Inhibition of Mycoparasitic Growth by Filamentous Nonpolyne Chitinolytic Producing Bacteria

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1 Inhibition of Mycoparasitic Growth by Filamentous Nonpolyne Chitinolytic Producing Bacteria Abdullah Y. Al-Mahdi, Qaees Y. M. Abdullah, Anas A. Al-Mahbashi, Abdulrahman A. Homed, Salwa H. Al-Kaeat and Saeed M. Al-Galibi. Abstract Fatty acids like ergosterol and tween 8 are depressing the inhibition of mycoparasitic activity of polyene antibiotics by neutralizing effect. Polyene antibiotics become bound to the fungal cell membrane and cause permeability changes with attendant depletion of essential cellular constituents. Loss of potassium and ammonium ions, inorganic phosphate, carboxylic acids, sugar phosphates, nucleotides and protein have been shown to occur in several fungal species after exposure to polyene antibiotics. Most polyene antibiotics are toxic to human and therefore used predominantly in topical applications. Kidney damage is a major problem when polyene is used systemically to treat several fungal infections. Since polyene macrolides are produced by actinomycetes, particularly Streptomyces, from 149 chitinolytic producing actinomycetes isolated where are 72% of isolates have been produce polyene antifungal antibiotics which their activity effected by ergosterol and tween 8, this method allows the analysis of a greater number of strains at an early stage of the screening procedure. We selected 2 isolates which showed maximum zone of inhibition, from both non-polyene and polyene antibiotic, which belonged Streptomyces or other genera for further investigation to detect novel antifungal antibiotics and novel chitinolytic producing actinomycetes. S I. INTRODUCTION ELECTIVE action against fungi alone was difficult to achieve due to many physiological features common to both eukaryotic microorganism and the infected host. Therefore, target directed screening has focused on certain loci that are specific to the fungal cell. Screens to detect inhibitors of chitin and glucan synthesis have been developed. Selitrennikoff, (198) described a novel method in which cell wall acting antibiotics are detected by their ability to block the regeneration of protoplasts of a temperature sensitive mutant of the OS-1 variant of Neurospora crassa. Chitin synthesis inhibitor screens were also used widely (Selitrennikoff, 198). Examination of plates for morphological abnormalities could differentiate between the responses of different organisms. Many known antifungal antibiotics induce abnormalities in fungi and yeasts. Morphological changes such as, sporulation inhibition, curling of hyphae, inhibition of branching and Abdullah Y. Al-Mahdi, Qaees Y. M. Abdullah, Anas A. Al-Mahbashi, Abdulrahman A. Homed, Salwa H. Al-Kaeat and Saeed M. Al-Galibi are at Microbiology Branch, Department of Biology, Faculty of Science, University of Sana'a, Sana'a, Yemen. regular and irregular swellings were some of the criteria used for screening of antifungal antibiotics (Coppoc, 2). There were two approaches to the discovery and screening of new antifungal antibiotics: the rational and empirical. It would be intellectually very satisfying to study the biochemistry or physiology of the fungi, identify the ways in which they differ from the host, and then design and synthesize a molecule that would selectively exploit that difference and kill the fungus without harming the host. Indeed mycologists have identified a number of potential targets viz., cell wall synthesis, membrane sterol biosynthesis, nucleic acid synthesis and nuclear division and have discovered antifungal agents whose mode of action can be explained by interference at these target sites. The discovery of the polyoxins which interfere with chitin synthesis, the echinocandins and papulocandins which interfere with glucan synthesis, the azoles which interfere with ergosterol biosynthesis, or griseofulvin and the benzimidazoles which interfere with micro-tubule-associated protein and tubulin polymerization respectively was, in each case, purely empirical and in no way rational; first came the discovery of antifungal activity and only then an explanation for that activity (Goodfellow, M. and O'Donnell, A. G. 1989; Ryley and Rathmell, 1984). Any laboratory screen must be a compromise. Different classes of compounds behave in different ways; this was particularly well illustrated by azoles. Activity against the yeast phase of Candida albicans was generally poor when considered in the light of in vivo potential, but this degree of activity or inactivity can be influenced considerably by the choice of media and conditions of assay. The mycelial phase of C. albicans, on the other hand, was very sensitive to the azoles, hence we include this in our screen but this difference in sensitivity between the two phases was not seen with many other classes of compounds. If one was to have a high throughput of samples, and sample size was in many cases limited, then an in vitro pre-screen was the only one realistic option. In vivo screening and evaluation is then restricted to compounds showing sufficient activity in vitro a difficult level to fix and to compounds which, for chemical reasons, seem worth testing. The cut-off points for the decision whether to test in vivo or not may be decided by the relative capacities of 18

2 the two screens (Ryley and Rathmell, 1984). Polyene family of antifungal antibiotics was poorly absorbed polyhydroxylated macrolides with characteristic conjugated olefinic chromophores has provided the majority of antifungal antibiotics of clinical importance. Polyenes characterized by a large ring of macrocyclic lactones, involving at least 26 atoms, 4 to 7 conjugated double bonds and hydrophobic region. They derive their action from their strong affinity towards sterols, particularly ergosterol which was present in the cell membrane of fungi. The hydrophobic polyene region binds to the hydrophobic sterol ring system within fungal cell membrane. In so doing, the hydroxylated portion of the polyene was pulled into the membrane interior, destabilizing the structure and causing leakage of cytoplasmic constituents. It was possible that polyene molecules associate together in the cell membrane to form aqueous channels. The pattern of leakage was progressive, with small metal ions such as K+ leaking first, followed by larger amino acids and nucleotides. The internal ph of the cell falls as K+ ions are released, macromolecules were degraded and the cells are killed. The selective antifungal activity of polyenes was poor, depending on the higher affinity for ergosterol than cholesterol. Kidney damage was a major problem when polyenes were used systemically to treat severe fungal infection. Human and animals cell membrane also contain a sterol (cholesterol), most polyenes were rather toxic to human and animals, therefore, used predominantly in topical applications. Amphotericin B and nystatin were prime examples of this family (Davies, 199). Ergosterol H H Cholesterol Non polyene family contains a large group of antifungal antibiotics and was differentiated by structure and mode of action. Griseofulvin, Azoles, and Flucoytosine groups were the most commonly used members of nonpolyene antifungal antibiotics (Coppoc, 2). H II. 1BMATERIAL AND METHODS: A. Bi- Isolation of chitinolytic producing bacteria: Isolation of actinomycetes were done by suspending 1 g of soil sample in 1 ml sterile water, which was vigorously shaken and allowed to settle for 5 min. The supernatant was serially diluted and plated on Starch Casein Agar (SCA) of ph 4.5, followed by incubation at 7 C for 14 days. The isolates were enumerated and selected for further study. The ph of the buffered medium was checked during incubation (Kanavade, 2). All the isolates were screened for chitinolytic activity on minimal salts medium supplemented with colloidal chitin. Isolates were spot inoculated on colloidal chitin agar (CCA) plates and incubated at 28 C for 7 days. Chitinase positive cultures were detected by clear zone of hydrolysis around colony against opaque background contributed by colloidal chitin (Hsu and Lockwood, 1975). B. Antifungal Activity: All 147 chitinolytic producing actinomycete isolates which showed strong activity against fungi are streaked on SCA, SYA, GAA, GYA, and NA of ph 4.5 and incubated at 28 C for 7 days. After 7 days, chitinolytic producing actinomycete isolates are screened for antifungal activity by ADD method. The seeded plates with agar discs of chitinolytic producing actinomycetes are further incubated at 28 C for 4 days. Zones of inhibition are recorded (Patel, 1985; Williams and Robinson, 1981). C. Polyene Antifungal Activity: Three Potato dextrose agar (PDA) plates, one of them without detergent as control, remaining two as test, out of which one supplemented with 4% Tween 8 and second supplemented with.% ergosterol. Each plate is spread with.4 ml of fungal suspension (2 1³ cfu ml-¹). Stock solution of each standard antifungal antibiotic (1 ul) is absorbed into 1 mm. sterile filter paper disc separately is placed on each (PDA) plate. Agar disc of each chitinolytic producing actinomycetes are grown on SCA for 7 days at 28 C is placed on each (PDA) plate, checked for polyene antifungal activity. The plates are kept for 2 hrs at 4 C and then incubated for 48 hrs at 28 C. Zone of inhibition around the agar disc of chitinolytic producing actinomycetes as well as standard antibiotic discs, is recorded (Chakrabarti, 1978). If the zone of inhibition around the agar disc of chitinolytic producing actinomycetes on the plates supplemented with ergosterol or tween 8 is similar to the zone of inhibition around the agar disc of chitinolytic producing actinomycetes in the control plate it indicates presence of nonpolyene antibiotic. This is because antifungal activity of non-polyene antibiotics is not antagonized by ergosterol and tween 8. 19

3 A. i- Isolation of chitinolytic: III. RESULTS AND DISCUSSION: For detection of chitinolytic activity, all 65 isolates were grown on colloidal chitin. Zones of chitin hydrolysis were observed within 8 days of incubation. On further incubation, zones of chitin hydrolysis increased in size. Zone of chitin hydrolysis was recorded. Out of all the isolates, 91% (1) produced chitinase. Amongst these, 95% (79) were strictly acidophilic actinomycetes and 84% (54), were neutrotolerant acidophilic actinomycetes. Streptomyces spp. were found to have good chitinase activity. B. Antifungal Activity on Different Media: In all 147 chitinolytic producing actinomycete isolates are grown on six different media viz., SCA, SCA*, SYA, GAA, GYA, NA and tested for their antifungal activity. SCA stimulated maximum antifungal activity amongst all media. The inhibition zone diameter ranged from cm. to.7 cm. against A.niger. The activity of the isolates is enhanced if they are grown on SCA with % NaCl (SCA*). The GAA medium was second and GYA third in stimulating antifungal activity. Least stimulation of antifungal activity is on NA medium (Fig. 1). C. Polyene Antifungal Activity: Of 147 chitinolytic producing actinomycetes, 71% produced polyene antifungal antibiotics and 29% of them produced non-polyene antibiotics. The activity of polyene antibiotics is affected by ergosterol and tween 8. Hamycin (5 μg/ml), Nystatin (4 μg/ml), and Cycloheximide (5 μg/ml) (Hi-Media company) are used as standard antibiotics. In absence of ergosterol and tween 8 the activity of Cycloheximide against A.niger is less ( cm.) and against C.albicans it is high (5.5 cm.). The activity of Hamycin and Nystatin is very less against A.niger and activity of Hamycin relatively high against C.albicans (2.8 cm.). In presence of ergosterol and tween 8 the antifungal activity of non-polyene antibiotic, Cycloheximide, is not affected. However, antifungal activity of Hamycin and Nystatin is affected which resulted in reduction of inhibition zone from 2.8 cm. to 1.2 cm. by Hamycin against C.albicans. Reduction in activity of Nystatin is insignificant. The results indicated that the polyene antibiotic activity was significantly affected by ergosterol than tween 8 (Fig. 2). There are 66 isolates which are active against A.niger and C.albicans. Seven of the cultures are active against C.albicans only and 74 cultures are active against A.niger only. Maximum zone of inhibition is.8 cm. by isolate no. 5/7 which is identified as Streptomyces sp. AK57, produced polyene antibiotics, and maximum zone of inhibition,. cm. for non-polyene antibiotics produced by isolate no. 4/9 which is identified as Catellatospora AK49. Total polyene antibiotic producing isolates are 16 and non-polyene antibiotic producing isolates are 41. There are 88 polyene antibiotic producing isolates 8% of them belonged to Streptomyces and 17% belonged to non-streptomyces. Non-polyene antibiotic producing isolates belonged to Streptomyces corresponding to 56% of the non-polyene antibiotic producing isolates and 18 belonged to non-streptomyces which corresponded to 44 % (Table 1)...4 Inhibition zone diam. (cm SCA SCA* GAA SYA GYA NA Aspergillus niger NRRL 129. Candida albicans NCIM 12. Cryptococcus humicolus MTCC 155. Fig. 1: Antifungal activity of acidophilic actinomycete grown on different media. 11

4 Inhibition zone diam. (cm.) A.niger (co.) A.niger (+E) A.niger (+T8) C.albicans (co) C.albicans (+E) C.albicans (+T8) Cycloheximide (Non-polyene) Hamycin (Polyene) Nystatin (Polyene) Fig. 2: Effect of detergents on antifungal activity of std. antibiotics (co, control, +E, with ergosterol, +T8, with tween 8). TABLE I EFFECT OF DETERGENT (ERGESTEROL AND TWEEN 8) ON ANTIFUNGAL ACTIVITY OF ACIDOPHILIC ACTINOMYCETES IN COMPARISON WITH STD. ANTIBIOTICS. Inhibition zone dima. (cm.) Antibiotics/ Isolate no. In absence of ergosterol & tween 8 A.nige C.albicans r In presence of ergosterol In presence of tween 8 A.nige C.albicans A.nige C.albicans r r Cycloheximide (5 μg/ml) Hamycin (5 μg/ml) Nystatin (4 μg/ml) / / / / / / / / / / / / / / / / / / / / Polyene antibiotic 111

5 In all 2 chitinolytic producing actinomycete isolates are selected for further investigation. Of them, 12 produced polyene antibiotics and 8 produced non-polyene antibiotics. Of them, ten belonged to Streptomyces and the rest non- Streptomyces (Fig. and Fig. 4). There are many important factors in the selection of fermentation medium i.e. the cost of the media or use of the simple material for fermentation in the large scale industry. Use of the whey medium (WM), is reported by Kanavade, et al., (22) for antifungal antibiotic production by chitinolytic producing actinomycetes. Tap water medium and soil extract medium are used as fermentation media (Lo, et al., 22). Acidic starch casein media with % NaCl is selected in this study as the best medium for production of antifungal antibiotics Isolates (%) Streptomyces Non-Streptomyces Polyene antibiotics Non-polyene antibiotics Fig. : Antifungal chitinolytic producing actinomycetes. Inhibition zone diam. (cm.) /4 4/1 4/1 5/7 5/22 9/18 9/25 9/28 9/1 14/1 15/7 19/1 / 4/9 7/1 9/9 9/47 14/9 18/ 29/4 Polyene antibiotics Non-polyene antibiotics Fig 4: Antagonic activity of chitinolytic producing actinomycetes which producing polyene and non-polyene antibiotics against A.niger. Chitinolytic producing actinomycetes are well known for their ability to produce a wide variety of antifungal compounds particularly polyene macrolide and non-polyene antibiotics. Different strategies can be employed to develop a profitable program to screen microbial metabolites for antifungal antibiotics while selecting against the polyene and non-polyene producing strains. For instance, screening can be restricted to the detection of cell wall acting antifungal compounds as polyene-ergosterol compound, detected by their ability to block this action in presence of ergosterol or tween 8 as easy targets available in the media. This new approach is to search for new antifungal antibiotics in primary screening stage and screening of large number of actinomycetes. This approach is based on reduction of inhibition zone in presence of ergosterol or tween 8. This approach has been modified by using ADD screening method. The results of this experiment gave typical results with control in presence of nystatin and hamycin as polyene antibiotics and cycloheximide as non- 112

6 polyene antibiotics. Ergosterol presented activity stronger than tween 8, due to structural difference (Gilles, et al., 1989). The other important observation is most of the acidophilus produce polyene antibiotics, (8%) and belonged to Streptomyces genus and non-polyene antibiotics producers belonged to non- Streptomyces genera. The maximum zone of inhibition is observed by polyene antibiotic producers. We selected 2 isolates which showed maximum zone of inhibition, from both non-polyene and polyene antibiotic, which belonged Streptomyces or other genera for further investigation to detect novel antifungal antibiotics and novel acidophilic actinomycetes. REFERENCES [1] Chakrabarti, S. Matai, S. and Chandra, A. L. (1978). Exocellular lipase production by a soil streptomycete. Current Science. 48; [2] Coppoc, G. L. (2). Action of antifungal agents; Antifungal agents. Academic press. London. UK. [] Davies, J. (199). What are antibiotics? Archaic functions for modern activities. Mol. Microbiol. 4; [4] Gilles, E. Elise, A. and Gerard, T. (1989). A screening method for antifungal substances using Saccharomyces cerevisiae strains resistant to polyene macrolides. Journal of Antibiotics, 11; [5] Goodfellow, M. and O'Donnell, A. G. (1989). Search and discovery of industrially-significant actinomycetes. Society for General Microbiology Symposia. 44; 4-8. [6] Hsu, S. C. and Lockwood, J. L. (1975). Powdered chitin as a selective medium for enumeration of actinomycetes in water and soil. Appl. Microbiol. 29; [7] Kanavade, V. L. (2). Use of Bio-Industrial Waste for Production of Microbial Biomass with Potential in Environmental Management. Ph.D. Thesis, University of Pune, India. [8] Kanavade, V. L. Almahdi, A. Y. and Kapadnis, B. P. (22). Use of whey for isolation and propagation of acidophilic actinomycetes with phosphate mobilization ability. Journal of Maharashtra Agricultural Universities. 27; [9] Lo, C. W. Lai, N. S. Cheah, H-Y. Wong, N. K. I. and Ho, C. C. (22). Actinomycetes isolates from soil samples from the Crocker range Sabah. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) [1] Patel, M. V. (1985). An agar plate method for the screening of antibiotics triggering autolytic enzymes. Journal of Antibiotics. 8; [11] Ryley, J. F. and Rathmell, W. G. (1984). Discovery of antifungal agents: In vitro and in vivo testing. Mode of action of antifungal agents. pp Symposium of British Mycological Society. U.K. [12] Selitrennikoff, C. P. (198). Use of a temperature-sensitive, protoplast forming N.crassa strain for the detection of antifungal antibiotics. Antimicrobial Agents and Chemotherapy. 2; [1] Williams, S. T. and Robinson C. S. (1981). The role of streptomycetes in decomposition of chitin in acidic soils. Journal of General Microbiology. 127;