DETECTION AND PREVALENCE OF EFFLUX PUMP-MEDIATED DRUG RESISTANCE IN CLINICAL ISOLATES OF MULTIDRUG-RESISTANT GRAM-NEGATIVE BACTERIA FROM NORTH KERALA, INDIA
Abstract
Objectives: The present study was carried out to detect the prevalence of efflux pump-mediated drug resistance in clinical isolates of multidrugresistant
(MDR) Gram-negative
bacteria
isolated
from
North Kerala.
Methods: Clinical isolates (n = 123) of MDR Gram-negative bacteria were collected from various clinical laboratories in North Kerala, and their effluxmediated
drug resistance
was
detected
by
two
simple phenotypic assays
-
ethidium
bromide
(EB)-agar
cartwheel
method and
efflux pump inhibitor
(EPI)-based
microplate
assay,
employing
phenylalanine-arginine
β-naphthylamide
as inhibitor.
Results: The 123 Gram-negative MDR strains tested comprised Escherichia coli, Pseudomonas aeruginosa, Acinetobacter spp., and Klebsiella spp. The
EB-agar cartwheel method of screening revealed efflux activity in 20% (n=25) of the strains with representatives from all 4 genera. The efflux activity
was revealed at a minimum concentration of EB at 1 mg/l. P. aeruginosa strains showed the highest activity, many folds higher up to a concentration
of 2.5 mg/l. The confirmatory EPI-based microplate assay showed efflux activity only in 15% (n=18) strains with 6% (n=7) active against more than
one antibiotic. Efflux pump-mediated drug resistance was found to be most prevalent in P. aeruginosa (34.8%, n=8 out of 23), followed by that in E. coli
(18.6%, n=8 out of 43), Acinetobacter spp. (9%, n=1out of 11), and Klebsiella spp. (2%, n=1 out of 46).
Conclusion: This study reports on the emergence of efflux pump-based multidrug-resistance in North Kerala. Our results showed that 15% of drug
resistance in Gram-negative MDR strains is attributable to efflux-related mechanisms, thereby emphasizing the need for inclusion of efflux-related
tests in the diagnostic regimen for MDR clinical bacteria.
Keywords: Gram-negative bacteria, Multidrug-resistance, Efflux pumps, Ethidium bromide, Efflux pump-inhibitor.
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References
. Martins A, Hunyadi A, Amaral L. Mechanisms of resistance in bacteria:
An evolutionary approach. Open Microbiol J 2013;7:53-8.
Sun J, Deng Z, Yan A. Bacterial multidrug efflux pumps: Mechanisms,
physiology and pharmacological exploitations. Biochem Biophys Res
Commun 2014;453(2):254-67.
Li XZ, Nikaido H. Efflux-mediated drug resistance in bacteria: An
update. Drugs 2009;69(12):1555-623.
Poole K. Efflux pumps as antimicrobial resistance mechanisms. Ann
Med 2007;39(3):162-76.
Piddock LJ. Multidrug-resistance efflux pumps - not just for resistance.
Nat Rev Microbiol 2006;4(8):629-36.
Putman M, van Veen HW, Konings WN. Molecular properties of bacterial
multidrug transporters. Microbiol Mol Biol Rev 2000;64(4):672-93.
Kumar A, Schweizer HP. Bacterial resistance to antibiotics: Active
efflux and reduced uptake. Adv Drug Deliv Rev 2005;57(10):1486-513.
Butaye P, Cloeckaert A, Schwarz S. Mobile genes coding for effluxmediated
antimicrobial
resistance
in
Gram-positive
and
Gram-negative
bacteria.
Int J Antimicrob
Agents
;22(3):205-10.
Baron EJ, Peterson LR, Finegold SM. Bailey and Scott’s Diagnostic
Microbiology. 9
ed. St. Louis: The C V Mosby Company; 1994.
Clinical and Laboratory Standards Institute. Performance Standards for
th
Antimicrobial Susceptibility Testing. 22
Informational Supplement.
M100-S22. Wayne, PA: CLSI; 2012.
nd
Martins M, Couto I, Viveiros M, Amaral L. Identification of effluxmediated
multi-drug
resistance in
bacterial
clinical
isolates
by
two
simple
methods. Methods Mol Biol 2010;642:143-57.
Pearson JP, Van Delden C, Iglewski BH. Active efflux and diffusion are
involved in transport of Pseudomonas aeruginosa cell-to-cell signals.
J Bacteriol 1999;181(4):1203-10.
Whalen KE, Poulson-Ellestad KL, Deering RW, Rowley DC, Mincer TJ.
Enhancement of antibiotic activity against multidrug-resistant bacteria
by the efflux pump inhibitor 3,4-dibromopyrrole-2,5-dione isolated
from a Pseudoalteromonas sp. J Nat Prod 2015;78(3):402-12.
Kourtesi C, Ball AR, Huang YY, Jachak SM, Vera DM, Khondkar P,
et al. Microbial efflux systems and inhibitors: Approaches to drug
discovery and the challenge of clinical implementation. Open Microbiol
J 2013;7:34-52.
Lomovskaya O, Warren MS, Lee A, Galazzo J, Fronko R, Lee M, et al.
Identification and characterization of inhibitors of multidrug resistance
efflux pumps in Pseudomonas aeruginosa: Novel agents for combination
therapy. Antimicrob Agents Chemother 2001;45(1):105-16.
Asian J Pharm Clin Res, Vol 9, Issue 3, 2016, 324-327
Suresh et al.
Misra R, Morrison KD, Cho HJ, Khuu T. Importance of real-time
assays to distinguish multidrug efflux pump-inhibiting and outer
membrane-destabilizing activities in Escherichia coli. J Bacteriol
;197(15):2479-88.
Lamers RP, Cavallari JF, Burrows LL. The efflux inhibitor
phenylalanine-arginine beta-naphthylamide (PAßN) permeabilizes
the outer membrane of Gram-negative bacteria. PLoS One
;8(3):e60666.
Xing L, Barnie PA, Su Z, Xu H. Development of efflux pumps and
inhibitors (EPIs) in A. baumanii. Clin Microbial 2014;3:135.
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