Detection and expression of methicillin/oxacillin resistance in multidrug-resistant and non-multidrug-resistant Staphylococcus aureus in Central Sydney, Australia

Abstract

Ninety clinical Staphylococcus aureus isolates from separate patients were examined phenotypically and genotypically for susceptibility to methicillin/oxacillin. Thirty were methicillin/oxacillin susceptible and 60 were methicillin and oxacillin resistant (MRSA). The 60 MRSA isolates examined were subdivided into two groups according to their antibiotic profiles and comprised 30 non-multidrug-resistant (NMDR) isolates, resistant to less than two non-beta-lactam antibiotics, and 30 multidrug-resistant (MDR) isolates, resistant to three or more non-beta-lactam antibiotics. Phenotypic and genotypic analysis of methicillin/oxacillin showed that despite use of the guidelines published by the NCCLS for the testing of S. aureus susceptibility to methicillin/oxacillin, MIC values of some NMDR MRSA isolates fell below the NCCLS-recommended breakpoints. Etest strips failed to detect two NMDR MRSA isolates tested with oxacillin and four tested with methicillin. Lowering the NCCLS-recommended oxacillin screen agar concentration from 6 to 2 mg/L and temperature of incubation to 30 degrees C, improved the specificity and sensitivity of NMDR MRSA detection from 87% to 100%. On PFGE analysis these NMDR MRSA strains were genotypically different. Genotypic tests, such as multiplex PCR for the mecA/nuc genes and DNA hybridization for the mecA gene, or phenotypic monoclonal antibody-based tests to detect penicillin-binding protein 2a (PBP2a) offer advantages for problematic isolates in detecting or confirming low-level phenotypic heterogeneous mecA expression of oxacillin and methicillin resistance in NMDR MRSA.

Molecular Analysis of Human and Canine Staphylococcus aureus Strains Reveals Distinct Extended-Host-Spectrum Genotypes Independent of Their Methicillin Resistance

“…Three isolates came from an unknown geographical origin. All isolates were verified as S. aureus, and methicillin resistance was detected according to the presence of mecA using a protocol described by Merlino et al (27).…”

Detection of Integrons and Staphylococcal Cassette Chromosome ( SCCmec ) Types in Staphylococcus Aureus Isolated from Burn and Non-Burn Patients

“…The PCR molecular test was performed to investigate the mecA gene prevalence for all isolates with specific primers as described previously by Merlino et al in 2002. The PCR program was performed as follows: initial denaturation at 94°C for 5 min for 40 cycles, consisting of denaturation (94°C for 30 sec), annealing (57°C for 45 sec), and extension (72°C for 30 sec), followed by a final extension at 72°C for 5 min (21) . Thereafter the SCCmec typing was carried out by a multiplex PCR method using specific primers as described by Boye et al in 2007.…”

Antimicrobial Potential of Plant Seed Extracts against Multidrug Resistant Methicillin Resistant Staphylococcus aureus (MDR-MRSA)

“…Five microlitres were then taken from the suspension and used directly for the PCR (Ubukata et al, 1992). Synthetic oligonuclotide used were mecA F primer 1282 (5'-AAA-ATC-GAT-GGT-AAA-GGT-TGG-C-3') and mecA R primer 1793 (5'-AGT-TCT-GCA-GTA-CCG-GAT-TTG-C-3') and reaction condition was described by Merlino et al, 2002. A Eppendrof mastercycler was programmed with the initial denaturation 5 min at 94ºC, 30 cycles with a 60 seconds denaturation at 94ºC, 30 seconds annealing at 50ºC and 90 seconds extension at 72ºC and 2 min final extension at 72ºC and a holding at 4ºC until the sample was analyzed.…”