1887

Abstract

complex (MABSC) is an environmental organism and opportunistic pathogen. MABSC pulmonary infections in people with cystic fibrosis are of growing clinical concern. Resistance data guide the use of macrolides and amikacin in MABSC pulmonary disease treatment. MABSC can acquire resistance against macrolides or amikacin via 23S or 16S rRNA gene mutations, respectively.

Current culture-based methods for MABSC detection and antibiotic resistance characterization are typically prolonged, limiting their utility to directly inform treatment or clinical trials. Culture-independent molecular methods may help address this limitation.

To develop real-time PCR assays for characterization of key 23S or 16S rRNA gene mutations associated with constitutive resistance in MABSC.

We designed two real-time PCR assays to detect the key 23S and 16S rRNA gene mutations. The highly conserved nature of rRNA genes was a major design challenge. To reduce potential cross-reactivity, primers included non-template bases and targeted single-nucleotide polymorphisms unique to MABSC. We applied these assays, as well as a previously developed real-time PCR assay for MABSC detection, to 968 respiratory samples from people with cystic fibrosis. The results from the molecular methods were compared to those for gold standard culture methods and 23S and 16S rRNA gene sequencing.

The real-time PCR MABSC detection assay provided a sensitivity of 83.8 % and a specificity of 97.8 % compared to culture. The results from the real-time PCR resistance detection assays were mostly concordant (>77.4 %) with cultured isolate sequencing. The real-time PCR resistance detection assays identified several samples harbouring both resistant and susceptible MABSC, while culture-dependent methods only identified susceptible MABSC in these samples.

Using the molecular methods described here, results for health care providers or researchers could be available days or weeks earlier than is currently possible via culture-based antibiotic susceptibility testing.

Funding
This study was supported by the:
  • Pathology Queensland SERC (Award 4946)
    • Principle Award Recipient: DavidM Whiley
  • Sasakawa Memorial Fund of the Children’s Hospital Foundation (Award 50222)
    • Principle Award Recipient: DavidM Whiley
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2021-04-28
2021-05-17
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References

  1. Harris KA, Kenna DTD. Mycobacterium abscessus infection in cystic fibrosis: molecular typing and clinical outcomes. J Med Microbiol 2014; 63:1241–1246 [CrossRef][PubMed]
    [Google Scholar]
  2. Esther CR, Esserman DA, Gilligan P, Kerr A, Noone PG. Chronic Mycobacterium abscessus infection and lung function decline in cystic fibrosis. J Cyst Fibros 2010; 9:117–123 [CrossRef][PubMed]
    [Google Scholar]
  3. Qvist T, Taylor-Robinson D, Waldmann E, Olesen HV, Hansen CR et al. Comparing the harmful effects of nontuberculous mycobacteria and Gram negative bacteria on lung function in patients with cystic fibrosis. J Cyst Fibros 2016; 15:380–385 [CrossRef][PubMed]
    [Google Scholar]
  4. Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007; 175:367–416 [CrossRef][PubMed]
    [Google Scholar]
  5. Floto RA, Olivier KN, Saiman L, Daley CL, Herrmann J-L et al. US Cystic fibrosis Foundation and European cystic fibrosis Society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis. Thorax 2016; 71 Suppl 1:i1–22 [CrossRef][PubMed]
    [Google Scholar]
  6. Haworth CS, Banks J, Capstick T, Fisher AJ, Gorsuch T et al. British thoracic Society guidelines for the management of non-tuberculous mycobacterial pulmonary disease (NTM-PD). Thorax 2017; 72:ii1–ii64 [CrossRef][PubMed]
    [Google Scholar]
  7. Nessar R, Cambau E, Reyrat JM, Murray A, Gicquel B. Mycobacterium abscessus: a new antibiotic nightmare. J Antimicrob Chemother 2012; 67:810–818 [CrossRef][PubMed]
    [Google Scholar]
  8. Whittier S, Hopfer RL, Knowles MR, Gilligan PH. Improved recovery of mycobacteria from respiratory secretions of patients with cystic fibrosis. J Clin Microbiol 1993; 31:861–864 [CrossRef][PubMed]
    [Google Scholar]
  9. Shin SH, Jhun BW, Kim S-Y, Choe J, Jeon K et al. Nontuberculous mycobacterial lung diseases caused by mixed infection with Mycobacterium avium complex and Mycobacterium abscessus complex. Antimicrob Agents Chemother 2018; 62:e01105–01118 [CrossRef][PubMed]
    [Google Scholar]
  10. Griffith DE, Philley JV, Brown-Elliott BA, Benwill JL, Shepherd S et al. The significance of Mycobacterium abscessus subspecies abscessus isolation during Mycobacterium avium complex lung disease therapy. Chest 2015; 147:1369–1375 [CrossRef][PubMed]
    [Google Scholar]
  11. Cowman SA, James P, Wilson R, Cookson WOC, Moffatt MF et al. Profiling mycobacterial communities in pulmonary nontuberculous mycobacterial disease. PLoS One 2018; 13:e0208018 [CrossRef][PubMed]
    [Google Scholar]
  12. Shaw LP, Doyle RM, Kavaliunaite E, Spencer H, Balloux F et al. Children with cystic fibrosis are infected with multiple subpopulations of Mycobacterium abscessus with different antimicrobial resistance profiles. Clin Infect Dis 2019; 69:1678–1686 [CrossRef][PubMed]
    [Google Scholar]
  13. National Committee for Clinical Laboratory Standards Susceptibility Testing Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; approved standard, 2nd ed. CLSI document M24-A2. 2011
    [Google Scholar]
  14. Brown-Elliott BA, Nash KA, Wallace RJ. Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clin Microbiol Rev 2012; 25:545–582 [CrossRef][PubMed]
    [Google Scholar]
  15. Bryant JM, Grogono DM, Greaves D, Foweraker J, Roddick I et al. Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study. Lancet 2013; 381:1551–1560 [CrossRef][PubMed]
    [Google Scholar]
  16. Bryant JM, Grogono DM, Rodriguez-Rincon D, Everall I, Brown KP et al. Emergence and spread of a human-transmissible multidrug-resistant nontuberculous Mycobacterium. Science 2016; 354:751–757 [CrossRef][PubMed]
    [Google Scholar]
  17. Brown-Elliott BA, Woods GL. Antimycobacterial susceptibility testing of nontuberculous mycobacteria. J Clin Microbiol 2019; 57:e00834–19 [CrossRef][PubMed]
    [Google Scholar]
  18. Jagielski T, Minias A, van Ingen J, Rastogi N, Brzostek A et al. Methodological and clinical aspects of the molecular epidemiology of Mycobacterium tuberculosis and other mycobacteria. Clin Microbiol Rev 2016; 29:239–290 [CrossRef][PubMed]
    [Google Scholar]
  19. Prammananan T, Sander P, Brown BA, Frischkorn K, Onyi GO et al. A single 16S ribosomal RNA substitution is responsible for resistance to amikacin and other 2-deoxystreptamine aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae . J Infect Dis 1998; 177:1573–1581 [CrossRef][PubMed]
    [Google Scholar]
  20. Wallace RJ, Meier A, Brown BA, Zhang Y, Sander P et al. Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus . Antimicrob Agents Chemother 1996; 40:1676–1681 [CrossRef][PubMed]
    [Google Scholar]
  21. Jhun BW, Yang B, Moon SM, Lee H, Park HY et al. Amikacin inhalation as salvage therapy for refractory nontuberculous mycobacterial lung disease. Antimicrob Agents Chemother 2018; 62:e00011-18–18 [CrossRef][PubMed]
    [Google Scholar]
  22. Olivier KN, Griffith DE, Eagle G, McGinnis JP, Micioni L et al. Randomized trial of liposomal amikacin for inhalation in nontuberculous mycobacterial lung disease. Am J Respir Crit Care Med 2017; 195:814–823 [CrossRef][PubMed]
    [Google Scholar]
  23. Kobayashi T, Tsuyuguchi K, Yoshida S, Kurahara Y, Ikegami N et al. Mycobacterium abscessus subsp. abscessus lung disease: drug susceptibility testing in sputum culture negative conversion. Int J Mycobacteriol 2018; 7:69–75 [CrossRef][PubMed]
    [Google Scholar]
  24. Guo Q, Chu H, Ye M, Zhang Z, Li B et al. The clarithromycin susceptibility genotype affects the treatment outcome of patients with Mycobacterium abscessus lung disease. Antimicrob Agents Chemother 2018; 62:e02360–17 [CrossRef][PubMed]
    [Google Scholar]
  25. Nash KA, Brown-Elliott BA, Wallace RJ. A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae . Antimicrob Agents Chemother 2009; 53:1367–1376 [CrossRef][PubMed]
    [Google Scholar]
  26. Brown-Elliott BA, Iakhiaeva E, Griffith DE, Woods GL, Stout JE et al. In vitro activity of amikacin against isolates of Mycobacterium avium complex with proposed MIC breakpoints and finding of a 16S rRNA gene mutation in treated isolates. J Clin Microbiol 2013; 51:3389–3394 [CrossRef][PubMed]
    [Google Scholar]
  27. Shallom SJ, Moura NS, Olivier KN, Sampaio EP, Holland SM et al. New real-time PCR assays for detection of inducible and acquired clarithromycin resistance in the Mycobacterium abscessus group. J Clin Microbiol 2015; 53:3430–3437 [CrossRef][PubMed]
    [Google Scholar]
  28. Sharma MK, La Y, Janella D, Soualhine H. A real-time PCR assay for rapid identification of inducible and acquired clarithromycin resistance in Mycobacterium abscessus . BMC Infect Dis 2020; 20:944 [CrossRef][PubMed]
    [Google Scholar]
  29. Mougari F, Loiseau J, Veziris N, Bernard C, Bercot B et al. Evaluation of the new genotype NTM-DR kit for the molecular detection of antimicrobial resistance in non-tuberculous mycobacteria. J Antimicrob Chemother 2017; 72:1669–1677 [CrossRef][PubMed]
    [Google Scholar]
  30. Duplancic C, Stockwell R, Wainwright C, Thomson R, Bell S. P156 nontuberculous mycobacteria infection in people with cystic fibrosis attending cystic fibrosis treatment clinics in Australia. J Cyst Fibros 2019; 18:S101–S102 [CrossRef]
    [Google Scholar]
  31. Syrmis MW, Pandey S, Tolson C, Carter R, Congdon J et al. Identification of Mycobacterium abscessus complex and M. abscessus subsp. massiliense culture isolates by real-time assays. J Med Microbiol 2015; 64:790–794 [CrossRef][PubMed]
    [Google Scholar]
  32. Trembizki E, Buckley C, Donovan B, Chen M, Guy R et al. Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance. J Antimicrob Chemother 2015; 70:3244–3249 [CrossRef][PubMed]
    [Google Scholar]
  33. Buckley C, Trembizki E, Donovan B, Chen M, Freeman K et al. A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae gyrA 91 locus associated with ciprofloxacin susceptibility. J Antimicrob Chemother 2016; 71:353–356 [CrossRef][PubMed]
    [Google Scholar]
  34. Rocchetti TT, Silbert S, Gostnell A, Kubasek C, Widen R. Validation of a multiplex real-time PCR assay for detection of Mycobacterium spp., Mycobacterium tuberculosis complex, and Mycobacterium avium complex directly from clinical samples by use of the BD Max open system. J Clin Microbiol 2016; 54:1644–1647 [CrossRef][PubMed]
    [Google Scholar]
  35. Centers for Disease Control and Prevention Multiplex Real-Time PCR Detection of Klebsiella pneumoniae Carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM-1) genes. Healthcare-associated Infections - Laboratory Resources; 2011
  36. Bialasiewicz S, Whiley DM, Buhrer-Skinner M, Bautista C, Barker K et al. A novel gel-based method for self-collection and ambient temperature postal transport of urine for PCR detection of Chlamydia trachomatis . Sex Transm Infect 2009; 85:102–105 [CrossRef][PubMed]
    [Google Scholar]
  37. Whiley DM, Buda PJ, Bayliss J, Cover L, Bates J et al. A new confirmatory Neisseria gonorrhoeae real-time PCR assay targeting the porA pseudogene. Eur J Clin Microbiol Infect Dis 2004; 23:705–710 [CrossRef][PubMed]
    [Google Scholar]
  38. Queensland Health Nontuberculous mycobacteria in Queensland; 2016
  39. Tortoli E. Microbiological features and clinical relevance of new species of the genus Mycobacterium. Clin Microbiol Rev 2014; 27:727–752 [CrossRef][PubMed]
    [Google Scholar]
  40. Forbes BA, Hall GS, Miller MB, Novak SM, Rowlinson M-C et al. Practice guidelines for clinical microbiology laboratories: mycobacteria. Clin Microbiol Rev 2018; 31:66 [CrossRef][PubMed]
    [Google Scholar]
  41. Stout JE, Koh W-J, Yew WW. Update on pulmonary disease due to non-tuberculous mycobacteria. Int J Infect Dis 2016; 45:123–134 [CrossRef][PubMed]
    [Google Scholar]
  42. Roux A-L, Catherinot E, Ripoll F, Soismier N, Macheras E et al. Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in France. J Clin Microbiol 2009; 47:4124–4128 [CrossRef][PubMed]
    [Google Scholar]
  43. Caverly LJ, Carmody LA, Haig S-J, Kotlarz N, Kalikin LM et al. Culture-independent identification of nontuberculous mycobacteria in cystic fibrosis respiratory samples. PLoS One 2016; 11:e0153876 [CrossRef][PubMed]
    [Google Scholar]
  44. Tran AC, Halse TA, Escuyer VE, Musser KA. Detection of Mycobacterium avium complex DNA directly in clinical respiratory specimens: opportunities for improved turn-around time and cost savings. Diagn Microbiol Infect Dis 2014; 79:43–48 [CrossRef][PubMed]
    [Google Scholar]
  45. Kim J-U, Ryu D-S, Cha C-H, Park S-H. Paradigm for diagnosing mycobacterial disease: direct detection and differentiation of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in clinical specimens using multiplex real-time PCR. J Clin Pathol 2018; 71:774–780 [CrossRef][PubMed]
    [Google Scholar]
  46. Lim J-H, Kim C-K, Bae MH. Evaluation of the performance of two real-time PCR assays for detecting Mycobacterium species. J Clin Lab Anal 2019; 33:e22645 [CrossRef][PubMed]
    [Google Scholar]
  47. Maurer FP, Bruderer VL, Castelberg C, Ritter C, Scherbakov D et al. Aminoglycoside-Modifying enzymes determine the innate susceptibility to aminoglycoside antibiotics in rapidly growing mycobacteria. J Antimicrob Chemother 2015; 70:1412–1419 [CrossRef][PubMed]
    [Google Scholar]
  48. Rominski A, Selchow P, Becker K, Brülle JK, Dal Molin M et al. Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes. J Antimicrob Chemother 2017; 72:2191–2200 [CrossRef][PubMed]
    [Google Scholar]
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