From multidrug-resistant to extensively drug-resistant tuberculosis in Lisbon, Portugal: the stepwise mode of resistance acquisition.
OBJECTIVE
The development and transmission of extensively drug-resistant (XDR) tuberculosis (TB) constitutes a serious threat to the effective control of TB in several countries. Here, in an attempt to further elucidate the dynamics of the acquisition of resistance to second-line drugs and investigate an eventual role for eis promoter mutations in aminoglycoside resistance, we have studied a set of multidrug-resistant (MDR)/XDR-TB isolates circulating in Lisbon, Portugal.


METHODS
Forty-four MDR-TB or XDR-TB isolates were genotyped and screened for mutations in genes associated with second-line drug resistance, namely tlyA, gyrA, rrs and eis.


RESULTS
The most prevalent mutations found in each gene were Ins755GT in tlyA, A1401G in rrs, G-10A in eis and S91P in gyrA. Additionally, two genetic clusters were found in this study: Lisboa3 and Q1. The characteristic mutational profile found among recent XDR-TB circulating in Lisbon was also found in MDR-TB strains isolated in the 1990s. Also investigated was the resistance level conferred by eis G-10A mutations, revealing that eis G-10A mutations may result in amikacin resistance undetectable by widely used phenotypic assays.


CONCLUSIONS
The analysis of the distribution of the mutations found by genetic clustering showed that in the Q1 cluster, two mutations, gyrA D94A and rrs A1401G, were enough to ensure development of XDR-TB from an MDR strain. Moreover, in the Lisboa3 cluster it was possible to elaborate a model in which the development of low-level kanamycin resistance was at the origin of the emergence of XDR-TB strains that can be discriminated by tlyA mutations.
Centro de Patogénese Molecular, URIA,  Faculdade de Farmácia da Universidade de Lisboa, Lisboa,  Portugal.
João
J
Perdigão
Rita
R
Macedo
Carla
C
Silva
Diana
D
Machado
Isabel
I
Couto
Miguel
M
Viveiros
Luisa
L
Jordao
Isabel
I
Portugal
D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011174:Portugal / Q000453:epidemiology; D018088:Tuberculosis, Multidrug-Resistant / Q000453:epidemiology / Q000235:genetics* / Q000382:microbiology*