Antimicrobial activity of various ethanolic plant extracts against pathogenic multi drug resistant Candida spp.

A total of 50 Candida isolates were isolated and identified from clinical specimens and these were tested for resistance to various antifungal drugs. It was observed multi-drug resistance in all candida isolates by 84%, 62%, 60%, 76%, 46, 30%, and 22% against fluconazole, clotrimazole, Amphotericin B, itraconazole, ketoconazole, miconazole and nystatin tested respectively. The isolates, which were found to be resistant to antifungal drugs were selected and subjected to antifungal testing against six ethanolic plants, extract namely Azadiracta indica, Allium sativum, Cordia dichotoma Ocimum sanctum, Syzygium cumini and Trigonella foenum grecum. All the plant extracts tested were found to effective against all MDR Candida isolates with inhibition zone ranging from 10- 18mm in diameter. Ethanolic extract of Allium sativum was observed most effective against the isolates among all the plants extracts tested. The minimum inhibitory concentration (MIC) of all ethanolic plant extract was recorded ranging from 1.56-25mg/ml against MDR candida isolates. Phytochemical analysis of the alcoholic plant extracts revealed the presence of alkaloid, flavanoid, glycosoid, phenol; phenol, tannins, saponins in all the plants studied. The present study may be successful in identifying the plants with different antimicrobial activity. These plants containing various phytochemicals may be exploited in the treatment of infectious diseases caused by drug-resistant microorganisms.


Preparation of plant extracts:
Hundred (100) g of dry powder of plant material was soaked in 100ml of ethanol for 5 days with intermittent shaking and at the end of extraction, the extract were filtered through Whatman filter paper No. 1 (Whatman Ltd., England) to make a crude ethanol extract. The filtered extract was left to dryness under reduced pressure on rotary evaporator at 40°c and stored at 4°c for further use [20]. Stock solution of crude extracts at different concentration (50 mg/ml, 100 mg/ml, 200 mg/ml, and 300mg/ml) was prepared for antimicrobial assay.

Phytochemical screening:
The extracts of the dry powdered leaves and seeds analyzed for the presence of various phytoconstituents like flavanoid, alkaloid, glycosoid, phenol, tannin and saponin [21].

Determination of antimicrobial resistance:
Pure isolates of identified Candida spp. were subjected to antimicrobial susceptibility testing using the disc diffusion method as recommended by Kirby Bauer method according to the recommendations of Clinical Laboratory Standard Institute [22], using the following antifungals discs Amphotricin B, Clotrimazole, Fluconazole, Itraconazoel, Ketoconazole, Miconazole, Nystatin obtained from Hi-Media Laboratories, India. The presence of a clear zone around the antibiotic disc is measured with meter rule in millimeter (mm).

Antimicrobial activity of plant extracts:
Antimicrobial activity of plant extract was carried out by agar well diffusion method [21]. 15 MDR isolates of Candida spp were selected for antimicrobial screening. 0.1 ml of diluted inoculums (105 CFU: ml) was spread on the SDA; wells were made on the medium by using 6 mm cork borer. The dried plant extracts were dissolved in dimethyl sulfoxide (DMSO) to make final extract concentration 300, 200, 100, 50, mg⁄ml. Each well was filled with 50 µl of plant extract, incubated at 37°C for 24 h. Zone of inhibition around of each well was measured in millimeter. DMSO and ethanol was used as a negative control and an antibiotic from which the isolates were sensitive was used as positive control.

Determination of MIC of plant extracts:
To determine MIC of plant extracts the broth micro-dilution method was performed [23] with some modifications. The inoculums of the tested isolates were prepared using the colony suspension method. Ninety-six-well culture plates were used, and serial two-fold dilutions of the extracts were dispensed into the plate wells. Two-fold dilutions of nystatin were used well along with 150µl of Mueller Hinton Broth. 30µl of broth culture was added to the wells. Three control wells were maintained for each test batch; the positive control (antibiotic, Mueller-Hinton broth and test organism) and sterility control (Mueller-Hinton broth and DMSO) and negative control (Mueller-Hinton broth, test organism and DMSO). The plates were incubated at 37 °C for 24 h. The fungal activity in the test wells was detected by adding 40 µL of 0.2 mg/ml of 2-(4-Iodo phenyl)-3-(4-nitro phenyl) 5phenyltetrazolium chloride (I.N.T.) (Himedia, India) solution dissolved in sterile distilled water to each well. The plates were incubated for further 30 min, and estimated visually for any change in color to pink indicating reduction of the dye due to bacterial growth. The lowest concentration (highest dilution) of the plant extract required to inhibit visible growth of the tested microorganism was designated as the MIC.

Conclusion:
It can be concluded that the alcoholic extracts of different plant extracts have a significant activity against multi-drug resistant pathogenic candida spp. The obtained data are also comparable to the commonly used antifungal antibiotics such as fluconazole, itraconazole, clotrimazole, miconazole, ketoconazole and Amphotericin-B. These plant extracts may be the potential alternatives of antibiotics to avoid their overuse and side effects on human health and environment. Further studies are also required for evaluating their clinical efficacy.
Lucknow, for providing the clinical samples and his cooperation with regard the research work.