Molecular Characterization of Carbapenemase-Producing Pseudomonas aeruginosa of Czech Origin and Evidence for Clonal Spread of Extensively Resistant Sequence Type 357 Expressing IMP-7 Metallo-β-Lactamase

Abstract

The objective of this study was to perform molecular surveillance for assessing the spread of carbapenemase-producing Pseudomonas aeruginosa in Czech hospitals. One hundred thirty-six carbapenemase-producing isolates were recovered from 22 hospitals located throughout the country. Sequence type 357 (ST357) dominated (n = 120) among carbapenemase producers. One hundred seventeen isolates produced IMP-type (IMP-7 [n = 116] and IMP-1 [n = 1]) metallo-β-lactamases (MβLs), 15 produced the VIM-2 MβL, and the remaining isolates expressed the GES-5 enzyme. The bla_IMP-like genes were located in three main integron types, with In-p110-like being the most prevalent (n = 115). The two other IMP-encoding integrons (In1392 and In1393) have not been described previously. bla_VIM-2-carrying integrons included In59-like, In56, and a novel element (In1391). bla_GES-5 was carried by In717. Sequencing data showed that In-p110-like was associated with a Tn4380-like transposon inserted in genomic island LESGI-3 in the P. aeruginosa chromosome. The other integrons were also integrated into the P. aeruginosa chromosome. These findings indicated the clonal spread of ST357 P. aeruginosa, carrying the IMP-7-encoding integron In-p110, in Czech hospitals. Additionally, the sporadic emergence of P. aeruginosa producing different carbapenemase types, associated with divergent or novel integrons, punctuated the ongoing evolution of these bacteria.

Keywords: GES; Illumina sequencing; ST111; ST235; VIM; class 1 integrons; genomic islands (GIs); integrative conjugative element (ICE).

FIG 1

Geographic map showing the locations of the hospitals as well as the number of the carbapenemase-producing P. aeruginosa isolates (CPP) collected during the study.

FIG 2

Minimal spanning tree of 287 P. aeruginosa isolates, recovered from Czech hospitals during 2015, showing sequence types (STs) versus carbapenemase content. Each circle corresponds to an ST. The area of each circle is proportional to the number of isolates. The style of the connecting lines between STs corresponds to the number of allelic differences: up to 3 differences, solid lines; above 3 allelic differences, dashed lines.

FIG 3

Linear maps of the genetic context of carbapenemase-encoding class 1 integrons In-p110, In1392, and In717 inserted in LESGI-3 (A) and In1393 integrated into the P. aeruginosa chromosome (B). Arrows show the direction of transcription of open reading frames (ORFs), while truncated ORFs appear as rectangles (arrows within rectangles indicate the direction of transcription). Resistance genes, IS elements, and transposases are shown in red, yellow, and green, respectively. intI1 genes are shaded orange. Sequences associated with GIs are shaded light gray; dark gray rectangles indicate the P. aeruginosa chromosome. The remaining genes are shown in white.

FIG 4

Linear maps of the genetic context of carbapenemase-encoding class 1 integrons In56, located in the novel PAGI-56 (A), In59-like, associated with ICE1 (B), and In1391, inserted in the PACS171b GI (C). Arrows show the direction of transcription of ORFs, while truncated ORFs appear as rectangles (arrows within rectangles indicate the direction of transcription). Resistance genes, IS elements, and transposases are shown in red, yellow, and green, respectively. intI1 genes are shaded orange, while the repA gene of pAMBL2 is shown in pink. Sequences associated with GIs are shaded light gray; dark gray rectangles indicate the P. aeruginosa chromosome; ICE1 is shown in blue. The remaining genes are shown in white.

FIG 5

Phylogenetic tree of P. aeruginosa isolates, which were sequenced using the Illumina platform, based on Bayesian statistics constructed from core genome alignment. The boxed portion shows the clade of ST357 isolates. Branch lengths indicate the number of base changes per site. STs and carbapenemase content are indicated as squares and circles, respectively, of different colors. Numbers at the branches represent their posterior probabilities.