Complete Genome Sequence of Highly Multidrug-Resistant Pseudomonas aeruginosa NCGM2.S1, a Representative Strain of a Cluster Endemic to Japan

Abstract

We report the completely annotated genome sequence of Pseudomonas aeruginosa NCGM2.S1, a representative strain of a cluster endemic to Japan with a high level of resistance to carbapenem (MIC ≥ 128 μg/ml), amikacin (MIC ≥ 128 μg/ml), and fluoroquinolone (MIC ≥ 128 μg/ml).

GENOME ANNOUNCEMENT

The emergence of multidrug-resistant (MDR) P. aeruginosa strains is a serious problem in Japan (2, 6, 7). The MDR P. aeruginosa strain NCGM2.S1 caused an outbreak of urinary tract infection at a hospital in Miyagi Prefecture, northern Japan (6). NCGM2.S1 harbors a metallo-β-lactamase gene, blaIMP-1, and an aminoglycoside 6′-N-acetyltransferase gene, aac(6′)-Iae, in the class I integron In113 (6). Epidemiological studies indicated that clonal expansion of NCGM2.S1 occurred in hospitals in this area (7) as well as other areas in Japan (4, 8). We developed kits to detect aac(6′)-Iae and AAC(6′)-Iae, which were used to survey MDR P. aeruginosa strains (3, 7).

The genome of P. aeruginosa was sequenced using a Roche FLX Titanium genome sequencer. We obtained a total of 532,063 reads, covering a total of 6,697,230 bp, or 28.9-fold coverage. Sequences were assembled into a total of 270 contigs. Gaps were filled by Sanger sequencing of PCR products by brute force amplification of the regions between contigs. Primary CDS extraction and initial functional assignment were performed using the RAST automated annotation servers (1). The results were compared to verify the annotation and were corrected manually by in silico molecular cloning (In Silico Biology, Inc., Kanagawa, Japan). The P. aeruginosa NCGM2.S1 genome consists of a single circular chromosome of 6,764,661 bp, with an average GC content of 66.1%. The chromosome was shown to contain a total of 6,271 protein-coding genes, 77 tRNA genes, 1 tmRNA for all amino acids, and 4 rrn operons. In addition, the chromosome harbors 6 prophage-like elements.

Although P. aeruginosa NCGM2.S1 is a representative strain of an endemic cluster showing a high level of multidrug resistance in Japan, it does not have any plasmids. Instead, the chromosome was shown to harbor the class I integron In113 carrying aac(6′)-Iae and blaIMP-1, which are responsible for high levels of resistance to aminoglycosides and β-lactams, respectively. Of note, In113 is inserted into the middle of oprD, resulting in complete disruption of the gene. OprD is responsible for sensitivity to imipenem, and its reduced expression increases the level of resistance (5). Analysis of the complete NCGM2.S1 genome sequence strongly suggested that P. aeruginosa acquires drug resistance not only by obtaining drug resistance genes but also by disrupting the genes involved in drug sensitivity.

Nucleotide sequence accession number.

The nucleotide sequence of the chromosome of P. aeruginosa NCGM2.S1 has been deposited in the DNA Database of Japan under accession no. AP012280.