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.

Evaluation of polymerase chain reaction, conventional and MRSA screen latex agglutination methods for detection of methicillin-resistant, -borderline and -susceptible Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), is difficult and expensive to treat, therefore early screening is essential. Several phenotypic and genotypic methods are used to detect MRSA; however, the method of choice remains problematic. We have evaluated four phenotypic methods, broth microdilution (MIC), oxacillin disk agar diffusion (ODD), oxacillin screening salt agar (OSS), and a new rapid phenotypic (MRSA screen latex agglutination, MSLA) with the genotypic gold standard of PCR mecA detection to determine the most appropriate method for routine laboratory use. We randomly collected 203 S. aureus isolates from patients and carriers at two hospitals in Thailand. Using MIC method, three sub-groups were differentiated from among these isolates, namely MRSA (106 isolates), borderline-resistant S. aureus (BRSA) (65 isolates), and methicillin-susceptible S. aureus (MSSA)(32 isolates). A total of 10 methicillin-resistant S. epidermidis (MRSE) isolates were also included. The sensitivity and specificity of MIC, ODD, OSS, and MSLA were 99 and 96, 100 and 97, 100 and 97, and 100 and 100%, respectively. Our study indicated that ODD is still appropriate for routine laboratory. MSLA had the highest sensitivity and specificity and is rapid but expensive, so is the most appropriate method for emergency cases. MIC method was better for BRSA detection and OSS method was more appropriate for screening clinical specimens and carriers.

Evaluation of different primers for detecting mecA gene by PCR in comparison with phenotypic methods for discrimination of methicillin-resistant Staphylococcus aureus.

Detection of the mecA gene by polymerase chain reaction (PCR) is the gold standard for identifying methicillin-resistant Staphylococcus aureus (MRSA). PCR assays, employing MR1-MR2 primers (primer set 1) and MR3-MR4 primers (primer set 2) to generate 154 and 533 bp fragment, respectively, are most widely used for amplification of mecA gene. The purpose of this study was to evaluate the presence of mecA gene in 100 clinical isolates of S. aureus using PCR with the two pairs of primers. The results were compared to the broth dilution MIC method, oxacillin salt screening method (OSS) and oxacillin disk agar diffusion method (ODD). Fifteen of the 100 isolates showed a discrepancy between the mecA primer sets 1 and 2. Three isolates (3%) without the mecA gene showed discrepancies with phenotypic methods. The sensitivity, specificity and positive and negative predictive values for the 154 and 533 bp products of mecA were 79, 85, 83, 81 and 94, 100, 100, 94%, respectively. The results indicated that primer set 2 was more appropriate than primer set 1 for the detection of mecA gene in MRSA. There was a good correlation among the mecA gene detection, ODD and OSS methods. The discrepancy of three isolates between PCR and phenotypic methods should be clarified for other resistant mechanisms.