Beyond Mueller–Hinton agar: comparative evaluation of agar media for antibiotic susceptibility testing and implications for resource-limited laboratory settings

Abstract

This study conducted a proof-of-concept risk assessment to compare the performance of available culture media with Mueller-Hinton agar (MHA) as the reference standard in antimicrobial susceptibility testing (AST) in resource-limited laboratories. Seven culture media were assessed using nine reference bacterial strains, tested against eight broad-spectrum antibiotics. Kirby-Bauer disc diffusion assay, zone diameter agreement (ZDA) of ±2 mm, similarity metrics, reproducibility indices, and multivariate approaches were performed. Data analysis was conducted in R-Studio using a comprehensive suite of statistical packages. Ward's hierarchical clustering and Euclidean distance metrics revealed clear media-specific differences in antimicrobial diffusion patterns. MHA consistently produced the largest inhibition zones, approximately 75%, serving as the reference anchor for comparison. Others exhibited moderately comparable zone diameters, whereas nutrient and plate count agars produced consistently smaller zones. ZDA ranged from 66.7% (plate count agar [PCA]) to 81.9% (nutrient agar [NA] and peptone water agar [PWA]), with no medium achieving the ≥90% threshold used in published disk diffusion media comparisons. Categorical agreement assessed using CLSI breakpoints ranged from 92.3% to 96.2% per medium; no very major errors were observed, while major errors (2.6%) and minor errors (3.8%) were concentrated in two specific organism-antibiotic combinations, indicating a clinically relevant risk of interpretive error when MHA is substituted. Correlation analysis showed the strongest concordance for Luria-Bertani (r = 0.834) and rapid sensitivity test agar (r = 0.791). Most media underestimated zone diameters, but some demonstrated partial similarity to MHA, with none fully replicating its performance characteristics. These findings underscore the diagnostic risks associated with media substitution and highlight the need for strengthened AST. IMPORTANCE: Antimicrobial susceptibility testing (AST) is essential for guiding treatment of bacterial infections, yet its reliability depends on standardized media. In many resource-limited laboratories, Mueller-Hinton agar (MHA) is often substituted with more readily available media due to cost and access constraints. However, the performance and risks associated with this widespread practice remain poorly characterized. Our study provides critical data on the performance of commonly substituted media, including the magnitude and direction of interpretive errors when standard clinical breakpoints are applied. It also identifies specific organism-antibiotic combinations with the highest risk of misclassification. While some alternative media showed partial similarity, none fully replicated MHA's performance. Our study does not advocate for modifying established standards or introducing new breakpoints, but documents a common practice, quantifies the associated diagnostic risks, and offers evidence-based guidance to reduce harm when standard methods are unavailable. These findings support quality improvement efforts and reinforce the need to strengthen diagnostic capacity in resource-limited settings.