Induction Signals and Functional Regulation of Antibiotic Tolerance in Escherichia coli.

摘要

Bacteria respond swiftly to environmental perturbations, often becoming insensitive to bactericidal antibiotics. The underlying basis of this tolerance phenomenon, which presumably involves physiological adaptation mechanisms that counteract antibiotic-induced lethality in bacteria, remains poorly-defined. In this study, the fundamental issues of antibiotic tolerance development were addressed, with a focus on elucidating the environmental cues and genetic determinants that regulate this phenotypic switching process.;By examining the relationship between exogenous nutrition status and antibiotic susceptibility in bacteria, amino acids deprivation was identified as a prerequisite condition for tolerance development, during which a repertoire of drug-sepcific phenotypes evolved according to the relative abundances of other key essential nutrients. Sustainability of tolerance was highly dependent on a lack of carbon source and the duration of nutrition stress. Importantly, organisms which experienced prolonged starvation (over 24 h) were found to harbor subpopulations which remained drug-tolerant in nutrient-rich medium, suggesting that antibiotic persisters originated from starvation-induced precursor organisms.;Apart from the nutrition factor, a threshold cell density of 10 8 cells per ml was established as an independent mediator which could elicit phenotypic tolerance under nutrient-rich conditions, producing phenotypes which were markedly different from those observable under starvation in terms of drug specificity. Such cell density effects could be attributed to (i) impeded diffusion of drug and nutrient molecules, which simultaneously suppressed the deleterious effects of antibiotics and elicited cellular protection responses, and (ii) a hitherto undefined quorum sensing-like induction signal which was detectable in spent media of nutrient-supplemented but not starving populations. This finding indicates that bacteria can initiate active defense through cell density sensing even in the absence of starvation stress.;Comparative transcriptomic analysis showed that transient tolerance elicited by amino acids starvation was characterized by global metabolic down-regulation, whereas emergence of sustainable phenotypes was tightly coupled to a metabolically active state. Gene knockout analysis on established tolerance determinants, such as hipA, phoU and glpD, revealed that their roles in tolerance development were condition and drug specific, suggesting that the cellular network governing starvation-mediated tolerance was highly complex. Studies on selected determinants further revealed the functional roles of multiple stress signaling and protection systems, including the stringent and SOS responses, heat shock proteins, oxidative defense enzymes, and several novel determinants. Among them, the SOS response was specifically required for development of tolerance to fluoroquinolones, whereas products of two novel genes, yhfZ and yqgB, were predominantly involved in protection against both fluoroquinolones and aminoglycosides. Taken together, results of gene expression and deletion studies depict the involvement of multiple protection systems in sustaining antibiotic stress for a prolonged period. This idea was supported by results of functional studies, which suggested that growth inhibition by bacteriostatic agents, impedance of antibiotic entry and neutralization of hydroxyl radicals were in each case not sufficient to produce significant phenotypic tolerance.;In conclusion, starvation and high cell density-mediated responses were identified as complementary tolerance induction factors in bacteria. Further elucidation of the core components of bacterial "multidrug tolerance regulon" should enable development of more effective strategies for combating resilient microbial infections.