Background: β-lactam resistance in Staphylococci is mediated by mec genes and usually diagnosed by Cefoxitin disk diffusion and PCR testing. Here, we report methicillin-resistant Staphylococcus lugdunensis and Staphylococcus aureus strains lacking mec gene misdiagnosed by reference methods. Since the strains are not β-lactamase hyperproducers we investigated the molecular basis of their methicillin resistance.
Methods: We tested 2 Staphylococcus lugdunensis (SL1, SL2) and 4 Staphylococcus aureus isolates (SA1, SA2, SA3, SA4): (i) by Cefoxitin disk diffusion (DDFOX), (ii) for agar dilution Oxacillin MIC (AD), (iii) by VITEK®2 (bioMérieux) for antimicrobial susceptibility testing containing Oxacillin MIC (V2OXA) and Cefoxitin Screen Test (V2OXSF), (iv) for mec (A,B,C) genes and (v) by whole genome sequencing (WGS).
Results: The 6 isolates were detected as methicillin susceptible by DDFOX and mec negative. However all isolates displayed variable results for V2OXA MIC (0,5 to >=4mg/L) and V2OXSF (POSITIVE or NEGATIVE). For S. lugdunensis isolates the V2OXSF growth curve atypical pattern has led to investigate the OXSF wells. The plates inoculated with the broth extracted from the OXSF well showed two colony morphotypes (small “P” and regular “G”) for both isolates. The small colonies (SL1P, SL2P) were Oxacillin resistant (V2OXA MIC≥4; AD MIC=4) and V2OXSF POSITIVE whereas the regular colonies (SL1G, SL2G) were Oxacillin susceptible (V2OXA MIC=2; AD MIC=0,5) and V2OXSF NEGATIVE. The 4 morphotypes were confirmed as cefoxitin susceptible by DDFOX and mec negative. Interestingly, WGS revealed, in phenotypically Oxacillin resistant isolates, a GdpP truncation in the N-terminal domain containing a diguanylate cyclase (GGDEF) motif for the subpopulation of small colonies of S. lugdunensis (SL1P, SL2P) and for S. aureus isolates SA1 and SA3. However, GdpP non synonymous mutations were detected in SA2 and SA4 isolates. GdpP is a cyclic diadenosine monophosphate phosphodiesterase enzyme which function is the hydrolysis of a signaling nucleotide (c-di-AMP).
Conclusions: We described mec negative S. lugdunenis and S. aureus strains detected by VITEK2 OXSF test expressing heterogeneous methicillin resistance due to a loss of function of GdpP previously described as associated with reduced growth which may arise as a result of the selective pressure of exposure to β-lactams.