PROKARYOTES
crossm Draft Genome Sequence of Mycobacterium chimaera Type Strain Fl-0169 Stacy Pfaller,a Vasily Tokarev,b Collin Kessler,b Christopher McLimans,b Vicente Gomez-Alvarez,c* Justin Wright,b Dawn King,a Regina Lamendellab Office of Research and Development, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio, USAa; Department of Biology, Juniata College, Huntingdon, Pennsylvania, USAb; Office of Research and Development, National Risk Management Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio, USAc
We report here the draft genome sequence of the type strain Mycobacterium chimaera Fl-0169, a member of the Mycobacterium avium complex (MAC). M. chimaera Fl-0169T was isolated from a patient in Italy and is highly similar to strains of M. chimaera isolated in Ireland, although Fl-0169T possesses unique virulence genes. ABSTRACT
T
he Mycobacterium avium complex (MAC) contains several clinically relevant closely related species. The MAC, including M. avium, M. intracellulare, and M. chimaera, contains the most frequently isolated mycobacteria from humans in the United States, surpassing isolations of Mycobacterium tuberculosis and other species (1, 2). While M. avium and M. intracellulare have been studied for decades, less is known about M. chimaera, which was recently described in 2004 (3). The type strain of M. chimaera, Fl-0169, was isolated in Italy from a 56-year-old female patient with bronchiectstasis (3). The strain produced discordant identification results based on phenotypic and genotypic comparisons to its close relatives in the MAC, and it was classified as a new species, for which the name M. chimaera was proposed (3). Evidence suggests that M. avium, M. intracellulare, and M. chimaera are differently virulent and require specieslevel identification for targeted treatment (4). Therefore, a comparative genomic analysis of MAC species is critically needed to identify diagnostic targets that reliably differentiate species of MAC. Prolonged outbreaks of M. chimaera from heater-cooler units used in open chest surgery also drive the need for better diagnostic tools (5, 6). With treatment costs for Mycobacterium infections estimated to be ⬎$1.8 billion annually in the United States (7), correct species identification may result in improved treatment selection, lower costs, and improved patient outcomes. M. chimaera strain Fl-0169T was obtained from the Leibniz Institute DSMZ (Braunschweig, Germany) and genomic DNA extracted using the MoBio PowerSoil DNA extraction kit (Mo Bio Laboratories, Solana Beach, CA). Paired-end 125-bp libraries were prepared using the Nextera DNA library preparation kit (Illumina, Inc., San Diego, CA) and sequenced on the Illumina HiSeq 2500 (Illumina, Inc.). Quality-filtered reads were de novo assembled using SOAPdenovo2 (8) and Velvet (9). Contigs from both assemblers were combined using CISA (10) and genomes annotated using the Joint Genome Institute’s (JGI) Integrated Microbial Genomics (IMG) (11), the U.S. Department of Energy Systems Knowledge Database (Kbase; http://www.kbase.us), and MG RAST (12) annotation pipelines. Upon submission to IMG (13), scaffolds were broken into contigs, and coding genes were identified with Prodigal (14) and assigned a locus tag. Compared to other M. chimaera strains (15), several genes are unique to strain Fl-0169T, including the putative virulence genes anthranilate phosphoribosyltransferase (EC Volume 5 Issue 8 e01620-16
Received 22 December 2016 Accepted 26 December 2016 Published 23 February 2017 Citation Pfaller S, Tokarev V, Kessler C, McLimans C, Gomez-Alvarez V, Wright J, King D, Lamendella R. 2017. Draft genome sequence of Mycobacterium chimaera type strain Fl-0169. Genome Announc 5:e01620-16. https:// doi.org/10.1128/genomeA.01620-16. Copyright © 2017 Pfaller et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Stacy Pfaller, [email protected]. * Present address: Vicente Gomez-Alvarez, Office of Pollution Prevention and Toxics, Risk Assessment Division, U.S. Environmental Protection Agency, Washington, District of Columbia, USA.
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Pfaller et al.
2.4.2.18), sporulation initiation inhibitor protein Soj, and a 3-oxoacyl-(acyl-carrierprotein) synthase, KASII (EC 2.3.1.41) (16). The Insert Species into Genome Tree application within KBase was used to compare M. chimaera strain Fl-0169T to other genomes and revealed that M. chimaera strain Fl-0169T is most similar to M. chimaera strain MCIMRL6. The draft genome of strain Fl-0169T consists of 304 contigs and an N50 of 33,938 bp, for a full assembly size of 6,308,407 bp. The average coverage per contig was 18.27⫻. The G⫹C content per assemblage of the CISA-assembled genomes was calculated using EMBOSS (17) and averaged 69.6%. Other features include the presence of 6,462 protein-coding genes (4,715 genes with and 1,747 genes without functional prediction), three rRNA genes, and 43 tRNA genes. Accession number(s). This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number MRBR00000000. The version described in this paper is version MRBR01000000. ACKNOWLEDGMENTS The opinions expressed are those of the authors and do not necessarily reflect the official positions and policies of the U.S. EPA. Any mention of product or trade names does not constitute recommendation for use by the U.S. EPA. The U.S. EPA through the Office of Research and Development funded and managed this research.
REFERENCES 1. Hoefsloot W, van Ingen J, Andrejak C, Angeby K, Bauriaud R, Bemer P, Beylis N, Boeree MJ, Cacho J, Chihota V, Chimara E, Churchyard G, Cias R, Daza R, Daley CL, Dekhuijzen PN, Domingo D, Drobniewski F, Esteban J, Fauville-Dufaux M, Folkvardsen DB, Gibbons N, Gómez-Mampaso E, Gonzalez R, Hoffmann H, Hsueh PR, Indra A, Jagielski T, Jamieson F, Jankovic M, Jong E, Keane J, Koh WJ, Lange B, Leao S, Macedo R, Mannsåker T, Marras TK, Maugein J, Milburn HJ. 2013. The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study. Eur Respir J 42:1604 –1613. https://doi.org/10.1183/09031936.00149212. 2. Johnson MM, Odell JA. 2014. Nontuberculous mycobacterial pulmonary infections. J Thorac Dis 6:210 –220. https://doi.org/10.3978/j.issn.2072 -1439.2013.12.24. 3. Tortoli E, Rindi L, Garcia MJ, Chiaradonna P, Dei R, Garzelli C, Kroppenstedt RM, Lari N, Mattei R, Mariottini A, Mazzarelli G, Murcia MI, Nanetti A, Piccoli P, Scarparo C. 2004. Proposal to elevate the genetic variant MAC-A included in the Mycobacterium avium complex, to species rank as Mycobacterium chimaera sp. nov. Int J Syst Evol Microbiol 54:1277–1285. https://doi.org/10.1099/ijs.0.02777-0. 4. Boyle DP, Zembower TR, Reddy S, Qi C. 2015. Comparison of clinical features, virulence, and relapse among Mycobacterium avium complex species. Am J Respir Crit Care Med 191:1310 –1317. https://doi.org/ 10.1164/rccm.201501-0067OC. 5. Kohler P, Kuster SP, Bloemberg G, Schulthess B, Frank M, Tanner FC, Rössle M, Böni C, Falk V, Wilhelm MJ, Sommerstein R, Achermann Y, Ten Oever J, Debast SB, Wolfhagen MJHM, Brandon Bravo Bruinsma GJ, Vos MC, Bogers A, Serr A, Beyersdorf F, Sax H, Böttger EC, Weber R, van Ingen J, Wagner D, Hasse B. 2015. Healthcare-associated prosthetic heart valve, aortic vascular graft, and disseminated Mycobacterium chimaera infections subsequent to open heart surgery. Eur Heart J 36:2745–2753. https://doi.org/10.1093/eurheartj/ehv342. 6. Sax H, Bloemberg G, Hasse B, Sommerstein R, Kohler P, Achermann Y, Rössle M, Falk V, Kuster SP, Böttger EC, Weber R. 2015. Prolonged outbreak of Mycobacterium chimaera infection after open-chest heart surgery. Clin Infect Dis 61:67–75. https://doi.org/10.1093/cid/civ198. 7. Thomson R, Donnan E, Unwin S. 2015. Nontuberculous mycobacterial lung disease: time to get a grip! Ann Am Thorac Soc 12:1425–1427. https://doi.org/10.1513/AnnalsATS.201508-524ED. 8. Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y, Tang J, Wu G, Zhang H, Shi Y, Liu Y, Yu C, Wang B, Lu Y, Han C, Cheung
Volume 5 Issue 8 e01620-16
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12.
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15.
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DW, Yiu SM, Peng S, Xiaoqian Z, Liu G, Liao X, Li Y, Yang H, Wang J, Lam TW, Wang J. 2012. SOAPdenovo2: an empirically improved memoryefficient short-read de novo assembler. GigaScience 1:18. https://doi.org/ 10.1186/2047-217X-1-18. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821– 829. https:// doi.org/10.1101/gr.074492.107. Lin SH, Liao YC. 2013. CISA: Contig Integrator for Sequence Assembly of bacterial genomes. PLoS One 8:e60843. https://doi.org/10.1371/ journal.pone.0060843. Markowitz VM, Chen IMA, Palaniappan K, Chu K, Szeto E, Grechkin Y, Ratner A, Jacob B, Huang J, Williams P, Huntemann M, Anderson I, Mavromatis K, Ivanova NN, Kyrpides NC. 2012. IMG: the integrated microbial genomes database and comparative analysis system. Nucleic Acids Res 40:D115–D122. https://doi.org/10.1093/nar/gkr1044. Wilke A, Bischof J, Gerlach W, Glass E, Harrison T, Keegan KP, Paczian T, Trimble WL, Bagchi S, Grama A, Chaterji S, Meyer F. 2016. The MG-RAST metagenomics database and portal in 2015. Nucleic Acids Res 44: D590 –D594. https://doi.org/10.1093/nar/gkv1322. Huntemann M, Ivanova NN, Mavromatis K, Tripp HJ, Paez-Espino D, Palaniappan K, Szeto E, Pillay M, Chen I-MA, Pati A, Markowitz VM, Kyrpides NC. 2015. The standard operating procedure of the DOE-JGI microbial genome annotation pipeline (MGAP v. 4) keywords. Stand Genomic Sci 10:86. https://doi.org/10.4056/sigs.632. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. https://doi.org/10.1186/1471 -2105-11-119. Aogáin M, Roycroft E, Raftery P, Mok S, Fitzgibbon M, Rogers TR. 2015. Draft genome sequences of three Mycobacterium chimaera respiratory isolates. Genome Announc 3(6):e01409-15. https://doi.org/10.1128/ genomeA.01409-15. Zhang YJ, Reddy MC, Ioerger TR, Rothchild AC, Dartois V, Schuster BM, Trauner A, Wallis D, Galaviz S, Huttenhower C, Sacchettini JC, Behar SM, Rubin EJ. 2013. Tryptophan biosynthesis protects mycobacteria from CD4 T cell-mediated killing. Cell 155:1296 –1308. https://doi.org/ 10.1016/j.cell.2013.10.045. Rice P, Longden I, Bleasby A. 2000. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16:276 –277. https://doi.org/ 10.1016/S0168-9525(00)02024-2.
genomea.asm.org 2
crossm Draft Genome Sequence of Mycobacterium chimaera Type Strain Fl-0169 Stacy Pfaller,a Vasily Tokarev,b Collin Kessler,b Christopher McLimans,b Vicente Gomez-Alvarez,c* Justin Wright,b Dawn King,a Regina Lamendellab Office of Research and Development, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio, USAa; Department of Biology, Juniata College, Huntingdon, Pennsylvania, USAb; Office of Research and Development, National Risk Management Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio, USAc
We report here the draft genome sequence of the type strain Mycobacterium chimaera Fl-0169, a member of the Mycobacterium avium complex (MAC). M. chimaera Fl-0169T was isolated from a patient in Italy and is highly similar to strains of M. chimaera isolated in Ireland, although Fl-0169T possesses unique virulence genes. ABSTRACT
T
he Mycobacterium avium complex (MAC) contains several clinically relevant closely related species. The MAC, including M. avium, M. intracellulare, and M. chimaera, contains the most frequently isolated mycobacteria from humans in the United States, surpassing isolations of Mycobacterium tuberculosis and other species (1, 2). While M. avium and M. intracellulare have been studied for decades, less is known about M. chimaera, which was recently described in 2004 (3). The type strain of M. chimaera, Fl-0169, was isolated in Italy from a 56-year-old female patient with bronchiectstasis (3). The strain produced discordant identification results based on phenotypic and genotypic comparisons to its close relatives in the MAC, and it was classified as a new species, for which the name M. chimaera was proposed (3). Evidence suggests that M. avium, M. intracellulare, and M. chimaera are differently virulent and require specieslevel identification for targeted treatment (4). Therefore, a comparative genomic analysis of MAC species is critically needed to identify diagnostic targets that reliably differentiate species of MAC. Prolonged outbreaks of M. chimaera from heater-cooler units used in open chest surgery also drive the need for better diagnostic tools (5, 6). With treatment costs for Mycobacterium infections estimated to be ⬎$1.8 billion annually in the United States (7), correct species identification may result in improved treatment selection, lower costs, and improved patient outcomes. M. chimaera strain Fl-0169T was obtained from the Leibniz Institute DSMZ (Braunschweig, Germany) and genomic DNA extracted using the MoBio PowerSoil DNA extraction kit (Mo Bio Laboratories, Solana Beach, CA). Paired-end 125-bp libraries were prepared using the Nextera DNA library preparation kit (Illumina, Inc., San Diego, CA) and sequenced on the Illumina HiSeq 2500 (Illumina, Inc.). Quality-filtered reads were de novo assembled using SOAPdenovo2 (8) and Velvet (9). Contigs from both assemblers were combined using CISA (10) and genomes annotated using the Joint Genome Institute’s (JGI) Integrated Microbial Genomics (IMG) (11), the U.S. Department of Energy Systems Knowledge Database (Kbase; http://www.kbase.us), and MG RAST (12) annotation pipelines. Upon submission to IMG (13), scaffolds were broken into contigs, and coding genes were identified with Prodigal (14) and assigned a locus tag. Compared to other M. chimaera strains (15), several genes are unique to strain Fl-0169T, including the putative virulence genes anthranilate phosphoribosyltransferase (EC Volume 5 Issue 8 e01620-16
Received 22 December 2016 Accepted 26 December 2016 Published 23 February 2017 Citation Pfaller S, Tokarev V, Kessler C, McLimans C, Gomez-Alvarez V, Wright J, King D, Lamendella R. 2017. Draft genome sequence of Mycobacterium chimaera type strain Fl-0169. Genome Announc 5:e01620-16. https:// doi.org/10.1128/genomeA.01620-16. Copyright © 2017 Pfaller et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Stacy Pfaller, [email protected]. * Present address: Vicente Gomez-Alvarez, Office of Pollution Prevention and Toxics, Risk Assessment Division, U.S. Environmental Protection Agency, Washington, District of Columbia, USA.
genomea.asm.org 1
Pfaller et al.
2.4.2.18), sporulation initiation inhibitor protein Soj, and a 3-oxoacyl-(acyl-carrierprotein) synthase, KASII (EC 2.3.1.41) (16). The Insert Species into Genome Tree application within KBase was used to compare M. chimaera strain Fl-0169T to other genomes and revealed that M. chimaera strain Fl-0169T is most similar to M. chimaera strain MCIMRL6. The draft genome of strain Fl-0169T consists of 304 contigs and an N50 of 33,938 bp, for a full assembly size of 6,308,407 bp. The average coverage per contig was 18.27⫻. The G⫹C content per assemblage of the CISA-assembled genomes was calculated using EMBOSS (17) and averaged 69.6%. Other features include the presence of 6,462 protein-coding genes (4,715 genes with and 1,747 genes without functional prediction), three rRNA genes, and 43 tRNA genes. Accession number(s). This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number MRBR00000000. The version described in this paper is version MRBR01000000. ACKNOWLEDGMENTS The opinions expressed are those of the authors and do not necessarily reflect the official positions and policies of the U.S. EPA. Any mention of product or trade names does not constitute recommendation for use by the U.S. EPA. The U.S. EPA through the Office of Research and Development funded and managed this research.
REFERENCES 1. Hoefsloot W, van Ingen J, Andrejak C, Angeby K, Bauriaud R, Bemer P, Beylis N, Boeree MJ, Cacho J, Chihota V, Chimara E, Churchyard G, Cias R, Daza R, Daley CL, Dekhuijzen PN, Domingo D, Drobniewski F, Esteban J, Fauville-Dufaux M, Folkvardsen DB, Gibbons N, Gómez-Mampaso E, Gonzalez R, Hoffmann H, Hsueh PR, Indra A, Jagielski T, Jamieson F, Jankovic M, Jong E, Keane J, Koh WJ, Lange B, Leao S, Macedo R, Mannsåker T, Marras TK, Maugein J, Milburn HJ. 2013. The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study. Eur Respir J 42:1604 –1613. https://doi.org/10.1183/09031936.00149212. 2. Johnson MM, Odell JA. 2014. Nontuberculous mycobacterial pulmonary infections. J Thorac Dis 6:210 –220. https://doi.org/10.3978/j.issn.2072 -1439.2013.12.24. 3. Tortoli E, Rindi L, Garcia MJ, Chiaradonna P, Dei R, Garzelli C, Kroppenstedt RM, Lari N, Mattei R, Mariottini A, Mazzarelli G, Murcia MI, Nanetti A, Piccoli P, Scarparo C. 2004. Proposal to elevate the genetic variant MAC-A included in the Mycobacterium avium complex, to species rank as Mycobacterium chimaera sp. nov. Int J Syst Evol Microbiol 54:1277–1285. https://doi.org/10.1099/ijs.0.02777-0. 4. Boyle DP, Zembower TR, Reddy S, Qi C. 2015. Comparison of clinical features, virulence, and relapse among Mycobacterium avium complex species. Am J Respir Crit Care Med 191:1310 –1317. https://doi.org/ 10.1164/rccm.201501-0067OC. 5. Kohler P, Kuster SP, Bloemberg G, Schulthess B, Frank M, Tanner FC, Rössle M, Böni C, Falk V, Wilhelm MJ, Sommerstein R, Achermann Y, Ten Oever J, Debast SB, Wolfhagen MJHM, Brandon Bravo Bruinsma GJ, Vos MC, Bogers A, Serr A, Beyersdorf F, Sax H, Böttger EC, Weber R, van Ingen J, Wagner D, Hasse B. 2015. Healthcare-associated prosthetic heart valve, aortic vascular graft, and disseminated Mycobacterium chimaera infections subsequent to open heart surgery. Eur Heart J 36:2745–2753. https://doi.org/10.1093/eurheartj/ehv342. 6. Sax H, Bloemberg G, Hasse B, Sommerstein R, Kohler P, Achermann Y, Rössle M, Falk V, Kuster SP, Böttger EC, Weber R. 2015. Prolonged outbreak of Mycobacterium chimaera infection after open-chest heart surgery. Clin Infect Dis 61:67–75. https://doi.org/10.1093/cid/civ198. 7. Thomson R, Donnan E, Unwin S. 2015. Nontuberculous mycobacterial lung disease: time to get a grip! Ann Am Thorac Soc 12:1425–1427. https://doi.org/10.1513/AnnalsATS.201508-524ED. 8. Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y, Tang J, Wu G, Zhang H, Shi Y, Liu Y, Yu C, Wang B, Lu Y, Han C, Cheung
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DW, Yiu SM, Peng S, Xiaoqian Z, Liu G, Liao X, Li Y, Yang H, Wang J, Lam TW, Wang J. 2012. SOAPdenovo2: an empirically improved memoryefficient short-read de novo assembler. GigaScience 1:18. https://doi.org/ 10.1186/2047-217X-1-18. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821– 829. https:// doi.org/10.1101/gr.074492.107. Lin SH, Liao YC. 2013. CISA: Contig Integrator for Sequence Assembly of bacterial genomes. PLoS One 8:e60843. https://doi.org/10.1371/ journal.pone.0060843. Markowitz VM, Chen IMA, Palaniappan K, Chu K, Szeto E, Grechkin Y, Ratner A, Jacob B, Huang J, Williams P, Huntemann M, Anderson I, Mavromatis K, Ivanova NN, Kyrpides NC. 2012. IMG: the integrated microbial genomes database and comparative analysis system. Nucleic Acids Res 40:D115–D122. https://doi.org/10.1093/nar/gkr1044. Wilke A, Bischof J, Gerlach W, Glass E, Harrison T, Keegan KP, Paczian T, Trimble WL, Bagchi S, Grama A, Chaterji S, Meyer F. 2016. The MG-RAST metagenomics database and portal in 2015. Nucleic Acids Res 44: D590 –D594. https://doi.org/10.1093/nar/gkv1322. Huntemann M, Ivanova NN, Mavromatis K, Tripp HJ, Paez-Espino D, Palaniappan K, Szeto E, Pillay M, Chen I-MA, Pati A, Markowitz VM, Kyrpides NC. 2015. The standard operating procedure of the DOE-JGI microbial genome annotation pipeline (MGAP v. 4) keywords. Stand Genomic Sci 10:86. https://doi.org/10.4056/sigs.632. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. https://doi.org/10.1186/1471 -2105-11-119. Aogáin M, Roycroft E, Raftery P, Mok S, Fitzgibbon M, Rogers TR. 2015. Draft genome sequences of three Mycobacterium chimaera respiratory isolates. Genome Announc 3(6):e01409-15. https://doi.org/10.1128/ genomeA.01409-15. Zhang YJ, Reddy MC, Ioerger TR, Rothchild AC, Dartois V, Schuster BM, Trauner A, Wallis D, Galaviz S, Huttenhower C, Sacchettini JC, Behar SM, Rubin EJ. 2013. Tryptophan biosynthesis protects mycobacteria from CD4 T cell-mediated killing. Cell 155:1296 –1308. https://doi.org/ 10.1016/j.cell.2013.10.045. Rice P, Longden I, Bleasby A. 2000. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16:276 –277. https://doi.org/ 10.1016/S0168-9525(00)02024-2.
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