Ibuprofen-Mediated Reversal of Fluconazole Resistance in Clinical Isolates of Candida
Monika Sharma1, Debasis Biswas2, Aarti Kotwal3, Bhaskar Thakuria4, Barnali Kakati5, Bhupendra Singh Chauhan6, Abhishek Patras7
1 Senior Resident, Department of Microbiology, Govt. Medical College, Jammu, Jammu & Kashmir, India.
2 Professor, Department of Microbiology, AIIMSBhopal, Bhopal, India.
3 Associate Professor, Department of Microbiology, Himalayan Institute of Medical Sciences, Jolly Grant, Dehradun, India.
4 Associate Professor, Department of Microbiology, Subharti Medical College, Meerut, Uttar Pradesh, India.
5 Associate Professor, Department of Microbiology, Himalayan Institute of Medical Sciences, Jolly Grant, Dehradun, India.
6 Lab Technician, Department of Microbiology, Himalayan Institute of Medical Sciences, Jolly Grant, Dehradun, India.
7 Lab Technician, Department of Microbiology, Himalayan Institute of Medical Sciences, Jolly Grant, Dehradun, India.
NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Debasis Biswas, Professor, Department of Microbiology, AIIMS Bhopal, Bhopal-402624, India. E-mail : dbiswas71@rediffmail.com
Introduction: In view of the increasing prevalence of invasive Candidiasis in today’s health-care scenario and the emergence of fluconazole resistance among clinical isolates of Candida, we sought to determine if Ibuprofen could elicit a reversal of fluconazole resistance and thereby offer a potential therapeutic breakthrough in fluconazole-resistant Candidiasis.
Materials and Methods: We selected 69 clinical isolates of Candida, which demonstrated an MIC of >32 μg/ml for fluconazole, and subjected them to broth microdilution in presence and absence of Ibuprofen.
Results: Forty two of the 69 isolates (60.9%) demonstrated reversal of Fluconazole resistance with concomitant use of Ibuprofen. This was characterized by significant species-wise variation (p=0.00008), with all the C. albicans isolates and none of the C. glabrata isolates demonstrating such reversal. Only 22.2% and 37.7% of C. krusei and C. tropicalis isolates respectively showed Ibuprofen-mediated reversal of Fluconazole resistance.
Conclusion: Since Ibuprofen is a known efflux pump inhibitor, our findings hint at the possible mechanism of Fluconazole resistance in most of our Candida isolates and suggest a potential therapeutic alternative that could be useful in the majority of Fluconazole-resistant clinical isolates of Candida.
Introduction
Invasive candidiasis has emerged as the commonest form of opportunistic mycoses throughout the world. Apart from its widespread occurrence, it is often acutely progressive, difficult to diagnose and associated with increased hospital stay and high mortality rates [1–5]. Treatment of this condition has become further complicated owing to the relative rise in the proportion of non-albicansCandida isolates, which often demonstrate intrinsic resistance towards specific antifungal agents [6–11]. Moreover, there has been a documented increase in fluconazole resistance even among other species of Candida, including C. albicans, C. lusitaniae, C. tropicalis and C. dubliniensis [12–16], which has been partially attributed to the popular use of fluconazole as empirical antifungal therapy since the 1990s [17].
Azole resistance calls for the use of alternative antifungal drugs like echinocandins, voriconazole, posaconazole, ravuconazole and Amphotericin B. But constraints like high costs or adverse effects associated with these agents often limit their usage. Some recent pioneering studies have reported the modulating effect of verapamil, oestradiol, progesterone and ibuprofen on resistance of Candida isolates. While fluconazole MIC decreased in most strains after exposure to these modulators, this effect was particularly remarkable for Ibuprofen. The molecular basis of this reversal of resistance has also been demonstrated recently [18]. Thus, drugs capable of reversing fluconazole resistance might offer novel breakthroughs in the treatment of resistant Candida infections. As we have previously observed a high prevalence of fluconazole resistance among Candida isolates recovered in our centre [19], in this study we were interested in exploring if the resistant phenotype could be reversed in a proportion of these isolates by the use of Ibuprofen.
Materials and Methods
The study was conducted over a period of 18 months, from December 2009 till May 2011, in a tertiary care teaching hospital in the Himalayan region of northern India. Candida species isolated from various clinical samples such as blood, CSF, ET secretions, indwelling devices, urine, sputum etc., were processed for mycological characterization and antifungal susceptibility testing by broth microdilution method recommended by CLSI [10]. Sixty nine isolates with MIC>32 μg/ml for fluconazole were included in the study. Standard ATCC strains, viz. C. albicans ATCC 5314 and C. krusei ATCC 6258, were used as controls.
For testing the modulation of fungal sensitivity to fluconazole with Ibuprofen, the fluconazole-resistant candida isolates were cultured with fluconozole (Sigma-Aldrich), in presence and absence of ibuprofen (Sigma-Aldrich). Briefly, the respective isolates were sub-cultured on Sabouraud’s Dextrose Agar to ensure purity and viability and inoculum was prepared by picking five distinct colonies of approximately 1 mm diameter. Final inoculums size of 0.5 x 103 to 2.5 x 103 cfu/ml was achieved in sterile, 96-well, U-bottom microtiter plates, after dispensing 2X concentrations of fluconazole into the wells of columns 1 to 10 in 100 μl volumes. Serial doubling dilutions of fluconazole was used, ranging between 0.12 and 64.0 μg/ml. In growth-control wells 100 μl of sterile drug-free medium was added to the inoculums, while sterility-control wells contained drug-free medium only. In a separate microtiter plate, a similar plan of inoculation was used for monitoring the effect of Ibuprofen, except that 5 μg Ibuprofen drug solution (from a stock solution of 4.0 mg/ml) was dispensed immediately after addition of the fungal inocula. Following incubation for 48 hours at 350C, the microtiter plates were scored and growth in each well was compared with that of the growth control well. The MIC was defined as the lowest concentration of fluconazole in which a prominent decrease in turbidity was observed. MIC ≤ 8.0 μg/ml was considered to be sensitive, MIC between 16.0 and 32.0 μg/ml was considered to be sensitive-dose dependent and that ≥ 64.0 μg/ml was considered resistant.
Statistical Analysis
Chi-square test was performed to determine if the proportion of isolates demonstrating Ibuprofen- mediated reversal of fluconazole resistance was significantly different across the various species of Candida. p-value < 0.05 was considered significant.
Results
A total of 69 fluconazole-resistant clinical isolates of Candida spp. were included in the study. Majority of the isolates were C. tropicalis (n=33), followed by C. parapsilosis (n=21), C. krusei (n=9), C. glabrata and C. albicans (3 isolates each). While 42 of the 69 selected fluconazole-resistant isolates (60.9%) demonstrated inhibition of growth with the concomitant use of Ibuprofen and Fluconazole, such reversal was characterized by significant species-wise variation (p=0.00008). All the isolates of C. albicans and 95.2% of C.parapsilosis demonstrated significant reduction in MIC after concomitant culture with fluconazole and ibuprofen. However, reversal of resistance was shown by only 37.7% of C. tropicalis isolates. On the other hand, no inhibition of growth was observed with the concomitant use of Ibuprofen among any isolate of C. glabrata. Similarly out of the 9 isolates of C. krusei tested for modulation of sensitivity to fluconazole, 7 isolates did not show any inhibition of growth with the use of Ibuprofen [Table/Fig-1].
Species-wise distribution of clinical isolates of Candida demonstrating reversal of Fluconazole resistance with Ibuprofen
| Species | Resistant to fluconazole | Reversal after Ibuprofen (%) |
|---|
| C. albicans | 3 | 3 (100) |
| C. tropicalis | 33 | 17 (51.5) |
| C. glabrata | 3 | 0 (0) |
| C. parapsilosis | 21 | 20 (95.2) |
| C. krusei | 9 | 2 (22.2) |
| Total | 69 | 42 (60.9) |
| Chi-square | 23.87 |
| Degrees of freedom | 4 |
| p-value | 0.00008 |
Discussion
We have previously reported increased levels of fluconazole resistance among Candida isolates recovered from our centre [19]. Despite the rising prevalence of azole-resistance in Candida species, technical difficulties in the genetic manipulation of this diploid species have impeded the delineation of precise contributions of individual resistance mechanisms among clinical isolates of Candida [20]. However, this information is likely to be helpful in identifying potentially useful novel drug targets and in understanding their relative importance in clinical practice. Moreover, some of the mechanisms of antifungal resistance contribute to cross- resistance to other antifungal agents and, hence, knowledge of the same might aid clinical decision-making.
In view of earlier studies demonstrating ibuprofen to be a potential efflux pump inhibitor, [18-21] we inferred that the reversal of fluconazole resistance with ibuprofen, as observed by us, hints at the possible role of efflux pumps mediating azole resistance in majority of our isolates. Similar to our study, several other authors have also observed an in vitro synergistic effect of ibuprofen with fluconazole in the pathogenic yeast C. albicans [18-22]. In one such study, Pina-Vaz et al., studied the modulating effect of verapamil, oestradiol, progesterone and ibuprofen on azole-resistance in Candida. They included 42 clinical isolates of Candida (38 fluconazole resistant, 2 ATCC type strains and C. albicans strains with known mechanisms of fluconazole resistance). Incubating these strains with sub-inhibitory concentrations of different modulators they observed that fluconazole MIC decreased in most strains after exposure to modulators, including control strains with documented over-expression of efflux pump. This modulatory effect was particularly remarkable for ibuprofen. Resistance to itraconazole and voriconazole was also reverted with the help of these modulators. However no significant MIC variation was observed for C.krusei isolates [18]. In another study by the same group, synergistic interaction between ibuprofen and fluconazole was analysed in 62 clinical isolates and 5 control strains of C. albicans. It was observed that resistant isolates that reverted to susceptible after incubation with ibuprofen showed over-expression of genes encoding efflux pumps, viz. CDR1 and CDR2. Conversely, strains that did not revert displayed a remarkable increase in expression of the azole target gene, ERG11, along with CDR genes [21]. Moreover, in view of the importance of prostaglandins in fungal colonization, additional in vivo therapeutic benefit with ibuprofen can also result from its inhibitory effect on prostaglandin synthesis [23].
Our study revealed a varying effect of ibuprofen among the different species of Candida, which is indicative of heterogeneity in the mechanism of azole resistance among the various species. Previous studies have also described species-wise differences in the molecular mechanisms responsible for azole resistance in Candida. Vandeputte et al., investigated the mechanism of acquired azole resistance in a clinical isolate of C.tropicalis and observed over-expression of the gene coding for target enzyme, lanosterol 14 α-demethylase, together with a missense mutation in this gene, though no over-expression of efflux protein gene CtMDR1 was found [24]. On the other hand, Sanguinetti et al., reported up-regulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps in a set of fluconazole resistant and susceptible dose- dependent clinical isolates of C. glabrata, while no mutation or upregulation of the target enzyme was observed in any of these isolates [25]. Analysing the molecular mechanisms of resistance in clinical isolates of C. albicans recovered from an HIV-infected patient with recurrent oropharyngeal candidiasis, Martinez et al., noted over-expression of MDR and CDR genes encoding efflux pumps in isolates with decreased fluconazole susceptibility. However, over-expression of ERG11 gene encoding for the target enzyme of azoles was not observed in these isolates [26].
Antifungal resistance, as measured in vitro by MIC determination, is a function of the several genetic resistance mechanisms operating in any strain [20]. Since efflux pumps are encoded in Candida species by 2 gene families of transporters, viz. the CDR genes of the ATP-binding cassette super family and the MDR genes of the major facilitators class [25,27], we need to validate our findings in future studies by demonstrating the effect of ibuprofen on the quantitative level of expression of these genes in clinical isolates of Candida exposed to fluconazole.
Conclusion
Our study presents a simple phenotypic assay that is adaptable to the workload of a clinical microbiology laboratory and hints at the mechanism of fluconazole resistance in a majority of clinical isolates of Candida. We need to validate our findings in future studies by demonstrating the effect of ibuprofen on the quantitative level of expression of genes encoding efflux pump system in clinical isolates of Candida exposed to fluconazole.
[1]. Abelson JA, Moore T, Bruckner D, Deville J, Nielsen K, Frequency of fungemia in hospitalized pediatric inpatients over 11 years at a tertiary care institution Pediatrics 2005 116:61-67. [Google Scholar]
[2]. Chakrabarti A, Drug resistance in fungi–an emerging problem Regional Health Forum 2011 15:97-103. [Google Scholar]
[3]. Aquino VR, Lunardi LW, Goldani LZ, Barth AL, Prevalence, susceptibility profile for fluconazole and risk factors for candidemia in a tertiary care hospital in southern Brazil Braz J Infect Dis 2005 9:411-18. [Google Scholar]
[4]. Sheng WH, Wang JT, Lin MS, Chang SC, Risk factors affecting in-hospital mortality in patients with nosocomial infections J Formos Med Assoc 2007 106:110-18. [Google Scholar]
[5]. Morgan J, Meltzer MI, Plikaytis BD, Sofair AN, Huie-White S, Wilcox S, Excess mortality, hospital stay, and cost due to candidemia: a case-control study using data from population-based candidemia surveillance Infect Control Hosp Epidemiol 2005 26:540-47. [Google Scholar]
[6]. Trick WE, Fridkin SK, Edwards JR, Hajjeh RA, Gaynes RP, National Nosocomial Infections Surveillance System Hospitals. Secular trend of hospital-acquired candidemia among intensive care unit patients in the United States during 1989-1999 Clin Infect Dis 2002 35:627-30. [Google Scholar]
[7]. Pfaller MA, Jones RN, Doern GV, Sader HS, Messer SA, Houston A, Bloodstream infections due to Candida species: SENTRY antimicrobial surveillance program in North America and Latin America, 1997-1998 Antimicrob Agents Chemother 2000 44:747-51. [Google Scholar]
[8]. Pfaller MA, Diekema DJ, International Fungal Surveillance Participant Group. Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconazole susceptibility of bloodstream isolates of Candida Clin Microbiol Infect 2004 10(Suppl 1):11-23. [Google Scholar]
[9]. Krcmery V, Barnes AJ, Non-albicans Candida spp. causing fungaemia: pathogenicity and antifungal resistance J Hosp Infect 2002 50:243-60. [Google Scholar]
[10]. Chai YA, Wang Y, Khoo AL, Chan FY, Chow C, Kumarasinghe G, Predominance of Candidatropicalis bloodstream infections in a Singapore teaching hospital Med Mycol 2007 45:435-39. [Google Scholar]
[11]. Shivaprakasha S, Radhakrishnan K, Karim PM, Candidaspp., other than Candidaalbicans: a major cause of fungaemia in a tertiary care centre Indian J Med Microbiol 2007 25:405-07. [Google Scholar]
[12]. Lewis RE, Viale P, Kontoyiannis DP, Douglas LJ, The potential impact of antifungal drug resistance mechanisms on the host immune response of Candida Virulence 2012 3(4):368-76. [Google Scholar]
[13]. Hajjeh RA, Sofair AN, Harrison LH, Lyon GM, Arthington-Skaggs BA, Mirza SA, Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program J Clin Microbiol 2004 42:1519-27. [Google Scholar]
[14]. Cuenca-Estrella M, Rodriguez D, Almirante B, Morgan J, Planes AM, Almela M, In vitro susceptibilities of bloodstream isolates of Candida species to six antifungal agents: results from a population-based active surveillance programme, Barcelona, Spain, 2002-2003 J Antimicrob Chemother 2005 55:194-99. [Google Scholar]
[15]. Pfaller MA, Diekema DJ, Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candidaalbicans and Aspergillus fumigatus J Clin Microbiol 2004 42:4419-31. [Google Scholar]
[16]. Pfaller MA, Antifungal drug resistance: Mechanisms, epidemiology and consequences for treatment Am J Med 2012 125(Suppl 1):S3-13. [Google Scholar]
[17]. Tan TY, Tan AL, Tee NW, Ng LS, A retrospective analysis of antifungal susceptibilities of Candida bloodstream isolates from Singapore hospitals Ann Acad Med Singapore 2008 37:835-40. [Google Scholar]
[18]. Pina-Vaz C, Rodrigues AG, Costa-de-Oliveira S, Ricardo E, Mårdh PA, Potent synergic effect between ibuprofen and azoles on Candida resulting from blockade of efflux pumps as determined by FUN-1 staining and flow cytometry J Antimicrob Chemother 2005 56:678-85. [Google Scholar]
[19]. Kotwal A, Biswas D, Sharma JP, Gupta A, Jindal P, An observational study on the epidemiological and mycological profile of Candidemia in ICU patients Med Sci Monit 2011 17:CR663-68. [Google Scholar]
[20]. MacCallum DM, Coste A, Ischer F, Jacobsen MD, Odds FC, Sanglard D, Genetic dissection of azole resistance mechanisms in Candidaalbicans and their validation in a mouse model of disseminated infection Antimicrob Agents Chemother 2010 54:1476-83. [Google Scholar]
[21]. Ricardo E, Costa-de-Oliveira S, Dias AS, Guerra J, Rodrigues AG, Pina-Vaz C, Ibuprofen reverts antifungal resistance on Candidaalbicans showing over expression of CDR genes FEMS Yeast Res 2009 9:618-25. [Google Scholar]
[22]. Arai R, Sugita T, Nishikawa A, Reassessment of the in vitro synergistic effect of fluconazole with the non-steroidal anti-inflammatory agent ibuprofen against Candidaalbicans Mycoses 2005 48:38-41. [Google Scholar]
[23]. Alem MA, Douglas LJ, Effects of aspirin and other nonsteroidal anti-inflammatory drugs on biofilms and planktonic cells of Candidaalbicans Antimicrob Agents Chemother 2004 48:41-47. [Google Scholar]
[24]. Vandeputte P, Larcher G, Bergès T, Renier G, Chabasse D, Bouchara JP, Mechanisms of azole resistance in a clinical isolate of Candidatropicalis Antimicrob Agents Chemother 2005 49:4608-15. [Google Scholar]
[25]. Sanguinetti M, Posteraro B, Fiori B, Ranno S, Torelli R, Fadda G, Mechanisms of azole resistance in clinical isolates of Candidaglabrata collected during a hospital survey of antifungal resistance Antimicrob Agents Chemother 2005 49:668-79. [Google Scholar]
[26]. Martínez M, López-Ribot JL, Kirkpatrick WR, Bachmann SP, Perea S, Ruesga MT, Heterogeneous mechanisms of azole resistance in Candidaalbicans clinical isolates from an HIV-infected patient on continuous fluconazole therapy for oropharyngeal candidosis J Antimicrob Chemother 2002 49:515-24. [Google Scholar]
[27]. Sanglard D, Ischer F, Monod M, Bille J, Cloning of Candidaalbicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene Microbiology 1997 143(Pt 2):405-16. [Google Scholar]