Journal articles: 'Mycobacterium tuberculosis sequencing'

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Sharma, Megha, Bharti Malhotra, Jitendra Tiwari, and Shipra Bhargava. "Profile of Nontuberculous Mycobacteria in Patients Suspected of Tuberculosis and Drug-Resistant Tuberculosis." Journal of Laboratory Physicians 12, no. 03 (November 23, 2020): 203–11. http://dx.doi.org/10.1055/s-0040-1721160.

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Abstract Objective Infections due to nontuberculous mycobacteria (NTM) is increasing globally and may present as drug-resistant tuberculosis (DRTB). In India, data on NTM prevalence and species diversity is limited. Present study was conducted to detect the prevalence and profile of NTM among patients suspected of DRTB using paraffin slide culture (PSC)and mycobacteria growth indicator tube (MGIT) culture methods for isolation of NTM. Material and Method A total of 2,938 samples suspected of TB/DRTB were cultured on PSC and MGIT960. Species identification of mycobacterial isolate was done by sequencing of 16s ribosomal RNA gene. Result Among 2938 samples, 35 (1.19%) were found positive for NTM by PSC and 9 (0.30%) were found positive by MGIT. The diversity of NTM species was high (13 species). Out of 35 NTM isolates by PSC, maximum 34.29% (12) isolates were found to be Mycobacterium fortuitum, followed by 11.43% (4) Mycobacterium abscessus and Mycobacterium chelonae, and 42.85% (15) were other species viz. 8.57% (3) were Mycobacterium intracellulare and Mycobacterium kansasii, 5.71% (2) were Mycobacterium peregrinum, and 2.85% (1) were Mycobacterium flavescens, Mycobacterium farcinogenes, Mycobacterium moriokanese, Mycobacterium wolinskyi, Mycobacterium simiae, Mycobacterium goodii, and Mycobacterium terrae each. Coinfection of Mycobacterium tuberculosis(MTB) and NTM was found in 60% (21) samples. Conclusion Prevalence of NTM was low among multidrug resistant tuberculosis/TB suspected patients, similar to other studies done in India. PSC was found better than MGIT for the isolation of NTM, though poor separation of NTM and MTB on subculture may have led to false negativity in cases of coinfection. About 13 species were isolated; M. fortuitum was the most common of all. Since coinfection of NTM and TB can also occur, samples of patients suspected of NTM should be cultured on PSC even if positive for MTB.

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Soini, Hanna, and James M. Musser. "Molecular Diagnosis of Mycobacteria." Clinical Chemistry 47, no. 5 (May 1, 2001): 809–14. http://dx.doi.org/10.1093/clinchem/47.5.809.

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Abstract Tuberculosis is one of the leading infectious diseases in the world and is responsible for more than 2 million deaths and 8 million new cases annually. Because of the slow growth rate of the causative agent Mycobacterium tuberculosis, isolation, identification, and drug susceptibility testing of this organism and other clinically important mycobacteria can take several weeks or longer. During the past several years, many molecular methods have been developed for direct detection, species identification, and drug susceptibility testing of mycobacteria. These methods can potentially reduce the diagnostic time from weeks to days. Currently, two nucleic acid amplification methods, the Enhanced Mycobacterium tuberculosis Direct Test (Gen-Probe) and the Amplicor Mycobacterium tuberculosis Test (Roche Diagnostic Systems), have been approved by the Food and Drug Administration for direct detection of M. tuberculosis from clinical specimens. PCR-based sequencing has become commonly used to identify many mycobacterial species. DNA probes have been widely used for species determination of the most commonly encountered mycobacteria. High-density oligonucleotide arrays (DNA microarrays) also have been applied to simultaneous species identification and detection of mutations that confer rifampin resistance in mycobacteria.

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De Smet, Koen A. L. "Mycobacterium tuberculosis: Beyond genome sequencing." Trends in Microbiology 5, no. 11 (November 1997): 429–31. http://dx.doi.org/10.1016/s0966-842x(97)01132-3.

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Ahmad, Suhail, and Eiman Mokaddas. "Diversity of Nontuberculous Mycobacteria in Kuwait: Rapid Identification and Differentiation of Mycobacterium Species by Multiplex PCR, INNO-LiPA Mycobacteria v2 Assay and PCR Sequencing of rDNA." Medical Principles and Practice 28, no. 3 (2019): 208–15. http://dx.doi.org/10.1159/000498910.

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Objective: Nontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait. Materials and Methods: DNA was extracted from MGIT cultures (n = 1,033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1,033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers. Results: mPCR identified 979 isolates as MTB, 53 as NTM and 1 isolate as mixed culture. LiPA and/or PCR sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in one culture and 5 isolates only at genus level. PCR sequencing yielded more specific identification for 22 isolates at the species/subspecies level. Conclusions: mPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at the species/species complex level and 2 mixed cultures. PCR sequencing yielded more specific identification at the species/subspecies level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR sequencing is suitable for the proper management of mycobacterial infections in Kuwait.

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Spitaleri, Andrea, Arash Ghodousi, Paolo Miotto, and Daniela Maria Cirillo. "Whole genome sequencing in Mycobacterium tuberculosis." Annals of Translational Medicine 7, S6 (September 2019): S197. http://dx.doi.org/10.21037/atm.2019.07.28.

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Cabibbe, Andrea M., Timothy M. Walker, Stefan Niemann, and Daniela M. Cirillo. "Whole genome sequencing of Mycobacterium tuberculosis." European Respiratory Journal 52, no. 5 (September 12, 2018): 1801163. http://dx.doi.org/10.1183/13993003.01163-2018.

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Miller, Kennon, Susan M. Harrington, and Gary W. Procop. "Acid-fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria." Archives of Pathology & Laboratory Medicine 139, no. 8 (January 9, 2015): 1020–23. http://dx.doi.org/10.5858/arpa.2013-0705-oa.

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Context New molecular diagnostic tests are attractive because of the potential they hold for improving diagnostics in microbiology. The value of these tests, which is often assumed, should be investigated to determine the best use of these potentially powerful tools. Objective To investigate the usefulness of broad-range polymerase chain reaction (PCR), followed by sequencing, in mycobacterial infections. Design We reviewed the test performance of acid-fast bacilli (AFB) PCR and traditional diagnostic methods (histopathology, AFB smear, and culture). We assessed the diagnostic effect and cost of the unrestricted ordering of broad-range PCR for the detection and identification of mycobacteria in clinical specimens. Results The AFB PCR was less sensitive than culture and histopathology and was less specific than culture, AFB smear, and histopathology. During 18 months, $93 063 was spent on 183 patient specimens for broad-range PCR and DNA sequencing for mycobacteria to confirm one culture-proven Mycobacterium tuberculosis infection that was also known to be positive by AFB smear and histopathology. In this cohort, there was a false-negative AFB PCR for M tuberculosis and a false-positive AFB PCR for Mycobacterium lentiflavum. Conclusion Testing of AFB smear–negative specimens from patients without an inflammatory response supportive of a mycobacterial infection is costly and has not been proven to improve patient care. Traditional diagnostics (histopathology, AFB smear, and culture) should remain the primary methods for the detection of mycobacteria in clinical specimens.

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Williams, K. J., C. L. Ling, C. Jenkins, S. H. Gillespie, and T. D. McHugh. "A paradigm for the molecular identification of Mycobacterium species in a routine diagnostic laboratory." Journal of Medical Microbiology 56, no. 5 (May 1, 2007): 598–602. http://dx.doi.org/10.1099/jmm.0.46855-0.

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The aim of this study was to improve the identification of Mycobacterium species in the context of a UK teaching hospital. Real-time PCR assays were established to enable the rapid differentiation between Mycobacterium tuberculosis (MTB) complex and Mycobacterium species other than tuberculosis (MOTT), followed by 16S rRNA gene sequencing for the speciation of MOTT. Real-time PCR assays gave comparable results to those from the reference laboratory. The implementation of these PCR assays using an improved bead extraction method has enhanced the mycobacterial diagnostic service at the Royal Free Hospital by providing a rapid means of differentiating between MTB complex and MOTT, and would be simple to implement in similar laboratories. Sequence analysis successfully identified a range of Mycobacterium spp. representative of those encountered in the clinical setting of the authors, including Mycobacterium avium complex, Mycobacterium fortuitum group, Mycobacterium chelonae–Mycobacterium abscessus group, Mycobacterium xenopi and Mycobacterium gordonae. It provides a useful tool for the identification of MOTT when clinically indicated.

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Moriconi, Patricia Rossi, Cássia Yumi Ikuta, Fábio Gregori, Gisele de Oliveira, Sheila de Oliveira, Paloma De Oliveira Tonietti, José Soares Ferreira Neto, Fernando Ferreira, Adriana Cortez, and Evelise Oliveira Telles. "Mycobacteria in Minas cheese commercialized in open fairs in São Paulo, Brazil." Brazilian Journal of Veterinary Research and Animal Science 55, no. 4 (December 26, 2018): e146525. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2018.146525.

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Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that affects dairy herds throughout the Brazilian territory, constituting a neglected zoonosis transmitted by raw milk and its derivatives. In this study, we evaluated the presence of M. bovis and other mycobacteria in Minas cheese obtained from open fairs in the city of São Paulo between 2012 and 2013. Samples (n = 133) were decontaminated using hexa-cetylpyridinium chloride and seeded on Stonebrink–Leslie medium. The isolates were submitted to molecular identification by TB Multiplex PCR targeting the 16S rRNA gene and amplicon nucleotide sequencing. From 16 cheese samples (12%), we obtained 26 putative colonies of Mycobacterium spp., none of which belonged to any of the Mycobacterium tuberculosis, Mycobacterium avium, or Mycobacterium intracellulare complexes. Phylogenetic analysis showed that sample sequences were grouped in a clade that includes only non-tuberculous mycobacteria with proximity to sequences obtained from Mycobacterium novocastrense (3 sequences), Mycobacterium holsaticum (1 sequence), andMycobacterium elephantis (2 sequences). Although no epidemiological evidence was found regarding the importance of oral transmission of mycobacteria in healthy people, their importance in the immunosuppressed population remains uncertain.

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Koentjoro, Maharani Pertiwi, Adyan Donastin, and Endry Nugroho Prasetyo. "A SIMPLE METHOD OF DNA EXTRACTION OF MYCOBACTERIUM TUBERCULOSIS FROM SPUTUM CULTURES FOR SEQUENCING ANALYSIS." African Journal of Infectious Diseases 15, no. 2s (September 1, 2021): 19–22. http://dx.doi.org/10.21010/ajid.v15i2s.2.

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Background: Concern has been raised about DNA extraction from Mycobacterium tuberculosis due to its complex procedure. This study demonstrates a simple and fast DNA extraction method of mycobacterial genome to subsequent molecular investigation, such as Polymerization Chain Reaction (PCR) amplification, with species-specific primers and sequencing. Materials and Methods: Total DNA was isolated from M. tuberculosis cultured by using boil method. DNA was evaluated via measures of DNA quantity and quality (absorbance at 230, 260 and 280 nm), DNA integrity (electrophoresis). Molecular tests were tested namely PCR and sequencing. Conclusions: The quality of DNA obtained is acceptable for PCR and sequencing analysis. These findings demonstrate that the method used is inexpensive and suitable for minimum infrastructure facilities.

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Boritsch, Eva C., Varun Khanna, Alexandre Pawlik, Nadine Honoré, Victor H. Navas, Laurence Ma, Christiane Bouchier, et al. "Key experimental evidence of chromosomal DNA transfer among selected tuberculosis-causing mycobacteria." Proceedings of the National Academy of Sciences 113, no. 35 (August 15, 2016): 9876–81. http://dx.doi.org/10.1073/pnas.1604921113.

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Horizontal gene transfer (HGT) is a major driving force of bacterial diversification and evolution. For tuberculosis-causing mycobacteria, the impact of HGT in the emergence and distribution of dominant lineages remains a matter of debate. Here, by using fluorescence-assisted mating assays and whole genome sequencing, we present unique experimental evidence of chromosomal DNA transfer between tubercle bacilli of the early-branching Mycobacterium canettii clade. We found that the obtained recombinants had received multiple donor-derived DNA fragments in the size range of 100 bp to 118 kbp, fragments large enough to contain whole operons. Although the transfer frequency between M. canettii strains was low and no transfer could be observed among classical Mycobacterium tuberculosis complex (MTBC) strains, our study provides the proof of concept for genetic exchange in tubercle bacilli. This outstanding, now experimentally validated phenomenon presumably played a key role in the early evolution of the MTBC toward pathogenicity. Moreover, our findings also provide important information for the risk evaluation of potential transfer of drug resistance and fitness mutations among clinically relevant mycobacterial strains.

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Ford, Chris, Karina Yusim, Tom Ioerger, Shihai Feng, Michael Chase, Mary Greene, Bette Korber, and Sarah Fortune. "Mycobacterium tuberculosis – Heterogeneity revealed through whole genome sequencing." Tuberculosis 92, no. 3 (May 2012): 194–201. http://dx.doi.org/10.1016/j.tube.2011.11.003.

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Amlerova, Jana, Ibrahim Bitar, and Jaroslav Hrabak. "Genotyping of Mycobacterium tuberculosis using whole genome sequencing." Folia Microbiologica 63, no. 5 (March 17, 2018): 537–45. http://dx.doi.org/10.1007/s12223-018-0599-y.

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Srivastava, Ranjana, D. Kumar, M. N. Waskar, Meera Sharma, V. M. Katoch, and Brahm S. Srivastava. "Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis." Journal of Medical Microbiology 55, no. 8 (August 1, 2006): 1071–77. http://dx.doi.org/10.1099/jmm.0.46379-0.

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A repetitive sequence specific to Mycobacterium tuberculosis was isolated from a λgt11 library of M. tuberculosis by DNA–DNA hybridization using genomic DNA of M. tuberculosis as probe followed by subtractive hybridization with a cocktail of other mycobacterial DNA. This led to identification of CD192, a 1291 bp fragment of M. tuberculosis containing repetitive sequences, which produced positive hybridization signals with M. tuberculosis DNA within 30 min. Nucleotide sequencing revealed the presence of several direct and inverted repeats within the 1291 bp fragment that belonged to a PPE family gene (Rv0355) of M. tuberculosis. The use of CD192 as a DNA probe for the identification of M. tuberculosis in culture and clinical samples was investigated. The 1291 bp sequence was present in M. tuberculosis, Mycobacterium bovis and M. bovis BCG, but was not present in many of the other mycobacterial strains tested, including M. tuberculosis H37Ra. More than 300 clinical isolates of M. tuberculosis were probed with CD192, and the presence of the 1291 bp sequence was observed in all the clinical strains, including those lacking IS6110. The sequence displayed RFLP among the clinical isolates. A PCR assay was developed which detected M. tuberculosis with 100 % specificity from specimens of sputum, cerebrospinal fluid and pleural effusion from clinically diagnosed cases of tuberculosis.

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Iketleng, Thato, Richard Lessells, Mlungisi Thabiso Dlamini, Tuelo Mogashoa, Lucy Mupfumi, Sikhulile Moyo, Simani Gaseitsiwe, and Tulio de Oliveira. "Mycobacterium tuberculosis Next-Generation Whole Genome Sequencing: Opportunities and Challenges." Tuberculosis Research and Treatment 2018 (December 9, 2018): 1–8. http://dx.doi.org/10.1155/2018/1298542.

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Mycobacterium tuberculosis drug resistance is a threat to global tuberculosis (TB) control. Comprehensive and timely drug susceptibility determination is critical to inform appropriate treatment of drug-resistant tuberculosis (DR-TB). Phenotypic drug susceptibility testing (DST) is the gold standard for M. tuberculosis drug resistance determination. M. tuberculosis whole genome sequencing (WGS) has the potential to be a one-stop method for both comprehensive DST and epidemiological investigations. We discuss in this review the tremendous opportunities that next-generation WGS presents in terms of understanding the molecular epidemiology of tuberculosis and mechanisms of drug resistance. The potential clinical value and public health impact in the areas of DST for patient management and tracing of transmission chains for timely public health intervention are also discussed. We present the current challenges for the implementation of WGS in low and middle-income settings. WGS analysis has already been adapted routinely in laboratories to inform patient management and public health interventions in low burden high-income settings such as the United Kingdom. We predict that the technology will be adapted similarly in high burden settings where the impact on the epidemic will be greatest.

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Mba Medie, Felix, Iskandar Ben Salah, Michel Drancourt, and Bernard Henrissat. "Paradoxical conservation of a set of three cellulose-targeting genes in Mycobacterium tuberculosis complex organisms." Microbiology 156, no. 5 (May 1, 2010): 1468–75. http://dx.doi.org/10.1099/mic.0.037812-0.

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The genome of the tuberculosis agent Mycobacterium tuberculosis encodes a putative cellulose-binding protein (CBD2), one candidate cellulase (Cel12), and one fully active cellulase (Cel6). This observation is puzzling, because cellulose is a major component of plant cell walls, whereas M. tuberculosis is a human pathogen without known contact with plants. In order to investigate the biological role of such cellulose-targeting genes in M. tuberculosis we report here the search for and transcription analysis of this set of genes in the genus Mycobacterium. An in silico search for cellulose-targeting orthologues found that only 2.5 % of the sequenced bacterial genomes encode the Cel6, Cel12 and CBD2 gene set simultaneously, including those of the M. tuberculosis complex (MTC) members. PCR amplification and sequencing further demonstrated the presence of these three genes in five non-sequenced MTC bacteria. Among mycobacteria, the combination of Cel6, Cel12 and CBD2 was unique to MTC members, with the exception of Mycobacterium bovis BCG Pasteur, which lacked CBD2. RT-PCR in M. tuberculosis H37Rv indicated that the three cellulose-targeting genes were transcribed into mRNA. The present work shows that MTC organisms are the sole mycobacteria among very few organisms to encode the three cellulose-targeting genes CBD2, Cel6 and Cel12. Our data point toward a unique, yet unknown, relationship with non-plant cellulose-producing hosts such as amoebae.

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Nopponpunth, Vanida, Worachart Sirawaraporn, Patricia J. Greene, and Daniel V. Santi. "Cloning and Expression of Mycobacterium tuberculosis and Mycobacterium leprae Dihydropteroate Synthase in Escherichia coli." Journal of Bacteriology 181, no. 21 (November 1, 1999): 6814–21. http://dx.doi.org/10.1128/jb.181.21.6814-6821.1999.

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ABSTRACT The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosisdihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain ofEscherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. lepraedihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from bothM. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.

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Dohal, M., I. Porvaznik, P. Kusnir, and J. Mokry. "Whole-Genome Sequencing in Relation to Resistance of Mycobacterium Tuberculosis." Acta Medica Martiniana 19, no. 1 (April 1, 2019): 12–21. http://dx.doi.org/10.2478/acm-2019-0002.

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Abstract Tuberculosis, a disease caused by Mycobacterium tuberculosis, represents one of the deadliest infections worldwide. The incidence of resistant forms is increasing year by year; therefore, it is necessary to involve new methods for rapid diagnostics and treatment. One of the possible solutions is the use of whole-genome sequencing (WGS). The WGS provides an identification of complete genome of the microorganism, including all genes responsible for resistance, in comparison with other genotypic methods (eg. Xpert MTB / RIF or Hain line-probes) that are capable to detect only basic genes. WGS data are available in 1-9 days and several online software tools (TBProfiler, CASTB, Mykrobe PredictorTB) are used for their interpretation and analysis, compared to 3-8 weeks in the case of classic phenotypic evaluation. Furthermore, WGS predicts resistance to the first-line antituberculotics with a sensitivity of 85-100% and a specificity of 85-100%. This review elucidates the importance and summarizes the current knowledge about the possible use of WGS in diagnosis and treatment of resistant forms of tuberculosis elucidates. WGS of M. tuberculosis brings new possibilities for rapid and accurate diagnostics of resistant forms of tuberculosis. Introducing WGS into routine practice can help to reduce the spread of resistant forms of tuberculosis as well as to increase the success rate of the treatment, especially through an appropriate combination of antituberculotics ATs. Introduction of WGS into routine diagnostics can, in spite of the financial difficulty, significantly improve patient care.

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Guimaraes, Ana M. S., and Cristina K. Zimpel. "Mycobacterium bovis: From Genotyping to Genome Sequencing." Microorganisms 8, no. 5 (May 3, 2020): 667. http://dx.doi.org/10.3390/microorganisms8050667.

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Mycobacterium bovis is the main pathogen of bovine, zoonotic, and wildlife tuberculosis. Despite the existence of programs for bovine tuberculosis (bTB) control in many regions, the disease remains a challenge for the veterinary and public health sectors, especially in developing countries and in high-income nations with wildlife reservoirs. Current bTB control programs are mostly based on test-and-slaughter, movement restrictions, and post-mortem inspection measures. In certain settings, contact tracing and surveillance has benefited from M. bovis genotyping techniques. More recently, whole-genome sequencing (WGS) has become the preferential technique to inform outbreak response through contact tracing and source identification for many infectious diseases. As the cost per genome decreases, the application of WGS to bTB control programs is inevitable moving forward. However, there are technical challenges in data analyses and interpretation that hinder the implementation of M. bovis WGS as a molecular epidemiology tool. Therefore, the aim of this review is to describe M. bovis genotyping techniques and discuss current standards and challenges of the use of M. bovis WGS for transmission investigation, surveillance, and global lineages distribution. We compiled a series of associated research gaps to be explored with the ultimate goal of implementing M. bovis WGS in a standardized manner in bTB control programs.

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Vázquez-Chacón, Carlos Arturo, Felipe de Jesús Rodríguez-Gaxiola, Cruz Fernando López-Carrera, Mayra Cruz-Rivera, Armando Martínez-Guarneros, Ricardo Parra-Unda, Eliakym Arámbula-Meraz, Salvador Fonseca-Coronado, Gilberto Vaughan, and Paúl Alexis López-Durán. "Identification of drug resistance mutations among Mycobacterium bovis lineages in the Americas." PLOS Neglected Tropical Diseases 15, no. 2 (February 16, 2021): e0009145. http://dx.doi.org/10.1371/journal.pntd.0009145.

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Identifying the Mycobacterium tuberculosis resistance mutation patterns is of the utmost importance to assure proper patient’s management and devising of control programs aimed to limit spread of disease. Zoonotic Mycobacterium bovis infection still represents a threat to human health, particularly in dairy production regions. Routinary, molecular characterization of M. bovis is performed primarily by spoligotyping and mycobacterial interspersed repetitive units (MIRU) while next generation sequencing (NGS) approaches are often performed by reference laboratories. However, spoligotyping and MIRU methodologies lack the resolution required for the fine characterization of tuberculosis isolates, particularly in outbreak settings. In conjunction with sophisticated bioinformatic algorithms, whole genome sequencing (WGS) analysis is becoming the method of choice for advanced genetic characterization of tuberculosis isolates. WGS provides valuable information on drug resistance and compensatory mutations that other technologies cannot assess. Here, we performed an analysis of the most frequently identified mutations associated with tuberculosis drug resistance and their genetic relationship among 2,074 Mycobacterium bovis WGS recovered primarily from non-human hosts. Full-length gene sequences harboring drug resistant associated mutations and their phylogenetic relationships were analyzed. The results showed that M. bovis isolates harbor mutations conferring resistance to both first- and second-line antibiotics. Mutations conferring resistance for isoniazid, fluoroquinolones, streptomycin, and aminoglycosides were identified among animal strains. Our findings highlight the importance of molecular surveillance to monitor the emergence of mutations associated with multi and extensive drug resistance in livestock and other non-human mammals.

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Saad, Jamal, Ahmed Loukil, and Michel Drancourt. "Bead-captured Mycobacterium tuberculosis for next-generation sequencing diagnosis of uncultured tuberculosis." European Journal of Clinical Microbiology & Infectious Diseases 39, no. 1 (October 14, 2019): 205–7. http://dx.doi.org/10.1007/s10096-019-03700-1.

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Takiff, Howard E., and Oscar Feo. "Clinical value of whole-genome sequencing of Mycobacterium tuberculosis." Lancet Infectious Diseases 15, no. 9 (September 2015): 1077–90. http://dx.doi.org/10.1016/s1473-3099(15)00071-7.

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Jaafar, Mohammad Maaruf, Mohd Zakihalani A. Halim, Mohamad Izwan Ismail, Lee Lian Shien, Teh Lay Kek, Ngeow Yun Fong, Norazmi Mohd Nor, et al. "Genome Sequencing and Annotation of Mycobacterium tuberculosis PR08 strain." Genomics Data 7 (March 2016): 119–20. http://dx.doi.org/10.1016/j.gdata.2015.12.030.

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Shivendra, Kumar Shahi. "MYCOBACTERIUM TUBERCULOSIS FROM EXTRA PULMONARY SITES NEXT-GENERATION WHOLE GENOME SEQUENCING: OPPORTUNITIES & CHALLENGES." Biological Markers in Fundamental and Clinical Medicine (collection of abstracts) 3, no. 1 (November 7, 2019): 90. http://dx.doi.org/10.29256/v.03.01.2019.escbm60.

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Slany, M., J. Svobodova, A. Ettlova, I. Slana, V. Mrlik, and I. Pavlik. "Mycobacterium arupense among the isolates of non-tuberculous mycobacteria from human, animal and environmental samples." Veterinární Medicína 55, No. 8 (September 15, 2010): 369–76. http://dx.doi.org/10.17221/2956-vetmed.

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Mycobacterium arupense is a non-tuberculous, potentially pathogenic species rarely isolated from humans. The aim of the study was to ascertain the spectrum of non-tuberculous mycobacteria within 271 sequenced mycobacterial isolates not belonging to M. tuberculosis and M. avium complexes. Isolates were collected between 2004 and 2009 in the Czech Republic and were examined within the framework of ecological studies carried out in animal populations infected with mycobacteria. A total of thirty-three mycobacterial species were identified. This report describes the isolation of M. arupense from the sputum of three human patients and seven different animal and environmental samples collected in the last six years in the Czech Republic: one isolate from leftover refrigerated organic dog food, two isolates from urine and clay collected from an okapi (Okapia johnstoni) and antelope bongo (Tragelaphus eurycerus) enclosure in a zoological garden, one isolate from the soil in an eagle's nest (Haliaeetus albicilla) band two isolates from two common vole (Microtus arvalis) livers from one cattle farm. All isolates were identified by biochemical tests, morphology and 16S rDNA sequencing. Also, retrospective screening for M. arupense occurrence within the collected isolates is presented.

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Eilertson, Brandon, Fernanda Maruri, Amondrea Blackman, Miguel Herrera, David C. Samuels, and Timothy R. Sterling. "High Proportion of Heteroresistance ingyrAandgyrBin Fluoroquinolone-Resistant Mycobacterium tuberculosis Clinical Isolates." Antimicrobial Agents and Chemotherapy 58, no. 6 (March 31, 2014): 3270–75. http://dx.doi.org/10.1128/aac.02066-13.

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ABSTRACTHeteroresistance is the coexistence of populations with differing nucleotides at a drug resistance locus within a sample of organisms. Although Sanger sequencing is the gold standard for sequencing, it may be less sensitive than deep sequencing for detecting fluoroquinolone heteroresistance inMycobacterium tuberculosis. Twenty-seven fluoroquinolone monoresistant and 11 fluoroquinolone-susceptibleM. tuberculosisisolates were analyzed by Sanger and Illumina deep sequencing. Individual sequencing reads were analyzed to detect heteroresistance in thegyrAandgyrBgenes. Heteroresistance to fluoroquinolones was identified in 10/26 (38%) phenotypically fluoroquinolone-resistant samples and 0/11 (P= 0.02) fluoroquinolone-susceptible controls. One resistant sample was excluded because of contamination with the laboratory strainM. tuberculosisH37Rv. Sanger sequencing revealed resistance-conferring mutations in 15 isolates, while deep sequencing revealed mutations in 20 isolates. Isolates with fluoroquinolone resistance-conferring mutations by Sanger sequencing all had at least those same mutations identified by deep sequencing. By deep sequencing, 10 isolates had a single fixed (defined as >95% frequency) mutation, while 10 were heteroresistant, 5 of which had a single unfixed (defined as <95% frequency) mutation and 5 had multiple unfixed mutations. Illumina deep sequencing identified a higher proportion of fluoroquinolone-resistantM. tuberculosisisolates with heteroresistance than did Sanger sequencing. The heteroresistant isolates frequently demonstrated multiple mutations, but resistant isolates with fixed mutations each had only a single mutation.

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Cabezas Vinueza, Leslie, and Patricia Jiménez Arias. "Distribution of Mycobacterium tuberculosis lineages in South America." Anatomía Digital 4, no. 3 (July 5, 2021): 34–58. http://dx.doi.org/10.33262/anatomiadigital.v4i3.1755.

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Molecular genotyping of Mycobacterium tuberculosis allows for the identification of circulating lineages and sublineages in the population and their relationship with migratory movements. The purpose of this review is to describe the phylogeography of Mycobacterium tuberculosis reported in South American countries that was analyzed using genotyping tools, analyze the Tuberculosis hotspots for the region and determine the impact of the COVID-19 pandemic on the Tuberculosis control program. The Latin American Mediterranean (LAM) sublineage belonging to the Euro-American lineage (Lineage 4) presents the highest prevalence in South America and is followed by the Beijing sublineage belonging to the East Asian lineage (Lineage 2). The Beijing sublineage is considered of worldwide interest because of its association with multidrug-resistant tuberculosis (MDR-TB), which is almost entirely distributed in South America, with Peru being the country with the highest prevalence for this sublineage. On the other hand, the Indo-Oceanic (Lineage 1), India-East Asia (Lineage 3) and West- African 2 (Lineage 6) sublineages have been reported with lower prevalence in South America. The molecular techniques used in the genotyping studies for Mycobacterium tuberculosis in South America were as follows: typing by complementary oligonucleotide spacer sequences (Spoligotyping), restriction-hybridization patterns (IS6110-RFLP, PGRS-RFLP), mycobacterial interspaced repeat units-variable number tandem repeats (MIRU-VNTR) and whole genome sequencing (WGS). At present, Brazil and Peru are the hotspots for tuberculosis and MDR-TB in South America, where the control of tuberculosis wholly affected by the COVID-19 pandemic. Thus, there have been significant impacts on containment programs and possible post-pandemic scenarios such that scientific contributions will need to be evaluated and implemented with new strategies for prevention, diagnosis, treatment and control of Tuberculosis.

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Kim, Hong, Sun-Hyun Kim, Tae-Sun Shim, Mi-na Kim, Gill-Han Bai, Young-Gil Park, Sueng-Hyun Lee, et al. "Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65)." International Journal of Systematic and Evolutionary Microbiology 55, no. 4 (July 1, 2005): 1649–56. http://dx.doi.org/10.1099/ijs.0.63553-0.

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The nucleotide sequences (604 bp) of partial heat-shock protein genes (hsp65) from 161 Mycobacterium strains containing 56 reference Mycobacterium species and 105 clinical isolates were determined and compared. hsp65 sequence analysis showed a higher degree of divergence between Mycobacterium species than did 16S rRNA gene analysis. Generally, the topology of the phylogenetic tree based on the hsp65 DNA sequences was similar to that of the 16S rRNA gene, thus revealing natural relationships among Mycobacterium species. When a direct sequencing protocol targeting 422 bp sequences was applied to 70 non-tuberculous mycobacterium (NTM) clinical isolates, all NTMs were clearly identified. In addition, an XhoI PCR restriction fragment length polymorphism analysis method for the differentiation of Mycobacterium tuberculosis complex from NTM strains was developed during this study. The results obtained suggest that 604 bp hsp65 sequences are useful for the phylogenetic analysis and species identification of mycobacteria.

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GK Madhavilatha, AK Anilkumar. "Mutations Associated with Pyrazinamide Resistance in the Clinical Isolates of Mycobacterium tuberculosis." Mapana - Journal of Sciences 12, no. 4 (July 1, 2013): 1–8. http://dx.doi.org/10.12723/mjs.27.1.

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Pyrazinamide is one of the four first line drugs for treatment of tuberculosis. It has been widely accepted, that pyrazinamide (PZA) resistance in Mycobacterium tuberculosis is correlated with mutations in the pncA gene. In the present study, pyrazinamide susceptibility was tested in 65 clinical isolates of Mycobacterium tuberculosis by pncA gene sequencing and was then correlated with pyrazinamidase activity. 68% of the resistant isolates showed mutation in the pncA gene which was further correlated with an assay for pyrazinamidase activity.

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Lee, Robyn S., and Madhukar Pai. "Real-Time Sequencing of Mycobacterium tuberculosis: Are We There Yet?" Journal of Clinical Microbiology 55, no. 5 (March 15, 2017): 1249–54. http://dx.doi.org/10.1128/jcm.00358-17.

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ABSTRACT Whole-genome sequencing has taken a leading role in epidemiologic studies of tuberculosis, but thus far, its real-time clinical utility has been low, in part because of the requirement for culture. In their report in this issue, Votintseva et al. (A. A. Votintseva, P. Bradley, L. Pankhurst, C. del Ojo Elias, M. Loose, K. Nilgiriwala, A. Chatterjee, E. G. Smith, N. Sanderson, T. M. Walker, M. R. Morgan, D. H. Wyllie, A. S. Walker, T. E. A. Peto, D. W. Crook, and Z. Iqbal, J Clin Microbiol 55:1285–1298, 2017, https://doi.org/10.1128/JCM.02483-16 ) present a new method for extracting Mycobacterium tuberculosis DNA directly from smear-positive respiratory samples, making it feasible to generate drug resistance predictions and phylogenetic trees in 44 h with the Illumina MiSeq. They also illustrate the potential for a <24-h turnaround time from DNA extraction to clinically relevant results with Illumina MiniSeq and Oxford Nanopore Technologies MinION. We comment on the promise and limitations of these approaches.

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Si, Jianwei, Zhe Wang, Zili Wang, and Haomin Li. "Sequencing-based detection of drug-resistant Mycobacterium tuberculosis in patients with spinal tuberculosis." Archives of Orthopaedic and Trauma Surgery 132, no. 7 (March 30, 2012): 941–45. http://dx.doi.org/10.1007/s00402-012-1506-7.

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Blackwood, Kym S., Cheng He, James Gunton, Christine Y. Turenne, Joyce Wolfe, and Amin M. Kabani. "Evaluation of recA Sequences for Identification of Mycobacterium Species." Journal of Clinical Microbiology 38, no. 8 (2000): 2846–52. http://dx.doi.org/10.1128/jcm.38.8.2846-2852.2000.

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16S rRNA sequence data have been used to provide a molecular basis for an accurate system for identification of members of the genusMycobacterium. Previous studies have shown thatMycobacterium species demonstrate high levels (>94%) of 16S rRNA sequence similarity and that this method cannot differentiate between all species, i.e., M. gastri and M. kansasii. In the present study, we have used the recAgene as an alternative sequencing target in order to complement 16S rRNA sequence-based genetic identification. The recA genes of 30 Mycobacterium species were amplified by PCR, sequenced, and compared with the published recA sequences of M. tuberculosis, M. smegmatis, and M. leprae available from GenBank. By recA sequencing the species showed a lower degree of interspecies similarity than they did by 16S rRNA gene sequence analysis, ranging from 96.2% betweenM. gastri and M. kansasii to 75.7% betweenM. aurum and M. leprae. Exceptions to this were members of the M. tuberculosis complex, which were identical. Two strains of each of 27 species were tested, and the intraspecies similarity ranged from 98.7 to 100%. In addition, we identified new Mycobacterium species that contain a protein intron in their recA genes, similar to M. tuberculosis and M. leprae. We propose thatrecA gene sequencing offers a complementary method to 16S rRNA gene sequencing for the accurate identification of theMycobacterium species.

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van Soolingen, Dick. "Whole-genome sequencing of Mycobacterium tuberculosis as an epidemiological marker." Lancet Respiratory Medicine 2, no. 4 (April 2014): 251–52. http://dx.doi.org/10.1016/s2213-2600(14)70049-9.

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Daum, L. T., P. B. Fourie, S. Bhattacharyya, N. A. Ismail, S. Gradus, N. E. Maningi, S. V. Omar, and G. W. Fischer. "Next-Generation Sequencing for Identifying Pyrazinamide Resistance in Mycobacterium tuberculosis." Clinical Infectious Diseases 58, no. 6 (December 12, 2013): 903–4. http://dx.doi.org/10.1093/cid/cit811.

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Zakham, Fathiah, Halima Bazoui, Mohammed Akrim, Sanae Lemrabet, Ouafae Lahlou, Mohamed Elmzibri, Abdelaziz Benjouad, My Mustapha Ennaji, and Rajae Elaouad. "Evaluation of conventional molecular diagnosis of Mycobacterium tuberculosis in clinical specimens from Morocco." Journal of Infection in Developing Countries 6, no. 01 (November 29, 2011): 40–45. http://dx.doi.org/10.3855/jidc.1857.

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Introduction: Tuberculosis is a major public health threat, annually affecting new individuals worldwide, especially those in developing countries. Rapid detection of the agent and effective treatment are two important factors in controlling this disease.Methodology: The present study aimed to evaluate polymerase chain reaction (PCR) as a rapid and direct molecular method for the diagnosis of Mycobacterium tuberculosis (MTB) in 70 clinical specimens (62 sputum samples, six cerebrospinal fluids, and two biopsies) using heat shock protein (hsp65) as the gene target. Automated sequencing of the same gene was used for the identification of MTB to the species level. Results: The sensitivity of PCR was 81.13%, with specificity of 88.24%; the positive and negative predictive values were 95.56% and 60%, respectively.Conclusion: Based on these results, the hsp65 gene sequence can be used to differentiate the members of MTB complex from non-tuberculosis mycobacteria (NTM).

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Dupont, Christian, Yushu Chen, Zhujun Xu, Françoise Roquet-Banères, Mickaël Blaise, Anne-Kathrin Witt, Faustine Dubar, et al. "A piperidinol-containing molecule is active against Mycobacterium tuberculosis by inhibiting the mycolic acid flippase activity of MmpL3." Journal of Biological Chemistry 294, no. 46 (September 27, 2019): 17512–23. http://dx.doi.org/10.1074/jbc.ra119.010135.

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Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis. Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane–associated mycolic acid flippase in M. tuberculosis. Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.

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Harkins, Kelly M., Jane E. Buikstra, Tessa Campbell, Kirsten I. Bos, Eric D. Johnson, Johannes Krause, and Anne C. Stone. "Screening ancient tuberculosis with qPCR: challenges and opportunities." Philosophical Transactions of the Royal Society B: Biological Sciences 370, no. 1660 (January 19, 2015): 20130622. http://dx.doi.org/10.1098/rstb.2013.0622.

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The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies.

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Poudel, Ajay, Chie Nakajima, Yukari Fukushima, Haruka Suzuki, Basu Dev Pandey, Bhagwan Maharjan, and Yasuhiko Suzuki. "Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolated in Nepal." Antimicrobial Agents and Chemotherapy 56, no. 6 (March 26, 2012): 2831–36. http://dx.doi.org/10.1128/aac.06418-11.

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ABSTRACTDespite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance inMycobacterium tuberculosisis required. In the present study, we investigated the prevalence of mutations inrpoBandkatGgenes and theinhApromoter region in 158M. tuberculosisisolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) ofrpoBwere identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in thekatGgene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in theinhApromoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance inM. tuberculosisin Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.

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Dohál, Matúš, Igor Porvazník, Kristián Pršo, Erik Michael Rasmussen, Ivan Solovič, and Juraj Mokrý. "Whole-genome sequencing and Mycobacterium tuberculosis: Challenges in sample preparation and sequencing data analysis." Tuberculosis 123 (July 2020): 101946. http://dx.doi.org/10.1016/j.tube.2020.101946.

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Li, Wei, Andrés Obregón-Henao, Joshua B. Wallach, E. Jeffrey North, Richard E. Lee, Mercedes Gonzalez-Juarrero, Dirk Schnappinger, and Mary Jackson. "Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3." Antimicrobial Agents and Chemotherapy 60, no. 9 (June 13, 2016): 5198–207. http://dx.doi.org/10.1128/aac.00826-16.

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ABSTRACTIn recent years, whole-cell-based screens for novel small molecule inhibitors active againstMycobacterium tuberculosisin culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, thatmmpL3is required for the replication and viability ofM. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencingmmpL3had a rapid bactericidal effect on actively replicating cellsin vitroand reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further renderedM. tuberculosishypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies.

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Dookie, Navisha, Nesri Padayatchi, Richard J. Lessells, Cherise L. Naicker, Sunitha Chotoo, and Kogieleum Naidoo. "Individualized Treatment of Multidrug-resistant Tuberculosis Using Whole-Genome Sequencing and Expanded Drug-Susceptibility Testing." Clinical Infectious Diseases 71, no. 11 (May 8, 2020): 2981–85. http://dx.doi.org/10.1093/cid/ciaa526.

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Abstract A case of multidrug-resistant tuberculosis is presented. It highlights the role of whole-genome sequencing, expanded phenotypic drug susceptibility testing, and enhanced case management, offering a more complete understanding of drug susceptibility to Mycobacterium tuberculosis. This approach guides an effective individualized treatment strategy that results in rapid sustained culture conversion.

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Schafbuch, Ryan, Stacy Tinkler, Chee Kin Lim, Rebecca Wolking, and José Ramos-Vara. "Disseminated mycobacteriosis caused by Mycobacterium kansasii in a pot-bellied pig." Journal of Veterinary Diagnostic Investigation 30, no. 4 (June 1, 2018): 646–50. http://dx.doi.org/10.1177/1040638718780189.

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A 1.5-y-old spayed female Juliana pot-bellied pig was presented to the Purdue University Veterinary Teaching Hospital with a history of wasting and anorexia. Enlarged and partially mineralized lymph nodes were identified on radiographs and computed tomography scan. Generalized lymphadenomegaly and disseminated nodules in the lungs, liver, spleen, and kidneys were identified on postmortem examination. Histologic examination revealed caseonecrotic granulomas with numerous intracellular, acid-fast bacilli. Mycobacterium kansasii type II was identified as the etiologic agent by PCR amplification using universal Mycobacterium primers, direct sequencing of the PCR amplicon, and comparison to sequences in GenBank. We describe a case in a pot-bellied pig of mycobacteriosis caused by an atypical mycobacterial species and highlight the important role of laboratory testing in suspected cases of tuberculosis.

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Dupont, Chris, Keith Thompson, Cord Heuer, Brigitte Gicquel, and Alan Murray. "Identification and characterization of an immunogenic 22 kDa exported protein of Mycobacterium avium subspecies paratuberculosis." Journal of Medical Microbiology 54, no. 11 (November 1, 2005): 1083–92. http://dx.doi.org/10.1099/jmm.0.46163-0.

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An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 316F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75 % identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 316F) compared to none from a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.

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Jalil, Asra'a A. Abdul, Zahra M. Al-khafaji, and Mushtak T. Al-ouqaili. "INVESTIGATION OF ACCD3 GENE OF MYCOBACTERIUM TUBERCULOSIS IRAQI ISOLATES." Asian Journal of Pharmaceutical and Clinical Research 11, no. 8 (August 7, 2018): 208. http://dx.doi.org/10.22159/ajpcr.2018.v11i8.25269.

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Objective: Mycobacterium tuberculosis, one of the deadliest human pathogens, causes several million new infections and about 2 million fatalities annually. The cell wall of M. tuberculosis is endowed with a highly impermeable, complex array of diverse lipids such as mycolic acids, which bestow the bacterium with not only virulence but also resistance to host immunity and antibiotics.Methods: Mycobacterial lipid metabolism has thus emerged as an attractive target for the design and development of novel antimycobacterial therapeutics. The first committed step in the biosynthesis of mycolic acid is the carboxylation of acetyl-CoA to malonyl-CoA which is catalyzed by acetyl-coenzyme A carboxylase carboxyl transferase beta subunit (accD3), a primer pairs were designed computationally and used for the amplification of accD3 gene using conventional polymerase chain reaction (PCR) and sequencing the PCR product and analyze the results.Results: Two sequences of the detection gene (LprM gene) and eight sequences of accD3 gene under study were deposited at NCBI – GenBank database with accession numbers (LC009881, LC009880.1, LC006979, LC008196, LC009412, LC009414, LC034168, LC038020, LC041163, and LC041368) and primer pairs deposited at Probe database/NCBI with accession number Pr032816836.Conclusion: AccD3 gene is a good drug target in MDR M. tuberculosis strains.

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Christianson, Sara, Joyce Wolfe, Hafid Soualhine, and Meenu K. Sharma. "Comparison of repetitive-sequence-based polymerase chain reaction with random amplified polymorphic DNA analysis for rapid genotyping of nontuberculosis mycobacteria." Canadian Journal of Microbiology 58, no. 8 (August 2012): 953–64. http://dx.doi.org/10.1139/w2012-068.

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Nontuberculosis mycobacteria (NTM) are an important cause of human disease and infections. Though less notorious than tuberculosis, these infections are clinically significant and have been associated with outbreaks in various settings. To accommodate outbreak investigations for the numerous species of NTM, we evaluated a DiversiLab repetitive-sequence-based PCR (rep-PCR) kit for genotyping of mycobacteria. This kit was used to genotype both rapidly and slowly growing mycobacteria and was compared with other PCR-based genotyping methods, including random amplified polymorphic DNA (RAPD) analysis, hsp65 gene sequencing, and mycobacterial interspersed repetitive unit – variable number of tandem repeat (MIRU–VNTR) analysis. Compared with RAPD analysis, rep-PCR achieved better reproducibility in testing. When compared with hsp65 gene sequencing and MIRU–VNTR for Mycobacterium avium , rep-PCR provided results that agreed with these less discriminatory genotyping methods but provided a higher level of discrimination for situations such as outbreak investigations. We also evaluated the kit for its ability to identify closely related rapidly growing NTM. While rep-PCR was informative in some cases, a much larger library of isolates would be necessary to truly evaluate it as an identification tool. Overall, rep-PCR was able to provide improved reproducibility over RAPD and a discriminatory genotyping method for the isolates evaluated in this study.

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Valdez-Palomares, Fernanda, Marcela Muñoz Torrico, Berenice Palacios-González, Xavier Soberón, and Eugenia Silva-Herzog. "Altered Microbial Composition of Drug-Sensitive and Drug-Resistant TB Patients Compared with Healthy Volunteers." Microorganisms 9, no. 8 (August 18, 2021): 1762. http://dx.doi.org/10.3390/microorganisms9081762.

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Mycobacterium tuberculosis infection has three discernible outcomes: active tuberculosis, latent tuberculosis, or clearance of the bacterium. The outcome of the infection depends on the interaction of the bacterium, the immune system, and the microbiome of the host. The current study uses 16S rRNA sequencing to determine the diversity and composition of the respiratory microbiome of drug-resistant and drug-sensitive tuberculosis patients as well as healthy volunteers. Tuberculosis patients exhibited increased microbial diversity and differentially abundant bacteria than healthy volunteers. Compositional differences were also observed when comparing drug-sensitive or -resistant tuberculosis patients. Finally, we defined and assessed the differences in the core sputum microbiota between tuberculosis patients and healthy volunteers. Our observations collectively suggest that in sputum, Mycobacterium tuberculosis infection is related to altered bacterial diversity and compositional differences of core members of the microbiome, with potential implications for the bacterial pulmonary ecosystem’s stability and function.

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Bespyatykh, Julia, Egor Shitikov, Dmitry Bespiatykh, Andrei Guliaev, Ksenia Klimina, Vladimir Veselovsky, Georgij Arapidi, et al. "Metabolic Changes of Mycobacterium tuberculosis during the Anti-Tuberculosis Therapy." Pathogens 9, no. 2 (February 18, 2020): 131. http://dx.doi.org/10.3390/pathogens9020131.

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Tuberculosis, caused by Mycobacterium tuberculosis complex bacteria, remains one of the most pressing health problems. Despite the general trend towards reduction of the disease incidence rate, the situation remains extremely tense due to the distribution of the resistant forms. Most often, these strains emerge through the intra-host microevolution of the pathogen during treatment failure. In the present study, the focus was on three serial clinical isolates of Mycobacterium tuberculosis Beijing B0/W148 cluster from one patient with pulmonary tuberculosis, to evaluate their changes in metabolism during anti-tuberculosis therapy. Using whole genome sequencing (WGS), 9 polymorphisms were determined, which occurred in a stepwise or transient manner during treatment and were linked to the resistance (GyrA D94A; inhA t-8a) or virulence. The effect of the inhA t-8a mutation was confirmed on both proteomic and transcriptomic levels. Additionally, the amount of RpsL protein, which is a target of anti-tuberculosis drugs, was reduced. At the systemic level, profound changes in metabolism, linked to the evolution of the pathogen in the host and the effects of therapy, were documented. An overabundance of the FAS-II system proteins (HtdX, HtdY) and expression changes in the virulence factors have been observed at the RNA and protein levels.

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Juréen, Pontus, Jim Werngren, Juan-Carlos Toro, and Sven Hoffner. "Pyrazinamide Resistance and pncA Gene Mutations in Mycobacterium tuberculosis." Antimicrobial Agents and Chemotherapy 52, no. 5 (March 3, 2008): 1852–54. http://dx.doi.org/10.1128/aac.00110-08.

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ABSTRACT Thirty-four pyrazinamide-resistant and 37 pyrazinamide-susceptible Mycobacterium tuberculosis complex strains were analyzed for pncA gene mutations. None of the sensitive strains had any mutations, apart from silent mutations, whereas all but one resistant strain showed pncA mutations. By using sequencing as a means of early resistance detection, the inconsistency of phenotypic pyrazinamide assays can be circumvented.

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Ameke, Selassie, Prince Asare, Samuel Yaw Aboagye, Isaac Darko Otchere, Stephen Osei-Wusu, Dorothy Yeboah-Manu, and Adwoa Asante-Poku. "Molecular epidemiology of Mycobacterium tuberculosis complex in the Volta Region of Ghana." PLOS ONE 16, no. 3 (March 17, 2021): e0238898. http://dx.doi.org/10.1371/journal.pone.0238898.

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Context Available molecular epidemiological data from recent studies suggest significant genetic variation between the different lineages of Mycobacterium tuberculosis complex (MTBC) and the MTBC lineages might have adapted to different human populations. Aim This study sought to determine the population structure of clinical MTBC isolates from the Volta Region of Ghana. Methods The MTBC isolates obtained from collected sputum samples were identified by PCR detecting of IS6110 and genotyped using spoligotyping. Non-tuberculous mycobacterial isolates were characterized by amplification of the heat shock protein 65 (hsp65) gene and sequencing. The drug susceptibility profiles of the MTBCs determined using GenoType MTBDRplus. Results One hundred and seventeen (117, 93.6%) out of 125 mycobacterial positive isolates were characterized as members of the MTBC of which M. tuberculosis sensu stricto (MTBss) and M. africanum (MAF) were respectively 94 (80.3%) and 23 (19.7%). In all, 39 distinct spoligotype patterns were obtained; 26 for MTBss and 13 for MAF lineages. Spoligotyping identified 89 (76%) Lineage 4, 16 (13.6%) Lineage 5, 7 (6.0%) Lineage 6, 3 (2.6%) Lineage 2, 1(0.9%) Lineage 3 and 1 (0.9%) Lineage 1. Among the Lineage 4 isolates, 62/89 (69.7%) belonged to Cameroon sub-lineage, 13 (14.7%) Ghana, 8 (9.0%) Haarlem, 2 (2.2%) LAM, 1 (1.1%) Uganda I, 1 (1.1%) X and the remaining two (2.2%) were orphan. Significant localization of MAF was found within the Ho municipality (n = 13, 29.5%) compared to the more cosmopolitan Ketu-South/Aflao (n = 3, 8.3%) (p-value = 0.017). Eight (8) non-tuberculous mycobacteria were characterized as M. abscessus (7) and M. fortuitum (1). Conclusion We confirmed the importance of M. africanum lineages as a cause of TB in the Volta region of Ghana.

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Walker, Timothy, Camilla L. Ip, Ruth H. Harrell, Jason T. Evans, Georgia Kapatai, Martin J. Dedicoat, David W. Eyre, et al. "A whole-genome sequencing approach to targeting Mycobacterium tuberculosis outbreak management." Lancet 380 (November 2012): S77. http://dx.doi.org/10.1016/s0140-6736(13)60433-x.

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Journal articles on the topic 'Mycobacterium tuberculosis sequencing'

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