Development of carbapenem resistance in Pseudomonas aeruginosa is associated with OprD polymorphisms, particularly the amino acid substitution at codon 170

Pan-susceptible Pseudomonas aeruginosa (PSPA) clinical isolates carrying an OprD with loop 7 shortening (the group-1A allele) were found to rapidly develop carbapenem resistance under continuous selection pressure. We further studied whether OprD polymorphisms are associated with the potential to develop carbapenem resistance. OprD amino acid sequences of 126 PSPA clinical isolates were analysed to determine their STs using P. aeruginosa strain PAO1 as the control strain. Site-directed mutagenesis was performed in PAO1 to generate polymorphisms of interest. A disc diffusion method was used to select carbapenem-resistant variants from the mutant strains. Expression levels of oprD were determined by quantitative RT-PCR. MICs of carbapenems were determined by Etest. Forty-eight (38.1%) of the tested isolates carried the group-1A allele. Another two major STs, C1 and C2, both of which harboured an F170L polymorphism, were found in 21 (16.7%) and 39 (31.0%) isolates, respectively. The PAO1 type was also found in 14 (11.1%) isolates. Under continuous selective pressure, isolates of most STs developed carbapenem resistance at different numbers of passaging events; only those belonging to the PAO1 type remained susceptible. However, PAO1 mutants carrying either the oprD group-1A allele or the OprD-F170L polymorphism were able to develop carbapenem resistance. Reduced oprD expression triggered by continuous imipenem challenge was found in PAO1 mutants, but not in the PAO1 WT strain. OprD polymorphisms, particularly the F170L substitution and the specific shortening in loop 7, appear to determine the potential for P. aeruginosa to develop carbapenem resistance.