Genomic Characterization of IMP-Producing Pseudomonas aeruginosa in Bulgaria Reveals the Emergence of IMP-100, a Novel Plasmid-Mediated Variant Coexisting with a Chromosomal VIM-4

Abstract

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections represent a major public health concern and require comprehensive understanding of their genetic makeup. This study investigated the first occurrence of imipenemase (IMP)-carrying P. aeruginosa strains from Bulgaria. Whole genome sequencing identified a novel plasmid-mediated IMP-100 allele located in a a novel In4886 integron embedded in a putative Tn7700 transposon. Two other closely related chromosomal IMP variants, IMP-13 and IMP-84, were also detected. The IMP-producers were resistant to last-line drugs including cefiderocol (CFDC) (two out of three) and susceptible to colistin. The IMP-13/84 cassettes were situated in a In320 integron inserted in a Tn5051-like transposon as previously reported. Lastly, the p4782-IMP plasmid rendered the PA01 transformant resistant to CFDC, suggesting a transferable CFDC resistance. A variety of virulence factors associated with adhesion, antiphagocytosis, iron uptake, and quorum sensing, as well as secretion systems, toxins, and proteases, were confirmed, suggesting significant pathogenic potential consistent with the observed strong biofilm formation. The emergence of IMP-producing MDR P. aeruginosa is alarming as it remains unsusceptible even to last-generation drugs like CFDC. Newly detected IMP-100 was even located in a CFDC-resistant XDR strain.

Keywords: Pseudomonas; blaIMP; metallo-beta-lactamase; novel allele; plasmid.

Figure 1

Genomic inference of resistance determinants. Columns represent strains, whereas AMR genes are in rows. The color scheme represents the affected antimicrobial class (right) in relation to the resistance genes (left).

Figure 2

Phylogenetic tree and resistance heatmap. Plasmids from the current study are highlighted in green. The different tree clusters (C1 ÷ C4) are distinguished by varying colors. The heatmap legend below provides information on the antibiotic class of the detected genes. The tree was generated with neighbor-joining after the NCBI blast with Max Seq Difference score ≥0.75. The heatmap and the tree were visualized with iToL 6.8 (https://itol.embl.de/about.cgi, accessed on 25 July 2023).

Figure 3

Closest plasmids with carbapenemases. The plasmid accession numbers are listed in the legend and colored differently. The most inner circle (blue) is p4782-IMP which serves as a reference for comparison. Only important gene annotations were visualized in blue text. The novel transposon (Tn7700) is shown in black. The figure was produced with the Gview Atlas server (https://server.gview.ca, accessed on 25 July 2023).

Figure 4

Phylogenetic tree of all available IMP variants. All NCBI available protein sequences were downloaded and aligned with Muscle alignment 3.8.425 in Geneious Prime 2022 and the phylogenetic tree was constructed with Geneious Tree builder with the Jukes–Cantor genetic distance model and the neighbor-joining build method with bootstrap 1000. The tree was visualized in iToL 6.8 (https://itol.embl.de/about.cgi, accessed on 25 July 2023). Cluster groups G1 ÷ G7 were colored differently in accordance to [14], and the new IMP-100 allele is colored in red with a red dot at the tip of the leaf. Bootstrap indices are represented as circles placed at each node ranging in size from 90 (small circle) to 100 (large circle) to show the degree of cluster consistency.

Figure 5

Genetic environment of bla_IMP-100. A hypothesized origin of the novel transposon Tn7700 is depicted. Matching genes from the different sources are connected and identically colored. Non-matching genes (either hypothetical proteins or not) are shown in white. Unlabeled genes are hypothetical. The figure was created with Clinker v0.0.28 (https://github.com/gamcil/clinker, accessed on 25 July 2023).