We performed Illumina whole-genome sequencing on a carbapenem-resistant Pseudomonas aeruginosa strain isolated from a cystic fibrosis patient with chronic airway colonization. The draft genome comprises 6,770,411 bp, including the carbapenemase blaNDM-1 and the extended-spectrum beta-lactamase blaPME-1.
ABSTRACT
We performed Illumina whole-genome sequencing on a carbapenem-resistant Pseudomonas aeruginosa strain isolated from a cystic fibrosis patient with chronic airway colonization. The draft genome comprises 6,770,411 bp, including the carbapenemase blaNDM-1 and the extended-spectrum beta-lactamase blaPME-1. This isolate harbors 3 prophages, 14 antibiotic resistance genes, and 257 virulence genes.
ANNOUNCEMENT
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently involved in nosocomial infections (1, 2). We isolated P. aeruginosa strain PA-81 from tracheal secretions of a cystic fibrosis patient admitted to a tertiary care hospital in Pakistan. The sample was plated on blood agar (Oxoid, UK) followed by substreaking of morphologically distinct colonies on Pseudomonas cetrimide agar (Oxoid). The isolate was transferred to St. Louis, MO, for further characterization. Distinct colonies of the isolate were used for identification by Vitek MS matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) with the library v2.3.3 (bioMérieux, Durham, NC) using default settings. Because the strain was found to be resistant to all available classes of antibiotics, including cefepime, meropenem, piperacillin-tazobactam, ceftolozane-tazobactam, ceftazidime-avibactam, ciprofloxacin, aztreonam, and trimethoprim-sulfamethoxazole, this strain was selected for further analysis from among more than 200 P. aeruginosa isolates collected in 2016.
Genomic DNA was extracted from overnight growth on blood agar (Hardy Diagnostics, Santa Maria, CA) using the QIAamp BiOstic bacteremia DNA kit (Qiagen, Germantown, MD). Illumina sequencing libraries were prepared using the Nextera DNA flex library prep kit (Illumina, San Diego, CA) with 0.5 ng genomic DNA (3). Whole-genome sequencing was performed on an Illumina NextSeq 500 instrument to obtain 2 × 150-bp reads. Raw reads were processed and analyzed on the High Performance Computing Center cluster at Washington University in St. Louis School of Medicine, St. Louis, MO. Adapter sequences were trimmed using Trimmomatic v0.36 and decontaminated using DeconSeq v0.4.3 (4, 5). SPAdes v3.11.0 was used for de novo assembly of paired-end reads totaling 6,588,272 bp to produce an assembly with a mean genome coverage of 22× (6). The quality of the resulting assembly was evaluated using QUAST v4.5 (7). The assembly produced 331 contigs (largest contig, 580,364 bp) with an N50 value of 265,381 bp. PATRIC v3.5.20 was used for annotation, which determined a total genome length of 6,770,411 bp with 6,607 coding sequences (CDSs). The strain had a 66% GC content, 65 tRNA genes, 3 rRNA genes, and the 16S, 23S, and 5S loci (8).
The genome of strain PA-81 contained 614 unique genes that were absent from the genome of the PAO1 reference strain (NCBI Reference Sequence number NC_002516). The strain belongs to sequence type 357 (ST357) (https://cge.cbs.dtu.dk/services/MLST/) and serotype O11 (https://cge.cbs.dtu.dk/services/PAst/). Antibiotic resistance gene (ARG) annotation using the ResFinder and Comprehensive Antibiotic Resistance Database (CARD) servers (9, 10) revealed a number of ARGs against multiple classes of antibiotics, including beta-lactams (blaOXA-10, blaOXA-50, blaPAO, blaAmp-C, blaNDM-1, and blaPME-1), aminoglycosides [aph(3′)-IIb, aph(3′)-VIa, ant(2ʺ)-Ia, and aadA1], fosfomycin (fosA), trimethoprim (dfrB2), amphenicols (catB7), and sulfonamides (sul1). The presence of blaNDM-1 provides a likely mechanism of carbapenem resistance (11, 12). Further analysis using PlasmidFinder v1.3 did not reveal any known plasmid replicons (13). Three prophage regions of 21 kb, 12.6 kb, and 9.6 kb with identity scores of 90%, 85%, and 10%, respectively, were identified using PHAge Search Tool-Enhanced Release (PHASTER) (14). Default settings were used for all the employed tools and software.
To the best of our knowledge, this is the first draft genome sequence of a blaNDM-1- and blaPME-1-harboring P. aeruginosa strain from Pakistan. A more detailed analysis of this genome will be reported in a future publication.
Data availability.
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number QXJN00000000 (version QXJN01000000) and SRA accession number SRR8510690.
ACKNOWLEDGMENTS
This work was supported in part by an International Research Support Initiative Program (IRSIP) award of the Higher Education Commission (HEC) of Pakistan to S.I. and a United States Agency for International Development award (3220-29047) to S.A., C.-A.D.B., and G.D. R.F.P. received support from an NIGMS training grant through award T32 GM007067 (primary investigator, James Skeath) and the Monsanto Excellence Fund graduate fellowship.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
We acknowledge the pathology lab staff of PIMS, Islamabad, Pakistan, for providing the clinical isolate and associated metadata. We also thank The Edison Family Center for Genome Sciences & Systems Biology staff members Eric Martin, Brian Koebbe, and Jessica Hoisington-López for their technical support and sequencing expertise.
REFERENCES
- 1.Gellatly SL, Hancock REW. 2013. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis 67:159–173. doi: 10.1111/2049-632X.12033. [DOI] [PubMed] [Google Scholar]
- 2.Balasubramanian D, Schneper L, Kumari H, Mathee K. 2013. A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res 41:1–20. doi: 10.1093/nar/gks1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Baym M, Kryazhimskiy S, Lieberman TD, Chung H, Desai MM, Kishony R. 2015. Inexpensive multiplexed library preparation for megabase-sized genomes. PLoS One 10:e0128036. doi: 10.1371/journal.pone.0128036. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Schmieder R, Edwards R. 2011. Fast identification and removal of sequence contamination from genomic and metagenomic datasets. PLoS One 6:e17288. doi: 10.1371/journal.pone.0017288. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.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 single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. doi: 10.1093/bioinformatics/btt086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Wattam AR, Abraham D, Dalay O, Disz TL, Driscoll T, Gabbard JL, Gillespie JJ, Gough R, Hix D, Kenyon R, Machi D, Mao C, Nordberg EK, Olson R, Overbeek R, Pusch GD, Shukla M, Schulman J, Stevens RL, Sullivan DE, Vonstein V, Warren A, Will R, Wilson MJC, Yoo HS, Zhang C, Zhang Y, Sobral BW. 2014. PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res 42:D581–D591. doi: 10.1093/nar/gkt1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640–2644. doi: 10.1093/jac/dks261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.McArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, Bhullar K, Canova MJ, De Pascale G, Ejim L, Kalan L, King AM, Koteva K, Morar M, Mulvey MR, O'Brien JS, Pawlowski AC, Piddock LJV, Spanogiannopoulos P, Sutherland AD, Tang I, Taylor PL, Thaker M, Wang W, Yan M, Yu T, Wright GD. 2013. The Comprehensive Antibiotic Resistance Database. Antimicrob Agents Chemother 57:3348–3357. doi: 10.1128/AAC.00419-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Teo JWP, La M-V, Jureen R, Lin RTP. 2015. Emergence of a New Delhi metallo-β-lactamase-1-producing Pseudomonas aeruginosa in Singapore. Emerg Microbes Infect 4:1. doi: 10.1038/emi.2015.72. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, Walsh TR. 2009. Characterization of a new metallo-β-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 53:5046–5054. doi: 10.1128/AAC.00774-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Carattoli A, Zankari E, García-Fernández A, Larsen MV, Lund O, Villa L, Aarestrup FM, Hasman H. 2014. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother 58:3895–3903. doi: 10.1128/AAC.02412-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Arndt D, Grant JR, Marcu A, Sajed T, Pon A, Liang Y, Wishart DS. 2016. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res 44:W16–W21. doi: 10.1093/nar/gkw387. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number QXJN00000000 (version QXJN01000000) and SRA accession number SRR8510690.