De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro - PubMed (original) (raw)
De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro
Jees Sebastian et al. Antimicrob Agents Chemother. 2017.
Abstract
Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.
Keywords: Mycobacterium tuberculosis; antibiotic resistance; hydroxyl radical; moxifloxacin; persistence phase cells; rifampicin.
Copyright © 2017 American Society for Microbiology.
Figures
FIG 1
RIF susceptibility profile of M. tuberculosis cells during extended exposure to lethal concentrations of RIF. (A) RIF susceptibility profile of M. tuberculosis cells exposed to 1 μg/ml (10× MBC) RIF for 18 days when plated on RIF-free plates (•; red line). The CFU of the cells on RIF-containing (50× MBC) plates (■; green line) and the concentration of RIF during the course of the experiment (right y axis; ▲; blue line) are shown. (B) RIF susceptibility profile of M. tuberculosis cells exposed to 2 μg/ml (20× MBC) of RIF (•; red line) and concentration of RIF during the course of the experiment (right y axis; ▲; blue line).
FIG 2
Schematic diagram of the RRDR of the rpoB gene and the list of mutations found in M. tuberculosis colonies isolated from the persistent phase of RIF treatment. (A) Genetic map of the RRDR of rpoB, with the location of the mutations found in RIF-resistant mutants obtained from the persistence phase during prolonged exposure of M. tuberculosis cells to RIF. The different-colored stars represent different kinds of mutations, and the number within the parentheses shows the percentage of that kind of mutation. The nature of the nucleotide changes are indicated. (B) The list of RIF-resistant mutations selected from the RIF persistence phase from three independent cultures (R1, R2, and R3) during RIF treatment. The letter “B” indicates the batch of the colony on the plate (batchwise colony formation was observed on the agar plate during the persistent phase, reflecting their growth rate, and the colonies formed at a given time were considered a batch). Five different kinds of mutations detected (Ser450-Leu, Ser450-Trp, His445-Asp, His445-Tyr, and Thr323-Met) in the RRDR of the rpoB gene are indicated.
FIG 3
Luria-Delbruck (modified) experiment showing M. tuberculosis RIF-resistant mutant generation and the response of the bacilli to RIF in the absence and presence of thiourea (TU). RIF-resistant mutant generation in M. tuberculosis cells upon exposure to RIF in the absence (A) and in the presence (B) of TU. Yellow coloration represents cultures exposed to TU. The numbers in the red boxes indicate the numbers of resistant colonies obtained from that particular culture upon plating the cells on 50× MBC RIF plates. (C) RIF susceptibility kinetics of RIF-exposed M. tuberculosis cells in the absence and presence of TU during the LD experiment. The right y axis represents the RIF concentration. Additional cultures (2 to 3) after day 17 of plating were used for RIF assay on day 18, and for this reason results for RIF assay are shown for 18 days, although the experiment was completed on day 17.
FIG 4
Schematic diagram of the mutations in the RRDR of rpoB and list of mutations found in colonies isolated from LD experiment. (A) Genetic map of the mutations in the RRDR of rpoB from mutants isolated from the LD experiment. The colored stars represent different kinds of mutations, and the number within the parentheses shows the percentage of each kind of mutation. (B) List of the mutations at the nucleotide and amino acid levels. Six different mutations were detected (Gln432-Leu, His445-Arg, His445-Asp, His445-Tyr, Ser450-Leu, and Ser450-Trp) in the RRDR of rpoB.
FIG 5
EPR spectra of DMPO-OH adduct in the lysates of RIF-exposed M. tuberculosis cells (n = 2). (A to C) Representative EPR profile of DMPO-OH adduct in the lysates of the mid-log phase (MLP), persister (RIF Per), and TU-treated RIF persister (RIF Per TU) cells. (D) Bar graph representing the levels of DMPO-OH adduct in the cell lysates of RIF-exposed M. tuberculosis cells in the absence and presence of TU and of MLP cells (control). An asterisk indicates a P value of ≤0.05. Statistical significance was calculated using Student's t test.
FIG 6
Flow cytometry analysis of intact HPF-stained M. tuberculosis cells during RIF exposure (n = 3 independent biological samples). Shown are representative density plots of HPF-stained M. tuberculosis cells from the RIF-unexposed mid-log phase (control) (A), RIF-exposed killing phase (B), day 12 of RIF persistence phase (C), and regrowth phase (D). (E) Histogram overlay of the HPF fluorescence of the respective density plots. (F) Bar graph representing the average median HPF fluorescence normalized [HPF median fluo. (Nor.)] to its respective autofluorescence control. (G) The overlay of the HPF fluorescence of the samples from different days of cell exposure to 10× MBC rifampin on the CFU graph (red line) depicted in Fig. 1A, showing high levels of fluorescence (high levels of OH radical generation) in the cells during the RIF persistence phase and reduced levels in the killing phase and regrowth phase. The y axis on the left represents CFU/ml, and the y axis on the right represents normalized median fluorescence of HPF by flow cytometry. An asterisk indicates a P value of ≤0.05. Statistical significance was calculated using the paired t test.
FIG 7
Detection of oxidative stress status in RIF-exposed M. tuberculosis cells using redox-sensitive roGFP2. (A to D) Representative density plots of the flow cytometry profile of the M. tuberculosis-Mrx1-roGFP2 integrant in the different samples showing redox changes in bacteria during RIF exposure. (E and F) Corresponding histogram overlay of fluorescence from Mrx1-roGFP2 in the reduced and oxidized states. Ex., excitation. (G) Bar graph showing the ratiometric changes in the median values of roGFP2 (excitation at 405 and 488 nm) for the same experiment. Double asterisks indicate a P value of ≤0.01. Statistical significance was calculated using the paired t test.
FIG 8
Luria-Delbruck (modified) experiment showing the emergence of RIF-resistant and MXF-resistant mutants from RIF persistence phase M. tuberculosis cells in the absence and presence of TU. (A and B) MXF-resistant mutants formed from the RIF persistence phase M. tuberculosis cells in the absence and presence of TU. The numbers in green boxes indicate the numbers of MXF-resistant mutants obtained from the respective cultures by plating the cells on a 4× MBC MXF plate. (C and D) RIF-resistant mutants formed from the RIF persistence phase M. tuberculosis cells in the absence and presence of TU, respectively. The numbers in the red boxes indicate the numbers of RIF-resistant mutants obtained from the respective culture.
FIG 9
Whole-genome sequencing analysis of four RIF-resistant mutants. (A) Overlaid Circos plot of four RIF-resistant mutants showing the genome-wide mutations with respect to the parental strain. (B) Bar graphs showing the kinds of base substitutions and their percentages in the RIF-resistant mutants, indicating A:T→C:G as the most prevalent mutation (69%), followed by the A:T→T:A (25%) changes. (C) Dot plot representing the position of common mutations in the four RIF-resistant mutants with respect to the parental strain, showing their independent origin. The y axis represents the position of the mutations, and the x axis indicates the number of common mutations. (D) Bar graph showing the number of genes mutated in each of the RIF-resistant mutants. (E and F) Growth curves of the RIF-resistant mutants with respect to that of the parental strain.
References
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