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

Abstract

A high rate of double point mutations in gyrA (56% of 87 ofloxacin-resistant Mycobacterium tuberculosis clinical isolates) indicates the emergence of fluoroquinolone resistance. This is the first report to describe denaturing high-pressure liquid chromatography analysis of mutations in gyrA of M. tuberculosis in a large number of clinical isolates.

FIG. 1.

Nucleotide sequence and missense mutations within the QRDRs of gyrA. All the isolates contain a naturally occurring polymorphism, codon 95 AGC→ACC. Seventy-three (84%) of the 87 ofloxacin-resistant clinical isolates were found to carry a codon 94 mutation, and 49 (56%) were found to harbor a double point mutation.

FIG. 2.

Ofloxacin MIC relative to the gyrA QRDR allele spectrum. Ofloxacin MICs are given above each panel. n, number in each MIC group. Bars indicate the percentage represented by each allele.

FIG. 3.

DHPLC patterns of gyrA genes of the 109 clinical isolates when M. tuberculosis H37Rv was used as a reference strain. Patterns A, B, and C are shown as examples (details are shown in Fig. 4). W, H37Rv wild type. When M. tuberculosis Erdman was used as a reference strain, A, B, and C were changed to A1, B1, and C1, respectively. W1 indicates the M. tuberculosis Erdman wild type. Isolates (MIC less than 2 μg/ml) that harbored only the codon 95 ACC natural polymorphism with no other mutation in gyrA QRDRs showed the wild-type pattern C1.

FIG. 4.

Specific DHPLC pattern of each gyrA QRDR mutation type.