BACKGROUND: Pseudomonas putida is a Gram-negative, non-fermenting bacterium frequently encountered in various environmental niches. P. putida rarely causes disease in humans, though serious infections and outbreaks have been reported from time to time. Some have suggested that P. putida functions as an exchange platform for antibiotic resistance genes (ARG), and thus represents a serious concern in the spread of ARGs to more pathogenic organisms within a hospital. Though poorly understood, the frequency of ARG exchange between P. putida and the more virulent Pseudomonas aeruginosa and its clinical relevance are particularly important for designing efficient infection control strategies, such as deciding whether high-risk patients colonized with a multidrug resistant but typically low pathogenic P. putida strain should be contact isolated or not. RESULTS: In this study, 21,373 screening samples (stool, rectal and throat swab) were examined to determine the presence of P. putida in a high-risk group of haemato-oncology patients during a 28-month period. A total of 89 P. putida group strains were isolated from 85 patients, with 41 of 89 (46.1%) strains harbouring the metallo-beta-lactamase gene bla VIM. These 41 clinical isolates, plus 18 bla VIM positive environmental P. putida isolates, and 17 bla VIM positive P. aeruginosa isolates, were characterized by whole genome sequencing (WGS). We constructed a maximum-likelihood tree to separate the 59 bla VIM positive P. putida group strains into eight distinct phylogenetic clusters. Bla VIM-1 was present in 6 clusters while bla VIM-2 was detected in 4 clusters. Five P. putida group strains contained both, bla VIM-1 and bla VIM-2 genes. In contrast, all P. aeruginosa strains belonged to a single genetic cluster and contained the same ARGs. Apart from bla VIM-2 and sul genes, no other ARGs were shared between P. aeruginosa and P. putida. Furthermore, the bla VIM-2 gene in P. aeruginosa was predicted to be only chromosomally located. CONCLUSION: These data provide evidence that no exchange of comprehensive ARG harbouring mobile genetic elements had occurred between P. aeruginosa and P. putida group strains during the study period, thus eliminating the need to implement enhanced infection control measures for high-risk patients colonized with a bla VIM positiv P. putida group strains in our clinical setting.