Performance of gradient diffusion strip and disk diffusion versus broth microdilution for lefamulin susceptibility testing of 422 Staphylococcus aureus isolates

TL;DR

This study compares simpler antibiotic susceptibility tests for lefamulin against a standard method using 422 Staphylococcus aureus isolates.

Contribution

The study evaluates the accuracy of gradient diffusion strips and disk diffusion for lefamulin susceptibility testing in clinical isolates.

Findings

Gradient diffusion strips showed 100% categorical agreement and 92.2% essential agreement with broth microdilution.

Disk diffusion achieved 99.5% categorical agreement but had a high very major error rate due to few non-susceptible isolates.

Both methods demonstrated high accuracy but require confirmation for borderline results.

Abstract

Lefamulin is a novel pleuromutilin antibiotic with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). While broth microdilution (BMD) is the reference method for susceptibility testing, simpler alternatives such as MIC Test Strip (gradient diffusion strip [GDS]) and disk diffusion (DD) are urgently needed for routine laboratories. We evaluated the accuracy of GDS and DD compared to BMD for lefamulin against S. aureus. A total of 422 non-duplicate clinical isolates of S. aureus (256 methicillin-susceptible S. aureus [MSSA] and 166 MRSA) collected between December 2023 and December 2024 at the Affiliated Tai’an Central Hospital of Qingdao University were tested by BMD, GDS, and DD. Interpretation followed CLSI M100-S34 breakpoints (MIC ≤ 0.25 mg/L or zone diameter ≥ 23 mm = susceptible). BMD served as the reference to calculate categorical agreement (CA), essential agreement (EA), major errors (ME), and very major errors (VME). Lefamulin exhibited potent activity against S. aureus, with 98.6% (416/422) of isolates classified as susceptible by BMD; six isolates (four MSSA and two MRSA) were non-susceptible. Compared with BMD, GDS showed 100% CA and 92.2% EA and yielded no VME and ME, and MICs were 0.49 log₂ lower than BMD. Disk diffusion with the 20 µg lefamulin disk achieved 99.5% CA (421/422), with a VME rate of 16.7% (1/6) and an ME rate of 0.2% (1/416). Lefamulin GDS fulfilled CLSI criteria but systematically underestimated MICs; BMD confirmation is advised for GDS MIC = 0.25 mg/L. Disk diffusion showed excellent agreement, yet its VME rate surpassed the CLSI threshold owing to scarce non-susceptible isolates; any non-susceptible result by disk should be verified by BMD.

Lefamulin is a new antibiotic active against methicillin-resistant Staphylococcus aureus, but simple susceptibility tests are lacking. We compared two easy-to-perform methods with reference broth microdilution for 422 S. aureus clinical isolates. Both showed high accuracy after on-site confirmation of borderline results and can be immediately implemented in routine laboratories to guide appropriate lefamulin therapy and help contain resistance.

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