# 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.

## Key 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.