Phenotypic and Genotypic Detection of Carbapenemase Producing Pseudomonas aeruginosa Recovered from Clinical Specimens

Abstract

The worldwide emergence of multi-drug resistant strains of Pseudomonas spp. and P. aeruginosa in particular is a growing concern. P. aeruginosa is an opportunistic pathogen with innate resistance to many antibiotics and disinfectants including anti-pseudomonal Penicillins, Ceftazidime, Carbapenems, Aminoglycosides and Ciprofloxacin. It constitutes a common pathogen in hospitals. Its treatment is a therapeutic challenge because of the intrinsic resistance and the ability to easily acquire resistance determinants. Carbapenem resistance in Pseudomonas spp. can result from different mechanisms, such as a decreased bacterial outer membrane permeability (e.g., loss or modification of the OprD2 porin or overexpression of efflux pumps), often associated with overexpression of β-lactamases possessing no significant carbapenemase activity (AmpCs) or to expression of true carbapenemases. Carbapenemases have been reported in Pseudomonas spp., including the Ambler class A KPC- and GES-type β-lactamases and most commonly the Ambler class B or metallo-β-lactamases (MBLs) of the VIM, IMP, SPM, GIM, AIM, DIM, FIM, and NDM types. Easy and rapid detection of carbapenemase producers in the clinical laboratory is of major importance for the determination of appropriate therapeutic schemes and the implementation of infection control measures. So, the aim of this study was to evaluate the ability of phenotypic methods (ChromID Carba agar chromogenic medium, Modified Hodge test (MHT) and P. aeruginosa -Modified Hodge test (PAE-MHT)) for detection of carbapenemase producing strains of P. aeruginosa and determine the carbapenamase gene classes associated with different P. aeruginosa carbapenem resistant isolates using conventional PCR (blaKPC) and conventional multiplex PCR (blaVIM, blaIMP, blaOXA-48). One hundred carbapenem resistant (imipenem and meropenem by disk diffusion) P. aeruginosa isolates, were collected from isolates recovered from different clinical specimens referred to Central Microbiology Laboratory of Ain Shams University hospials for routine culture and sensitivity. All isolates were subjected to antimicrobial susceptibility testing by Kirby Bauer disk diffusion method using the following anti pseudomonal drugs: (Antipseudomonal penicillin (ticarcillin and piperacillin / tazobactam), Aminoglycosides (amikacin, tobramycin and gentamycin), Carbapenem (imipenem, meropenam, doripenem), Flouroquinolones (ciprofloxacin, levofloxacin), Azotreonam and Cephalosporins (ceftazdime and maxipeme). Then phenotypic detection of carbapenemases production was assessed by ChromID carba agar, Modified Hodge test (MHT), Pseudomonas aeruginosa modified Hodge test (PAE-MHT). Lastly genotypic detection of some carbapenemase genes using conventional PCR (blaKPC) and conventional multiplex PCR (blaVIM, blaIMP, blaOXA-48) was performed. All of the 100 CRPA isolates (100%) were resistant to both imipenem and meropenem. Regarding doripenem, 3 (3%) isolates gave intermediate sensitivity and 95 (95%) of the isolates were resistant. All of CRPA isolates were resistant to ticarcillin (100%), 56 (56%) were resistant to pipracillin/ tazobactam, 58 (58%) to amikicin, 91 (91%) to tobramycin, 98 (98%) to gentamycin, 90 (90%) to ciprofloxacin, 99 (99%) to levofloxacin, 63 (63%) to aztreonam, 91 (91%) to ceftazidime and 89 (89%) to maxipem. All of CRPA strains (100%) were MDR. All (100%) the CRPA isolates grow on ChromID Carba agar showing 100% senestivity. Out of 100 CRPA, 54 (54%) isolates gave positive results by MHT, three (3%) gave indeterminate results (inhibition of growth of the indicator strain produced by the test isolate) and 43 (43%) were negative with 58.9% sensitivity and 80% specificity. While by PAE-MHT, 91% of the strains were positive, three (3%) gave indeterminate results and 6 (6%) were negative with 97.9% sensitivity and 80% specificity. By PCR, blaKPC was the most prevalent gene as it was detected in 81 (81%) of the isolates, followed by blaVIM in 74 (74%) of the isolates. blaIMP was detected in only one (1%) isolate, and blaOXA-48 in 34 (34%) of the isolates. Genes for carbapenemase production were detected in 95 (95%) of CRPA isolates. Five strains were negative for all the four carbapenemase genes included in the study. Fifty seven (57%) strains contained two genes; (41%: blaKPC + blaVIM), (1%: blaKPC + blaIMP), (11%: blaKPC + blaOXA-48) and (4%: blaVIM + bla OXA-48). Nineteen strains (19%) were positive for three genes (blaKPC+ blaVIM+ blaOXA-48).