Antifunagal activity

1. Antifungal activity Presented by Khajaphir M M Pharma Part 1 G C P Bangalore-560027 1

2. An  antimicrobial  is an agent that kills  microorganisms  or inhibits their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibacterials are used against  bacteria  and antifungals are used against  fungi . They can also be classified according to their function. Agents that kill microbes are called  microbicidal , while those that merely inhibit their growth are called  microbiostatic. The main classes of antimicrobial agents are  disinfectants  ("nonselective antimicrobials" such as  bleach ), which kill a wide range of microbes on non-living surfaces to prevent the spread of illness,  antiseptics  (which are applied to living tissue and help reduce infection during surgery), and  antibiotics  (which destroy microorganisms within the body). 2

3. The term "antibiotic" originally described only those formulations derived from living organisms but is now also applied to  synthetic  antimicrobials, such as the  sulphonamides , or fluoroquinolones . The term also used to be restricted to antibacterials (and is often used as a synonym for them by medical professionals and in medical literature), but its context has broadened to include all antimicrobials. Antibacterial agents can be further subdivided into  bactericidal  agents, which kill bacteria, and  bacteriostatic agents , which slow down or stall bacterial growth . Antifungals are used to kill or prevent further growth of  fungi . In medicine, they are used as a treatment for infections such as  athlete's foot ,  ringworm  and  thrush  and work by exploiting differences between mammalian and fungal cells. They kill off the fungal organism without dangerous effects on the host. 3

4. Unlike bacteria, both fungi and humans are eukaryotes . Thus, fungal and human  cells  are similar at the molecular level, making it more difficult to find a target for an antifungal drug to attack that does not also exist in the infected organism. Consequently, there are often  side effects  to some of these drugs. Some of these side effects can be life-threatening if the drug is not used properly . Fungi lack  chloroplasts  and are  heterotrophic  organisms and so require preformed  organic compounds  as energy sources . Fungi have a cell wall and   vacuoles . They reproduce by both sexual and asexual means, and like  basal  plant groups (such as  ferns  and  mosses ) produce spores . Similar to mosses and algae, fungi typically have  haploid   nuclei. Some species grow as unicellular yeasts that reproduce by  budding  or  binary fission .  Dimorphic fungi  can switch between a yeast phase and a hyphal phase in response to environmental conditions . 4

5. Most fungi grow as  hyphae , which are cylindrical, thread-like structures 2–10  µm  in diameter and up to several centimeters in length. Fig: 1.  hypha   2. conidiophore 3.   phialide 4. conidia 5.   septa Hyphae grow at their tips (apices); new hyphae are typically formed by emergence of new tips along existing hyphae by a process called  branching , or occasionally growing hyphal tips fork, giving rise to two parallel-growing hyphae 5

6. The combination of apical growth and branching/forking leads to the development of a  mycelium , an interconnected network of hyphae .  Hyphae can be either  septate  or  coenocytic . Septate hyphae are divided into compartments separated by cross walls with each compartment containing one or more nuclei; coenocytic hyphae are not compartmentalized. Septa have  pores  that allow  cytoplasm ,  organelles , and sometimes nuclei to pass through; an example is the dolipore septum in fungi of the phylum Basidiomycota . Antifungal activity of natural extracts and pure compounds can be detected by inhibition of various fungi, yeast or filamentous, by samples that are placed in contact with them. Several methods for detecting activity are available, but since they are not equally sensitive or not based upon the same principle, results will be profoundly influenced by the method 6

7. Dermatophytes are responsible for the majority of fungal infections involving skin, hair and nails. They comprise a phylogenetically closely related group of genera with numerous species They attack the keratinized tissues and cause a wide spectrum of clinical manifestations that vary from mild to severe. Dermatophytosis is one of the most common fungal infections worldwide. Materials and methods The isolates : The clinical dermatophyte isolates were isolated from patients suspected of having a dermatophytosis who were referred to the medical mycology laboratory, Samples were taken from infected areas and cultivated on Mycosel agar media, that was incubated at 25°C for 2-3 weeks. Identification of the isolates was based on gross colony characteristics and microscopic morphology of their micro- and macro conidia and accessory structures. The isolates were then transferred to sterile distilled water (DW) in vials and stored at room temperature as stocks. 7

8. Preparation of the antifungal disks All antifungal drugs were obtained as standard powder. The stocks were prepared by dissolving the powders in their specific solvents (DMSO, water, ethanol), after which, they were loaded into blank paper disks at the following potencies : clotrimazole (10µg/disk), terbinafine(30µg/disk ), griseofulvin(25µg/disk ), fluconazole(25µg/disk ), ketoconazole (10µg/disk) and miconazole (10µg/disk) according to antifungal disks potency of Rosco Diagnostica Company (Neo- sensitabs, Denmark). Amphotericin B and clotrimazole disks and standard strain of T. rubrum were used as controls. 8

9. Preparation of the inocula The isolates were transferred from DW stocks to Mycosel agar and then sub- cultured to potato dextrose agar (Merck, Germany) to enhance sporulation. Seven day-old cultures were covered with 1ml DW and the colonies were probed with the tip of a sterile Pasteur pipette to obtain a mixture of mycelium and conidia. The suspensions were transferred to sterile tubes and allowed to sediment for 30 minutes and then adjusted with a spectrophotometer set at 65% transmittance and 530nm. Disk diffusion assay All the tests were performed according to Esteban et al. The inoculum was evenly spread on the surface of 10cm Petri dishes containing Sabouraud dextrose agar medium (Merck, Germany) and exposed to air dry. 9

10. Then, the antifungal disks were applied to the plates, after which the plates were incubated at 25°C for 5-10 days. After the colonies grew, the zones of inhibition around the disks were measured and recorded . Criteria of susceptibility and resistance of antifungal disks were measured according to following table, 1. All tests were performed in duplicate and Microsoft SPSS was used for data analysis. Table: Criteria of susceptibility and resistance of antifungal disks 10 Antifungal drugs potency Zone diameter sensitive In mm intermediate resistance Clotrimazole 10 µg ≥20 19-12 ≤11 Fluconazole 25 µg ≥22 21-15 ≤14 Griseofulvin 25 µg ≥10 21-15 No zone Ketoconazole 15 µg ≥30 29-23 ≤22 Miconazole 10 µg ≥20 19-12 ≤11 Terbinafine 30 µg ≥20 19-12 ≤11

11. Results A total of forty species of dermatophytes were isolated and identified. The isolates belong to three genera and eight species as follows: T. mentagrophytes 13(32.5%), T. rubrum 8(20%), E. floccosum 7(17.5%), T. violaceum 4(10%), M. gypseum 3(7.5%), T. verrucosum 2(5%), T. tonsurans 2(5%), T. schoenleinii 1(2.5%) and Trichophyton species 1(2.5 %). Test results of the susceptibility to antifungal drugs were as follows: Ketoconazole: 31 (77.5%) susceptible, 4(10 %) intermediate, 5 (12.5%) resistant. Griseofulvin: 3 (7.5%) resistant, 37 (92.5%) sensitive. Miconazole: 36 (90%) sensitive, 4 (10%) intermediate. Terbinafine: 1 (2.5%) resistant, 39 (97.5%) sensitive. Clotrimazole: 39 (97.5%) susceptible, 1 (2.5%) intermediate. Fluconazole: 39 (97.5%) resistant, 1 (2.5%) intermediate . Regarding the data, it was revealed that clotrimazole and terbinafine were the most effective antifungal drugs and fluconazole had the poorest activity (Fig. 1). 11

12. Fig(1)Diffusion bioassays for flumequine standard solutions: agar disc (on the left)and agar cylinder (on the right) method. Test bacteria : Bacillus subtilis . Fig(2) Sensitivity T. mentagrophytes to tested antifungals drugs g, Griseofulvin; k, Ketoconazole; c, Clotrimazole; m, Miconazole; f, Fluconazole; t, Terbinafine Antifungal susceptibility testing is a dynamic field of medical mycology . Development and standardization of antifungal susceptibility tests have shown remarkable progress in the field of medical mycology. 12

13. MIC MEASERMENTS MIC is the lowest concentration which resulted in maintenance or reduction of inoculums viability. Serial tube dilution technique was used to determine of MIC of the extracts against four gram-positive and four gram-negative bacteria. The plant extract (0.512 mg) was dissolved in 2 ml distilled water (2 drops tween-80 was added to facilitate dissolution) to obtain stock solution . After preparation of suspensions of test organisms (107 organism per ml), 1 drop of suspension (0.02 ml) was added to each broth dilution. After 18 h incubation at 37°C, the tubes were then examined for the growth. The MIC of the extract was taken as the lowest concentration that showed no growth . Growth was observed in those tubes where the concentration of the extract was below the inhibitory level and the broth medium was observed turbid (cloudy). Distilled water with 2 drops of tween-80 and kanamycin were used as negative and positive control ,respectively 13

14. BIO-AUTOGRAPHY METHODS Antimicrobial activity can be used by bioautography that localizes on a chromatogram using three approaches : (a) direct bioautography , where the microorganism grows directly on the thin-layer-chromatographic (TLC) plate (b) contact bio- autography , where the antimicrobial compounds are transferred from the TLC plate to an inoculated agar plate through direct contact (c) agar-overlay bioautography , where a seeded agar medium is applied directly onto the TLC plate. The fungal organisms used in this study were moulds ( Aspergillus fumigatus and   Microsporum canis ), yeasts( Candida albicans  and  Cryptococcus neoformans ) and a thermally dimorphic fungus ( Sporothrix schenckii ). All fungal organisms were isolated from clinical cases that were not treated prior to sampling in the Department of Veterinary Tropical Diseases.   A. fumigatus  was isolated from a chicken,  C. albicans  from a Goldian finch,  C. neoformans  from a cheetah,  M. canis  from a cat suffering from dermatophytosis and  Sporothrix schenckii  from a horse with cutaneous lymphangitis . These fungi represent the most common and important disease-causing fungi of animals. 14

15. Medium : Sabouraud dextrose (SD) agar (Oxoid, Basingstoke, UK) was used as the maintenance growth medium for all the fungal strains used, and the fungi were cultured in SD broth Method Ten µl (10 mg/ml) of each extract were loaded onto TLC plates in a narrow band and eluted using the three different mobile solvent systems (CEF, BEA and EMW ). The developed plates were dried under a stream of fast moving air for 5 days to remove traces of solvent on the plates . One week old cultures of fungal organisms grown on SD agar were each transferred into 250 ml of freshly prepared SD broth using a sterile swab 15

16. Densities of fungal cultures used for  A. fumigatus, C. albicans, C. neoformans, M. canis  and  S. schenckii  were approximately 8 ×10 6 , 3 ×10 6 , 3 ×10 6 , 2 ×10 5  and 1 ×10 5  cells/ml respectively. 16

17. The prepared chromatograms were sprayed with the fungal or bacterial suspension until wet. This process was carried out in a biosafety Class II cabinet (Labotec, SA) for fungi, and Laminar flow cabinet (Labotec, SA) for bacteria. Thereafter, the plates were incubated overnight at 35°C and 100% relative humidity in the dark and then sprayed with a 2 mg/ml solution of  p -iodonitrotetrazolium violet and further incubated overnight or longer in the case of  S. schenckii  and  M. canis . White bands indicate where reduction of INT to the coloured formazan did not take place due to the presence of compounds that inhibited the growth of tested organisms . 17

18. It is important to realize that bioautography is not a quantitative measure of antimicrobial activity. It only indicates the number of compounds that were separated with antimicrobial activity . The fact that of the bacteria tested,  S. aureus  had the highest number of inhibition bands does not mean that this was the most susceptible organism. Similarly,  C. neoformans  with the most inhibition bands against fungi does not necessarily have the highest susceptibility among fungal organisms . Some of the compounds are active against both bacteria and fungi, while others are selective in their activity. It is possible that compounds that have activity against all the tested organisms possess a broad antimicrobial action or they may even be general metabolic toxins that could be toxic to animals as well. 18

19. 3. Dilution methods The main advantage of the dilution method is possibility to estimate the concentration of the test compound in the agar medium or in the broth suspension; for this reason they are commonly used in determination of MIC values. The application range includes complex exracts,pure substances ,and both polar and nonpolar samples. In the agar dilution procedure ,various concentrations of the tested compound are mixed with nutrient agar.the agar plates are inoculated and then incubated. The concentration of the antimicrobial substance ,at which no microorganism growth is detected ,gives the MIC value. In the tube assay ,various concentrations of the tested are mixed with bacterial suspension in series of tubes-the lowest concentration causing inhibition in microorganism growth corresponds to the MIC value. 19

20. In the broth micro-dilution assay,the microorganisms are grown in the plate walls,to which various concentrations of the tested compounds are added. The growth of the microorganisms is indicated by the presence of turbidity in the wells. 20

21. References International journals of microbiology. Internet sourses 21

22. Thank you 22