Research article open access mycobacterium complex. Tuberculosis tb is a major global health problem and drug resistance compromises the efforts to control this disease. Research article open access mycobacterium complex from non. This report was revised and renamed handbook on tb laboratory. New cdc program for rapid genotyping of mycobacterium tuberculosis isolates in january 2004, the cdc tuberculosis genotyping program was initiated to enable rapid genotyping of isolates from every patient in the united states with culturepositive tuberculosis tb. Dna microarray gene expression profile of mycobacterium. We genotyped and sequenced the complete genomes of 68 m. Mycobacterium tuberculosis contains at least nine small rna families in its genome. Wholegenome sequencing wgs is a promising tool for distinguishing between reinfection and persistent infection in isolates taken at different times from a single patient, but. Availability of an assay for detecting mycobacterium. Resistance patterns of mycobacterium tuberculosis isolates.
This study aimed to determine resistance patterns of mycobacterium tuberculosis isolated from pulmonary samples. Tuberculosis remains a major global health problem. Original article resistance patterns of mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in nairobi perpetual wangui ndungu1, samuel kariuki2, zipporah nganga1, gunturu revathi3 1institute of tropical medicine and infectious diseases itromid, jomo kenyatta university of agriculture and technology jkuat, nairobi, kenya. Molecular characterization of multidrugresistant mycobacterium. Differentiation of mycobacterium tuberculosis complex from nontubercular mycobacteria by nested multiplex pcr targeting is6110, mtp40 and 32kd alpha antigen encoding gene fragments pallavi sinha1, anamika gupta1, pradyot prakash1, shampa anupurba1, rajneesh tripathi1 and g. Bacterial dna gyrase is a validated target for antibacterial chemotherapy.
Yet, the rise of extensively resistant strains paints a picture of a microbe with an impressive adaptive potential. In addition, determining the essential gene cluster is important for both understanding a survival mechanism and finding novel molecular targets for antituberculosis drugs. Mycobacterium tuberculosis has caused tuberculosis tb in humans for thousands of years 1, 2, and the world health organization who estimates that one third of the global population is infected with m. An effective way to treat this disease is through the use of quinolones, which target type ii topoisomerase, a protein crucial for regulation of the bacteriums dna topology and dna regulation. Polymerase chain reaction pcr techniques are now widely used for early detection and species differentiation of mycobacteria, but mostly with their own limitations. Pyrazinamide pza is an important drug used in both first and second line treatment regimes. Only some genetic markers associated with drugresistant tb are known. Wholegenome sequencing analysis of serially isolated multi. Same as taqvet mycobacterium tuberculosis complex realtime pcr kittaqman probes dna simplex two well reactionrealtime pcr enables sensitive and specific detection of pathogen nucleic acid in animal samples, allowing for reliable and rapid screening and detection of infected animals. A 165 bp conserved region of a gene coding for 65 k dalton antigen protein of m. On the one hand, as a single gene nonsequencing biomarker. There have been concerted efforts by whole genome sequencing and bioinformatics analysis to identify. To determine the sensitivity of this method, we analyzed 35 rifr strains and 11 rifampinsusceptible rifs strains, using the dna sequencing of the core region of rpob for. Handbook on tuberculosis laboratory diagnostic methods in.
A portion of the mycobacterium tuberculosis gene encoding the beta subunit of rna polymerase rpob was amplified by pcr using degenerate oligonucleotides and used as a hybridization probe to isolate plasmid clones carrying the entire rpob gene of m. Tuberculosis remains a major public health problem. Evolution of extensively drugresistant mycobacterium tuberculosis. Restricted structural gene polymorphism in the mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Mycobacterium tuberculosis is detected by the five overlapping molecular probes probes ae that collectively are complementary to the entire 81 bp rpob core region8, 9. Classical genotyping suggests a genetically similar mycobacterium tuberculosis mtb strain. Bovine tuberculosis is a chronic granulomatous disease mainly affecting lymph node and lung tissues of cattle. Deciphering the biology of mycobacterium tuberculosis from. Molecular characterization of the rpob gene mutations of. Get a printable copy pdf file of the complete article 1. Same as taqvet mycobacterium tuberculosis complex realtime pcr kit taqman probes dna simplex two well reaction realtime pcr enables sensitive and specific detection of pathogen nucleic acid in animal samples, allowing for reliable and rapid screening and detection of infected animals.
A new single gene differential biomarker for mycobacterium. Indicators and targets for laboratory strengthening under the end tb strategy. Introduction of xpert mtbrif assay has revolutionalised the diagnosis of tuberculosis tb by simultaneously detecting the bacteria and resistance to rifampicin rif, a surrogate marker for multidrug resistant tb mdrtb as well as one of the principal firstline antituberculosis drugs. The small rna srna families were identified through rnomics the direct analysis of rna molecules isolated from cultures of mycobacterium tuberculosis. A portion of the mycobacterium tuberculosis gene encoding the beta subunit of rna polymerase. It consists of two subunits, gyra and gyrb, which form an a2b2 complex in the active enzyme.
However, knowledge of strain genotype is no less clinically and epidemiologically relevant. While mycobacterium tuberculosis complex members are the most commonly known human pathogens, non tuberculosis mycobacteria also known as environmental mycobacteria, atypical mycobacteria and mycobacteria other than tuberculosis mott are also implicated in tuberculosis like disease, localized lymphadenitis, gastrointestinal disease, and disseminated infections. Rapid detection of mycobacterium bovis dna in cattle lymph. An immediate excellent contribution of ngs with regard to tuberculosis treatment and control is its capacity to rapidly screen for multiple gene targets linked to the development of drug resistance. The dutch tb research policy, global health themes and research areas. This coating makes the cells impervious to gram staining, and as a. Among mdrtb patients s531 l was the most common mutation detected in rpob gene. Also, mycobacterium species has a peculiar cell wall, made of an impermeable complex structure rich in mycolate, making the lyses of its cell difficult. The emergence of drug resistant strains of mycobacterium tuberculosis complex has made the management of tuberculosis difficult.
Additionally, inaccurate diagnosis can lead to therapeutic approaches consistent with mycobacterium tuberculosis infection that are useless. All datasets generated for this study are included in the manuscript andor the supplementary files. Identification of nontuberculous mycobacteria by partial gene. Molecular genetic tests offer considerable time advantages in the identification of mycobacteria, enabling a more. Tuberculosis, a global health problem and highly prevalent in india, has always been a serious problem with respect to definitive diagnosis. S315t1 was the most common mutation noted in katg gene. Combating the spread of drug resistant tuberculosis is a global health priority.
In east greenland, a dramatic increase of tuberculosis tb incidence has been observed in recent years. Identification of nontuberculous mycobacteria by partial. Objective to detect the specific mutations in rpob gene of mycobacterium. Yet mycobacterium tuberculosis mtb, the aetiological agent of tb, is still responsible for 1. Quantitative genomic dna from mycobacterium tuberculosis.
Evolution of extensively drugresistant mycobacterium. Similarly, a 365 bp region between sequence position 771741 of dnaj gene of m. Sequence alignment of mycobacterium tuberculosis gyrb with other bacterial gyrbs predicts the presence of 40 potential additional amino acids at the gyrb nterminus. Specific and rapid detection of mycobacterium tuberculosis.
Mycobacterium tuberculosis whole genome sequencing and. Gene expression profile of mycobacterium tuberculosis in a. Improved molecular diagnostic methods for detection drug resistance in mycobacterium tuberculosis mtb strains are required. A portion of the mycobacterium tuberculosis gene encoding the beta subunit of rna. The goal of this paper is to describe the advances made in the understanding of the molecular basis of m. Characterization of rpob mutations in rifampicinresistant mycobacterium tuberculosis isolated in china. The bacterium mycobacterium tuberculosis is the cause of tuberculosis tb, one of the most common curable infectious diseases in the world. Drugresistant tuberculosis tb is a major health threat in myanmar. Using an example of the emerging multidrugresistant strain of mycobacterium tuberculosis, we showed the value of informed understanding when in silico prediction from ngs data achieved with. Sep 12, 2016 in east greenland, a dramatic increase of tuberculosis tb incidence has been observed in recent years. Mycobacterium tuberculosis tu delft research information portal. In this regard, atypical clinical presentation of tuberculosis in individuals who have a deficient.
All relevant data are within the paper and its supporting information files. Since the 1880s, laboratory diagnosis of tuberculosis has relied primarily on microscopy and culture. The worldwide emergence and spread of drug resistant strains of mycobacterium tuberculosis has adverse effects on tuberculosis tb control programs. The use of polymerase chain reactionsinglestranded conformational polymorphism pcrsscp to study rpob gene mutations in rifampinresistant rifr mycobacterium tuberculosis has yielded contradictory results. Algorithms for the diagnosis of tuberculosis pan american health. The number of effective drugs for the prevention and control of tuberculosis is very limited. Mycobacterium tuberculosis is characterized by a low mutation rate. Rifampicin resistance mutations in the 81 bp rrdr of rpob. Nextgeneration sequencing ngs technology is becoming more affordable and is increasingly being widely used for highresolution molecular epidemiology of tuberculosis. Virulence life cycle of mycobacterium tuberculosis and. The nonthaburi nb genotype, belongingtotheindooceaniclineagelineage1,hasauniquespoligotypeandis6110. Pulmonary tuberculosis patients in nairobi were randomly sampled after informed consent and recruited into the study using a structured questionnaire. While mycobacterium tuberculosis complex members are the most commonly known human pathogens, nontuberculosis mycobacteria also known as environmental mycobacteria, atypical mycobacteria and mycobacteria other than tuberculosis mott are also implicated in tuberculosislike disease, localized lymphadenitis, gastrointestinal disease, and disseminated. It is caused by mycobacterium bovis, a member of the mycobacterium tuberculosis complex group of bacteria.
Tuberculosis tb is a major cause of morbidity and mortality worldwide. A multiple genome analysis of mycobacterium tuberculosis. The quantitative format allows for the generation of a standard curve. Cultureindependent detection and characterisation of. Characterization of rpob mutations in rifampinresistant clinical mycobacterium tuberculosis isolates from greece article pdf available in journal of clinical microbiology 361.
Laboratory diagnostic methods that allow effective, early detection of cases are central to management of tuberculosis in the individual patient and in the community. Pulmonary tuberculosis still in on the list of the world major health problems. In order to apply a radiolabelledprobe based detection of mutations in selected. The srnas were characterised through race mapping and northern blot experiments. Drugresistant tuberculosis tb, one of the leading causes of death worldwide, arises mainly from spontaneous mutations in the genome of mycobacterium tuberculosis. In 1921, calmette and guerin introduced the vaccine against tb, and between 1944 and 1966, streptomycin, isoniazid inh, ethambutol, rifampin and pyrazinamide were discovered as remedies for tb. Mutations in rpob and katg genes in mycobacterium isolates. Deciphering drug resistance in mycobacterium tuberculosis using wholegenome sequencing.
In the present study, the performance of public databases in the accurate identification of ntm by sequence analysis of 16s rrna and hsp65 genes were evaluated and compared. Clinical relevance of mycobacterium tuberculosis plcd gene. Evaluation of costeffective total nucleic acids extraction. Screening essential genes of mycobacterium tuberculosis. In order to apply a radiolabelledprobe based detection of mutations in selected genes leading to drug resistance. There have been concerted efforts by whole genome sequencing and. Mycobacterium tuberculosis is characterized by a low mutation rate and a lack of genetic recombination. The aim of this study was to analyze mutations occurring in the rpob gene of mycobacterium tuberculosis mtb isolates from clinical samples of extrapulmonary tuberculosis eptb. Nextgeneration sequencing of mycobacterium tuberculosis. Shengfen wang, bing zhao, yuanyuan song, yang zhou, yu pang, xichao ou, qiang li, hui xia, yanlin zhao national tuberculosis reference laboratory, national center for tuberculosis control and prevention, chinese center for disease. Pdf characterization of rpob mutations in rifampin. Lsi vetmax mycobacterium tuberculosis realtime pcr kit. Resistance levels and rpob gene mutations among in vitroselected rifampinresistant mycobacterium tuberculosis mutants. Wholegenome sequencing wgs is a promising tool for distinguishing between reinfection and persistent infection in isolates taken at different times from a single patient, but has not yet been.
To identify mycobacterium tuberculosis virulence factors, we integrated comparative genomics and epidemiologic data analysis to investigate the relationship between certain genomic insertions and deletions in the phospholipasec gene d plcd with the clinical presentation of tuberculosis tb. Mycobacterium tuberculosis radiology reference article. However, these snps can vary between mtb lineages therefore local data is required to describe different strain populations. Here we describe the first documented case of extensively drugresistant tuberculosis evolved from a susceptible ancestor within a single patient. Overexpression of g2 srna, for example, prevented growth of m. Tuberculosis has not been eradicated yet, from developing countries. Study of genetic evolution in mycobacterium tuberculosis. Rpob gene of mycobacterium tuberculosis pdf tuberculosis rpob gene was cloned into the shuttle plasmid pmv261 and electroporated into the lr223 strain of mycobacterium smegmatis, which is. Lsi vetmax mycobacterium tuberculosis realtime pcr kit, complex. Clinical tuberculosis manifests often as pulmonary and occasionally as extrapulmonary tuberculosis. Wholegenome sequencing analysis of serially isolated.
Whole genome association studies are being applied to identify genetic determinants of resistance to antituberculosis drugs. Resistance to first and second line antituberculous drugs has been associated with single nucleotide polymorphisms snps in particular genes. The global resurgence of tb highlights the need for an improved understanding of its epidemiology and its. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular. Mycobacterium tuberculosis is a thin, slightly curved bacillus. The lack of an effective vaccine to prevent those who are latently infected with mycobacterium tuberculosis from developing active disease, the variable results obtained in bacillus calmetteguerin vaccine efficacy trials, the emergence of multidrugresistant tb worldwide, and the increasing coinfection of hiv with m. A member of the mycobacterium tuberculosis complex, it is an obligate aerobic bacterium that is the etiologic agent of the majority of tuberculosis cases. Characterization of rpsl gene mutations in streptomycin. Wholegenome sequencing of multidrugresistant mycobacterium. Mutations in the welldefined 81bp region of the rpob gene, known as the rifresistancedetermining region rrdr, have been detected in. Mycobacterium tuberculosis complex mtbc and nontuberculosis mycobacteria. Along with clinical, radio graphic, and other laboratory findings, results of the assay aid in the diagnosis of pulmonary tuberculosis tb.
Nontuberculous mycobacteria dna university of washington. Virulence life cycle of mycobacterium tuberculosis and openi. Asdes srna is thought to be a cisacting regulator of a fatty acid desaturase desa2 while aspks is found with the open reading frame for polyketide synthase12 pks12 and is an antisense regulator of pks12 mrna. Furthermore, its incidence is increasing in the industrialized world, due to the human immunodeficiency virus hiv epidemic. The transcriptome of mycobacterium tuberculosis request pdf. However, its complete mechanism of action and resistance remains unclear. Diversity of mycobacterium tuberculosis across evolutionary scales. Identification of the likely translational start of. Nontuberculosis mycobacterium infections are caused by germs which are usually found in stagnated water and soil. Molecular characterization of the rpob gene mutations of mycobacterium tuberculosis isolated from china. Novel mutation in 16s rrna associated with streptomycin dependence in mycobacterium tuberculosis. Therefore, highthroughput screening for mycobacterium tuberculosis drug targets is critical. Fourteen multidrugresistant mycobacterium tuberculosis isolates were sequenced. It is still unclear as to why this germ causes infections for a few people and does not affect others but physicians are of the opinion that people who already have a disease condition or any sort of damage to the lungs are more prone to get.
Genetic heterogeneity revealed by sequence analysis of. Multidrug and extensively drugresistant tuberculosis mdr and xdrtb are problems that threaten public health worldwide. Pdf detection of mutations associated with isoniazid and. Gene replacement in mycobacterium tuberculosis and. However, these snps can vary between mtb lineages therefore local data is required to.
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