Abstract
Acinetobacter baumannii is a Gram-negative bacterium capable of causing hospital-acquired infections that has been grouped with Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species as ESKAPE pathogens because of their extensive drug resistance phenotypes and increasing risk to human health. Twenty-four multidrug-resistant A. baumannii strains isolated from wounded military personnel were sequenced and annotated.
GENOME ANNOUNCEMENT
The Gram-negative coccobacillus Acinetobacter baumannii is an opportunistic human pathogen causing myriad human diseases, including pneumonia, bacteremia, urinary tract infections, meningitis, and wound infections. A. baumannii is the fifth most common Gram-negative pathogen associated with nosocomial infections (1, 2). Of concern is the increasing multidrug resistance of A. baumannii isolates, which has caused this bacterium to be included as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen, underscoring its ability to “escape” antimicrobials (3). In fact, A. baumannii strains resistant to all known antibiotics have been encountered, demonstrating the paramount impact of this pathogen on public health (2). The genomes of 24 A. baumannii strains isolated from wounded warriors at Walter Reed Army Medical Center (WRAMC) and San Antonio Military Medical Center (SAMMC), Fort Sam Houston, San Antonio, TX, were sequenced using next-generation sequencing for future analyses to investigate the resistance and virulence mechanisms of this emerging pathogen.
As described previously, strains were routinely stored at -80°C in 10% glycerol (4). DNA was isolated 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) standard curve method in a black 96-well plate (Corning, Tewksbury, MA, USA) using a FilterMax F5 spectrophotometer with multimode analysis software version 3.4.0.25 (Molecular Devices, Sunnyvale, CA, USA). Whole DNA was sheared to approximately 500 bp in a microTUBE-50 using an M220 focused ultrasonicator (Covaris, Woburn, MA, USA). Fragmentation of the 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 the MiSeq version 3 600-cycle kit (Illumina, San Diego, CA, USA) to perform 300-bp paired-end sequencing on a MiSeq instrument (Illumina), per the manufacturer’s instructions. De novo assembly was performed using Genomics Workbench 8.0 with the Bacterial Genome Finishing module (CLC bio, Boston, MA, USA) run on a workstation with an AMD Opteron 2.10 GHz 16-core processor with 128 Gb DDR3 ECC random access memory (RAM). Genomes were annotated with Prokka version 1.10 on a quad-core i7 workstation with 32 Gb DDR3 running Ubuntu 14.04 LTS (5). The de novo assembly statistics for 24 A. baumannii isolates are shown in Table 1.
TABLE 1 .
Assembly metrics and accession numbers of A. baumannii genomes
| Strain ID | No. of contigs | N50 contig size (bp) | Total size (bp) | Coverage (×) | G+C content (%) | No. of ORFsa | No. of RNAs | Accession no. |
|---|---|---|---|---|---|---|---|---|
| AB2828 | 107 | 124,070 | 4,426,896 | 30 | 39.21 | 4,274 | 53 | LRDT00000000 |
| AB3340 | 76 | 132,604 | 4,010,248 | 28 | 38.86 | 3,864 | 49 | LRDU00000000 |
| AB3560 | 58 | 247,914 | 4,012,126 | 30 | 38.92 | 3,894 | 59 | LRDV00000000 |
| AB967 | 27 | 401,652 | 3,795,032 | 29 | 38.84 | 3,633 | 62 | LRDS00000000 |
| AB3785 | 70 | 134,647 | 3,894,584 | 29 | 39.01 | 3,745 | 58 | LRDX00000000 |
| AB3638 | 78 | 108,414 | 4,294,582 | 31 | 38.72 | 4,113 | 62 | LRDW00000000 |
| AB3806 | 86 | 96,852 | 4,295,294 | 33 | 38.75 | 4,117 | 59 | LRDY00000000 |
| AB3927 | 45 | 227,995 | 4,113,781 | 30 | 38.82 | 3,978 | 58 | LRDZ00000000 |
| AB4026 | 67 | 160,728 | 3,905,198 | 30 | 38.99 | 3,749 | 50 | LREB00000000 |
| AB4027 | 72 | 152,887 | 3,903,961 | 32 | 39.00 | 3,749 | 54 | LREC00000000 |
| AB4025 | 69 | 152,887 | 3,902,672 | 29 | 39.00 | 3,741 | 55 | LREA00000000 |
| AB4456 | 58 | 182,799 | 4,001,807 | 27 | 38.92 | 3,857 | 47 | LREF00000000 |
| AB4052 | 43 | 262,160 | 3,921,338 | 33 | 39.00 | 3,739 | 51 | LRED00000000 |
| AB4448 | 43 | 369,360 | 3,992,257 | 28 | 38.92 | 3,854 | 58 | LREE00000000 |
| AB4490 | 98 | 84,980 | 3,947,403 | 31 | 38.99 | 3,786 | 60 | LREG00000000 |
| AB4498 | 76 | 128,212 | 3,905,177 | 32 | 39.00 | 3,753 | 57 | LREH00000000 |
| AB4795 | 78 | 113,293 | 3,882,341 | 33 | 39.03 | 3,727 | 62 | LREI00000000 |
| AB4878 | 45 | 223,470 | 3,862,567 | 26 | 38.98 | 3,685 | 50 | LREJ00000000 |
| AB4957 | 50 | 223,470 | 3,882,040 | 33 | 38.97 | 3,722 | 60 | LREL00000000 |
| AB4932 | 39 | 237,199 | 3,865,974 | 33 | 38.99 | 3,703 | 60 | LREK00000000 |
| AB5001 | 33 | 223,470 | 3,789,469 | 30 | 38.99 | 3,586 | 52 | LREN00000000 |
| AB4991 | 52 | 310,788 | 3,877,107 | 28 | 39.09 | 3,686 | 58 | LREM00000000 |
| AB5674 | 34 | 419,504 | 3,869,253 | 29 | 39.03 | 3,679 | 52 | LREP00000000 |
| AB5197 | 58 | 184,472 | 3,959,484 | 33 | 39.04 | 3,799 | 58 | LREO00000000 |
ORFs, 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 U.S. Department of Defense W81XWH-12-2-0035 award to L.A.A.
We are grateful to Daniel V. Zurawski from Walter Reed Army Institute of Research for providing the A. baumannii strains listed in Table 1. We also 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 the Department of Defense.
Footnotes
Citation Arivett BA, Ream DC, Fiester SE, Kidane D, Actis LA. 2016. Draft genome sequences of Acinetobacter baumannii isolates from wounded military personnel. Genome Announc 4(4):e00773-16. doi:10.1128/genomeA.00773-16.
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