Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity

Edson Machado Sidra Ezidio Gonçalves Vasconcellos Camillo Cerdeira Lia Lima Gomes Ricardo Junqueira Luciana Distasio de Carvalho Jesus Pais Ramos Paulo Redner Carlos Eduardo Dias Campos Paulo Cesar de Souza Caldas Ana Paula Chaves Sobral Gomes Telma Goldenberg Fatima Fandinho Montes Fernanda Carvalho de Queiroz Mello Vinicius de Oliveira Mussi Elena Lasunskaia Dick van Soolingen Antonio Basílio de Miranda Leen Rigouts Bouke C de Jong Conor J Meehan Marcos Catanho Philip N Suffys About the authors

Abstract

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

Key words:
Mycobacterium kansasii; genomes; lung disease; genotype I; diversity; drug resistance


Nontuberculous Mycobacterium (NTM) species are widespread in the (man-made) environment and some species cause opportunistic infections in humans. Mycobacterium kansasii, frequently isolated from tap water, is a slow-growing photochromogenic NTM and a pathogen that is commonly isolated from patients with pre-existing lung disease, similar to other NTM clinical species such as M. abscessus, M. avium complex, M. malmoense and M. xenopi.11. Wassilew N, Hoffmann H, Andrejak C, Lange C. Pulmonary disease caused by non-tuberculous mycobacteria. Respiration. 2016; 91(5): 386-402. In the USA and South America M. kansasii is the second most isolated NTM after M. avium complex22. Hoefsloot W, van Ingen J, Andrejak C, Ängeby K, Bauriaud R, Bemer P, et al. The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: An NTM-NET collaborative study. Eur Respir J. 2013; 42(6): 1604-13.,33. Kaur P, Fishman JA, Misdraji J, Varma MC, Kotton CN. Disseminated Mycobacterium kansasii infection with hepatic abscesses in a renal transplant recipient. Transpl Infect Dis. 2011; 13(5): 531-5. while in Rio de Janeiro, it is the most frequent NTM to cause pulmonary disease.44. de Mello KG, Mello FC, Borga L, Rolla V, Duarte RS, Sampaio EP, et al. Clinical and therapeutic features of pulmonary nontuberculous mycobacterial disease, Brazil, 1993-2011. Emerg Infect Dis. 2013; 19(3): 393-9. Besides chronic bronchopulmonary disease, M. kansasii causes other clinical manifestations such as lymphadenitis55. Choi YG, Cho SY, Lee DG, Yim E, Joo H, Ryu S, et al. Mycobacterium kansasii pneumonia with mediastinal lymphadenitis in a patient with acute myeloid leukemia?: successful treatment to stem cell transplantation. Infect Chemother. 2017; 49(1): 78-83., skin and soft tissue infection66. Han SH, Kim KM, Chin BS, Choi SH, Lee HS, Kim MS, et al. Disseminated Mycobacterium kansasii infection associated with skin lesions: a case report and comprehensive review of the literature. J Korean Med Sci. 2010; 25(2): 304-8., tenosynovitis, arthritis, osteomyelitis and disseminated infection in patients co-infected with human immunodeficiency virus.77. Menashe L, Kerr LD, Hermann G. Mycobacterium kansasii causing chronic monoarticular synovitis in a patient with HIV/AIDS. J Radiol Case Rep. 2015; 9(9): 26-35.

Seven major genotypes (I to VII) of M. kansasii have been described and while human isolates are mainly of types I and II (with type II mainly HIV-related)88. Santin M, Alcaide F, Benitez MA, Salazar A, Ardanuy C, Podzamczer D, et al. Incidence and molecular typing of Mycobacterium kansasii in a defined geographical area in Catalonia, Spain. Epidemiol Infect. 2004; 132(3): 425-32.,99. Taillard C, Greub G, Weber R, Pfyffer GE, Bodmer T, Zimmerli S, et al. Clinical implications of Mycobacterium kansasii species heterogeneity?: Swiss National Survey. J Clin Microbiol. 2003; 41(3): 1240-4., environmental isolates are mostly of the other subtypes.1010. Li Y, Pang Y, Tong X, Zheng H, Zhao Y, Wang C. Mycobacterium kansasii subtype I is associated with clarithromycin resistance in China. Front Microbiol. 2016; 7: 2097. Type I has been described as a heterogeneous group, incompletely characterized on the genomic level.1111. Alcaide F, Richter I, Bernasconi C, Springer B, Hagenau C, Schulze-Röbbecke R, et al. Heterogeneity and clonality among isolates of Mycobacterium kansasii: implications for epidemiological and pathogenicity studies. J Clin Microbiol. 1997; 35(8): 1959-64.,1212. Bakula Z, Safianowska A, Nowacka-Mazurek M, Bielecki J, Jagielski T. Short communication: subtyping of Mycobacterium kansasii by PCR-restriction enzyme analysis of the hsp65 gene. Biomed Res Int. 2013; 2013: 178725. Recently, the genome sequence of the ATCC strain 12478 Hauduroy isolated in Kansas in 1955 has been compared with that of the M. tuberculosis strain H37Rv1313. Wang J, McIntosh F, Radomski N, Dewar K, Simeone R, Enninga J, et al. Insights on the emergence of Mycobacterium tuberculosis from the analysis of Mycobacterium kansasii. Genome Biol Evol. 2015; 7(3): 856-70., while other M. kansasii genomes are available from environmental1414. Strapagiel D, Borówka P, Marciniak B, Bakula Z, van Ingen J, Safianowska A, et al. Draft genome sequences of Mycobacterium kansasii strains 1010001454, 1010001458, 1010001468, 1010001493, 1010001495, and 1010001469, isolated from environmental sources. Genome Announc. 2016; 4(3): e00456-16., human1515. Borówka P, Lach J, Bakula Z, van Ingen J, Safianowska A, Brzostek A, et al. Draft genome sequences of Mycobacterium kansasii clinical strains. Genome Announc. 2017; 5(22): e00406-17. and simian1616. Panda A, Nagaraj S, Zhao X, Tettelin H, DeTolla LJ. Complete genome sequences of Mycobacterium kansasii strains isolated from Rhesus macaques. Genome Announc. 2017; 5(16): e00187-17. sources.

Here, we report the genome sequence of 12 clinical M. kansasii isolates belonging to genotype I, as determined by hsp65 sequencing. The strains were isolated from human Brazilian patients with pulmonary disease and possibly earlier tuberculosis. Nine isolates were from sputum, two from bronchoalveolar lavage (7287 and 10742) and one of unknown origin (1580). Isolates were from residents of the state of Rio de Janeiro (n = 7), Pernambuco (n = 3), Rio Grande do Sul (n = 1), and Santa Catarina (n = 1). Genomic DNA libraries were constructed using the Nextera XT DNA library kit and whole-genome shotgun sequencing was performed on the Illumina HiSeq2500 platform, generating paired end reads of 2 × 100 bp. Genomes were de novo assembled using SPAdes software version 3.11.11717. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012; 19(5): 455-77., and annotated with RAST1818. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST server: rapid annotations using subsystems technology. BMC Genomics. 2008; 9: 75. (Table). No evidence for the presence of plasmids was observed.

TABLE
Genome features, predicted genes, and GenBank accession numbers of Mycobacterium kansasii strains isolated in Brazil from patients with pulmonary disease

To evaluate the overall similarity of the genomes of the isolates from Brazil with that of the isolate ATCC 12478, we also compared the 12 genomes with the ATCC reference genome using the BLAST Ring Image Generator (BRIG) program1919. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011; 12: 402. (Fig. 1).

We observed many deletions spread in the genomes that were either shared by all or part of the isolates. Three isolates (1580, 3657 and 4404) presented a region of difference of about 27 kb causing the loss of five helicases, a restriction endonuclease and two hypothetical proteins. We also observed multiple deletions shared by the isolates 1580, 3657, 4404, 8835, 8837 and 8839 (Fig. 1).

Fig. 1:
BLAST ring image of the 12 Brazilian genomes against the ATCC 12478 reference genome.

In addition, a reference-based single-nucleotide polymorphism (SNP) calling was performed against the genome of the M. kansasii ATCC 12478 strain using both Snippy (version 3.2; https://github.com/tseemann/snippy) and the wgSNP module of BioNumerics v7.6 (Applied Maths, Sint-Martens-Latem, Belgium). Most isolates presented homogeneously distributed SNPs over the entire genome, except for isolates 6849 and 6498 which clearly presented regions of higher SNP frequency (data not shown). As demonstrated by a Neighbour-Joining phylogenetic tree based on 5,607,341 sites and 1000 bootstrap replicates (Fig. 2), three groups were observed when compared to the genome of the ATCC strain, one presenting less than 100 SNP (n = 4) (middle part), a second with either less than 5,000 SNPs (n = 1) and more than 10,000 SNPs (n = 1) (lower part) and a third group with more than 10,000 SNPs (n = 6) (upper part). We observed an association between SNP-based grouping and phylogeny on basis of large deletions, that led us suggest that the third group has evolved from the common ancestor.

From two patients, we performed whole genome sequencing of two isolates each; the pair of isolates from a patient from Rio de Janeiro (10742 and 10953) had 0 SNPs difference; the second pair from a patient from Recife (8837 and 8839) presented 277 mutations (data not shown).

Fig. 2:
Neighbor-Joining SNP-based tree of the 12 Brazilian isolates and the ATCC 12478 reference genome.

Additionally, two isolates (6498 and 8835) presented a Minimal Inhibitory Concentration of > 1 µg/mL for rifampicin in vitro and both presented a non-synonymous SNP, localized respectively at position 4267612 (C to G) and 4267613 (A to C), causing an amino acid change in codon 411 of rpoB of respectively Gln to Glu and of Gln to Pro. We as such confirm the scarce existing data on the association between resistance against rifampicin and presence of point mutations in M. kansasii rpoB gene. Indeed, point mutations in rpoB gene are the main reason for resistance against rifampicin and this has been confirmed in several bacteria. Also, molecular determinants of drug resistance in M. kansasii has been recently suggested by Bakuła et al.2020. Bakula Z, Modrzejewska M, Penningsb L, Proboszczc M, Safianowskac A, Bieleckia J, et al. Drug susceptibility profiling and genetic determinants of drug resistance in Mycobacterium kansasii. Antimicrob Agents Chemother. 2018; 62(4): e01788-17.. Among the present isolates, we observed considerable differences of in vitro virulence in macrophages as measured by growth and induction of necrosis and of cytokine production (data not shown).

In conclusion, these are preliminary data on the variability within the M. kansasii genotype 1 in Brazil and, even on the basis of this small sample number, we evidenced three genetic groups that are separated by the presence of large number of SNPs throughout the genome and by the pattern of large deletions. One grouping of strains from Rio de Janeiro is highly similar to the ATCC strain Hauduroy (< 100 SNPs) that was isolated more than 70 years ago in the US. A second group of human isolates from Rio de Janeiro and Pernambuco presented over 10,000 SNPs when compared to the ATCC strain. We also observed an association between genotypes and geographic origin of isolates, separating those from the states of Rio de Janeiro, Pernambuco and Santa Catarina. Our observation of considerable differences of in vitro and in vivo virulence and their differences on the genome level is under investigation.

ACKNOWLEDGEMENTS

To the platforms at Fiocruz (Plataformas de Bioinformática RPT04-A/RJ and Sequenciamento de Ácidos Nuclêicos de Nova Geração RPT01-J/RJ). To Applied Maths for offering a one and a half year free access to the Bionumerics software.

REFERENCES

  • 1
    Wassilew N, Hoffmann H, Andrejak C, Lange C. Pulmonary disease caused by non-tuberculous mycobacteria. Respiration. 2016; 91(5): 386-402.
  • 2
    Hoefsloot W, van Ingen J, Andrejak C, Ängeby K, Bauriaud R, Bemer P, et al. The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: An NTM-NET collaborative study. Eur Respir J. 2013; 42(6): 1604-13.
  • 3
    Kaur P, Fishman JA, Misdraji J, Varma MC, Kotton CN. Disseminated Mycobacterium kansasii infection with hepatic abscesses in a renal transplant recipient. Transpl Infect Dis. 2011; 13(5): 531-5.
  • 4
    de Mello KG, Mello FC, Borga L, Rolla V, Duarte RS, Sampaio EP, et al. Clinical and therapeutic features of pulmonary nontuberculous mycobacterial disease, Brazil, 1993-2011. Emerg Infect Dis. 2013; 19(3): 393-9.
  • 5
    Choi YG, Cho SY, Lee DG, Yim E, Joo H, Ryu S, et al. Mycobacterium kansasii pneumonia with mediastinal lymphadenitis in a patient with acute myeloid leukemia?: successful treatment to stem cell transplantation. Infect Chemother. 2017; 49(1): 78-83.
  • 6
    Han SH, Kim KM, Chin BS, Choi SH, Lee HS, Kim MS, et al. Disseminated Mycobacterium kansasii infection associated with skin lesions: a case report and comprehensive review of the literature. J Korean Med Sci. 2010; 25(2): 304-8.
  • 7
    Menashe L, Kerr LD, Hermann G. Mycobacterium kansasii causing chronic monoarticular synovitis in a patient with HIV/AIDS. J Radiol Case Rep. 2015; 9(9): 26-35.
  • 8
    Santin M, Alcaide F, Benitez MA, Salazar A, Ardanuy C, Podzamczer D, et al. Incidence and molecular typing of Mycobacterium kansasii in a defined geographical area in Catalonia, Spain. Epidemiol Infect. 2004; 132(3): 425-32.
  • 9
    Taillard C, Greub G, Weber R, Pfyffer GE, Bodmer T, Zimmerli S, et al. Clinical implications of Mycobacterium kansasii species heterogeneity?: Swiss National Survey. J Clin Microbiol. 2003; 41(3): 1240-4.
  • 10
    Li Y, Pang Y, Tong X, Zheng H, Zhao Y, Wang C. Mycobacterium kansasii subtype I is associated with clarithromycin resistance in China. Front Microbiol. 2016; 7: 2097.
  • 11
    Alcaide F, Richter I, Bernasconi C, Springer B, Hagenau C, Schulze-Röbbecke R, et al. Heterogeneity and clonality among isolates of Mycobacterium kansasii: implications for epidemiological and pathogenicity studies. J Clin Microbiol. 1997; 35(8): 1959-64.
  • 12
    Bakula Z, Safianowska A, Nowacka-Mazurek M, Bielecki J, Jagielski T. Short communication: subtyping of Mycobacterium kansasii by PCR-restriction enzyme analysis of the hsp65 gene. Biomed Res Int. 2013; 2013: 178725.
  • 13
    Wang J, McIntosh F, Radomski N, Dewar K, Simeone R, Enninga J, et al. Insights on the emergence of Mycobacterium tuberculosis from the analysis of Mycobacterium kansasii. Genome Biol Evol. 2015; 7(3): 856-70.
  • 14
    Strapagiel D, Borówka P, Marciniak B, Bakula Z, van Ingen J, Safianowska A, et al. Draft genome sequences of Mycobacterium kansasii strains 1010001454, 1010001458, 1010001468, 1010001493, 1010001495, and 1010001469, isolated from environmental sources. Genome Announc. 2016; 4(3): e00456-16.
  • 15
    Borówka P, Lach J, Bakula Z, van Ingen J, Safianowska A, Brzostek A, et al. Draft genome sequences of Mycobacterium kansasii clinical strains. Genome Announc. 2017; 5(22): e00406-17.
  • 16
    Panda A, Nagaraj S, Zhao X, Tettelin H, DeTolla LJ. Complete genome sequences of Mycobacterium kansasii strains isolated from Rhesus macaques. Genome Announc. 2017; 5(16): e00187-17.
  • 17
    Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012; 19(5): 455-77.
  • 18
    Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST server: rapid annotations using subsystems technology. BMC Genomics. 2008; 9: 75.
  • 19
    Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011; 12: 402.
  • 20
    Bakula Z, Modrzejewska M, Penningsb L, Proboszczc M, Safianowskac A, Bieleckia J, et al. Drug susceptibility profiling and genetic determinants of drug resistance in Mycobacterium kansasii. Antimicrob Agents Chemother. 2018; 62(4): e01788-17.

  • Financial support: CNPq (scholarships 207422/2014-1, 500769/2014-1, 311554/2013-0; grants 407624/2012-0, 459100/2014-9)

Publication Dates

  • Publication in this collection
    25 June 2018
  • Date of issue
    2018

History

  • Received
    15 Feb 2018
  • Accepted
    05 June 2018
Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
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