Draft Genome Sequence of a Pseudomonas sp. Strain Carrying bla_IMP-25 and bla_VIM-2 Carbapenemase Genes from Hospital Sewage

GENOME ANNOUNCEMENT

The genus Pseudomonas comprises 231 species (http://www.bacterio.net/pseudomonas.html), many of which are important opportunistic pathogens for plants and animals. Pseudomonas sp. strain WCHP16 was recovered from the influx mainstream of the wastewater treatment plant in West China Hospital, Chengdu, western China, in January 2015. Strain WCHP16 exhibited high-level resistance to meropenem (MIC, >512 µg/ml). Screening for acquired carbapenem-hydrolyzing β-lactamase (carbapenemase) genes bla_GES (including noncarbapenemase variants), bla_IMP, bla_IMI, bla_KPC, bla_NDM, bla_OXA-48-like, and bla_VIM was performed using PCR as described previously (1–4). Strain WCHP16 was positive to both bla_IMP and bla_VIM and was therefore subjected to whole-genome sequencing.

Genomic DNA of strain WCHP16 was prepared using the QIAamp DNA minikit (Qiagen, Hilden, Germany) and then was sequenced using the Hiseq 2500 Sequencer (Illumina, San Diego, CA, USA) with the 150-bp paired-end protocol and 100× coverage. A total of 4,244,316 reads and 636,647,400 clean bases were generated. The Spades program (version 2.8) (5) was used for de novo assembly and generated 141 contigs ≥1,000 bp in length (N₅₀, 111,079 bp) with a 59.53% G+C content. The genome size was about 5.7 Mb. Annotation of the genomic sequence was carried out using the Prokka program (version 1.11) (6). The genome of strain WCHP16 contained 5,504 coding sequences and 67 tRNA genes. Antimicrobial resistance genes were predicted using ResFinder from the Center for Genomic Epidemiology (http://genomicepidemiology.org/). Strain WCHP16 had two carbapenemase genes, i.e., bla_IMP-25 and bla_VIM-2. bla_IMP-25 was a variant of bla_IMP-1 and was first found in a Pseudomonas aeruginosa from South Korea (GenBank accession number EU541448). bla_IMP-25 was rarely reported and had only been identified in P. aeruginosa in China and South Korea before (7). Furthermore, to our knowledge, the co-existence of bla_IMP-25 and bla_VIM-2 in a single strain has not been reported before.

Other antimicrobial resistance genes identified in strain WCHP16 included aacA4 (mediating resistance to aminoglycosides), arr-2 (to rifampin), bla_OXA-1 (to penicillins), catB3 (to phenicol), dfrA22 and dfrB1 (to trimethoprim), qnrVC1 (to quinolones), and sul1 (to sulfonamides).

The 16S rRNA gene sequence of strain WCHP16 had the highest identity with those of Pseudomonas sp. 5 (GenBank accession number NZ_JYOC00000000; 99.48% identity), Pseudomonas putida UASWS0946 (NZ_JXOG00000000; 99.35% identity), and Pseudomonas sp. NBRC 111117 (NZ_BCAT00000000; 99.35% identity).

Average nucleotide identity (ANI) has been increasingly employed to determine bacterial species and the ≥95% ANI cutoff has been proposed to define Pseudomonas species (8, 9) previously. The ≥95% ANI cutoff corresponds to the ≥70% DNA-DNA hybridization value for defining a bacterial species (10). Pair-wise ANI between strain WCHP16 and the three Pseudomonas strains was therefore determined using the JSpecies web program (http://imedea.uib-csic.es/jspecies/) with the default settings (11). The genome of strain WCHP16 shared only 79.99% to 81.06% ANI with the three Pseudomonas strains, which were far below the ≥95% ANI cutoff. The ANI results suggest that strain WCHP16 is likely to belong to a new species of the Pseudomonas genus.