Background: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.

The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.

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 streptomycinsusceptible 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.

The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB)

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.Streptomycin (STR), the first antibiotic used to control tuberculosis, acts by binding to the 30S ribosomal subunit, inhibiting polypeptide synthesis (5, 24). Mutations in genes rpsL and rrs, encoding the ribosomal protein S12 and the 16S rRNA, respectively, are responsible for most of the high-level STR resistance in Mycobacterium tuberculosis (7,9,24). However, a low level of resistance is found in approximately one-third of the clinical isolates resistant to STR without mutations in these genes (7,11,17). Recently, a new STR resistance locus (gidB) that encodes a conserved 7-methylguanosine methyltransferase specific for the 16S rRNA was found to confer a low level of STR resistance (12).Also possibly involved with a low level of STR resistance is the efflux system. This system can grant to bacteria a mechanism which favors their survival in a hostile environment (e.g., the presence of antibiotics), and those bacteria that overexpress efflux pumps (EPs) can be selected (23). It has been shown that the M. tuberculosis genome encodes multiple putative EPs (2, 6), and reports have suggested that EPs may also be involved in transporting fluoroquinolones, aminoglycosides, tetracycline, and possibly isoniazid and ethambutol (4,14,18,22).In this study we have evaluated the possible role of the efflux mechanism as a molecular basis of STR resistance in clinical isolates of M. tuberculosis, using carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil as efflux pump inhibitors (EPI) (1, 10).A total of 79 clinical isolates from the state of Rio Grande do Sul, Brazil, were studied. Resistance to drugs other than STR was found in 62 isolates (see Table S1 in the supplemental material). The resazurin microtiter assay (13) was used for MIC determination. Microplates were divided into three equal parts: one with 7H9-oleic acid-albumin-dextrose-catalase medium, one with medium and verapamil (100 M), and one with medium and CCCP (11 M). Serial 1:2 dilutions of STR (250 g/ml to 0.25 g/ml) were performed in each column. The breakpoint to determine STR resistance was a MIC of Ն8 g/ml.DNA was isolated from mycobacterial cultures by the lysozyme/proteinase K cetyltrimethylammonium bromide procedure (21). A 306-bp fragment of the M. tuberculosis rpsL gene (GenBank accession number L08011) and the 530 loop (238 bp) and 912 region (238 bp) of the rrs gene (GenBank accession number X52917) were amplified as described by Tracevska et al. (20). A 675-bp fragment of the gene gidB (GenBank accession number AAK48404) was amplified using primers gidB1 (5ЈGTCCCTCCACTCGCCATC3Ј) and gidB2 (5ЈGCGGAGTGCGTAATGTCTC3Ј). PCR amplification was performed by the following steps: initial denaturation at 92°C ...

Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.

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.