Hufnagel, D. A. | bioRxiv Science

bioRxiv2025

Metabolic heterogeneity coupled with a resistance gene generates antibiotic heteroresistance

Heteroresistance is a form of antibiotic resistance in which a phenotypically unstable subpopulation of resistant cells exists within a majority population of susceptible cells. The impact of the extracellular environment on mechanisms of heteroresistance is unclear. Studying fosfomycin heteroresistance in an Enterobacter cloacae complex isolate, we observed that glucose availability greatly increased the frequency of the resistant subpopulation. Glucose downregulated the glycerol and fosfomycin importer GlpT, whose expression was heterogenous at the single cell level. This heterogeneous expression of GlpT, in combination with the expression of the fosfomycin resistance gene fosA, which acted as a resistance enhancer, led to the generation of heteroresistance. Correspondingly, the frequency of the fosfomycin resistant subpopulation was increased in murine models of hyperglycemia/diabetes. These data demonstrate how metabolic heterogeneity and carbon source availability can impact antibiotic resistance phenotypes in the infection environment.

microbiology↗

bioRxiv2023

Pre-existing heterogeneity facilitates development of heteroresistance upon gene acquisition

Antibiotic resistance causes 1.27 million global deaths annually and is predicted to worsen. Heteroresistance is a form of resistance in which only a minor and unstable subpopulation of cells of a bacterial isolate are resistant to a given antibiotic, and are therefore often undetected by clinical diagnostics. These infrequent and undetected resistant cells can be selected during antibiotic therapy, expand in number, and cause unexplained treatment failures. A major question is how heteroresistance evolves. Here, studying the antibiotic fosfomycin, we report that heteroresistance can develop from a pre-existing state of phenotypic heterogeneity in which an isolate harbors a subpopulation with increased minimum inhibitory concentration (MIC), but below the clinical resistance breakpoint. We call this phenomenon heterosusceptibility and demonstrate that acquisition of a resistance gene, fosA, increases the MIC of the subpopulation beyond the breakpoint, making the isolate heteroresistant. Conversely, deletion of fosA from a heteroresistant isolate led to reduction of the MIC of the resistant subpopulation without a loss of heterogeneity, thus generating heterosusceptibility. A survey of 103 carbapenem-resistant Enterobacterales (CRE) revealed that the Escherichia sp. isolates lacked the fosA gene and uniformly exhibited fosfomycin heterosusceptibility, whereas the Klebsiella and Enterobacter encoded the fosA gene and were almost exclusively heteroresistant. Furthermore, some isolates exhibited heterosusceptibility to other antibiotics, demonstrating that this is a widespread phenomenon. These results highlight a mechanism for the evolution of heteroresistance and suggest that surveillance for heterosusceptibility may facilitate the prediction of impending heteroresistance before it evolves.

microbiology↗