BACKGROUNDMycobacterium abscessus (M. abscessus) can cause a variety of human infections, involving the lung, skin and soft tissues, and is generally believed to be acquired from environmental sources. The aim of this study was to investigate the molecular diversity and antibiotic susceptibility of M. abscessus isolates as the basis for strategies to improve control and management of infection.

METHODSSeventy M. abscessus isolates from patients attending the Guangzhou Thoracic Hospital were identified from 2003 to 2005 by biochemical tests, gas chromatography, polymerase chain reaction (PCR)-restriction analysis (PRA) of heat shock protein gene hsp65, and sequencing of the quinolone resistance determining regions (QRDRs) of gyrA. Susceptibilities to six antibiotics were determined by micro-broth dilution. Isolates were genotyped using randomly amplified polymorphic DNA (RAPD) analysis.

RESULTSMost isolates (63/70; 90%) were susceptible to amikacin but rates of susceptibility to other antibiotics varied from moderate, clarithromycin (60%) and imipenem (43%), to low for ciprofloxacin and ofloxacin (3%), and 87% of isolates had intermediate susceptibility to cefoxitin. RAPD analysis showed that the 70 clinical isolates displayed 69 unique RAPD patterns.

CONCLUSIONSThe high genetic diversity of isolates suggests that they are not transmitted from person to person but, presumably, are acquired independently from environmental sources. M. abscessus isolates displayed variable levels of susceptibility to all antibiotics tested, other than amikacin, indicating a need for routine susceptibility testing to guide treatment.

Amikacin ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Cefoxitin ; pharmacology ; China ; Chromatography, Gas ; Ciprofloxacin ; pharmacology ; Clarithromycin ; pharmacology ; Imipenem ; pharmacology ; Microbial Sensitivity Tests ; Mycobacterium ; drug effects ; genetics ; Polymerase Chain Reaction ; Random Amplified Polymorphic DNA Technique

As the incidence of nontuberculous mycobacterial infection has been increasing recently in Korea, the importance of drug susceptibility test for clinical isolates of mycobacteria has become larger. In this study we determined the antimicrobial susceptibility patterns of clinical isolates of M. fortuitum and M. abscessus in Korea, and evaluated the efficacy of a modified broth microdilution method using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC), in terms of its ability to provide accurate and easy-to-read minimal inhibitory concentration (MIC) endpoints for the susceptibility testing of rapidly growing mycobacteria. Most isolates of M. fortuitum and M. abscessus in Korea are susceptible or intermediately susceptible to amikacin, cefoxitin, ciprofloxacin, and clarithromycin. Many isolates of M. fortuitum are susceptible to doxycycline, sulfamethoxazole, and imipenem, while many M. abscessus isolates are resistant to these drugs. In the present study, the modified broth microdilution method using STC was found to be reliable, easy to read, and inexpensive for M. fortuitum and M. abscessus susceptibility testing. The modified colorimetric MIC testing method using STC was proven to be a useful surrogate for RGM antibiotic susceptibility testing.
Anti-Bacterial Agents/pharmacology ; Cefoxitin/pharmacology ; Chemistry, Pharmaceutical/methods ; Ciprofloxacin/pharmacology ; Clarithromycin/pharmacology ; Colorimetry/methods ; Drug Resistance, Bacterial ; Korea ; *Microbial Sensitivity Tests ; Mycobacterium/*metabolism ; Mycobacterium fortuitum/*metabolism ; Tetrazolium Salts/*pharmacology

BACKGROUND: Susceptibility testing of Staphylococcus aureus often requires cumbersome supplementary tests. MicroScan Pos Breakpoint Combo Panel Type 28 (PBC28) (Siemens, USA) includes cefoxitin screening to detect methicillin-resistant Staphylococcus aureus (MRSA), inducible clindamycin resistance detection (ICD), and determination of low-range minimum inhibitory concentration of vancomycin (0.5-16 microgram/mL). The purpose of this study was to evaluate the performance of PBC28 in comparison with that of Pos Combo Type 1A (PC1A) (Siemens). METHODS: From December 2009 to March 2010, 500 non-duplicate clinical isolates of S. aureus were tested with PC1A and PBC28. Categorical agreements (CA) between the interpretations of the 2 panels were estimated. The presence of the mecA gene was determined by PCR, and double-disk diffusion test (D-test) was performed on the isolates resistant to erythromycin but susceptible or intermediately resistant to clindamycin. Ninety-six isolates representing various vancomycin minimum inhibitory concentrations (MICs) were tested in parallel with repeat PBC28, broth macrodilution, and epsilometer test (E test). RESULTS: The CA was 99.3% with a very major error (VME) of 0.2%, major error (ME) of 0.1%, and minor error (mE) of 0.4% in total. PBC28 showed 100% CA for 1 isolate with vancomycin MIC of 4 microgram/mL and 35 isolates (7.0%) with MIC of 2 microgram/mL. However, only 15, 27, and 35 isolates with vancomycin MIC of 2 microgram/mL showed 100% CA in repeat PBC28, broth macrodilution, and E test, respectively. PC1A and PBC28 detected all 314 mecA-positive isolates. Among the 63 isolates tested with the D-test, 58 (92.1%) were positive, and the results were 100% concordant with those of ICD. CONCLUSIONS: PBC28 can be appropriate susceptibility testing of S. aureus, including MRSA detection and ICD. However, the lower-range vancomycin MIC test was not reproducible enough to reliably differentiate MIC of 2 microgram/mL from MIC< or =1 microgram/mL.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; Cefoxitin/*pharmacology ; Clindamycin/*pharmacology ; Drug Resistance, Bacterial ; Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; *Microbial Sensitivity Tests ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Staphylococcus aureus/*drug effects/genetics/isolation & purification ; Vancomycin/*pharmacology

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

BACKGROUND: Plasmid-mediated AmpC beta-lactamases (PABLs) have been detected in the strains of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Salmonella spp. PABLs may be difficult to detect and might interfere in the therapeutic and infection-control processes. Although several PABL-detection methods based on phenotypes have been reported, the Clinical and Laboratory Standards Institute currently does not recommend a routine detection method for PABLs. The aim of this study is to compare the performances of 3 phenotypic PABL detection methods. METHODS: Total 276 non-duplicated clinical isolates of E. coli (N=97), K. pneumoniae (N=136), and P. mirabilis (N=43) were collected from 14 hospitals in Korea between April and June 2007 in a non-consecutive and non-random manner. Multiplex PCR was performed to detect the PABL genes. Further, 3 phenotypic detection methods-cephamycin-Hodge test, Tris-EDTA (TE) disk test, and combination-disk test with 3-aminophenylboronic acid (BA)-were performed using cefoxitin and cefotetan disks. RESULTS: PABL genes were detected by multiplex PCR in 122/276 isolates, including 14/97 E. coli, 105/136 K. pneumoniae, and 3/43 P. mirabilis isolates. The combination-disk test with BA showed higher sensitivity (98.4%), specificity (92.2%), and efficiency (96.3%) than the cephamycin-Hodge (76.2%, 96.1%, and 88.6%, respectively) and the TE-disk (80.3%, 91.6%, and 87.9%, respectively) tests. CONCLUSIONS: The combination-disk test with BA is a simple, efficient, and interpretable test that can be applicable in clinical laboratories involved in the detection of PABLs in clinical isolates of E. coli, K. pneumoniae, and P. mirabilis.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*analysis ; Cefotetan/pharmacology ; Cefoxitin/pharmacology ; Disk Diffusion Antimicrobial Tests/*methods ; Escherichia coli/genetics/*isolation & purification ; Humans ; Klebsiella pneumoniae/genetics/*isolation & purification ; Phenotype ; Plasmids ; Proteus mirabilis/genetics/*isolation & purification ; Sensitivity and Specificity ; beta-Lactamases/*analysis

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: Bacteroides fragilis group organisms are the most frequently isolated anaerobes in human infections. Increasing resistance to various antimicrobial agents is a significant problem in choosing appropriate antimicrobial agents to treat anaerobic infections. Periodic monitoring of the regional resistance trends of B. fragilis group isolates is needed. METHODS: A total of 466 nonduplicate clinical isolates of B. fragilis group organisms (276 B. fragilis, 106 Bacteroides thetaiotaomicron, and 84 other B. fragilis group organisms) were collected during the 8-yr period from 1997 to 2004 in a Korean university hospital. Minimum inhibitory concentrations to various antimicrobial agents were determined by the CLSI agar dilution method. RESULTS: Eight isolates were resistant to imipenem. Additionally, the resistance rates to cefotetan were decreased in B. thetaiotaomicron, while those for clindamycin were significantly increased compared to the rates found in previous studies. Depending on species, resistance rates were 1-4% for imipenem, 1-6% for piperacillin-tazobactam, 4-11% for cefoxitin, 33-49% for piperacillin, 14-60% for cefotetan, and 51-76% for clindamycin. No isolates were resistant to chloramphenicol or metronidazole. CONCLUSIONS: Piperacillin-tazobactam, cefoxitin, imipenem, chloramphenicol, and metronidazole are still active against B. fragilis group isolates, while clindamycin no longer has a value as an empirical therapeutic agent in Korea. Furthermore, this study identified the first imipenem-resistant B. fragilis group isolates in Korea.
Anti-Bacterial Agents/pharmacology ; Bacteroides/classification/*drug effects/isolation & purification ; Bacteroides fragilis/drug effects/isolation & purification ; Cefoxitin/pharmacology ; Chloramphenicol/pharmacology ; *Drug Resistance, Multiple, Bacterial ; Humans ; Imipenem/pharmacology ; Metronidazole/pharmacology ; Microbial Sensitivity Tests ; Penicillanic Acid/analogs & derivatives/pharmacology ; Piperacillin/pharmacology ; Republic of Korea