Use of a high-density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis.
A Mycobacterium high-density DNA probe array designed to detect rpoB mutations conferring rifampicin resistance in Mycobacterium tuberculosis was evaluated. The rpoB hybridisation patterns produced by 41 susceptible (RifS) and 59 rifampicin-resistant (RifR) clinical isolates of M. tuberculosis were compared with the results of conventional dideoxynucleotide sequencing of the rpoB gene. For all the RifR isolates, the rpoB hybridisation patterns correlated with the rpoB sequencing results. Among the 59 isolates, 11 distinct amino-acid changes were detected by the DNA probe array. Of these, 36 (61%) corresponded to replacement of the serine residue found in position 531 (S531L in 34 isolates and S531W in two isolates), 16 (27%) affected histidine 526 (five H526D, five H526Y, four H526L, one H526N and one H526R), four (6.8%) replaced aspartate 516 with a valine, and one (1.7%) replaced glutamine 513 with a leucine. Deletion of the asparagine residue at position 519 was detected in one isolate susceptible to rifampicin, but yielding c. 0.1% resistant colonies on rifampicin-containing medium. No mutation was detected in the rpoB region from one isolate yielding c. 5% of resistant colonies on rifampicin-containing medium. Finally, a D516Y substitution was detected in association with an unexpected mutation, G523W, not tiled on the DNA probe array, but which could be detected by analysing the hybridisation pattern obtained with the wild-type probes covering codon 523. In conclusion, the Mycobacterium probe array is a promising approach to rapid detection of mutations involved in rifampicin resistance in M. tuberculosis.
Sougakoff
W
W
Laboratoire de Bactériologie-Hygiène, Faculté de Médecine Pitié-Salpêtrière, Université Pierre et Marie Curie, Paris, France. sougakof@chups.jussieu.fr
Rodrigue
M
M
Truffot-Pernot
C
C
Renard
M
M
Durin
N
N
Szpytma
M
M
Vachon
R
R
Troesch
A
A
Jarlier
V
V
D019943:Amino Acid Substitution; D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D015342:DNA Probes; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods; D012293:Rifampin / Q000494:pharmacology*; D014397:Tuberculosis, Pulmonary / Q000382:microbiology
D000904:Antibiotics, Antitubercular; D015342:DNA Probes; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin