Genetic analysis of extensively drug-resistant Mycobacterium tuberculosis strains in Lisbon, Portugal.
OBJECTIVE
Extensively drug-resistant (XDR) tuberculosis (TB) threatens the global control of TB worldwide. Lisbon has a high XDR-TB rate [50% of the multidrug-resistant tuberculosis (MDR-TB)], which is mainly associated with Lisboa family strains. Few studies have addressed the identification of mutations associated with resistance to second-line injectable drugs, and the relative frequency of such mutations varies geographically. The aim of this study was to characterize the genetic changes associated with the high number of XDR-TB cases in Lisbon.


METHODS
In the present study we analysed 26 XDR-TB clinical isolates. The gyrA, tlyA and rrs genes were screened for mutations that could be responsible for resistance to fluoroquinolones and second-line injectable drugs. Moreover, the strains under analysis were also genotyped by MIRU-VNTR ('mycobacterial interspersed repetitive unit-variable number of tandem repeats').


RESULTS
The mutational analysis identified the most frequent mutations in the resistance-associated genes: S91P in gyrA (42.3%); A1401G in rrs (30.8%); and Ins755GT in tlyA (42.3%). The occurrence of mutations in rrs was associated with the non-occurrence of mutations in tlyA. The genotypic analysis revealed that the strains were highly clonal, belonging to one of two MIRU-VNTR clusters, with the largest belonging to the Lisboa family. Association between mutations in gyrA and rrs or tlyA was verified.


CONCLUSIONS
The association of specific mutations highlighted the strains' high clonality and indicates recent XDR-TB transmission. In addition, the identification of the most frequent resistance-associated mutations will be invaluable in applying XDR-TB molecular detection tests in the region in the near future.
Perdigão
João
J
Centro de Patogénese Molecular, URIA, Universidade de Lisboa, 1649-003 Lisbon, Portugal.
Macedo
Rita
R
Malaquias
Ana
A
Ferreira
Ana
A
Brum
Laura
L
Portugal
Isabel
I
D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D016000:Cluster Analysis; D016172:DNA Fingerprinting; D027081:DNA Gyrase / Q000235:genetics; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D054908:Extensively Drug-Resistant Tuberculosis / Q000382:microbiology*; D005838:Genotype; D006801:Humans; D016254:Mutagenesis, Insertional; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D017354:Point Mutation; D011174:Portugal; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C112817:TlyA protein, Mycobacterium tuberculosis; D027081:DNA Gyrase