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.     

Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis

New and better drugs are needed for tuberculosis (TB), particularly for the multi-drug resistant (MDR) disease. However, the highly infectious nature of MDR Mycobacterium tuberculosis restricts its use for large scale screening of probable drug candidates. We have evaluated the potential of a screen based on a 'fast grower' mycobacterium to shortlist compounds which could be active against MDR M. tuberculosis. Sensitivity profiles of M. smegmatis, M. phlei and M. fortuitum as well as MDR clinical isolates of M. tuberculosis were determined against anti-TB drugs isoniazid and rifampicin. Among the three fast growers, M. smegmatis was found to display a profile similar to MDR M. tuberculosis. Subsequently we evaluated the performance of M. smegmatis as a 'surrogate' screen for 120 compounds which were synthesized for anti-TB activity. Fifty of these molecules were active against M. tuberculosis H(37)Rv at a minimum inhibitory concentration (MIC) cutoff of 相似文献   

Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae

The co-emergence of multidrug resistant pathogenic bacterial strains and the Human Immunodeficiency Virus pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life was identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth.     

Comparative analyses of actinobacterial genomic fragments from Lake Kinneret

The high genomic G+C group of Actinobacteria possesses a variety of physiological and metabolic properties, and exhibits diverse lifestyles and ecological distribution. In recent years, Actinobacteria have been found to frequently dominate samples obtained from freshwater samples. Furthermore, phylogenetic analyses have shown that 16S rRNA genes from uncultured actinobacterial freshwater samples cluster in four distinct lineages. While these lineages are abundant, little is known about them and currently no pure‐culture representatives or genomic fragments of them are available. In a screen of a genomic library from the moderately eutrophic freshwater Lake Kinneret, five fosmid clones containing actinobacterial genomic fragments were found. Three ～40 kb genomic fragments were chosen for sequencing. Fosmids K003 and K005 showed high similarity and were affiliated with the acIV actinobacterial freshwater lineage. Fosmid K004 was affiliated with the highly abundant acI lineage. A comparative genomic analysis revealed high synteny between the two freshwater clones K003 and K005 but a lower synteny between these two and the K004 fosmid. Fosmids K003 and K005 share an identical arrangement of arginine biosynthesis gene while K004 showed a slightly different arrangement by lacking the argF gene. Fosmid Ant4E12, an Antarctic actinobacterial clone, showed a higher synteny with K003/5 than K004 and a similar arginine operon, but in a different genomic context. The Clusters of Orthologous Groups categories assignment of the three fosmids yielded genes that were mostly involved in amino acid and nucleotide metabolism, as well as transport and ribosomal RNA translation, structure and biogenesis. These genomic fragments represent the first sequences to be published from these lineages, providing a cornerstone for future work on this environmentally dominant group.     

Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis

A thorough understanding of Mycobacterium tuberculosis pathogenesis in humans has been elusive in part because of imperfect surrogate laboratory hosts, each with its own idiosyncrasies. Mycobacterium marinum is the closest genetic relative of the M. tuberculosis complex and is a natural pathogen of ectotherms. In this review, we present evidence that the similar genetic programmes of M. marinum and M. tuberculosis and the corresponding host immune responses reveal a conserved skeleton of Mycobacterium host–pathogen interactions. While both species have made niche-specific refinements, an essential framework has persisted. We highlight genetic comparisons of the two organisms and studies of M. marinum in the developing zebrafish. By pairing M. marinum with the simplified immune system of zebrafish embryos, many of the defining mechanisms of mycobacterial pathogenesis can be distilled and investigated in a tractable host/pathogen pair.     

Comparative genomic structures of Mycobacterium CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR) are inheritable genetic elements of many archaea and bacteria, conferring acquired immunity against invading nucleic acids. CRISPR might be indicative of the bacterial niche adaptation and evolutionary. Mycobacterium is an important genus occupying diverse niches with profound medical and environmental significance. To present a comparative genomic landscape of the Mycobacterium CRISPR, the feature of mycobacterium CRISPR structures with sequenced complete genomes were bioinformatically analyzed. The results show that CRISPR structures can be found among 14 mycobacteria, and all loci are chromosomally located. Long CRISPRs present in three species, namely M. tuberculosis, M. bovis, and M. avium. Integrated CRISPR-Cas system can only be found in M. tuberculosis and M. bovis, with highly conserved repeat sequences, very short leaders, and promoterless. M. tuberculosis and M. bovis repeat sequences cannot form stable RNA secondary structure, consistent with a Cas6-binding sequence. M. avium repeat sequences can form classical stem-loop structure. A three-step model of M. tuberculosis CRISPR-Cas system action was put forward based on the composition and function of cas genes cluster. M. tuberculosis and M. bovis CRISPRs might interfere with the invading nucleic acids, but have somehow lost the capacity to incorporate new spacers and co-evolve with corresponding mycobacteriophages.     

Unraveling Mycobacterium tuberculosis genomic diversity and evolution in Lisbon,Portugal, a highly drug resistant setting

Background

Multidrug- (MDR) and extensively drug resistant (XDR) tuberculosis (TB) presents a challenge to disease control and elimination goals. In Lisbon, Portugal, specific and successful XDR-TB strains have been found in circulation for almost two decades.

Results

In the present study we have genotyped and sequenced the genomes of 56 Mycobacterium tuberculosis isolates recovered mostly from Lisbon. The genotyping data revealed three major clusters associated with MDR-TB, two of which are associated with XDR-TB. Whilst the genomic data contributed to elucidate the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of Mycobacterium tuberculosis clinical isolates, revealed two major clades responsible for M/XDR-TB in the region: Lisboa3 and Q1 (LAM).The data presented by this study yielded insights on microevolution and identification of novel compensatory mutations associated with rifampicin resistance in rpoB and rpoC. The screening for other structural variations revealed putative clade-defining variants. One deletion in PPE41, found among Lisboa3 isolates, is proposed to contribute to immune evasion and as a selective advantage. Insertion sequence (IS) mapping has also demonstrated the role of IS6110 as a major driver in mycobacterial evolution by affecting gene integrity and regulation.

Conclusions

Globally, this study contributes with novel genome-wide phylogenetic data and has led to the identification of new genomic variants that support the notion of a growing genomic diversity facing both setting and host adaptation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-991) contains supplementary material, which is available to authorized users.     

A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.     

Comparative structural analyses of the alpha-glucan and glycogen from Mycobacterium bovis

Pathogenic mycobacteria such as Mycobacterium tuberculosis, the causative agent of tuberculosis, are surrounded by a noncovalently bound capsule, whose major carbohydrate constituent is a glycogen-like alpha-glucan. In the present study we compared the structures of the extracellular polysaccharide to that of the ubiquitous intracellular glycogen. The alpha-glucan was isolated from the culture medium of Mycobacterium bovis Bacille Calmette Guérin, the vaccine strain, in which it is released whereas the intracellular glycogen was obtained after the disruption of cells. The two purified polysaccharides were eluted from permeation gel at a similar position but glycogen was less soluble and gave a more opalescent solution in water than alpha-glucan. Combination of gas chromatography-mass spectrometry analysis of partially O-methylated, partially O-acetylated alditols and NMR analysis confirmed that both polysaccharides were composed of -->4-alpha-D-Glcp-1--> core, substituted at some six positions with short chains. Degradation of polysaccharides with pullulanase, followed by mass spectrometry analysis of the resulting products, also showed that the two polysaccharides do not differ in terms of lengths of branching. Interestingly, application of analytical ultracentrifugation and dynamic light scattering to the mycobacterial alpha-glucan and glycogen and their enzymatic degradative products indicated that the alpha-glucan possessed a higher molecular mass and was more compact than the glycogen from the same species, allowing the formulation of working structural models for the two polysaccharides. Consistent with the models, the alpha-glucan was found to be less accessible to pullulanase, a debranching enzyme, than glycogen.     

Genome comparison of Mycobacterium tuberculosis and other bacteria

The availability of the complete genome sequence of Mycobacterium tuberculosis allows its phylogenetic analysis based on the whole genome rather than single genes. As a genome-based tree is more representative of whole organisms and less inconsistent than single-gene trees, it could provide a better index for interpretation and inference about the origin and nature of species. The standard bacterial phylogeny based on 16S ribosomal RNA sequence comparison shows that M. tuberculosis is more related to Gram-positive than to Gram-negative bacteria. Our results based on genome comparison in terms of shared orthologous genes challenge this implication. We demonstrate that M. tuberculosis is more related to Gram-negative than to Gram-positive bacteria by a quantitative analysis on the genome tree. The numerical distance data derived from genome comparison and those from 16S rRNA comparison show high significant correlation, implying that conserved gene content carries a strong phylogenetic signature in evolution.     

人型结核杆菌全染色体DNA探针鉴别结核杆菌和其安分枝杆菌

The dot-blots containing DNA isolated from nonmycobacterial and mycobacterial microorganisms were hybridized with 32P-labeled M. tuberculosis whole chromosomal DNA at the various temperatures. The probe did not cross-hybridize to DNA of nonmycobacterial microorganisms (E. coli, Plasmid pUC19, Nocardia asteriodes), nor with DNA from all mycobacteria tested except M. bovis BCG under the higher temperature conditions. Microorganisms could also be directly spotted and lysed on nitrocellulose filters and used for hybridization thus making this technique suitable for clinical diagnosis.     

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG), and within the inhA promoter and/or in structural gene. A small percentage (approximately 10%) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh). Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.     

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.     

两个糙皮侧耳菌株的比较基因组学研究

随着糙皮侧耳基因组测序的完成,基因组数据的深入挖掘和利用成为研究重点。本研究基于较高质量的糙皮侧耳CCMSSC00389-1和CCMSSC03989-1基因组图谱,开展了种内比较基因组学分析。CCMSSC00389-1和CCMSSC03989-1的11条染色体序列表现出良好的共线性,且分别含有89.92%和91.68%的保守基因。对菌株特有基因的GO富集分析表明,两菌株各自分化出一些独特的调控方式或途径。CCMSSC00389-1和CCMSSC03989-1之间共存在931 542个单核苷酸多态性位点,231 654个插入/缺失和9 221个结构变异。对与遗传变异重叠基因的GO富集分析表明,碳水化合物降解、物质运输/催化和调控/蛋白活性相关的基因分别容易发生SNP、In/Del和SV变异。CCMSSC00389-1中50.326kb缺失序列的断点两侧含有双链断裂修复（DSB）信号,推测DSB介导50.326kb序列的缺失。     

Comparative Genomic Analysis of Mycobacterium tuberculosis Drug Resistant Strains from Russia

Tuberculosis caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains is a growing problem in many countries. The availability of the complete nucleotide sequences of several MTB genomes allows to use the comparative genomics as a tool to study the relationships of strains and differences in their evolutionary history including acquisition of drug-resistance. In our work, we sequenced three genomes of Russian MTB strains of different phenotypes – drug susceptible, MDR and XDR. Of them, MDR and XDR strains were collected in Tomsk (Siberia, Russia) during the local TB outbreak in 1998–1999 and belonged to rare KQ and KY families in accordance with IS6110 typing, which are considered endemic for Russia. Based on phylogenetic analysis, our isolates belonged to different genetic families, Beijing, Ural and LAM, which made the direct comparison of their genomes impossible. For this reason we performed their comparison in the broader context of all M. tuberculosis genomes available in GenBank. The list of unique individual non-synonymous SNPs for each sequenced isolate was formed by comparison with all SNPs detected within the same phylogenetic group. For further functional analysis, all proteins with unique SNPs were ascribed to 20 different functional classes based on Clusters of Orthologous Groups (COG). We have confirmed drug resistant status of our isolates that harbored almost all known drug-resistance associated mutations. Unique SNPs of an XDR isolate CTRI-4^XDR, belonging to a Beijing family were compared in more detail with SNPs of additional 14 Russian XDR strains of the same family. Only type specific mutations in genes of repair, replication and recombination system (COG category L) were found common within this group. Probably the other unique SNPs discovered in CTRI-4^XDR may have an important role in adaptation of this microorganism to its surrounding and in escape from antituberculosis drugs treatment.     

Comparative genomic analyses of the bacterial phosphotransferase system.

We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer.     

Comparative testing of skin reactions to PPD mycobacterins from Mycobacterium tuberculosis and Mycobacterium scrofulaceum in school-age children

Comparative skin tests with 2 TU PPD-RT 23 with Tween 80 (prepared from M. tuberculosis) and 5 TU PPD-RS 95 with Tween 80 (prepared from M. scrofulaceum) were intradermally given to a total of 1,140 7-year-old children in two towns of Karviná district (340 and 255 children) and in Teplice (267 children) and Prague (278 children). In the two groups of Karviná district children the percentages of small-sized reactions (6-9 mm) to PPD-RT 23 were 13.6 and 22.3% compared to 7.1% in Teplice and 5.7% in Prague. The prevalence of small reactions to the PPD-RS 95 test in district of Karviná was 14.4 and 16.9%, in Teplice 4.5% and in Prague 6.8%. In the two towns of Karviná district the percentages of children whose reaction to PPD-RS 95 was larger than to PPD-RT 23 were 56.4 and 42.8%, in Teplice 24% and in Prague 24%. The hypothesis is advanced that the higher degree of skin hypersensitivity to the M. scrofulaceum mycobacterin which was found among the Karviná district children tested is due to sensitization with environmental mycobacteria which are common in this area.     

Physiological responses of marine Dendryphiella species from different geographical locations

The saprobic, cosmopolitan, marine fungi Dendryphiella arenaria and Dendryphiella salina, isolated from various plant and algal substrates from different geographical locations and climatic zones, were studied for their adaptations to the abiotic and biotic parameters commonly found in their natural marine habitats. All the tested strains of D. arenaria and D. salina grew optimally on culture media with added marine salts, at pH values between 6.5 and 8.0 and at an incubation temperature of 25°C. The D. arenaria strains had faster mean colony extension rates under all conditions of culture. All strains exhibited an increased salt optimum with increasing incubation temperature. The TLC profiles of strains of the two species were similar. The culture extracts were antimicrobial, though production of the biologically active metabolites was strain-specific. There were no significant correlations between source of origin and responses to the investigated parameters. These results demonstrate phenotypic plasticity and the ability of each isolate to adapt to diverse biotopes.     

Comparative genomic and proteomic analyses of PE/PPE multigene family of Mycobacterium tuberculosis H37Rv and H37Ra reveal novel and interesting differences with implications in virulence

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease taking one human life every 15 s globally. The two well-characterized strains H₃₇Rv and H₃₇Ra, derived from the same parental strain M. tuberculosis H₃₇, show dramatically different pathogenic phenotypes. PE/PPE gene family, comprising of 176 open reading frames and present exclusively in genus Mycobacterium, accounts for ∼10% of the M. tuberculosis genome. Our comprehensive in silico analyses of PE/PPE family of H₃₇Ra and virulent H₃₇Rv strains revealed genetic differences between these strains in terms of several single nucleotide variations and InDels and these manifested in changes in physico-chemical properties, phosphorylation sites, and protein: protein interacting domains of the corresponding proteomes. Similar comparisons using the 13 sigma factor genes, 36 members of the mammalian cell entry family, 13 mycobacterial membrane protein large family members and 11 two-component signal transduction systems along with 5 orphaned response regulators and 2 orphaned sensor kinases failed to reveal very significant difference between H₃₇Rv and H₃₇Ra, reinforcing the importance of PE/PPE genes. Many of these changes between H₃₇Rv and H₃₇Ra can be correlated to differences in pathogenesis and virulence of the two strains.