Uncovering the Resistance Mechanisms in Extended-Drug-Resistant <i>Pseudomonas aeruginosa</i> Clinical Isolates: Insights from Gene Expression and Phenotypic Tests

(1) Background: The purpose of the study was to describe the activity of <i>mex</i> efflux pumps in Multidrug-Resistant (MDR) clinical isolates of <i>Pseudomonas aeruginosa</i> and to compare the carbapenem-resistance identification tests with PCR; (2) Methods: Sixty MDR <i>P. aeruginosa</i> were analyzed for detection of carbapenemase by disk diffusion inhibitory method, carbapenem inactivation method and Modified Hodge Test. Endpoint PCR was used to detect 7 carbapenemase genes (<i>bla</i>_KPC, <i>bla</i>_OXA48-like, <i>bla</i>_NDM, <i>bla</i>_GES-2, <i>bla</i>_SPM, <i>bla</i>_IMP, <i>bla</i>_VIM) and <i>mcr-1</i> for colistin resistance. The expression of <i>mex</i>A, <i>mex</i>B, <i>mex</i>C, <i>mex</i>E and <i>mex</i>X genes corresponding to the four main efflux pumps was also evaluated; (3) Results: From the tested strains, 71.66% presented at least one carbapenemase gene, with <i>bla</i>_GES-2 as the most occurring gene (63.3%). Compared with the PCR, the accuracy of phenotypic tests did not exceed 25% for <i>P. aeruginosa</i>. The efflux pump genes were present in all strains except one. In 85% of the isolates, an overactivity of <i>mex</i>A, <i>mex</i>B and mostly <i>mex</i>C was detected. Previous treatment with ceftriaxone increased the activity of <i>mex</i>C by more than 160 times; (4) Conclusions: In our MDR <i>P. aeruginosa</i> clinical isolates, the carbapenem resistance is not accurately detected by phenotypic tests, due to the overexpression of <i>mex</i> efflux pumps and in a lesser amount, due to carbapenemase production.