A novel multiplex real-time PCR for the identification of mycobacteria associated with zoonotic tuberculosis

Background

Tuberculosis (TB) is the leading cause of death worldwide from a single infectious agent. An ability to detect the Mycobacterium tuberculosis complex (MTC) in clinical material while simultaneously differentiating its members is considered important. This allows for the gathering of epidemiological information pertaining to the prevalence, transmission and geographical distribution of the MTC, including those MTC members associated with zoonotic TB infection in humans. Also differentiating between members of the MTC provides the clinician with inherent MTC specific drug susceptibility profiles to guide appropriate chemotherapy.

Methodology/Principal Findings

The aim of this study was to develop a multiplex real-time PCR assay using novel molecular targets to identify and differentiate between the phylogenetically closely related M. bovis, M. bovis BCG and M. caprae. The lpqT gene was explored for the collective identification of M. bovis, M. bovis BCG and M. caprae, the lepA gene was targeted for the specific identification of M. caprae and a Region of Difference 1 (RD1) assay was incorporated in the test to differentiate M. bovis BCG. The multiplex real-time PCR assay was evaluated on 133 bacterial strains and was determined to be 100% specific for the members of the MTC targeted.

Conclusions/Significance

The multiplex real-time PCR assay developed in this study is the first assay described for the identification and simultaneous differentiation of M. bovis, M. bovis BCG and M. caprae in one internally controlled reaction. Future validation of this multiplex assay should demonstrate its potential in the rapid and accurate diagnosis of TB caused by these three mycobacteria. Furthermore, the developed assay may be used in conjunction with a recently described multiplex real-time PCR assay for identification of the MTC and simultaneous differentiation of M. tuberculosis, M. canettii resulting in an ability to differentiate five of the eight members of the MTC.     

The genome of Mycobacterium africanum West African 2 reveals a lineage-specific locus and genome erosion common to the M. tuberculosis complex

Background

M. africanum West African 2 constitutes an ancient lineage of the M. tuberculosis complex that commonly causes human tuberculosis in West Africa and has an attenuated phenotype relative to M. tuberculosis.

Methodology/Principal Findings

In search of candidate genes underlying these differences, the genome of M. africanum West African 2 was sequenced using classical capillary sequencing techniques. Our findings reveal a unique sequence, RD900, that was independently lost during the evolution of two important lineages within the complex: the “modern” M. tuberculosis group and the lineage leading to M. bovis. Closely related to M. bovis and other animal strains within the M. tuberculosis complex, M. africanum West African 2 shares an abundance of pseudogenes with M. bovis but also with M. africanum West African clade 1. Comparison with other strains of the M. tuberculosis complex revealed pseudogenes events in all the known lineages pointing toward ongoing genome erosion likely due to increased genetic drift and relaxed selection linked to serial transmission-bottlenecks and an intracellular lifestyle.

Conclusions/Significance

The genomic differences identified between M. africanum West African 2 and the other strains of the Mycobacterium tuberculosis complex may explain its attenuated phenotype, and pave the way for targeted experiments to elucidate the phenotypic characteristic of M. africanum. Moreover, availability of the whole genome data allows for verification of conservation of targets used for the next generation of diagnostics and vaccines, in order to ensure similar efficacy in West Africa.     

Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms

Background

Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii.

Methodology/Principal Findings

Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10⁻⁶) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10⁴ CFU/mL) than M. tuberculosis (42×10¹ CFU/mL) and M. bovis (35×10¹ CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.

Conclusions/Significance

These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.     

Transmission of Mycobacterium tuberculosis between Farmers and Cattle in Central Ethiopia

Background

Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia.

Methodology/Principal Findings

A study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by “Vordermeier et al. (2002)” was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively.

Conclusions/Significance

The results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows with tuberculous mastitis.     

Simple and rapid method for detection of nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium canettii grown in the Bactec MGIT960 system

Mycobacterium tuberculosis reduces nitrate very strongly as compared to Mycobacterium bovis and M. bovis BCG. Nitrate reductase, in conjunction with niacin accumulation, constitutes one of the major biochemical tests used in clinical microbiology laboratories to differentiate M. tuberculosis from other members of the M. tuberculosis complex, as well as nontuberculous Mycobacteria. Determination of nitrate reductase activity is currently performed using cultures grown on solid media with a slow detection time and the need for large quantities of bacilli, as otherwise the test is not reliable. Hereby, we propose a nitrate reduction test coupled to Bactec MGIT960 system as a simple, rapid and economic method with a total gain of time of about 3 to 4 weeks over the conventional solid medium. In our study, almost all the M. tuberculosis and Mycobacterium canettii strains gave a strongly positive nitrate reductase result within 1 day of positive detection by the MGIT960 system. In contrast, M. bovis, M. bovis BCG and M. africanum strains remained negative even after 14 days of incubation. The possibility to detect nitrate reductase within 1 to 3 days of a positive culture using MGIT960 opens new perspectives with the possibility of confirming M. tuberculosis — starting directly from pathological specimens.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of mycobacteria in routine clinical practice

Background

Non-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed.

Methodology

We set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 10⁵ colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25 M. avium and 12 non-tuberculosis clinical isolates with identification scores ≥2 within 2.5 hours.

Conclusions

Our data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heat-inactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories in both developed and developing countries.     

Genotypic diversity and drug susceptibility patterns among M. tuberculosis complex isolates from South-Western Ghana

Objective

The aim of this study was to use spoligotyping and large sequence polymorphism (LSP) to study the population structure of M. tuberculosis complex (MTBC) isolates.

Methods

MTBC isolates were identified using standard biochemical procedures, IS6110 PCR, and large sequence polymorphisms. Isolates were further typed using spoligotyping, and the phenotypic drug susceptibility patterns were determined by the proportion method.

Result

One hundred and sixty-two isolates were characterised by LSP typing. Of these, 130 (80.25%) were identified as Mycobacterium tuberculosis sensu stricto (MTBss), with the Cameroon sub-lineage being dominant (N = 59/130, 45.38%). Thirty-two (19.75%) isolates were classified as Mycobacterium africanum type 1, and of these 26 (81.25%) were identified as West-Africa I, and 6 (18.75%) as West-Africa II. Spoligotyping sub-lineages identified among the MTBss included Haarlem (N = 15, 11.53%), Ghana (N = 22, 16.92%), Beijing (4, 3.08%), EAI (4, 3.08%), Uganda I (4, 3.08%), LAM (2, 1.54%), X (N = 1, 0.77%) and S (2, 1.54%). Nine isolates had SIT numbers with no identified sub-lineages while 17 had no SIT numbers. MTBss isolates were more likely to be resistant to streptomycin (p<0.008) and to any drug resistance (p<0.03) when compared to M. africanum.

Conclusion

This study demonstrated that overall 36.4% of TB in South-Western Ghana is caused by the Cameroon sub-lineage of MTBC and 20% by M. africanum type 1, including both the West-Africa 1 and West-Africa 2 lineages. The diversity of MTBC in Ghana should be considered when evaluating new TB vaccines.     

Genomic diversity among Beijing and non-Beijing Mycobacterium tuberculosis isolates from Myanmar

Background

The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M. tuberculosis genome and may account for the variation of virulence among M. tuberculosis isolates. Till date there are no studies that have examined the genomic composition of M. tuberculosis isolates from the high TB-burden country, Myanmar.

Methodology/Principle Findings

Twenty-two M. tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c [RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M. bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates.

Conclusion

This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M. tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Myanmar.     

Evaluation of molecular markers for the diagnosis of Mycobacterium bovis

Mycobacterium tuberculosis complex (MTC) comprises a group of bacteria that have a high degree of genetic similarity. Two species in this group, Mycobacterium tuberculosis and Mycobacterium bovis, are the main cause of human and bovine tuberculosis, respectively. M. bovis has a broader host range that includes humans; thus, the differentiation of mycobacterium is of great importance for epidemiological and public health considerations and to optimize treatment. The current study aimed to evaluate primers and molecular markers described in the literature to differentiate M. bovis and M. tuberculosis by PCR. Primers JB21/22, frequently cited in scientific literature, presented in our study the highest number of errors to identify M. bovis or M. tuberculosis (73 %) and primers Mb.400, designed to flank region of difference 4 (RD4), were considered the most efficient (detected all M. bovis tested and did not detect any M. tuberculosis tested). Although also designed to flank RD4, primers Mb.115 misidentified eight samples due to primer design problems. The results showed that RD4 is the ideal region to differentiate M. bovis from other bacteria classified in MTC, but primer design should be considered carefully.     

Genetic diversity of Mycobacterium tuberculosis isolates from Tibetans in Tibet, China

Background

Tuberculosis (TB) is a serious health problem in Tibet where Tibetans are the major ethnic group. Although genotyping of Mycobacterium tuberculosis (M. tuberculosis) isolates is a valuable tool for TB control, our knowledge of population structure of M. tuberculosis circulating in Tibet is limited.

Methodology/Principal Findings

In our study, a total of 576 M. tuberculosis isolates from Tibetans in Tibet, China, were analyzed via spoligotyping and 24-locus MIRU-VNTR. The Beijing genotype was the most prevalent family (90.63%, n = 522). Shared-type (ST) 1 was the most dominant genotype (88.89%, n = 512). We found that there was no association between the Beijing genotype and sex, age and treatment status. In this sample collection, 7 of the 24 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. An informative set of 12 loci had similar discriminatory power with 24 loci set.

Conclusions/Significance

The population structure of M. tuberculosis isolates in Tibetans is homogeneous and dominated by Beijing genotype. The analysis of 24-locus MIRU-VNTR data might be useful to select appropriate VNTR loci for the genotyping of M. tuberculosis.     

A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis

Background

Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis.

Methodology/Principal Findings

Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4×10⁻¹⁰ M.

Conclusions/Significance

A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.     

Construction of eukaryotic expression vectors encoding CFP-10 and ESAT-6 genes and their potential in lymphocyte proliferation

Background:

Mycobacterium (M.) bovis is the agent of bovine tuberculosis (TB) in a range of animal species, including humans. Recent advances in immunology and the molecular biology of Mycobacterium have allowed identification of a large number of antigens with the potential for the development of a new TB vaccine. The ESAT-6 and CFP-10 proteins of M. bovis are important structural and functional proteins known to be important immunogens.

Methods:

In the current study, the DNAs encoding these genes were utilized in the construction of pcDNA 3.1+/ESAT-6 and pcDNA3.1+/CFP-10 plasmids. After intramuscular injection of BALB/c mice with these plasmids, ESAT-6 and CFP-10 mRNA expression was assessed by RT-PCR. Mice were inoculated and boosted with the plasmids to evaluate their effects on lymphocyte proliferation.

Results:

Our results indicate the plasmids are expressed at the RNA level and can induce lymphocyte proliferation.

Conclusion:

Further study is needed to characterize the effect of these antigens on the immune system and determine whether they are effective vaccine candidates against M. bovis. Key Words: Mycobacterium bovis, DNA vaccine, ESAT-6, CFP-10, PPD, Proliferation assay, BALB/c mice     

Mycobacterium africanum Is Associated with Patient Ethnicity in Ghana

Mycobacterium africanum is a member of the Mycobacterium tuberculosis complex (MTBC) and an important cause of human tuberculosis in West Africa that is rarely observed elsewhere. Here we genotyped 613 MTBC clinical isolates from Ghana, and searched for associations between the different phylogenetic lineages of MTBC and patient variables. We found that 17.1% (105/613) of the MTBC isolates belonged to M. africanum, with the remaining belonging to M. tuberculosis sensu stricto. No M. bovis was identified in this sample. M. africanum was significantly more common in tuberculosis patients belonging to the Ewe ethnic group (adjusted odds ratio: 3.02; 95% confidence interval: 1.67–5.47, p<0.001). Stratifying our analysis by the two phylogenetic lineages of M. africanum (i.e. MTBC Lineages 5 and 6) revealed that this association was mainly driven by Lineage 5 (also known as M. africanum West Africa 1). Our findings suggest interactions between the genetic diversity of MTBC and human diversity, and offer a possible explanation for the geographical restriction of M. africanum to parts of West Africa.     

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non‐synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species‐specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia‐related genes between M. bovis and M. tuberculosis.     

Comparative genomic analysis of Mycobacterium tuberculosis clinical isolates

Background

Due to excessive antibiotic use, drug-resistant Mycobacterium tuberculosis has become a serious public health threat and a major obstacle to disease control in many countries. To better understand the evolution of drug-resistant M. tuberculosis strains, we performed whole genome sequencing for 7 M. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them.

Results

We observed that all 7 M. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of SNPs, ranging from 1409–1464. The numbers of insertion/deletions (Indels) identified in the 7 isolates were also similar, ranging from 56 to 101. A total of 39 types of mutations were identified in drug resistance-associated loci, including 14 previously reported ones and 25 newly identified ones. Sixteen of the identified large Indels spanned PE-PPE-PGRS genes, which represents a major source of antigenic variability. Aside from SNPs and Indels, a CRISPR locus with varied spacers was observed in all 7 clinical isolates, suggesting that they might play an important role in plasticity of the M. tuberculosis genome. The nucleotide diversity (Л value) and selection intensity (dN/dS value) of the whole genome sequences of the 7 isolates were similar. The dN/dS values were less than 1 for all 7 isolates (range from 0.608885 to 0.637365), supporting the notion that M. tuberculosis genomes undergo purifying selection. The Л values and dN/dS values were comparable between drug-susceptible and drug-resistant strains.

Conclusions

In this study, we show that clinical M. tuberculosis isolates exhibit distinct variations in terms of the distribution of SNP, Indels, CRISPR-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. Our study provides evidence strengthening the notion that the evolution of drug resistance among clinical M. tuberculosis isolates is clearly a complex and diversified process.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-469) contains supplementary material, which is available to authorized users.     

Association between Mycobacterium tuberculosis Complex Phylogenetic Lineage and Acquired Drug Resistance

Background

Development of resistance to antituberculosis drugs during treatment (i.e., acquired resistance) can lead to emergence of resistant strains and consequent poor clinical outcomes. However, it is unknown whether Mycobacterium tuberculosis complex species and lineage affects the likelihood of acquired resistance.

Methods

We analyzed data from the U.S. National Tuberculosis Surveillance System and National Tuberculosis Genotyping Service for tuberculosis cases during 2004–2011 with assigned species and lineage and both initial and final drug susceptibility test results. We determined univariate associations between species and lineage of Mycobacterium tuberculosis complex bacteria and acquired resistance to isoniazid, rifamycins, fluoroquinolones, and second-line injectables. We used Poisson regression with backward elimination to generate multivariable models for acquired resistance to isoniazid and rifamycins.

Results

M. bovis was independently associated with acquired resistance to isoniazid (adjusted prevalence ratio = 8.46, 95% CI 2.96–24.14) adjusting for HIV status, and with acquired resistance to rifamycins (adjusted prevalence ratio = 4.53, 95% CI 1.29–15.90) adjusting for homelessness, HIV status, initial resistance to isoniazid, site of disease, and administration of therapy. East Asian lineage was associated with acquired resistance to fluoroquinolones (prevalence ratio = 6.10, 95% CI 1.56–23.83).

Conclusions

We found an association between mycobacterial species and lineage and acquired drug resistance using U.S. surveillance data. Prospective clinical studies are needed to determine the clinical significance of these findings, including whether rapid genotyping of isolates at the outset of treatment may benefit patient management.     

First description of natural and experimental conjugation between Mycobacteria mediated by a linear plasmid

Background

In a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. The transfer mechanism of this insertion sequence to M. kansasii was investigated here.

Methodology/Principal Findings

A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. The linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau.

Conclusions/Significance

Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.     

First Insights into the Phylogenetic Diversity of Mycobacterium tuberculosis in Nepal

Background

Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown.

Methods and Findings

We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status.

Conclusions

We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.     

Effect of attenuation of Treg during BCG immunization on anti-mycobacterial Th1 responses and protection against Mycobacterium tuberculosis

Background

The functional equilibrium between natural regulatory T cells (Treg) and effector T cells can affect the issue of numerous infections. In unvaccinated mice, the influence of Treg in the control of primary infection with mycobacteria remains controversial.

Methodology

Here, we evaluated the role of Treg during prophylactic vaccination with Mycobacterium bovis BCG (Bacillus Calmette-Guérin) on the induction of T cell responses and on the protective effect against subsequent M. tuberculosis challenge in mice.

Principal Findings

We demonstrated that, subsequent to BCG injection, Treg were recruited to the draining lymph nodes and negatively control anti-mycobacterial CD4⁺ — but not CD8⁺ — T-cell responses. Treatment of BCG-immunized mice with an anti-CD25 mAb (PC61) induced an increase IFN-γ response against both subdominant and immunodominant regions of the protective immunogen TB10.4. In Treg-attenuated, BCG-immunized mice, which were then infected with M. tuberculosis, the lung mycobacterial load was significantly, albeit moderately, reduced compared to the control mice.

Conclusions

Our results provide the first demonstration that attenuation of Treg subset concomitant to BCG vaccination has a positive, yet limited, impact on the protective capacity of this vaccine against infection with M. tuberculosis. Thus, for rational design of improved BCG, it should be considered that, although the action of Treg does not represent the major cause of the limited efficiency of BCG, the impact of this cell population on the subsequent control of M. tuberculosis growth is significant and measurable.