Complete Genome Sequence of Pseudomonas aeruginosa CMC-115, a Clinical Strain from an Acute Ventilator-Associated Pneumonia Patient

We report the complete genome of clinical strain Pseudomonas aeruginosa CMC-115, which was isolated from an acute ventilator-associated pneumonia patient. Illumina sequencing reads were assembled using Geneious to yield a 6,375,262-bp circular chromosome that exhibited an unusual ferrichrome receptor in the pyoverdine synthesis locus and the absence of type 3 secretion system genes.

P seudomonas aeruginosa is an increasingly common opportunistic pathogen causing infection in immunocompromised and hospitalized patients, leading to health care-associated pneumonia. Patients on ventilators are particularly vulnerable; P. aeruginosa invades the lower respiratory tract and leads to ventilator-associated pneumonia (VAP), increasing the risk of death by as much as 30% in intensive care units (1)(2)(3). The complete genome of a Pseudomonas strain from an acute VAP case provides useful information for identifying novel virulence factors and antimicrobial resistance (AMR) genes.
A prospective study was conducted in 2010 to 2012 with approval from the Carilion Clinic Institutional Review Board. In that study, CMC-115 was obtained from the Quest Diagnostics Microbiology Laboratory at Carilion Clinic. The strain was isolated from a tracheal aspirate sample from an acute VAP patient, and its identity was confirmed by biochemical tests. Antimicrobial susceptibility testing was performed using a Vitek 2 automated system (bioMérieux) according to Clinical and Laboratory Standards Institute guidelines (4). The isolate was grown on a blood agar plate and transported to the Carilion Basic Science Laboratory (CBSL); glycerol stocks were prepared and stored in a Ϫ80°C freezer. The patient was treated at Carilion Roanoke Memorial Hospital and survived.
A single colony of CMC-115 was grown for 18 h in 25 ml lysogeny broth at 37°C, at 200 rpm. The cell pellet was used for genomic DNA isolation with a Genomic-tip 20/G (Qiagen) (5). The genome was sequenced on the Illumina NextSeq platform at the Virginia Tech Genomics Resource Center, with a library constructed using the Illumina TruSeq DNA preparation kit. Sequencing generated 68,209,004 paired-end reads with a length of 150 bp, which were assembled with the Geneious v11.0.4 (Biomatters, Ltd.) de novo algorithm to produce 40 contigs with a length of Ͼ1,000 bp and an N 50 value of 423,776 bp. The Geneious map to reference algorithm, with fine tuning set to iterate up to 10 times, was then used to iteratively map reads to the ends of the 40 contigs until the contigs overlapped and closed into a circular genome. A final use of the Geneious map to reference algorithm aligned Ͼ95% of the reads to the complete circular genome, with a uniform average coverage of 1,500ϫ. All Geneious assemblies and alignments were performed with the default low-sensitivity/fastest settings, which allow at most 10% base mismatches. The complete assembly of CMC-115 resulted in a circular genome of 6,375,262 bp with a GϩC content of 66.4%. The NCBI Prokaryotic Genome Annotation Pipeline v4.2 (6, 7) identified 5,930 genes, including 5,850 protein-coding genes and 80 RNA genes (64 tRNAs, 12 rRNAs, and 4 noncoding RNAs), along with 68 pseudogenes and 2 CRISPR arrays.
Data availability. The annotated complete genome assembly of strain Pseudomonas aeruginosa CMC-115 is available in GenBank under accession numbers CP046602, SRR11592953, PRJNA593818, and SAMN13451386.

ACKNOWLEDGMENTS
We acknowledge the faculty members of the Division of Infectious Disease, Carilion Clinic, and the Carilion Clinical Microbiology Laboratory for their assistance in collecting patient samples in the Carilion Clinic institutional review board-approved study. Experiments were carried out at the CBSL and Carilion Roanoke Community Hospital. We are thankful to Sarah Cox, Radford University Carilion, for her valuable support in manuscript reading and suggestions.
This study was supported by a Carilion Clinic grant to D.C.G. and J.R., a Graduate Student Research Supply grant to A.A. was funded by the Virginia Polytechnic Institute and State University (VT), and the Illumina sequencing was completed with the support of the Fralin Life Science Institute, VT, to R.V.J.