PREVALENCE OF PLASMID MEDIATED AMINOGLYCOSIDE MODIFYING ENZYMES IN PSEUDOMONAS AERUGINOSA IN HOSPITALIZED PATIENTS AT A TERTIARY CARE CENTRE

Santanu Hazra 1 , Partha Roy 2 and Anubha Patel 1 . 1. Resident, Department of Microbiology, Armed Forces Medical College, Pune 411040, India. 2. Senior Consultant (Microbiology and Virology), Oncquest Laboratories Limited, New Delhi 110029, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 02 December 2018 Final Accepted: 04 January 2019 Published: February 2019


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in hospitals with poor infection control practice. HAIs are most common in Intensive care units (ICU), acute surgical and orthopedic wards. The most frequent HAIs are infections of surgical wounds, urinary tract infections. Bloodstream infection and infections of lower respiratory tract including ventilator-associated pneumonia (VAP) are also very frequent in hospitalized patients. However, bloodstream infections and lower respiratory tract infections are the most lethal. [2] Enterobacteriaceae family being the most common identifiable groups overall, multidrugresistant organisms, including P. aeruginosa are increasingly being reported worldwide and associated with a high mortality rate. [3,4] P. aeruginosa are commonly associated with septicemia in burn patients, urinary tract infections in the catheterized patient, surgical site infections and chronic debilitating respiratory infections in cystic fibrosis patients. [2,4,5] In addition being intrinsically resistant to several antimicrobial agents, P. aeruginosa can easily develop resistance to all conventional antipseudomonal antibiotics, limiting the choice of effective antibiotics and complicating treatment. [4,6] The aminoglycosides are still very effective in the treatment of infections caused by aerobic gramnegative bacilli, including P. aeruginosa. Aminoglycosides act synergistically with other antibiotics active against the cell-wall like the β-lactam, thus facilitates cell killing and reduces the risk of development of resistance.
[6] Infections with P. aeruginosa are commonly treated with an aminoglycoside and antipseudomonal penicillin (e.g., Piperacillin) or antipseudomonal cephalosporin (e.g., Ceftazidime). Three mechanisms are responsible for aminoglycoside resistance: alteration of outer membrane permeability or diminished inner membrane transport or active efflux leading to decreased intracellular accumulation; target modification by 16S rRNA mutation or methylation or ribosomal protein-coding gene mutation; enzyme-mediated drug modification [7,8]. In the clinical setting, Aminoglycoside resistance is mainly due to AMEs causing modification of amino-or hydroxyl groups. This, in turn, reduces or inhibits binding of the aminoglycoside molecule to the ribosome and failure in trigging energydependent phase II. [8] The three main classes of AMEs are aminoglycoside N-acetyltransferases (AACs), aminoglycoside O-phosphotransferases (APHs), and aminoglycoside O-nucleotidyltransferases (ANTs). aac(6')-Ib is most frequently found in P. aeruginosa and a major contributor to aminoglycoside resistance. The genes coding for aac(6') is often part of mobile genetic elements. aph (3')-I and aph (3')-II are well documented in the clinical specimen of P. aeruginosa. [8] The genes encoding the APHs have been found on Rplasmid, transposons, integrons and responsible for multiple drug resistance. ant (2")-Ia is best known to confer resistance to gentamicin, kanamycin, and tobramycin. It is encoded on the plasmid or integrons in many Gram-negative bacteria, including P. aeruginosa. [8] However, little is known about the prevalence of these AMEs-coding genes among P. aeruginosa. Our effort in this study was to explore the plasmid-mediated AMEs-coding genes responsible for aminoglycoside resistance in P. aeruginosa from clinical isolates.

Isolation & identification
This prospective study was carried out from December 2013 to November 2014. One hundred and forty consecutive, non-repeat clinical isolates of P. aeruginosa were collected from various clinical samples of hospitalized patients at a tertiary care hospital and were identified by conventional phenotypic methods.

Antimicrobial susceptibility testing
Isolates were tested for their susceptibility to four aminoglycosides; amikacin, gentamicin, tobramycin, and netilmicin by Kirby-Bauer disc diffusion method. Results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines, 2014 (M100 -S24). [9] Isolates resistant to any tested aminoglycosides; amikacin, gentamicin, tobramycin, and netilmicin were defined as aminoglycoside resistant. P. aeruginosa (ATCC 27853) served as a control.
Genotypic Detection of Aminoglycoside Modifying Enzymes 4-5 colonies of isolate were dissolved in 2 ml Luria Bertani broth. The broth was incubated for 48 hours at 37ºC. Cells were harvested, and Plasmid DNA was extracted by the spin column method (Hi-PURA Plasmid Miniprep, Hi-MEDIA). (2")-I were selected for screening. PCR for detection of the AMEscoding genes was carried out using five sets of primers, designed from sequence deposited in the GeneBank database ( Table 1).

Five AMEscoding genes, aac (6')-I, aph (3')-I, aac (3)-I, aac (3)-II and ant
The PCR assay was carried out in a final volume of 25 μl, containing 12.5 μl of 2X Dream TaqTM Green PCR Master Mix (Fermentas, USA), 1 μl (25 picomoles) of each primer (Integrated DNA Technologies, IDT), 1 μl of plasmid DNA and molecular grade water. Various amplification conditions were used for each primer set. Amplification products were detected by electrophoresis on 1% w/v agarose gel containing 0.5% ethidium bromide and visualized on UV transilluminator. Amplicon size was determined using ready-to-use 100 bp Plus DNA ladder (Fermentas, USA) run along with samples every time.

DNA Sequencing
Ten amplicons representative of the two sets of primers were verified by Sanger's capillary sequencing. The nucleotide sequences of the amplicons were analyzed using the open access software of National Center of Biotechnology Information website (http://www.ncbi.nlm.nih.gov).

Conclusion:-
This study provided information about resistance pattern of different aminoglycosides against P. aeruginosa and resistance determinants providing useful comparative data for future study. Because of synergism with other drugs and post antibactericidal effect, aminoglycosides remain useful antipseudomonal agents. However, the high prevalence of plasmid-mediated aminoglycoside resistance in P. aeruginosa among hospitalized patients was observed which could easily spread and be disseminated among other bacteria. Proper infection control practices and periodic monitoring of resistance to aminoglycoside antibiotics are required to minimize further dissemination and preserve the usefulness of this important class of antibiotics for the treatment of complicated infection.

Funding:
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.