Genomic context of azole resistance mutations in Aspergillus fumigatus determined using whole-genome sequencing

mBio

Volumn 6, Issue 3, 2015, Pages

  Abdolrasouli, Alireza ^a,b   Rhodes, Johanna ^c   Beale, Mathew A ^c,d   Hagen, Ferry ^e   Rogers, Thomas R ^f,g   Chowdhary, Anuradha ^h   Meis, Jacques F ^e,i   Armstrong James, Darius ^a   Fisher, Matthew C ^c  

^a NATIONAL HEART AND LUNG INSTITUTE   (United Kingdom)

^b CHARING CROSS HOSPITAL   (United Kingdom)

^c IMPERIAL COLLEGE LONDON   (United Kingdom)

^d ST GEORGE'S UNIVERSITY OF LONDON   (United Kingdom)

^e CANISIUS WILHELMINA HOSPITAL   (Netherlands)

^f TRINITY COLLEGE DUBLIN   (Ireland)

^g ST JAMES S HOSPITAL   (Ireland)

^h UNIVERSITY OF DELHI   (India)

ⁱ RADBOUD UNIVERSITY MEDICAL CENTER   (Netherlands)

Author keywords

[No Author keywords available]

Indexed keywords

AMPHOTERICIN B; ANIDULAFUNGIN; ANTIFUNGAL AGENT; CASPOFUNGIN; ISAVUCONAZOLE; ITRACONAZOLE; MICAFUNGIN; POSACONAZOLE; TRIAZOLE DERIVATIVE; VORICONAZOLE; CYTOCHROME P-450 CYP51A, ASPERGILLUS; CYTOCHROME P450; FUNGAL DNA; FUNGAL PROTEIN; PYRROLE DERIVATIVE;

AMINO ACID SUBSTITUTION; ANTIFUNGAL RESISTANCE; ANTIFUNGAL SUSCEPTIBILITY; ARTICLE; ASPERGILLUS FUMIGATUS; CODON; CONTROLLED STUDY; CYP51A GENE; FUNGAL GENE; GENE MUTATION; GENETIC RECOMBINATION; GENETIC VARIABILITY; GENOME ANALYSIS; INDIA; NETHERLANDS; NONHUMAN; POPULATION GENETIC PARAMETERS; PRIORITY JOURNAL; PROMOTER REGION; SELECTIVE SWEEP; SEQUENCE ANALYSIS; SINGLE NUCLEOTIDE POLYMORPHISM; SPECIES IDENTIFICATION; TANDEM REPEAT; UNITED KINGDOM; WHOLE GENOME SEQUENCING; ASPERGILLOSIS; CHEMISTRY; DNA SEQUENCE; DRUG EFFECTS; GENETICS; GENOME-WIDE ASSOCIATION STUDY; HUMAN; MICROBIOLOGY; MOLECULAR GENETICS; MUTATION;

ASPERGILLUS FUMIGATUS; FUNGI;

ANTIFUNGAL AGENTS; ASPERGILLOSIS; ASPERGILLUS FUMIGATUS; AZOLES; CYTOCHROME P-450 ENZYME SYSTEM; DNA, FUNGAL; DRUG RESISTANCE, FUNGAL; ENVIRONMENTAL MICROBIOLOGY; FUNGAL PROTEINS; GENOME-WIDE ASSOCIATION STUDY; HUMANS; INDIA; MOLECULAR SEQUENCE DATA; MUTATION; NETHERLANDS; POLYMORPHISM, SINGLE NUCLEOTIDE; SEQUENCE ANALYSIS, DNA; UNITED KINGDOM;

EID: 84931270515     PISSN: 21612129     EISSN: 21507511     Source Type: Journal    
DOI: 10.1128/mBio.00536-15     Document Type: Article

References (73)

1. Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. 2012. Hidden killers: human fungal infections. Sci Transl Med 4:165rv13. http://dx.doi.org/10.1126/scitranslmed.3004404.
2. Armstrong-James D, Meintjes G, Brown GD. 2014. A neglected epidemic: fungal infections in HIV/AIDS. Trends Microbiol 22:120-127. http://dx.doi.org/10.1016/j.tim.2014.01.001.
3. Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ. 2012. Emerging fungal threats to animal, plant and ecosystem health. Nature 484:186-194. http://dx.doi.org/10.1038/nature10947.
4. Segal BH. 2009. Aspergillosis. N Engl J Med 360:1870-1884. http:// dx.doi.org/10.1056/NEJMra0808853.
5. Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr KA, Morrison VA, Segal BH, Steinbach WJ, Stevens DA, van Burik J-A, Wingard JR, Patterson TF, Infectious Diseases Society of America. 2008. Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 46:327-360. http://dx.doi.org/10.1086/525258.
6. Vermeulen E, Lagrou K, Verweij PE. 2013. Azole resistance in Aspergillus fumigatus: a growing public health concern. Curr Opin Infect Dis 26: 493-500. http://dx.doi.org/10.1097/QCO.0000000000000005.
7. Chowdhary A, Sharma C, van den Boom M, Yntema JB, Hagen F, Verweij PE, Meis JF. 2014. Multi-azole-resistant Aspergillus fumigatus in the environment in Tanzania. J Antimicrob Chemother 69:2979-2983. http://dx.doi.org/10.1093/jac/dku259.
8. Badali H, Vaezi A, Haghani I, Yazdanparast SA, Hedayati MT, Mousavi B, Ansari S, Hagen F, Meis JF, Chowdhary A. 2013. Environmental study of azole-resistant Aspergillus fumigatus with TR34/L98H mutations in the cyp51A gene in Iran. Mycoses 56:659-663. http://dx.doi.org/10.1111/ myc.12089.
9. Chowdhary A, Kathuria S, Xu J, Meis JF. 2013. Emergence of azoleresistant Aspergillus fumigatus strains due to agricultural azole use creates an increasing threat to human health. PLoS Pathog 9:e1003633. http:// dx.doi.org/10.1371/journal.ppat.1003633.
10. Pham CD, Reiss E, Hagen F, Meis JF, Lockhart SR. 2014. Passive surveillance for azole-resistant Aspergillus fumigatus, United States, 2011-2013. Emerg Infect Dis 20:1498-1503. http://dx.doi.org/10.3201/ eid2009.140142.
11. Howard SJ, Cerar D, Anderson MJ, Albarrag A, Fisher MC, Pasqualotto AC, Laverdiere M, Arendrup MC, Perlin DS, Denning DW. 2009. Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis 15:1068-1076. http:// dx.doi.org/10.3201/eid1507.090043.
12. Snelders E, van der Lee HA, Kuijpers J, Rijs AJ, Varga J, Samson RA, Mellado E, Donders AR, Melchers WJ, Verweij PE. 2008. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med 5:e219. http://dx.doi.org/10.1371/ journal.pmed.0050219.
13. van der Linden JW, Snelders E, Kampinga GA, Rijnders BJ, Mattsson E, Debets-Ossenkopp YJ, Kuijper EJ, Van Tiel FH, Melchers WJ, Verweij PE. 2011. Clinical implications of azole resistance in Aspergillus fumigatus 2007-2009. Emerg Infect Dis 17:1846-1854. http://dx.doi.org/10.3201/ eid1710.110226.
14. Howard SJ, Arendrup MC. 2011. Acquired antifungal drug resistance in Aspergillus fumigatus: epidemiology and detection. Med Mycol 49(Suppl 1):S90-S95. http://dx.doi.org/10.3109/13693786.2010.508469.
15. Verweij PE, Snelders E, Kema GH, Mellado E, Melchers WJ. 2009. Azole resistance in Aspergillus fumigatus: a side-effect of environmental fungicide use? Lancet Infect Dis 9:789-795. http://dx.doi.org/10.1016/S1473-3099(09)70265-8.
16. Snelders E, Camps SM, Karawajczyk A, Schaftenaar G, Kema GH, van der Lee HA, Klaassen CH, Melchers WJ, Verweij PE. 2012. Triazole fungicides can induce cross-resistance to medical triazoles in Aspergillus fumigatus. PLoS One 7:e31801. http://dx.doi.org/10.1371/ journal.pone.0031801.
17. Alcazar-Fuoli L, Mellado E. 2012. Ergosterol biosynthesis in Aspergillus fumigatus: its relevance as an antifungal target and role in antifungal drug resistance. Front Microbiol 3:439. http://dx.doi.org/10.3389/ fmicb.2012.00439.
18. Chowdhary A, Sharma C, Hagen F, Meis JF. 2014. Exploring azole antifungal drug resistance in Aspergillus fumigatus with special reference to resistance mechanisms. Future Microbiol 9:697-711. http://dx.doi.org/ 10.2217/fmb.14.27.
19. Mellado E, Diaz-Guerra TM, Cuenca-Estrella M, Rodriguez-Tudela JL. 2001. Identification of two different 14-α sterol demethylase-related genes (cyp51A and cyp51B) inAspergillus fumigatus and other Aspergillus species. J Clin Microbiol 39:2431-2438. http://dx.doi.org/10.1128/JCM.39.7.2431-2438.2001.
20. Nascimento AM, Goldman GH, Park S, Marras SA, Delmas G, Oza U, Lolans K, Dudley MN, Mann PA, Perlin DS. 2003. Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resis-tance to itraconazole. Antimicrob Agents Chemother 47:1719-1726. http://dx.doi.org/10.1128/AAC.47.5.1719-1726.2003.
21. Mann PA, Parmegiani RM, Wei SQ, Mendrick CA, Li X, Loebenberg D, Didomenico B, Hare RS, Walker SS, McNicholas PM. 2003. Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome P450 14α-demethylase. Antimicrob Agents Chemother 47:577-581. http:// dx.doi.org/10.1128/AAC.47.2.577-581.2003.
22. Bader O, Weig M, Reichard U, Lugert R, Kuhns M, Christner M, Held J, Peter S, Schumacher U, Buchheidt D, Tintelnot K, Groß U, MykoLabNet-D Partners. 2013. cyp51A-Based mechanisms of Aspergillus fumigatus azole drug resistance present in clinical samples from Germany. Antimicrob Agents Chemother 57:3513-3517. http://dx.doi.org/10.1128/ AAC.00167-13.
23. Mellado E, Garcia-Effron G, Alcazar-Fuoli L, Cuenca-Estrella M, Rodríguez-Tudela JL. 2004. Substitutions at methionine 220 in the 14α-sterol demethylase (cyp51A) of Aspergillus fumigatus are responsible for resistance in vitro to azole antifungal drugs. J Clin Microbiol 48: 2747-2750.
24. Mellado E, Garcia-Effron G, Alcázar-Fuoli L, Melchers WJ, Verweij PE, Cuenca-Estrella M, Rodríguez-Tudela JL. 2007. A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations. Antimicrob Agents Chemother 51:1897-1904. http://dx.doi.org/10.1128/AAC.01092-06.
25. Van der Linden JW, Camps SM, Kampinga GA, Arends JP, Debets-Ossenkopp YJ, Haas PJ, Rijnders BJ, Kuijper EJ, Van Tiel FH, Varga J, Karawajczyk A, Zoll J, Melchers WJ, Verweij PE. 2013. Aspergillosis due to voriconazole highly resistant Aspergillus fumigatus and recovery of genetically related resistant isolates from domiciles. Clin Infect Dis 57: 513-520. http://dx.doi.org/10.1093/cid/cit320.
26. Vermeulen E, Maertens J, Schoemans H, Lagrou K. 2012. Azoleresistant Aspergillus fumigatus due to TR46/Y121F/T289A mutation emerging in Belgium, July 2012. Euro Surveill 17:1-3.
27. Astvad KM, Jensen RH, Hassan TM, Mathiasen EG, Thomsen GM, Pedersen UG, Christensen M, Hilberg O, Arendrup MC. 2014. First detection of TR46/Y121F/T289A and TR34/L98H alterations in Aspergillus fumigatus isolates from azole-naive patients in Denmark despite negative findings in the environment. Antimicrob Agents Chemother 58: 5096-5101. http://dx.doi.org/10.1128/AAC.02855-14.
28. Fischer J, van Koningsbruggen-Rietschel S, Rietschel E, Vehreschild MJ, Wisplinghoff H, Krönke M, Hamprecht A. 2014. Prevalence and molecular characterization of azole resistance in Aspergillus spp. isolates from German cystic fibrosis patients. J Antimicrob Chemother 69:1533-1536. http://dx.doi.org/10.1093/jac/dku009.
29. Chowdhary A, Sharma C, Kathuria S, Hagen F, Meis JF. 2014. Azoleresistant Aspergillus fumigatus with the environmental. TR46/Y121F/ T289A mutation in India. J Antimicrob Chemother 69:555-557.
30. Vanhee LM, Symoens F, Jacobsen MD, Nelis HJ, Coenye T. 2009. Comparison of multiple typing methods for Aspergillus fumigatus. Clin Microbiol Infect 15:643-650. http://dx.doi.org/10.1111/j.1469-0691.2009.02844.x.
31. Rhodes J, Beale MA, Fisher MC. 2014. Illuminating choices for library prep: a comparison of library preparation methods for whole genome sequencing of Cryptococcus neoformans using Illumina HiSeq. PLoS One 9:e113501. http://dx.doi.org/10.1371/journal.pone.0113501.
32. Peacock S. 2014. Bring microbial sequencing to hospitals. Nature 509: 557-559. http://dx.doi.org/10.1038/509557a.
33. Köser CU, Ellington MJ, Peacock SJ. 2014. Whole-genome sequencing to control antimicrobial resistance. Trends Genet 30:401-407. http:// dx.doi.org/10.1016/j.tig.2014.07.003.
34. Reuter S, Ellington MJ, Cartwright EJ, Köser CU, Török ME, Gouliouris T, Harris SR, Brown NM, Holden MT, Quail M, Parkhill J, Smith GP, Bentley SD, Peacock SJ. 2013. Rapid bacterial whole-genome sequencing to enhance diagnostic and public health microbiology. JAMA Intern Med 173:1397-1404. h t t p : / / d x . d o i . o r g / 1 0 . 1 0 0 1 / jamainternmed.2013.7734.
35. Snitkin ES, Zelazny AM, Thomas PJ, Stock F, Henderson DK, Palmore TN, Segre JA, Segre JA. 2012. Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing. Sci Transl Med 4:148ra116. http://dx.doi.org/10.1126/ scitranslmed.3004129.
36. Köser CU, Bryant JM, Becq J, Török ME, Ellington MJ, Marti-Renom MA, Carmichael AJ, Parkhill J, Smith GP, Peacock SJ. 2013. Wholegenome sequencing for rapid susceptibility testing of M. tuberculosis. N Engl J Med 369:290-292. http://dx.doi.org/10.1056/NEJMc1215305.
37. Litvintseva AP, Hurst S, Gade L, Frace MA, Hilsabeck R, Schupp JM, Gillece JD, Roe C, Smith D, Keim P, Lockhart SR, Changayil S, Weil MR, MacCannell DR, Brandt ME, Engelthaler DM. 2014. Wholegenome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections. J Clin Microbiol 52:3216-3222. http:// dx.doi.org/10.1128/JCM.00936-14.
38. Etienne KA, Gillece J, Hilsabeck R, Schupp JM, Colman R, Lockhart SR, Gade L, Thompson EH, Sutton DA, Neblett-Fanfair R, Park BJ, Turabelidze G, Keim P, Brandt ME, Deak E, Engelthaler DM. 2012. Whole genome sequence typing to investigate the Apophysomyces outbreak following a tornado in Joplin, Missouri, 2011. PLoS One 7:e49989. http:// dx.doi.org/10.1371/journal.pone.0049989.
39. Engelthaler DM, Chiller T, Schupp JA, Colvin J, Beckstrom-Sternberg SM, Driebe EM, Moses T, Tembe W, Sinari S, Beckstrom-Sternberg JS, Christoforides A, Pearson JV, Carpten J, Keim P, Peterson A, Terashita D, Balajee SA. 2011. Next-generation sequencing of Coccidioides immitis isolated during cluster investigation. Emerg Infect Dis 17:227-232. http:// dx.doi.org/10.3201/eid1702.100620.
40. Janbon G, Ormerod KL, Paulet D, Byrnes EJ, Yadav V, Chatterjee G, Mullapudi N, Hon C-C, Billmyre RB, Brunel F, Bahn Y-S, Chen W, Chen Y, Chow EW, Coppée J-Y, Floyd-Averette A, Gaillardin C, Gerik KJ, Goldberg J, Gonzalez-Hilarion S, Gujja S, Hamlin JL, Hsueh Y-P, Ianiri G, Jones S, Kodira CD, Kozubowski L, Lam W, Marra M, Mesner LD, Mieczkowski PA, Moyrand F, Nielsen K, Proux C, Rossignol T, Schein JE, Sun S, Wollschlaeger C, Wood IA, Zeng Q, Neuvéglise C, Newlon CS, Perfert JR, Lodge, Idnurm A, Stajich JE, Kronstad JW, Sanyal K, Heitman J, Fraser JA, Cuomo CA, Dietrich FS. 2014. Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation. PLoS Genet 10:e1004261. http://dx.doi.org/10.1371/ journal.pgen.1004261.
41. Stamatakis A. 2006. RAxML-VI HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688-2690. http://dx.doi.org/10.1093/bioinformatics/btl446.
42. Mcvean GA, Myers SR, Hunt S, Deloukas P, Bentley DR, Donnelly P. 2004. The fine-scale structure of recombination rate variation in the human genome. Science 304:581-585. http://dx.doi.org/10.1126/ science.1092500.
43. Korte A, Vilhjálmsson BJ, Segura V, Platt A, Long Q, Nordborg M. 2012. A mixed-model approach for genome-wide association studies of correlated traits in structured populations. Nat Genet 44:1066-1071. http://dx.doi.org/10.1038/ng.2376.
44. Lockhart SR, Frade JP, Etienne KA, Pfaller MA, Diekema DJ, Balajee SA. 2011. Azole resistance in Aspergillus fumigatus isolates from the ARTEMIS global surveillance study is primarily due to the TR/L98H mutation in the cyp51A gene. Antimicrob Agents Chemother 55:4465-4468. http://dx.doi.org/10.1128/AAC.00185-11.
45. Selmecki A, Forche A, Berman J. 2006. Aneuploidy and isochromosome formation in drug-resistant Candida albicans. Science 313:367-370. http://dx.doi.org/10.1126/science.1128242.
46. Sionov E, Lee H, Chang YC, Kwon-Chung KJ. 2010. Cryptococcus neoformans overcomes stress of azole drugs by formation of disomy in specific multiple chromosomes. PLoS Pathog 6:e1000848. http://dx.doi.org/ 10.1371/journal.ppat.1000848.
47. Fraczek MG, Bromley M, Buied A, Moore CB, Rajendran R, Rautemaa R, Ramage G, Denning DW, Bowyer P. 2013. The cdr1B efflux transporter is associated with non-cyp51A-mediated itraconazole resistance in Aspergillus fumigatus. J Antimicrob Chemother 68:1486-1496. http:// dx.doi.org/10.1093/jac/dkt075.
48. Camps SM, Dutilh BE, Arendrup MC, Rijs AJ, Snelders E, Huynen MA, Verweij PE, Melchers WJ. 2012. Discovery of a HapE mutation that causes azole resistance in Aspergillus fumigatus through whole genome sequencing and sexual crossing. PLoS One 7:e50034. http://dx.doi.org/ 10.1371/journal.pone.0050034.
49. Buied A, Moore CB, Denning DW, Bowyer P. 2013. High-level expression of cyp51B in azole-resistant clinical Aspergillus fumigatus isolates. J Antimicrob Chemother 68:512-514. http://dx.doi.org/10.1093/jac/ dks451.
50. Calo S, Shertz-Wall C, Lee SC, Bastidas RJ, Nicolás FE, Granek JA, Mieczkowski P, Torres-Martínez S, Ruiz-Vázquez RM, Cardenas ME, Heitman J. 2014. Antifungal drug resistance evoked via RNAi-dependent epimutations. Nature 513:555-558. http://dx.doi.org/10.1038/ nature13575.
51. Müller S, Baldin C, Groth M, Guthke R, Kniemeyer O, Brakhage AA, Valiante V. 2012. Comparison of transcriptome technologies in the pathogenic fungus Aspergillus fumigatus reveals novel insights into the genome and MpkA dependent gene expression. BMC Genomics 13:519. http://dx.doi.org/10.1186/1471-2164-13-519.
52. Schoustra SE, Debets AJ, Slakhorst M, Hoekstra RF. 2007. Mitotic recombination accelerates adaptation in the fungus Aspergillus nidulans. PLoS Genet 3:e68. http://dx.doi.org/10.1371/journal.pgen.0030068.
53. Sheppard SK, Didelot X, Meric G, Torralbo A, Jolley KA, Kelly DJ, Bentley SD, Maiden MC, Parkhill J, Falush D. 2013. Genome-wide association study identifies vitamin B5 biosynthesis as a host specificity factor in Campylobacter. Proc Natl Acad Sci U S A 110:11923-11927. http://dx.doi.org/10.1073/pnas.1305559110.
54. Klaassen CH, Gibbons JG, Fedorova ND, Meis JF, Rokas A. 2012. Evidence for genetic differentiation and variable recombination rates among Dutch populations of the opportunistic human pathogen Aspergillus fumigatus. Mol Ecol 21:57-70. http://dx.doi.org/10.1111/j.1365-294X.2011.05364.x.
55. Paoletti M, Rydholm C, Schwier EU, Anderson MJ, Szakacs G, Lutzoni F, Debeaupuis J-P, Latgé J-P, Denning DW, Dyer PS. 2005. Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus. Curr Biol 15:1242-1248. http://dx.doi.org/10.1016/j.cub.2005.05.045.
56. Pringle A, Baker DM, Platt JL, Wares JP, Latgé JP, Taylor JW. 2005. Cryptic speciation in the cosmopolitan and clonal human pathogenic fungus Aspergillus fumigatus. Evolution 59:1886-1899. http://dx.doi.org/ 10.1111/j.0014-3820.2005.tb01059.x.
57. Rydholm C, Szakacs G, Lutzoni F. 2006. Low genetic variation and no detectable population structure in Aspergillus fumigatus compared to closely related Neosartorya species. Eukaryot Cell 5:650-657. http:// dx.doi.org/10.1128/EC.5.4.650-657.2006.
58. Henk DA, Shahar-Golan R, Ranjana DK, Zhan N, Fedorova N, Nierman WC, Hsueh PR, Yuen KY, Wertheim H, Day J, Vanittanakom N, Andrianopoulos A, Fisher MC. Clonality despite sex, the evolution of sexual neighborhoods in the pathogenic fungus Penicillium marneffei. PLoS Pathogens 8:e1002851.
59. Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, Arroyo J, Berriman M, Abe K, Archer DB, Bermejo C, Bennett J, Bowyer P, Chen D, Collins M, Coulsen R, Davies R, Dyer PS, Farman M, Fedorova N, Fedorova N, Feldblyum TV, Fischer R, Fosker N, Fraser A, García JL, García MJ, Goble A, Goldman GH, Gomi K, Griffith-Jones S, Gwilliam R, Haas B, Haas H, Harris D, Horiuchi H, Huang J, Humphray S, Jiménez J, Keller N, Khouri H, Kitamoto K, Kobayashi T, Konzack S, Kulkarni R, Kumagai T, Lafon A, Lafton A, Latgé J-P, Li W, Lord A, et al. 2005. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438:1151-1156. http://dx.doi.org/ 10.1038/nature04332.
60. Denning DW, Venkateswarlu K, Oakley KL, Anderson MJ, Manning NJ, Stevens DA, Warnock DW, Kelly SL. 1997. Itraconazole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother 41:1364-1368.
61. Chowdhary A, Kathuria S, Xu J, Sharma C, Sundar G, Singh PK, Gaur SN, Hagen F, Klaassen CH, Meis JF. 2012. Clonal expansion and emergence of environmental multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR34/L98H mutations in the cyp51A gene in India. PLoS One 7:e52871. http://dx.doi.org/10.1371/journal.pone.0052871.
62. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard, 2nd ed. CLSI document M38-A2. CLSI, Wayne, PA.
63. Verweij PE, Howard SJ, Melchers WJ, Denning DW. 2009. Azoleresistance in Aspergillus: proposed nomenclature and breakpoints. Drug Resist Updat 12:141-147. http://dx.doi.org/10.1016/j.drup.2009.09.002.
64. Pfaller JB, Messer SA, Hollis RJ, Diekema DJ, Pfaller MA. 2003. In vitro susceptibility testing of Aspergillus spp.: comparison of Etest and reference microdilution methods for determining voriconazole and itraconazole MICs. J Clin Microbiol 41:1126-1129. http://dx.doi.org/10.1128/ JCM.41.3.1126-1129.2003.
65. Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754-1760. http:// dx.doi.org/10.1093/bioinformatics/btp324.
66. Van der Auwera GA, Feldgarden M, Kolter R, Mahillon J. 2013. Wholegenome sequence of 94 environmental isolates of Bacillus cereus sensu lato. Genome Announc 1:1-4.
67. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ. 2011. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43:491-498. http://dx.doi.org/10.1038/ng.806.
68. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to singlecell sequencing. J Comput Biol 19:455-477. http://dx.doi.org/10.1089/ cmb.2012.0021.
69. Bradburd GS, Ralph PL, Coop GM. 2013. Disentangling the effects of geographic and ecological isolation on genetic differentiation. Evolution 67:3258-3273. http://dx.doi.org/10.1111/evo.12193.
70. R Development Core Team. 2008. R:A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.
71. Kamvar ZN, Tabima JF, Grünwald NJ. 2014. Poppr: an R package for genetic analysis of populations with clonal, partially clonal, and/or sexual reproduction. PeerJ 2:e281. http://dx.doi.org/10.7717/peerj.281.
72. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. 2009. Circos: an information aesthetic for comparative genomics. Genome Res 19:1639-1645. http://dx.doi.org/10.1101/ gr.092759.109.
73. Agapow PM, Burt A. 2001. Indices of multilocus disequilibrium. Mol Ecol Notes 1(1-2):101-102.