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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 11  |  Issue : 2  |  Page : 150-158

Mycobacterium smegmatis strains genetically resistant to moxifloxacin emerge de novo from the moxifloxacin-surviving population containing high levels of superoxide, H2O2, hydroxyl radical, and Fe (II)


Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru, Karnataka, India

Date of Submission11-Feb-2022
Date of Decision11-Apr-2022
Date of Acceptance20-Apr-2022
Date of Web Publication14-Jun-2022
Date of Print Publicaton14-Jun-2022

Correspondence Address:
Parthasarathi Ajitkumar
Department of Microbiology and Cell Biology, Indian Institute of Science, Bengalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_58_22

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  Abstract 


Background: The antibiotic-exposed bacteria often contain the reactive oxygen species (ROS), hydroxyl radical, which inflicts genome-wide mutations, causing the de novo formation of antibiotic-resistant strains. Hydroxyl radical is generated by Fenton reaction of Fe (II) with the ROS, H2O2, which, in turn, is formed by the dismutation of the ROS, superoxide. Therefore, for the emergence of bacterial strains genetically resistant to antibiotics, increased levels of superoxide, H2O2, hydroxyl radical, and Fe (II) should be present in the antibiotic-exposed bacteria. Here, we verified this premise by finding out whether the in vitro cultures of M. smegmatis, exposed to MBC of moxifloxacin for a prolonged duration, contain significantly high levels of superoxide, H2O2, hydroxyl radical, and Fe (II). Methods: Biological triplicate cultures of M. smegmatis, were exposed to MBC of moxifloxacin for 84 h. The colony-forming units (CFUs) of the cultures were determined on moxifloxacin-free and moxifloxacin-containing plates for the entire 84 h at a regular interval of 6 h. The cultures were analyzed at specific time points of killing phase (KP), antibiotic-surviving phase (ASP), and regrowth phase (RGP) for the presence of superoxide, H2O2, hydroxyl radical, and Fe (II) using the ROS- and Fe (II)-detecting fluorescence probes. The experimental cultures were grown in the presence of ROS and Fe (II) quenchers also and determined the levels of fluorescence corresponding to the ROS- and Fe (II)-specific probes. This was performed to establish the specificity of detection of ROS and Fe (II). Biological triplicate cultures, unexposed to moxifloxacin but cultured for 84 h, were used as the control for the measurement of ROS and Fe (II) levels. The CFUs of the cultures were determined on moxifloxacin-free and moxifloxacin-containing plates for the entire 84 h at regular intervals of 6 h. Flow cytometry analyses were performed for the detection and quantitation of the levels of fluorescence of the ROS-and Fe (II)-specific probes. The experimental cultures were grown in the presence of thiourea and bipyridyl as the ROS and Fe (II) quenchers, respectively, for the determination of the levels of fluorescence corresponding to the ROS- and Fe (II)-specific probes. Paired t-test was used to calculate statistical significance (n = 3). Results: The moxifloxacin-exposed cultures, but not the cultures unexposed to moxifloxacin, showed a triphasic response with a KP, ASP, and RGP. The cells in the late KP and ASP contained significantly elevated levels of superoxide, H2O2, hydroxyl radical, and Fe (II). Thus, high levels of the ROS and Fe (II) were found in the small population (in the ASP) of M. smegmatis cells that survived the moxifloxacin-mediated killing. From this moxifloxacin-surviving population (in the ASP), moxifloxacin-resistant genetic resisters emerged de novo at high frequency, regrew, divided, and populated the cultures. The levels of these ROS, Fe (II), and the high moxifloxacin resister generation frequency were quenched in the cultures grown in the presence of the respective ROS and Fe (II) quenchers. The cultures unexposed to moxifloxacin did not show any of these responses, indicating that the whole response was specific to antibiotic exposure. Conclusions: Significantly high levels of superoxide, H2O2, hydroxyl radical, and Fe (II) were generated in the M. smegmatis cultures exposed to moxifloxacin for a prolonged duration. It promoted the de novo emergence of genetic resisters to moxifloxacin at high frequency.

Keywords: Antibiotic resistance, antibiotic-surviving population, hydrogen peroxide, hydroxyl radical, moxifloxacin, Mycobacterium smegmatis, superoxide


How to cite this article:
Paul A, Nair RR, Jakkala K, Ajitkumar P. Mycobacterium smegmatis strains genetically resistant to moxifloxacin emerge de novo from the moxifloxacin-surviving population containing high levels of superoxide, H2O2, hydroxyl radical, and Fe (II). Int J Mycobacteriol 2022;11:150-8

How to cite this URL:
Paul A, Nair RR, Jakkala K, Ajitkumar P. Mycobacterium smegmatis strains genetically resistant to moxifloxacin emerge de novo from the moxifloxacin-surviving population containing high levels of superoxide, H2O2, hydroxyl radical, and Fe (II). Int J Mycobacteriol [serial online] 2022 [cited 2022 Sep 27];11:150-8. Available from: https://www.ijmyco.org/text.asp?2022/11/2/150/347520




  Introduction Top


Genetic resistance to antibiotics is the major impediment in the treatment of bacterial diseases. Bacterial strains, which are genetically resistant to antibiotics, can get selected against both lethal and nonlethal concentrations of antibiotics.[1] Normally, minimum bactericidal concentration (MBC) of antibiotics will kill a substantial proportion of the population. However, a very small proportion of the population remains unkilled and survives. This is a heterogeneous population that contains several different phenotypes of cells that adopt diverse mechanisms for survival against antibiotics.[2] Some of these include classical “Persisters”[3],[4] and persisters that grow and divide slowly like the isoniazid-tolerant persister cells of Mycobacterium smegmatis,[5] cells that are tolerant to antibiotics,[6],[7] and the cells that show phenotypic resistance.[8],[9] A very small proportion of this milieu of cells from this antibiotic-surviving population acquires genetic mutations and gets selected against the antibiotics as resisters.[10]

Most often, the antibiotic-exposed bacteria have been found to contain high levels of the reactive oxygen species (ROS), hydroxyl radical.[11],[12],[13],[14],[15],[16] It inflicts genome-wide mutations, and the mutants carrying nucleotide changes in the antibiotic targets get selected and emerge as strains that are genetically resistant to the antibiotic. For example, resisters and cross-resisters to different antibiotics emerge at high frequency from the in vitro cultures of Escherichia coli and Staphylococcus aureus surviving in the presence of ampicillin.[12],[15],[17],[18] Similarly, we have found the de novo formation of genetic resisters to antibiotics from the population surviving in the presence of MBC of rifampicin/moxifloxacin in the in vitro cultures of the human pathogen, Mycobacterium tuberculosis, and of the saprophyte-cum-opportunistic pathogen,[19],[18],[19],[20],[21] M. smegmatis.[14],[16],[22] These mutant strains carried oxidative stress-induced antibiotic-target-specific mutations, which were qualitatively and positionally identical as in the rifampicin/moxifloxacin-resistant clinical isolates of M. tuberculosis.[23],[24],[25],[26],[27],[28],[29],[30],[31] The nucleotide changes were characteristic of those inflicted by hydroxyl radicals.[32] Genetic resisters, not only to rifampicin or moxifloxacin but also to different antibiotics, also could be selected from the respective antibiotic-surviving population of M. tuberculosis and M. smegmatis.[14],[16] Selection of resisters and cross-resisters from the population surviving against a single antibiotic[12],[14],[15],[16],[17],[18] implied that genome-wide mutations would have been inflicted by the most potent DNA-nonspecific highly reactive ROS, hydroxyl radical.[32]

Superoxide also causes mutations indirectly by way of dismutation to H2O2,[33],[34] which in turn reacts with Fe (II) to form the mutagenic ROS, hydroxyl radical.[35] Most of the studies on the bacteria of diverse genera exposed to antibiotics had shown the formation of only either superoxide, or H2O2, or hydroxyl radical.[12],[13],[14],[15],[16],[17],[18],[22] Since there is a chemically necessary sequential formation of superoxide-to-H2O2-to-hydroxyl radical (in the Fe (II)'s presence), the formation of high levels of one of these three ROS in the bacteria exposed to antibiotics alluded to the formation of all the three ROS, and high levels of Fe (II). Confirming this premise, here, we show evidence for the presence of significantly high levels of superoxide, H2O2, hydroxyl radical, and Fe (II) in the small population of M. smegmatis cultures surviving against MBC of moxifloxacin and the emergence of moxifloxacin resisters from this population.


  Methods Top


Bacterial strains and culture conditions

M. smegmatis mc2155[37] was cultured in Middlebrook 7H9 broth (BD Biosciences) containing 0.2% glycerol (Fisher Scientific) and 0.05% Tween 80 (Sigma) at 170 rpm and 37°C. The colony-forming units (CFU) were determined from the Middlebrook 7H11 agar plates (BD Biosciences) of the cultures incubated at 37°C for 3–4 days. The antibiotic resister CFUs were determined on Petri plates containing 3.75 × MBC of moxifloxacin. The antibiotic resisters' CFU was divided by the total population CFU to calculate the resister generation frequency of M. smegmatis cultures against moxifloxacin. In parallel, M. smegmatis cultures were grown without moxifloxacin for 84 h and plated on moxifloxacin-free and moxifloxacin-containing plates for the time points and intervals used for the moxifloxacin-exposed cultures. The CFU profile and the very low frequency of resister generation from the moxifloxacin-unexposed cultures were the control samples used to show the specific effect of the exposure to moxifloxacin.

Determination of the colony-forming units profile in the cultures with thiourea and diphenyleneiodonium chloride

M. smegmatis cultures (108 cells/ml) were exposed to moxifloxacin (0.5 μg/ml; 3.75× MBC) for 84 h, with and without the ROS scavenger, thiourea (TU)[38] (TU, Sigma), and the total population's and the antibiotic resisters' CFUs were determined by plating at frequent intervals on moxifloxacin-free and moxifloxacin-containing plates, as described.[16] Different concentrations of TU (0.25 mM to 50 mM and 0.05 mM to 4 mM) were used to determine the nonlethal concentration of TU. The nontoxic concentration of TU in the presence of moxifloxacin was that for which the TU-exposed and TU-unexposed cultures' CFU profiles were comparable till the end of the killing phase (KP). The moxifloxacin concentrations of 0.375 ×, 3.75 × and 37.5 × MBC were used to find out the effect of different concentrations of moxifloxacin on the CFU profile. Diphenyleneiodonium chloride (DPI, Sigma), which is the inhibitor of nicotinamide adenine dinucleotide hydrogen (NADH) oxidase that produces superoxide,[39] was added at 100 nM (final concentration) to 108 cells per ml cultures, as reported.[40] Moxifloxacin was added to the DPI-exposed cultures at the mid-log phase (MLP) stage of the culture and plated at 6-h intervals throughout the exposure (84 h).

Determination of the levels of hydroxyl radical, H2O2, superoxide, and Fe (II)

Moxifloxacin was added to M. smegmatis MLP cultures, with and without TU/DPI, and grown for 84 h. The antibiotic-unexposed cultures were also grown in an identical manner. The levels of hydroxyl radical, H2O2, Fe (II), and superoxide were determined in these cultures. While hydroxyl radical levels were determined using 5 μM 3'-(p-hydroxyphenyl) fluorescein (HPF; Thermo Fisher Scientific),[41] the H2O2 levels were assayed using Amplex Red (AR).[42],[43] Superoxide levels were detected using 0.5 μM CellRox Green (CRG; Thermo Fisher Scientific).[44],[45] The levels of Fe (II) were determined in the moxifloxacin-exposed and -unexposed M. smegmatis cultures using the Fe (II) detecting fluorochrome, FeRhoNox™-1 (10 μM final concentration).[46] The detailed protocol of staining and flow cytometry analysis for the determination of the ROS levels is given under “Supplemental Materials and Methods” in the Supplemental Material.


  Results Top


The colony-forming units of Mycobacterium smegmatis cultures against moxifloxacin shows three phases

Three clear phases of CFU were observed from the antibiotic-free plates for the M. smegmatis MLP cultures exposed to moxifloxacin (0.5 μg/ml; ~3.75 × MBC) for 84 h [[Figure 1]a, blue line]. This profile was like the response of M. tuberculosis and M. smegmatis cultures against moxifloxacin reported by us earlier.[14],[16],[22] The first phase was the KP, with severe reduction in the CFU. The second phase, called antibiotic-surviving phase (ASP), showed much less change in the CFU. The third phase was the regrowth phase (RGP), showing a steady increase in CFU. The steady increase in the CFU in the RGP indicated proliferation of the cells that were surviving in the presence of the antibiotic. The concentration of moxifloxacin in the culture medium was found to remain consistently and reproducibly at the MBC levels without any appreciable change in 84 h during our earlier studies.[16],[22] Hence, the concentration of moxifloxacin in these cultures was not measured in this study. Nevertheless, this ruled out the possibility of any change in the antibiotic concentration as a probable reason for the proliferation of the cells in the RGP. A similar triphasic profile was observed for CFU against different concentrations (multiples or fractions of MBC) of moxifloxacin, indicating the robustness of the bacterial response over different concentrations of the antibiotic [Figure S1].
Figure 1: CFU profile of Mycobacterium smegmatis cultures during prolonged exposure to moxifloxacin and under moxifloxacin-unexposed condition (a). Triphasic response of Mycobacterium smegmatis to 0.5 μg/ml (3.75 × MBC) MXF for 84 h (b). CFU profile of Mycobacterium smegmatis cultures unexposed to moxifloxacin for 84 h. CFU/ml on antibiotic-free plates (blue line) and antibiotic-containing plates (green line) (n = 3 biological replicates in each case). KP: Killing phase, ASP: Antibiotic-surviving phase, RGP: Regrowth phase, CFU: Colony-forming units, MXF: Moxifloxacin

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The CFUs on the antibiotic plates revealed the emergence of antibiotic resisters, which started emerging toward the end of KP (~18 h) [lFigure 1]a, green line]. Post KP, the CFUs went on increasing steadily in the subsequent phases (ASP and RGP). The best-fit linear plot of the CFUs indicated that the first antibiotic resister clone arose at ~16–17 h toward the end of KP [Figure S2]. It indicated that the resister clone did not emerge from preexisting natural mutant, if any, in the culture but came up de novo. The CFU profiles from the moxifloxacin-free and moxifloxacin-containing plates showed statistical significance between the CFU for any selected time point with the CFU of another time point [Figure S3a and b]. The ASP's and RGP's CFU profiles showed differences between the moxifloxacin (±) plates. It indicated that these phases might contain different proportions of the cells that could become moxifloxacin-resistant, as reported,[47] or continue to remain as moxifloxacin tolerant, both contributing to the CFU rise in the RGP when plated in the moxifloxacin (-) plates. Contrary to these features of the moxifloxacin-exposed cultures, the moxifloxacin-unexposed cultures did not show either triphasic response or emergence of antibiotic resister clones [Figure 1]b. These observations indicated that the three phases of CFU and the emergence of moxifloxacin resisters occurred only in response to antibiotic exposure.



High levels of hydroxyl radical in the moxifloxacin-exposed cultures

Genome-wide mutagenesis occurs in antibiotic-exposed bacteria, and the antibiotic target-specific mutants get selected against the antibiotic.[12],[13],[14],[15],[16],[17],[18],[22] Such extensive mutagenesis is inflicted by the highly reactive DNA-nonspecific ROS, hydroxyl radical.[32] Thus, the CFUs on the moxifloxacin plates indicated the emergence of antibiotic resisters probably through the generation of ROS. The hydroxyl radical detecting HPF's fluorescence[41] was observed in the moxifloxacin-exposed M. smegmatis cultures [[Figure 2]a and [Figure S4] for statistical significance]. The hydroxyl radical generation peaked during the ~30–40 h of the exposure. Significant quenching of the HPF fluorescence by the nontoxic concentration of TU (TU; the ROS scavenger)[48],[49] [Figure S5], throughout the exposure and at specific time points in the late KP/early ASP (30 h), mid-ASP (36 h), and late ASP (54 h), confirmed hydroxyl radical detection [Figure 2]a and [Figure 2]b.
Figure 2: Formation of hydroxyl radical, H2O2, Fe (II), and superoxide in Mycobacterium smegmatis cultures upon prolonged exposure to moxifloxacin in the absence and presence of TU. (a, c, and h) Line graphs showing the median fluorescence of HPF (for hydroxyl radical), Amplex Red (for H2O2), and CRG (for superoxide), respectively. (b, d, and i) Bar graphs of the quantitation of median fluorescence of HPF, Amplex Red, and CRG for the 30 h, 36 h, or 54 h of the ASP. (e) Line graph showing the Fe (II) levels (μM/cell) determined using FeRhoNox-1 assay. (f) Quantitation of Fe (II) levels for the 30 h and 36 h ASP cells. (g) Quantitation of Fe (II) levels for the 30 h and 36 h (ASP cells) in the presence of 100 μM bipyridyl (iron chelator). The black box shows the peak period of the levels of the fluorescence (due to ROS levels) of HPF, Amplex Red, FeRhoNox-1 (as Fe [II] concentration per cell), and CRG, corresponding to the CFU/ml. Statistical significance was calculated using paired t-test (n = 3 biological replicates in each case). ROS: Reactive oxygen species, HPF: 3'-(p-Hydroxyphenyl) fluorescein CFU: Colony-forming units. CRG: CellRox Green. ASP: Antibiotic-surviving phase, TU: Thiourea, MXF: Moxifloxacin

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Elevated H2O2 and Fe (II) levels in the moxifloxacin-exposed bacteria

Fenton reaction between H2O2,[35] the dismutation product from superoxide,[33],[34] and Fe (II) that gets released from the ROS-damaged 4Fe-4S proteins,[36],[50] generates hydroxyl radical. Thus, the generation of significant levels of hydroxyl radical indicated the presence of H2O2. The moxifloxacin-exposed cultures contained significantly elevated levels of H2O2, as quantitated using AR assay[42],[43] [Figure 2]c and [Figure 2]d. The H2O2 formation peaked during the ~30–40 h of the exposure [[Figure 2]c and [Figure S6] for statistical significance]. The AR fluorescence quenching by TU throughout the exposure, especially at the H2O2 peak periods (30 h and 36 h), confirmed the presence of H2O2 [Figure 2]c and [Figure 2]d.



Damage to 4Fe-4S proteins/enzymes, resulting in the release of Fe (II), is known to be caused by hydroxyl radical and H2O2.[36],[50] Hence, the elevated levels of these ROS in the ASP of the moxifloxacin-exposed cultures implied the presence of elevated levels of Fe (II). The peak levels of Fe (II) were found to be in the ~28–44 h period [Figure 2]e and [Figure 2]f. The Fe (II) levels were significantly reduced by the Fe (II) scavenger, bipyridyl, indicating the specificity of the detection of Fe (II) [Figure 2]e. TU also significantly reduced the levels of Fe (II) throughout the exposure, especially at the Fe (II) peak periods (30 h and 36 h) [[Figure 2]g; [Figure S7] for statistical significance]. The decrease of H2O2 levels by TU would have reduced the damage to 4Fe-4S proteins and hence of Fe (II) levels. Thus, the peak levels of Fe (II) at 30 h and 36 h (in the peak ~28–42 h window) overlapped with the formation of the two ROS, hydroxyl radical and H2O2, the levels of which peaked in the ~30–40 h window.



Presence of elevated levels of superoxide in the moxifloxacin-exposed cultures

Since superoxide dismutation generates H2O2,[33],[34] the presence of high levels of H2O2 indicated the formation of elevated levels of superoxide. The superoxide detecting CRG fluorescence was found peaking in the ~20–30 h window during the exposure of the cultures to moxifloxacin [[Figure 2]h and [Figure S8]a, [Figure S8]b, for statistical significance]. The superoxide peaked during early ASP/late KP (30 h) [Figure S9] and [Figure 1]. TU reduced the levels of CRG fluorescence,[38] indicating the quenching of superoxide levels [Figure 2]h, [Figure 2]i and [Figure S9]. An overlap was found between the periods of the peak presence of superoxide (~20–30 h) and H2O2 (~30–40 h). Thus, the three ROS (superoxide, H2O2, and hydroxyl radical) were formed in overlapping periods, as would be expected due to the chemically necessary sequential formation of H2O2 from superoxide and hydroxyl radical from H2O2 and Fe (II). The frequency of moxifloxacin resister generation moxifloxacin was 10−3, which was ~5-log10-fold higher than the experimentally found and earlier reported natural mutation frequency of 10−8 of the MLP culture of M. smegmatis.[51],[52]



Superoxide inhibition reduced H2O2, Fe (II), and hydroxyl radical levels

Due to the sequential formation of superoxide-to-H2O2-to-hydroxyl radical in the presence of Fe (II), inhibition of superoxide formation should reduce the levels of H2O2, hydroxyl radical, and Fe (II). Further, since one of the sources of superoxide production in mycobacteria is NADH oxidase,[39] the presence of DPI (DPI; 100 nM for 108 cells/ml) that inhibits NADH oxidase activity[39] should reduce the levels of superoxide, H2O2, hydroxyl radical, and Fe (II). In accordance with this premise, the DPI-exposed moxifloxacin cultures contained significantly reduced levels of the fluorescence of CRG, AR, FeRhoNox1, and HPF that detect superoxide, H2O2, Fe (II), and hydroxyl radical, respectively [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e, [Figure 3]f, [Figure 3]g, [Figure 3]h, respectively, and [Figure S10], [Figure S11], [Figure S12], [Figure S13], respectively, for statistical significance]. These experiments confirmed that it was the production of superoxide (at least in part by NADH oxidase) at significantly high levels that had triggered the serial synthesis of H2O2 and then hydroxyl radical in the presence of Fe (II). These molecular processes were depicted in a model [Figure 4].
Figure 3: Generation of superoxide, H2O2, and hydroxyl radical, and Fe (II) in the Mycobacterium smegmatis cultures exposed to moxifloxacin for prolonged duration in the presence and absence of DPI. (a, c, and g) Line graphs showing the median fluorescence of CRG, Amplex Red, and HPF, respectively. (b, d, and h) Bar graphs of the quantitation of fluorescence of CRG, Amplex Red, and HPF for the 30 h and 36 h of the ASP during the exposure to moxifloxacin. (e) Line graph showing the Fe (II) levels (μM/cell) determined using FeRhoNox-1 assay. (f) Quantitation of Fe (II) levels for the 30 h and 36 h (ASP cells). The black box shows the peak period of the fluorescence levels corresponding to the CFU/ml. Statistical significance was calculated using paired t-test (n = 3 biological replicates in each case). CRG: CellRox Green, HPF: 3'-(p-Hydroxyphenyl) Fluorescein, CFU: Colony-forming units, DPI: Diphenyleneiodonium chloride, ASP: Antibiotic-surviving phase, Msm: M. smegmatis, MXF: Moxifloxacin, AR: Amplex red, FRN: FeRhoNox-1

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Figure 4: Model depicting the sequential and overlapping generation of superoxide, H2O2, and hydroxyl radical, and Fe (II) upon prolonged exposure of Mycobacterium smegmatis to moxifloxacin. The CFU profile and the sequential generation of three ROS upon prolonged exposure of Mycobacterium smegmatis cultures to moxifloxacin. Formation of superoxide, H2O2, and Fe (II), lead to the production of hydroxyl radical that inflict moxifloxacin target-specific mutations, causing the emergence of strains genetically resistant to moxifloxacin. The presence of a ROS scavenger, TU, or NADH oxidase (Nox) inhibitor, DPI significantly reduces the levels of ROS and the number of resister cells emerging from the population surviving in the presence of MBC of moxifloxacin. The images were drawn not to scale. MBC: Minimum bactericidal concentration, KP: Killing phase, ASP: Antibiotic-surviving phase, RGP: Regrowth phase, TU: Thiourea, NADH: Nicotinamide adenine dinucleotide hydrogen, DPI: Diphenyleneiodonium chloride, ROS: Reactive oxygen species

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  Discussion Top


Antibiotic-surviving population

Earlier studies on bacterial antibiotic response reported the emergence of antibiotic resisters in patients,[53] in macrophages,[54] in animal models,[55] and from the ASP in in vitro cultures.[14],[16],[56] Thus, whether it be in the in vitro cultures, in animal models, or in patients, a small population of the antibiotic-exposed bacteria survived from which emerged genetic resisters to the antibiotic. We and others had reported the emergence of resisters of E. coli and the ESKAPE pathogen, S. aureus, against several antibiotics having diverse targets.[12],[15],[57],[58] We had further shown that the antibiotic resisters of E. coli and S. aureus emerged from the respective ASPs.[58] These antibiotic-surviving populations, which were formed against MBC of antibiotics, showed a robust response, starting with a KP, and valid for different concentrations of the antibiotics.[58] In fact, genetic resisters to antibiotics have been found to emerge against different concentrations of antibiotics.[1],[59] Thus, the antibiotic survival of M. smegmatis to moxifloxacin, like the one shown by E. coli and S. aureus, stands as yet another example of the emergence of antibiotic-resistant strains from the bacterial populations surviving against antibiotics by diverse means. Since bacteria exhibit different modes of response to different antibiotic concentrations,[60],[61],[62] we used moxifloxacin at MBC levels to maintain the antibiotic concentration always high, with negligible changes in its concentration during the exposure, reported earlier.[16] We did not study antibiotic response at single-cell level as antibiotic-exposed single cells in isolation respond differently to antibiotics, unlike the single cells in a population, depending on the antibiotic concentration and exposure duration.[63]

Formation of three reactive oxygen species in the moxifloxacin-exposed Mycobacterium smegmatis cultures

The genetic resisters to moxifloxacin were formed from the cells in the ASP containing the three ROS and Fe (II) at significantly elevated levels. Almost all the earlier studies on the antibiotic-exposed mycobacteria,[13],[14],[16] and other bacteria[12],[15],[17],[18] showed the generation of mostly hydroxyl radical. Besides this ROS, DNA damage has been reported to be caused by superoxide also,[36] which must have been through the formation of hydroxyl radical. This is quite possible as activation of molecular oxygen could result in the formation of mostly superoxide, from which H2O2 and hydroxyl radical could be formed.[64] The DNA-nonspecific, highly reactive hydroxyl radical, even at very small levels, would inflict mutations facilitating resister generation. Advancing these observations, here, we showed the temporally overlapping formation of superoxide, H2O2, and hydroxyl radical in the moxifloxacin-exposed M. smegmatis cultures.



Besides hydroxyl radical, another ROS, peroxynitrite (ONOO¯), which is a product of superoxide and nitric oxide,[65],[66] also inflicts DNA damage and facilitates antibiotic resister formation.[11],[67],[68] However, since nitric oxide production requires nitrite,[69] which is absent in the Middlebrook 7H9 growth medium, peroxynitrite would not have formed in the moxifloxacin-exposed M. smegmatis cultures. Contrary to these observations on the formation of ROS upon antibiotic exposure of bacteria of diverse genera, including M. smegmatis, high levels of ROS were not generated in the M. smegmatis cultures exposed to kanamycin, streptomycin, or norfloxacin.[45] Thus, bacterial exposure to antibiotics necessarily needs not always generate high levels of ROS. Further, a recent report showed that the persisters of M. smegmatis against ciprofloxacin, and to a lesser extent against rifampicin, contained sustained high levels of the error-prone DNA polymerase DnaE2, which was suggested to have caused mutagenesis across the genome facilitating antibiotic resister generation.[70] However, it needs to be resolved as to how the cells manage to tolerate and survive despite mutation infliction by an ROS and an enzyme. Nevertheless, bacteria of diverse genera of different habitats seemed to have shown a comparable response to MBC of different types of antibiotics upon prolonged exposure.

Conclusion

A small proportion of the M. smegmatis population surviving in the presence of MBC of moxifloxacin contained three ROS, namely superoxide, H2O2, and hydroxyl radical, and Fe (II) at significantly elevated levels. This population formed the source of genetic resisters to moxifloxacin emerging at significantly high frequency. This phenomenon has clinical relevance in the emergence of antibiotic resisters against anti-tuberculosis drugs.

Ethical clearance

This article does not contain any experiments with human participants or animal models or with any pathogenic bacteria performed by any of the authors. Hence ethical clearance was not required.

Acknowledgments

With highest respects and regards, P.A. dedicates this work as a tribute to T. Ramakrishnan (late), who led the pioneering, fundamental, and foundation-laying work on the biochemistry and molecular biology of Mycobacterium tuberculosis at the Indian Institute of Science, Bangalore, India. We acknowledge Soumalya Seal for supporting in computational analysis of the linear plot, DBT-supported FACS facility for providing FACS facility, DST-FIST for providing infrastructure and equipment facility, UGC-Centre for Advanced Study for providing infrastructure and equipment facility, and ICMR-Centre for Advanced Study for providing equipment facility.

Financial support and sponsorship

This study was financially supported by DBT-IISc Partnership Programme (2012–2020) and Indian Institute of Science.

Conflicts of interest

There are no conflicts of interest.

Patient declaration of consent statement

The study reported in the work was not performed on any human patients. Hence, patients' declaration of consent was not required.

Data availability statement

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Data availability within the article and supplementary materials

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  Supplemental Materials Top



  Supplementary Materials And Methods Top


Determination of hydroxyl radical and superoxide levels using flow cytometry

Mycobacterium smegmatis mid-log phase (MLP) cultures were grown in the presence of nontoxic concentrations of thiourea/diphenyleneiodonium chloride (TU/DPI) containing medium till MLP. Then, moxifloxacin was added, and culturing continued for 84 h. The nontoxic concentrations of TU/DPI were determined through standardization as mentioned in the Materials and Methods in the main text. At each time point, from 4 × 600 μl aliquots collected, 100 μl each was used for CFU determination. The 500 μl remaining from each sample was used for flow cytometry. Among the four aliquots of 500 μl each, two aliquots were used unstained for checking autofluorescence. The remaining two aliquots were stained with 5 μM 3′-(p-Hydroxyphenyl) fluorescein (HPF; Thermo Fisher Scientific) and 0.5 μM CellRox Green (CRG; Thermo Fisher Scientific), for determining respectively hydroxyl radical and superoxide levels.[1],[2],[3] The period of incubations of the HPF or CRG stained cells was 30 min and 2 h, respectively, with mixing in the dark at 37°C. The TU/DPI-exposed samples were also processed similarly. The respective unstained autofluorescence samples and the antibiotic-unexposed cultures were also processed in an identical manner. Flow cytometry analyses were performed using ~ 10000 cells for gating with a solid-state laser (488 nm) and a GFP emission filter (527/32 nm) in FACS Verse (Becton Dickinson). The PMT voltage for the FACSuite cytometer was 208 (FSC) and 333 (SSC). The instrument calibration was performed using tracking beads (CS&T, Becton Dickinson). The FACSuite software was used to process and analyze the flow cytometry data. Paired t-test (n = 3 biological triplicates each time) was used for determining the statistical significance of HPF/CRG median fluorescence.

H2O2 levels' determination using flow cytometry

The of H2O2 levels' determination, in the (+/−) TU/DPI moxifloxacin-exposed cultures, was performed using Amplex Red assay,[4],[5] as mentioned for the analysis of HPF and CRG fluorescence. Since the moxifloxacin-exposed cultures, which were grown in the (−) TU/DPI would have higher cell density, they were diluted ~10-fold at all the time points after KP, for the quantitative matching of the cell numbers with the (+) TU/DPI moxifloxacin-exposed cultures. The cultures (1100 μl) were collected from each time point, centrifuged at ~5000 × g, at 4°C, 10 min. The cells were resuspended in the same volume of the growth medium. Each of these samples was divided at 1:5:5 v/v/v ratio into three tubes. The CFU of each sample was determined using the entire 100 μl sample in the first tube. The 500 μl sample in one of the remaining two tubes was sonicated for 1 s pulse at a time at 1 s pulse interval at 30% amplitude for a total duration of 2 min. The sonicate was centrifuged (~8800 × g) for 15 min at 4°C for filtration using a spin filter (3 kDa cut-off; Amicon). The remaining 500 μl sample was sonicated, serially diluted and plated, to determine the live cells % after sonication (sonication efficiency).

Two different standard sets of concentrations (0.1–10 μM) of H2O2 (Invitrogen) were prepared using a growth medium. From these standards, technical triplicates of 3 × 50 μl were added into a black multi-well plate. Fifty microliters of the Mycobacterium smegmatis cell lysates was also added into different wells. Fifty microliter each of the H2O2-detecting dye (Amplex Red-HRP at the final concentration: 100 μM Amplex Red and 0.2 U/ml HRP (Invitrogen) was used for the fluorescence detection by incubation for 30 min at 25°C in the dark. The fluorescence values were recorded at Ex530/Em590 nm in a microplate reader (Tecan Infinite 200 Pro). The i-control software was used to analyze the values. The H2O2 concentration was determined per cell. Paired t-test was used to calculate statistical significance.

FeRhoNox™-1 assay to determine Fe (II) levels

The processing of the sample was identical to that described above except that 100 mM sodium acetate buffer (pH 5.2) (1100 μl), instead of a growth medium, was used for the resuspension of the samples from an equivalent volume of the cultures. A parallel set of the samples was exposed to the Fe (II) ion chelator, 2,2'-bipyridyl (100 μM), to determine the specificity of Fe (II) detection. The ferrous ammonium sulfate (0.25 μM to 16 μM) in 100 mM sodium acetate buffer (pH 5.2) was used as the standards for Fe (II) estimation. The samples (200 μl each), followed by 10 μM final concentration FeRhoNoxTM-1,[6],[7] were added into 96-well plates (Costar, transparent). The incubation was for 60 min at 37°C. The fluorescence values were recorded at Ex540/Em575 nm in a microplate reader (Tecan Infinite 200 Pro). The i-control software was used for the analysis of the values. The Fe (II) levels were determined per cell using total CFU for the normalization of the fluorescence values, exactly for the determination of HPF and CRG fluorescence. Paired t-test was used to calculate the statistical significance.


  Supplementary References Top


  1. Setsukinai K, Urano Y, Kakinuma K, Majima HJ, Nagano T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 2003;278:3170-5.
  2. Mukherjee P, Sureka K, Datta P, Hossain T, Barik S, Das KP, et al. Novel role of Wag31 in protection of mycobacteria under oxidative stress. Mol Microbiol 2009;73:103-19.
  3. McBee ME, Chionh YH, Sharaf ML, Ho P, Cai MW, Dedon PC. Production of superoxide in bacteria is stress- and cell state-dependent: A gating-optimized flow cytometry method that minimizes ROS measurement artifacts with fluorescent dyes. Front Microbiol 2017;8:459.
  4. Votyakova TV, Reynolds IJ. Detection of hydrogen peroxide with Amplex Red: Interference by NADH and reduced glutathione auto-oxidation. Arch Biochem Biophys 2004;431:138-44.
  5. Mishin V, Gray JP, Heck DE, Laskin DL, Laskin JD. Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes. Free Radic Biol Med 2010;48:1485-91.
  6. Hirayama T, Okuda K, Nagasawa H. A highly selective turn-on fluorescent probe for iron (II) to visualize labile iron in living cells. Chem Sci 2013;4:1250-6.
  7. Vijay S, Nair RR, Sharan D, Jakkala K, Mukkayyan N, Swaminath S, et al. Mycobacterial cultures contain cell size and density specific sub-populations of cells with significant differential susceptibility to antibiotics, oxidative and nitrite stress. Front Microbiol 2017;8:463.




 
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