Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis clinical isolates in the area of Barcelona.

Objectives: To determine the proportion and type of mutations in Mycobacterium tuberculosis isolates resistant to streptomycin, and their relationship with the level of resistance and with the epidemiological molecular pattern of the isolates.

Methods: Sixty-nine streptomycin-resistant isolates from a M. tuberculosis strain collection (1995-2005) from Barcelona were studied. The MIC of streptomycin for each isolate was determined using the proportions method with Middlebrook 7H11 medium. The entire rpsL gene and two specific fragments of the rrs gene (the 530 loop and the 912 region) were sequenced. IS6110-restriction fragment length polymorphism and spoligotyping were performed in each isolate.

Results: Twenty-six (26/69, 37.7%) streptomycin-resistant isolates presented a mutation in either the rpsL gene and/or the rrs530 loop, with no mutation in the rrs912 region. Seventeen (24.6%) isolates showed rpsL mutations (codons 43 and 88) associated with high MIC levels. Nine (13.0%) isolates had alterations in the rrs gene (A513T, A513C and C516T). Nineteen isolates (19/64, 29.7%) were classified into seven clusters (containing 2-5 isolates per cluster). Nineteen different spoligotype patterns were found. All the LAM3 spoligotype isolates (10/67, 14.9%) were associated with a C491T change in the rrs gene, being also observed in all LAM3 streptomycin-susceptible isolates.

Conclusions: Mutations in the rpsL and rrs genes were detected in 37.7% of streptomycin-resistant M. tuberculosis isolates. High-level resistance was associated with mutations in the rpsL gene, whereas wild-type isolates showed low MIC levels. The presence of the C491T substitution in the rrs gene in streptomycin-susceptible and -resistant isolates demonstrates that this change is an epidemiological marker associated with LAM3 sublineage.