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Antimicrobial Agents and Chemotherapy, September 2008, p. 3369-3376, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.00309-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Convergent Evolutionary Analysis Identifies Significant Mutations in Drug Resistance Targets of Mycobacterium tuberculosis ^,

Manzour Hernando Hazbón ,^1,, Alifiya S. Motiwala,^1, Magali Cavatore,¹ Michael Brimacombe,² Thomas S. Whittam,³ and David Alland^1*

Division of Infectious Disease, Department of Medicine and the Ruy V. Lourenço Center for the Study of Emerging and Re-emerging Pathogens, New Jersey Medical School,¹ Department of Preventive Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103,² Microbial Evolution Laboratory, National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824³

Received 5 March 2008/ Returned for modification 10 June 2008/ Accepted 25 June 2008

Mycobacterium tuberculosis adapts to the environment by selecting for advantageous single-nucleotide polymorphisms (SNPs). We studied whether advantageous SNPs could be distinguished from neutral mutations within genes associated with drug resistance. A total of 1,003 clinical isolates of M. tuberculosis were related phylogenetically and tested for the distribution of SNPs in putative drug resistance genes. Drug resistance-associated versus non-drug-resistance-associated SNPs in putative drug resistance genes were compared for associations with single versus multiple-branch outcomes using the chi-square and Fisher exact tests. All 286 (100%) isolates containing isoniazid (INH) resistance-associated SNPs had multibranch distributions, suggestive of multiple ancestry and convergent evolution. In contrast, all 327 (100%) isolates containing non-drug-resistance-associated SNPs were monophyletic and thus showed no evidence of convergent evolution (P < 0.001). Convergence testing was then applied to SNPs at position 481 of the iniA (Rv0342) gene and position 306 of the embB gene, both potential drug resistance targets for INH and/or ethambutol. Mutant embB306 alleles showed multibranch distributions, suggestive of convergent evolution; however, all 44 iniA(H481Q) mutations were monophyletic. In conclusion, this study validates convergence analysis as a tool for identifying mutations that cause INH resistance and explores mutations in other genes. Our results suggest that embB306 mutations are likely to confer drug resistance, while iniA(H481Q) mutations are not. This approach may be applied on a genome-wide scale to identify SNPs that impact antibiotic resistance and other types of biological fitness.

Published ahead of print on 30 June 2008.

Supplemental material for this article may be found at http://aac.asm.org/.

M.H.H. and A.S.M. contributed equally to this study.

Present address: Walter Reed Army Institute of Research, Silver Spring, Maryland.