Assessment of phenotypic heterogeneity in Salmonella Typhimurium preadapted to ciprofloxacin and tetracycline.

Abstract

Bacterial population exposed to stressful antibiotic conditions consists of various subpopulations such as tolerant, persister, and resistant cells. The aim of this study was to evaluate the phenotypic heterogeneity of Salmonella Typhimurium preadapted to sublethal concentrations of antibiotics. Salmonella Typhimurium cells were treated with 1/2×MIC of antibiotics for the first 48h and successively 1×MIC for the second 24h at 37°C, including untreated control (CON), no antibiotic and 1×MIC ciprofloxacin (NON-CIP), 1/2×MIC ciprofloxacin and 1×MIC ciprofloxacin (CIP-CIP), 1/2×MIC tetracycline and 1×MIC ciprofloxacin (TET-CIP), no antibiotic and 1×MIC tetracycline (NON-TET), 1/2×MIC ciprofloxacin and 1×MIC tetracycline (CIP-TET), and 1/2×MIC tetracycline and 1×MIC tetracycline (TET-TET). All treatments were evaluated by antibiotic susceptibility, ATP level, relative fitness, cross-resistance, and persistence. S. Typhimurium cells were more susceptible to non-adapted NON-CIP and NON-TET (>3-log reduction) than pre-adapted CIP-CIP, TET-CIP, CIP-TET, and TET-TET. CON exhibited the highest ATP level, corresponding to the viable cell number. The relative fitness levels were more than 0.95 for all treatments, except for NON-CIP (0.78). The resistance to ciprofloxacin and tetracycline was increased at all treatments with the exception of NON-TET. The persister cells were noticeably induced at CIP-TET treatment, showing more than 5 log CFU mL-1. The results suggest that the antibiotic preadaptation led to heterogeneous populations including persisters that can develop to resistance. This study provides new insight in the bacterial persistence associated with their potential risk and paves the way to design antibiotic therapy targeting dormant bacteria.