Pseudomonas aeruginosa is a major cause of severe, hospital-acquired infections. It is intrinsically resistant to multiple antimicrobial classes, and the global emergence of carbapenem-resistant strains, particularly those belonging to high-risk sequence types ST111, ST235, and ST175, is a source of concern. Infections caused by such strains are associated with increased morbidity and mortality in healthcare facilities. Carbapenem resistance in these strains often arises through the production of metallo-beta-lactamase enzymes, and Verona Integron-encoded Metallo-beta-lactamase, or VIM, is the most frequently encountered MBL in P. aeruginosa worldwide, typically encoded as a mobile gene cassette on a class 1 integron.

At Erasmus MC, VIM-positive P. aeruginosa clones have emerged since 2003, causing colonisations and infections in patients, with isolates belonging primarily to the international high-risk clones ST446 and ST111. Environmental investigations identified sinks as a persistent reservoir within the hospital. Although blaVIM-encoding integrons are usually located on the P. aeruginosa chromosome, a previous study reported a blaVIM-2-containing conjugative plasmid in P. aeruginosa strain S04 90, an ST446 clone isolated from a patient at the hospital in 2009. That observation was worrisome because plasmids transfer easily between bacteria of the same species and even within the same genus. During the years-long outbreak, several VIM-positive non-aeruginosa Pseudomonas isolates were also recovered from the patient environment and, sporadically, from patients.

In the new study, the team screened both VIM-positive P. aeruginosa and non-aeruginosa Pseudomonas isolates for blaVIM-2-containing plasmids, genetically characterized the plasmids they found, and compared them with publicly available plasmid sequences to trace their origin. Environmental sampling at the hospital began in 2011 after the outbreak was recognized. Sink culture samples were taken from wash basins, countertops, faucet aerators, and sink drains, incubated in enrichment broth containing ceftazidime and vancomycin, and screened for blaVIM using real-time PCR. Positive broths were subcultured, and isolates were identified by MALDI-TOF mass spectrometry and stored at minus 80 degrees Celsius. Additional isolates came from sink drain samples cultured using a microbial culturomics technique, and clinical cultures were obtained through routine diagnostic pathways.

Whole-genome sequencing was performed with Illumina technology, and plasmid reconstruction relied on hybrid assemblies combining long-read MinION sequencing with short Illumina reads. The researchers first re-sequenced the original P. aeruginosa strain S04 90, stored as VIM-PA R-340, and confirmed the presence and identity of its plasmid with only minor differences: seven of 159,166 nucleotides differed from the published sequence, a 99.996 percent identity match, with four of those differences located within the blaVIM-2-encoding integron.

The central result concerned six VIM-positive non-aeruginosa Pseudomonas isolates that carried an identical plasmid sequence: one P. oleovorans, two P. carnis, and three isolates of a novel Pseudomonas species. Five of these strains had been cultured from the sink environment in the same intensive care unit, ICU-2, but from different patient rooms between 2013 and 2016; the sixth, a novel Pseudomonas species, had been isolated from a patient staying in ICU-2 in 2014. Notably, the two P. carnis isolates had originally been identified as P. alcaligenes and P. gessardii by MALDI-TOF mass spectrometry and were reclassified only after sequencing, while the three novel-species isolates had been called P. putida. The earliest non-aeruginosa carrier, P. carnis strain VIM-PC R-7810, was recovered from the ICU environment in 2013, and its plasmid, designated pR7810, differed from the previously reported P. aeruginosa plasmid.

Alignment of the two plasmid sequences revealed a high overall similarity with minor differences. The most obvious difference was a rearrangement of a 4.7-kilobase pair region encoding a hypothetical protein. A 2.6-kilobase insertion sequence 99 percent identical to ISpst3 of the IS21 family was found in the plasmid of VIM-PA R-340 but only in the chromosome of VIM-PC R-7810, while a 1.2-kilobase insertion sequence 98 percent identical to IS222 was found only in the VIM-PA R-340 plasmid. Apart from these differences, the two plasmid sequences were identical, including a class 1, blaVIM-2-encoding integron identical to a recently described integron type carried by the majority of VIM-2-positive P. aeruginosa isolates in the Netherlands. Analysis with MOB-Typer showed that both plasmids represent conjugative plasmids of MPF type I, rep cluster 339, containing a MOB relaxase type Q.

On the P. aeruginosa side, three VIM-positive isolates carried a plasmid identical to that of VIM-PA R-340, and three others carried a plasmid identical to pR7810. All six P. aeruginosa strains belonged to the ST111 high-risk clone and were shown by core-genome multilocus sequence typing to be closely related. They were isolated between 2016 and 2018 from ICU-2 and the surgical ward, from sink drain samples and clinical cultures. One P. aeruginosa strain carrying the pR7810-identical plasmid was isolated in 2017 from a patient in the very ICU room where P. carnis VIM-PC R-7810 had been recovered in 2013, and in 2018 two further strains came from sink drains in the same surgical ward room to which that patient had been transferred. In all isolates studied, the blaVIM-2 gene was located only on plasmids and not on the chromosome.

To place these plasmids in an international context, the researchers performed a BLAST search and retrieved thirteen plasmid sequences with more than 50 percent coverage to their reference plasmid. All fifteen plasmids shared a highly similar backbone structure, but k-mer-based clustering showed considerable diversity, and the two Erasmus MC plasmids clustered closely together with the published S04 90 sequence while remaining notably distinct from other deposited sequences. The nearest neighbour was a plasmid from a Belgian P. aeruginosa strain that carried the carbapenemase gene blaIMP-13 on its chromosome but no carbapenemase on its plasmid. The authors conclude that the two plasmids appear unique to their hospital and, in a wider context, to the Netherlands, and that their source remains unknown.

The study’s authors hypothesize that these two blaVIM-2-encoding plasmids likely transferred between high-risk P. aeruginosa clones and other Pseudomonas species, using sink drains as a primary environmental reservoir within the hospital. To their knowledge, this is the first report of VIM-2-positive P. carnis and the second report of P. oleovorans carrying a blaVIM-2-encoding plasmid. The authors note limitations: not all VIM-positive P. aeruginosa strains were sequenced, environmental sampling was not routine and was guided by suspected transmission or intervention monitoring, so other carriers may have been missed, and conjugation experiments were not performed to verify transmissibility directly, although the high similarity to the previously confirmed conjugative S04 90 plasmid and the presence of identical sequences in multiple species strongly suggest plasmid transfer. The authors argue that non-aeruginosa Pseudomonas species should not be ignored during outbreak investigations and should be screened for carbapenemases, and that plasmid analysis is essential to fully understand how resistance genes move through hospitals.

Subject of Research: Molecular comparison of blaVIM-2-containing plasmids in environmental and clinical Pseudomonas isolates at a Dutch hospital

Article Title: Co-occurrence of VIM-positive non- aeruginosa Pseudomonas spp. and Pseudomonas aeruginosa in the hospital: A detailed molecular comparison of bla VIM-2 -containing plasmids from environmental and clinical isolates

Article References: Pirzadian, J., Zandijk, W. H. A., Heikema, A. P., Koene, H. H. H. T., Goessens, W. H. F., Vos, M. C., Klaassen, C. H. W., & Severin, J. A. (2026). Co-occurrence of VIM-positive non-aeruginosa Pseudomonas spp. and Pseudomonas aeruginosa in the hospital: A detailed molecular comparison of blaVIM-2-containing plasmids from environmental and clinical isolates. Heliyon, 12(16), Article e45547. https://doi.org/10.1016/j.heliyon.2026.e45547

Image Credits: AI Generated

DOI: 10.1016/j.heliyon.2026.e45547

Keywords: Pseudomonas aeruginosa, non-aeruginosa Pseudomonas, blaVIM-2, plasmid, carbapenem resistance, metallo-beta-lactamase, sink drains, hospital environment, whole-genome sequencing, ST111, Erasmus MC, infection control