1887

Abstract

We report on the combination of chemical mutagenesis, azithromycin selection and next-generation sequencing (Mut-Seq) for the identification of small nucleotide variants that decrease the susceptibility of to the macrolide antibiotic azithromycin. Mutations in the 23S ribosomal RNA or in ribosomal proteins can confer resistance to macrolides and these were detected by Mut-Seq. By concentrating on recurrent variants, we could associate mutations in genes implicated in the metabolism of glutamine with decreased azithromycin susceptibility among mutants. Glutamine synthetase catalyses the transformation of glutamate and ammonium into glutamine and its chemical inhibition is shown to sensitize to antibiotics. A mutation affecting the ribosomal-binding site of a putative ribonuclease J2 is also shown to confer low-level resistance. Mut-Seq has the potential to reveal chromosomal changes enabling high resistance as well as novel events conferring more subtle phenotypes.

Funding
This study was supported by the:
  • Canadian Institutes of Health Research
    • Principle Award Recipient: Marc Ouellette
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2020-10-19
2021-07-29
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References

  1. Hampton LM, Farley MM, Schaffner W, Thomas A, Reingold A et al. Prevention of antibiotic-nonsusceptible Streptococcus pneumoniae with conjugate vaccines. J Infect Dis 2012; 205:401–411 [View Article]
    [Google Scholar]
  2. Richter SS, Diekema DJ, Heilmann KP, Dohrn CL, Riahi F et al. Changes in pneumococcal serotypes and antimicrobial resistance after introduction of the 13-valent conjugate vaccine in the United States. Antimicrob Agents Chemother 2014; 58:6484–6489 [View Article]
    [Google Scholar]
  3. Tomczyk S, Lynfield R, Schaffner W, Reingold A, Miller L et al. Prevention of antibiotic-nonsusceptible invasive pneumococcal disease with the 13-Valent pneumococcal conjugate vaccine. Clin Infect Dis 2016; 62:1119–1125 [View Article]
    [Google Scholar]
  4. Sader HS, Mendes RE, Le J, Denys G, Flamm RK et al. Antimicrobial susceptibility of Streptococcus pneumoniae from North America, Europe, Latin America, and the Asia-Pacific region: results from 20 years of the SENTRY antimicrobial surveillance program (1997–2016). Open Forum Infect Dis 2019; 6:S14–S23 [View Article]
    [Google Scholar]
  5. Schroeder MR, Chancey ST, Thomas S, Kuo W-H, Satola SW et al. A population-based assessment of the impact of 7- and 13-Valent pneumococcal conjugate vaccines on macrolide-resistant invasive pneumococcal disease: emergence and decline of Streptococcus pneumoniae serotype 19A (CC320) with dual macrolide resistance mechanisms. Clin Infect Dis 2017; 65:990–998 [View Article]
    [Google Scholar]
  6. Schroeder MR, Stephens DS. Macrolide resistance in Streptococcus pneumoniae . Front Cell Infect Microbiol 2016; 6:98 [View Article]
    [Google Scholar]
  7. Canu A, Malbruny B, Coquemont Maëlle, Davies TA, Appelbaum PC et al. Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae . Antimicrob Agents Chemother 2002; 46:125–131 [View Article]
    [Google Scholar]
  8. Davies TA, Bush K, Sahm D, Evangelista A. Predominance of 23S rRNA mutants among non-erm, non-mef macrolide-resistant clinical isolates of Streptococcus pneumoniae collected in the United States in 1999-2000. Antimicrob Agents Chemother 2005; 49:3031–3033 [View Article]
    [Google Scholar]
  9. Farrell DJ, Douthwaite S, Morrissey I, Bakker S, Poehlsgaard J et al. Macrolide resistance by ribosomal mutation in clinical isolates of Streptococcus pneumoniae from the PROTEKT 1999-2000 study. Antimicrob Agents Chemother 2003; 47:1777–1783 [View Article]
    [Google Scholar]
  10. Nagai K, Davies TA, Dewasse BE, Pankuch GA, Jacobs MR. In vitro development of resistance to ceftriaxone, cefprozil and azithromycin in Streptococcus pneumoniae . J Antimicrob Chemother 2000; 46:909–915 [View Article]
    [Google Scholar]
  11. Tait-Kamradt A, Davies T, Appelbaum PC, Depardieu F, Courvalin P et al. Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumoniae from eastern Europe and North America. Antimicrob Agents Chemother 2000; 44:3395–3401 [View Article]
    [Google Scholar]
  12. Tait-Kamradt A, Davies T, Cronan M, Jacobs MR, Appelbaum PC et al. Mutations in 23S rRNA and ribosomal protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage. Antimicrob Agents Chemother 2000; 44:2118–2125 [View Article]
    [Google Scholar]
  13. Wierzbowski AK, Nichol K, Laing N, Hisanaga T, Nikulin A et al. Macrolide resistance mechanisms among Streptococcus pneumoniae isolated over 6 years of Canadian respiratory organism susceptibility study (cross) (1998 2004). J Antimicrob Chemother 2007; 60:733–740 [View Article]
    [Google Scholar]
  14. Fyfe C, Grossman TH, Kerstein K, Sutcliffe J. Resistance to macrolide antibiotics in public health pathogens. Cold Spring Harb Perspect Med 2016; 6:a025395 [View Article]
    [Google Scholar]
  15. Gregory ST, Dahlberg AE. Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA. J Mol Biol 1999; 289:827–834 [View Article]
    [Google Scholar]
  16. Palmer AC, Chait R, Kishony R. Nonoptimal gene expression creates latent potential for antibiotic resistance. Mol Biol Evol 2018; 35:2669–2684
    [Google Scholar]
  17. Stokes JM, Lopatkin AJ, Lobritz MA, Collins JJ. Bacterial metabolism and antibiotic efficacy. Cell Metab 2019; 30:251–259 [View Article]
    [Google Scholar]
  18. Ouellette M, Bhattacharya A. Exploiting antimicrobial resistance: better knowledge of resistance mechanisms can inform the search for and development of new antibiotics. EMBO Rep 2020; 21:e50249
    [Google Scholar]
  19. Tomasz A, Hotchkiss RD. Regulation of the transformability of pneumococcal cultures by macromolecular cell products. Proc Natl Acad Sci U S A 1964; 51:480–487 [View Article]
    [Google Scholar]
  20. Gingras H, Patron K, Bhattacharya A, Leprohon P, Ouellette M. Gain- and loss-of-function screens coupled to next-generation sequencing for antibiotic mode of action and resistance studies in Streptococcus pneumoniae . Antimicrob Agents Chemother 2019; 63: [View Article]
    [Google Scholar]
  21. Li H, Durbin R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 2010; 26:589–595 [View Article]
    [Google Scholar]
  22. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010; 20:1297–1303 [View Article]
    [Google Scholar]
  23. Fani F, Leprohon P, Zhanel GG, Bergeron MG, Ouellette M. Genomic analyses of DNA transformation and penicillin resistance in Streptococcus pneumoniae clinical isolates. Antimicrob Agents Chemother 2014; 58:1397–1403 [View Article]
    [Google Scholar]
  24. El Khoury JY, Boucher N, Bergeron MG, Leprohon P, Ouellette M. Penicillin induces alterations in glutamine metabolism in Streptococcus pneumoniae . Sci Rep 2017; 7:14587 [View Article]
    [Google Scholar]
  25. Lisher JP, Tsui H-CT, Ramos-Montañez S, Hentchel KL, Martin JE et al. Biological and chemical adaptation to endogenous hydrogen peroxide production in Streptococcus pneumoniae D39. mSphere 2017; 2: [View Article]
    [Google Scholar]
  26. Härtel T, Klein M, Koedel U, Rohde M, Petruschka L et al. Impact of glutamine transporters on pneumococcal fitness under infection-related conditions. Infect Immun 2011; 79:44–58 [View Article]
    [Google Scholar]
  27. Kloosterman TG, Hendriksen WT, Bijlsma JJ, Bootsma HJ, van Hijum SA et al. Regulation of glutamine and glutamate metabolism by GlnR and GlnA in Streptococcus pneumoniae . J Biol Chem 2006; 281:25097–25109 [View Article]
    [Google Scholar]
  28. Brusilow WS, Peters TJ. Therapeutic effects of methionine sulfoximine in multiple diseases include and extend beyond inhibition of glutamine synthetase. Expert Opin Ther Targets 2017; 21:461–469 [View Article]
    [Google Scholar]
  29. Ronzio RA, Rowe WB, Meister A. Mechanism of inhibition of glutamine synthetase by methionine sulfoximine. Biochemistry 1969; 8:1066–1075 [View Article]
    [Google Scholar]
  30. Rowe WB, Ronzio RA, Meister A. Inhibition of glutamine synthetase by methionine sulfoximine. Studies on methionine sulfoximine phosphate. Biochemistry 1969; 8:2674–2680 [View Article]
    [Google Scholar]
  31. Fani F, Brotherton MC, Leprohon P, Ouellette M. Genomic analysis and reconstruction of cefotaxime resistance in Streptococcus pneumoniae. J Antimicrob Chemother 2013; 68:1718–1727 [View Article]
    [Google Scholar]
  32. Melese M, Chidambaram JD, Alemayehu W, Lee DC, EH Y et al. Feasibility of eliminating ocular Chlamydia trachomatis with repeat mass antibiotic treatments. JAMA 2004; 292:721–725
    [Google Scholar]
  33. Solomon AW, Holland MJ, Alexander ND, Massae PA, Aguirre A et al. Mass treatment with single-dose azithromycin for trachoma. N Engl J Med 2004; 351:1962–1971 [View Article]
    [Google Scholar]
  34. Keenan JD, Arzika AM, Maliki R, Boubacar N, Elh Adamou S et al. Longer-Term assessment of azithromycin for reducing childhood mortality in Africa. N Engl J Med 2019; 380:2207–2214 [View Article]
    [Google Scholar]
  35. Kelly C, Chalmers JD, Crossingham I, Relph N, Felix LM et al. Macrolide antibiotics for bronchiectasis. Cochrane Database Syst Rev 2018; 3:CD012406
    [Google Scholar]
  36. Doan T, Hinterwirth A, Worden L, Arzika AM, Maliki R et al. Gut microbiome alteration in MORDOR I: a community-randomized trial of mass azithromycin distribution. Nat Med 2019; 25:1370–1376 [View Article]
    [Google Scholar]
  37. O'Brien KS, Emerson P, Hooper PJ, Reingold AL, Dennis EG et al. Antimicrobial resistance following mass azithromycin distribution for trachoma: a systematic review. Lancet Infect Dis 2019; 19:e14–e25 [View Article]
    [Google Scholar]
  38. Depardieu F, Courvalin P. Mutation in 23S rRNA responsible for resistance to 16-membered macrolides and streptogramins in Streptococcus pneumoniae. Antimicrob Agents Chemother 2001; 45:319–323 [View Article]
    [Google Scholar]
  39. Wolter N, von Gottberg A, du Plessis M, de Gouveia L, Klugman KP. Molecular basis and clonal nature of increasing pneumococcal macrolide resistance in South Africa, 2000–2005. Int J Antimicrob Agents 2008; 32:62–67 [View Article]
    [Google Scholar]
  40. Schuwirth BS, Borovinskaya MA, Hau CW, Zhang W, Vila-Sanjurjo A et al. Structures of the bacterial ribosome at 3.5 A resolution. Science 2005; 310:827–834 [View Article]
    [Google Scholar]
  41. Wolter N, Smith AM, Farrell DJ, Klugman KP. Heterogeneous macrolide resistance and gene conversion in the pneumococcus. Antimicrob Agents Chemother 2006; 50:359–361 [View Article]
    [Google Scholar]
  42. Carvalho SM, Kuipers OP, Neves AR. Environmental and nutritional factors that affect growth and metabolism of the pneumococcal serotype 2 strain D39 and its nonencapsulated derivative strain R6. PLoS One 2013; 8:e58492 [View Article]
    [Google Scholar]
  43. Gustafson J, Strassle A, Hachler H, Kayser FH, Berger-Bachi B. The femC locus of Staphylococcus aureus required for methicillin resistance includes the glutamine synthetase operon. J Bacteriol 1994; 176:1460–1467 [View Article]
    [Google Scholar]
  44. Binh TT, Shiota S, Suzuki R, Matsuda M, Trang TT et al. Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using next-generation sequencing. J Antimicrob Chemother 2014; 69:1796–1803 [View Article]
    [Google Scholar]
  45. Zampieri M, Zimmermann M, Claassen M, Sauer U. Nontargeted metabolomics reveals the multilevel response to antibiotic perturbations. Cell Rep 2017; 19:1214–1228 [View Article]
    [Google Scholar]
  46. Wang Z, Soni V, Marriner G, Kaneko T, Boshoff HIM et al. Mode-of-action profiling reveals glutamine synthetase as a collateral metabolic vulnerability of M. tuberculosis to bedaquiline. Proc Natl Acad Sci U S A 2019; 116:19646–19651 [View Article]
    [Google Scholar]
  47. Britton RA, Wen T, Schaefer L, Pellegrini O, Uicker WC et al. Maturation of the 5′ end of Bacillus subtilis 16S rRNA by the essential ribonuclease YkqC/RNase J1. Mol Microbiol 2007; 63:127–138 [View Article]
    [Google Scholar]
  48. Linder P, Lemeille S, Redder P. Transcriptome-wide analyses of 5′-ends in RNase J mutants of a gram-positive pathogen reveal a role in RNA maturation, regulation and degradation. PLoS Genet 2014; 10:e1004207 [View Article]
    [Google Scholar]
  49. Song W, Kim YH, Sim SH, Hwang S, Lee JH et al. Antibiotic stress-induced modulation of the endoribonucleolytic activity of RNase III and RNase G confers resistance to aminoglycoside antibiotics in Escherichia coli . Nucleic Acids Res 2014; 42:4669–4681 [View Article]
    [Google Scholar]
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