Nationwide genome surveillance of carbapenem-resistant Pseudomonas aeruginosa in Japan

Abstract

Japan is a country with an approximate 10% prevalence rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Currently, a comprehensive overview of the genotype and phenotype patterns of CRPA in Japan is lacking. Herein, we conducted genome sequencing and quantitative antimicrobial susceptibility testing for 382 meropenem-resistant CRPA isolates that were collected from 78 hospitals across Japan from 2019 to 2020. CRPA exhibited susceptibility rates of 52.9%, 26.4%, and 88.0% against piperacillin-tazobactam, ciprofloxacin, and amikacin, respectively, whereas 27.7% of CRPA isolates was classified as difficult-to-treat resistance P. aeruginosa. Of the 148 sequence types detected, ST274 (9.7%) was predominant, followed by ST235 (7.6%). The proportion of urine isolates in ST235 was higher than that in other STs (P = 0.0056, χ² test). Only 4.1% of CRPA isolates carried the carbapenemase genes: bla_GES (2) and bla_IMP (13). One ST235 isolate carried the novel bla_IMP variant bla_IMP-98 in the chromosome. Regarding chromosomal mutations, 87.1% of CRPA isolates possessed inactivating or other resistance mutations in oprD, and 28.8% showed mutations in the regulatory genes (mexR, nalC, and nalD) for the MexAB-OprM efflux pump. Additionally, 4.7% of CRPA isolates carried a resistance mutation in the PBP3-encoding gene ftsI. The findings from this study and other surveillance studies collectively demonstrate that CRPA exhibits marked genetic diversity and that its multidrug resistance in Japan is less prevailed than in other regions. This study contributes a valuable data set that addresses a gap in genotype/phenotype information regarding CRPA in the Asia-Pacific region, where the epidemiological background markedly differs between regions.

Keywords: DTR-P. aeruginosa; IMP; SE; carbapenemase; integron.

Fig 1

(A) Minimum spanning tree of 382 meropenem-resistant CRPA isolates based on MLST profiles. Each node represents ST, otherwise clonal group (CG). STs with one allele difference were collapsed into one node as CG. Node size is proportional to the number of isolates. The number on the branch denotes the number of different alleles at the 7 MLST loci between two nodes. (B) Proportions of 10 globally most common CGs in Japan and other regions. Proportions of CGs in the USA, China, and South and Central America are based on Reyes et al.’s report (8).

Fig 2

Venn diagram showing the simultaneous occurrence of resistance mutations in meropenem-resistant P. aeruginosa. Numbers in circles represent the numbers of isolates having an acquired carbapenemase, a point mutation registered in AMRFinderPlus (30), or inactivating mutations. The “Mex” category indicates the presence of a mutation in at least one of three loci (mexR, nalC, and nalD) encoding a negative regulator for the mexAB operon. The “PBP3” category indicates the presence of a mutation in ftsI. The acquired carbapenemase/MBLs detected are GES-5, IMP-1, IMP-10, IMP-7, IMP-34, and IMP-98.

Fig 3

(A) Alignment of carboxyl-terminal regions of IMP variants. H, helix; e, sheet. IMP-98 differs from IMP-1 only at P213 indicated by an arrowhead. (B) Genetic context of bla_IMP-98. tni genes in green are associated with the transposition of the Tn402-related element. tniA* indicates disrupted tniA. tnpA and tnpR genes are associated with the transposition of the TnAs3-related transposon. The left four genes in the insertion in JBBCAEG-19-0032 are SE core genes: intA, encoding tyrosine recombinase; tfp, tyrosine recombinase fold protein; intB, large tyrosine recombinase; srap, SE-associated recombination auxiliary protein (32). bla_OXA-10 and aac(6′)-Ib in integron overlapped by 23 bp. intI and attC were identified by IntegronFinder2.0 (33). (C) Comparison between TnAs3 and the TnAs3-related transposon in the JBBCAEG-19-0032 chromosome. Integron-associated genes are shown in light-green. attI and attC sites were not found in TnAs3. mer genes in purple are mercury resistance genes. (D) Similarity of SE core genes between SE-PaeJB0032 and SE-6945 (34). The number indicates percentage identity in BLASTp. (E) Comparison of border regions between SE-PaeJB0032 and chromosome (attL, attR) with unoccupied integration site (attB) in PAO1. Sequences in orange are putative SE regions. The underlined sequence is a putative 6-bp footprint generated by SE integration (32, 34). Sequences are obtained from the following accession numbers: JBBCAEG-19-0032 chromosome, AP029374; PAO1 chromosome, NC_002516.2; pSEA1, AP024167; and TnAs3, CP000645.