Decreased cyclic-AMP caused by ATP contributes to fosfomycin heteroresistance in avian Escherichia coli

Abstract

Abstract Background Fosfomycin is an important broad-spectrum bactericidal antibiotic to treat multidrug-resistant bacteria infections. It is generally accepted that heteroresistant bacteria are an intermediate stage in the formation of drug resistance, but there are few studies on the formation mechanism underlying fosfomycin heteroresistance (FHR). Objectives To reveal the characteristics and formation mechanisms of FHR in Escherichia coli isolates obtained from chickens. Methods We identified the FHR according to the population analysis profile (PAP) test and in vitro time–kill assay. Growth curves for FHR E. coli and their subpopulations were measured. Also, the subpopulations were repeatedly cultured in fosfomycin-free medium for 5–20 overnight incubation periods. The formation mechanisms of FHR in E. coli isolates were identified through accumulation assay, carbohydrate utilization testing, real-time relative quantitative PCR analysis, DNA sequencing, transcriptomic analysis, intracellular ATP and cAMP-level assessment. Results Four of six E. coli strains were confirmed to show FHR, with a total of six subpopulations. The subpopulations restored phenotypic susceptibilities to fosfomycin within 5–20 overnight incubation sessions, but four of six subpopulations still maintained FHR characteristics. Differing from their parental isolates, the uptake of fosfomycin in the subpopulations through GlpT was reduced remarkably. Further studies identified that the low expression of glpT was due to the decrease of intracellular cAMP levels in the subpopulations, which was caused by the decreased ATP levels in cells. Conclusions Our findings revealed the formation mechanism of E. coli isolates showing FHR obtained from chicken in China and characterized the dynamic change traits in vitro of the subpopulations.