Abstract
Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital-acquired infections, is grouped as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen because of its extensive drug resistance phenotypes and effects on human health worldwide. Five multidrug resistant P. aeruginosa strains isolated from wounded military personnel were sequenced and annotated in this work.
GENOME ANNOUNCEMENT
Pseudomonas aeruginosa is a common environmental Gram-negative bacillus bacterium often associated with nosocomial infections including chronic lung infections in cystic fibrosis patients and bacteremia in burn victims. Human infections with P. aeruginosa can likely be traced back to 1862 when Luke observed rod-shaped particles in the blue-green pus of infections allowing this bacterium the opportunity to develop into a formidable human pathogen (1). Nosocomial pathogens, such as P. aeruginosa, have developed sophisticated resistance mechanisms since the introduction of antibiotics into the clinical setting (2). P. aeruginosa is currently the second most prevalent Gram-negative nosocomial pathogen preceded by Escherichia coli with as many as 2% of P. aeruginosa isolates specifically presenting with carbapenem-resistance (3). P. aeruginosa is referred to as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen due to its ability to escape the lethal action of antibiotics (4). In order to develop a broader understanding of the mechanisms by which nosocomial P. aeruginosa strains escape death by antibiotics, the genome sequences of five P. aeruginosa strains isolated from wounded soldiers at the Walter Reed Army Medical Center (WRAMC) were determined using next-generation sequencing methods for future bioinformatic analyses.
Strains routinely stored at −80°C in 10% glycerol (5) were used to isolate total DNA from overnight LB cultures grown with agitation at 37°C using the DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA). Absorption at 260 nm and 280 nm was measured for each sample to determine quantity and quality using the Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA). DNA concentrations for library preparation were determined by the SYBR green (Life Technologies, Grand Island, NY, USA) standard curve method in black 96-well plates (Corning, Tewksbury, MA, USA) using a FilterMax F5 spectrophotometer with Multi-Mode Analysis software version 3.4.0.25 (Molecular Devices, Sunnyvale, CA, USA). Whole DNA was sheared to approximately 500 bp in microTUBE-50 using M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). Fragmentation of resultant libraries was examined with a Bioanalyzer 2100 High Sensitivity DNA analysis kit (Agilent Technologies, Santa Clara, CA, USA) using version B.02.08.SI648 software. Individual libraries were normalized, pooled, and then sequenced using MiSeq v3 600-cycle kit (Illumina, San Diego, CA, USA) to perform 300-bp paired-end sequencing on a MiSeq instrument (Illumina) per manufacturer’s instructions. De novo assembly was performed using Genomics Workbench 8.0 with the bacterial genome finishing module (CLC bio, Boston, MA, USA) on a workstation with an AMD Opteron 2.10 GHz 16-core processor with 128 GB DDR3 ECC RAM. Genomes were annotated with Prokka version 1.10 on a quadcore i7 workstation with 32 GB DDR3 running Ubuntu 14.04 LTS (6). The de novo assembly statistics for the five P. aeruginosa sequenced isolates are shown in Table 1.
TABLE 1 .
Assembly metrics and accession numbers of Pseudomonas aeruginosa genomes
| Strain ID | No. of contigs | N50 contigs (bp) | Total size (bp) | Coverage (×) | G+C content (%) | No. of ORFsa | No. of RNAs | Accession no. |
|---|---|---|---|---|---|---|---|---|
| 105777 | 105 | 179,475 | 7,408,561 | 30 | 65.33 | 7,012 | 67 | LODH00000000 |
| 105819 | 63 | 302,533 | 7,208,927 | 26 | 65.65 | 6,703 | 68 | LOHH00000000 |
| 105880 | 86 | 215,191 | 6,914,271 | 17 | 65.98 | 6,490 | 60 | LOHI00000000 |
| 105857 | 93 | 304,460 | 6,933,765 | 27 | 65.99 | 6,563 | 67 | LOHJ00000000 |
| 105738 | 137 | 102,664 | 6,783,146 | 39 | 66.06 | 6,269 | 67 | LOHK00000000 |
Open reading frames.
Accession number(s).
The whole-genome shotgun projects were deposited into GenBank under Bioproject ID PRJNA261239 with accession numbers listed in Table 1.
ACKNOWLEDGMENTS
This work was supported by funds from Miami University and the United States Department of Defense grant W81XWH-12-2-0035 awarded to L.A.A.
We are grateful to Daniel V. Zurawski from Walter Reed Army Institute of Research for providing the P. aeruginosa strains listed in Table 1. We would also like to thank Andor Kiss and the Miami University Center for Bioinformatics and Functional Genomics for assistance in sequence acquisition.
The findings and opinions expressed herein belong to the authors and do not necessarily reflect the official views of the WRAIR, the U.S. Army, or Department of Defense.
Footnotes
Citation Arivett BA, Ream DC, Fiester SE, Kidane D, Actis LA. 2016. Draft genome sequences of Pseudomonas aeruginosa isolates from wounded military personnel. Genome Announc 4(4):e00829-16. doi:10.1128/genomeA.00829-16.
REFERENCES
- 1.Lyczak JB, Cannon CL, Pier GB. 2000. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2:1051–1060. doi: 10.1016/S1286-4579(00)01259-4. [DOI] [PubMed] [Google Scholar]
- 2.Breidenstein EB, de la Fuente-Núñez C, Hancock RE. 2011. Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol 19:419–426. doi: 10.1016/j.tim.2011.04.005. [DOI] [PubMed] [Google Scholar]
- 3.Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK, National Healthcare Safety Network Team, Participating National Healthcare Safety Network Facilities . 2008. Antimicrobial-resistant pathogens associated with health care-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect Control Hosp Epidemiol 29:996–1011. doi: 10.1086/591861. [DOI] [PubMed] [Google Scholar]
- 4.Rice L. 2008. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis 197:1079–1081. doi: 10.1086/533452. [DOI] [PubMed] [Google Scholar]
- 5.Arivett BA, Ream DC, Fiester SE, Mende K, Murray CK, Thompson MG, Kanduru S, Summers AM, Roth AL, Zurawski DV, Actis LA. 2015. Draft genome sequences of Klebsiella pneumoniae clinical type strain ATCC 13883 and three multidrug-resistant clinical isolates. Genome Announc 3(1):e01385-01314. doi: 10.1128/genomeA.01385-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. BioInformatics 30:2068–2069. doi: 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]