Beijing Strains Of Mycobacterium Tuberculosis Research Articles

Predominance of the Mycobacterium tuberculosis Beijing strain amongst children from a high tuberculosis burden township in South Africa

The objective was to determine the molecular epidemiology and drug susceptibility patterns of Mycobacterium tuberculosis (MTB) of children and their household contacts (HHC) in Umlazi, a high TB-burden township in South Africa. Sixty eight MTBRifPLUS positive TB-infected children (TIC) (≤14 years) and 111 HHC were enrolled. Drug susceptibility testing (DST) was performed on sputum samples using the proportion method and GenoType® MTBDR. Genotyping of MTB was conducted using IS6110-restriction fragment length polymorphism (RFLP) and spoligotyping. Rifampicin (RIF) susceptibility was observed in 67/68 TIC. GenoType® MTBDRplus and phenotypic DST identified drug resistant strains in five of 16 culture-confirmed TIC. The Beijing strain was identified in six and the F15/LAM4/KZN strain in one of the 13 TIC respectively. Four patients with unknown RFLP strains belonged to spoligoclades S, T1, T3 variant and X2. The S-lineage and an unknown strain were identified in two HHC. MDR-TB and pre-XDR-TB were identified in one HHC each. Household transmission could not be determined as none of the culture-confirmed TIC resided with the six culture-confirmed contacts. The predominance of the hypervirulent Beijing strain and presence of drug-resistant strains must be considered in the implementation of effective TB control strategies and development of efficacious vaccines.

Prediction and analyses of HLA-II restricted Mycobacterium tuberculosis CD4+ T cell epitopes in the Chinese population.

The Bacillus Calmette-Guérin (BCG) vaccine has been used to prevent tuberculosis (TB), but it cannot prevent adults against TB. The Mycobacterium tuberculosis Beijing strain is the most popular strain in China, but no vaccine is designed for the Beijing strain. It is vital to design a multiepitopes-based vaccine against the Beijing strain for the Chinese population. The bioinformatics tools were used to predict CD4+ T-cell epitopes in five protective antigens based on the Chinese population-specific alleles. The antigenicity, allergenicity, toxicity, IFN-γ level, population coverage, and three-dimensional structure were predicted using Vaxijen, AllerTOP, ToxinPred, IFN-γ epitope server, IEDB, and I-TASSER, respectively. One-hundred one promiscuous epitopes were obtained from Rv1813c, Rv2608, Rv3131, and Rv3628 proteins. After screening with antigenicity, allergenicity, toxicity, and IFN-γ level, seven epitopes from Rv2608 and Rv3131 proteins were selected to be vaccine candidates. Further study determined their three-dimensional structure and the coverage in the Chinese population as high as 99%. Our study predicted seven CD4+ T-cell dominant epitopes from the proteins Rv2608 and Rv3131 of M. tuberculosis Beijing strain for the first time, which may provide a basis for improving the design of multiepitopes-based vaccines for TB.

Screening for Mycobacterium tuberculosis Infection Using Beijing/K Strain-Specific Peptides in a School Outbreak Cohort.

BackgroundThe Beijing strain of Mycobacterium tuberculosis (M. tb) has been most frequently isolated from TB patients in South Korea, and the hyper-virulent Beijing/K genotype is associated with TB outbreaks. To examine the diagnostic potential of Beijing/K-specific peptides, we performed IFN-γ release assays (IGRA) using a MTBK antigen tube containing Beijing/K MTBK_24800, ESAT-6, and CFP-10 peptides in a cohort studied during a school TB outbreak.MethodsA total of 758 contacts were investigated for M. tb infection, and 43 contacts with latent TB infection (LTBI) and 25 active TB patients were enrolled based on serial screening with QuantiFERON-TB Gold In-Tube tests followed by clinical examinations. Blood collected in MTBK antigen tubes was utilized for IGRA and multiplex cytokine bead arrays. Immune responses were retested in 24 patients after TB treatment, and disease progression was investigated in subjects with LTBI.ResultsTotal proportions of active disease and LTBI during the outbreak were 3.7% (28/758) and 9.2% (70/758), respectively. All clinical isolates had a Beijing/K M. tb genotype. IFN-γ responses to the MTBK antigen identified M. tb infection and distinguished between active disease and LTBI. After anti-TB treatment, IFN-γ responses to the MTBK antigen were significantly reduced, and strong TNF-α responses at diagnosis were dramatically decreased.ConclusionsMTBK antigen-specific IFN-γ has diagnostic potential for differentiating M. tb infection from healthy controls, and between active TB and LTBI as well. In addition, TNF-α is a promising marker for monitoring therapeutic responses. These data provide informative readouts for TB diagnostics and vaccine studies in regions where the Beijing/K strain is endemic.

Proteomic analysis of drug-susceptible and multidrug-resistant nonreplicating Beijing strains of Mycobacterium tuberculosis cultured in vitro

The existence of latent tuberculosis infection (LTBI) is one of the main obstacles hindering eradication of tuberculosis (TB). To better understand molecular mechanisms and explore biomarkers for the pathogen during LTBI, we cultured strains of Mycobacterium tuberculosis (Mtb) under stress conditions, mimicking those in the host granuloma intracellular environment, to induce entry into the non-replicating persistence stage. The stresses included hypoxia, low pH (5.0), iron deprivation (100 μM of 2, 2’˗dipyridyl) and nutrient starvation (10% M7H9 medium). Three Mtb strains were studied: two clinical isolates (drug-susceptible Beijing (BJ) and multidrug-resistant Beijing (MDR-BJ) strains) and the reference laboratory strain, H37Rv. We investigated the proteomics profiles of these strains cultured in stressful conditions and then validated the findings by transcriptional analysis. NarJ (respiratory nitrate reductase delta chain) was significantly up-regulated at the protein level and the mRNA level in all three Mtb strains. The narJ gene is a member of the narGHJI operon encoding all nitrate reductase subunits, which play a role in nitrate metabolism during the adaptation of Mtb to stressful intracellular environments and the subsequent establishment of latent TB. The identification of up-regulated mRNAs and proteins of Mtb under stress conditions could assist development of biomarkers, drug targets and vaccine antigens.

Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain

Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction.

Genetic profiling of Mycobacterium tuberculosis revealed "modern" Beijing strains linked to MDR-TB from Southwestern Colombia.

Beijing strains of Mycobacterium tuberculosis (lineage 2) have been associated with drug-resistance and transmission of tuberculosis worldwide. Most of the Beijing strains identified in the Colombian Pacific coast have exhibited a multidrug resistant (MDR) phenotype. We sought to evaluate the clonality and sublineage of Beijing strains circulating in Southwestern Colombia. Thirty-seven Beijing strains were identified through spoligotyping out of 311 clinical isolates collected in 9 years from 2002–2010. Further analysis by MIRU-VNTR 24 loci was conducted for the Beijing strains. For sublineage classification, deletions of RD105, RD207, and RD131 and point mutations at fbpB, mutT2, and acs were evaluated. Drug-resistance associated mutations to first- and second-line anti-TB drugs were also evaluated. Additionally, two Beijing strains were Illumina-whole genome sequenced (one MDR and one drug-susceptible). Among the 37 Beijing strains characterized, 36 belonged to the SIT190 type from which 28 were MDR, four pre-extensively drug resistant (XDR) TB, and four XDR-TB. The remaining strain was SIT1 and drug susceptible. MIRU-VNTR analysis allowed the identification of three Beijing clusters and two unique strains. Beijing strains were confirmed as “modern” sublineage. The mutations rpoB S531L and katG S315T were the most common among MDR strains. Moreover, the two strains evaluated by whole genome sequencing (WGS) shared most of the genetic features with the sublineage 2.2.1 “modern” Beijing previously characterized from Asian strains. WGS analysis of the MDR strain revealed the presence of eight SNPs previously reported in other MDR “Beijing-like” strains from Colombia. The presence of “modern” Beijing strains in Southwestern Colombia, most of them with MDR phenotype, suggests a different origin of this M. tuberculosis sublineage compared to other Beijing strains found in neighboring South American countries. This work may serve as a genetic baseline to study the evolution and spread of M. tuberculosis Beijing strains in Colombia, which play an important role in the propagation of MDR-TB.

ACID-FAST BACILLI CONVERSION OF BEIJING AND NON-BEIJING STRAIN OF PULMONARY TUBERCULOSIS IN SOUTH SULAWESI

Beijing strains are a major part of the Mycobacterium tuberculosis Asian phylogenetic lineage. Beijing strains represent about 50% of all TB strains in East Asia and at least 13% of strains worldwide. Beijing strain of Mycobacterium tuberculosis is presumed as the factor of the increase in bacteria virulence and drug resistance, and the contributor in treatment failure. The aim of this study was to analyze the association between acid-fast bacilli conversion with strain genotipe Beijing and non-Beijing of pulmonary tuberculosis in South Sulawesi. The design of research was observational analytic with prospective approach. The sampling technique used consecutive sampling. Data were taken from active pulmonary tuberculosis patients’ medical record in Balai Besar Kesehatan Paru Masyarakat Makassar (Pulmonary Health Center of Makassar) and Community Health Center in Gowa Regency, South Sulawesi from March to June 2018. Collected sputum samples were screened for AFB and identified as Beijing strain and non Beijing strains using Multiplex PCR in Tropical Disease Institute of Universitas Airlangga. The results is showed that the characteristics of the respondents consisted of 12 respondents (33.3%) aged 56-65 years, 25 respondents (69.4%) men and 28 respondents (77.8%) had low category gradation of AFB smear. Univariate analysis showed 6 respondents (16.7%) with Beijing strains, 30 respondents (83.3%) with non-Beijing strains, 32 respondents (88.9%) conversion sputum AFB and 4 respondents (11.1%) non conversion sputum AFB. Bivariate analysis with Chi-Square statistical test shows that p value 0.022 < 0,05, that means there was association of Beijing strains with BTA conversion. Microscopic examination of BTA can be used to monitor and evaluate the treatment of new pulmonary TB patients undergoing treatment and the Beijing Mycobacterium tuberculosis strain has a significant correlation with the treatment failure of anti-tuberculosis drugs in South Sulawesi.

A Mycobacterium tuberculosis Beijing strain persists at high rates and extends its geographic boundaries 20 years after importation

Transmission of Beijing Mycobacterium tuberculosis can be investigated based on genotypic analysis of clinical isolates. A Beijing strain began to spread on Gran Canaria Island, Spain, at the end of the last century. In 1996, only 3 years after its importation to the island, its frequency had increased to 27.1% of all the isolates. The strain was tracked during the following years, and the most recent data obtained corresponded to 2007-8, when its presence continued to be alarming (21%). In the current study, we updated data on the distribution of this strain 20 years (2013–2014) after it was first detected on the island and extended the analysis for the first time to all the mycobacteriology laboratories covering the population of the Canary Island archipelago. Rapid updating was enabled by means of 2 different strain-specific PCRs: one targeting a peculiar feature of the strain, which was identified based on an IS6110 copy mapping in the Rv2180c gene, and a newly defined strain-specific single nucleotide polymorphism, which was identified by whole-genome sequencing. The results showed that the strain has remained highly prevalent (20.90% of all isolates), has spread throughout the neighbouring islands, and has also reached high representativeness in them (11–32%).

CLONING AND EXPRESSION OF MCE1A GENE FROM MYCOBACTERIUM TUBERCULOSIS BEIJING AND H37RV STRAIN FOR VACCINE CANDIDATE DEVELOPMENT.

Background:Tuberculosis remains the leading cause of death in the world, especially wherever poverty, malnutrition and poor housing prevail. Mycobacterium tuberculosis Beijing strain is the most common strain that causes tuberculosis in Indonesia. The wide spread of tuberculosis has been further aggravated by HIV-AIDS and drug resistance. Unfortunately, Bacille Calmette-Guerin (BCG) as the current vaccine has different protection function and efficacy. According to function analysis, mce1A gene was predicted to have a role in host invasion and survival of Mycobacterium tuberculosis in human macrophages.Materials and Methods:We performed cloning and protein expression of Mce1A gene of Mycobacterium tuberculosis Beijing strain as local isolate and standard strain H37Rv as a comparison on the expression system Escherichia coli BL21(DE3). Mce1A gene from the strains were amplified by PCR and inserted into the vector pET28a. Each resulting recombinant plasmid was subsequently transformed into E. coli BL21(DE3) and Mce1A protein was expressed with IPTG induction.Results:E. coli BL21(DE3) was succesfully transformed with a recombinant plasmid that contains the Mce1A gene insert with correct orientation and reading frame. There was no mutation found in the amino acids sequence for B and T cell epitope. Mce1A expression in E. coli BL21(DE3) showed a protein band that was higher than expected. The protein was confirmed with Western blotting using anti-His detector.Conclusion:We assumed that Mce1A recombinant protein that has been expressed in E. coli BL21(DE3) is in their dimeric form or alternatively formed aggregates of different sizes.

Host immune responses to antigens derived from a predominant strain of Mycobacterium tuberculosis

Beijing strain of Mycobacterium tuberculosis (M. tb) is characterized by a high incidence of transmission, relapse, and drug resistance. This study aimed to determine host immune responses to antigens derived from the Beijing/K strain which has been highly prevalent in tuberculosis (TB) outbreaks in South Korea. We recruited 52TB patients and 96 healthy subjects comprising 31 individuals with latent TB infection (LTBI) and 65TB-naïve controls based on QuantiFERON-TB Gold In-Tube (QFT-IT) tests. Blood samples were obtained for diluted whole-blood assays, multiplex bead arrays, ELISpot assays, and HLA typing. Molecular genotyping of M. tb was performed using clinical isolates. Active TB and LTBI groups were differentiated by TNF-α concentrations induced by the Beijing/K strain-derived antigens, MTBK_24790 and MTBK_24800 (P<0.001). MTBK_24800-induced IFN-γ and CXCL10 concentrations discriminated the TB-infected groups from TB-naïve controls (P<0.001). IFN-γ-producing T cells were generated in 87.2% of TB patients in response to MTBK_24800 peptide antigens. The major immunogenic epitope was at C-terminal of the antigen, and predominantly recognized by HLA-DR4 and -DQ4. Measurement of IFN-γ, CXCL10, and TNF-α concentrations induced by MTBK_24790 and MTBK_24800 may contribute to improved diagnosis of TB and vaccine development in regions where the Beijing/K strain is endemic.

Pulmonary immunity and durable protection induced by the ID93/GLA-SE vaccine candidate against the hyper-virulent Korean Beijing Mycobacterium tuberculosis strain K

The majority of tuberculosis (TB) vaccine candidates advanced to clinical trials have been evaluated preclinically using laboratory-adapted strains. However, it has been proposed that challenge with clinical isolates in preclinical vaccine testing could provide further and more practical validation. Here, we tested the ID93/GLA-SE TB vaccine candidate against the clinical Mycobacterium tuberculosis (Mtb) strain K (Mtb K) belonging to the Beijing family, the most prevalent Mtb strain in South Korea. Mice immunized with ID93/GLA-SE exhibited a significant reduction in bacteria and reduced lung inflammation against Mtb K when compared to non-immunized controls. In addition, we analyzed the immune responses in the lungs of ID93/GLA-SE-immunized mice, and showed that ID93/GLA-SE was able to elicit sustained Th1-biased immune responses including antigen-specific multifunctional CD4+ T cell co-producing IFN-γ, TNF-α, and IL-2 as well as a high magnitude of IFN-γ response for up to 10 weeks post-challenge. Notably, further investigation of T cell subsets in the lung following challenge showed remarkable generation of CD8+ central memory T cells by ID93/GLA-SE-immunization. Our findings showed that ID93/GLA-SE vaccine confers a high level of robust protection against the hypervirulent Mtb Beijing infection which was characterized by pulmonary Th1-polarized T-cell immune responses. These findings may also provide relevant information for potential utility of this vaccine candidate in East-Asian countries where the Beijing genotype is highly prevalent.

Ultrafast Assessment of the Presence of a High-Risk Mycobacterium tuberculosis Strain in a Population.

A persistent 8-year infection by a Beijing Mycobacterium tuberculosis strain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission-despite opportunities-and challenged some of the advantages associated with Beijing strains.

Protection by novel vaccine candidates, Mycobacterium tuberculosis ΔmosR and ΔechA7, against challenge with a Mycobacterium tuberculosis Beijing strain

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion people, claiming around 1.5 million lives annually. The only vaccine approved for clinical use against this disease is the Bacillus Calmette–Guérin (BCG) vaccine. Unfortunately, BCG has limited efficacy against the adult, pulmonary form of tuberculosis. This vaccine was developed from M. bovis with antigen expression and host specificity that differ from M. tuberculosis. To address these problems, we have designed two novel, live attenuated vaccine (LAV) candidates on an M. tuberculosis background: ΔmosR and ΔechA7. These targeted genes are important to M. tuberculosis pathogenicity during infection. To examine the efficacy of these strains, C57BL/6 mice were vaccinated subcutaneously with either LAV, BCG, or PBS. Both LAV strains persisted up to 16 weeks in the spleens or lungs of vaccinated mice, while eliciting minimal pathology prior to challenge. Following challenge with a selected, high virulence M. tuberculosis Beijing strain, protection was notably greater for both groups of LAV vaccinated animals as compared to BCG at both 30 and 60 days post-challenge. Additionally, vaccination with either ΔmosR or ΔechA7 elicited an immune response similar to BCG. Although these strains require further development to meet safety standards, this first evidence of protection by these two new, live attenuated vaccine candidates shows promise.

Is the Beijing strain of Mycobacterium tuberculosis associated with cavitary lung disease?

We conducted a cross-sectional study to describe clinical characteristics of patients with pulmonary tuberculosis with and without evidence of pulmonary cavitation on chest radiography and assess whether cavitation is associated with infection with Mycobacterium tuberculosis Beijing strain. Cases were selected from the Tuberculosis Registry (January 1, 2008–November 1, 2011) of the Florida Department of Health (FDOH). Molecular characterization was performed by spoligotyping and MIRU-VNTR. We analyzed 975 cases, where 144 (14.8%) were infected with the Beijing strain. Cavitation was not associated with disease caused by the Beijing strain. Alcohol use (OR=1.7; 95%CI: 1.249–2.313) was associated with increased risk of cavitation in the unadjusted analyses. Multivariable analyses showed that older age (⩾65years) (OR=0.5; 95%CI: 0.233–0.871), Hispanic ethnicity (OR=0.6; 95%CI: 0.312–0.962), and co-infection with HIV (OR=0.1; 95%CI: 0.068–0.295) demonstrated protective effects to cavitation. Understanding the factors associated with cavitation among pulmonary cases is essential toward improved tuberculosis management and control.

The pattern of cytokine production in vitro induced by ancient and modern Beijing Mycobacterium tuberculosis strains.

It is unclear to what extent the host-responses elicited by Beijing versus non-Beijing strains of Mycobacterium tuberculosis (MTB) contribute to the predominance of modern Beijing strains in Taiwan and some other Asian countries. The purpose of this study was to compare the expression profiles of virulence-related genes in human monocyte-derived macrophages infected in vitro with Beijing (ancient and modern strains) and non-Beijing strains (EAI strains) of MTB that are epidemic in Taiwan. We found that modern Beijing strains induced lower levels of pro-inflammatory cytokines, whereas EAI strains induced higher levels. Notably, the most prevalent modern Beijing sub-lineage, possessing intact RD150 and RD142 chromosomal regions, induced very low levels of pro-inflammatory cytokines, especially interleukin-1β. Moreover, in an intracellular growth assay, the survival of the same modern Beijing strain in human monocyte-derived macrophages was significantly higher than that of an ancient Beijing strain and an EAI strain. Taken together, these results may explain why modern Beijing strains of MTB predominate in Taiwan.

Heterogeneity of phenotypic characteristics of the modern and ancestral Beijing strains of Mycobacterium tuberculosis.

The Beijing strain of Mycobacterium tuberculosis (MTB) is of great concern because this hypervirulent strain has caused numerous tuberculosis outbreaks. However, the mechanisms that allow the MTB Beijing strain to be highly pathogenic remain unclear and previous studies have revealed heterogeneity within this family. To determine the association between some phenotypic characteristics and phylogroups of the Beijing strain of MTB. Eight Beijing strains, 5 modern and 3 ancestral sublineages, were selected from the phylogroups of MTB. The selection was based on copy number of IS6110 at NTF, region of differences, and single nucleotide polymorphisms. The abilities of these strains to grow intracellularly in THP-1 macrophages, to induce apoptosis, necrosis, and cytokines production were examined using quantitative real-time PCR and commercially available ELISA kits, respectively. There were some significant differences between the two sublineages of the Beijing strain of MTB. The ancestral Beijing sublineages showed higher intracellular growth rates (p < 0.05) and necrosis induction rates (p < 0.01) than the modern Beijing sublineages. By contrast, the modern Beijing sublineages induced lower apoptosis and protective cytokine responses, i.e., TNF-α (p < 0.05) and IL-6 (p < 0.01) and higher non-protective IL-10 response. The modern Beijing sublineages may have evolved so that they have greater ability to diminish host defense mechanisms. The slower growth rate and reduced necrosis induction in host cells might allow the bacteria to cause a persistent infection. The results revealed a phylogroup-associated heterogeneity of phenotypes among MTB Beijing sublineages.

In vivo characteristics of Korean Beijing Mycobacterium tuberculosis strain K1 in an aerosol challenge model and in the Cornell latent tuberculosis model

The Beijing Mycobacterium tuberculosis family is widely distributed and is the most common M. tuberculosis strain in East Asia. The highly transmissible and predominant Beijing M. tuberculosis strain in Korea, M. tuberculosis K1, was characterized using an aerobic challenge mouse model and a latent tuberculosis model with M. tuberculosis H37Rv as a reference. M. tuberculosis K1 multiplied over ten times more rapidly than M. tuberculosis H37Rv during the early stage of infection and induced high levels of histopathology in the lung. Low levels of T helper cell (Th) Th1 [interferon (IFN)-γ, interleukin (IL)-12p40] and Th2 cytokines (IL-4, IL-10) were induced in the lungs of M. tuberculosis K1-infected mice. In the latent model, mice infected with M. tuberculosis K1 exhibited more frequent relapse from the latent state than did mice infected with M. tuberculosis H37Rv. In conclusion, M. tuberculosis K1, a prevalent Beijing strain in Korea, is expected to spread due to its rapid growth during the early stages of infection, low-level induction of the immune response and high relapse rates from a latent state.

Role of phospholipase C in pathogenesis of Beijing and Non-Beijing strains of Mycobacterium tuberculosis

The Beijing strain of Mycobacterium tuberculosis bear high transmission potential and there is a significant correlation between Beijing strain and multidrug resistance. Phospholipase of M. tuberculosis plays an important role in its pathogenesis through breaking up phospholipids and production of diacylglycerol (DAG) as an important signaling molecule in infected macrophages as well as a precursor for synthesis of triacylglycerol that acts as an energy store for utilization during long-term dormancy of M.tuberculosis. DNA extraction was performed by using CTAB (cetyltrimethylammonium bromide) method from positive culture specimens of tuberculosis patients (Van Soolingen et al., 1991). Spoligotyping was done for differentiation of Beijing strains from non-Beijing strains. Finally Phospholipase C region was detected by polymerase chain reaction (PCR). The current study showed that, 19 (9.5%) of 200 strains were Beijing strain and 181 (90.5%) were non-Beijing strains, corresponding to spoligotyping. Based on PCR assays for plcA, plcB, plcC genes, of Beijing strains, 16(84.2%) strains were positive forplcA, and 17 (89.4%) strains were positive for plcB and 17(89.4%) strains were positive for plcC genes (Goudarzi et al., 2010). In non-Beijing strains 17 strains (9.4 %), 18 (9.9%), 18 (9.9%) were positive for plcA, plcB, plcC, respectively. Considering the majority of Beijing strains possess phospholipase C genes, it is possible that plc serves a role in pathogenesis and thereby severity of tuberculosis diseases. However, confirmative studies will be needed to verify the exact role of phospholipase C in the pathogenecity ofM. tuberculosis. Key words: Tuberculosis, phospholipase c, spoligotyping, polymerase chain reaction.

A novel method for the rapid and prospective identification of Beijing Mycobacterium tuberculosis strains by high-resolution melting analysis

Genotypic analysis of Mycobacterium tuberculosis (MTB) has enabled the definition of several lineages. The Beijing family, which is considered highly virulent and transmissible, has been associated with resistance in certain settings and involved in severe outbreaks, making it one of the most closely-monitored lineages. Therefore, rapid prospective identification of Beijing MTB strains could be relevant. In the present study, we evaluate a real-time PCR followed by high-resolution melting (HRM) based on the identification of a single nucleotide polymorphism (SNP) in the Rv2629 gene which defines Beijing lineage (A191C for Beijing genotype and A191A for non-Beijing genotype). This combined methodology efficiently differentiated Beijing and non-Beijing strains in 100% of the isolates from a collection of reference strains without requiring specific DNA probes. Additionally, HRM was able to assign a Beijing/non-Beijing genotype in 90.9% of the respiratory specimens assayed. Its applicability was tested on a Peruvian sample of circulating MTB strains, in which it identified 10.7% as belonging to the Beijing genotype; this proportion reached 20% in the North Lima area. HRM analysis of the A191C SNP is a rapid, reliable, and sensitive method for the efficient prospective survey of high-risk Beijing MTB strains, even in developing settings where MTB culture is often not available.

Deciphering the role of IS6110 in a highly transmissible Mycobacterium tuberculosis Beijing strain, GC1237

The capacity of infection and the ability of Mycobacterium tuberculosis strains belonging to the Beijing family to spread rapidly probably result from genetic advantages and unidentified mechanisms of virulence not yet thoroughly investigated. Among the mechanisms proposed to be responsible for the varying virulence phenotypes of M. tuberculosis strains we find IS6110 insertions, genetic reorganizations and deletions, which have strong influences on fitness. Beijing family is one of the lineages with the highest number of copies of IS6110. By studying genetic markers characteristic for this lineage, here we have characterized the clinical isolate M. tuberculosis GC1237 strain responsible for important epidemic outbreaks in the Gran Canary Island. We have identified and analyzed each point of insertion of IS6110 using a bacterial artificial chromosome (BAC) library of this strain, in addition to the use of other approximations. Nineteen copies of IS6110 have been localized in GC1237 genome of which, four copies of IS6110 can act as a promoter and we have focused in the characterization of one copy located 31 bp upstream of the essential gene Rv2179c and compared to the reference strain H37Rv.