Delineation of a bacterial starvation stress response network which can mediate antibiotic tolerance development

Abstract

This study aimed at elucidating the physiological basis of bacterial antibiotic tolerance. By use of a combined phenotypic and gene knockout approach, exogenous nutrient composition was identified as a crucial environmental factor which could mediate progressive development of tolerance with markedly varied drug specificity and sustainability. Deprivation of amino acids was a prerequisite for tolerance formation, conferring condition-specific phenotypes against inhibitors of cell wall synthesis and DNA replication (ampicillin and ofloxacin, respectively), according to the relative abundances of ammonium salts, phosphate, and nucleobases. Upon further depletion of glucose, this variable phase consistently evolved into a sustainable mode, along with enhanced capacity to withstand the effect of the protein synthesis inhibitor gentamicin. Nevertheless, all phenotypes produced during spontaneous nutrient depletion lacked the sustainable, multidrug-tolerant features exhibited by the stationary-phase population and were attributed to complex interaction between starvation-mediated metabolic and stress protection responses on the basis of the following reasons: (i) the nutrition-dependent tolerance characteristics observed suggested that adaptive biosynthetic mechanisms could suppress but not fully avert tolerance under transient starvation conditions; (ii) formation of specific phenotypes could be inhibited by suppressing protein synthesis prior to nutrient depletion; (iii) bacteriostatic drugs produced only weak tolerance in the absence of starvation signals; and (iv) the attenuation of the stringent and SOS responses, as well as the functionality of other putative tolerance determinants, including rpoS, hipA, glpD, and phoU, could alter the induction requirement and drug specificity of the resultant phenotypes. These data reveal the common physiological grounds characteristic of starvation responses and the onset of antibiotic tolerance in bacteria.

FIG. 1.

Effects of differential nutrient compositions on the induction and sustainability of antibiotic tolerance. Exponentially growing E. coli BW25113 bacteria were washed, resuspended, and incubated at 37°C for 2 h in a panel of defined media composed of various combinations of five major nutrient classes (glucose, ammonium salts, inorganic phosphate, amino acid mixture, and nucleobases), starting from full RDM, which contained all the five components, to the MOPS base, which was deprived of all nutrients. The preincubated cells were either untreated or subjected to antibiotic treatments using 100 μg ml⁻¹ ampicillin, 0.75 μg ml⁻¹ ofloxacin, or 6.25 μg ml⁻¹ gentamicin for up to 48 h. The degree of population survival was assessed by comparing the log-based population size prior to drug treatment to those determined upon 3 and 48 h of treatment, which reflected short-term and long-term tolerance, respectively. The nutrition recipes are categorized according to the drug specificity and level of sustainability of tolerance phenotypes inducible under each test condition. Category A, undetectable tolerance to all the three antibiotics; category B, tolerance to ofloxacin only; category C, tolerance to both ampicillin and ofloxacin (this category is subdivided on the basis of sustainability of tolerance to these two drugs); category D, tolerance to all the three test drugs. Population growth/viability in the untreated control was depicted by the specific growth rate (μ), which was determined on the basis of changes in cell density within the first 3 h of incubation upon the switch to the test medium.

FIG. 2.

Analysis of the tolerance induction effect of cell-free supernatant of multidrug-tolerant stationary-phase culture on a log-phase, drug-sensitive population. Exponentially growing cells were preincubated in filter-sterilized supernatant of the stationary-phase culture for 2 h (OCSN), followed by treatment with 100 μg ml⁻¹ ampicillin, 0.75 μg ml⁻¹ ofloxacin, or 6.25 μg ml⁻¹ gentamicin for 3 and 48 h and assessment of population survival. The following controls were included in the assay: stationary-phase culture (OC), log-phase populations reconstituted in RDM, and MOPS base.

FIG. 3.

Induction of drug tolerance by bacteriostatic agents. Exponentially growing cells were washed and preincubated in full RDM containing different classes of bacteriostatic agents (Tet, tetracycline [2 μg/ml]; Rif, rifampin [16 μg/ml]; Azide, sodium azide [5 mM]; Cyanide, potassium cyanide [5 mM]) for 2 h before drug treatment and assessment for survival. Exponentially growing cells resuspended in RDM and MOPS base were included as a control.

FIG. 4.

Effects of protein synthesis inhibitors on MOPS-induced antibiotic tolerance. Tetracycline (4 μg ml⁻¹) was added to an RDM-grown log-phase culture, incubated at 37°C for 2 h, and then reconstituted in MOPS basal medium containing the same concentration of tetracycline before being subjected to drug treatment and assessment of survival rate.

FIG. 5.

Assessment of the relative role of stringent response in nutrient depletion-mediated drug tolerance. Exponentially growing CF1943 (WT; ppGpp+) and CF1946 (ΔrelA ΔspoT; ppGpp⁰) populations were preincubated in RDM, RDM without amino acids (RDM-AA), RDM without amino acids and nucleobases (RDM-AA-Nuc), RDM without amino acids and a carbon source (RDM-AA-C), or MOPS base, followed by antibiotic challenge and evaluation of survival rate.

FIG. 6.

Evaluation of effects of stress response regulators and putative antibiotic tolerance genes on nutrient starvation-induced drug tolerance. BW25113 (WT) and its isogenic gene deletion mutants (ΔhipA, ΔphoU, ΔglpD, ΔrecA, and ΔrpoS mutants) were either grown to log phase and preincubated for 2 h in different tolerance-inducing medium backgrounds, including RDM, RDM minus amino acids (RDM-AA), RDM minus amino acids and a carbon source (RDM-AA-C), and MOPS base, or grown overnight to the stationary phase (ONC) prior to antibiotic treatment and assessment of population survival. An asterisk (*) denotes mutants which exhibited ≥4-fold reductions in tolerant-population size in comparison to the wild type.