Fitness cost associated with the chromosomal integron In70.2 in Pseudomonas aeruginosa clinical isolates

An epidemiologic survey performed at the Trieste University Hospital (northeastern Italy) between 1999 and 2002 revealed a remarkable spread of an MDR Pseudomonas aeruginosa strain, named TS-832035, which carried the chromosomal integron In70.2 containing four gene cassettes (blaVIM-1, aacA4, aphA15 and aadA1) in its variable region and conferring resistance to ß-lactams, including carbapenems, and to several aminoglycosides. Moreover, some other P. aeruginosa isolates, strictly related to TS-832035 but lacking in the integron In70.2, were detected, but they remained a minor component within the cluster during the three years of surveillance.They showed an MDR phenotype like TS- 832035, differing only for the susceptibility level to carbapenems. The genomic relatedness between TS-832035 and TS-103 was investigated by random amplification of polymorphic DNA (RAPD) typing, pulsed-field gel electrophoresis (PFGE) analysis of SpeI-digested genomic DNA, and multilocus sequence typing (MLST). The cost of the integron In70.2 on the fitness of TS-832035 was determined by performing growth kinetics and direct competition assays against the clonal isolate TS-103 in three media differing for nutrient availability: a rich medium (Luria Bertani (LB) Broth) and a minimal medium (28 g/l K2HPO4, 12 g/l KH2PO4, 0.4 g/l MgSO4, 7H2O, 4 g/l (NH4)2SO4) added with a rich carbon source (0.4% w/v glucose) or with a poorer carbon source (0.4% w/v sodium acetate). Growth kinetic data were obtained by measuring optical density at 600 nm (OD600). For competition assays, the number of CFU/ml of each isolate was estimated by colony-hybridization. We proved the clonality of the two isolates by molecular investigations.The results of the growth kinetics showed the existence of a significant in vitro fitness cost associated with the integron In70.2, more evident in a poorer medium.The sensitivity of the two isolates to the antimicrobial agents tested was the same, except for the different levels of resistance to carbapenems (MIC 16 μg/ml versus 64-128 μg/ml). Although we can not exclude that other factors may have favoured the in vivo spread of TS-832035, our results suggest that the increased level of resistance to carbapenems has conferred on this isolate a selective advantage able to compensate metabolic cost associated to the integron.