Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, February 2004, p. 596-601, Vol. 48, No. 2
0066-4804/04/$08.00+0 DOI: 10.1128/AAC.48.2.596-601.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Multiplex PCR Amplimer Conformation Analysis for Rapid Detection of gyrA Mutations in Fluoroquinolone-Resistant Mycobacterium tuberculosis Clinical Isolates
Augustine F. B. Cheng,1* Wing W. Yew,2 Edward W. C. Chan,1 Miu L. Chin,1 Mamie M. M. Hui,1 and Raphael C. Y. Chan1
Department of Microbiology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories,1
Tuberculosis and Chest Unit, Grantham Hospital, Aberdeen, Hong Kong, China2
Received 6 July 2003/
Returned for modification 22 August 2003/
Accepted 27 October 2003
A new strategy known as multiplex PCR amplimer conformation was developed for detection of mutation in the gyrA gene of 138 clinical isolates of Mycobacterium tuberculosis. The method generated a single-stranded and heteroduplex DNA banding pattern of multiplex PCR amplimers of the region of interest that was extremely sensitive to specific mutations, thus enabling much more sensitive and reliable mutation analysis compared to the standard single-stranded conformation polymorphism technique. The genetic profiles of the gyrA gene of the 138 isolates as detected by MPAC were confirmed by nucleotide sequencing and were found to correlate strongly with the in vitro susceptibilities of the mutant strains to six fluoroquinolones (ofloxacin, levofloxacin, sparfloxacin, moxifloxacin, gatifloxacin, and sitafloxacin). All 32 isolates that contained gyrA mutations exhibited cross-resistance to the six fluoroquinolones (ofloxacin MIC for 90% of strains > 16 mg/liter), although moxifloxacin, gatifloxacin, and sitafloxacin (MIC for 90% of strains
4 mg/liter) were apparently more active than ofloxacin, levofloxacin, and sparfloxacin (MIC for 90% of strains
16 mg/liter). All gyrA mutations were clustered in codons 90, 91, and 94, and aspartic acid 94 was most frequently mutated. Twenty-three isolates without gyrA mutations were also found to exhibit reduced susceptibility to ofloxacin (MIC for 90% of strains = 4 mg/liter), but largely remained susceptible to other drugs (MIC for 90% of strains
1 mg/liter). Another 83 isolates without mutations were fully susceptible to all six fluoroquinolones (ofloxacin MIC for 90% of strains = 1 mg/liter). In conclusion, high-level phenotypic resistance to fluoroquinolones among M. tuberculosis clinical isolates, which appears to be predominantly due to gyrA mutations, may be readily detected by genotyping techniques such as multiplex PCR amplimer conformation.
* Corresponding author. Mailing address: Department of Microbiology, Chinese University of Hong Kong, Prince of Wales Hospital, 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong SAR, China. Phone: (852) 2632 3333. Fax: (852) 2647 3227. E-mail:
a-cheng{at}cuhk.edu.hk.
Antimicrobial Agents and Chemotherapy, February 2004, p. 596-601, Vol. 48, No. 2
0066-4804/04/$08.00+0 DOI: 10.1128/AAC.48.2.596-601.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
This article has been cited by other articles:
-
Villar, R., Vicente, E., Solano, B., Perez-Silanes, S., Aldana, I., Maddry, J. A., Lenaerts, A. J., Franzblau, S. G., Cho, S.-H., Monge, A., Goldman, R. C.
(2008). In vitro and in vivo antimycobacterial activities of ketone and amide derivatives of quinoxaline 1,4-di-N-oxide. J Antimicrob Chemother
62: 547-554
[Abstract]
[Full Text]
-
Almeida, D., Nuermberger, E., Tyagi, S., Bishai, W. R., Grosset, J.
(2007). In Vivo Validation of the Mutant Selection Window Hypothesis with Moxifloxacin in a Murine Model of Tuberculosis. Antimicrob. Agents Chemother.
51: 4261-4266
[Abstract]
[Full Text]
-
Chan, R. C. Y., Hui, M., Chan, E. W. C., Au, T. K., Chin, M. L., Yip, C. K., AuYeang, C. K. W., Yeung, C. Y. L., Kam, K. M., Yip, P. C. W., Cheng, A. F. B.
(2007). Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong. J Antimicrob Chemother
59: 866-873
[Abstract]
[Full Text]
-
Matrat, S., Veziris, N., Mayer, C., Jarlier, V., Truffot-Pernot, C., Camuset, J., Bouvet, E., Cambau, E., Aubry, A.
(2006). Functional Analysis of DNA Gyrase Mutant Enzymes Carrying Mutations at Position 88 in the A Subunit Found in Clinical Strains of Mycobacterium tuberculosis Resistant to Fluoroquinolones. Antimicrob. Agents Chemother.
50: 4170-4173
[Abstract]
[Full Text]
-
Shi, R., Zhang, J., Li, C., Kazumi, Y., Sugawara, I.
(2006). Emergence of Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates from China as Determined by gyrA Mutation Analysis Using Denaturing High-Pressure Liquid Chromatography and DNA Sequencing. J. Clin. Microbiol.
44: 4566-4568
[Abstract]
[Full Text]
-
Aubry, A., Veziris, N., Cambau, E., Truffot-Pernot, C., Jarlier, V., Fisher, L. M.
(2006). Novel Gyrase Mutations in Quinolone-Resistant and -Hypersusceptible Clinical Isolates of Mycobacterium tuberculosis: Functional Analysis of Mutant Enzymes. Antimicrob. Agents Chemother.
50: 104-112
[Abstract]
[Full Text]
-
Huang, T.-S., Kunin, C. M., Shin-Jung Lee, S., Chen, Y.-S., Tu, H.-Z., Liu, Y.-C.
(2005). Trends in fluoroquinolone resistance of Mycobacterium tuberculosis complex in a Taiwanese medical centre: 1995-2003. J Antimicrob Chemother
56: 1058-1062
[Abstract]
[Full Text]
-
Giannoni, F., Iona, E., Sementilli, F., Brunori, L., Pardini, M., Migliori, G. B., Orefici, G., Fattorini, L.
(2005). Evaluation of a New Line Probe Assay for Rapid Identification of gyrA Mutations in Mycobacterium tuberculosis. Antimicrob. Agents Chemother.
49: 2928-2933
[Abstract]
[Full Text]
-
Mohapatra, P. R.
(2004). Fluoroquinolones in Multidrug-Resistant Tuberculosis. Am. J. Respir. Crit. Care Med.
170: 920-921
[Full Text]
-
Di Perri, G., Bonora, S.
(2004). Which agents should we use for the treatment of multidrug-resistant Mycobacterium tuberculosis?. J Antimicrob Chemother
54: 593-602
[Abstract]
[Full Text]