Journal of Genetics

, Volume 96, Issue 4, pp 599–612 | Cite as

Fitness-compensatory mutations facilitate the spread of drug-resistant F15/LAM4/KZN and F28 Mycobacterium tuberculosis strains in KwaZulu-Natal, South Africa

  • Charissa C. Naidoo
  • Manormoney PillayEmail author
Research Article


While the acquisition of drug resistance is often accompanied by fitness costs, Mycobacterium tuberculosis has developed mechanisms to overcome these costs in the form of compensatory mutations. In an attempt to dissect strain-specific differences in biological fitness, 10 M. tuberculosis genomes, representing F15/LAM4/KZN, Beijing, F11 and F28 genotypes were sequenced on the Illumina MiSeq platform. Drug-susceptible F15/LAM4/KZN strains differed by 43 SNPs, demonstrating that heterogeneity exists even among closely-related strains. We found unique, nonsynonymous single-nucleotide polymorphisms (SNPs) in the sigA and grcC1 genes of multidrug resistant (MDR) and XDR F15/LAM4/KZN strains, respectively. The F28 MDR strain harboured a novel ubiA mutation in combination with its embB M306I mutation, which may be related to ethambutol resistance. In addition, it possessed a low-frequency rpoC mutation, suggesting that this strain was in the process of developing compensation. In contrast, no compensatory mutations were identified in Beijing and F11 MDR strains, corroborating its low in vitro fitness. Clinical strains also harboured unique SNPs in a number of important genes associated with virulence, highlighting the need for future studies which examine the correlation of genetic variations with phenotypic diversity. In summary, whole-genome sequencing revealed the presence of fitness-compensatory mutations in F15/LAM4/KZN and F28 genotypes which predominate in MDR and/or extensively drug resistant (XDR) forms in KwaZulu-Natal, South Africa.


whole-genome sequencing Illumina MiSeq single-nucleotide polymorphisms CLC genomics workbench drug resistance Mycobacterium tuberculosis 



This study was supported by the National Research Foundation of South Africa (Grant no. 88959 and 90508) and the College of Health Sciences, University of KwaZulu-Natal.

Supplementary material

12041_2017_805_MOESM1_ESM.pdf (311 kb)
Supplementary material 1 (pdf 311 KB)


  1. Albrethsen J., Agner J., Piersma S. R., Hojrup P., Pham T. V., Weldingh K. et al. 2013 Proteomic profiling of Mycobacterium tuberculosis identifies nutrient-starvation-responsive toxin-antitoxin systems. Mol. Cell. Proteom. 12, 1180–1191.CrossRefGoogle Scholar
  2. Andersson D. I. and Levin B. R. 1999 The biological cost of antibiotic resistance. Curr. Opin. Microbiol. 2, 489–493.CrossRefPubMedGoogle Scholar
  3. Ando H., Miyoshi-Akiyama T., Watanabe S. and Kirikae T. 2014 A silent mutation in mabA confers isoniazid resistance on Mycobacterium tuberculosis. Mol. Microbiol. 91, 538–547.CrossRefPubMedGoogle Scholar
  4. Billington O. J., McHugh T. D. and Gillespie S. H. 1999 Physiological cost of rifampicin resistance induced in vitro in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 43, 1866–1869.PubMedPubMedCentralGoogle Scholar
  5. Bina X. R., Philippart J. A. and Bina J. E. 2009 Effect of the efflux inhibitors 1-(1-naphthylmethyl)-piperazine and pheny-arginine-beta-naphthylamide on antimicrobial susceptibility and virulence factor production in Vibrio cholera. J. Antimicrob. Chemother. 63, 103–108.CrossRefPubMedGoogle Scholar
  6. Black P. A., de Vos M., Louw G. E., van der Merwe R. G., Dippenaar A., Streicher E. M. et al. 2015 Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates. BMC Genomics 16, 857.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Casali N., Nikolayevskyy V., Balabanova Y., Harris S. R., Ignatyeva O., Kontsevaya I. et al. 2014 Evolution and transmission of drug-resistant tuberculosis in a Russian population. Nat. Genet. 46, 279–286.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cohen K. A., Abeel T., McGuire A. M., Desjardins C. A., Munsamy V., Shea T. P. et al. 2015 Evolution of extensively drug-resistant tuberculosis over four decades: whole genome sequencing and dating analysis of Mycobacterium tuberculosis isolates from KwaZulu-Natal. PLoS Med. 12, 1–22.CrossRefGoogle Scholar
  9. Comas I., Borrell S., Roetzer A., Rose G., Malla B., Kato-Maeda M. et al. 2012 Whole genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains identified compensatory mutations in RNA polymerase. Nat. Genet. 44, 106–110.CrossRefGoogle Scholar
  10. Coscolla M. and Gagneux S. 2014 Consequences of genomic diversity in Mycobacterium tuberculosis. Semin. Immunol. 26, 431–444.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cox H. S., McDermid C., Azevedo V., Muller O., Coetzee D., Simpson J. et al. 2010 Epidemic levels of drug resistant tuberculosis (MDR and XDR-TB) in a high HIV prevalence setting in Khayelitsha, South Africa. PLoS One 5, e13901.CrossRefPubMedPubMedCentralGoogle Scholar
  12. de Vos M., Müller B., Borrell S., Black P. A., van Helden P. D., Warren R. M. et al. 2013 Putative compensatory mutations in the rpoC gene of rifampin resistant Mycobacterium tuberculosis are associated with ongoing transmission. Antimicrob. Agents Chemother. 57, 827–832.CrossRefPubMedPubMedCentralGoogle Scholar
  13. Fenner L., Egger M., Bodmer T., Altpeter E., Zwahlen M., Jaton K. et al. 2012 Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 56, 3047–3053.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gagneux S., Burgo M. V., DeRiemer K., Encisco A., Munoz S., Hopewell P. C. et al. 2006 Impact of bacterial genetics on the transmission of isoniazid-resistant Mycobacterium tuberculosis. PLoS Pathol. 2, e61.CrossRefGoogle Scholar
  15. Gandhi N. R., Moll A., Sturm A. W., Pawinski R., Govender T., Lalloo U. et al. 2006 Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 368, 1–6.CrossRefGoogle Scholar
  16. Garton N. J., Waddell S. J., Sherratt A. L., Lee S., Smith R. J., Senner C. et al. 2008 Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum. PLoS Med. 5, e75.CrossRefPubMedPubMedCentralGoogle Scholar
  17. Gengenbacher M. and Kaufmann S. H. 2012 Mycobacterium tuberculosis: success through dormancy. FEMS Microbiol. Rev. 36, 514–532.CrossRefPubMedPubMedCentralGoogle Scholar
  18. Gillespie S. H. 2002 Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob. Agents Chemother. 46, 267–274.CrossRefPubMedPubMedCentralGoogle Scholar
  19. Gomez J. E. and McKinney J. D. 2004 M. tuberculosis persistence, latency, and drug tolerance. Tuberculosis 84, 29–44.CrossRefPubMedGoogle Scholar
  20. Goude R., Amin A. G., Chatterjee D. and Parish T. 2008 The critical role of embC in Mycobacterium tuberculosis. J. Bacteriol. 190, 4335–4341.CrossRefPubMedPubMedCentralGoogle Scholar
  21. He L., Wang X., Cui P., Juin J., Chen J., Zhang W. et al. 2015 ubiA (Rv3806c) encoding DPPR synthase involved in cell wall synthesis is associated with ethambutol resistance in Mycobacterium tuberculosis. Tuberculosis 95, 149–154.CrossRefPubMedGoogle Scholar
  22. Ioerger T. R., Koo S., No E., Chen X., Larsen M. H., Jacobs Jr W. R. et al. 2009 Genome analysis of multi- and extensively-drug-resistant tuberculosis from KwaZulu-Natal, South Africa. PLoS One 4, 1–9.CrossRefGoogle Scholar
  23. Ioerger T. R., Feng Y., Chen X., Dobos K. M., Victor T. C., Streicher E. M. et al. 2010 The non-clonality of drug resistance in Beijing genotype isolates of Mycobacterium tuberculosis from the Western Cape of South Africa. BMC Genomics 11, 670.CrossRefPubMedPubMedCentralGoogle Scholar
  24. Iwamoto T., Yoshida S., Suzuki K. and Wada T. 2008 Population structure analysis of the Mycobacterium tuberculosis Beijing Family indicates an association between certain sublineages and multidrug-resistance. Antimicrob. Agents Chemother. 52, 3805–3809.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kim S. Y., Park Y. J., Kim W. I., Lee S. H., Ludgerus Chang C., Kang S. J. et al. 2003 Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea. Diagn. Microbiol. Infect. Dis. 47, 497–502.CrossRefPubMedGoogle Scholar
  26. Knapp G. S., Lyubetskaya A., Peterson M. W., Gomes A. L. C., Ma Z., Galagan J. E. et al. 2015 Role of intragenic binding of Camp responsive protein (CRP) in regulation of the succinate dehydrogenase genes Rv0249c-Rv0247c in TB complex mycobacteria. Nucleic Acids Res. 43, 5377–5393.CrossRefPubMedPubMedCentralGoogle Scholar
  27. Larsen M. H., Biermann K., Tandberg S., Hsu T. and Jacobs Jr W. R. 2007 Genetic manipulation of Mycobacterium tuberculosis. Curr. Protoc. Microbiol. 10A.2.1–10a.2.21.Google Scholar
  28. Lew J. M., Kapopoulou A., Jones L. M. and Cole S. T. 2011 TubercuList—10 years after. Tuberc. Edinb. Scotl. 91, 1–7.Google Scholar
  29. Liu F., Hu Y., Wang Q., Li H. M., Gao G. F., Liu C. H. et al. 2014 Comparative genomic analysis of Mycobacterium tuberculosis clinical isolates. BMC Genomics 15, 469.CrossRefPubMedPubMedCentralGoogle Scholar
  30. Manca C., Tsenova L., Bergtold A., Freeman S., Tovey M., Musser J. M. et al. 2001 Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha/beta. Proc. Natl. Acad. Sci. USA 98, 5752–5757.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Mann F. M., Xu M., Davenport E. K. and Peters R. J. 2012 Functional characterisation and evolution of the isotuberclosinol operon in Mycobacterium tuberculosis and related mycobacteria. Front. Microbiol. 3, 368.CrossRefPubMedPubMedCentralGoogle Scholar
  32. Muller B., Chihota V. N., Pillay M., Klopper M., Streicher E. M., Coetzee G. et al. 2013 Programmatically selected multidrug-resistant strains drive the emergence of extensively drug-resistant tuberculosis in South Africa. PLoS One 8, 1–9.Google Scholar
  33. Naidoo C. C. and Pillay M. 2014 Increased in vitro fitness of multi- and extensively drug-resistant F15/LAM4/KZN strains of Mycobacterium tuberculosis. Clin. Microbiol. Infect. 20, O361–O369.CrossRefPubMedGoogle Scholar
  34. Piddock L. J. 2006 Multidrug-resistance efflux pumps? Not just for resistance. Nat. Rev. Microbiol. 4, 629–636.CrossRefPubMedGoogle Scholar
  35. Pillay M. and Sturm A. W. 2007 Evolution of the extensively drug-resistant F15/LAM4/KZN strain of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa. Clin. Infect. Dis. 45, 1409–1414.CrossRefPubMedGoogle Scholar
  36. Safi H., Lingaraju S., Amin A., Kim S., Jones M., Holmes M. et al. 2013 Evolution of high-level ethambutol-resistant tuberculosis through interacting mutations in decaprenylphosphoryl-\(\upbeta \)-D-arabinose biosynthetic and utilization pathway genes. Nat. Genet. 45, 1190–1197.CrossRefPubMedGoogle Scholar
  37. Sampson S. L. 2011 Mycobacterial PE/PPE proteins at the host-pathogen interface. Clin. Dev. Immunol. 2011, 1–11.Google Scholar
  38. Sandgren A., Strong M., Muthukrishnan P., Weiner B. K., Church G. M. and Murray M. B. 2009 Tuberculosis drug resistance mutation database. PLoS Med. 6, 0132–0136.CrossRefGoogle Scholar
  39. Sharpe M. L., Gao C., Kendall S. L., Baker E. N. and Lott J. S. 2008 The structure and unusual protein chemistry of hypoxic response protein 1, a latency antigen and highly expressed member of the DosR regulon in Mycobacterium tuberculosis. J. Mol. Biol. 383, 822–836.CrossRefPubMedGoogle Scholar
  40. Sherman D. R., Mdluli K., Hickey M. J., Arain T. M., Morris S. L., Barry 3rd C. E. et al. 1996 Compensatory ahpC gene expression in isoniazid-resistant Mycobacterium tuberculosis. Science 272, 1641–1643.CrossRefPubMedGoogle Scholar
  41. Smith I. 2003 Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin. Microbiol. Rev. 16, 463–496.CrossRefPubMedPubMedCentralGoogle Scholar
  42. Spies F. S., von Groll A., Ribeiro A. W., Ramos D. F., Ribeiro M. O., Costa E. R. D. et al. 2013 Biological cost in Mycobacterium tuberculosis with mutations in the rpsL, rrs, rpoB and katG genes. Tuberculosis 94, 150–154.CrossRefGoogle Scholar
  43. Sreevatsan S., Pan X., Stockbauer K. E., Connell K. D., Kreiswirth B. N., Whittam T. S. et al. 1997 Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionary recent global dissemination. Proc. Natl. Acad. Sci. USA 94, 9869–9874.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Stucki D., Ballif M., Bodmer T., Coscolla M., Maurer A., Droz S. et al. 2015 Strain-specific single nucleotide polymorphism typing combined with targeted whole-genome sequencing. J. Infect. Dis. 211, 1306–1316.CrossRefPubMedGoogle Scholar
  45. Unissa A. N., Narayanan S. and Selvakumar N. 2011 Virulence in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from South India. Int. J. Mol. Clin. Microbiol. 1, 87–96.Google Scholar
  46. van der Spuy G. D., Kremer K., Ndabambi S. L., Beyers N., Dunbar R., Marais B. J. et al. 2009 Changing Mycobacterium tuberculosis population highlights clade-specific pathogenic characteristics. Tuberculosis 89, 120–125.CrossRefPubMedGoogle Scholar
  47. Victor T. C., Streicher E. M., Kewley C., Jordaan A. M., van der Spuy G. D., Bosman M. et al. 2007 Spread of an emerging Mycobacterium tuberculosis drug-resistant strain in the Western Cape of South Africa. Int. J. Tuberc. Lung. Dis. 11, 195–201.PubMedGoogle Scholar
  48. WHO 2014 Global Tuberculosis Report.
  49. Wu S., Howard S. T., Lakey D. L., Kipnis A., Samten B., Safi H. et al. 2004 The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo. Mol. Microbiol. 51, 1551–1562.CrossRefPubMedGoogle Scholar

Copyright information

© Indian Academy of Sciences 2017

Authors and Affiliations

  1. 1.Medical Microbiology and Infection Control, School of Laboratory Medicine and Medical SciencesUniversity of KwaZulu-NatalCongellaSouth Africa
  2. 2.DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, and SAMRC Centre for Tuberculosis ResearchDivision of Molecular Biology and Human Genetics, Faculty of Medicine and Health SciencesCape TownSouth Africa

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