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Antimicrobial Agents and Chemotherapy, June 2005, p. 2210-2217, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2210-2217.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

Mark T. McCammon,^1* John S. Gillette,¹ Derek P. Thomas,¹ Srinivas V. Ramaswamy,^3, Ishmael I. Rosas,¹ Edward A. Graviss,³ Jan Vijg,² and Teresa N. Quitugua¹

Department of Microbiology and Immunology,¹ Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas,² Houston Tuberculosis Initiative, Department of Pathology, Baylor College of Medicine, Houston, Texas³

Received 13 December 2004/ Returned for modification 12 January 2005/ Accepted 8 February 2005

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90°C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis.

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* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, STCBM Building, 15355 Lambda Dr., San Antonio, TX 78245. Phone: (210) 562-5037. Fax: (210) 562-5041. E-mail: mccammon{at}uthscsa.edu.

Present address: Channing Laboratory, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts.

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Antimicrobial Agents and Chemotherapy, June 2005, p. 2210-2217, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2210-2217.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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