Exposure to sub-lethal doses of aminoglycoside and fluroquinolone reveals physiological adaptation to antibiotics. The growth curve of M. smegmatis challenged with either 1x, ½x, or ¼x MBC99 of streptomycin (A) or with norfloxacin (B). Data points represent the mean of at least three independent replicates ± SD. 1x MBC99 of streptomycin (250 ng/ml) and 1x MBC99 of norfloxacin (2 µg/ml). Bacterial viability at the start was 600,000 CFU/ml, corresponding to OD600nm of 0.0025/ml.

Differential expression analysis of total proteome upon norfloxacin and streptomycin treatment.

Volcano plot analysis of the proteins temporally quantified at 7.5 hr, 15 hr, and 25 hr time points for streptomycin (A) norfloxacin treatment (B). Each dot represents one protein. Fold change cut off ±2 and a t-test significance cut-off of ≤ 0.05 were applied to identify differentially expressed proteins, shown in different colours.

Norfloxacin and streptomycin have common mechanism of action.

Heat map analysis represents the log2 fold changes of proteins involved in (A) DNA damage response and proteostasis network, (B) DNA replication and transcription, (C) translation, and (D) cell cycle and cell division processes, identified from proteomics data. Colours in the heat map depicts the extent of differential expression, where EE demonstrates exclusively expressed, ER demonstrates exclusively repressed/absent in response to antibiotics, and Ab represents proteins that were not identified or quantified with high significance. (E) Scatter plot of the Pearson correlation analysis of common proteins identified upon NOR and STR treatment. Each dot represents a single protein with its log2 fold difference for NOR (x-axis) and STR (y-axis) treatment at t = 15 hour time point. (F) Heat map depicting the log2 fold changes of proteins involved in central carbon metabolism (CCM) of M. smegmatis. Figure (G) illustrates the CCM pathway of M. smegmatis and denotes the expression status of indicated enzymes with colour coding similar to (F).

Norfloxacin and streptomycin generate reactive oxygen species as part of their lethality.

The heat map (A) depicts the log2 fold differences of proteins involved in antioxidant response. EE demonstrates proteins that are exclusively expressed in response to NOR or STR. Temporal (B and C) and dose-dependent (D and G) ratiometric response of M. smegmatis expressing Mrx1-roGFP2 redox biosensor upon STR and NOR treatment at 3 hour. The biosensor fluorescence measurements were recorded at the excitation of 405 nm and 488 nm, for a common emission at 520 nm. Figures demonstrate the effect of 10 mM glutathione on the survival of M. smegmatis treated with lethal dose (10x MBC99) of STR (E) and NOR (H). Figures (F) and (I) presents the effect of bipyridyl on the survival of M. smegmatis treated with lethal dose of STR and NOR, respectively. Data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated by students’ t-test (unpaired), *p < 0.05, **p < 0.01, ***p < 0.001.

Norfloxacin and Streptomycin treatment induces a lethal burst in ATP levels.

Time-course of relative luminescence measurements representing ATP levels in M. smegmatis in response to ¼x (A & D), ½x (B & E) 1x MBC99 (C & F) of streptomycin & norfloxacin, respectively. The right y-axis represents the viability of cells measured just before the measurement of ATP. Bar graph (G) depicting PHR/mCherry ratios (ATP/ADP) of the ATP biosensor in M. smegmatis exposed to increasing concentrations of NOR and STR for 3 hours; 50 micromolar CCCP was used as a control. 1x MBC99 for STR and NOR are 1 µg/ml and 16 µg/ml, respectively for OD600 = 0.8. All data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated by students’ t-test (unpaired), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.

Inhibition of excess ATP production mitigates antibiotic lethality.

Bar graphs reports the dose-dependent reduction in ATP levels upon CCCP co-treatment with STR (A and B) and NOR (D and E). The effect of increasing concentrations of CCCP on survival of M. smegmatis challenged with lethal dose of STR (C) and NOR (F), respectively. Bar graphs represent the relative luminescence measurements indicating ATP levels in wild-type and ΔatpD in response to 1x MBC99 of STR and NOR for 3 hours (G). Time kill curves demonstrate the survival differences of wild-type and ΔatpD in response to 1x MBC99 of STR (H) and NOR (I). All data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated by students’ t-test (unpaired), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.

Norfloxacin and streptomycin induced ROS is insufficient to mediate cell death.

Ratiometric response of M. smegmatis expressing Mrx1-roGFP2 redox biosensor (left y-axis) and the cell viability (right y-axis) in response to STR (A & B) and NOR (C & D) with and without the co-supplementation with CCCP post 3 hours and 6 hours of treatments. Data points represent the mean of at least three independent replicates ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by students’ t-test (unpaired).

Antibiotic induced ATP levels chelates divalent metal ions.

(A) Effect of increasing concentrations of bipyridyl (BP) for 6 hours on the survival of M. smegmatis (0.8 OD/ml). Bar graphs indicate the effect of increasing concentrations of MgSO4 (B), MnCl2 (C), ZnSO4 (D), and FeSO4 (E) on the survival of M. smegmatis (0.8 OD/ml) challenged with lethal dose of the antibiotics for 6 hours. Growth curve (F) showing the effect of 5 mM of FeSO4, MgSO4, MnCl2, ZnSO4 on the survival of M. smegmatis. Data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated using students’ t-test (unpaired). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

13C metabolomics suggests increased metabolic flexibility and bifurcation of TCA cycle flux as bacterial adaptive mechanism.

(A) Percent 13C enrichment in CCM metabolites of M. smegmatis challenged with ¼x MBC99 of antibiotics at 25 hour time point (recovery phase). “M” denotes the molecular mass of the metabolites, where M+1(n) denotes the incorporation of 13C labelled carbon in the metabolites, leading to increase in the mass by that extent. Data for each isotopologue are represented as mean ± SD from independent triplicates. PEP, phosphoenolpyruvate carboxykinase; OXG, alpha-ketoglutarate; Glu, glutamate, Gln, glutamine, OAA, oxaloacetate; Asp, aspartate; GABA, γ-Aminobutyric acid, SSA, succinic semialdehyde. Line graphs represent the ratios of NADH/NAD+ and NADPH/NADP+ with time (B,D) and dose-dependent manner (C,E), respectively, after antibiotic treatments. Data points represent the mean of at least three independent replicates ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 were calculated by student’s t-test (unpaired).

Sub-lethal antibiotic exposure can potentiate development of antibiotic resistance.

Heat map (A) showing the log2 fold differences of protein Eis in response to antibiotic treatment. Figure (B) depicts the mutational frequency of M. smegmatis grown in ¼ x MBC99 of the antibiotics for 25 hours; each dot represents the mutational frequency of one replicate (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001 were calculated by student’s t-test (unpaired). (C) Proposed extended model of antibiotic lethality in Mycobacteria. Figure on left explains the interaction among the central dogma processes and central carbon metabolism (CCM), where, depending on the need for energy, flux through CCM is modulated leading to optimal growth and division; Figure on right explains the effect of inhibition of essential cellular processes by antibiotics, resulting in activation of CCM and respiration, leading to ROS generation and ATP burst. Increase in ATP levels is also contributed by the inhibition of ATP consuming central dogma & cell division processes by antibiotics. Elevated ATP levels in part chelate essential divalent metal ions and can deny them as co-factors for various proteins, eventually leading to cell death. The thickness of arrows indicate the impact of the processes.