Analysis of three phenotyping methods and pulsed-field gel electrophoresis for differentiation of methicillin resistant Staphylococcus aureus isolated from two hospitals in Thailand.

Methicillin resistant Staphylococcus aureus (MRSA) is an important hospital and community-acquired pathogen. Rapid and reliable epidemiologic typing is necessary for controlling the spread of MRSA outbreak. The objective of this study was to compare the phenotyping with the genotyping method to differentiate MRSA isolates obtained from the two hospitals in Thailand (central and northeastern). Seventy-four MRSA isolates were randomly collected and confirmed by the presence of mecA gene. Antibiogram, phage typing and enterotoxin production were used for the phenotyping analysis. Pulsed-field gel electrophoresis (PFGE) with Smal digestion of chromosomal DNA was used for the genotyping analysis. We found 17 distinct profiles by the 3 phenotypic typing methods and 18 PFGE types designated as 5 major types (A-E) and 13 subtypes. The most frequent PFGE types and their related subtypes found in both hospitals were A and C, comprising 54 and 27%, respectively. The antibiogram could differentiate 6 different types. All isolates were resistant to the majority of antimicrobial agents tested, but were susceptible to vancomycin and fosfomycin. Ten (13.5%) MRSA isolates produced enterotoxin A. Nontypable phage and phage type 77 were found predominantly in MRSA isolated from the northeast and central hospital, respectively. A significant correlation was found between the phenotyping and the genotyping methods and there was a good correlation between antibiogram and PFGE. Antibiogram typing alone can be used as a useful epidemiological marker for practical purposes. PFGE types A and C were the common endemic MRSA clones in both hospitals in Thailand.

Occurrence of extended-spectrum beta-lactamase in clinical isolates of Klebsiella pneumoniae in a University Hospital, Thailand.

The occurrence and antimicrobial susceptibility of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients attending Siriraj Hospital in Bangkok from August 2000 to January 2001 were determined. ESBL-producing isolates were screened with four different methods: disk diffusion according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines, Etest ESBL (CT/CTL and TZ/TZL), Oxoid combination discs and MIC Etest strip. Antimicrobial susceptibility testing were determined by a microdilution automatic method (VITEX system, bioMerieux). Of 22,178 clinical specimens, 400 (1.8%) K. pneumoniae were isolated Of 26% (104/400) of these isolates were suspected to be ESBL-producing. Rates of detection of ESBL-producing K. pneumoniae were 18.67%, 30% and 23.78% for blood, sputum and urine samples, respectively. Susceptibility testing has revealed that all 70 tested isolates including 53 isolates from blood and sputum and 17 isolates from urine samples were susceptible to imipenem (MIC< or =4 mg/L). None of the tested isolates were susceptible to cephalosporins, cephamycin and aztreonam. Rate of susceptibility to ciprofloxacin, levofloxacin, gentamicin and tobramycin were 60%, 64%, 28% and 9%, respectively, for isolates from blood and sputum; 71%, 71%, 18% and 6% for urinary isolates. The present findings revealed a high occurrence rate of multi-drug resistance ESBL-producing K. pneumoniae in patients attending the university hospital. Imipenem was highly active against ESBL-producing K. pneumoniae.

Detection of Clostridium difficile toxin A and B genes from stool samples of Thai diarrheal patients by polymerase chain reaction technique.

The prevalence of Clostridium difficile isolated from stools of Thai adult patients with suspected antibiotic-associated diarrhea (AAD) was 18.64 per cent. The recovery rate of toxin genes (tcdA and tcdB) by polymerase chain reaction (PCR) from stool samples yielded almost the same compared to the recovery rate of the toxin detection by enzyme immunoassay (EIA), which were 44.9 per cent and 46.7 per cent, respectively. Correlation of toxin gene detection by PCR and toxin detection by EIA was 90.6 per cent. All but one stool sample, the tcdA gene was detected together with the tcdB gene. Both genes were always detected together from tox gene-positive strains. Although, there were some discrepancy results for certain samples, the direct PCR-based-detection of C. difficile tox genes in stool samples seems to be the appropriate method for the diagnosis of C. difficile diarrhea. The PCR assay should be a recommended technique to be used routinely in laboratories. Further optimization of the technique to increase the sensitivity of the PCR assays is still needed. However, a quantitative isolation of the organism from stools of suspected antibiotic-associated diarrhea (AAD) or antibiotic-associated colitis (AAC) patients may give some evidence for clinicians in hospitals who cannot perform PCR-based or EIA-based techniques, since 48.6 per cent of the isolates were demonstrated as toxigenic strains.