|Year : 2015 | Volume
| Issue : 1 | Page : 84-86
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 Choudhury1, A Das Talukdar1, AP Maurya2, M Dutta Choudhury1, D Dhar (Chanda)3, A Chakravarty3, A Bhattacharjee2
1 Department of Life Science and Bioinformatics, Assam University, Silchar, India
2 Department of Microbiology, Assam University, Silchar, India
3 Silchar Medical College and Hospital , Silchar, Assam, India
|Date of Submission||19-Aug-2013|
|Date of Acceptance||24-Mar-2014|
|Date of Web Publication||5-Jan-2015|
Department of Microbiology, Assam University, Silchar
Source of Support: Department of Biotechnology, Govt. of India,, Conflict of Interest: None
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.
Keywords: Efflux pump, fluroquinolone, MexAB-OprM
|How to cite this article:|
Choudhury D, Talukdar A D, Maurya A P, Choudhury M D, Dhar (Chanda) D, Chakravarty A, Bhattacharjee A. 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
|How to cite this URL:|
Choudhury D, Talukdar A D, Maurya A P, Choudhury M D, Dhar (Chanda) D, Chakravarty A, Bhattacharjee A. 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 [serial online] 2015 [cited 2019 Nov 19];33:84-6. Available from: http://www.ijmm.org/text.asp?2015/33/1/84/148388
| ~ Introduction|| |
Pseudomonas aeruginosa, being one of the leading opportunistic pathogens, represents solemn threat for treatment of nosocomial infections.  The situation has been further intricated by its ability to acquire resistance against series of antimicrobial agents.  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 (DNA gyrase and/or topoisomerases) and overexpression of antibiotic efflux pumps (EPs). 
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. 
Increasing rate of FQ resistance in nosocomial isolates of P. aeruginosa are reported by large- scale surveillance studies.  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|| |
A total of 234 non-duplicate, consecutive clinical isolates of P. aeruginosa were obtained from stool, pus, urine, and throat swab of the patients who were admitted or attended OPDs of a tertiary referral hospital of north east India from March 2012 to February 2013.
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).  The MIC (Minimum inhibitory concentration) reduction assay was performed with ciprofloxacin alone and in combination with CCCP at concentration of 20 μg/ml. 
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.  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. 
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.  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). 
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. 
| ~ Results|| |
Out of the 234 isolates, 56 (23%) were found to be positive for EP mediated fluroquinolone resistance. A sharp 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].
|Figure 1: Polymerase chain reaction confirms the presence of mexA gene in the isolates|
Click here to view
All the isolates showed multi-drug resistance (MDR) phenotypes, whereas polymixin B was found to have moderate activity.
The MIC 50 and MIC 90 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.
|Table 2: MIC50 and MIC90 of efflux-mediated FQ resistant P. aeruginosa isolates|
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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.  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 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;  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.  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.  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.  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 the work. We also acknowledge the help from Assam University biotech hub for providing laboratory facility to complete this work.
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[Table 1], [Table 2]