Detection of ethambutol-resistant Mycobacterium tuberculosis strains by multiplex allele-specific PCR assay targeting embB306 mutations

We describe a multiplex allele-specific (MAS)-PCR assay to detect simultaneously mutations in the first and third bases of the embB gene codon 306ATG. These mutations are known to confer ethambutol (EMB) resistance in the majority of clinical Mycobacterium tuberculosis isolates worldwide. The mutated bases are revealed depending on the presence or absence of the respective indicative fragments amplified from the embB306 wild-type allele. Initially optimized on purified DNA samples, the assay was tested on crude cell lysates and auramine-stained sputum slide DNA preparations with the same reproducibility and interpretability of the generated profiles in agarose gel electrophoresis. Since EMB resistance is generally linked to multiple-drug resistance (MDR), the MAS-PCR assay for EMB resistance detection can be used in clinical laboratory practice in areas with a high prevalence and a high transmission rate of MDR-EMB-resistant tuberculosis.     

Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations

A study set comprised 44 Mycobacterium tuberculosis strains of the Beijing family selected for their representativeness among those previously characterized by IS6110-RFLP and spoligotyping (Northwest Russia, 1997 to 2003). In the present study, these strains were subjected to mycobacterial interspersed repetitive units (MIRU) typing to assess a discriminatory power of the 12-MIRU-loci scheme (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). The 44 Russian Beijing strains were subdivided into 12 MIRU types with identical profiles: 10 unique strains and two major types shared by 10 and 24 strains. Thus, basically, two distinct sublineages appear to shape the evolution of the Beijing strains in Russia. Most of the MIRU loci were found to be (almost) monomorphic in the Russian Beijing strains; the Hunter-Gaston discriminatory index (HGDI) for all 12 loci taken together was 0.65, whereas MIRU26 (the most variable in our study) showed a moderate level of discrimination (0.49). The results were compared against all available published MIRU profiles of Beijing strains from Russia (3 strains) and other geographic areas (51 strains in total), including South Africa (38 strains), East Asia (7 strains), and the United States (4 strains). A UPGMA (unweighted pair-group method with arithmetic averages)-based tree was constructed. Interestingly, no MIRU types were shared by Russian and South African strains (the two largest samples in this analysis), whereas both major Russian types included also isolates from other locations (United States and/or East Asia). This implies the evolution of the Beijing genotype to be generally strictly clonal, although a possibility of a convergent evolution of the MIRU loci cannot be excluded. We propose a dissemination of the prevailing local Beijing clones to have started earlier in South Africa rather than in Russia since more monomorphic loci were identified in Russian samples than in South African samples (mean HGDI scores, 0.08 versus 0.17). To conclude, we suggest to use a limited number of MIRUs for preliminary subdivision of Beijing strains in Russian (loci 26 + 31), South African (10 + 26 + 39), and global settings (10 + 26 + 39).     

PCR-Based Methodology for Detecting Multidrug-Resistant Strains of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Beijing Family Circulating in Russia

The Beijing genotype of Mycobacterium tuberculosis has been identified in 40–50% of the clinical isolates studied in Russia during the last decade. This genotype has been reported to be associated with multiple drug resistance and possesses some significant pathogenic properties. Therefore, early identification of such strains is of extreme importance in the timely detection of drug resistance. The present study was performed on 354 strains isolated in Russia from 1996 to 2002 and previously characterised by IS6110-restriction fragment length polymorphism (RFLP) typing and spoligotyping. These strains included 198 Beijing family strains and 156 strains of other genotypes (IS6110-RFLP profiles). A subsequent polymerase chain reaction (PCR) analysis with IS6110-derived outwardly oriented primers (IS6110-PCR) easily discriminated the Beijing strains from non-Beijing strains. The multiplex allele-specific (MAS)-PCR assays were further used to detect mutations in katG315 and rpoB531, associated with resistance to isoniazid and rifampin, respectively. The katG315 and rpoB531 mutations were found to be more prevalent among Beijing (96.8% and 77.3%) than among non-Beijing strains (85.7% and 28%). Consequently, we propose a two-step methodology based on routine PCR and simple agarose gel electrophoresis in order to detect (i) a Beijing family strain using IS6110-PCR, and, (ii) its possible resistance to the major anti-tuberculosis drugs using specific MAS-PCR assays.     

Origin and primary dispersal of the Mycobacterium tuberculosis Beijing genotype: clues from human phylogeography

We suggest that the evolution of the population structure of microbial pathogens is influenced by that of modern humans. Consequently, the timing of hallmark changes in bacterial genomes within the last 100,000 yr may be attempted by comparison with relevant human migrations. Here, we used a lineage within Mycobacterium tuberculosis, a Beijing genotype, as a model and compared its phylogeography with human demography and Y chromosome-based phylogeography. We hypothesize that two key events shaped the early history of the Beijing genotype: (1) its Upper Palaeolithic origin in the Homo sapiens sapiens K-M9 cluster in Central Asia, and (2) primary Neolithic dispersal of the secondary Beijing NTF::IS6110 lineage by Proto-Sino-Tibetan farmers within east Asia (human O-M214/M122 haplogroup). The independent introductions of the Beijing strains from east Asia to northern Eurasia and South Africa were likely historically recent, whereas their differential dissemination within these areas has been influenced by demographic and climatic factors.     

Dietary polyamines promote the growth of azoxymethane-induced aberrant crypt foci in rat colon

We have examined whether dietary polyamines influence the formation and initial growth of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rat colon. Effects of a combination of dietary polyamines at three dose levels (putrescine: 50, 280, 740 nmol/g; spermidine: 10, 261, 763 nmol/g; spermine: 1, 31, 91 nmol/g) in the polyamine-poor AIN-76A diet were studied in animals in two different experimental situations: animals treated with AOM alone and animals treated with AOM + difluoromethylornithine (DFMO), a specific inhibitor of endogenous polyamine synthesis. In both experimental situations, dietary polyamines enhanced the growth of ACF, expressed as the number of large ACF (foci with three or more aberrant crypts, ACF > or = 3), whereas the formation of ACF, expressed as the number of ACF, was apparently not altered. In animals treated with AOM alone, maximal growth enhancing effect on ACF was nearly obtained with the median level of dietary polyamine. In rats fed a low polyamine diet, basic AIN-76A, DFMO reduced the growth of AOM-induced ACF by 83%. This inhibitory effect of DFMO was counteracted by dietary polyamines in a dose- dependent manner, and it was abolished at the highest level of polyamines. In conclusion, it was demonstrated that dietary polyamines are able to enhance the growth of AOM-induced ACF. Further, dietary polyamines reversed the DFMO-caused inhibition of ACF growth, probably by compensating for the DFMO-reduced endogenous polyamine synthesis.      

Russian “Successful” Clone B0/W148 of Mycobacterium tuberculosis Beijing Genotype: a Multiplex PCR Assay for Rapid Detection and Global Screening

We describe a multiplex PCR assay to detect the Mycobacterium tuberculosis Beijing genotype variant B0/W148, which is considered a “successful” clone of M. tuberculosis, widespread in Russia. Specificity and sensitivity of the assay were 100% based on the analysis of a collection of 516 M. tuberculosis isolates of different genotypes and origins. This assay may be used for accurate and simple detection and surveillance of this clinically and epidemiologically important variant of M. tuberculosis.     

Real-Time PCR Assay for Rapid Detection of Epidemiologically and Clinically Significant Mycobacterium tuberculosis Beijing Genotype Isolates

Mycobacterium tuberculosis Beijing genotype strains are rapidly disseminating, frequently hypervirulent, and multidrug resistant. Here, we describe a method for their rapid detection by real-time PCR that targets the specific IS6110 insertion in the dnaA-dnaN genome region. The method was evaluated with a geographically and genetically diverse collection representing areas in East Asia and the former Soviet Union in which the Beijing genotype is endemic and epidemic (i.e., major foci of its global propagation) and with clinical specimens.     

应用反向线性杂交技术快速检测结核分枝杆菌利福平耐药性研究

探讨反向线性杂交技术(reverse line blot assay,RLB)在结核分枝杆菌利福平(RFP)耐药性快速检测中的应用价值.采用RLB将包含rpoB基因核心区的扩增产物与标记特异性探针的膜进行杂交,共对121株结核分枝杆菌进行RFP耐药性检测,并将结果与常规药敏实验进行比较.结果发现,121株中有71株对RFP耐药,56株为多重耐药株;采用RLB共检测到65株RFP耐药株rpoB基因核心区存在突变,其灵敏度为91.5%(65/71);50株RFP敏感株中均未检测到突变,则特异性为100%(51/51);56株多耐药菌株中,92.9%(52/56)存在rpoB基因核心区突变.因此,用RLB检测结核分枝杆菌RFP耐药株具有快速,高效,特异性和灵敏度高的优点,且可用于多耐药菌株的筛选,具有推广和潜在的临床应用价值.     

Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing, China

Background China is one of the high burden countries of Mycobacterium tuberculosis （TB） infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin （RIF） and isoniazid （INH） resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance. Methods Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide （RIFO） assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA （-15C/T） and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism （RFLP） method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups. Results Sixty-five （91.5%） of 71 RIF resistant and 52 （92.9%） of 56 multidrug-resistant （MDR, i.e. resistant to at least RIF and INH） strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively, katG315 AGC〉ACC and inhA-15C〉T mutations were found in 40 （60.6%） and 10 （15.2%） of 66 INH resistant strains, respectively; 7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into ＂modern＂ （76.6%） and ＂ancestral＂ （23.4%） group. There is no significant difference between ＂ancestral＂ and ＂modern＂ group in prevalance of drug resistance-associated gene mutations. Conclusions The hot-spot region of rpoB gene can be used as genetic marker for detection of RIF resistant strains; a combined use of both katG315 and inhA-15 can improve the detection rate of I NH resistant strains; the Beijing genotype is prevalent in Beijing, China; the modified RIFO assay can be a practical tool for rapid detection of RIF resistant and MDR isolates in the routine diagnostic work.     

Efficient Differentiation of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Strains of the W-Beijing Family from Russia using Highly Polymorphic VNTR Loci

The W-Beijing family is a widespread Mycobacterium tuberculosis clonal lineage that frequently causes epidemic outbreaks. This family is genetically homogeneous and conserved, so ETR-VNTR (exact tandem repeat-variable number of tandem repeats) typing is insufficient for strain differentiation, due to a common ETR-A to E profile (42435). This leads to the false clustering in molecular epidemiological studies, especially in the regions of predominance of the W-Beijing family. In this study, we searched for VNTR loci with a high evolutionary rate of polymorphism in the W-Beijing genome. Here we further evaluated VNTR typing on a set of 99 Mycobacterium tuberculosis clinical isolates and reference strains. These isolates were characterized and classified into several genotype families based on three ETR loci (A, C, E) and eight additional loci [previously described as QUB (Queen’s University Belfast) or MIRU (Mycobacterial Interspersed Repetitive Units) or Mtubs]. Ninety-nine strains were divided into 74 VNTR-types, 51 isolates of the W-Beijing family identified by IS6110 RFLP-typing (the restriction fragment length polymorphism-typing) and/or spoligotyping were subdivided into 30 VNTR-types. HGDI (the Hunter–Gaston discriminatory index) for all studied loci was close to that of IS6110 RFLP typing, a “gold standard” method for subtyping M. tuberculosis complex strains. The QUB 26 and QUB 18 loci located in the PPE genes were highly polymorphic and more discriminative than other loci (HGDI is 0.8). Statistically significant increase of tandem repeats number in loci ETR-A, -E, QUB 26, QUB 18, QUB 11B, Mtub21 was revealed in the W-Beijing group compared to genetically divergent non-W-Beijing strains. Thirty-six isolates were subjected to IS6110 RFLP typing. The congruence between results of the IS6110 RFLP typing and 11-loci VNTR typing was estimated on 23 isolates of the W-Beijing family. These isolates were subdivided into 9 IS6110-RFLP types and 13 VNTR types. The poor profiles correlation (0.767) reflects the differences in the rate and type of evolution between genome regions targeted by IS6110-RFLP and VNTR typing. VNTR typing in proposed format is powerful tool for discrimination of M. tuberculosis strains with different level of genetic relationship.