Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR

Abstract

In Staphylococcus aureus, mecA and femA are the genetic determinants of methicillin resistance. By using a multiplex PCR strategy, 310-and 686-bp regions of the mecA and femA genes, respectively, were coamplified to identify susceptible (lacking mecA) and resistant (mecA ؉) staphylococci and to differentiate S. aureus (femA ؉) from coagulase-negative staphylococci (lacking femA). A third staphylococcal genomic sequence, corresponding to IS431 and spanning 444 bp, was used as a PCR control. One hundred sixty-five staphylococcal strains were tested. All 72 methicillin-resistant strains were found to be mecA ؉ , and 92 of the 93 susceptible isolates lacked mecA. Only one coagulase-negative Staphylococcus isolate carrying the mecA gene was highly susceptible to oxacillin. The femA determinant was a unique feature of S. aureus; it was found in 100% of the S. aureus strains tested but was undetectable in all of the coagulase-negative staphylococci tested. The possibility of directly detecting the mecA and femA genes in blood samples was also investigated. After two amplification steps, a sensitivity of 50 microorganisms per ml of freshly collected spiked blood was achieved. In conclusion, coamplification of mecA and femA determinants proved to be very reliable both for rapid detection of methicillin resistance and differential diagnosis between S. aureus and other staphylococci. This technique, which can be successfully performed with blood samples, could be a useful tool in the diagnosis and treatment monitoring of staphylococcal infections.

Cross‐sectional study of antimicrobial‐resistant bacteria in horses. Part 1: Prevalence of antimicrobial‐resistantEscherichia coliand methicillin‐resistantStaphylococcus aureus

“…Isolates consistent with staphylococcal species were prepared for antimicrobial sensitivity testing according to BSAC guidelines using 10 antimicrobial discs: methicillin (5 mg), gentamicin (10 mg), rifampicin (2 mg), tetracycline (10 mg), vancomycin (5 mg), teicoplanin (30 mg), ciprofloxacin (1 mg), co-trimoxazole (25 mg), fusidic acid (10 mg), mupirocin (5 mg). All unique staphylococcal isolates were subjected to previously reported PCR assays for the mecA gene for methicillin resistance, and the femA and nuc genes for identification of S. aureus (Brakstad et al 1992;Vannuffel et al 1995). All isolates confirmed as S. aureus were subjected to molecular characterisation by spa gene typing and SCCmec typing in accordance with previously published methods (Oliveira and de Lencastre 2002;Harmsen et al 2003).…”

Clonal spread of SCCmec type IV methicillin-resistant Staphylococcus aureus between community and hospital

“…Antimicrobial susceptibilities were determined using the disk-diffusion method, performed according to CLSI guidelines [19], for oxacillin, clindamycin, erythromycin, gentamicin, minocycline, trimethoprim-sulphamethoxazole, vancomycin, ciprofloxacin and rifampicin. Resistance to oxacillin was confirmed by detecting the presence of the mecA gene by PCR [20].…”

A NOVEL MULTIPLEX PCR SYSTEM FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B, TSST, NUC AND FEM GENES OF STAPHYLOCOCCUS AUREUS IN FOOD SYSTEM

“…The Fem gene of S. aureus encodes a protein that influences the level of methicillin resistance of S. aureus. This gene too has been reported to be specific to S. aureus (Vannuffel et al 1995).…”