The Gram-negative bacterium Pseudomonas aeruginosa is often multidrug resistant, associated with global epidemic outbreaks, and responsible for significant morbidity and mortality in hospitalized patients. Here, we present the draft genome sequence of BWH047, a multidrug-resistant P. aeruginosa clinical isolate belonging to the epidemic sequence type 235 and demonstrating high levels of colistin resistance.
ABSTRACT
The Gram-negative bacterium Pseudomonas aeruginosa is often multidrug resistant, associated with global epidemic outbreaks, and responsible for significant morbidity and mortality in hospitalized patients. Here, we present the draft genome sequence of BWH047, a multidrug-resistant P. aeruginosa clinical isolate belonging to the epidemic sequence type 235 and demonstrating high levels of colistin resistance.
ANNOUNCEMENT
Pseudomonas aeruginosa frequently causes severe health care-associated infections (1) that are associated with high mortality. Multidrug-resistant (MDR) P. aeruginosa infections are consistently associated with poor clinical outcomes (2). The global P. aeruginosa population includes several recognized high-risk epidemic clones belonging to multilocus sequence type 111 (ST-111), ST-175, or ST-235 (3, 4). ST-235, a globally distributed multidrug-resistant high-risk clone associated with nosocomial infections, is known to harbor a variety of antimicrobial resistance genes, including those for β-lactamases and carbapenemases, as well as horizontally acquired antibiotic resistance elements (4).
In this study, we determined the draft genome sequence of the ST-235 MDR P. aeruginosa clinical isolate BWH047, an isolate collected from a patient at Brigham and Women’s Hospital (BWH) in 2016. BWH047 was isolated from a bronchoalveolar lavage fluid specimen from an immunosuppressed 57-year-old patient who had undergone allogeneic hematopoietic cell transplantation in 2009 and bilateral lung transplantation in April 2015. After persistent recovery of P. aeruginosa with escalating antimicrobial resistance, inhaled colistin therapy was initiated in August 2015. Antibiotic susceptibilities were determined by Kirby-Bauer (KB) testing (5). BWH047 was initially sensitive to amikacin, cefepime, ceftazidime, colistin, and piperacillin, resistant to ciprofloxacin, levofloxacin, gentamicin, tobramycin, and meropenem, and intermediate to aztreonam. Broth microdilution (MBD) resistance testing (6) was completed, and BWH047 was found to be sensitive to aztreonam, ceftazidime, and piperacillin-tazobactam, intermediate to cefepime, and resistant to ciprofloxacin, colistin, gentamicin, and meropenem. Despite initial KB testing reports of colistin sensitivity, MBD testing demonstrated considerable colistin resistance.
Genomic DNA isolated from a single colony of BWH047 was sequenced on the HiSeq 4000 platform (Illumina, Inc., San Diego, CA), generating 2 × 150-bp paired-end reads. A total of 4,957,758 reads were generated, for a total of 743,663,700 bases. Adapter sequences were removed, and reads were quality trimmed using Trimmomatic v0.36 (7), resulting in 3,941,146 paired reads totaling 569,414,246 bases. De novo assembly was performed using SPAdes v3.9.1 (8). Quality control was performed by aligning trimmed reads to assembly contigs using bwa v0.7.15 (9). All contigs shorter than 200 bp or with an average fold coverage of <5× per base were removed. The resulting assembly consisted of 138 contigs totaling 6,742,224 bases of sequence, and the average fold coverage was 84×. The assembly N50 value was 192,392 bp, and the average GC content was 66.15%. Annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline v4.8 (10, 11) and identified 6,368 coding sequences. Default parameters were used for all software, unless otherwise specified.
To examine the antibiotic resistance profile of BWH047, antibiotic resistance genes were identified using ResFinder v3.1.0 (12) and the CARD database v3.0.1 (13). We identified two β-lactamase genes, blaOXA-488 and PDC-2 (14). Also identified were two aminoglycoside resistance genes [aph(3′)-IIb and ant(2″)-Ia], a single nucleotide insertion in oprD resulting in a frameshift in the C-terminal portion of the gene likely conferring carbapenem resistance, mutations in gyrA (T83I) and parC (S87L) conferring fluoroquinolone resistance, one fosfomycin resistance gene (fosA), one phenicol resistance gene (catB7), one sulfonamide resistance gene (sul1), and one trimethoprim resistance gene (dfrA10). No canonical colistin resistance mutations were observed (15), and homology to plasmid-borne colistin resistance genes mcr1 to mcr9 was not detected (16, 17). All identified resistance genes had a nucleotide identity of 97.47 to 100% over 85 to 100% of the reference gene lengths.
Data availability.
Illumina sequencing reads were deposited in the NCBI Sequence Read Archive (SRA) with accession numbers PRJNA533806 and SRR9109023. This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number SUPJ00000000. The version described in this paper is version SUPJ01000000.
ACKNOWLEDGMENTS
We acknowledge the Brigham and Women’s Hospital (BWH) clinical microbiology laboratory for assistance in collecting patient samples under institutional review board (IRB) protocol 2015-P-001829.
This research was supported in part through the computational resources and staff contributions provided by the Genomics Compute Cluster at Northwestern University, which is jointly supported by the Feinberg School of Medicine, the Center for Genetic Medicine, Feinberg’s Department of Biochemistry and Molecular Genetics, the Office of the Provost, the Office for Research, and Northwestern Information Technology. The Genomics Compute Cluster is part of Quest, Northwestern University’s high-performance computing facility, which serves to advance research in genomics.
This work was supported by the American Cancer Society (grants 130602-PF-17-107-01-MPC to K.E.R.B. and MRSG-13-220-01 to E.A.O.) and the National Institute of Allergy and Infectious Diseases (NIAID) (grant T32 AI007061-36A1 to K.E.R.B. and grants RO1 AI118257, U19 AI135964, and R21 129167 to A.R.H.).
REFERENCES
- 1.Vincent JL. 2003. Nosocomial infections in adult intensive-care units. Lancet 361:2068–2077. doi: 10.1016/S0140-6736(03)13644-6. [DOI] [PubMed] [Google Scholar]
- 2.Peña C, Suarez C, Gozalo M, Murillas J, Almirante B, Pomar V, Aguilar M, Granados A, Calbo E, Rodriguez-Bano J, Rodriguez F, Tubau F, Martinez-Martinez L, Oliver A, Spanish Network for Research in Infectious Diseases (REIPI) . 2012. Prospective multicenter study of the impact of carbapenem resistance on mortality in Pseudomonas aeruginosa bloodstream infections. Antimicrob Agents Chemother 56:1265–1272. doi: 10.1128/AAC.05991-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Cabot G, Ocampo-Sosa AA, Domínguez MA, Gago JF, Juan C, Tubau F, Rodríguez C, Moyà B, Peña C, Martínez-Martínez L, Oliver A, Spanish Network for Research in Infectious Diseases (REIPI) . 2012. Genetic markers of widespread extensively drug-resistant Pseudomonas aeruginosa high-risk clones. Antimicrob Agents Chemother 56:6349–6357. doi: 10.1128/AAC.01388-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Oliver A, Mulet X, López-Causapé C, Juan C. 2015. The increasing threat of Pseudomonas aeruginosa high-risk clones. Drug Resist Updat 21–22:41–59. doi: 10.1016/j.drup.2015.08.002. [DOI] [PubMed] [Google Scholar]
- 5.Clinical and Laboratory Standards Institute. 2018. Performance standards for antimicrobial susceptibility testing: 28th informational supplement. CLSI document M100-S28. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 6.Wiegand I, Hilpert K, Hancock RE. 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3:163–175. doi: 10.1038/nprot.2007.521. [DOI] [PubMed] [Google Scholar]
- 7.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]
- 8.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]
- 9.Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. doi: 10.1093/bioinformatics/btp324. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. Nucleic Acids Res 44:6614–6624. doi: 10.1093/nar/gkw569. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V, O’Neill K, Li W, Chitsaz F, Derbyshire MK, Gonzales NR, Gwadz M, Lu F, Marchler GH, Song JS, Thanki N, Yamashita RA, Zheng C, Thibaud-Nissen F, Geer LY, Marchler-Bauer A, Pruitt KD. 2018. RefSeq: an update on prokaryotic genome annotation and curation. Nucleic Acids Res 46:D851–D860. doi: 10.1093/nar/gkx1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.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]
- 13.Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, Lago BA, Dave BM, Pereira S, Sharma AN, Doshi S, Courtot M, Lo R, Williams LE, Frye JG, Elsayegh T, Sardar D, Westman EL, Pawlowski AC, Johnson TA, Brinkman FS, Wright GD, McArthur AG. 2017. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res 45:D566–D573. doi: 10.1093/nar/gkw1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Rodríguez-Martínez JM, Poirel L, Nordmann P. 2009. Extended-spectrum cephalosporinases in Pseudomonas aeruginosa. Antimicrob Agents Chemother 53:1766–1771. doi: 10.1128/AAC.01410-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.López-Causapé C, Cabot G, Del Barrio-Tofino E, Oliver A. 2018. The versatile mutational resistome of Pseudomonas aeruginosa. Front Microbiol 9:685. doi: 10.3389/fmicb.2018.00685. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Liu Y-Y, Wang Y, Walsh TR, Yi L-X, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, Yu L-F, Gu D, Ren H, Chen X, Lv L, He D, Zhou H, Liang Z, Liu J-H, Shen J. 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 16:161–168. doi: 10.1016/S1473-3099(15)00424-7. [DOI] [PubMed] [Google Scholar]
- 17.Carroll LM, Gaballa A, Guldimann C, Sullivan G, Henderson LO, Wiedmann M. 2019. Identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible Salmonella enterica serotype Typhimurium isolate. mBio 10:e00853-19. doi: 10.1128/mBio.00853-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
Illumina sequencing reads were deposited in the NCBI Sequence Read Archive (SRA) with accession numbers PRJNA533806 and SRR9109023. This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number SUPJ00000000. The version described in this paper is version SUPJ01000000.