Indian Journal of Medical Microbiology, (2015) 33(1): 84-86 1

Original Article

Contribution of efflux pumps in fluroquinolone resistance in multi-drug resistant

nosocomial isolates of Pseudomanas aeruginosa from a tertiary referral hospital in
north east India
D Choudhury, A Das Talukdar, AP Maurya, M Dutta Choudhury, D Dhar (Chanda), A Chakravarty, *A Bhattacharjee

Abstract
Background: Pseudomonas aeruginosa is one of the leading opportunistic pathogen and its ability to acquire resistance
against series of antimicrobial agents confine treatment option for nosocomial infections. Increasing resistance to
fluroquinolone (FQ) agents has further worsened the scenario. The major mechanism of resistance to FQs includes
mutation in FQs target genes in bacteria (DNA gyrase and/or topoisomerases) and overexpression of antibiotic efflux
pumps. Objective: We have investigated the role of efflux pump mediated FQ resistance in nosocomial isolates of
P. aeruginosa from a tertiary referral hospital in north eastern part of India. Materials and Methods: A total of 234
non-duplicate, consecutive clinical isolates of P. aeruginosa were obtained from a tertiary referral hospital of north-east
India. An efflux pump inhibitor (EPI), carbonyl cyanide m-chlorophenylhydrazone (CCCP) based method was used for
determination of efflux pump activity and multiplex polymerase chain reaction (PCR) was performed for molecular
characterisation of efflux pump. Minimum inhibitory concentration (MIC) reduction assay was also performed for
all the isolates. Results and Conclusion: A total number of 56 (23%) have shown efflux mediated FQ resistance.
MexAB-OprM efflux system was predominant type. This is the first report of efflux pump mediated FQ resistance from
this part of the world and the continued emergence of these mutants with such high MIC range from this part of the
world demands serious awareness, diagnostic intervention, and proper therapeutic option.

Key words: Efflux pump, fluroquinolone, MexAB-OprM

Introduction (DNA gyrase and/or topoisomerases) and overexpression of

Pseudomonas aeruginosa, being one of the leading
opportunistic pathogens, represents solemn threat for
treatment of nosocomial infections.[1] The situation has been
further intricated by its ability to acquire resistance against
series of antimicrobial agents.[2] Fluoroquinolones (FQs)
are broad-spectrum antibacterial agents commonly used to
treat P. aeruginosa infections. But unfortunately, increasing
resistance to FQ agents has worsened the scenario and limits
the treatment options. The major mechanism of resistance
to FQs includes mutation in FQs target genes in bacteria
*Corresponding author (email: <ab0404@gmail.com>)
Department of Life Science and Bioinformatics (DC, ADT, MDC),
and Department of Microbiology (APM, AB), Assam University,
Silchar, Silchar Medical College and Hospital (DDC, AC), Silchar,
Assam, India
Received: 19-08-2013
Accepted: 24-03-2014
antibiotic efflux pumps (EPs).[3]
It has been previously reported that in P. aeruginosa,
interplay of MexAB-OprM and target mutations are
responsible for conferring high-level FQ resistance. Cells
carrying double mutation in gyrA and mexR that encode
DNA gyrase and repressor for MexAB-OprM operon
exhibited 1,024 times higher FQ resistance than the cells
lacking MexAB-OprM.[4]
Increasing rate of FQ resistance in nosocomial isolates
of P. aeruginosa are reported by large- scale surveillance
studies.[4] However, mechanism of FQ resistance are still
not available from this region of the world. Hence, the
present study was undertaken to investigate EP-mediated
FQ resistance and also the possibility of other acquired
resistance mechanism in nosocomial isolates of
P. aeruginosa from a tertiary referral hospital of north east
India.

Materials and Methods

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A total of 234 non-duplicate, consecutive clinical
PMID:
*** isolates of P. aeruginosa were obtained from stool, pus,
urine, and throat swab of the patients who were admitted
DOI: or attended OPDs of a tertiary referral hospital of north east
10.4103/0255-0857.148388
India from March 2012 to February 2013.
January - March 2015 Choudhury, et al.: Efflux pump mediated fluroquinolone resistance in Pseudomanasaeruginosa
Phenotypic detection of efflux pump activity
Efflux pump activity of the strains were
phenotypically detected by double disc synergy test
using ciprofloxacin (10 μg, Himedia, Mumbai) and
carbonyl cyanide m-chlorophenylhydrazone (CCCP, 100
mM, Himedia, Mumbai, India).[5] The MIC (Minimum
inhibitory concentration) reduction assay was performed
with ciprofloxacin alone and in combination with CCCP at
concentration of 20 μg/ml.[2]
Molecular characterisation of efflux pump system
The presence of the MexAB-OprM, MexCD-OprJ,
and MexEF-OprN efflux system was determined using
polymerase chain reaction (PCR). The primers used were as
described previously.[6] PCR amplifications were performed
using 25 μl of total reaction volume. Reactions were run
under the following conditions: initial denaturation at 95°C
for 2 min, 32 cycles of 95°C for 25 sec, 60°C for 45 sec,
72°C for 1 min and final extension at 72°C for 3 min. PCR
products were examined on 1.0% (w/v) agarose gels.
Investigation of plasmid mediated quinolone resistance
The plasmid mediated FQ resistant determinants, qnrA,
qnrB, qnrS, and qepA were detected by PCR. The reaction
condition and primers used were as described earlier.[7]
Antibiotic susceptibility
Antibiotic susceptibility testing was performed on
Mueller-Hinton agar (Himedia, Mumbai, India) plates
by Kirby Bauer disc diffusion method and interpreted
as per Clinical and Laboratory Standards Institute
(CLSI) recommendations.[8] The antibiotics tested were,
ciprofloxacin (10 μg), amikacin (30 μg), gentamicin
(10 μg), imipenem (10 μg), faropenem (10 μg), carbenicillin
(10 μg), polymixin B (300 μg), tigecyclin (15 μg),
ceftazidime (30 μg), pipericillin-tazobactum (100/10 μg)
(Himedia, Mumbai, India).
Determination of minimum inhibitory concentration
MIC was determined on Muller-Hinton agar plates by
agar dilution method against levofloxacin, ofloxacin, and
ciprofloxacin (Himedia, Mumbai, India).[8]
DNA fingerprinting by ERIC (enterobacteriaceae repetitive
intergenic consensus sequence) PCR
Enterobacteriaceae repetitive intergenic consensus
sequence (ERIC) PCR was performed to determine clonal
relatedness of the isolates. The primers and reaction
conditions used were as described previously.[9]
Results
Out of the 234 isolates, 56 (23%) were found to be
positive for EP mediated fluroquinolone resistance. A sharp
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85
reduction in MIC was observed against ciprofloxacin when
CCCP was added at a fixed concentration of 20 μg/ml
[Table 1].
All the positive isolates were further subjected for
molecular characterisation by multiplex PCR assay and
it was found that MexAB-OprM was the predominant
type (n = 38) responsible for EP activity as no other
Mex-based mechanism could be detected by our target
primers. A previously confirmed strain of P. aeruginosa with
hyperexpression of MexAB-OprM efflux pump was used as
positive control [Figure 1].
All the isolates showed multi-drug resistance (MDR)
phenotypes, whereas polymixin B was found to have
moderate activity.
The MIC50 and MIC90 of the isolates were showed in
Table 2. It was found that majority of the isolates were
above the breakpoint for all the FQ drugs tested, whereas
for ciprofloxacin none of them were within the susceptible
range.
PCR results could not establish the presence of any
plasmid-mediated quinolone resistance in the isolates.
ERIC PCR revealed that all the isolates were clonally
non-related.
Discussion
The present study demonstrates a high prevalence of
EP mediated FQ resistance (23%) among clinical isolates
of P. aeruginosa from a tertiary care hospital of north east
India. It has been documented by various workers that
FQ-resistant organisms often exhibit MDR phenotype and
the MDR EP plays a crucial role in FQ resistance.[10] In our
study, we noticed that the EP mediated FQ resistant strains
are coupled with cross-resistance to structurally unrelated
antimicrobial agents. Hence, in this study, correlation
Table 1: Reduced MIC against ciprofloxacin with
the addition of CCCP
Antibiotics MIC50 (μg/ml) MIC90 (μg/ml)
Ciprofloxacin 32 128
Ciprofloxacin+CCCP 8 32
MIC: Minimum inhibitory concentration, CCCP: Carbonyl cyanide
m-chlorophenylhydrazone
Table 2: MIC50 and MIC90 of efflux-mediated FQ
resistant P. aeruginosa isolates
Antibiotics MIC50 (μg/ml) MIC90 (μg/ml)
Levofloxacin 16 128
Ciprofloxacin 32 >128
Ofloxacin 32 <128
MIC: Minimum inhibitory concentration, FQ: Fluroquinolone
86 Indian Journal of Medical Microbiology
Figure 1: Polymerase chain reaction confirms the presence of mexA
gene in the isolates
between FQ resistance and MDR has been established
through phenotypic and genotypic confirmation of EP
overexpression. It has been also observed that increased
efflux of FQ was the sole mechanism for conferring
resistance as acquired mechanisms were absent in study
isolates. MexAB-OprM is known to express constitutively
in P. aeruginosa under normal laboratory condition;[3]
however, in this study, it has been observed that some of the
strains are lacking this system.
It has been previously reported that EP selected mutants
exhibit four to eight fold higher MIC.[10] Present study supports
this fact as it was observed that for ciprofloxacin, MIC of
all the strains was above breakpoint range, which could be
significantly reduced when an EPI (CCCP) was added.
To the best of our knowledge, this study presents the first
report emphasizing the role of MDR EPs and FQ resistance
in clinical isolates of P. aeruginosa from India. These EP
mediated resistant strains often exhibit FQ and b-lactam
resistance and thereby limiting treatment options.[10] Further,
diagnostic microbiology laboratories must screen this
intrinsic resistance mechanism in routine basis, so that proper
antimicrobial agent can be selected minimizing the treatment
failure. Less attention has been paid on efflux mediated
FQ resistance where many authors take it as of secondary
concern for resistant phenotype.[1] However, in the current
study, it is proved that efflux pump plays a significant role
in FQ resistance in this hospital setting. The broad substrate
specificity of mex pumps and FQ being their universal
substrate, it is obvious that increased use of FQ will lead to
selection of efflux- mediated mutants and the continued
emergence of these mutants with such high MIC range from
this part of the world demands solemn awareness, diagnostic
intervention, and proper therapeutic option.
Acknowledgment
We would like to acknowledge the help of HOD, Department
of Microbiology, Assam University for providing infrastructure.
We sincerely acknowledge the financial support provided by
Department of Biotechnology (DBT), Govt. of India to carry out
www.ijmm.org
vol. 33, No. 1
the work. We also acknowledge the help from Assam University
biotech hub for providing laboratory facility to complete this work.
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How to cite this article: Choudhury D, Talukdar AD, Maurya AP,
Choudhury MD, Dhar (Chanda) D, Chakravarty A, et al. Contribution
of efflux pumps in fluroquinolone resistance in multi-drug resistant
nosocomial isolates of Pseudomanas aeruginosa from a tertiary referral
hospital in north east India. Indian J Med Microbiol 2015;33:84-6.
Source of Support: Department of Biotechnology, Govt. of India,
Conflict of Interest: None declared.
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