Role of the Outer Membrane Protein OprD(2) in Carbapenem-Resistance Mechanisms of Pseudomonas aeruginosa

We investigated the relationship between the outer membrane protein OprD(2) and carbapenem-resistance in 141 clinical isolates of Pseudomonas aeruginosa collected between January and December 2013 from the First Affiliated Hospital of Anhui Medical University in China. Agar dilution methods were employed to determine the minimum inhibitory concentration of meropenem (MEM) and imipenem (IMP) for P. aeruginosa. The gene encoding OprD(2) was amplified from141 P. aeruginosa isolates and analyzed by PCR and DNA sequencing. Differences between the effects of IMP(R) and IMP(S) groups on the resistance of the P. aeruginosa were observed by SDS-poly acrylamide gel electrophoresis (SDS-PAGE). Three resistance types were classified in the 141 carbapenem-resistant P. aeruginosa (CRPA) isolates tested, namely IMP(R)MEM(R) (66.7%), IMP(R)MEM(S) (32.6%), and IMP(R)MEM(S) (0.7%). DNA sequencing revealed significant diverse gene mutations in the OprD(2)-encoding gene in these strains. Thirty-four strains had large fragment deletions in the OprD(2)gene, in 6 strains the gene contained fragment inserts, and in 96 resistant strains, the gene featured small fragment deletions or multi-site mutations. Only 4 metallo-β-lactamase strains and 1 imipenem-sensitive (meropenem-resistant) strain showed a normal OprD(2) gene. Using SDS-PAGE to detect the outer membrane protein in 16 CRPA isolates, it was found that 10 IMP(R)MEM(R) strains and 5 IMP(R)MEM(S) strains had lost the OprD(2) protein, while the IMP(S)MEM(R) strain contained a normal 46-kDa protein. In conclusion, mutation or loss of the OprD(2)-encoding gene caused the loss of OprD(2), which further led to carbapenem-resistance of P. aeruginosa. Our findings provide insights into the mechanism of carbapenem resistance in P. aeruginosa.