Correlation of Penicillin Binding Protein 2a Detection with Oxacillin Resistance in Staphylococcus aureus and Discovery of a Novel Penicillin Binding Protein 2a Mutation

Abstract

We compared a rapid slide latex agglutination test (LAT; Oxoid, Basingstoke, United Kingdom) that detects penicillin binding protein 2a (PBP2a) with MicroScan conventional panels (Dade Behring, West Sacramento, CA) for detection of oxacillin resistance in Staphylococcus aureus. The PBP2a LAT demonstrated 99% agreement with MicroScan oxacillin MIC results for 388 isolates of S. aureus. All 249 oxacillin-resistant isolates gave strong positive reactions in the LAT (100% sensitivity). Three of the 139 oxacillin-susceptible isolates were also strongly positive and one was weakly positive in the LAT (97.1% specificity). The three oxacillin-susceptible isolates with strongly positive reactions were further characterized. The mecA gene was detected in all three by PCR; one isolate was determined to be resistant to oxacillin by reference broth microdilution testing (MIC, 8 g/ml), one isolate was inducibly resistant to oxacillin (MIC of 16 g/ml after overnight induction), and one isolate remained susceptible regardless of the method used for testing. Sequence analysis of a 2.1-kb gene fragment of the mecA gene from the susceptible isolate revealed a one-base substitution at nucleotide position 1449 which results in a Met-to-Ile change for amino acid residue 483. This amino acid substitution has not been previously reported and may be associated with a change in the function of PBP2a resulting in oxacillin susceptibility. An additional 487 isolates were tested in parallel with the both the LAT and MicroScan panels using criteria in which only strong (3 to 4؉) or repeatedly weak (1 to 2؉) LAT reactions were considered positive, and the results showed 99.4% agreement. The PBP2a LAT provided rapid and reliable detection of oxacillin resistance and proved a useful adjunct to the phenotypic method. Both methods provided reliable detection of oxacillin-resistant S. aureus and facilitated the discovery of a novel, functionally impaired form of PBP2a.

Rapid Differentiation of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus by Flow Cytometry after Brief Antibiotic Exposure

“…The promise of flow cytometry is that it can detect far fewer microorganisms and that the technology could potentially be developed to be able to identify and determine resistance of microorganisms directly in body fluids. A test based on detection of a PBP2a protein could mistakenly identify MSSA as MRSA if the bacterium is susceptible to methicillin but carries a mecA gene producing a product with impaired function (1).…”

Evaluation of Susceptibility Test Breakpoints Used to Predict mecA-Mediated Resistance in Staphylococcus Pseudintevmedius Isolated from Dogs

“…The sequence of the entire mecA gene was obtained from selected samples as previously described. 5 The mecA screening was done using primers designed in the current study: GAP-FWD-TCAGAGTTAATGGGACCAAC and GAP-REV-GACTGAACGTCCGATAAAAA. These primers amplified a 562-bp fragment of the mecA gene.…”

Molecular epidemiology and antimicrobial susceptibilities of 273 exfoliative toxin-encoding-gene-positive Staphylococcus aureus isolates from patients with impetigo in Japan

“…Oxacillin-susceptible S. aureus with mecA has been reported in the past (Bressler Petinaki et al, 2002). The oxacillin-susceptible strains with mecA were thought to be caused by a single amino acid substitution in PBP29 (Bressler et al, 2005). Additionally, the mecRI gene, which encodes a transmembrane b-lactam-sensing signal transducer, of SCCmec types IV and V is defective (Deurenberg et al, 2007).…”