Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria

Abstract

Bacteria become highly tolerant to antibiotics when nutrients are limited. The inactivity of antibiotic targets caused by starvation-induced growth arrest is thought to be a key mechanism producing tolerance. Here we show that the antibiotic tolerance of nutrient-limited and biofilm Pseudomonas aeruginosa is mediated by active responses to starvation, rather than by the passive effects of growth arrest. The protective mechanism is controlled by the starvation-signaling stringent response (SR), and our experiments link SR-mediated tolerance to reduced levels of oxidant stress in bacterial cells. Furthermore, inactivating this protective mechanism sensitized biofilms by several orders of magnitude to four different classes of antibiotics and markedly enhanced the efficacy of antibiotic treatment in experimental infections.

Figure 1. SR inactivation impairs starvation-induced, stationary phase, and biofilm antibiotic tolerance

A. Detection of (p)ppGpp by thin layer chromatography. The E.coli relA+ strain expresses an inducible relA. B. Growth curves of wild type (➄) and ΔrelA spoT (➉) strains, with and without SHX treatment. C. Ofloxacin tolerance of log-phase bacteria following SHX-induced starvation. Error bars indicate SD. P ≤0.001 (*) versus wild type. D. Ofloxacin tolerance of stationary-phase wild-type (➄), ΔrelA spoT (➉) and ΔrelA spoT +SR (➂) strains. Error bars indicate SD. P ≤0.05 (*) or ≤0.001 (**) versus wild type. E. Antibiotic killing of biofilms treated with ofloxacin (30 µg/mL), meropenem (300 µg/mL), colistin (300 µg/mL) and gentamicin (50 µg/mL). Error bars indicate SD. P ≤0.0005 (*) or ≤0.05 (**) versus wild type. F. Rates of protein and DNA synthesis in biofilms measured by S³⁵-methionine and H³-adenine incorporation. Error bars indicate SD. P ≤0.05 (*) versus wild type.

Figure 2. HAQs mediate antibiotic susceptibility in the ΔrelA spoT mutant

A. Endogenous levels of hydroxyl radicals (OH•) in biofilms. OH• was measured using the probe HPF (3’-p-hydroxyphenyl fluorescein). Error bars indicate SD. P ≤0.005 (*) versus wild type. B. Autolysis occurs in the ΔrelA spoT mutant after prolonged growth on agar (arrow). Scale bar indicates 2.5 mm. C. Spontaneous cell death in ΔrelA spoT biofilms detected by viability staining (live cells are green, dead cells red). Images were acquired with the same microscope settings. D. HAQ measurements by LC/MS. Error bars indicate SD. P ≤0.01 (*) versus wild type. E. Antibiotic killing of biofilms treated with ofloxacin (30 µg/mL), meropenem (300 µg/mL), colistin (300 µg/mL) and gentamicin (50 µg/mL). Error bars indicate SD. P ≤0.0005 (*) or ≤0.05 (**) versus wild type. F. Relationship between HAQ levels, ofloxacin tolerance, and [OH•] in wild type and ΔrelA spoT biofilms. Strains producing graded HAQ expression in the wild type include: a) ΔpqsA ctrl, b) wild type ctrl, c) ΔpqsA pqsA-E+. Strains producing graded HAQ expression in ΔrelA spoT include: d) ΔrelA spoT pqsA ctrl, e) ΔrelA spoT pqsA pqsA-C+, f) ΔrelA spoT ctrl, g) ΔrelA spoT pqsA pqsA-E+. Error bars indicate SD. Biofilm killing P ≤0.001 (*) versus ΔpqsA ctrl; OH• levels P ≤0.05 (**) versus ΔpqsA ctrl.

Figure 3. SR inactivation impairs oxidative defenses

A, B and C. SOD and catalase activity in biofilms as measured by native-protein activity gel staining (A) and biochemical assays (B and C). Error bars indicate SD. An image of intact gels from (A) are shown in Fig S11. P ≤0.001 (*), P ≤0.05 (**) versus the wild type. D. Biofilms lacking HAQs show similar ofloxacin tolerance with or without an intact SR. Error bars indicate SD. P ≤0.001 (*) versus ΔpqsA. E. Antibiotic tolerance in E.coli biofilms treated with ofloxacin (30 µg/mL) and tobramycin (50 µg/mL). Error bars indicate SD. P ≤0.005 (*) versus wild type.

Figure 4. SR inactivation improves antibiotic efficacy in murine infections, and blocks the emergence of resistant mutants

A. Ofloxacin treatment is more effective against lethal infections produced by the ΔrelA spoT strain than in infections caused by wild-type or ΔrelA spoT pqsA P. aeruginosa. Graphs represent pooled data from three independent experiments, with at least 15 mice per group. P ≤0.005 (*) versus treated wild-type infections. B. Ofloxacin treatment is more effective in subcutaneous biofilm infections if the SR is inactivated. Graphs represent pooled data from two independent experiments, with at least six mice per group. Error bars indicate SEM. P ≤0.001 (*) versus treated wild-type infections. C. Resistant mutants emerge after prolonged exposure to ofloxacin in the wild-type (➄), but not the ΔrelA spoT strain (➉). P ≤0.005 (*) versus wild type.