Slow growth determines nonheritable antibiotic resistance in Salmonella enterica

Abstract

Bacteria can withstand killing by bactericidal antibiotics through phenotypic changes mediated by their preexisting genetic repertoire. These changes can be exhibited transiently by a large fraction of the bacterial population, giving rise to tolerance, or displayed by a small subpopulation, giving rise to persistence. Apart from undermining the use of antibiotics, tolerant and persistent bacteria foster the emergence of antibiotic-resistant mutants. Persister formation has been attributed to alterations in the abundance of particular proteins, metabolites, and signaling molecules, including toxin-antitoxin modules, adenosine triphosphate, and guanosine (penta) tetraphosphate, respectively. Here, we report that persistent bacteria form as a result of slow growth alone, despite opposite changes in the abundance of such proteins, metabolites, and signaling molecules. Our findings argue that transitory disturbances to core activities, which are often linked to cell growth, promote a persister state regardless of the underlying physiological process responsible for the change in growth.

Fig 1.. Effects of low cytosolic Mg²⁺ on Salmonella antibiotic tolerance.

(A) Quantification of surviving wild-type (14028s), Δ12TA (MP1422), and relA::Tn10 spoT (MP342) Salmonella following 2.5 h exposure to cefotaxime (200 μg/mL) or ciprofloxacin (2 μg/mL) following 2 or 5 h of growth in low Mg²⁺ medium. Error bars represent standard deviations, derived from at least 8 biological samples and 3 independent experiments. Note log scale of y axis. (B) Representative dilution series of wild-type (14028s), Δ12TA (MP1422), and relA::Tn10 spoT (MP342) Salmonella following 2.5 h exposure to cefotaxime or ciprofloxacin following 2 or 5 h of growth in low Mg²⁺ medium. Each spot represents a 5 μL aliquot.

Fig 2.. Effects toxin-antitoxin modules, (p)ppGpp biosynthetic and degradation genes, or acidic pH on Salmonella persistence.

(A) Quantification of surviving wild-type (14028s), Δ12TA (MP1422), and relA::Tn10 spoT (MP342) Salmonella following 24 h exposure to cefotaxime or ciprofloxacin in LB medium. (B) Quantification of surviving wild-type (14028s), Δ12TA (MP1422), and relA::Tn10 spoT (MP342) Salmonella following acid shock (30 min in LB pH 4.5) prior to a 24 h exposure to cefotaxime or ciprofloxacin in LB medium. (C) Quantification of surviving wild-type (14028s), ΔTA3 (MP1454), ΔTA5 (MP1455), ΔTA7 (MP1456), ΔTA9 (MP1457), and ΔTA10 (MP1458) Salmonella following a 24 h exposure to cefotaxime or ciprofloxacin in LB medium. Error bars represent standard deviations (N = 8 biological replicates). Note log scale of y axis. For (A) and (B), two-tailed t-test between populations at the edge of brackets. For (C), two-tailed t-tests paired with wild-type populations: **p<0.01, ***p<0.001 and N.S. for no significance.

Fig 3.. Effect of Shx on bacterial growth and antibiotic tolerance.

(A) Growth curves of wild-type Salmonella (14028s) in LB medium in the absence (black lines) or presence (green lines) of Shx (100 μg/mL). Shx was added for 30 min prior to outgrowth (top panel) or at 120 min of growth (bottom panel). (B) Concentration of viable bacteria (Log colony forming units (CFU)/mL) in cultures of wild-type Salmonella (14028s) gown as described in (A). Where indicated, bacteria were exposed to cefotaxime (200 μg/mL) or ciprofloxacin (2 μg/mL) for either 24 h (top panel) or 22 h (bottom panel). Error bars represent standard deviations (N = 6 biological replicates). Rightmost figure shows enlarged portion of left part of figure shaded in pink.

Fig 4.. Effects of ATP depletion and inhibition of protein synthesis on antibiotic tolerance.

(A) Fraction of surviving wild-type Salmonella (14028s) carrying pATPase or the pVector control following 24 h exposure to cefotaxime (200 μg/mL) or ciprofloxacin (2 μg/mL). (B) Bacterial growth (left y-axis, solid lines) and ATP levels (right y-axis, dashed lines) of wild-type Salmonella (14028s) exposed to chloramphenicol at 120 min (red lines) or not exposed to chloramphenicol (black lines). Fraction of surviving (C) wild-type (14028s) or (D) relA::Tn10 spoT (MP342) Salmonella following cefotaxime or ciprofloxacin exposure in the presence or absence of chloramphenicol (50 μg/mL). Error bars represent standard deviations (N = 8 biological replicates). For (A), (C) and (D), note log scale of y axis. Wilcoxon rank-sum test between pVector and pATPase (A) or untreated and untreated populations (C) and (D): ***p<0.001, ****p<0.0001.

Fig 5.. Relationship between growth and antibiotic tolerance in nutritional auxotrophs and relA spoT strains.

(A) Fraction of surviving wild-type (14028s), manA (MP50) and trpEDCBA::Tn10 (AS301) Salmonella following 24 h treatment with either cefotaxime (200 μg/mL) or ciprofloxacin (2 μg/mL) under growth-permissive and growth-restrictive conditions (see Materials and Methods). Fraction of surviving (B) wild-type (14028s) and relA::Tn10 spoT trpA::kan (MP1494), or (C) wild-type (14028s) and relA::Tn10 spoT (MP342) Salmonella following 24 h treatment with cefotaxime (200 μg/mL) under growth-permissive and growth-restrictive conditions (see Materials and Methods). Error bars represent standard deviations (N = 8 biological replicates). Note log scale of y axis. Wilcoxon rank-sum test between populations at the edge of brackets: ****p<0.0001 and N.S. for no significance.