Comparison of PCR detection of mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci

Abstract

Approximately 75% of coagulase-negative staphylococci are resistant to methicillin, but it is suspected that even more resistance exists that is not detected by standard susceptibility assays. To determine the most accurate assay for measuring resistance, we compared the detection of mecA by PCR with detection by National Committee for Clinical Laboratory Standards methods using oxacillin as the class drug. Strains from 11 species of coagulase-negative staphylococci were selected such that 84% were susceptible by the broth microdilution method. Of 45 mecA-positive strains, 1 strain was unable to express the mecA gene product after induction and was not included in further analyses. For microdilution with 2% NaCl, the disk test without salt, and agar screen containing 4% NaCl plus 6 g of oxacillin per ml, the sensitivities in detecting the 44 mecA-positive strains were 50, 84, and 70%, respectively, at 24 h and 77, 82, and 100%, respectively, at 48 h. The specificities of microdilution, disk, and agar screen in detecting the 97 strains lacking mecA were 100, 89, and 100%, respectively, at 24 h. Only the disk test proved to be less specific at 48 h (81%). Furthermore, for 10 of the mecA-positive strains plus an additional 8 strains subsequently added to the analyses, the MICs were 2 g/ml at 24 h by the broth microdilution method; all 18 strains were positive for mecA by PCR. Thus, an oxacillin MIC of >2 g/ml indicated resistance and is probably a more appropriate breakpoint than the current National Committee for Clinical Laboratory Standards breakpoint of 4 g/ml for coagulase-negative staphylococci. Strains for which MICs are <2 g/ml may be methicillin resistant and should be verified as susceptible by oxacillin agar screening with incubation for 48 h.

Methicillin resistance study in clinical isolates of coagulase-negative staphylococci and determination of their susceptibility to alternative antimicrobial agents

“…It is likely that resistance is not expressed in these strains, despite the presence of sequences corresponding to the mecA gene in the bacterial chromosome. On the contrary, speci®cities obtained with the same tests were lower than those reported by other groups (Geha et al 1994;Mulder 1996;York et al 1996;Kohner et al 1999). This observation suggests that our CNS population has an abnormally high prevalence of mecA-independent methicillin resistance.…”

“…When compared with several previous studies (Geha et al 1994;York et al 1996;Kohner et al 1999), both the disc diffusion and the agar dilution tests showed signi®cant increases in sensitivity (Table 2). These improvements are largely due to the use of the new NCCLS guidelines (NCCLS 2000a, b) which provide more accurate criteria to evaluate methicillin resistance through these phenotypic assays.…”

“…Our results agree well with previous reports indicating that testing of Staph. saprophyticus in the disc diffusion assay, or even for b-lactamase production, was problematic (York et al 1996). On the other hand, both the mecA gene and PBP2a have been found in Staph.…”

Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol

“…The detection of methicillin resistance in staphylococci is complex and di⁄culties exist in accurately identifying MRS mainly because the resistance is often heterogeneous and its expression is a¡ected by di¡erent factors [3]. A number of methods have been studied to detect methicillin resistance in both S. aureus and S. epidermidis, but contradictory results have been reported [4,18,19]. In this study, most isolates (95.3%) showed complete concordance among E-test, mecA PCR (revealed by generation of a single 533-bp fragment) and PBP2P (Tables 1 and 2).…”

Differentiation BetweenStaphylococcus AureusandCoagulase-Negative StaphylococcusSpecies by Real-Time PCR Including Detection of Methicillin Resistants in Comparison to Conventional Microbiology Testing

“…The presence of an additional low-affinity penicillinbinding protein 2a (PBP2a), which is encoded by the mecA gene, is associated with methicillin resistance in both S. aureus and CoNS (12,(15)(16)(17)(18)(19). Polymerase chain reaction (PCR) to detect the mecA gene as a rapid method for the identification of MRSA has been amply demonstrated (11,(20)(21)(22)(23)(24)(25)(26)(27). Some even consider PCR to be the method of choice for identifying MRSA and other methicillinresistant staphylococci (8,(28)(29)(30)(31)(32)(33)(34).…”