|Iranian Journal of Pharmaceutical Research (2006)
Received: May 2005
Accepted: September 2005
Copyright ? 2005 by School of Pharmacy
Antimicrobial Activity of Indigenous Strains of Aureobasidium Isolated From Santalum Album Leaves
Enayat Kalantara*, Rajendra Deopurkarb and Balu Kapadnisb
aDepartment of Laboratory Medicine, School of Paramedical Sciences, Ahvaz, Jundi-shapour University of Medical Sciences, Ahvaz, Iran. bDepartment of Microbiology, University of Pune, Pune, India.
Although more than 3000 antibiotics have been reported from Actinomycetes/ higher fungi; biotechnological potential of yeasts and yeast-like fungi with respect to production of antimicrobial compounds has not been sufficiently investigated. We examined the antimicrobial activity of 11 strains of Aureobasidium pullulans (two new isolates and nine standard strains). All the strains of Aureobasidium pullulans inhibited Ps. fluorescen s, but none of these strains could inhibit Candida albicans and S. cerevisiae. Interestingly, the yeast Pichia angusta was inhibited by six of the A. pullulans strains used in the present investigation. Two indigenous isolates of Aureobasidium Natural Isolate 1 (NI. 1) and Natural Isolate 2 (NI. 2) showed antibacterial activity against the Gram-negative cultures; most of which were resistant to Gentamicin. This study provides evidence that A. pullulans is a promising producer of antimicrobial agents for better chemotherapeutic agents, possibly against Pseudomonals infections.
Aureobasidium pullulans (de Bary) Arn. is a cosmopolitan yeast- like fungus that occurs in diverse habitats, including the phyllosphere of many plants and also on various tropical fruits (1, 2). Aureobasidium pullulans is industrially important because of its? capacity to produce the polysaccharide ?pullulan? (3-6). In addition, it produces various potentially useful products such as xylanase (7-8), β-D- fructofuranoside fructohydrolase (4, 9), sucrase (10), esterase (11) and β-galactosidase (4). Moreover, World Health Organization has placed A. pullulans within ?risk ?Group I? (12), where there is no possibility of infection to either society or laboratory workers. There are relatively few reports on antimicrobial compounds obtained from various yeasts and yeast-like fungi (13), and very little work has been carried out on antibacterial activity of A. pullulans.
McCormack et al. (14) reported for the first time the inhibition of Ps. aeruginosa and Staphylococcus aureus by compounds obtained from A. pullulans. Takesako et al, (15) reported a group of antifungal antibiotics, named aureobasidins, from A. pullulans. Aureobasidium appears to be a promising organism for development of newer antimicrobial agents, both for chemotherapy as well as non-medical applications.
This article reports on the isolation of two natural isolates of Aureobasidium pullulans strains from the leaf surface of Santalum album. Studies on antimicrobial activity of these two isolates and nine standard cultures of Aureobasidium pullulans were carried out.
Organism and their cultivation:
Target Organisms: Pseudomonas aeruginosa, Pseudomonas Putida, Pseudomonas fluorescens, Klebsiella pneumoniae, Acinetobacter calcoaceticus,
Escherichia coli, Salmonella typhi, Staphylococcus aureus, and Bacillus spp were maintained on nutrient agar slants containing 20% glycerol and stored at 4-10?C. Candida albicans, Saccharomyces cerevisiae, Pichia angusta and A. Pullulans strains were? maintained on SDA medium containing 20% glycerol and stored at? 4-10?C (Table 1).
Isolation of Aureobasidium Pullulans from aerial surfaces of plants:
Aureobasidium pullulans strains were isolated from Santalum album leaves, essentially based on a procedure described by Pollock et.al. (6). Briefly, Leaves of? Santalum album were washed gently with water? and cut into small pieces, 0.5 g of which was soaked in 10 ml of sterile distilled water and then incubated for three days at 28? C on a shaker set at 120 rpm. An aliquot of 0. 1 ml was transferred to a 10 ml medium, which included the following (per liter of deionised water) ingredients: 2.0g of yeast extract, 0.5 g of (NH4)2Hpo4, 1.0 g of NaCl, 0.2 g of MgSO4. 7H2O, 3.0g of K2HPO4, 0.01 g each of FeSO4, MnSO4 and ZnSO4 pH7, 20.0g Sucrose and 10 mg ml-1 of chloramphenicol. After two days of shaking at 28? C, the turbid culture was allowed to stand stationary to settle down filaments and aggregates. bout 100 ?l of the diluted sample was spread on the same medium, and looked upon for isolated colonies after 2- 6 days. The cultures were observed periodically, under a phase contrast microscope, to examine their morphology.
Biochemical characterization of natural isolates of A. pullulans:
Our natural isolates were compared to standard strains of A. pullulans, using the auxanogram method, in order to determine the utilization of Carbon and Nitrogen sources (16). Based on these, tests similarity coefficients of natural isolates with the standard A. pullulans ATCC 16625 were calculated as S=a / a+b+c, where a= number of positive characteristics shared by the two strains; b= number of positivecharacteristics? in the first strain when the second strain was negative; c= number of positivecharacteristics in the second strain when first strain was negative. Cultural characteristics of all the A. pullulans strains were studied on potato dextrose agar (PDA) and Sabouraud dextrose agar (SDA) after incubation for 2-6 days.
Growth conditions and fermentation:
A loopful of cells (108 cells / ml) taken from the slant culture of A. pullulans were was suspended in three ml of sterile distilled water.? From this suspension one ml was transferred into 10 ml of Sabouraud Dextrose Broth (SDB), incubated for 48 h on a shaker setat 120 rpm, in order to prepare inoculum for all the experiments. One ml of the inoculum was added to 100 ml of malt extract broth and incubated at 28?C on a shaker set at 120 rpm for three days. The entire content of the flask was centrifuged at 5000 rpm for 20 min and the supernatant was extracted three times with 10 ml of ethyl acetate, concentrated up to 2 ml under a stream of nitrogen gas and then used for testing the antimicrobial activity.
Testing the antimicrobial activity:
Antimicrobial activity of the above extract was determined by the disc assay procedure (17). The concentrated ethyl acetate extract (100 ?l) was added to an ampoule containing 10 sterile (Whatman paper number 1; 5 mm dia.) disks and kept in a refrigerator for 24 h. Control disks were prepared using an ethyl acetate extract of the un-inoculated medium.
Results and Discussion
Identification of natural isolates:
On the basis of morphological characteristics and assimilation of different carbon and nitrogen sources, natural isolates were identified as A. pullulans. Further details on the growth behavior of the two isolates and ATCC 16625 are given in Table 2. In the early stage of their growth, oval budding cells were predominant and at a later stage elongated cells with blastospores present on swollen assimilation of adonitol, tips and chlamydospores could be seen. Similarity coefficients of NI.1 and NI.2 with A. pullulans (ATCC 16625) were estimated to be 87 and 92% respectively. Isolates NI.1 and NI.2 isolates belonged to the same species, as could be seen from the similarity coefficient with the ATCC strain of A. pullulans 16625. These isolates were taken to be different strains, as they showed variations from the ATCC 16625 with respect to assimilation of adonitol, ammonium sulphate and protease activity (NI.1); assimilation of adonitol and xylanase activity (NI.2). At the beginning, both the strains produced creamy white colonies, which turned black during the latter stages of growth. Strain NI.1 formed mucoid colonies (Table 3).
Almost all the A. pullulans strains showed activity against Gram-negative bacteria, but none of them inhibited Staphylococcus aureus and Bacillus spp. Strikingly, Pseudomonals seem to be apparently much more sensitive to A. pullulans. Ps. fluorescens, Ps. aeruginosa and Ps. putida were inhibited by 11, 10 and eight strains of A. pullulans, respectively. Similarly, out of the 11 strains of A. pullulans, eight strains showed inhibition of A. calcoaceticus, an emerging nosocomial pathogen. Three/ four of the A. pullulans strains showed inhibition of E. coli, S. typhi and K. pneumoniae. Though P angusta was inhibited by six strains of A pullulans, none could inhibit C. albicans and S. cerevisiae (Table 4). The natural isolates were compared with nine standard strains of A. pullulans and identified as A. pullulans on the basis of morphological, physiological and cultural characteristics. Production of antibiotic has been reported most often from the eubacteria-like Bacillus, Actinimycetes, e.g. Streptomycetes and lower fungi e.g. Cephalosporium. It should be pointed out that more than 3000 antibiotics have been isolated from actinomycetes and? relativelyfew from fungi (18). Mac William (19) has found that yeasts and yeast-like fungi, as compared to other microorganisms, are not a promising source of novel antibiotics.
Because of the emergence of pathogens resistant to most currently available antimicrobial agents and a concomitant increase in the number of immunosuppressed patients, screening of antimicrobial compounds is becoming increasingly important. This study provides comprehensive evidence that A. pullulans, a safe non-pathogenic (12) and ubiquitous organism (1), could be a promising producer of antimicrobial compounds. In this study the ntimicrobial activity of A. pullulans strains was predominantly against Gram- negative bacteria and Pseudomonas spp.. In particular and unlike the study of McCormack et al (14), no activity was seen against Staphylococcus aureus. Studies in our laboratory indicate that there are intracellular compounds inhibiting Staphylococcus aureus (Data not shown).
It is worth noting that Pseudomonas aeruginosa has been a notorious organism for chemotherapy .It is highly resistant to β-lactam antibiotics and intractable to treatment with most potent antipseudomonal agents (20-21). It is also an opportunistic pathogen responsible for a wide range of infections.
Taken together, the results described here provide hope for obtaining better chemotherapeutic agents, possibly against Pseudomonas infections.
Authors are thankful to Jawaharlal Nehru Memorial Fund, New Delhi, for providing a Research fellowship to Enayat Kalantar.
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