the antibiotics with the lowest resistance rate were vancomycin (0.9%), teicoplanin (1.1%), nitrofurantoin (6.9%), amikacin (20.1%), chloramphenicol (30.7%), and cefoperazone/sulbactam (31.5%). The methecillin-resistant rates of S. aureu , S. epidermidis, and S. haemolyticus were 57.1%, 65.0%, and 66.0%. For Candida isolates, the most sensitive antibiotics were amphotericin B (0.3%), nystain (0.3%), itraconazole (5.6%), fluconazole (9.4%), and fluorocytosine (9.4%).

CONCLUSIONThe results suggest high rate of ESBL production and oxacillin resistance of the bacteria isolated in the hospital. More rational use of antimicrobial agents is crucial for reducing the drug-resistance of the bacteria, and effective measures must be taken to reduce dissemination of multidrug-resistant bacteria.

Anti-Infective Agents ; pharmacology ; Bacteria ; drug effects ; isolation & purification ; Candida ; drug effects ; isolation & purification ; Drug Resistance, Bacterial ; Drug Resistance, Fungal ; Microbial Sensitivity Tests ; Oxacillin ; pharmacology ; beta-Lactamases ; biosynthesis

BACKGROUND: Cinical and Laboratory Standards Institute (CLSI) recommends the use of cefoxitin disks instead of long-used oxacillin disks for screening methicillin-resistant isolates of staphylococci. The frequency of discrepant results and accuracy of the tests were evaluated by detecting mecA gene. METHODS: A total of 3,123 Stapylococci isolates from patients in Severance Hospital were tested during September 2005 to August 2006 by the CLSI-recommended test using both cefoxitin and oxacillin disks. The mecA gene was detected by PCR and the oxacillin minimum inhibitory concentration (MIC) was determined by using agar dilution method for the isolates with discrepant tests. RESULTS: Among 1,915 S. aureus islolates tested, one isolate was resistant to oxacillin disk but susceptible to cefoxitin disk; the isolate did not have mecA gene. Another isolate susceptible to oxacillin but resistant to cefoxitin had mecA gene. Among 1,208 coagulase-negative staphylococcal isolates, 15 isolates were resistant to oxacillin disk but susceptible to cefoxitin disk; the isolates did not have mecA genes. Two isolates susceptible to oxacillin disk but resistant to cefoxitin disk had mecA genes. Among the 16 Staphylococcus isolates that did not have mecA gene, 15 isolates had the oxacillin MICs of < or =2 microgram/mL and were considered as methicillin-susceptible, while 1 isolate with the MIC of 4 microgram/mL was considered as methicillin-resistant. CONCLUSIONS: Overall, 1.9% of staphylococcal isolates showed discrepant results when the screening tests were performed by using oxacillin and cefoxitin disks. None of the isolates resistant to oxacillin disk but susceptible to cefoxitin disk had mecA gene. In conclusion, the cefoxitin disk test is more reliable than oxacillin disk test in screening methicillin-resistant staphylococcal isolates.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/analysis/*genetics ; Cefoxitin/*pharmacology ; *Disk Diffusion Antimicrobial Tests ; Humans ; Methicillin/pharmacology ; Methicillin Resistance ; Oxacillin/*pharmacology ; Polymerase Chain Reaction ; Staphylococcal Infections/microbiology ; Staphylococcus aureus/*drug effects/isolation & purification

BACKGROUND: Cinical and Laboratory Standards Institute (CLSI) recommends the use of cefoxitin disks instead of long-used oxacillin disks for screening methicillin-resistant isolates of staphylococci. The frequency of discrepant results and accuracy of the tests were evaluated by detecting mecA gene. METHODS: A total of 3,123 Stapylococci isolates from patients in Severance Hospital were tested during September 2005 to August 2006 by the CLSI-recommended test using both cefoxitin and oxacillin disks. The mecA gene was detected by PCR and the oxacillin minimum inhibitory concentration (MIC) was determined by using agar dilution method for the isolates with discrepant tests. RESULTS: Among 1,915 S. aureus islolates tested, one isolate was resistant to oxacillin disk but susceptible to cefoxitin disk; the isolate did not have mecA gene. Another isolate susceptible to oxacillin but resistant to cefoxitin had mecA gene. Among 1,208 coagulase-negative staphylococcal isolates, 15 isolates were resistant to oxacillin disk but susceptible to cefoxitin disk; the isolates did not have mecA genes. Two isolates susceptible to oxacillin disk but resistant to cefoxitin disk had mecA genes. Among the 16 Staphylococcus isolates that did not have mecA gene, 15 isolates had the oxacillin MICs of < or =2 microgram/mL and were considered as methicillin-susceptible, while 1 isolate with the MIC of 4 microgram/mL was considered as methicillin-resistant. CONCLUSIONS: Overall, 1.9% of staphylococcal isolates showed discrepant results when the screening tests were performed by using oxacillin and cefoxitin disks. None of the isolates resistant to oxacillin disk but susceptible to cefoxitin disk had mecA gene. In conclusion, the cefoxitin disk test is more reliable than oxacillin disk test in screening methicillin-resistant staphylococcal isolates.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/analysis/*genetics ; Cefoxitin/*pharmacology ; *Disk Diffusion Antimicrobial Tests ; Humans ; Methicillin/pharmacology ; Methicillin Resistance ; Oxacillin/*pharmacology ; Polymerase Chain Reaction ; Staphylococcal Infections/microbiology ; Staphylococcus aureus/*drug effects/isolation & purification

OBJECTIVE: To evaluate the reliability and clinical practicability of cefoxitin disk diffusion test in the detection of methicillin-resistant staphylococcus (MRS) heterogenic drug-resistant strains. METHODS: Three hundred and ten strains of staphylococcus isolated from clinics were detected by the oxacillin disk diffusion test, the cefoxitin disk diffusion test as well as the oxacillin agar dilution test according to the standard operation procedures of NCCLS, and the detection of mecA gene of staphylococcus was used as a criterion. The sensitivities and specitivities of the 4 methods were compared. RESULTS: By the detection of mecA gene, the ratio for MRSA was 57.1%(113/198) and the ratio for MRCNS was 62.5%(70/112). Both the sensitivity and specificity of cefoxitin disk diffusion test in the detection of MRS were 100%, and those in the detection of MRCNS were 98.6% and 100%. CONCLUSION Cefoxitin disk diffusion test is reliable, simple and convenient, and it can be used as a conventional method for the detection of MRS in clinical laboratories.
Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Cefoxitin ; pharmacology ; Humans ; Methicillin ; pharmacology ; Methicillin Resistance ; genetics ; Microbial Sensitivity Tests ; instrumentation ; methods ; Oxacillin ; pharmacology ; Penicillin-Binding Proteins ; Reproducibility of Results ; Staphylococcus aureus ; drug effects ; genetics ; isolation & purification