The sampling bottleneck in antimicrobial susceptibility testing: consequences and paths forward.

Conventional antimicrobial susceptibility testing (AST) is based on inocula derived from a small number of colonies, creating a substantial sampling bottleneck relative to the complexity of within-host bacterial populations. Although this limitation is well recognized, its clinical impact is likely limited in many immunocompetent hosts, where effective immune responses and adequate antimicrobial exposure buffer against the influence of low-frequency resistant subpopulations. However, genomic and mechanistic studies show that such subpopulations can exist at low frequencies and may become clinically relevant in high-risk settings, including persistent or high-burden infections and in profoundly immunocompromised patients. This article outlines the biological basis of the sampling constraint, clarifies when it is most likely to matter, and discusses targeted population-based and sequencing-informed adjunct approaches that may complement reference AST in select scenarios. Improving transparency around these limitations-without undermining confidence in standardized methods-supports antimicrobial stewardship and more nuanced interpretation in defined clinical contexts.