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Draft Genome Sequence of a Fluconazole-Resistant Candida auris Strain from a Candidemia Patient in India Cheshta Sharma,a Nitin Kumar,b
Jacques F. Meis,c,d Rajesh Pandey,e
Anuradha Chowdharya
Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, Indiaa; Host-Microbiota Interactions Laboratory, Wellcome Trust Sanger Institute, Hinxton, United Kingdomb; Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlandsc; Department of Medical Microbiology, Radboudumc, Nijmegen, The Netherlandsd; CSIR Ayurgenomics Unit-TRISUTRA, Council of Scientific & Industrial Research–Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi, Indiae
Received 26 May 2015 Accepted 16 June 2015 Published 16 July 2015 Citation Sharma C, Kumar N, Meis JF, Pandey R, Chowdhary A. 2015. Draft genome sequence of a fluconazole-resistant Candida auris strain from a candidemia patient in India. Genome Announc 3(4):e00722-15. doi:10.1128/genomeA.00722-15. Copyright © 2015 Sharma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Anuradha Chowdhary, [email protected]
C
andida auris is a multidrug-resistant yeast in the Metschnikowiaceae clade, which was first reported in 2009 from the external auditory canal of a Japanese patient (1). In the last 5 years, a wide spectrum of clinical manifestations due to this unusual yeast, ranging from fungemia to deep-seated infections with high mortality rates, have been reported (2). Further, multidrug-resistant clonal strains of C. auris are widespread in hospitals, suggesting nosocomial transmission (2, 3). Recent reports of this pathogen from Kuwait, India, South Korea, and South Africa highlight the problem of increasing treatment failures in infections due to C. auris (3–6). Candida auris is often misidentified by commercial automated systems as Candida haemulonii, and it is mainly resistant to fluconazole and exhibits elevated MICs to voriconazole, amphotericin B, and caspofungin (2, 7). Thus, the accurate identification of C. auris by molecular methods and antifungal susceptibility testing using reference methods is of paramount importance. The lack of wholegenome data of C. auris prompted us to undertake the draft genome sequencing of C. auris VPCI 479/P/13 obtained from a blood culture from a patient with fungemia in Delhi, India. The isolate was identified by amplification and sequencing of the ITS, D1D2, RPB1, and RPB2 genes and showed 99% homology with South Korean C. auris isolates (KCTC 17809 and KCTC 17810). The isolate was resistant to fluconazole with an MIC of ⬎64 g/ml. Genomic DNA was extracted using a column-based method with the QIAamp DNA minikit (Qiagen, Hilden, Germany). One nanogram of genomic DNA (gDNA) in the Nextera XT DNA sample preparation protocol (Illumina, Inc., San Diego, CA, USA) was used to generate sequencing-ready libraries, according to the manufacturer’s instructions. The sequencing library was quantified using Qubit 2.0 (Invitrogen, Waltham, MA, USA) and qualified by Bioanalyzer (Agilent Technologies, Richardson, TX, USA). The genome of C. auris VPCI 479/P/13 was sequenced using the Illumina MiSeq platform with a MiSeq version 3 protocol (paired end, 300 ⫻ 2 bp). The adaptor contamination was removed from
July/August 2015 Volume 3 Issue 4 e00722-15
the reads, followed by trimming from both sides using the modified Mott trimming algorithm to reach a Q30 score. The FASTQ files were then imported in the CLC Genomics Workbench software (CLC bio-Qiagen). Genome assembly was achieved by the combination of Velvet version 1.2.08 (8), SSPACE 2.0, and GapFiller version 1.10 (9, 10). Repetitive sequences were masked using RepeatMasker version 4.0.5 (http://www.repeatmasker.org), followed by ab initio gene prediction using GeneMark-ES 2.0 (11). Next, rRNAs and tRNAs were predicted with RNAmmer and tRNAscan-SE version 1.21, respectively (12, 13). The draft genome of C. auris VPCI 479/P/13 is 12.3 Mb, with a G⫹C content of 44.8%, distributed on 533 scaffolds (ⱖ1,000 bp) with an N50 length of 37,205 bp. The complete genome sequence of C. auris VPCI 479/P/13 contains 6,675 coding sequences (CDSs), one 5.8S rRNA, 184 tRNAs, and 3,262 repetitive elements. The draft genome sequence presented here will facilitate further genomic studies on the biology and virulence of C. auris. Nucleotide sequence accession number. The draft genome sequence of C. auris strain VPCI 479/P/13 has been deposited at ENA under accession number CVRJ00000000. ACKNOWLEDGMENTS C.S. was supported by a research grant from University Grants Commission Research Fellowship, India (F.2-15/2003 SA-I). J.F.M. received grants from Astellas, Basilea, and Merck. J.F.M. has been a consultant to Astellas, Basilea, and Merck and received speaker’s fees from Merck and Gilead. All other authors declare no potential conflicts of interest. We alone are responsible for the content and writing of the paper.
REFERENCES 1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K, Yamaguchi H. 2009. Candida auris sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital. Microbiol Immunol 53:41– 44. http://dx.doi.org/10.1111/j.1348-0421.2008.00083.x. 2. Chowdhary A, Anil Kumar V, Sharma C, Prakash A, Agarwal K, Babu R, Dinesh KR, Karim S, Singh SK, Hagen F, Meis JF. 2014. Multidrug-
Genome Announcements
genomea.asm.org 1
Downloaded from http://genomea.asm.org/ on September 28, 2015 by guest
Candida auris is a multidrug-resistant yeast incriminated in a wide spectrum of invasive infections, especially in intensive care settings. The first draft genome sequence of C. auris, VPCI 479/P/13, from a case with fungemia was sequenced using the Illumina MiSeq platform. The estimated genome size is 12.3 Mb, with 6,675 coding sequences.
Sharma et al.
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4.
5. 6.
2 genomea.asm.org
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9.
10. 11.
12.
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microdilution, and Etest method. J Clin Microbiol 53:1823–1830. http:// dx.doi.org/10.1128/JCM.00367-15. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821– 829. http:// dx.doi.org/10.1101/gr.074492.107. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27: 578 –579. http://dx.doi.org/10.1093/bioinformatics/btq683. Boetzer M, Pirovano W. 2012. Toward almost closed genomes with GapFiller. Genome Biol 13:R56. http://dx.doi.org/10.1186/gb-2012-13-6-r56. Ter-Hovhannisyan V, Lomsadze A, Chernoff YO, Borodovsky M. 2008. Gene prediction in novel fungal genomes using an ab initio algorithm with unsupervised training. Genome Res 18:1979 –1990. http://dx.doi.org/ 10.1101/gr.081612.108. Lagesen K, Hallin P, Rødland EA, Staerfeldt HH, Rognes T, Ussery DW. 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35:3100 –3108. http://dx.doi.org/10.1093/ nar/gkm160. Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964. http://dx.doi.org/10.1093/nar/25.5.0955.
Genome Announcements
July/August 2015 Volume 3 Issue 4 e00722-15
Downloaded from http://genomea.asm.org/ on September 28, 2015 by guest
7.
resistant endemic clonal strain of Candida auris in India. Eur J Clin Microbiol Infect Dis 33:919 –926. http://dx.doi.org/10.1007/s10096-013 -2027-1. Chowdhary A, Sharma C, Duggal S, Agarwal K, Prakash A, Singh PK, Jain S, Kathuria S, Randhawa HS, Hagen F, Meis JF. 2013. New clonal strain of Candida auris, Delhi, India. Emerg Infect Dis 19:1670 –1673. http://dx.doi.org/10.3201/eid1910.130393. Lee WG, Shin JH, Uh Y, Kang MG, Kim SH, Park KH, Jang HC. 2011. First three reported cases of nosocomial fungemia caused by Candida auris. J Clin Microbiol 49:3139 –3142. http://dx.doi.org/10.1128/ JCM.00319-11. Magobo RE, Corcoran C, Seetharam S, Govender NP. 2014. Candida auris-associated candidemia, South Africa. Emerg Infect Dis 20: 1250 –1251. http://dx.doi.org/10.3201/eid2007.131765. Emara M, Ahmad S, Khan Z, Joseph L, Al-Obaid I, Purohit P, Bafna R. 2015. Candida auris candidemia in Kuwait, 2014. Emerg Infect Dis 21: 1091–1092. http://dx.doi.org/10.3201/eid2106.150270. Kathuria S, Singh PK, Sharma C, Prakash A, Masih A, Kumar A, Meis JF, Chowdhary A. 2015. Multidrug-resistant Candida auris misidentified as Candida haemulonii: characterization by matrix-assisted laser desorption ionization–time of flight mass spectrometry and DNA sequencing and its antifungal susceptibility profile variability by Vitek 2, CLSI broth
Draft Genome Sequence of a Fluconazole-Resistant Candida auris Strain from a Candidemia Patient in India Cheshta Sharma,a Nitin Kumar,b
Jacques F. Meis,c,d Rajesh Pandey,e
Anuradha Chowdharya
Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, Indiaa; Host-Microbiota Interactions Laboratory, Wellcome Trust Sanger Institute, Hinxton, United Kingdomb; Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlandsc; Department of Medical Microbiology, Radboudumc, Nijmegen, The Netherlandsd; CSIR Ayurgenomics Unit-TRISUTRA, Council of Scientific & Industrial Research–Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi, Indiae
Received 26 May 2015 Accepted 16 June 2015 Published 16 July 2015 Citation Sharma C, Kumar N, Meis JF, Pandey R, Chowdhary A. 2015. Draft genome sequence of a fluconazole-resistant Candida auris strain from a candidemia patient in India. Genome Announc 3(4):e00722-15. doi:10.1128/genomeA.00722-15. Copyright © 2015 Sharma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Anuradha Chowdhary, [email protected]
C
andida auris is a multidrug-resistant yeast in the Metschnikowiaceae clade, which was first reported in 2009 from the external auditory canal of a Japanese patient (1). In the last 5 years, a wide spectrum of clinical manifestations due to this unusual yeast, ranging from fungemia to deep-seated infections with high mortality rates, have been reported (2). Further, multidrug-resistant clonal strains of C. auris are widespread in hospitals, suggesting nosocomial transmission (2, 3). Recent reports of this pathogen from Kuwait, India, South Korea, and South Africa highlight the problem of increasing treatment failures in infections due to C. auris (3–6). Candida auris is often misidentified by commercial automated systems as Candida haemulonii, and it is mainly resistant to fluconazole and exhibits elevated MICs to voriconazole, amphotericin B, and caspofungin (2, 7). Thus, the accurate identification of C. auris by molecular methods and antifungal susceptibility testing using reference methods is of paramount importance. The lack of wholegenome data of C. auris prompted us to undertake the draft genome sequencing of C. auris VPCI 479/P/13 obtained from a blood culture from a patient with fungemia in Delhi, India. The isolate was identified by amplification and sequencing of the ITS, D1D2, RPB1, and RPB2 genes and showed 99% homology with South Korean C. auris isolates (KCTC 17809 and KCTC 17810). The isolate was resistant to fluconazole with an MIC of ⬎64 g/ml. Genomic DNA was extracted using a column-based method with the QIAamp DNA minikit (Qiagen, Hilden, Germany). One nanogram of genomic DNA (gDNA) in the Nextera XT DNA sample preparation protocol (Illumina, Inc., San Diego, CA, USA) was used to generate sequencing-ready libraries, according to the manufacturer’s instructions. The sequencing library was quantified using Qubit 2.0 (Invitrogen, Waltham, MA, USA) and qualified by Bioanalyzer (Agilent Technologies, Richardson, TX, USA). The genome of C. auris VPCI 479/P/13 was sequenced using the Illumina MiSeq platform with a MiSeq version 3 protocol (paired end, 300 ⫻ 2 bp). The adaptor contamination was removed from
July/August 2015 Volume 3 Issue 4 e00722-15
the reads, followed by trimming from both sides using the modified Mott trimming algorithm to reach a Q30 score. The FASTQ files were then imported in the CLC Genomics Workbench software (CLC bio-Qiagen). Genome assembly was achieved by the combination of Velvet version 1.2.08 (8), SSPACE 2.0, and GapFiller version 1.10 (9, 10). Repetitive sequences were masked using RepeatMasker version 4.0.5 (http://www.repeatmasker.org), followed by ab initio gene prediction using GeneMark-ES 2.0 (11). Next, rRNAs and tRNAs were predicted with RNAmmer and tRNAscan-SE version 1.21, respectively (12, 13). The draft genome of C. auris VPCI 479/P/13 is 12.3 Mb, with a G⫹C content of 44.8%, distributed on 533 scaffolds (ⱖ1,000 bp) with an N50 length of 37,205 bp. The complete genome sequence of C. auris VPCI 479/P/13 contains 6,675 coding sequences (CDSs), one 5.8S rRNA, 184 tRNAs, and 3,262 repetitive elements. The draft genome sequence presented here will facilitate further genomic studies on the biology and virulence of C. auris. Nucleotide sequence accession number. The draft genome sequence of C. auris strain VPCI 479/P/13 has been deposited at ENA under accession number CVRJ00000000. ACKNOWLEDGMENTS C.S. was supported by a research grant from University Grants Commission Research Fellowship, India (F.2-15/2003 SA-I). J.F.M. received grants from Astellas, Basilea, and Merck. J.F.M. has been a consultant to Astellas, Basilea, and Merck and received speaker’s fees from Merck and Gilead. All other authors declare no potential conflicts of interest. We alone are responsible for the content and writing of the paper.
REFERENCES 1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K, Yamaguchi H. 2009. Candida auris sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital. Microbiol Immunol 53:41– 44. http://dx.doi.org/10.1111/j.1348-0421.2008.00083.x. 2. Chowdhary A, Anil Kumar V, Sharma C, Prakash A, Agarwal K, Babu R, Dinesh KR, Karim S, Singh SK, Hagen F, Meis JF. 2014. Multidrug-
Genome Announcements
genomea.asm.org 1
Downloaded from http://genomea.asm.org/ on September 28, 2015 by guest
Candida auris is a multidrug-resistant yeast incriminated in a wide spectrum of invasive infections, especially in intensive care settings. The first draft genome sequence of C. auris, VPCI 479/P/13, from a case with fungemia was sequenced using the Illumina MiSeq platform. The estimated genome size is 12.3 Mb, with 6,675 coding sequences.
Sharma et al.
3.
4.
5. 6.
2 genomea.asm.org
8.
9.
10. 11.
12.
13.
microdilution, and Etest method. J Clin Microbiol 53:1823–1830. http:// dx.doi.org/10.1128/JCM.00367-15. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821– 829. http:// dx.doi.org/10.1101/gr.074492.107. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27: 578 –579. http://dx.doi.org/10.1093/bioinformatics/btq683. Boetzer M, Pirovano W. 2012. Toward almost closed genomes with GapFiller. Genome Biol 13:R56. http://dx.doi.org/10.1186/gb-2012-13-6-r56. Ter-Hovhannisyan V, Lomsadze A, Chernoff YO, Borodovsky M. 2008. Gene prediction in novel fungal genomes using an ab initio algorithm with unsupervised training. Genome Res 18:1979 –1990. http://dx.doi.org/ 10.1101/gr.081612.108. Lagesen K, Hallin P, Rødland EA, Staerfeldt HH, Rognes T, Ussery DW. 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35:3100 –3108. http://dx.doi.org/10.1093/ nar/gkm160. Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964. http://dx.doi.org/10.1093/nar/25.5.0955.
Genome Announcements
July/August 2015 Volume 3 Issue 4 e00722-15
Downloaded from http://genomea.asm.org/ on September 28, 2015 by guest
7.
resistant endemic clonal strain of Candida auris in India. Eur J Clin Microbiol Infect Dis 33:919 –926. http://dx.doi.org/10.1007/s10096-013 -2027-1. Chowdhary A, Sharma C, Duggal S, Agarwal K, Prakash A, Singh PK, Jain S, Kathuria S, Randhawa HS, Hagen F, Meis JF. 2013. New clonal strain of Candida auris, Delhi, India. Emerg Infect Dis 19:1670 –1673. http://dx.doi.org/10.3201/eid1910.130393. Lee WG, Shin JH, Uh Y, Kang MG, Kim SH, Park KH, Jang HC. 2011. First three reported cases of nosocomial fungemia caused by Candida auris. J Clin Microbiol 49:3139 –3142. http://dx.doi.org/10.1128/ JCM.00319-11. Magobo RE, Corcoran C, Seetharam S, Govender NP. 2014. Candida auris-associated candidemia, South Africa. Emerg Infect Dis 20: 1250 –1251. http://dx.doi.org/10.3201/eid2007.131765. Emara M, Ahmad S, Khan Z, Joseph L, Al-Obaid I, Purohit P, Bafna R. 2015. Candida auris candidemia in Kuwait, 2014. Emerg Infect Dis 21: 1091–1092. http://dx.doi.org/10.3201/eid2106.150270. Kathuria S, Singh PK, Sharma C, Prakash A, Masih A, Kumar A, Meis JF, Chowdhary A. 2015. Multidrug-resistant Candida auris misidentified as Candida haemulonii: characterization by matrix-assisted laser desorption ionization–time of flight mass spectrometry and DNA sequencing and its antifungal susceptibility profile variability by Vitek 2, CLSI broth
