The Genotypic Population Structure of Mycobacterium tuberculosis Complex from Moroccan Patients Reveals a Predominance of Euro-American Lineages

Background

Tuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating Mycobacterium tuberculosis genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level.

Methodology/Principal Findings

Spoligotyping was performed on a panel of 592 M. tuberculosis complex strains covering a 2-year period (2004–2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1– ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2– modern/TbD1−/PGG1 group (Beijing, CAS), group 3– evolutionary recent/TbD1−/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%.

Conclusion

The M. tuberculosis population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the “evolutionary recent” TbD1−/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the M. tuberculosis population structure for an enhanced TB management program in Morocco.     

Genotypic Diversity Analysis of Mycobacterium tuberculosis Strains Collected from Beijing in 2009, Using Spoligotyping and VNTR Typing

Background

Tuberculosis (TB) is a serious problem in China. While there have been some studies on the nationwide genotyping of Mycobacterium tuberculosis (M. tuberculosis), there has been little detailed research in Beijing, the capital of China, which has a huge population. Here, M. tuberculosis clinical strains collected in Beijing during 2009 were genotyped by classical methods.

Methodology/Principal Findings

Our aim was to analyze the genetic diversity of M. tuberculosis strains within the Beijing metropolitan area. We characterized these strains using two standard methods, spoligotyping (n = 1585) and variable number of tandem repeat (VNTR) typing (n = 1053). We found that the most prominent genotype was Beijing family genotype. Other genotypes included the MANU, T and H families etc. Spoligotyping resulted in 137 type patterns, included 101 unclustered strains and 1484 strains clustered into 36 clusters. In VNTR typing analysis, we selected 12-locus (QUB-11b, MIRU10, Mtub21, MIRU 23, MIRU39, MIRU16, MIRU40, MIRU31, Mtub24, Mtub04, MIRU20, and QUB-4156c) and named it 12-locus (BJ) VNTR. VNTR resulted in 869 type patterns, included 796 unclustered strains and 257 strains clustered into 73 clusters. It has almost equal discriminatory power to the 24-locus VNTR.

Conclusions/Significance

Our study provides a detailed characterization of the genotypic diversity of M. tuberculosis in Beijing. Combining spoligotyping and VNTR typing to study the genotyping of M. tuberculosis gave superior results than when these techniques were used separately. Our results indicated that Beijing family strains were still the most prevalent M. tuberculosis in Beijing. Moreover, VNTR typing analyzing of M. tuberculosis strains in Beijing was successfully accomplished using 12-locus (BJ) VNTR. This method used for strains genotyping from the Beijing metropolitan area was comparable. This study will not only provide TB researchers with valuable information for related studies, but also provides guidance for the prevention and control of TB in Beijing.     

Lineage specific histories of Mycobacterium tuberculosis dispersal in Africa and Eurasia

Mycobacterium tuberculosis (M.tb) is a globally distributed, obligate pathogen of humans that can be divided into seven clearly defined lineages. An emerging consensus places the origin and global dispersal of M.tb within the past 6,000 years: identifying how the ancestral clone of M.tb spread and differentiated within this timeframe is important for identifying the ecological drivers of the current pandemic. We used Bayesian phylogeographic inference to reconstruct the migratory history of M.tb in Africa and Eurasia and to investigate lineage specific patterns of spread from a geographically diverse sample of 552 M.tb genomes. Applying evolutionary rates inferred with ancient M.tb genome calibration, we estimated the timing of major events in the migratory history of the pathogen. Inferred timings contextualize M.tb dispersal within historical phenomena that altered patterns of connectivity throughout Africa and Eurasia: trans‐Indian Ocean trade in spices and other goods, the Silk Road and its predecessors, the expansion of the Roman Empire, and the European Age of Exploration. We found that Eastern Africa and Southeast Asia have been critical in the dispersal of M.tb. Our results further reveal that M.tb populations have grown through range expansion, as well as in situ, and delineate the independent evolutionary trajectories of bacterial subpopulations underlying the current pandemic.     

MLVA Based Classification of Mycobacterium tuberculosis Complex Lineages for a Robust Phylogeographic Snapshot of Its Worldwide Molecular Diversity

Multiple-locus variable-number tandem repeat analysis (MLVA) is useful to establish transmission routes and sources of infections for various microorganisms including Mycobacterium tuberculosis complex (MTC). The recently released SITVITWEB database contains 12-loci Mycobacterial Interspersed Repetitive Units – Variable Number of Tandem DNA Repeats (MIRU-VNTR) profiles and spoligotype patterns for thousands of MTC strains; it uses MIRU International Types (MIT) and Spoligotype International Types (SIT) to designate clustered patterns worldwide. Considering existing doubts on the ability of spoligotyping alone to reveal exact phylogenetic relationships between MTC strains, we developed a MLVA based classification for MTC genotypic lineages. We studied 6 different subsets of MTC isolates encompassing 7793 strains worldwide. Minimum spanning trees (MST) were constructed to identify major lineages, and the most common representative located as a central node was taken as the prototype defining different phylogenetic groups. A total of 7 major lineages with their respective prototypes were identified: Indo-Oceanic/MIT57, East Asian and African Indian/MIT17, Euro American/MIT116, West African-I/MIT934, West African-II/MIT664, M. bovis/MIT49, M.canettii/MIT60. Further MST subdivision identified an additional 34 sublineage MIT prototypes. The phylogenetic relationships among the 37 newly defined MIRU-VNTR lineages were inferred using a classification algorithm based on a bayesian approach. This information was used to construct an updated phylogenetic and phylogeographic snapshot of worldwide MTC diversity studied both at the regional, sub-regional, and country level according to the United Nations specifications. We also looked for IS6110 insertional events that are known to modify the results of the spoligotyping in specific circumstances, and showed that a fair portion of convergence leading to the currently observed bias in phylogenetic classification of strains may be traced back to the presence of IS6110. These results shed new light on the evolutionary history of the pathogen in relation to the history of peopling and human migration.     

Acetylome Analysis Reveals Diverse Functions of Lysine Acetylation in Mycobacterium tuberculosis

The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD⁺-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.Mycobacterium tuberculosis was responsible for 1.3 million deaths and 8.6 million new cases of tuberculosis (TB)¹ worldwide in 2012 (1). This global public health crisis remains a serious problem, with the emergence of drug-resistant M. tuberculosis, especially multidrug-resistant and extensively drug-resistant M. tuberculosis, and also the emergence of coinfections of TB and human immunodeficiency virus (2, 3). To counter the increasing threat of TB, it is critical to understand fundamental aspects of TB-related biology. Such studies will not only provide new drug targets for the design of novel therapeutic agents, but also facilitate the development of novel diagnostic tools and new vaccines.Acetylation is one of the important protein modifications and occurs both co- and post-translationally on the α-amino group at the N terminus of the protein, so-called “N-terminal acetylation,” or on the ε-amino group on the side chain of lysine (4). Lysine acetylation is one of the most common post-translational modifications to proteins in both eukaryotes and prokaryotes. As a dynamic and reversible process, protein acetylation plays important roles in many cellular physiological processes, including cell-cycle regulation and apoptosis, cell morphology (5), metabolic pathways (6–8), protein interactions (9), and enzymatic activity (8, 10). In recent years, great advances have been made in proteomic studies, and a large number of lysine-acetylated proteins have been identified in many eukaryotes, including human (5, 11, 12), rat (13), mouse (11), Drosophila (14), Arabidopsis (15, 16), Saccharomyces cerevisiae (17), and protozoans (18, 19). The global analysis of lysine acetylation has also been reported in bacteria, including Escherichia coli (20–22), Erwinia amylovora (23), Bacillus subtilis (24), and Salmonella enterica (6). These acetylome studies have generated large datasets of bacterial proteins acetylated on lysine residues and have demonstrated the diverse cellular functions of lysine acetylation in bacteria.Increasing evidence shows that protein acetylation occurs and plays an important regulatory role in mycobacteria (8, 25–31). For example, Lange et al. reported the N-terminal acetylation of early secreted antigenic target 6 (ESAT-6) protein (31). Rv1151c is reported to be an NAD⁺-dependent protein deacetylase in M. tuberculosis that deacetylates and thus regulates the activity of acetyl-CoA synthase (25, 32). Two cyclic adenosine monophosphate (cAMP)-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) show similarity to the GNAT family of acetyltransferases and could acetylate a universal stress protein (USP, MSMEG_4207) (30). Subsequent structural studies revealed the fine mechanisms of how cAMP regulates the protein lysine acetyltransferase in mycobacteria (27, 28). Very recently, reversible lysine acetylation was shown to regulate the activity of several fatty acyl-CoA synthetases in M. tuberculosis (8, 26), and also to regulate acetate and propionate metabolism in M. smegmatis (8, 26). However, to the best of our knowledge, only a few acetylated proteins in M. tuberculosis have been identified, presenting a major obstacle to further understanding the regulatory roles of reversible lysine acetylation in this life-threatening pathogen.To fill this gap in our knowledge, we undertook a systematic study of the functional roles of lysine acetylation in M. tuberculosis. We performed an acetylomic analysis of M. tuberculosis H37Ra using high-accuracy MS combined with the identification of 226 unique lysine acetylation sites on 137 proteins. This set of M. tuberculosis proteins acetylated on lysine residues supports the emerging view that lysine acetylation is a general and fundamental regulatory process, and is not restricted to eukaryotes. It also opens the way for its detailed functional and evolutionary analysis of lysine acetylation in M. tuberculosis. The identified acetylated proteins that are involved in several important biological processes were functionally categorized into an interaction map. This is the first time that an interaction network of acetylated proteins in M. tuberculosis has been constructed, and should allow us to better understand the significance of acetylation in key cellular mechanisms in M. tuberculosis. To further explore the effects of lysine acetylation on the physiology of M. tuberculosis H37Ra, MRA_1161, the gene encoding the only known protein deacetylase in this bacterium, was deleted. The roles of MRA_1161 in the colony morphology, carbon source utilization, heat stress tolerance, and biofilm formation of M. tuberculosis were analyzed. The effect of lysine acetylation on the immunogenicity of a known immunogen, HspX, was also tested.     

Genotypic Analysis of Multidrug-Resistant Mycobacterium tuberculosis Isolates Recovered From Central China

DNA sequencing analysis was used to investigate genetic alterations in the rpoB, katG, and inhA regulatory region and embB in 66 Mycobacterium tuberculosis isolates recovered from Central China. Of the 36 multidrug-resistant isolates, 33 (92%) had mutations in the amplified region of rpoB. The most frequent mutation (58%, 19/36) was S531L (TCG→TTG). At least one mutation was found in the katG and inhA regulatory region in 83% (30/36) of the multidrug-resistant isolates, and mutations at katG codon 315 were identified in 78% (28/36). Alterations at embB306 may not confer resistance to EMB, and embB306 mutants were more frequently accompanied by rpoB mutations (100%, 16/16) than by katG 315 mutations (75%, 12/16). Our results show that geographic variation in the molecular genetic mechanism is responsible for drug resistance in multidrug-resistant M. tuberculosis. This observation will facilitate the development of a rapid molecular drug resistance screening approach for drug-resistant M. tuberculosis.     

Spatiotemporal Clustering of Mycobacterium tuberculosis Complex Genotypes in Florida: Genetic Diversity Segregated by Country of Birth

BackgroundTuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). Although the MTBC is highly clonal, between-strain genetic diversity has been observed. In low TB incidence settings, immigration may facilitate the importation of MTBC strains with a potential to complicate TB control efforts.MethodsWe investigated the genetic diversity and spatiotemporal clustering of 2,510 MTBC strains isolated in Florida, United States, between 2009 and 2013 and genotyped using spoligotyping and 24-locus MIRU-VNTR. We mapped the genetic diversity to the centroid of patient residential zip codes using a geographic information system (GIS). We assessed transmission dynamics and the influence of immigration on genotype clustering using space-time permutation models adjusted for foreign-born population density and county-level HIV risk and multinomial models stratified by country of birth and timing of immigration in SaTScan.Conclusions/SignificanceAlmost five percent of TB cases reported in Florida during 2009–2013 were potentially due to recent transmission. Improvements to TB screening practices among the prison population and recent immigrants are likely to impact TB control. Due to the monomorphic nature of available markers, whole genome sequencing is needed to conclusively delineate recent transmission events between U.S. and foreign-born persons.     

Widespread Environmental Contamination with Mycobacterium tuberculosis Complex Revealed by a Molecular Detection Protocol

Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI₉₅ 0.70–0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.     

PbGP43 Genotyping Using Paraffin-Embedded Biopsies of Human Paracoccidioidomycosis Reveals a Genetically Distinct Lineage in the Paracoccidioides brasiliensis Complex

Mycopathologia - Paracoccidioidomycosis (PCM) is a systemic mycosis caused by a group of cryptic species embedded in the Paracoccidioides brasiliensis complex and Paracoccidioides lutzii. Four...     

Pyrazinamide Resistance,Mycobacterium tuberculosis Lineage and Treatment Outcomes in San Francisco,California

Background

Pyrazinamide (PZA) is a first line agent for the treatment of active tuberculosis. PZA is also considered a potent companion drug for newer regimens under development. There are limited data on the demographic, clinical, and pathogen characteristics of PZA resistant tuberculosis.

Methods

Using a retrospective cohort study design, we evaluated all PZA resistant M. tuberculosis (M.tb) and M. bovis cases reported in San Francisco from 1991 to 2011. Demographic, clinical, and molecular data were analyzed. M.tb lineage was determined for all PZA resistant strains and compared to PZA susceptible strains.

Results

PZA resistance was identified in 1.8% (50 of 2,842) of mycobacterial isolates tested, corresponding to a case rate of 0.3 per 100,000 in the population. Monoresistant PZA infection was associated with the Hispanic population ([OR], 6.3; 95% [CI], 1.97–20.16) and 48% of cases were due to M. bovis. Infection with monoresistant PZA was also associated with extrapulmonary disease ([OR], 6.0; 95% [CI], 2.70–13.26). There was no statistically significant difference between treatment failure and mortality rates in patients infected with PZA monoresistance compared to pansusceptible controls (4% vs. 8%, p = 0.51), or those with PZA and MDR resistance (PZA-MDR) compared to MDR controls (18% vs. 29%, p = 0.40). PZA resistance was not associated with M.tb lineage.

Conclusions

Across two decades of comprehensive epidemiologic data on tuberculosis in San Francisco County, PZA resistance was uncommon. PZA resistance caused predominantly extrapulmonary disease and was more common in Hispanics compared to other ethnicities, with nearly half the cases attributed to M. bovis. No association was found between PZA monoresistance and M.tb lineage. Treatment outcomes were not adversely influenced by the presence of PZA resistance.     

The Diversity of Coliphages and Coliforms in Horse Feces Reveals a Complex Pattern of Ecological Interactions

The diversity of coliphages and indigenous coliform strains (ICSs) simultaneously present in horse feces was investigated by culture-based and molecular methods. The richness of coliforms (as estimated by the Chao1 method) is about 1,000 individual ICSs distinguishable by genomic fingerprinting present in a single sample of feces. This unexpectedly high value indicates that some factor limits the competition of coliform bacteria in the horse gut microbial system. In contrast, the diversity of phages active against any selected ICS is generally limited to one to three viral genotypes present in the sample. The sensitivities of different ICSs to simultaneously present coliphages overlap only slightly; the phages isolated from the same sample on different ICSs are usually unrelated. As a result, the titers of phages in fecal extract as determined for different Escherichia coli strains and ICSs may differ by several orders of magnitude. Summarizing all the data, we propose that coliphage infection may provide a selection pressure that maintains the high level of coliform diversity, restricting the possibility of a few best competitors outgrowing other ICSs. We also observed high-magnitude temporal variations of coliphage titers as determined using an E. coli C600 test culture in the same animal during a 16-day period of monitoring. No correlation with total coliform count was observed. These results are in good agreement with our hypothesis.     

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.     

Genotypic Analysis of Klebsiella pneumoniae Isolates in a Beijing Hospital Reveals High Genetic Diversity and Clonal Population Structure of Drug-Resistant Isolates

Background

The genetic diversity and the clinical relevance of the drug-resistant Klebsiella pneumoniae isolates from hospital settings are largely unknown. We thus conducted this prospective study to analyze the molecular epidemiology of K. pneumoniae isolates from patients being treated in the 306 Hospital in Beijing, China for the period of November 1, 2010–October 31, 2011.

Methodology/Principal Findings

Antibiotic susceptibility testing, PCR amplification and sequencing of the drug resistance-associated genes, and multilocus sequence typing (MLST) were conducted. A total of 163 isolates were analyzed. The percentage of MDR, XDR and PDR isolates were 63.8% (104), 20.9 (34), and 1.8% (3), respectively. MLST results showed that 60 sequence types (STs) were identified, which were further separated by eBURST into 13 clonal complexes and 18 singletons. The most dominant ST was ST15 (10.4%). Seven new alleles and 24 new STs were first identified in this study. Multiple logistic regression analysis revealed that certain clinical characteristics were associated with those prevalent STs such as: from ICU, from medical ward, from community acquired infection, from patients without heart disease, from patients with treatment success, susceptible to extended spectrum cephalosporin, susceptible to cephamycins, susceptible to fluoroquinolones, and with MDR.

Conclusions/Significance

Our data indicate that certain drug-resistant K. pneumoniae clones are highly prevalent and are associated with certain clinical characteristics in hospital settings. Our study provides evidence demonstrating that intensive nosocomial infection control measures are urgently needed.     

Structure of the Sensor Domain of Mycobacterium tuberculosis PknH Receptor Kinase Reveals a Conserved Binding Cleft

Since their discovery over 20 years ago, eukaryotic-like transmembrane receptor Ser/Thr protein kinases (STPKs) have been shown to play critical roles in the virulence, growth, persistence, and reactivation of many bacteria. Information regarding the signals transmitted by these proteins, however, remains scarce. To enhance understanding of the basis for STPK receptor signaling, we determined the 1.7-Å-resolution crystal structure of the extracellular sensor domain of the Mycobacterium tuberculosis receptor STPK, PknH (Rv1266c). The PknH sensor domain adopts an unanticipated fold containing two intramolecular disulfide bonds and a large hydrophobic and polar cleft. The residues lining the cleft and those surrounding the disulfide bonds are conserved. These results suggest that PknH binds a small-molecule ligand that signals by changing the location or quaternary structure of the kinase domain.     

Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

Background

Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB). The aim of this study was to identify the presence of Isoniazid (INH) and Rifampicin(RIF) drug resistance in new and previously treated tuberculosis (TB) cases using DNA sequencing.

Methods

This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli.

Results

Of the 153 patients, 105 (68.6%) were new cases and 48 (31.4%) were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases) and INH resistant specimens (26 cases) to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9%) of RIF resistance were having rpoB gene mutation versus 3 cases (13.1%) having no mutation with a high statistical significant difference between them (P < 0.001). Direct sequencing of Kat G gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG i.e. no evidence of mutation with a high statistical significant difference between them (P < 0.001).

Conclusion

We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.     

VRC34-Antibody Lineage Development Reveals How a Required Rare Mutation Shapes the Maturation of a Broad HIV-Neutralizing Lineage

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Genetic Diversity of Mycobacterium tuberculosis in Peru and Exploration of Phylogenetic Associations with Drug Resistance

Background

There is limited available data on the strain diversity of M tuberculosis in Peru, though there may be interesting lessons to learn from a setting where multidrug resistant TB has emerged as a major problem despite an apparently well-functioning DOTS control programme.

Methods

Spoligotyping was undertaken on 794 strains of M tuberculosis collected between 1999 and 2005 from 553 community-based patients and 241 hospital-based HIV co-infected patients with pulmonary tuberculosis in Lima, Peru. Phylogenetic and epidemiologic analyses permitted identification of clusters and exploration of spoligotype associations with drug resistance.

Results

Mean patient age was 31.9 years, 63% were male and 30.4% were known to be HIV+. Rifampicin mono-resistance, isoniazid mono-resistance and multidrug resistance (MDR) were identified in 4.7%, 8.7% and 17.3% of strains respectively. Of 794 strains from 794 patients there were 149 different spoligotypes. Of these there were 27 strains (3.4%) with novel, unique orphan spoligotypes. 498 strains (62.7%) were clustered in the nine most common spoligotypes: 16.4% SIT 50 (clade H3), 12.3% SIT 53 (clade T1), 8.3% SIT 33 (LAM3), 7.4% SIT 42 (LAM9), 5.5% SIT 1 (Beijing), 3.9% SIT 47 (H1), 3.0% SIT 222 (clade unknown), 3.0% SIT1355 (LAM), and 2.8% SIT 92 (X3). Amongst HIV-negative community-based TB patients no associations were seen between drug resistance and specific spoligotypes; in contrast HIV-associated MDRTB, but not isoniazid or rifampicin mono-resistance, was associated with SIT42 and SIT53 strains.

Conclusion

Two spoligotypes were associated with MDR particularly amongst patients with HIV. The MDR-HIV association was significantly reduced after controlling for SIT42 and SIT53 status; residual confounding may explain the remaining apparent association. These data are suggestive of a prolonged, clonal, hospital-based outbreak of MDR disease amongst HIV patients but do not support a hypothesis of strain-specific propensity for the acquisition of resistance-conferring mutations.     

Comparative Whole-Genome Analysis of Clinical Isolates Reveals Characteristic Architecture of Mycobacterium tuberculosis Pangenome

The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.