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Antimicrobial Agents and Chemotherapy, June 2005, p. 2200-2209, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2200-2209.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Detection of rpoB Mutations Associated with Rifampin Resistance in Mycobacterium tuberculosis Using Denaturing Gradient Gel Electrophoresis

Mark T. McCammon,1* John S. Gillette,1 Derek P. Thomas,1 Srinivas V. Ramaswamy,3,{dagger} Edward A. Graviss,3 Barry N. Kreiswirth,4 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, Texas,3 Public Health Research Institute, Newark, New Jersey4

Received 18 November 2004/ Returned for modification 22 December 2004/ Accepted 23 February 2005

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of rpoB were developed. These primer sets were used to scan rpoB for DNA alterations in 296 M. tuberculosis patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within rpoB were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable rpoB mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in M. tuberculosis.

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

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

Antimicrobial Agents and Chemotherapy, June 2005, p. 2200-2209, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2200-2209.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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