Beyond Mueller–Hinton Broth: A Scoping Review of Metabolic Context, Conditional Antibiotic Susceptibility, and Metabolism-Aware Antimicrobial Susceptibility Testing

Abstract

Background. Standard antimicrobial susceptibility testing (AST) in Mueller–Hinton broth samples a single metabolic state, yet bacterial susceptibility in vivo depends on nutrient availability, host-derived metabolites, and spatial structure. The extent to which this mismatch produces clinically relevant errors in susceptibility classification has not been systematically mapped. Objective. To map the evidence linking metabolic context to conditional antibiotic susceptibility and to identify gaps between mechanistic insight, physiologically relevant models, and clinical validation. Methods. We conducted a scoping review following PRISMA-ScR. The search combined PubMed, Semantic Scholar, Web of Science, and Embase with AI-assisted discovery and screening using Consensus AI and large language models, with full human verification at every inclusion decision. AI use is reported per PRISMA-trAIce. Records were classified by mechanism (porin regulation, efflux, stringent response, metabolic rewiring, biofilm structure) and by infection-relevant context (gut-mimicking media, bicarbonate and host factors, single-cell and heterogeneity, in vivo models, clinical validation). Results. Of 1,543,377 records identified, 100 studies met inclusion criteria. Mechanistic evidence was dense across all seven mechanistic foci. In contrast, evidence in physiologically relevant contexts was sparse: gut-mimicking AST appeared in only one mechanism category, and clinical validation was absent in five of seven. The most prominent gap was the absence of prospective clinical validation linking host-mimicking AST results to patient outcomes. Conclusions. Metabolic state is a primary determinant of antibiotic susceptibility, and standard Mueller–Hinton AST samples only one such state. The field has established mechanisms but not clinical translation. Prospective validation of host-mimicking AST across pathogen and drug classes is the highest-priority next step.