OS-MRSA are commonly borderline oxacillin susceptible
Vitek2 system is one of the automated susceptibility testing (AST) systems widely used in clinical microbiological laboratories, however, with mecA gene PCR as the gold standard, we noticed that Vitek2-Oxacillin miscategorized an especially high proportion (33%, 15/46) of S. aureus isolates for which the Vitek2-Oxacillin MIC were 1 or 2 mg/L, furthermore, CLSI-recommended reference broth microdilution (rBMD) confirmed the oxacillin-susceptibility of 13 out of the 15 miscategorized mecA-positive isolates (Table 1). Another 4 mecA-positive S. aureus isolates with Vitek2-OXA MIC of ≤0.25 mg/L and 0.5 mg/L were further validated as oxacillin susceptible by reference broth microdilution, thus a total of 17 OS-MRSA strains was identified (Table 2).
Oxacillin-MIC of more than half of these OS-MRSA strains (11/17) were determined to be 1-2 mg/L by rBMD (close to CLSI oxacillin-susceptible breakpoint), namely borderline oxacillin susceptible (Table 2). Additionally, OS-MRSA were more frequently found in S. aureus isolates with Vitek2-oxacillin MIC of 1 or 2 mg/L (13/46, 28.26%), whereas less frequently isolated among isolates with Vitek2- oxacillin MIC of ≤0.25 mg/L and at 0.5 mg/L (0.32% and 1.25%, respectively) (Figure 1).
The frequent occurrence of OS-MRSA in S. aureus isolates with Vitek2-oxacillin MIC of 1 or 2 mg/L) partly explain why false susceptible results by Vitek2 system were predominately found in these
borderline susceptible population, while with the exception of these borderline susceptible isolates (1-2 mg/L), categorical results of Vitek2-oxacillin highly agree with those of mecA PCR, cefoxitin disk and oxacillin MIC by rBMD (99.2%-100%) (Table 1).
Genotypic and phenotypic characteristics of OS-MRSA
ST59 clones represented the predominant clone for 58.8% (10/17) of OS-MRSA based on MLST, followed by ST965 (3/17, 17.6%) and ST630 (2/17, 11.8%) (Table 2, Figure 2A). In addition to MLST, spa typing revealed that 7 of 10 ST59 OS-MRSA isolates were t172, one of four remaining isolates was t163, and the other two isolates were t437. Of 17 OS-MRSA isolates, 11 carried SCCmec type IV, other 5 carried SCCmec type V (Table 2). Taking all typing methods into account, ST59-t172-IV was the major contributor of OS-MRSA (Table 2).
In addition, large parts of OS-MRSA were recovered from pediatric patients (11/17; 64.7%) (Figure 2B), yet only 23.3% (223/956) of total isolates were from pediatric patients.
Though susceptible to oxacillin, 15 out of 17 OS-MRSA were classified as cefoxitin-resistant by cefoxitin disk diffusion (Figure 2C). Further antimicrobial susceptibility tests revealed that unlike oxacillin-resistant MRSA (OR-MRSA) that commonly exhibited a multi-drug resistant (MDR) phenotype, OS-MRSA isolates displayed a significantly lower MDR rate and were less likely to be MDR (p<0.05), and so were MSSA (Table 3). Moreover, resistant rates of OS-MRSA were near to those of MSSA for all antibiotics tested (p>0.05) but were obviously lower relative to that of OR-MRSA for several kinds of antibiotics (p<0.05) (Table 3). What’s more, the remarkably differential MIC pattern between OS-MRSA and OR-MRSA further suggested that OS-MRSA appeared to be less susceptible in vitro to a majority of representative antibiotics, including cefazolin, one of the first-generation cephalosporin (Figure 2D).
Notably, oxacillin resistances of all identified OS-MRSA strains were inducible, as prior incubation with oxacillin in vitro could convert all OS-MRSA isolates to highly oxacillin-resistant strains (MIC ≥32mg/L), in which the presence of mecA perhaps play a part (Table 2).
Optimized detection procedures for OS-MRSA
OS-MRSA is a particular type of MRSA and inherently oxacillin-susceptible. Its phenotypic trait makes OS-MRSA easier to be misinterpreted as MSSA by conventional phenotypic tests based on oxacillin susceptibility (Table 1 and Table 2).
Accordingly, special attention should be paid to borderline susceptible S. aureus population (OXA MICs of 1-2 mg/L) in routine tests, considering the frequent appearance of OS-MRSA in borderline oxacillin susceptible group (Table 1, Figure 1). Though the Vitek2 AST card incorporates oxacillin MIC as well as cefoxitin screening to provide simultaneous determination for S. aureus, 11 of 17 OS-MRSA strains still couldn’t be adjusted to MRSA by the AES software on the basis of the cefoxitin result (Table 2, Figure 2C). By contrast, 15 of 17 OS-MRSA could be corrected to cefoxitin-resistant MRSA by disk diffusion test (Table 2, Figure 2C). Generally, the results of cefoxitin disk diffusion highly correlated with mecA gene PCR; it was found to be 100% specific and demonstrated higher sensitivity (98.4%) in detecting mecA positive isolates than the Vitek2 system (Table 4). However, the PBP2a test yielded better correlation with mecA PCR (Table 4) as compared to the cefoxitin disk diffusion test, whereas mecA PCR, as well as PBP2a test, are costly and not accessible for all clinical microbiological laboratories.
For these reasons, it’s recommended that oxacillin-susceptible S. aureus with borderline MIC (1-2 g/mL) and negative cefoxitin screen by Vitek2 should be retested in routine management by cefoxitin disk diffusion, which is accessible to nearly all microbiological laboratories. These supplementary tests are not laborious as borderline oxacillin-susceptible population only represent a small part of all (46/956, 5%). Notably, in case the results obtained by cefoxitin disk diffusion test remain negative, final reports should be based on detection of mecA or PBP2a.