Carbapenemase-producing gram-negative bacteria are an emerging problem worldwide. ^{Reference Baxter, Hastings, Law and Glass1} In low-endemicity countries, the spread of carbapenemase-producing microorganisms is frequently related to outbreaks in hospitals, Pseudomonas aeruginosa is frequently transmitted by contact with the hands of healthcare workers or environmental sources. ^{Reference Voor, Severin, Hagenaars, De, Gommers and Vos2-Reference Kossow, Kampmeier and Willems5} To our knowledge, we report the first well-documented in-hospital patient-to-patient-transmission of a bla _VIM-2 integron between isolates belonging to 2 different species, P. aeruginosa and P. alcaligenes, with indirect transmission to patients, likely by environmental contamination.

A 72-year-old male (patient Z) was transferred to our hospital for diabetic foot syndrome and chronic osteomyelitis after being previously hospitalized in Southeast Asia. He had received multiple antibiotic treatment courses, not recorded in the transfer protocol. At day 15 of the hospitalization in at the University Hospital Basel, a first screening for carbapenemase-producing gram-negatives, consisting of swabs of the throat and rectum as well as a urine sample, showed colonization with several isolates: a P. aeruginosa harboring a bla _VIM-2 carbapenemase-encoding gene; Citrobacter koseri and Klebsiella pneumoniae, both with a bla _NDM-1 carbapenemase-encoding genes; and Escherichia coli with a bla _OXA-181 carbapenemase-encoding gene. In addition to these strains, culture of the bone biopsy revealed isolates of Proteus mirabilis, Enterococcus faecalis, and Morganella morganii. P. alcaligenes was not identified in either the screening or the biopsies. An amputation of the right lower limb was performed for treatment of the chronic polymicrobial osteomyelitis. Antibiotic treatment was stopped after cultures of the bone from the proximal part of the amputation site remained negative, and there were no signs of osteomyelitis in the pathology specimen. The patient was put under strict contact precautions. All contact patients who shared the same room during or after hospitalization of the index case were screened for carbapenemase-producing gram-negative pathogens using the chromID CARBA SMART Bi-Plate Agar (bioMérieux, Marcy-l’Étoile, France) and, if cultures were positive, the Xpert Carba-R (Cepheid, Sunnyvale, CA) was used to identify carbapenemases.

A 90-year-old women (patient Y) was hospitalized in room 1 five days after the index patient Z had moved to a different room (Fig. 1). The patient was hospitalized for a soft-tissue infection of her left lower leg with bacteremia with Staphylococcus aureus and Enterococcus faecalis secondary to a leg injury. She was treated with debridement of the left lower leg. After a negative rectal screening for carbapenemase-producing microorganisms on day 4 of her hospitalization, she tested positive for bla _VIM-2 containing P. alcaligenes in a rectal swab taken 5 days later. The species was routinely determined by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) and later confirmed by 16S rRNA gene identity and ribosomal multilocus sequence typing (rMLST). She did not experience any infection due to Pseudomonas alcaligenes, and they are rarely reported in the literature as well. ^{Reference Suzuki, Suzuki, Matsui, Hiraki, Kawano and Shibayama6} She lived at home with her husband and denied any recent travel or hospitalization abroad or even within Switzerland. She had a history of myelodysplastic syndrome treated symptomatically with erythropoietin and danazolum. All other contact patients were negative for carbapenemase-producing gram-negative microorganisms.

Fig. 1. Room occupancy of patient Z and patient Y during hospitalization.

In our institution, the first isolated strains from each patient harboring carbapenemases are routinely typed by whole-genome sequencing. We sequenced isolates 807875-6-18 and 808084-18 on both an Illumina NextSeq500 device (Illumina, San Diego, CA; 56x and 87x mean coverage, respectively) and an Oxford Nanopore MinION device (Oxford Nanopore, Oxford, UK; 104x and 128x mean coverage, respectively) in a hybrid assembly approach using Unicycler v 0.3.0b software ^{Reference Wick, Judd, Gorrie and Holt7} to reconstruct the genomes into 30 and 9 scaffolds, respectively. All reads have been submitted to the European Nucleotide Archive (project no. PRJEB34600). We scanned the 2 assemblies for resistance genes using ABRicate software ^{Reference Wick, Judd, Gorrie and Holt7} to search the NCBI AMR gene database (PRJNA313047). In both genomes, we identified a bla _VIM-2 gene. A closer comparison of the genes and their genomic surroundings using Artemis software ^{Reference Rutherford, Parkhill and Crook8} showed that the bla _VIM-2 genes are located in a 2,264-bp integron in both strains. The integron carries an integrase and a bla _VIM-2 gene (Fig. 2). The sequence comparison, performed in SeaView ^{Reference Gouy, Guindon and Gascuel9} shows that the elements in the 2 strains differ by 2 single-nucleotide polymorphisms (SNPs). The bla _VIM-2 genes are identical. By searching the NCBI database, we found that the found integron is almost identical (1 SNP difference) to the integron ln76 that was described in the context of the discovery of the first bla _VIM-2 gene in a P. aeruginosa isolate in Latin America. ^{Reference Rutherford, Parkhill and Crook8} We found that the integron in P. aeruginosa isolate 807875-6-18 (patient Z) is located on a 1,238,389-bp contig (chromosome fragment), and in P. alcaligenes isolate 808084-18 (patient Y) it is located on a 77,160-bp circular contig, representing a plasmid (rep_cluster_777, accession number JQ432564 (https://github.com/jrober84/mob-typer)). This contig shows identity (58% coverage and 98% sequence identity) to the pKS208 plasmid previously described in an uncultured bacterium that does not carry the bla _VIM-2 gene. ^{Reference Gouy, Guindon and Gascuel9}

Fig. 2. Coding sequences of the integron carrying bla_VIM-2.

The evidence of horizontal gene transfer between 2 species is supported by (1) a very strong epidemiological link between the 2 patients, and (2) detection of identical bla _VIM-2 that are extremely rare in our patient population. ^{Reference Erb, Frei, Dangel and Widmer10} In 2018, bla _VIM-2 occurred in only 16 cases in Switzerland, of which 15 were in non–Escherichia coli or non–Klebsiella pneumoniae bacteria. ¹¹ Molecular typing to detect transmission is commonly performed within species, but it may be extended to detect transmission of key factors between species if the epidemiological link is strong. A strong epidemiological link should trigger an extended workup with not only next-generation sequencing of the core genome but also include transposons to prove or to exclude nosocomial transmission.