Application of six multiplex PCR's among 200 clinical isolates of Pseudomonas aeruginosa for the detection of 20 drug resistance encoding genes

Abstract

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.

Identification of multi-drug resistant genes in <i>P. aeruginosa</i> isolates from patients under mechanical ventilation and respiratory support in an intensive care unit

“…In this study significantly higher number of isolates (70.8 %) was positive for bla TEM gene. This is similar to current findings in a study in India which reported that bla TEM was the predominant gene present among the P. aeruginosa isolates (72.5 %) [9]. In that study [9], the bla OXA (33.5 %) and bla VIM (11.5%) genes were comparable.…”

“…A comparable resistance to imipenem (41.1 %) and meropenem (38.9 %) was reported in China [4]. The study also reported comparable resistance values for ceftazidime (40.3 %), cefepime (38.5%) and piperacillin/tazobactam (38.9 %), while a study in India reported much lower resistance (25 %) for meropenem and comparable resistance (25 %) for piperacillin/tazobactam [9]. In contrast, a study in Brazil reported much higher degrees of resistance (64.8 %) to meropenem and imipenem in P. aeruginosa isolated from ICU patients [19].Although the majority of the isolates were resistant to gentamicin, antibiotic resistance did not differ significantly among the isolates (p = 1.00).…”

“…A study in India reported comparable resistance (18 %) to amikacin, but much lower resistance values to gentamicin (38 %), ceftazidime (26 %), cefepime (5.5 %), meropenem (18.5 %), imipenem (20.5 %) and ciprofloxacin (25.5 %) [9], when compared to the 37.2 % obtained in the present study. In contrast, a study in Romania reported higher resistance to amikacin (46.9 %), imipenem (49.8 %), piperacillin/tazobactam (49.7 %), meropenem (50.5 %), ceftazidime (53.1 %), ciprofloxacin (60.3 %) and gentamicin (64.2 %) [20].…”

“…In addition, due to geographical variations and participating centers, the SENTRY antimicrobial surveillance program was developed to track antimicrobial resistance trends nationally and internationally, fail to report the true prevalence of MDR P. aeruginosa [8]. With the lack of safe therapeutic antibiotics against MDR and pandrug resistant strains, continued presence of such strains would pose serious challenges in infection control management [9]. Hence, it is extremely vital to understand the distribution of pathogens and antibiotic resistance patterns of pneumonia so as to achieve optimal antibiotic therapy.…”

“…DNA was extracted from pure cultures by alkali lysis method and stored at -20 ⁰C until used for PCR. Multiplex PCR was used to determine extended-spectrum β-lactamases (ESBL)-MBL resistant genes such as bla TEM , bla OXA , bla CTX-M-15 , bla VIM [9]. The PCR was performed using a 50-μL master mix containing 5 μL of template DNA, 5 pmoL of each primer (Table 1), dNTPs (2 mM), 3 units of Taq polymerase enzyme, 5 μL of 10x reaction buffer, and molecular grade PCR water in a total volume of 50 μL.…”