Abstract: As a part of a research program that aimed to identify antibacterial and antifungal substances from fungus specimen of North Iranian soil samples, four penicillium species were identified as a source of secondary metabolites possessing antibiotic activities. These microorganisms were cultured in a liquid medium for 10 days. The antimicrobial disc assay activity of these extracts towards Candida albicans, Bacillus subtillis, Staphylococcus aureus, Salmonella typhi and Escherichia coli was performed. Supernatants and cell extracts of P. viridicatum Westling, P. citrinum Thom., P. aurantiogriseum Dierckx and P. waksmanii Zaleski showed distinguished antimicrobial activities. TLC assay of supernatants and cell extracts showed production of citrinin by P. citrinum and P. aurantiogriseum and penicillic acid by P. viridicatum and P. aurantiogriseum; while two unknown metabolites from P. waksmanii needed more examinations. These mycotoxins could be examined for other biological activities such as antineoplastic property.
INTRODUCTION
Microorganisms have been traditionally used to produce a variety of important substances for the pharmaceutical and food industries. The discovery and development of antibiotics was one of the most significant advances in medicine in the 20th century (Uchida et al., 2006; Silva et al., 2004).
Despite of the huge expectative on synthetic molecules with effective antimicrobial properties, natural products are still a worth promise. Screening of novel strains are bringing about microorganisms, not yet assayed for their antibacterial activity that can produce innovative molecules or useful templates for new antibiotics development (Du et al., 2009; Takahashi et al., 2008).
A large number of fungal extracts and/or extracellular products have been found to have antimicrobial activity, mainly from the filamentous fungus penicillium sp. (Petit et al., 2009; Rancic et al., 2006). The filamentous fungi of the genus Penicillium that belong to Ascomycetes are recognized to be ubiquitous in the environment (Nakashima et al., 2008).
Since, the discovery of penicillin, the micromycetes have been famous as producers of antibiotics and other secondary metabolites with biological activity (Sonjak et al., 2005; Rancic et al., 2006).
Previously unexplored environments have their own ecosphere, interactions and evolution that might contain new producer organisms (Bertinetti et al., 2009). Agricultural fields in North of Iran hold a soil rich in nutritional compounds. There are various genuses of micromycetes which compete together in such habitats, so it is expected that fungus isolated from these areas should develop a sophisticated metabolism, most probably molecules of odd chemical structures possessing interesting biological properties (Montematini et al., 2000). Those facts motivated the development of a research program which aimed to perform the isolation of antimicrobial substances from North Iranian soil fungi. The various species of Penicillium can colonise many different environments. They are common in soils, in foods, in drinks and in indoor air (Rundberget et al., 2004; Leitao, 2009).
As a part of this program, twenty isolates of penicillium were investigated for antimicrobial as well as antifungal activities.
MATERIALS AND METHODS
Microorganisms: Penicillium species were isolated from a soil sample collected at agricultural soils of Northern Iran. Small portions of soil were serially diluted and plated on solid media containing (g L-1): KH2PO4 (1.0), MgSO4.7H2O (0.5), Peptone (5.0), Dextrose (10.0) and Rose Bengal (0.033). Upon growth, individual colonies were transferred to other plates successively until the isolation of pure cultures (Lucas et al., 2007). Penicillium species was identified by microscopic observation of micro-structure (Pitt, 2000). The fungal strains were maintained on Potato Dextrose Agar (PDA) and refrigerated at 7°C (Lucas et al., 2007).
There were used the following Persian Type Culture Collection (PTCC) strains: Candida albicans PTCC 5027, Bacillus subtillis PTCC 1023, Staphylococcus aureus PTCC 1337, Salmonella typhi PTCC 1609 and Escherichia coli PTCC 1533 for antimicrobial assay.
Culture condition: For metabolites production, Penicillim species were inoculated into twenty conical flasks, containing each, 500 mL of aqueous medium containing (g L): Dextrose (20.0), Peptone (5.0), KH2PO4 (1.0), MgSO4.7H2O (0.5) and NaCl (5.0) and then incubated at the temperature of 28°C and shaking at 200 rpm. After 10 days, the mycelium was separated from the culture broth by filtration. The mycelia were extracted 3 times with 300 mL of methanol each. The extracts were dried by evaporation using a rotator vacuum distilling apparatus (Lucas et al., 2007).
Antimicrobial assay: The antimicrobial activity of extracts was evaluated against Candida albicans, Bacillus subtilis, Staphylococcus aureus, Salmonella typhi and Escherichia coli by qualitative disc test assay.
Two milligram of each extract were solubilized in 1 mL of chloroform. Fifty microliter of each solution were quantitatively loaded on a paper to make a final concentration of 100 μL disc-1. The solvent was removed by dry air. The test plates were prepared with 20 mL of nutrient agar and sterilized in autoclave by 15 min at 121°C. The 0.4 mL of the diluted bacteria inoculum (500 μL of stock culture in 2.0 mL of nutrient broth let 18 h at 37°C were transferred to 4.5 mL of saline solution) and homogenized using a vortex. A negative control was set using a disc impregnated with 50 μL of methanol and chloramphenicol (30 μg disc-1) was used as positive control of antibacterial test; to antifungal test miconazol (50 μg disc-1) was used. Experiments were run in duplicate. The result was ridden after incubation by 24 h at 37 μC (Lucas et al., 2007).
Metabolites assay: Thin Layer Chromatography (Kieselguhr 60 F254 TLC) was performed for identification of metabolites. Standard mycotoxins used for reference of Rf were citrinin and penicillic acid. 10 mL of each supernatant, methanol extract and 10 mL of standard solutions (1 mg mL-1) were spotted on TLC plates. The elution systems used were as follows: Chloroform-acetone-2-propanol (85/15/20, v/v/v). The plates were developed under darkness and examined at daylight, 365 and 254 nm. Spots with Rf different from standard on either elution systems were not considered (Khaddor et al., 2007).
RESULTS
The initial antibacterial activity screening of soil isolates was made against six test microorganisms in agar medium. 80% of the investigated strains possessed antimicrobial activity at least against one of the test microorganisms (Table 1, 2).
Four strains showed high inhibition potential against Candida albicans, Bacillus subtilis, Staphylococcus aureus, Salmonella typhi and Escherichia coli over mm sterile zone.
It should be pointed out that from all Penicillium species isolated from agricultural soils of Northern Iran, P. aurantiogriseum (species No.1164) showed to be one of the most active isolates (p<0.05). At the same time, other isolates including: P. viridicatum (species No.1193), P. citrinum (species No.1154) and P. waksmanii (species No.1099) also showed considerable antimicrobial activities (p<0.05). The values of inhibition zones are shown in Table 1 and 2.
Identification of mycotoxins: Two different toxins were identified in P. aurantiogriseum culture: Penicillic acid and citrinin and their Rf were 0.77 and 0.85, respectively.
| Table 1: | Diameter of inhibition zones (mm) of tested supernatants on disc diffusion test |
| Control: Miconazol (C. albicans), chloramphenicol (bacteria), a: Absence of inhibition zone | |
| Table 2: | Diameter of inhibition zones (mm) of tested methanol extracts on disc diffusion test |
| Control: Miconazol (C. albicans), chloramphenicol (bacteria), a: Absence of inhibition zone | |
Citrinin and penicillic acid were also characterized in the cultures of P. citrinum and P. viridicatum, respectively. P. waksmanii could produce two metabolites with Rf 0.08 and 0.15 and these two metabolites were purified for further evaluation.
DISCUSSION
Microorganisms produce many bioactive compounds as secondary metabolites including antibiotics and cytotoxic compounds (Nakashima et al., 2008). To identify the biological activities of the metabolites produced by the Penicillium strains, isolated from North of Iran, we screened these species for antibiotic activities. This is the first report of the biological activity of Penicillium strains from soils of Northern Iran.
Some of the most well known metabolites are produced by species of Penicillium, the most famous and economically important being penicillins produced by Penicillium chrysogenum (Raper and Thom, 1949), mycophenolic acid produced by Penicillium brevicompactum (Bentley, 2000) and compactins produced by Penicillium solitum (Frisvad and Filtenborg, 1989). Penicillium strains are also well known because a large number of them produce mycotoxins (Frisvad et al., 2004).
Many of these mycotoxins such as citrinin and penicillic acid have also antimicrobial activity (Wang et al., 2004; Kang et al., 2007).
Among the Penicillium strains in our study, P. aurantiogriseum showed to be one of the most active isolates. These potency may be due to the simultaneous production of citrinin and penicillic acid in the cultures of this strain. There are few reports of the production of citrinin by P. aurantiogriseum, but Varnaite et al. (2006) also reported the production of citrinin by P. aurantiogriseum(Varnaite et al., 2006). Few reports are available about simultaneous production of citrinin and penicillic acid by P. aurantiogriseum.
Citrinin is the only mycotoxin produced by P. citrinum. P. citrinum is the major producer of this toxin, but production by P. expansum and P. verrucosum has also been reliably reported. Literature citations indicate that at least 22 Penicillium species have been reported to produce citrinin, but the great majority of these are either regarded as synonyms, or require confirmation (Pitt and Leistner, 1988).
The antibacterial activity of P. aurantiogriseum and P. viridicatum, has been reported by Khaddor et al. (2007). They found that P. aurantiogriseum produces penicillic acid, terrestric acid and aurantiamine, while penicillic acid, terrestric acid, brevianamide A and xanthomegnin were produced by P. viridicatum.
We detected penicillic acid in the culture of P. viridicatum. The potential for production of penicillic acid by P. viridicatum also reported by Frisvad and Filtenborg (1983).
P. waksmanii is a ubiquitous anamorphic fungus, widespread at once in land and in various aquatic environments such as sewage sludge or algae (Petita et al., 2004).
Actually, different metabolites have been isolated from cultures of P. waksmanii: two alkaloids (Kozlovskii et al., 1997), four pyrones (pyrenocines A, B, D and E with significant cytotoxicity) and three sulfur-containing dioxopiperazines (Amagata et al., 1998). Furthermore, Petita et al. (2004) has also detected griseofulvin in a marine strain of Penicillium waksmanii in 2004. P. waksmanii is an important producer of cellulase and hemicellulase as well (Han et al., 2009).
Present finding about antimicrobial activity of terrestrial Penicillium waksmanii was not reported yet. The TLC analysis of metabolites from Penicillium waksmanii with dragendorff reagent detected that these two metabolites were not nitrogenous compounds like alkaloids and should be examined for further elucidations.
Malmstrom et al. (2000) have confirmed that P. citrinum (25 isolates) consistently produced citrinin and tanzawaic acid A, Varnaite et al. (2006) also reported the production of citrinin by P. aurantiogriseum.
Xin et al. (2005) also found a new cytotoxic compound from P. aurantiogriseum.
CONCLUSION
The aim of this study was to evaluate the biotechnological potential of North Iranian soil fungal species, as a source of antibacterial compounds that could be used as lead compounds to develop new antibiotics for clinical/therapeutic use.
As a conclusion, the selected species should further examine for the characterization of their metabolites and optimize for the culture conditions in large scale production.
ACKNOWLEDGMENT
The authors are grateful to Mazandaran University of Medical Sciences for financial support (grant No. 87-87).