Pseudomonas aeruginosa (P. aeruginosa) is a menacing opportunistic, nosocomial pathogen; become a growing concern as conventional antimicrobial therapy is now futile against it. Multi‐drug resistant P. aeruginosa (MDRPA) has distinctive resistance mechanisms such as production of β‐lactamases, repression of porin genes and over‐expression of efflux pumps. The focus of this study is to standardize and application of multiplex PCR (mPCR) to detect the presence of betalactamase genes encoding blaTem, blaOXA, blaCTX‐M‐15, blaVim, blaGes, blaVeb, blaDIM, AmpC and Efflux pump genes encoding Mex A,B‐oprM, Mex C,D‐oprJ, Mex X,Y‐oprN, oprD, nfxB, MexR. A total of 200 clinical isolates of P. aeruginosa were tested for the presence of the above mentioned genes genotypically through mPCR and characterized by phenotypic methods for ESBL and MBL production. Out of 200 isolates, 163 (81.5%) nfxB regulator gene, 102 (51%) MexA, 96 (48%) MexC, 93 (46.5%) MexB, 86 (43%) MexD, 81 (40.5%) OprM, 74 (37%) OprJ, 72 (36%) OprD and MexR, 53 (26.5%) Mex X and OprN, 49 (24.5%) MexY gene. Betalactamase genes 145 (72.5%) blaTem, 67 (33.5%) blaOXA, 35 (17.5%) blaVim, 25(12.50%), 23 (11.50%) blaVeb, 21 (11.5%) blaGes, 14 (7%) Ctx‐m and 10 (5%) AmpC and 5 (2.5%) blaDim‐1 gene were tested positive by mPCR. Phenotypically 38 (19%) and 29 (14.5%) out of 200 tested positive for ESBL and MBL production. Application of this mPCR on clinical specimens is fast, accurate, specific and low‐cost reliable tool for the screening, where culture negative Eubacterial PCR positive cases for an early molecular detection of drug resistance mechanism assisting the clinician to treat the disease with appropriate antibiotic selection.

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Nested PCRs are superior than uniplex and multiplex PCR. PCRs proved to be a powerful tool in the diagnosis of endophthalmitis, especially for detecting uncultured microbes.

Pseudomonas aeruginosa isolates harboring acquired drug-resistant genes lead to increased mortality. Here, we have sequenced and annotated the genomes of two multidrug-resistant (MDR) P. aeruginosa isolates and a susceptible P. aeruginosa clinical isolate evidencing divergent antibiotic susceptibilities. Genomic analysis showed insight on the different genomic strategies adapted by P. aeruginosa to combat antimicrobial effects.