The disk diffusion (DD) method remains the most popular manual technique for antibiotic susceptibility testing (AST) in clinical microbiology laboratories. This is because of its simplicity, reproducibility, and limited cost compared to (automated) microdilution systems, which are usually less sensitive at detecting certain important mechanisms of resistance. Here, we evaluate the PREVI® Isola automated seeder system using a new protocol for spreading bacterial suspensions (eight deposits of calibrated inocula of bacteria, followed by two rounds of rotation) in comparison with manual DD reference testing on a large series of clinical and reference strains. The average time required for seeding one agar plate for DD with this new protocol was 51 s per plate, i.e., 70 agar plates/h. Reproducibility and repeatability was assessed on three reference and three randomly chosen clinical strains, as usually requested by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and was excellent compared to the manual method. The standard deviations of zones of growth inhibition showed no statistical discrimination. The correlation between the two methods, assessed using 294 clinical isolates and a panel of six antibiotics (n = 3,528 zones of growth inhibition measured), was excellent, with a correlation coefficient of 0.977. The new PREVI® Isola protocol adapted for DD had a sensitivity of 99 % and a specificity of 100 % compared to the manual technique for interpreting DD as recommended by the EUCAST.

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BackgroundB-lactam resistance in Staphylococci is mediated by mec genes usually diagnosed by disc diffusion Cefoxitin test (DDFOX) 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 B-lactamase hyperproducers we investigated the molecular basis of the methicillin resistance.MethodsWe tested 2 S. lugdunensis isolates (SL1, SL2) collected from distinct blood cultures of the same patient and 2 S. aureus isolates (SA1, SA2): (i) by DDFOX, (ii) for Oxacillin MIC by agar dilution (AD), (iii) by VITEK®2 (bioMérieux) for Oxacillin MIC (V2 OXA) and Cefoxitin Screen Test (V2 OXSF), (iv) for mecA, B, C genes by PCR and (v) by whole-genome sequencing (WGS).ResultsThe 4 isolates were methicillin susceptible by DD FOX and mec negative. However, all the isolates displayed variable results for V2 OXA MIC (0.5 to ≥4 mg/L) and for V2 OXSF (POSITIVE, NEGATIVE). For SL1 and SL2 isolates, the V2 OXSF growth curve atypical pattern has led to investigating the OXSF wells. The plates inoculated with the broth extracted from the OXSF well showed 2 colony morphotypes (small “P” and regular “G”) for both isolates. The small colonies (SL1P, SL2P) were Oxacillin resistant (V2 OXA MIC≥4; AD MIC = 4) and V2 OXSF POSITIVE whereas the regular colonies (SL1G, SL2G) were Oxacillin susceptible (V2 OXA MIC = 2; AD MIC = 0.5) and V2 OXSF NEGATIVE. The 4 morphotypes were cefoxitin susceptible by DDFOX and mec negative. Interestingly, WGS revealed a GdpP truncation in the N-terminal domain only found in S. lugdunensis small colonies (SL1P, SL2P) phenotypically resistant to Oxacillin. GdpP is a cyclic diadenosine monophosphate phosphodiesterase enzyme which function is the hydrolysis of a signaling nucleotide.ConclusionWe described mec negative S. lugdunensis and S. aureus strains expressing heterogeneous methicillin resistance detected by the VITEK2 OXSF test. S. lugdunensis subpopulation of small colonies resistant to oxacillin is associated with a truncation of GdpP protein previously described in S. aureus. Interestingly GdpP loss of function in Staphylococci is associated with a reduced growth and may arise as a result of the selective pressure of exposure to B Lactams.Disclosures All authors: No reported disclosures.

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