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. 2005 Mar;43(3):1220-7.
doi: 10.1128/JCM.43.3.1220-1227.2005.

Systematic molecular characterization of multidrug-resistant Mycobacterium tuberculosis complex isolates from Spain

S Samper  1 , M J Iglesias, M J Rabanaque, L I Gómez, M C Lafoz, M S Jiménez, A Ortega, M A Lezcano, D Van Soolingen, C Martín; Spanish Working Group on MDR-TB
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Systematic molecular characterization of multidrug-resistant Mycobacterium tuberculosis complex isolates from Spain

S Samper et al. J Clin Microbiol. 2005 Mar.

Abstract

We used spoligotyping and restriction fragment length polymorphism (RFLP) of the IS6110-insertion sequence to study the molecular epidemiology of multidrug-resistant (MDR) tuberculosis in Spain. We analyzed 180 Mycobacterium tuberculosis complex isolates collected between January 1998 and December 2000. Consecutive isolates from the same patients (n = 23) always had identical genotypes, meaning that no cases of reinfection occurred. A total of 105 isolates (58.3%) had unique RFLP patterns, whereas 75 isolates (41.7%) were in 20 different RFLP clusters. Characterization of the katG and rpoB genes showed that 14 strains included in the RFLP clusters did not actually cluster. Only 33.8% of the strains isolated were suggestive of MDR transmission, a frequency lower than that for susceptible strains in Spain (46.6%). We found that the Beijing/W genotype, which is prevalent worldwide, was significantly associated with immigrants. The 22 isolates in the largest cluster corresponded to the Mycobacterium bovis strain responsible for two nosocomial MDR outbreaks in Spain.

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Figures

FIG. 1.
FIG. 1.
Dendrogram based on the 20 different IS6110-RFLP patterns of the clustered M. tuberculosis complex isolates. CN, cluster name assigned by the University of Zaragoza; EU, name of the cluster in the European Database in the RIVM; NI, number of isolates in each RFLP cluster.
FIG. 2.
FIG. 2.
Dendrogram showing the redefined clusters (excluding the B strain cluster) based on the results of the three genetic techniques: IS6110-RFLP, rifotyping, and PCR-RFLP of the katG gene. Isolates with identical IS6110-RFLP patterns, but different mutations conferring resistance, were transmitted before the acquisition of drug resistance. RFLP clusters tb11, tb12, tb14, and tb17 disappeared, and tb9 was divided into tb9a and tb9b. The methods are shown in the following order: RFLP-IS6110, rpoB mutations (lanes 1 through 5, wild-type sequence of the gene; lane 6, Asp516Gly mutant; lane 7, 515-to-516 insertion; lane 8, His526Tyr mutant; lane 9, Arg529Gln mutant; lane 10, Ser531Leu mutant; lane 11, Ser531Tyr mutant), and katG (mutations are represented by numerical values, as follows: 0, wild-type; 1, Ser315Thr; 2, Arg463Leu; 3, Ser315Thr and Arg463Leu). CN, number of the RFLP cluster. CB, country of birth (1, Spain; 2, foreign country).
FIG. 3.
FIG. 3.
MspI restriction fragments of the 620-bp portion of the katG gene of six MDR M. tuberculosis isolates. Lanes: M, 10-bp marker; w, wild type (bands at 228, 153, 137, and 65 bp); 1, codon Ser315Thr (bands at 228, 137, 132, and 65 bp); 2, codon Arg463Leu (bands at 228, 202, and 153 bp); 3, codons 315Thr and 463Leu (bands at 228, 202, and 132 bp).
FIG. 4.
FIG. 4.
Age distribution of the 167 MDR TB patients (for whom age was known) according to transmission status. We considered transmission cases to be those with the same RFLP pattern and the same mutations in the katG and rpoB genes.
FIG. 5.
FIG. 5.
Genotyping analysis of strains with the Beijing/W genotype. The dendrogram based on RFLP-IS6110 patterns shared 65% similarity. The 11 strains harbored leucine at codon 463 of the katG gene (2, codon 463Leu; 3, codons 315Thr and 463Leu). Three isolates had identical RFLP patterns, but one of them (asterisked) had different mutations conferring resistance to rifampin.
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