Emergence of Carbapenem-Resistant ST244, ST292, and ST2446 Pseudomonas aeruginosa Clones in Burn Patients in Yunnan Province

Abstract

Introduction: The prevalence of carbapenem-resistant Pseudomonas aeruginosa is increasing persistently, particularly in burn ward isolates. Here, we investigate the prevalence of carbapenem-resistant Pseudomonas aeruginosa in a burn ward of a provincial-level hospital at Kunming, Yunnan province, China.

Methods: A total of 118 P. aeruginosa strains were isolated from 57 hospitalized patients, and their MICs were measured. Carbapenem-resistant isolates were selected for multilocus sequence typing (MLST). Carbapenem-resistance mechanisms were identified by examining carbapenemase genes and OprD protein and Carba-NP testing. Representative isolates were further characterized by de novo sequencing for carbapenemase molecular background.

Results: Among 118 P. aeruginosa isolates, 54 (54/118,45.8%) were carbapenem-resistant Pseudomonas aeruginosa, and 3 genotypes were found (ST292, ST244, and ST2446). Non-carbapenemase-producing ST292 was the most prevalent ST, followed by ST2446 and ST244. A novel 13-bp oprD deletion was found in the ST292 clone, which formed the truncated outer membrane protein and may cause carbapenem resistance. ST244 and ST2446 harbored blaIMP-45 and blaIMP-87, respectively. blaIMP-45 is located in a megaplasmid, together with aac(6')-Ib3, blaOXA-1, catB3, qnrVC6, armA, msr(E), mph(E), aph(3')-Ia, tetC/tetR, aac(6')-Ib3, floR, mexC-mexD-oprJ, fosA and lead to extensive drug resistance. ST2446 contains a carbapenem-resistant gene blaIMP-87 on the chromosome and is acquired by a novel gene cassette array (blaIMP-87-ant(2")-Ia-blaOXA-10-aac(6')-Ib3) of class 1 integron.

Discussion: For the first time, ST244, ST292 and ST2446 are reported emerging in burn patients, with distinctive carbapenem-resistance mechanisms, respectively. The obtained results highlight the need to surveillance carbapenem-resistant isolates in burn patients.

Keywords: P. aeruginosa; blaIMP; carbapenem-resistance; oprD.

Figure 1

The OprD mutations of KB_ST292 (ST292 clones in Kunming burn ward) and compared with PAO1. (A) Deletion of 1088_1100del13 leads to a frame-shift mutation and early termination at 1050–1052, *=Terminor; (B) Locations of amino acid substitutions (14 sites) and a 12-amino acid mutation.

Figure 2

Out membrane profiling determined by SDS-PAGE; PAO1, reference P. aeruginosa strain; ST292, ST244, ST2446, representative clinical isolates. M, molecular size marker (Thermo). The arrow on the upper right indicates the banding position of OprD.

Figure 3

Mauve alignment for chromosomes of KB-PA_3, PAO1 and KB-PA_F19; Chromosomes shows locally colinear blocks of matched colored regions. The gap between locally colinear blocks shows the additional regions.

Figure 4

Phylogenetic tree of blaIMP-87 and blaIMP-45 with 31 sub-types of blaIMP; Phylogenetic tree was constructed based on neighbor-Joining by MEGA 6.0. blaIMP-45 is labeled with blue and blaIMP-87 with red.

Figure 5

Schematic representation of blaIMP-87 gene context of KB-PA_3, and intI1 comparison between of pEC732-IMP, KB-PA_3 and S27-KUU; Open arrows indicate coding sequences and direction of transcription. Resistance genes (Red); transposon module (yellow); integron module (sky blue); insertion sequence of IS1017 (green). S27-KUU only reported the gene cassette array, and we add the possible integron module in the gray box. Shaded areas between the genetic elements indicate homology (≥95% identity).

Figure 6

Resistance region comparison between pKB-PA_F19-4 and pBM413; Open arrows indicate coding sequences and direction of transcription. Resistance genes (Red); transposon module (yellow); integron module (sky blue); repM and DNA-binding protein gene (dark blue). Shaded areas between the genetic elements indicate homology (≥95% identity).