Studies of In-Vitro Susceptibility of Clinical Fungal Isolates from Immunocompromised Patients

Abstract

Clinical fungi were isolated from the blood samples of immunocompromised patients from Subhash Chandra Medical College Jabalpur in SDA with chloramphenicol @ 30μg/ml.the common isolates were Candida albicans, Aspergillus fumigatus, A. glaucus, A.terreus, A. niger, A. flavus, Penicillium sps. and Curvularia sps. The isolates were then subjected to in vitro susceptibility testing against different antifungals viz Amphotericin B, Ketocaonazole, itraconazole, clotrimazoleand fluconazole and also against natural herbs like neem, tulsi, garlic ginger, aloevra, turmeric etc by DDM method. Ketocaonazole was found to be highly effective amongst all antifungals and garlic was most effective amongst all natural antifungals.

Keywords

in vitro susceptibility testing, antifungals, immunocompromised

Introduction

During the past twenty five years there has been alarming increase in the fungal diseases with increasing use of chemotherapeutic agents, irradiation, immunosuppressive agents, broad spectrum antibiotics etc. there are many antimycotics which are used in treating fungal infections. The isolates collected in this program can be used to assess the activities of new antimicrobial agents and to aid in the development and validation of new susceptibility methods [1, 2, 3]. The treatment of choice for infected patients remains amphotericin B, itraconazole, voriconazole etc. the appropriate susceptibility is decided by various in vitro susceptibility methods [4,5,6,7,8]. The studies to date that have documented the efficacies of agar based methods for the testing of susceptibilities to fluconazole or voriconazole have generally included adequate number of Candida albicans species but few Candida glabrata isolates. From susceptibility testing one can determine the resistivity of the clinical isolate to various antifungals by determining the MIC range against a sufficiently large number of isolates.

The resistance towards the drugs is increasing day by day. The overuse of antifungal agents makes the fungi resistant towards it and simultaneously leads to chronic conditions in the patients. The drugs may even cause fatal problems. In order to avoid these effects alternative therapy has to be adopted which includes treatment through natural products. The antifungal activities of some spices and herbs have been reported by Pai et. al. 1995, Christina et. al, 2003, Anupama et. al. Apart from the synthetic antifungal antibiotic the naturally occurring herbal products and spices are known to have powerful antifungal properties. Research has proved their antifungal activities. Herbs like goldenseal, myrrh, walnut, licorice, lemongrass and spices like turmeric, cinnamon, clove, ajwain and medicinal plants like neem, tulsi, eucalyptus etc. have a great antifungal profile. Even the combination of these works in synergy and gives better results than a single herb [9].

Materials and Methods

Organisms

A total of 36 clinical isolates of filamentous fungi were isolated from blood samples collected from Subhash Chandra Medical College Jabalpur from 2004 to 2008. The clinical isolates included 10 isolates of Aspergillus fumigatus, 5 of A.flavus, 10 of A. niger, 3 of A. tereus, 4 of A. glaucus, 2 of Penicillium and 2 of Curvularia sps.

Antifungal agents

Voriconazole (Pfizer), fluconazole (Pfizer), itraconazole (Jansen), flucytosine (Sigma), amphotericin B (Hi media). Serial two fold dilutions were prepared exactly as outlined in NCCLS document M 27 A [11]. Final dilutions were prepared in RPMI 1640 medium (Sigma) buffered to pH 7.0 with 0.165M morpholine propane sulphonic acid (MOPS) buffer (Sigma). The dilutions were kept in Eppendorf’s tubes until used and stored at -4°C in Quick freezer.

Natural products

Turmeric, cinnamon, clove, ginger, garlic, neem, tulsi were used for the present study. The natural products were dried, powdered and then alcoholic extracts were prepared. The discs of natural products were prepared by soaking the discs in alcoholic extracts for a period of 24 hours and then dried in air.

Susceptibility testing

Reference antifungal Susceptibility testing of clinical isolates were performed by disc diffusion method as described by Barry et al [10]. MIC’s were determined with RPMI 1640 agar. An inoculum suspension adjusted to the turbidity of a 0.5 McFarland standard (~106cells/ml), and incubation at 35°C for 48 hours. MIC interpretive criteria were those published by Rex et al and the NCCLS document M 27 A and were as follows: susceptible, MIC <8μg/ml; susceptible-dose dependent, MIC = 16 to 32μg/ml; resistant, MIC > 64μg/ml. The interpretive criteria for disc test were those published by Barry et al [10]. and the NCCLS document M 27 A (11): susceptible, zone diameter of > 19mm; susceptible-dose dependent, zone diameter of 15 – 18 mm; resistant, zone diameter of < 14mm.

Quality control

QC was performed for disc diffusion method in accordance with NCCLS document M 27 A (11) by using C. albicans 3809 as reference culture.

Analysis of results

The diameters of the zone of inhibition for the test antifungals were measured in mm by disc diffusion method and their respective BMD MIC’s were also studied. The errors were identified as susceptibility by one method and resistivity by other method.

Results and Discussion

Amongst the 75 clinical samples studied 36 were positive for fungal growth. The common fungi isolated were Candida albicans, Aspergillus fumigatus, A. glaucus, A.terreus, A. niger, A. flavus, Penicillium sps. and Curvularia sps . The in vitro sensitivity results of the clinical isolates obtained by disc diffusion method against antifungals are expressed in table no. 1.The size of zone indicates the sensitivity of the culture to the antifungal used. From the table it is evident that ketoconazole is the most effective antifungal against all the clinical isolates. Amphotericin B is the most effective antifungal against Penicillium spp.followed by A. niger and Candida albicans and least effective to A. flavus and A. fumigatus. Ketoconazole is effective against Candida albicans followed by A. fumigatus, A. flavus and A. niger. Fluconazole is effective against Penicillium spp. and Curvularia sps followed by Candida albicans, A. niger and A. fumigatus. A. flavus was found to be resistant against fluconazole. 5FC was effective against, A. glaucus followed by Candida albicans, A.terreus and A. fumigatus. A. flavus and A. niger was found to be slightly sensitive to 5FC.

Table 1: Results of in vitro sensitivity tests of clinical isolates against antifungals expressed as zone of inhibition in cms.

Table no. 2: In vitro susceptibility of fungal isolates to plant extracts by disc diffusion method (Kirby Bauer) expressed as zone of inhibition in cms.

The clinical isolates were also subjected to in vitro sensitivity tests against spices and the results obtained are expressed in table 2. The extent of zone of inhibition of the fungal growth indicates the sensitivity of the fungi to the plant extract studied. It is evident from the results that garlic is most effective antifungal agent as the zone of inhibition is largest with the garlic disc followed by onion, ginger, cinnamon, neem, tulsi, aloe vera. Lemon grass was the least effective plant with antifungal property against all the clinical isolates tested. The present results are similar to those obtained by Ali et. al. 1999. From the table it is clear that garlic is the most effective spice against the clinical isolates tested and turmeric is the least effective spice. The order of susceptibility of Candida albicans to various spices are garlic > turmeric > ginger > onion > cinnamon. Though from zone of inhibition obtained it is evident that Candida albicans is susceptible to all the spices tested. The orders of susceptibility of A. fumigatus to various spices are garlic > onion > cinnamon >ginger >turmeric. A. fumigatus is highly susceptible to garlic, intermediately susceptible to onion, cinnamon and ginger while weakly susceptible to turmeric. The orders of susceptibility of A. flavus to various spices are garlic>onion>cinnamon >ginger. A. flavus is resistant to turmeric. The orders of susceptibility of A. niger to various spices are garlic>onion>cinnamon = ginger> turmeric. Though from zone of inhibition obtained it is evident that A. niger is susceptible to all the spices tested. The orders of susceptibility of Penicillium spp. to various spices are onion >garlic>cinnamon>turmeric>ginger.

It is clearly illustrated from the table that the common spices used in our daily life and the extracts of natural herbs effects the fungal growth adversely and therefore might be considered to control fungal diseases. The bioactive compounds in these plant extracts must be analyzed, purified and then used as drug for controlling fungal pathogens without the development of drug resistance in pathogens and also without any side effects.

The results obtained are in accordance with those obtained by Abraham et al 1999, Bassiouny et al 1986, and Stern et al, 1987. The antifungal activities of some spices have also been reported by Anupan et. al 2005 [12], Dorman et. al. 2000 [13], Erdogan et. al.2004 [14], Nuzrat et al 2013[15] and Hammer et. al. 1999 [16] which are coherent with the results obtained in the present investigation.

Conclusion

It is clearly illustrated from the study that the most effective antifungal against the mycotic agents studied is ketoconazole and therefore is of clinical relevance in controlling mycosis. In vitro susceptibility profile of mycotic agents obtained against spices suggests that the common spices used in our daily life affects fungal growth adversely and therefore might be considered to control fungal diseases. The bioactive compounds in these spices must be analyzed, purified and then used as drug for controlling fungal pathogens without the development of drug resistance in pathogens and also without any side effects.

Acknowledgement

This work was supported by University Grants Commission.

References

1. Jones, R. N., and the MYSTIC Advisory Board. Detection of emerging resistance patterns within longitudinal surveillance systems: data sensitivity and microbial susceptibility. J Antimicrob Chemother. 2000;46(Topic T2): 1-8.
2. Morace G, G Amato, F Bistoni, G Fadda, P Marone, MT Montagna, et al. Multicenter comparitive evaluation of six commercial systems and the National Committee for Clinical Laboratory Standards M27-A broth microdilution method for fluconazole susceptibility testing of Candida species. J Clin Microbial. 2002;40: 2953-2958
3. Goldman M, JC Pottage, DC Weaver. Candida krusei fungemia. Medicine (Baltimore). 1993:72:143-150.
4. Arendrup M, B Lundgren, IM Jensen, BS Hensen, N Frimondt Mofller. Comparison of E-test and a tablet diffusion test with the NCCLS broth microdilution method for fluconazole and amphotericin B susceptibility testing of Candida isolates. J Antimicrob Chemother. 2001;47: 521-526.
5. Kronvall G, I Karlson. Fluconazole and voriconazole multi disc testing of Candida species for disc calibration and MIC estimation. J Clin Microbial. 2001;39:1422-1428.
6. Masterton RG. Surveillance studies: how can they help the management of infection? J Antimicrob Chemother. 2000;46(Topic T2): 53-58.
7. Pfaller MA, DJ Diekema, SA Messer, L Boyken, RJ Hollis. The ARTEMIS Global Antifungal susceptibility program participants group. Activity of Fluconazole and voriconazole Determined by broth microdilution, E test and disc diffusion methods against 1586 recent clinical species: report from the Global Antifungal susceptibility program, J Clin Microbial. 2001;40:852-856.
8. Pfaller MA, SA Messer, RJ Hollis, RN Jones, GV Doein, ME Brandt, et al. Trends in species distribution and susceptibility to fluconazole among blood stream isolates of Candida species in the United States. Diagn Microbial Infect Dis. 1999;33:217-222.
9. Ansam F Sawalha. Complementary and Alternative medicine in Palestine. Use ans safety implications. J Alt Compl Med. 2007;13:2,263.
10. Barry AL, MA Pfaller, RP Rennie, PC Fuchs, SD Brown. Precision and accuracy of fluconazole susceptibility testing by broth microdilution,Etest and disc diffusion methods. Antimicrob Agents Chemother. 2002;46: 1781-1784.
11. National Committee for Clinical Laboratory Standards. 1997. Reference methods for broth dilution antifungal susceptibility testing of yeasts. Approved Standards M 27-A. National Committee for Clinical Standards, Wayne. Pa.
12. Anupan N Devkatte, Gajanan B Zore, S Mohan Karuppayil. Potential of plant oil as inhibitors of C. albicans growth. FEMS Yeast Res. 2005;5(a):863-873.
13. HJD Dorman, SG Deans. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J App Microbiol. 2000;88(2):308 ? 316.
14. Erdogan Ceylan, Daniel YC Fung.. Antimicrobial activity of spices. J Rapid Methods Automation Microbiol. 2004;12(1):1-55.
15. Nuzrat Tabassum, GM Vidyasagar. Antifungal investigations in plant essential oils: A review. Int J Pharm Pharm Sci. 2013;5 Suppl 2:19-28.
16. KA Hammer, CF Larson, TV Riley. Antimicrobial activity of essential oils and other plant extracts. J App Microbiol. 1999. 86: 6, 985 ? 990.