Streptomycin resistance and lineage-specific polymorphisms in Mycobacterium tuberculosis gidB gene.

Mutations related to streptomycin resistance in the rpsL and rrs genes are well known and can explain about 70% of this phenotypic resistance. Recently, the gidB gene was found to be associated with low-level streptomycin resistance in Mycobacterium tuberculosis. Mutations in gidB have been reported with high frequency, and this gene appears to be very polymorphic, with frameshift and point mutations occurring in streptomycin-susceptible and streptomycin-resistant strains. In this study, mutations in gidB appeared in 27% of streptomycin-resistant strains that contained no mutations in the rpsL or rrs genes, and they were associated with low-level streptomycin resistance. However, the association of certain mutations in gidB with streptomycin resistance needs to be further investigated, as we also found mutations in gidB in streptomycin-susceptible strains. This occurred only when the strain was resistant to rifampin and isoniazid. Two specific mutations appeared very frequently in this and other studies of streptomycin-susceptible and -resistant strains; these mutations were not considered related to streptomycin resistance, but as a polymorphism. We stratified the strains according to the different phylogenetic lineages and showed that the gidB(16) polymorphism (16G allele) was exclusively present in the Latin American-Mediterranean (LAM) genotype, while the gidB(92) polymorphism (92C allele) was associated with the Beijing lineage in another population. In the sample studied, the two characterized single-nucleotide polymorphisms could distinguish LAM and Beijing lineages from the other lineages.

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay.

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Correlations of mutations in katG, oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America.

BACKGROUND: Mutations associated with resistance to rifampin or streptomycin have been reported for W/Beijing and Latin American Mediterranean (LAM) strain families of Mycobacterium tuberculosis. A few studies with limited sample sizes have separately evaluated mutations in katG, ahpC and inhA genes that are associated with isoniazid (INH) resistance. Increasing prevalence of INH resistance, especially in high tuberculosis (TB) prevalent countries is worsening the burden of TB control programs, since similar transmission rates are noted for INH susceptible and resistant M. tuberculosis strains. RESULTS: We, therefore, conducted a comprehensive evaluation of INH resistant M. tuberculosis strains (n = 224) from three South American countries with high burden of drug resistant TB to characterize mutations in katG, ahpC and inhA gene loci and correlate with minimal inhibitory concentrations (MIC) levels and spoligotype strain family. Mutations in katG were observed in 181 (80.8%) of the isolates of which 178 (98.3%) was contributed by the katG S315T mutation. Additional mutations seen included oxyR-ahpC; inhA regulatory region and inhA structural gene. The S315T katG mutation was significantly more likely to be associated with MIC for INH >or=2 microg/mL. The S315T katG mutation was also more frequent in Haarlem family strains than LAM (n = 81) and T strain families. CONCLUSION: Our data suggests that genetic screening for the S315T katG mutation may provide rapid information for anti-TB regimen selection, epidemiological monitoring of INH resistance and, possibly, to track transmission of INH resistant strains.

Identification of mutations related to streptomycin resistance in clinical isolates of Mycobacterium tuberculosis and possible involvement of efflux mechanism.

The MIC for streptomycin in the presence of efflux pump (EP) inhibitors and the sequencing of rpsL, rrs, and gidB genes provided evidence for the possible participation of EP in low-level streptomycin (STR) resistance of some isolates without mutations. Mutation in the gidB gene and an EP could act synergistically to confer low STR resistance.

In house reverse line hybridization assay for rapid detection of susceptibility to rifampicin in isolates of Mycobacterium tuberculosis.

We developed a Reverse Hybridization Assay (RHA) slightly modified from the Rifoligotyping assay and analyzed the presence of mutations in a specific region of rpoB gene in 157 isolates (90 rifampin-resistant and 67 rifampin sensitive) of Mycobacterium tuberculosis from patients attended in South and Southeast region of Brazil. Comparing to standardized drug susceptibility testing results, the sensitivity and specificity of the RHA was respectively 93% (95% IC: 86.6%-97.2%) and 100% respectively. Additionally, a high agreement (kappa coefficient 95%) between the RHA assay and sequencing was obtained. Among the 90 rifampicin-resistant isolates, RHA identified point mutations in the following codons: 42 isolates (46.6%) in 531; 29 isolates (32.2%) in 526, 6 isolates (6.7%) in 516, 3 isolates (3.3%) in 522, 2 isolates (2.2%) in 515, 514, 513 and 1 isolate (1.1%) in 511, 524 and 525. Mutations in different codons were simultaneously identified in 8 isolates (8.9%). The RHA used in the present study had a high accuracy and can be rapidly performed. However, more reproducible hybridization conditions should be looked for to increase reliability of mutant probe interpretation.

Outbreaks due to Mycobacterium abscessus subsp. bolletii in southern Brazil: persistence of a single clone from 2007 to 2011.

Outbreaks associated with rapidly growing mycobacteria (RGM) have been increasingly reported worldwide, including in Brazil. Among the RGM, the Mycobacterium abscessus complex is the most pathogenic and related to multidrug resistance. The aim of this study was to evaluate the antimicrobial susceptibility and molecular profile of RGM isolates involved in new postsurgical infection outbreaks in Brazil since 2007. Of the 109 cases reported in the state of Rio Grande do Sul between 2007 and 2011, 43 (39 %) had confirmed mycobacterial growth in culture. Clinical isolates were obtained from biopsy specimens or abscess aspirates. PRA-hsp65 restriction pattern identified the isolates as M. abscessus type 2, and partial rpoB sequencing confirmed the identification as M. abscessus subsp. bolletii. All isolates were susceptible to amikacin and resistant to ciprofloxacin, doxycycline, sulfamethoxazole, moxifloxacin and tobramycin. Most isolates (72 %) were fully susceptible to cefoxitin but six isolates (14 %) were fully resistant to clarithromycin. The latter differed from the susceptibility profiles of the previously described BRA100 clone from other Brazilian regions. Nevertheless, pulsed-field gel electrophoresis analysis revealed that these isolates belonged to a single BRA100 clone. In conclusion, our study reports the persistence of an emergent single and highly resistant clone of M. abscessus subsp. bolletii for several years even after national implementation of infection control measures.

Biological cost in Mycobacterium tuberculosis with mutations in the rpsL, rrs, rpoB, and katG genes.

When bacteria develop drug-resistant mutations, there is often an associated biological cost; however, some strains can exhibit low- or no-cost mutations. In the present study, a quantitative resazurin reduction assay was used to measure the biological cost of Mycobacterium tuberculosis isolates that contained different mutations in the rpsL, rrs, rpoB, and katG genes, and showed different resistance profiles. Biological costs were determined by comparing the growth curves of drug-resistant isolates with drug-susceptible strains. Some strains, such as those with rpoB mutations other than S531L and strains with mutations in all of the studied genes, grew more slowly than did drug-susceptible strains. However, some strains grew more quickly than drug-susceptible strains, such as those that had only the rpsL K43R mutation. Strains with the mutation katG S315T presented heterogeneous biological costs. When analyzed individually, strains with the mutations rpsL43/katG315, rpoB531, and rpoB531/katG315 grew faster than drug-susceptible strains. The results suggest that some strains with the most common mutations correlated to a high resistance toward streptomycin, isoniazid and rifampicin can grow as well as or better than susceptible strains.

Characteristics of multidrug-resistant Mycobacterium tuberculosis in southern Brazil.

A major threat to tuberculosis (TB) control programs is the emergence of drug resistant Mycobacterium tuberculosis strains that cause TB that cannot be cured by standard anti-TB drug regimens. Because few data exist on MDR-TB in this region of the country, we performed an epidemiologic study that combined conventional and molecular analysis of MDR-TB cases from Rio Grande do Sul (RS) that were diagnosed in this period and included cases that were under treatment with second line drug schemes. Included were 121 MDR cases and sequencing of rpoB and katG showed that 106 (87.6%) strains were mutated in rpoB and 97 (80.2%) in katG. Spoligotyping demonstrated that the LAM genotype was predominant (n = 70, 57.8%) and included the largest group composed by 22 (18.1%) strains with the LAM5 ST93 genotype. Other main genotypes belonged to the families T (n = 22, 18.2%), U family (n = 16, 13.2%), Haarlem (n = 5, 4.1%) and X (n = 1, 0.8%). Genotyping by IS6110-RFLP analysis showed 51 distinct fingerprints, 38 (31.4%) of these observed only once and the other 13 patterns being shared among the rest of the isolates (n = 83, 68.6%). Among the 22 strains that were LAM5 ST93, only two had different IS6110-RFLP genotypes. In conclusion, there exists a high degree of M. Tuberculosis genotype clustering among MDR-TB cases in Rio Grande do Sul. Moreover, we observed a large MDR-TB outbreak.

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3 percent and 98.1 percent, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6 percent and 100 percent, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100 percent consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Mutations in katG, inhA, and ahpC genes of Brazilian isoniazid-resistant isolates of Mycobacterium tuberculosis.

The presence of mutations in specific regions of the katG, inhA, and ahpC genes was analyzed with 69 Mycobacterium tuberculosis isoniazid-resistant isolates from three Brazilian states. Point mutations in codon 315 of the katG gene were observed in 87.1, 60.9, and 60% of the isolates from Rio Grande do Sul, Rio de Janeiro, and São Paulo, respectively. Mutations in the inhA gene were identified only in one isolate from RJ State, and the ahpC promoter region revealed mutations in distinct positions in 12.9, 21.7, and 6.7% of the isolates from RS, RJ and SP, respectively.