Cost versus benefit of Pseudomonas aeruginosa hypermutation in the absence or presence of antibiotic treatment

Stressful conditions in patients with cystic fibrosis (CF) are thought to select P. aeruginosa hypermutable clones with mutations in genes of the mismatch repair system (MRS) by hitchhiking with adaptive mutations. Whether the increased mutation rate of P. aeruginosa hypermutable strains is associated with a biological benefit or cost for the colonization of secondary environments is not known. We have previously showed that P. aeruginosa increased mutagenesis is associated with an important biological cost reducing the potential for the colonization of new environments in the absence of selective pressure when tested in a mouse model of chronic infection (Montanari et al, Pediatric Pulmonology 28, 2005; abstract nr 287). Here, we compared those results in the presence of selective pressure such as antibiotic treatments. Couples of clinical hypermutable/wild type clonally related P. aeruginosa strains, the laboratory strains PAO1∆mutS/PAO1 and the strains complemented with plasmids containing genes involved in the MRS (mutS, mutL and uvrD) were subjected to competition in vitro and in vivo in the presence or absence of antibiotics. In vitro, in the presence of streptomycin (STR 100g/ml) or nalidixic acid (NAL 200g/ml), the hypermutable outcompeted the wild type and its complemented strains in the laboratory strain PAO1 (competition index (CI) (PAO1∆mutS/PAO1)/ (PAO1∆mutS mutS+/PAO1): 1.4 in streptomycin and 1.25 in nalidixic acid) and in the P. aeruginosa clinical clonal pairs tested in NAL (CI (BST44/BST2)/(BST44mutS+/BST2): 2.3; (RP74/RP73)/(RP74mutL+/RP73): 7.1; (MF2/MF1)/(MF2uvrD+/MF1): 10.1). In the absence of antibiotic pressure, three hypermutable strain(s) were disadvantaged (CI (PAO1∆mutS/PAO1)/ (PAO1∆mutS mutS+/PAO1): 0.25; (BST44/BST2)/(BST44mutS+/BST2): 0.4; (RP74/RP73)/(RP74mutL+/RP73): 0.54) and one decreased its advantage (MF2/MF1)/(MF2uvrD+/MF1): 4.1). To show that the advantage of the P. aeruginosa strains is not caused by increased resistance, the MICs for STR and NAL were determined. Although the hypermutable strains showed a higher level of resistance to the antibiotics, the MICs returned back to the level of the wild type strains when complemented with the wild type copy of MRS gene. These data indicated that the resistant phenotype was developed in vitro after antibiotic exposure and was not caused by stable mutations during previous exposures in the CF patient’s lung. Competition experiments between PAO1∆mutS/PAO1 were performed in the agar beads mouse model for chronic airway infection. Under streptomycin treatment for 14 days, the hypermutable strain PAO1∆mutS was more efficient to establish a murine chronic infection when compared to its wild type parent strain (CI PAO1∆mutS/PAO1: 2.97) while in the absence of antibiotic treatment the results were opposed (CI: 0.002). The results suggest that P. aeruginosa increased mutagenesis, associated with an important biological cost in the absence of selective pressure, turn to a benefit in the presence of strong selective pressure such as antibiotic treatments. Supported by the Italian CF Research Foundation and Associazione Lombarda FC.