1887

Abstract

Bacterial antibiotic persister cells tolerate lethal concentrations of antibiotics but emerge as the antibiotic-sensitive population upon antibiotics withdrawal. However, the possibility of antibiotic-resistant genetic mutants emerging from the antibiotic persister population in the continued exposure to microbicidal concentrations of antibiotics needed investigation. We explored this possibility using the fast-growing as a model organism for biology, as it is known to incur antibiotic-resistant mutations identical to and at identical target positions as found in the clinical isolates of . Here we report that the moxifloxacin (MXF) persister population generate significantly elevated levels of hydroxyl radical. Hydroxyl radical being a sequence-non-specific mutagen, resulted in the emergence of moxifloxacin-resistant genetic mutants at 8-log higher frequency from the persister population. Luria–Delbruck experiment (in modified format) confirmed that MXF-resistant mutants emerged from the persister population and were not pre-existent. The nature of the mutations in the quinolone resistance determining region indicated that they were generated due to oxidative stress. These mutations were identical to and at identical positions as found in the clinical isolates of MXF-resistant . Interestingly, from the MXF persister population, resisters to microbicidal concentrations of ethambutol and isoniazid could also be selected. These observations implied that the significantly high levels of hydroxyl radical might have generated genome-wide mutations, creating a pool of mutants in the MXF persister population, facilitating selection of resisters to other antibiotics also. These findings may be of clinical relevance to the emergence of drug-resistant strains during prolonged tuberculosis treatment regimen with high doses of multiple antibiotics.

Funding
This study was supported by the:
  • Department of Biotechnology and Indian Institute of Science (Award DBT-IISc Partnership programme)
    • Principle Award Recipient: Parthasarathi Ajitkumar
  • Department of Biotechnology, Ministry of Science and Technology, Govt of India (Award BT-PR23219-MED-29-1184-2017)
    • Principle Award Recipient: Parthasarathi Ajitkumar
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2019-11-20
2021-10-28
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References

  1. Bigger J. Treatment of Staphylococcal infections with penicillin by intermittent sterilisation. The Lancet 1944; 244:497–500 [View Article]
    [Google Scholar]
  2. Beck F, Yegian D. A study of the tubercle Bacillus in resected pulmonary lesions. Am Rev Tuberc 1952; 66:44–51 [View Article]
    [Google Scholar]
  3. Medlar EM, Bernstein S, Steward DM. A bacteriologic study of resected tuberculous lesions. Am Rev Tuberc 1952; 66:36–43 [View Article]
    [Google Scholar]
  4. Opie EL, Aronson JD. Tubercle bacilli in latent tuberculosis lesions and in lung tissue without tuberculous lesions. Arch Pathol Lab Med 1927; 4:1–21
    [Google Scholar]
  5. Robertson HE. The persistence of tuberculous infections. Am J Pathol 1933; 9:711–718
    [Google Scholar]
  6. Jindani A, Aber VR, Edwards EA, Mitchison DA. The early bactericidal activity of drugs in patients with pulmonary tuberculosis. Am Rev Respir Dis 1980; 121:939–949 [View Article]
    [Google Scholar]
  7. Khomenko AG. The variability of Mycobacterium tuberculosis in patients with cavitary pulmonary tuberculosis in the course of chemotherapy. Tubercle 1987; 68:243–253 [View Article]
    [Google Scholar]
  8. McCune RM, Feldmann FM, Lambert HP, McDermott W. Microbial persistence. I. The capacity of tubercle bacilli to survive sterilization in mouse tissues. J Exp Med 1966; 123:445–468 [View Article]
    [Google Scholar]
  9. Hoff DR, Ryan GJ, Driver ER, Ssemakulu CC, De Groote MA et al. Location of intra- and extracellular M. tuberculosis populations in lungs of mice and guinea pigs during disease progression and after drug treatment. PLoS One 2011; 6:e17550 [View Article]
    [Google Scholar]
  10. Hu Y, Mangan JA, Dhillon J, Sole KM, Mitchison DA et al. Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis induced by exposure to rifampin or pyrazinamide. J Bacteriol 2000; 182:6358–6365 [View Article]
    [Google Scholar]
  11. Keren I, Minami S, Rubin E, Lewis K. Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio 2011; 2:e00100–00111 [View Article]
    [Google Scholar]
  12. Borrell S, Teo Y, Giardina F, Streicher EM, Klopper M et al. Epistasis between antibiotic resistance mutations drives the evolution of extensively drug-resistant tuberculosis. Evol Med Public Health 2013; 2013:65–74 [View Article]
    [Google Scholar]
  13. Grant SS, Kaufmann BB, Chand NS, Haseley N, Hung DT. Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals. Proc Natl Acad Sci U S A 2012; 109:12147–12152 [View Article]
    [Google Scholar]
  14. Mor N, Simon B, Mezo N, Heifets L. Comparison of activities of rifapentine and rifampin against Mycobacterium tuberculosis residing in human macrophages. Antimicrob Agents Chemother 1995; 39:2073–2077 [View Article]
    [Google Scholar]
  15. Dickinson JM, Aber VR, Allen BW, Ellard GA, Mitchison DA. Assay of rifampicin in serum. J Clin Pathol 1974; 27:457–462 [View Article]
    [Google Scholar]
  16. Bhaskar A, Chawla M, Mehta M, Parikh P, Chandra P et al. Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection. PLoS Pathog 2014; 10:e1003902 [View Article]
    [Google Scholar]
  17. Roy S, Narayana Y, Balaji KN, Ajitkumar P. Highly fluorescent GFPm 2+-based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages. Microb Biotechnol 2012; 5:98–105 [View Article]
    [Google Scholar]
  18. Nair RR, Sharan D, Sebastian J, Swaminath S, Ajitkumar P. Heterogeneity of ROS levels in antibiotic-exposed mycobacterial subpopulations confers differential susceptibility. Microbiology 2019; 165:668–682 [View Article]
    [Google Scholar]
  19. Sambrook J, Russell DW. Molecular Cloning: a Laboratory Manual, 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001
    [Google Scholar]
  20. Tadolini B, Cabrini L. On the mechanism of OH. scavenger action. Biochem J 1988; 253:931–932 [View Article]
    [Google Scholar]
  21. Piccaro G, Pietraforte D, Giannoni F, Mustazzolu A, Fattorini L. Rifampin induces hydroxyl radical formation in Mycobacterium tuberculosis . Antimicrob Agents Chemother 2014; 58:7527–7533 [View Article]
    [Google Scholar]
  22. Setsukinai K-ichi, 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; 278:3170–3175 [View Article]
    [Google Scholar]
  23. Mukherjee P, Sureka K, Datta P, Hossain T, Barik S et al. Novel role of Wag31 in protection of mycobacteria under oxidative stress. Mol Microbiol 2009; 73:103–119 [View Article]
    [Google Scholar]
  24. Luria SE, Delbrück M. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 1943; 28:491–511
    [Google Scholar]
  25. Heym B, Cole ST. Isolation and characterization of isoniazid-resistant mutants of Mycobacterium smegmatis and M. aurum . Res Microbiol 1992; 143:721–730 [View Article]
    [Google Scholar]
  26. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007; 130:797–810 [View Article]
    [Google Scholar]
  27. Wang X, Zhao X. Contribution of oxidative damage to antimicrobial lethality. Antimicrob Agents Chemother 2009; 53:1395–1402 [View Article]
    [Google Scholar]
  28. Liu Y, Liu X, Qu Y, Wang X, Li L et al. Inhibitors of reactive oxygen species accumulation delay and/or reduce the lethality of several antistaphylococcal agents. Antimicrob Agents Chemother 2012; 56:6048–6050 [View Article]
    [Google Scholar]
  29. Sampson TR, Liu X, Schroeder MR, Kraft CS, Burd EM et al. Rapid killing of Acinetobacter baumannii by polymyxins is mediated by a hydroxyl radical death pathway. Antimicrob Agents Chemother 2012; 56:5642–5649 [View Article]
    [Google Scholar]
  30. Jensen Peter Ø, Briales A, Brochmann RP, Wang H, Kragh KN et al. Formation of hydroxyl radicals contributes to the bactericidal activity of ciprofloxacin against Pseudomonas aeruginosa biofilms. Pathog Dis 2014; 70:440–443 [View Article]
    [Google Scholar]
  31. Kotova VI, Mironov AS, Zavigel'skiĭ GB. [Role of reactive oxygen species in the bactericidal action of quinolones – inhibitors of DNA gyrase]. Mol Biol 2014; 48:990–998
    [Google Scholar]
  32. Liu X, Marrakchi M, Jahne M, Rogers S, Andreescu S. Real-time investigation of antibiotics-induced oxidative stress and superoxide release in bacteria using an electrochemical biosensor. Free Radic Biol Med 2016; 91:25–33 [View Article]
    [Google Scholar]
  33. Van Acker H, Gielis J, Acke M, Cools F, Cos P et al. The role of reactive oxygen species in antibiotic-induced cell death in Burkholderia cepacia complex bacteria. PLoS One 2016; 11:e0159837 [View Article]
    [Google Scholar]
  34. Sebastian J, Swaminath S, Nair RR, Jakkala K, Pradhan A et al. De novo emergence of genetically resistant mutants of Mycobacterium tuberculosis from the persistence phase cells formed against antituberculosis drugs in vitro . Antimicrob Agents Chemother 2017; 61:e01343–16 [View Article]
    [Google Scholar]
  35. Sakai A, Nakanishi M, Yoshiyama K, Maki H. Impact of reactive oxygen species on spontaneous mutagenesis in Escherichia coli . Genes Cells 2006; 11:767–778 [View Article]
    [Google Scholar]
  36. Nair RR, Sharan D, Ajitkumar P. A minor subpopulation of mycobacteria inherently produces high levels of reactive oxygen species that generate antibiotic resisters at high frequency from itself and enhance resister generation from its major kin subpopulation. Front Microbiol 2019; 10:10 [View Article]
    [Google Scholar]
  37. Takiff HE, Salazar L, Guerrero C, Philipp W, Huang WM et al. Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations. Antimicrob Agents Chemother 1994; 38:773–780 [View Article]
    [Google Scholar]
  38. Xu C, Kreiswirth BN, Sreevatsan S, Musser JM, Drlica K. Fluoroquinolone resistance associated with specific gyrase mutations in clinical isolates of multidrug-resistant Mycobacterium tuberculosis . J Infect Dis 1996; 174:1127–1130 [View Article]
    [Google Scholar]
  39. Von Groll A, Martin A, Jureen P, Hoffner S, Vandamme P et al. Fluoroquinolone resistance in Mycobacterium tuberculosis and mutations in gyrA and gyrB . Antimicrob Agents Chemother 2009; 53:4498–4500 [View Article]
    [Google Scholar]
  40. Chien JY, Chiu WY, Chien ST, Chiang CJ, Yu CJ et al. Mutations in gyrA and gyrB among fluoroquinolone- and multidrug-resistant Mycobacterium tuberculosis isolates. Antimicrob Agents Chemother 2016; 60:2090–2096 [View Article]
    [Google Scholar]
  41. Devasia R, Blackman A, Eden S, Li H, Maruri F et al. High proportion of fluoroquinolone-resistant Mycobacterium tuberculosis isolates with novel gyrase polymorphisms and a gyrA region associated with fluoroquinolone susceptibility. J Clin Microbiol 2012; 50:1390–1396 [View Article]
    [Google Scholar]
  42. Cadet J, Wagner JR. Dna base damage by reactive oxygen species, oxidizing agents, and UV radiation. Cold Spring Harb Perspect Biol 2013; 5:a0125591–18 [View Article]
    [Google Scholar]
  43. Buchmeier NA, Newton GL, Koledin T, Fahey RC. Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. Mol Microbiol 2003; 47:1723–1732 [View Article]
    [Google Scholar]
  44. Ung KSE, Av-Gay Y. Mycothiol-dependent mycobacterial response to oxidative stress. FEBS Lett 2006; 580:2712–2716 [View Article]
    [Google Scholar]
  45. Van Laer K, Buts L, Foloppe N, Vertommen D, Van Belle K et al. Mycoredoxin-1 is one of the missing links in the oxidative stress defence mechanism of mycobacteria. Mol Microbiol 2012; 86:787–804 [View Article]
    [Google Scholar]
  46. Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA et al. Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 2004; 279:13044–13053 [View Article]
    [Google Scholar]
  47. López-Mirabal HR, Winther JR. Redox characterstics of eukaryotic cytosol. Biochem Biophys Acta 1783; 2008:629–640
    [Google Scholar]
  48. Sarathy JP, Dartois V, Lee EJD. The role of transport mechanisms in Mycobacterium tuberculosis drug resistance and tolerance. Pharmaceuticals 2012; 5:1210–1235 [View Article]
    [Google Scholar]
  49. Nyström T. Stationary-Phase physiology. Annu Rev Microbiol 2004; 58:161–181 [View Article]
    [Google Scholar]
  50. Wood ML, Dizdaroglu M, Gajewski E, Essigmann JM. Mechanistic studies of ionizing radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome. Biochemistry 1990; 29:7024–7032 [View Article]
    [Google Scholar]
  51. Ramaswamy SV, Amin AG, Göksel S, Stager CE, Dou SJ et al. Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis . Antimicrob Agents Chemother 2000; 44:326–336 [View Article]
    [Google Scholar]
  52. Musser JM, Kapur V, Williams DL, Kreiswirth BN, van Soolingen D et al. Characterization of the catalase-peroxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance. J Infect Dis 1996; 173:196–202 [View Article]
    [Google Scholar]
  53. Kandler JL, Mercante AD, Dalton TL, Ezewudo MN, Cowan LS et al. Validation of novel Mycobacterium tuberculosis isoniazid resistance mutations not detectable by common molecular tests. Antimicrob Agents Chemother 2018; 62:e00974–18 [View Article]
    [Google Scholar]
  54. Lyu LD, Tang BK, Fan XY, Ma H, Zhao GP. Mycobacterial MazG safeguards genetic stability via housecleaning of 5-OH-dCTP. PLoS Pathog 2013; 9:e1003814 [View Article]
    [Google Scholar]
  55. Jindani A, Doré CJ, Mitchison DA. Bactericidal and sterilizing activities of antituberculosis drugs during the first 14 days. Am J Respir Crit Care Med 2003; 167:1348–1354 [View Article]
    [Google Scholar]
  56. Ahmad Z, Klinkenberg LG, Pinn ML, Fraig MM, Peloquin CA et al. Biphasic kill curve of isoniazid reveals the presence of drug-tolerant, not drug-resistant, Mycobacterium tuberculosis in the guinea pig. J Infect Dis 2009; 200:1136–1143 [View Article]
    [Google Scholar]
  57. Adams KN, Takaki K, Connolly LE, Wiedenhoft H, Winglee K et al. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 2011; 145:39–53 [View Article]
    [Google Scholar]
  58. Barrett TC, Mok WWK, Murawski AM, Brynildsen MP. Enhanced antibiotic resistance development from fluoroquinolone persisters after a single exposure to antibiotic. Nat Commun 2019; 10:1177 [View Article]
    [Google Scholar]
  59. Windels EM, Michiels JE, Fauvart M, Wenseleers T, Van den Bergh B et al. Bacterial persistence promotes the evolution of antibiotic resistance by increasing survival and mutation rates. ISME J 2019; 13:1239–1251 [View Article]
    [Google Scholar]
  60. Maisonneuve E, Gerdes K. Molecular mechanisms underlying bacterial persisters. Cell 2014; 157:539–548 [View Article]
    [Google Scholar]
  61. Kohanski MA, DePristo MA, Collins JJ. Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Mol Cell 2010; 37:311–320 [View Article]
    [Google Scholar]
  62. Nair CG, Chao C, Ryall B, Williams HD. Sub-lethal concentrations of antibiotics increase mutation frequency in the cystic fibrosis pathogen Pseudomonas aeruginosa . Lett Appl Microbiol 2013; 56:149–154 [View Article]
    [Google Scholar]
  63. Choudhuri BS, Bhakta S, Barik R, Basu J, Kundu M et al. Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis . Biochem J 2002; 367:279–285 [View Article]
    [Google Scholar]
  64. Pasca MR, Guglierame P, Arcesi F, Bellinzoni M, De Rossi E et al. Rv2686c-Rv2687c-Rv2688c, an ABC fluoroquinolone efflux pump in Mycobacterium tuberculosis . Antimicrob Agents Chemother 2004; 48:3175–3178 [View Article]
    [Google Scholar]
  65. Gupta S, Tyagi S, Almeida DV, Maiga MC, Ammerman NC et al. Acceleration of tuberculosis treatment by adjunctive therapy with verapamil as an efflux inhibitor. Am J Respir Crit Care Med 2013; 188:600–607 [View Article]
    [Google Scholar]
  66. Chuang Y-M, Bandyopadhyay N, Rifat D, Rubin H, Bader JS et al. Deficiency of the novel exopolyphosphatase Rv1026/PPX2 leads to metabolic downshift and altered cell wall permeability in Mycobacterium tuberculosis . mBio 2015; 6:e02428-14 [View Article]
    [Google Scholar]
  67. Hui J, Gordon N, Kajioka R. Permeability barrier to rifampin in mycobacteria. Antimicrob Agents Chemother 1977; 11:773–779 [View Article]
    [Google Scholar]
  68. Nguyen L. Antibiotic resistance mechanisms in M. tuberculosis: an update. Arch Toxicol 2016; 90:1585–1604 [View Article]
    [Google Scholar]
  69. Karunakaran P, Davies J. Genetic antagonism and hypermutability in Mycobacterium smegmatis . J Bacteriol 2000; 182:3331–3335 [View Article]
    [Google Scholar]
  70. Saumaa S, Tover A, Tark M, Tegova R, Kivisaar M. Oxidative DNA damage defense systems in avoidance of stationary-phase mutagenesis in Pseudomonas putida . J Bacteriol 2007; 189:5504–5514 [View Article]
    [Google Scholar]
  71. Vidales LE, Cárdenas LC, Robleto E, Yasbin RE, Pedraza-Reyes M. Defects in the error prevention oxidized guanine system potentiate stationary-phase mutagenesis in Bacillus subtilis . J Bacteriol 2009; 191:506–513 [View Article]
    [Google Scholar]
  72. Sareen D, Newton GL, Fahey RC, Buchmeier NA. Mycothiol is essential for growth of Mycobacterium tuberculosis Erdman. J Bacteriol 2003; 185:6736–6740 [View Article]
    [Google Scholar]
  73. Jothivasan VK, Hamilton CJ. Mycothiol: synthesis, biosynthesis and biological functions of the major low molecular weight thiol in actinomycetes. Nat Prod Rep 2008; 25:1091–1117 [View Article]
    [Google Scholar]
  74. Newton GL, Rawat M, La Clair JJ, Jothivasan VK, Budiarto T et al. Bacillithiol is an antioxidant thiol produced in Bacilli. Nat Chem Biol 2009; 5:625–627 [View Article]
    [Google Scholar]
  75. Knöppel A, Näsvall J, Andersson DI. Evolution of antibiotic resistance without antibiotic exposure. Antimicrob Agents Chemother 2017; 61:e01495–17 [View Article]
    [Google Scholar]
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