Possible Role of Curcumin as an Efflux Pump Inhibitor in Multi Drug Resistant Clinical Isolates of Pseudomonas aeruginosa
Nidhi Negi1, Pradyot Prakash2, Mohan Lal Gupta3, Tribhuban Mohan Mohapatra4
1 Junior Resident, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, (U.P.), India.
2 Assistant Professor, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, (U.P.), India.
3 Junior Resident, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, (U.P.), India.
4 Professor, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, (U.P.), India.
NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Nidhi Negi, Junior Resident, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, (U.P.), India. Phone : 9456513444, E-mail : nidhinegi28@gmail.com
Introduction: Multidrug resistant non-fermenters are continuously increasing in hospital and ICU settings. One of the mechanisms of developing drug resistance is possession of efflux pump through which bacteria extrude antimicrobial agents and other toxic substances. If these efflux channels are blocked or inhibited, increased drug concentration can be achieved in a bacterial cell with optimal drug dose. Present study was aimed to investigate role of curcumin as efflux pump inhibitor (EPI) and to compare its activity with a known EPI like phe-arg-beta-naphthylamide (PAβN).
Materials and Methods: A total of 170 clinical isolates of Pseudomonas aeruginosa were taken, antimicrobial susceptibility was performed by disc diffusion test and minimum inhibitory concentration (MIC) against selected drugs before and after adding known synthetic EPI, PAβN (20mg/L). Out of these, 30 multidrug resistant strains were taken and MIC was performed with curcumin (50mg/L) with and without selected drugs.
Results : Significant reduction in MIC was observed after adding curcumin (50mg/L) with selected antimicrobial agents in 9/30 (30%) of multi drug resistant (MDR) isolates of Pseudomonas aeruginosa, while no change in MIC was observed when curcumin (50mg/L) was used alone, indicating its efflux pump inhibitor activity.
Conclusion: This study suggests role of efflux pump in development of drug resistance which can be overcome by use of an efflux pump inhibitor, with more emphasis on compound like curcumin which will have less or no adverse effects if used in vivo.
Introduction
Pseudomonas aeruginosa is increasingly recognized as an emerging opportunistic pathogen especially in hospital settings and well known for its drug resistance. The intrinsic multidrug resistance of Pseudomonas aeruginosa is a result of combination of factors which include an outer membrane with low permeability and the expression of efflux systems [1]. Efflux means transport of a substance out of cell. Multidrug resistance (MDR) can be defined as resistance to three or more antimicrobial classes [2]. Efflux pumps attribute to multidrug resistance by extruding various classes of antimicrobial agents and various other substances such as dyes, detergents, biocides and molecules required for cell to cell signalling [3,4]. Bacterial efflux systems generally fall into five classes, the major facilitator (MF) superfamily, the ATP-binding cassette (ABC) family, the resistance-nodulation-division (RND) family, the small multidrug resistance (SMR) family and the multidrug and toxic compound extrusion (MATE) family [5].
Recently, it was observed that the use of efflux pump inhibitors (EPIs) is helpful to potentiate the activity of antimicrobial agents as many efflux pumps possess structural homology with them [6]. Hence to develop an effective EPI, there is a need to understand the basic structural and physiological mechanisms of the efflux pumps associated with drug resistance [7]. The mechanism of action of EPIs is through competitive inhibition where these become the substrate of efflux pumps instead of the target antibiotics. As long as these inhibitors are extruded outside the cells by the efflux pumps, the concentration of antibiotic increases intracellularly, eventually leading to cell death. Peptidomimetic compounds with phenylalanine arginyl beta naphthylamide (PAβN) also known as MC-207,110 have been used as broad spectrum EPI for Pseudomonas aeruginosa [8,9]. In addition to inhibition of efflux pump activity, PAβN permeabilizes bacterial membranes [10]. Among other synthetic compounds, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,4-dinitrophenol (DNP) and verapamil has been investigated as potential inhibitor of efflux pump activity in Pseudomonas aeruginosa and Mycobacterium tuberculosis [8,11].
Curcumin is a natural extract derived from rhizomes of the plant Curcuma longa (Zingiberaceae) grown in regions of India and South East Asia. Extensive research in this field has shown its activity as an anti-inflammatory, antiviral, antioxidant, and anticancer agent[12–16]. It has been observed that curcumin potentiated the effect of various antibiotics against Staphylococcus aureus and reduced the virulence in animal pathogenicity models [17–18].
Among other natural compounds, plant alkaloid reserpine was shown in a study as an efflux pump inhibitor of Bacillus subtilis and Streptococcus pneumoniae [19,20]. Other studies have also identified a group of cationic berberine alkaloids as a potential efflux pump inhibitor [21]. For the first time, in present study, we investigated the role of curcumin as EPI by observing decrease in MIC values of selected antimicrobial agents against multidrug resistant (MDR) Pseudomonas aeruginosa strains tested and compared its activity with a known synthetic EPI.
Materials and Methods
A total of 170 isolates of Pseudomonas aeruginosa, from clinical samples were processed in the Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University from June 2011 to May 2012. Antimicrobial susceptibility testing was done by modified Kirby Bauer method with meropenem (10μg/disc) (Astra Zenica Pharma India), carbenicillin (100μg/disc), ceftazidime (30μg/disc), gentamicin (10μg/disc) and ciprofloxacin (5μg/disc) Hi-Media, Mumbai, India. Further MIC testing was carried out by agar dilution method.
Preparation of agar dilution plates: A series of dilution of antibiotics ranging from 2048 μg/ml to 2μg/ml were made from stock solution in Mueller Hinton agar plates as shown in [Table/Fig-1], which was allowed to equilibrate in water bath at 48oC- 50oC. The agar and antibiotics were mixed thoroughly and poured into petri dish in a depth of 4mm. The agar was allowed to solidify at room temperature and stored at 4-8oC. Pseudomonas aeruginosa ATCC (27853) was used as control.
Preparation of antibiotic dilution range
| Amount of antibiotic solution added (μl)† (Original stock solution concentration of 10mg/ml) | Volume of Mueller Hinton agar (ml) | Final concentration of antibiotic in 20 ml of medium used in each plate (μg/ml) |
|---|
| 8192 | 11.808 | 4096 |
| 4096 | 15.904 | 2048 |
| 2048 | 17.952 | 1024 |
| 1024 | 18.976 | 512 |
| 512 | 19.488 | 256 |
| 256 | 19.744 | 128 |
| 128 | 19.872 | 64 |
| 64 | 19.936 | 32 |
| 32 | 19.968 | 16 |
| 16 | 19.984 | 8 |
| 8 | 19.992 | 4 |
| 4 | 19.996 | 2 |
Interpretation
The results were interpreted according to the CLSI guidelines 2013, for MIC break points to be sensitive, intermediate and resistant [22].
MIC of antimicrobial agents with PAβN: Two different concentrations of PABN were taken to check the appropriate concentration for this study. PAβN (M.P biomedicals India) (20 mg/L and 50mg/L) [8], along with each antibotic of appropriate dilutions (ranging from 4096 μg/ml to 2 μg/ml), were added to the corresponding amount of molten Mueller Hinton agar. This was allowed to equilibrate in water bath at 48o-50oC. Media was poured in petridishes, allowed to settle and MIC testing were carried out on it.
MIC of antimicrobial agents with curcumin: Curcumin (Hi Media, Mumbai, India) was dissolved in dimethyl sulfoxide (Sigma Aldrich, India), and a stock concentration of 10 mg/ml was stored at -20°C. Final test concentrations consisted of 50, 30, 20, 15, 10, and 5μg/ml of curcumin solution [23]. Appropriate dilutions (ranging from 4096 μg/ml to 2 μg/ml) of each antimicrobial solution were added to the corresponding amount of molten Mueller Hinton agar which was allowed to equilibrate in water bath at 48o - 50oC along with curcumin at concentration mentioned above to determine its EPI activity. Mueller Hinton agar was prepared even with variable concentrations of curcumin, without antimicrobial, so as to find whether it has any antimicrobial effect when used alone.
Results
Out of total 170 strains, 86 (50.58%) were resistant to atleast one antimicrobial agent tested and 30 (17.64%) were observed to be resistant against all the selected antimicrobial agents namely meropenem, carbenicillin, gentamicin, ceftazidime and ciprofloxacin according to MIC studies. It was observed that after increasing the concentration of PAβN from 20mg/L to 50 mg/L with those selected antimicrobials namely meropenem and carbenicillin the increase in sensitivity was only 3% and 13.33% respectively, which was not significant (p = 0.77). So the concentration of 20mg/L was considered appropriate for the present study.
Isolates which were resistant earlier and after addition of PAβN (20 mg/L) whose MIC dropped within the sensitive range, were considered to have efflux pump mediated resistance mechanism [Table/Fig-2]. No major effect of curcumin with antibiotics was observed till 15μg/ml but when concentration was increased from 20- 50μg/ml a considerable increase in sensitivity of strains was noted [Table/Fig-3a,b,c,d,e]. Best results were obtained at concentration of 50μg/ml which has been included in our study. None of the 30 MDR strains including ATCC strain of Pseudomonas aeruginosa was found susceptible to curcumin alone at any of the above concentration.
Susceptibility pattern of Pseudomonas aeruginosa as determined by MIC using selected antimicrobial agents with and without adding PAβN
| Name of antimi-crobial agent | Total no of sensitive strains after MIC testing % n=170 | Total no of resistant strains after MIC testing % | Total number of sensitive strains after addition of PAβN (%) | Number of strains showing resistance due to efflux pump(%) |
|---|
| Meropenem | 120(70.58%) | 50(29.41%) | 133(78.23%) | 13(13/50=26%) |
| Carbenicillin | 109(64.44%) | 61(35.88%) | 121(71.17%) | 12(12/61=19.67%) |
| Ceftazidime | 99(58.23%) | 71(41.76%) | 114(67.05%) | 15(15/71=21.12%) |
| Gentamicin | 83(48.82%) | 87(51.17%) | 96(58.77%) | 13(13/ 87=14.9%) |
| Ciprofloxacin | 74(43.52%) | 98(57.64%) | 83(48.82%) | 9(9/98=9.18%) |
Note: As per CLSI guidelines 2013 for meropenem and gentamicin: Sensitive ≤ 4μg/ml; Intermediate susceptibility 8μg/ml; Resistance ≥ 16μg/ml; for carbenicillin: Sensitive ≤ 128 μg/ml; Intermediate susceptibility 256 μg/ml; Resistance ≥ 512 μg/ml; for ceftazidime: Sensitive ≤ 8μg/ml; Intermediate susceptibility 16μg/ml; Resistance ≥ 32μg/ml; for ciprofloxacin: Sensitive ≤ 1μgm/ml; Intermediate susceptibility 2μgm/ml; Resistance ≥ 4μgm/ml. Note: In this data intermediate susceptibility was considered as sensitive
Multi drug resistant strains which became sensitive after adding curcumin with antimicrobial agents (meropenem) at its various concentrations
| Antimicrobial agent (μg/L) | Curcumin | Total no of resistant strains after MIC testing % |
|---|
| Meropenem | 5 | 0 |
| 8-16 | 10 | 0 |
| 15 | 0 |
| 20 | 3 |
| 30 | 3 |
| 50 | 5 |
Multi drug resistant strains which became sensitive after adding curcumin with antimicrobial agents (carbenicillin) at its various concentrations
| Antimicrobial agent (μg/L) | Curcumin (μg/L) | No of sensitive strains |
|---|
| Carbenicillin | 5 | 0 |
| 16 - 64 | 10 | 0 |
| 15 | 1 |
| 20 | 3 |
| 30 | 5 |
| 50 | 8 |
Multi drug resistant strains which became sensitive after adding curcumin with antimicrobial agents (gentamicin) at its various concentrations
| Antimicrobial agent (μg/L) | Curcumin | Total no of resistant strains after MIC testing % |
|---|
| Ceftazidime | 5 | 0 |
| 8-16 | 10 | 0 |
| 15 | 2 |
| 20 | 2 |
| 30 | 5 |
| 50 | 6 |
Multi drug resistant strains which became sensitive after adding curcumin with antimicrobial agents (ceftazidime) at its various concentrations
| Antimicrobial agent (μg/L) | Curcumin | Total no of resistant strains after MIC testing % |
|---|
| Gentamicin | 5 | 0 |
| 8-16 | 10 | 0 |
| 15 | 0 |
| 20 | 1 |
| 30 | 1 |
| 50 | 3 |
Multi drug resistant strains which became sensitive after adding curcumin with antimicrobial agents (ciprofloxacin) at its various concentrations
| Antimicrobial agent (μg/L) | Curcumin | Total no of resistant strains after MIC testing % |
|---|
| Ciprofloxacin | 5 | 0 |
| 2-4 | 10 | 0 |
| 15 | 0 |
| 20 | 0 |
| 30 | 1 |
| 50 | 3 |
On interpretation of results, the EPI activity of curcumin was seen best with carbenicillin, followed by ceftazidime and meropenem. Finally out of the 30 drug resistant strains 9 became sensitive after adding curcumin (50μg/ml) with one or the other antimicrobial agents [Table/Fig-4].
Multi drug resistant strains which became sensitive after adding PAβN and curcumin
| Name of antimicrobials | Strains which became sensitive after adding PAβN(20μg/ml) n=30 | Strains which became sensitive after adding Curcumin (50μg/ml) |
|---|
| Meropenem | 7 (23.33%) | 5 (16.6%) |
| Carbenicillin | 2 (6.6%) | 8 (26.66%) |
| Ceftazidime | 6 (20%) | 6 (20%) |
| Gentamicin | 0 (0%) | 3 (10%) |
| Ciprofloxacin | 0 (0%) | 3 (10%) |
Discussion
Pseudomonas aeruginosa is an important pathogen frequently associated with healthcare associated infections, particularly in critically ill or immunocompromised patients. The true prevalence of MDR Pseudomonas aeruginosa is not yet well established. However, rates of resistance increased for imipenem, quinolones by 15-23%, 15-32% and for third generation cephalosporins 16-25% [24].
A recent study showed that PAβN may be used for phenotypic screening for presence of efflux pump activity in clinical isolates of Pseudomonas aeruginosa along with genotypic methods [25]. Further, another study observed 4 to 8-fold reduction of meropenem MICs among Acinetobacter baumanii using PAβN as efflux pump inhibitor [26].
Among 170 strains of Pseudomonas aeruginosa included in the present study, antimicrobial resistance was observed to be contributed by efflux pump for one drug or the other drug in 27% (23/85) strains. Further among 30 MDR isolates, curcumin was responsible for reduction of MIC of 30% (9/30) of MDR Pseudomonas aeruginosa isolates to the level of susceptibility towards one or the other 5 drugs employed. In the present study 3 resistant isolates to gentamicin and ciprofloxacin became sensitive after adding curcumin and not after adding PAβN. The above observation indicates that curcumin is inhibiting the expression of efflux pump which are not inhibited by PAβN. This indicates the potential use of curcumin as an adjunct to antimicrobials in treatment of drug resistant bacterial infection.
Conclusion
The mechanism by which curcumin has potentiated the effect of antimicrobial compounds in the present study appears to be due to inhibition of bacterial efflux pump which needs further study for its genotypic validation.
Note: As per CLSI guidelines 2013 for meropenem and gentamicin: Sensitive ≤ 4μg/ml; Intermediate susceptibility 8μg/ml; Resistance ≥ 16μg/ml; for carbenicillin: Sensitive ≤ 128 μg/ml; Intermediate susceptibility 256 μg/ml; Resistance ≥ 512 μg/ml; for ceftazidime: Sensitive ≤ 8μg/ml; Intermediate susceptibility 16μg/ml; Resistance ≥ 32μg/ml; for ciprofloxacin: Sensitive ≤ 1μgm/ml; Intermediate susceptibility 2μgm/ml; Resistance ≥ 4μgm/ml. Note: In this data intermediate susceptibility was considered as sensitive
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