Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (sensu stricto), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defence systems and show that the MKC species harbours unique and shared genomic signatures. High frequency of presence of prophages and different types of defence systems were observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the twentieth century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasii's increased virulence and drug resistance.

Department of Biosciences Nottingham Trent University Nottingham UK

Department of Diagnostic Mycobacterioses Regional Institute of Public Health Ostrava Czech Republic

Department of Microbiological Pathology Sefako Makgatho Health Sciences University Pretoria South Africa

German Center for Infection Research Partner Site Hamburg Lübeck Borstel Riems Borstel Germany

Global Institute of Health University of Antwerp Antwerp Belgium

Laboratório de Bacteriologia e Bioensaios Instituto Nacional de Infectologia Fiocruz Rio de Janeiro RJ Brazil

Laboratório de Biologia do Reconhecer Universidade Estadual do Norte Fluminense Darcy Ribeiro Campos dos Goytacazes RJ Brazil

Laboratório de Biologia Molecular Aplicada a Micobactérias Instituto Oswaldo Cruz Fiocruz Rio de Janeiro RJ Brazil

Laboratório de Genética Molecular de Microrganismos Instituto Oswaldo Cruz Fiocruz Rio de Janeiro RJ Brazil

Laboratório de Referência Nacional para Tuberculose Centro de Referência Professor Hélio Fraga Escola Nacional de Saúde Pública Fiocruz Rio de Janeiro RJ Brazil

Laboratory of Molecular Epidemiology and Evolutionary Genetics St Petersburg Pasteur Institute St Petersburg Russia

Molecular and Experimental Mycobacteriology Research Center Borstel Borstel Germany

Mycobacteria Reference Laboratory Croatian National Institute of Public Health Zagreb Croatia

Mycobacteriology Section of Microbiology Laboratory North Estonia Medical Centre Tallinn Estonia

National Health Laboratory Service Dr George Mukhari Tertiary Laboratory Medical Microbiology Pretoria South Africa

National Institute for Public Health and the Environment Bilthoven Netherlands

National Reference Laboratory for Mycobacteria University Clinic of Respiratory and Allergic Diseases Golnik Slovenia

Núcleo de Doenças Infecciosas Universidade Federal do Espírito Santo Vitória ES Brazil

One Health Research Group Universidad de Las Américas Quito Ecuador

Serviço de Pesquisa Clínica Centro de Referência Professor Hélio Fraga Escola Nacional de Saúde Pública Fiocruz Rio de Janeiro RJ Brazil

St Petersburg Research Institute of Phthisiopulmonology St Petersburg Russia

TB and Mycobacteria Unit Institut Pasteur de Guadeloupe Guadeloupe France

Tuberculosis Department Servicio Autónomo Instituto de Biomedicina Dr Jacinto Convit Universidad Central de Venezuela Caracas Venezuela

Unit of Mycobacteriology Institute of Tropical Medicine Antwerp Belgium

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Machado E, Vasconcellos S, Gomes L, Catanho M, Ramos J, et al. Online resource. Microbiology Society; 2024. DOI

Tagini F, et al. Phylogenomics reveal that Mycobacterium Kansasii subtypes are species-level lineages. Description of Mycobacterium pseudokansasii sp. nov., Mycobacterium innocens sp. nov. and mycobacterium attenuatum sp. nov. Int J Syst Evol Microbiol. 2019 doi: 10.1099/ijsem.0.003378. PubMed DOI

Jagielski T, Borówka P, Bakuła Z, Lach J, Marciniak B, et al. Genomic insights into the Mycobacterium kansasii complex: an update. Front Microbiol. 2020;10:2918. doi: 10.3389/fmicb.2019.02918. PubMed DOI PMC

Taillard C, Greub G, Weber R, Pfyffer GE, Bodmer T, et al. Clinical implications of Mycobacterium kansasii species heterogeneity: Swiss National Survey. J Clin Microbiol. 2003;41:1240–1244. doi: 10.1128/JCM.41.3.1240-1244.2003. PubMed DOI PMC

Mussi VO, Simão TLBV, Almeida FM, Machado E, de Carvalho LD, et al. A murine model of Mycobacterium kansasii infection reproducing necrotic lung pathology reveals considerable heterogeneity in virulence of clinical isolates. Front Microbiol. 2021;12:718477. doi: 10.3389/fmicb.2021.718477. PubMed DOI PMC

Tagini F, Pillonel T, Bertelli C, Jaton K, Greub G. Pathogenic determinants of the Mycobacterium kansasii complex: an unsuspected role for distributive conjugal transfer. Microorganisms. 2021;9:348. doi: 10.3390/microorganisms9020348. PubMed DOI PMC

Luo T, Xu P, Zhang Y, Porter JL, Ghanem M, et al. Population genomics provides insights into the evolution and adaptation to humans of the waterborne pathogen Mycobacterium kansasii. Nat Commun. 2021;12:2491. doi: 10.1038/s41467-021-22760-6. PubMed DOI PMC

Wang J, McIntosh F, Radomski N, Dewar K, Simeone R, et al. Insights on the emergence of Mycobacterium tuberculosis from the analysis of Mycobacterium kansasii. Genome Biol Evol. 2015;7:856–870. doi: 10.1093/gbe/evv035. PubMed DOI PMC

Guo Y, Cao Y, Liu H, Yang J, Wang W, et al. Clinical and microbiological characteristics of Mycobacterium kansasii pulmonary infections in China. Microbiol Spectr. 2022;10:e0147521. doi: 10.1128/spectrum.01475-21. PubMed DOI PMC

Goldenberg T, Gayoso R, Mogami R, Lourenço MC, Ramos JP, et al. Clinical and epidemiological characteristics of M. kansasii pulmonary infections from Rio de Janeiro, Brazil, between 2006 and 2016. J Bras Pneumol. 2020;46:1–7. doi: 10.36416/1806-3756/e20190345. PubMed DOI

Bakuła Z, Modrzejewska M, Pennings L, Proboszcz M, Safianowska A, et al. Drug susceptibility profiling and genetic determinants of drug resistance in Mycobacterium kansasii. Antimicrob Agents Chemother. 2018;62:e01788-17. doi: 10.1128/AAC.01788-17. PubMed DOI PMC

Machado E, Vasconcellos SEG, Cerdeira C, Gomes LL, Junqueira R, et al. Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity. Mem Inst Oswaldo Cruz. 2018;113:e180085. doi: 10.1590/0074-02760180085. PubMed DOI PMC

Borówka P, Lach J, Bakuła Z, van Ingen J, Safianowska A, et al. Draft genome sequences of Mycobacterium kansasii clinical strains. Genome Announc. 2017;5:1–3. doi: 10.1128/genomeA.00406-17. PubMed DOI PMC

Baym M, Kryazhimskiy S, Lieberman TD, Chung H, Desai MM, et al. Inexpensive multiplexed library preparation for megabase-sized genomes. PLoS One. 2015;10:e0128036. doi: 10.1371/journal.pone.0128036. PubMed DOI PMC

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–477. doi: 10.1089/cmb.2012.0021. PubMed DOI PMC

Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27:722–736. doi: 10.1101/gr.215087.116. PubMed DOI PMC

Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25:1043–1055. doi: 10.1101/gr.186072.114. PubMed DOI PMC

Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun. 2018;9:1–8. doi: 10.1038/s41467-018-07641-9. PubMed DOI PMC

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068–2069. doi: 10.1093/bioinformatics/btu153. PubMed DOI

Page AJ, Cummins` CA, Hunt M, Wong VK, Reuter S, et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 2015;31:3691–3693. doi: 10.1093/bioinformatics/btv421. PubMed DOI PMC

Tonkin-Hill G, MacAlasdair N, Ruis C, Weimann A, Horesh G, et al. Producing polished prokaryotic pangenomes with the Panaroo pipeline. Genome Biol. 2020;21:180. doi: 10.1186/s13059-020-02090-4. PubMed DOI PMC

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Hadfield J, Croucher NJ, Goater RJ, Abudahab K, Aanensen DM, et al. Phandango: an interactive viewer for bacterial population genomics. Bioinformatics. 2018;34:292–293. doi: 10.1093/bioinformatics/btx610. PubMed DOI PMC

Tonkin-Hill G, Gladstone RA, Pöntinen AK, Arredondo-Alonso S, Bentley SD, et al. Robust analysis of prokaryotic pangenome gene gain and loss rates with Panstripe. Genome Res. 2023;33:129–140. doi: 10.1101/gr.277340.122. PubMed DOI PMC

Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35:4453–4455. doi: 10.1093/bioinformatics/btz305. PubMed DOI PMC

Mostowy R, Croucher NJ, Andam CP, Corander J, Hanage WP, et al. Efficient inference of recent and ancestral recombination within bacterial populations. Mol Biol Evol. 2017;34:1167–1182. doi: 10.1093/molbev/msx066. PubMed DOI PMC

Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49:W293–W296. doi: 10.1093/nar/gkab301. PubMed DOI PMC

Page AJ, Taylor B, Delaney AJ, Soares J, Seemann T, et al. SNP-sites: rapid efficient extraction of SNPs from multi-FASTA alignments. Microb Genom. 2016;2:e000056. doi: 10.1099/mgen.0.000056. PubMed DOI PMC

Croucher NJ, Page AJ, Connor TR, Delaney AJ, Keane JA, et al. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Res. 2015;43:e15. doi: 10.1093/nar/gku1196. PubMed DOI PMC

Tonkin-Hill G, Lees JA, Bentley SD, Frost SDW, Corander JR. RhierBAPS: an R implementation of the population clustering algorithm hierBAPS. Wellcome Open Res. 2018;3:93. doi: 10.12688/wellcomeopenres.14694.1. PubMed DOI PMC

Didelot X, Croucher NJ, Bentley SD, Harris SR, Wilson DJ. Bayesian inference of ancestral dates on bacterial phylogenetic trees. Nucleic Acids Res. 2018;46:e134. doi: 10.1093/nar/gky783. PubMed DOI PMC

Schwengers O, Barth P, Falgenhauer L, Hain T, Chakraborty T, et al. Platon: identification and characterization of bacterial plasmid contigs in short-read draft assemblies exploiting protein sequence-based replicon distribution scores. Microb Genom. 2020;6:1–12. doi: 10.1099/mgen.0.000398. PubMed DOI PMC

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. PubMed DOI

Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:1–9. doi: 10.1186/1471-2105-10-421. PubMed DOI PMC

Garcillán-Barcia MP, Redondo-Salvo S, Vielva L, Cruz F. Methods in Molecular Biology (Clifton, N.J.) vol. 2075. 2020. MOBscan: automated annotation of MOB relaxases; pp. 295–308. PubMed

Guo J, Bolduc B, Zayed AA, Varsani A, Dominguez-Huerta G, et al. VirSorter2: a multi-classifier, expert-guided approach to detect diverse DNA and RNA viruses. Microbiome. 2021;9:1–13. doi: 10.1186/s40168-020-00990-y. PubMed DOI PMC

Nayfach S, Camargo AP, Schulz F, Eloe-Fadrosh E, Roux S, et al. CheckV assesses the quality and completeness of metagenome-assembled viral genomes. Nat Biotechnol. 2021;39:578–585. doi: 10.1038/s41587-020-00774-7. PubMed DOI PMC

Shaffer M, Borton MA, McGivern BB, Zayed AA, La Rosa SL, et al. DRAM for distilling microbial metabolism to automate the curation of microbiome function. Nucleic Acids Res. 2020;48:8883–8900. doi: 10.1093/nar/gkaa621. PubMed DOI PMC

Lanza VF, Baquero F, de la Cruz F, Coque TM. AcCNET (Accessory Genome Constellation Network): comparative genomics software for accessory genome analysis using bipartite networks. Bioinformatics. 2017;33:283–285. doi: 10.1093/bioinformatics/btw601. PubMed DOI

Russell DA, Hatfull GF. PhagesDB: the actinobacteriophage database. Bioinformatics. 2017;33:784–786. doi: 10.1093/bioinformatics/btw711. PubMed DOI PMC

Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498. doi: 10.1101/gr.1239303. PubMed DOI PMC

Gibson MK, Forsberg KJ, Dantas G. Improved annotation of antibiotic resistance determinants reveals microbial resistomes cluster by ecology. ISME J. 2015;9:207–216. doi: 10.1038/ismej.2014.106. PubMed DOI PMC

Eddy SR. Accelerated Profile HMM Searches. PLoS Comput Biol. 2011;7:e1002195. doi: 10.1371/journal.pcbi.1002195. PubMed DOI PMC

Payne LJ, Todeschini TC, Wu Y, Perry BJ, Ronson CW, et al. Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types. Nucleic Acids Res. 2021;49:10868–10878. doi: 10.1093/nar/gkab883. PubMed DOI PMC

Tesson F, Hervé A, Touchon M, d’Humières C, Cury J, et al. Systematic and quantitative view of the antiviral arsenal of prokaryotes. bioRxiv. 2021 doi: 10.1101/2021.09.02.458658. PubMed DOI PMC

Abby SS, Néron B, Ménager H, Touchon M, Rocha EPC. MacSyFinder: a program to mine genomes for molecular systems with an application to CRISPR-Cas systems. PLoS One. 2014;9:e110726. doi: 10.1371/journal.pone.0110726. PubMed DOI PMC

Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007;35:W52–W57. doi: 10.1093/nar/gkm360. PubMed DOI PMC

Bespiatykh D, Bespyatykh J, Mokrousov I, Shitikov E. A comprehensive map of Mycobacterium tuberculosis complex regions of difference. mSphere. 2021;6:e0053521. doi: 10.1128/mSphere.00535-21. PubMed DOI PMC

McElroy KE, Luciani F, Thomas T. GemSIM: general, error-model based simulator of next-generation sequencing data. BMC Genomics. 2012;13:74. doi: 10.1186/1471-2164-13-74. PubMed DOI PMC

Engel HWB, Berwald LG, Grange JM, Kubin M. Phage typing of Mycobacterium kansasii. Tubercle. 1980;61:11–19. doi: 10.1016/0041-3879(80)90054-9. PubMed DOI

Zhang Y, Yu C, Jiang Y, Zheng X, Wang L, et al. Drug resistance profile of Mycobacterium kansasii clinical isolates before and after 2-month empirical antimycobacterial treatment. Clin Microbiol Infect. 2023;29:353–359. doi: 10.1016/j.cmi.2022.10.002. PubMed DOI

Rajendran P, Padmapriyadarsini C, Nagarajan N, Samyuktha R, Govindaraju V, et al. Molecular characterisation of M. kansasii isolates by whole-genome sequencing. Pathogens. 2023;12:1249. doi: 10.3390/pathogens12101249. PubMed DOI PMC

Marin MG, Wippel C, Quinones-Olvera N, Behruznia M, Jeffrey BM, et al. Analysis of the limited M. tuberculosis accessory genome reveals potential pitfalls of pan-genome analysis approaches. bioRxiv. 2024:2024.03.21.586149. doi: 10.1101/2024.03.21.586149. DOI

Guan Q, Ummels R, Ben-Rached F, Alzahid Y, Amini MS, et al. Comparative genomic and transcriptomic analyses of Mycobacterium kansasii subtypes provide new insights into their pathogenicity and taxonomy. Front Cell Infect Microbiol. 2020;10:122. doi: 10.3389/fcimb.2020.00122. PubMed DOI PMC

Rivera-Calzada A, Famelis N, Llorca O, Geibel S. Type VII secretion systems: structure, functions and transport models. Nat Rev Microbiol. 2021;19:567–584. doi: 10.1038/s41579-021-00560-5. PubMed DOI

Ates LS, Brosch R. Discovery of the type VII ESX-1 secretion needle? Mol Microbiol. 2017;103:7–12. doi: 10.1111/mmi.13579. PubMed DOI

DeStefano MS, Shoen CM, Cynamon MH. Therapy for Mycobacterium kansasii infection: beyond 2018. Front Microbiol. 2018;9:2271. doi: 10.3389/fmicb.2018.02271. PubMed DOI PMC

Bohr LL, Youngblom MA, Eldholm V, Pepperell CS. Genome reorganization during emergence of host-associated Mycobacterium abscessus. Microb Genom. 2021;7:000706. doi: 10.1099/mgen.0.000706. PubMed DOI PMC

Doron S, Melamed S, Ofir G, Leavitt A, Lopatina A, et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science. 2018;359:eaar4120. doi: 10.1126/science.aar4120. PubMed DOI PMC

Tock MR, Dryden DT. The biology of restriction and anti-restriction. Curr Opin Microbiol. 2005;8:466–472. doi: 10.1016/j.mib.2005.06.003. PubMed DOI

Molineux IJ. Host-parasite interactions: recent developments in the genetics of abortive phage infections. New Biol. 1991;3:230–236. PubMed

Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol. 1997;35:907–914. doi: 10.1128/jcm.35.4.907-914.1997. PubMed DOI PMC

Zhang Q, Ye Y. Not all predicted CRISPR-Cas systems are equal: isolated cas genes and classes of CRISPR like elements. BMC Bioinformatics. 2017;18:92. doi: 10.1186/s12859-017-1512-4. PubMed DOI PMC

Shi S, Ehrt S. Dihydrolipoamide acyltransferase is critical for Mycobacterium tuberculosis pathogenesis. Infect Immun. 2006;74:56–63. doi: 10.1128/IAI.74.1.56-63.2006. PubMed DOI PMC

Forrellad MA, Klepp LI, Gioffré A, Sabio y García J, Morbidoni HR, et al. Virulence factors of the Mycobacterium tuberculosis complex. Virulence. 2013;4:3–66. doi: 10.4161/viru.22329. PubMed DOI PMC

Nair J, Rouse DA, Bai GH, Morris SL. The rpsL gene and streptomycin resistance in single and multiple drug-resistant strains of Mycobacterium tuberculosis. Mol Microbiol. 1993;10:521–527. doi: 10.1111/j.1365-2958.1993.tb00924.x. PubMed DOI

Sreevatsan S, Stockbauer KE, Pan X, Kreiswirth BN, Moghazeh SL, et al. Ethambutol resistance in Mycobacterium tuberculosis: critical role of embB mutations. Antimicrob Agents Chemother. 1997;41:1677–1681. doi: 10.1128/AAC.41.8.1677. PubMed DOI PMC

Martini MC, Hicks ND, Xiao J, Alonso MN, Barbier T, et al. Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis. PLoS Pathog. 2022;18:e1010705. doi: 10.1371/journal.ppat.1010705. PubMed DOI PMC

Kreutzfeldt KM, Jansen RS, Hartman TE, Gouzy A, Wang R, et al. CinA mediates multidrug tolerance in Mycobacterium tuberculosis. Nat Commun. 2022;13:2203. doi: 10.1038/s41467-022-29832-1. PubMed DOI PMC