Variability and interpretability of benzylpenicillin inhibition zone edges in Staphylococcus aureus - a multi-center study in Austria

PURPOSE: Benzylpenicillin is regaining attention as a treatment option for susceptible S. aureus, including in severe invasive diseases such as blood stream infections. Timely and reliable susceptibility determination is essential to support its use in clinical practice. In this study, we assessed the EUCAST-recommended methodology of interpreting zone edges in a national multicenter trial.

METHODS: In total, nine microbiology laboratories in Austria participated. Each center received 10 isolates in blinded duplicates, all with inhibition zones of ≥ 26 mm. Three were blaZ-positive with sharp edges and seven were blaZ-negative with fuzzy edges. Benzylpenicillin susceptibility testing according to EUCAST guidelines using 1 unit discs was performed by two independent technicians in duplicate on two separate days. All plates were interpreted by two different assessors generating a total of 1440 data points.

RESULTS: Overall, 85.5% of all interpretations were correct. Both, major and very major errors occurred. There was high variability between laboratories with overall accuracy ranging from 61.9% to 100%. There were statistically significant differences in the average inhibition zone between MH agars from different manufacturers. Laboratories using agars giving rise to a smaller average inhibition zone (i.e. lower diffusion of benzylpenicillin) performed less well. Laboratories with the methodology implemented performed better than those without experience.

CONCLUSION: In summary, our study demonstrates that benzylpenicillin susceptibility testing using the EUCAST-recommended methodology is feasible, but accurate and reproducible assessment of zone edges requires both experience and optimal materials. Thorough validation of locally used materials is therefore essential before implementing this approach for routine use.