Abstract
Soil cyanobacteria isolated from the paddy fields of seven provinces in Iran was evaluated for antimicrobial activity. Aqueous, petroleum ether, and methanol extracts from 76 microalgae were examined for antimicrobial properties against four bacteria and two fungi. Of total microalgae, 22.4% (17 cyanobacteria) exhibited antimicrobial effects. Selected cyanobacteria with positive antimicrobial activities were members of the families Stigonemataceae, Nostocaceae, Oscillatoriaceae, and Chroococcaceae. Growth of Bacillus subtilis Persian Type Culture Collection (PTCC) 1204 and Staphylococcus epidermidis PTCC 1114 were inhibited by 12 and 14 species of cyanobacteria, respectively. Also, eight cyanobacteria inhibited the growth of Escherichia coli PTCC 1047, and two species inhibited the growth of Salmonella typhi PTCC 1108. Considering fungi, six species inhibited the growth of Candida kefyr ATCC 1140, and one cyanobacterium species inhibited the growth of Candida albicans ATCC 14053. Furthermore, one of the paddy fields (Khozestan) showed no antimicrobial activity.
Introduction
Cyanoacteria are a major component of microbial flora in rice paddy fields and contribute to the fertility of such ecosystems (Roger & Kulasooriya, Citation1980). Producing active biocide components could be another important selective advantage (Mundt et al., Citation2001). Terresterial and marine cyanobacteria have proved to be an extremely valuable source of novel bioactive agents (Jaki et al., Citation2000; Hirata et al., Citation2000).
Screening of cyanobacteria for antibiotics and other pharmacologically active compounds has recently received considerable attention, cyanobacteria are as novel bioactive natural producers (Borowitzka, Citation1995; Borowitzka & Borowitzka, Citation1988). Many active substances with antibacterial, antiviral, fungicide, enzyme inhibiting, immunosuppressive, cytotoxic, and algicide activity have been isolated from cyanobacterial biomass or in some cases from the medium of laboratory cultures (Knubel et al., Citation1990; Mule et al., Citation1991; Gerwork et al., Citation1994; Falch et al., Citation1995; Jaki et al., Citation1999).
Cyanobacteria of local habitats have rarely been screened for biological effects. Ghasemi et al. (Citation2003) have surveyed antibacterial activities of microalgae of northern Iran. Also, a novel antimicrobial substance named Parsiguine has been identified (Ghasemi et al., Citation2004). Therefore, in a continuing investigation, the biological activities of various species and strains of terrestrial cyanobacteria in different provinces of Iran were studied. The ability to produce antimicrobial substances may be noticed not only as a defensive instrument for the strains but also as a good source of new bioactive compounds from a pharmaceutical point-of-view. Accordingly, 228 lipophilic and hydrophilic extracts obtained from 76 samples of cultured soil cyanobacteria were investigated to detect their antimicrobial effects.
Materials and Methods
Materials
All of the chemicals and reagents used in the current study were purchased from Merck (Darmstadt, Germany). Gentamicin and nystatin were obtained from Sigma (St. Louis, MO, USA).
Methods
Isolation and culture conditions
Soil algae were isolated from paddy fields of different provinces in Iran: Golestan, Mazandaran, and Gillan (north); Isfahan and Fars (central); Ilam (west) and Khoozestan (south). Soil samples were cultured by usual methods (Kaushik, Citation1987). Algae were grown in 250-ml conical flasks containing 100 ml of BG11 medium adjusted to pH 7.4. The cultures were illuminated continuously (50 µE m−2 s−1) supplied by six fluorescent lamps and following incubation at 30±1°C. Identification of cyanobacteria with antimicrobial activity was done according to Desikachary (Citation1959), Prescott (Citation1962), Anagnostidis & Komarek (Citation1990), and John et al. (Citation2003).
Antimicrobial bioassay
Cells were harvested by centrifugation at 5000 × g for 15 min, followed by extraction with petroleum ether and methanol. The aqueous supernatants and solvent extracts were concentrated under reduced pressure at 40°C. The antibacterial and fungicide activities of algal extracts were evaluated by agar plate diffusion test (Lorain, Citation1996).
The following bacteria and fungi were used as test organisms: Gram-positive bacteria, Staphylococcus epidermidis. (Winslow & Winslow) Schleifer & Kloos PTCC 1114 and Bacillus subtilis. (Ehrenberg) Cohn PTCC 1204; Gram-negative bacteria, Escherichia coli. (Migula) Castellani & Chalmers PTCC 1047 and Salmonella typhi. (Schroeter) Warren & Scott PTCC 1108; Fungi, Candida kefyr. (Beij.) Uden & H. R. Buckley ex S. A. Mey. & Ahearn ATCC 38296, and Candida albicans. (C. P. Robin) Berkhout ATCC 14053.
Filter-paper disks (6.4 mm) were saturated with 60 µl of the test solution, dried, and placed on Müller-Hinton agar plates for bacteria and Saubouraud's dextrose agar plates for fungi inoculated with a lawn of the test microorganisms.
Plates were incubated at an appropriate temperature for bacteria (37°C) and fungi (25°C) for a period of 18–24 h in dark. Gentamicin and nystatin disks (10 µg) were used as positive controls. The distinct and circular radii of inhibition zones were measured.
The following formula was used for comparison of the antimicrobial activity of the sample with that of the standard (antimicrobial index):
Results
Soil cyanobacteria for this study were isolated from different geographical locations, mainly north of Iran due to high distribution of paddy fields in these regions. Specification of species and their original habitats are listed in .
Table 1.. Investigated species with antimicrobial activity and their geographical origin.
Seventy-six strains were isolated. All of them were cultured in an inorganic medium (BG110) at 30°C with illumination. The harvested cells were successively extracted with solvents of increasing polarity (petroleum ether and methanol, respectively). The supernatants were extracted separately. Antimicrobial activity was shown by 17 isolated species. Five species belonged to the genus Nostoc.. Nine of them were included in the family Stigonemataceae (three Stigonema., four Hapalosiphon., two Fischerella.). Others belonged to the genera Plectonema., Oscillatoria., and Chroococcus. ().
Table 2.. List of cyanobacteria with antimicrobial activity.
Between the fungi, Candida albicans. ATCC 14053 was more resistant. It was inhibited by only one species. The cell extracts and culture medium of 10 strains such as Oscillatoria., Hapalosiphon., Fischerella., and Stigonema. species showed antifungal activities against C. kefyr. ATCC 38296. But among all strains, only the petroleum ether and methanol extracts of H. hibernicus. West & West FS 33 showed antifungal activity against C. albicans. ATCC 14053. The same results were achieved in this cyanobacterium in relation to C. kefyr. ATCC 38296 ().
Table 3.. Antimicrobial activity of different extracts from cyanobacteria.
Also, H. hibernicus. FS 33 indicated a widespread spectrum of antimicrobial activities. Only two cyanobacteria indicated antibacterial activity against Salmonella typhi. PTCC 1108, and these results were detected in petroleum ether extracts.
Results showed that in species with activity against S. epidermidis. PTCC 1114, the Ai (Antimicrobial index) varied between 32 and 112. This is comparable with the results of Ghasemi et al. (Citation2003). It was different in other cases, for instance 38–78 against B. subtilis. PTCC 1204 and 32–86 against E. coli. PTCC 1047. With respect to S. typhi. PTCC 1108, the activity index was around 84 and changed to 83–175 in experiments with C. kefyr. ATCC 38296. But it decreased to 80 in relation to C. albicans. ATCC 14053. On the other hand, Fischerella ambigua. (Naeg.) Gomont FS18 has the highest Ai (112) in the methanol extract against S. epidermidis. PTCC 1114. It seems this species may have potent antimicrobial activity and can be the subject of future experiments.
Discussion
In the past decades, screening programs have revealed that cyanobacteria are a potential source of new active substances for medicine and pharmacy, and numerous active compounds have been isolated (Moore et al., Citation1989; Falch et al., Citation1995; Jaki et al., Citation1999). But few have been done with respect to cyanobacteria from paddy fields. Possibly, the synthesis of highly active toxin can be a defense option of cyanobacteria in these environments against other organisms like bacteria, fungi, viruses, and eukaryotic microalgae (Mundt et al., Citation2001).
Cyanobacteria from the paddy fields of northern Thailand have produced bioactive substances with antibiotic activity against B. subtilis. (Chetsumon et al., Citation1993). This is in agreement with results obtained in the current project in which cell extracts and culture media of 11 species had activity against B. subtilis..
Also, extracts of terrestrial Fischerella ambigua. were the most active in the test systems used (Falch et al., Citation1995). The polychlorinated aromatics, ambigols A and B, were isolated as the agents responsible for the pronounced antimicrobial activities of the lipophilic extracts. These activities were also well established in the current observations. Petroleum ether, methanol, and supernatant extracts of F. ambigua. FS18 had inhibitony activity against B. subtilis. PTCC 1204, S. epidermidis. PTCC 1114, and E. coli. PTCC 1047. In addition, cell extracts of this strain had antifungal activity against C. kefyr. ATCC 38296.
As mentioned, few studies have been done in relation to cyanobacteria of Iran (Ghasemi et al., Citation2003), and this study follows a screening program for isolation and identification of antimicrobial-producing species from Iranian paddy fields. No inhibition was shown against E. coli. PTCC 1047 and no antimicrobial activity was detected in nonpolar extracts (Ghasemi et al., Citation2003). In spite of that, data obtained from this research demonstrated antimicrobial activity against E. coli. PTCC 1047 in six methanol, three petroleum ether, and one aqueous extract. A diterpenoid with antibacterial activity from Nostoc commune. Vaucher against E. coli. who isolated by Jaki et al. (Citation1999).
No inhibition was observed against C. albicans. ATCC 14053 by a methanol extract of Nostoc muscorum. C. A. Agardh FS 13. This disagreed with the observations of Cano et al. (Citation1990), who have evaluated the antifungal activity of terrestrial cyanobacterium N. muscorum. against the same fungus by 20.83%. Mule et al. (Citation1991) also indicated that phenolic compounds of N. muscorum. exert an inhibitory effect on C. albicans..
The antibacterial effect against S. typhi. was only found in more lipophilic extracts (petroleum ether). The aqueous extracts were ineffective, a phenomenon that has already been described by Mundt et al. (Citation2001).
The antimicrobial activities of algal extracts are shown in and Citation3. Data in show the cyanobacteria with antimicrobial effects belong to the following families. Stigonemataceae: Stigonema ocellatum. (Dillw.) Thuret FS 23, S. turfaceum. (Berkeley) Cooke FS 14, S. ocellatum. FS 30, H. brasiliensis. Borge FS 6, H. aureus. West & West FS 3, H. fontinalis. (Agardh) Bornet FS 7, H. hiberinicus. FS 33, Fischerella ambigua. FS 2, and F. ambigua. FS 18; Nostocaceae: Nostoc muscorum. C. A. Agardh FS 13, Nostoc. sp. FS 4, N. linckia. (Roth) Bornet & Thuret FS 8, N. microscopicum. Carmichael FS 21, and N. punctiforme. C. A. Agardh FS 5; Oscillatoriaceae: Plectonema notatum. Schmidle FS 28 and Oscillatoria angusta. Koppe FS 19; Chroococcaceae:Chroococcus. sp. FS 1.
Apparently, the potent antibacterial cyanobacteria from different localities of Iran belong to Stigonemataceae and Nostocaceae. These results are in agreement with other experiments in which cyanobacteria such as H. fontinalis. (Moore et al., 1987), F. ambigua. (Falch et al., Citation1995), F. musciola. (Borzi) Gomont (Hagmann & Jüttner, Citation1996), N. spongiaeforme. C. A. Agardh (Hirata et al., Citation2000), and N. commune. (Jaki et al., 2000) produce antimicrobial substances.
In a few cases, species like Plectonema notatum. FS 28 (Golestan Province) and Oscillatoria angusta. FS 19 (Mazandaran Province) showed antibacterial effects against B. subtilis. PTCC 1204 and S. epidermidis. PTCC 1114. Antimicrobial activity of Chroococcus. sp. FS 1 was observed in one paddy field in Mazandaran Province against S. epidermidis. PTCC 1114 (Tables and ).
A variety of solvents with different polarities were used for the extraction of algal bioactive materials. All extracts showed antimicrobial activity, probably because of the different polarity of active components. But the percentage of methanol extracts with positive effects was higher, indicating a higher chance of finding antimicrobial activity in methanol extracts. It was found that active compounds was higher against Gram-positive bacteria (S. epidermidis. PTCC 1114, B. subtilis. PTCC 1204) in comparison with Gram-negative bacteria (E. coli. PTCC 1047, S. typhi. PTCC 1108), the same results that Ghasemi et al. (Citation2003) have observed. Gram-positive bacteria (S. epidermidis. PTCC 1114, S. aureus. Rosenbach PTCC 1112, S. haemolyticus. Schleifer & Kloos ATCC 29970) are more sensitive than Gram-negative bacteria [Shigella sonnei. (Levine) Weldin PTCC 1325, Escherichia coli. PTCC 1047, and Proteus vulgaris. Hauser PTCC 1312].
Seventeen species from different localities showed antimicrobial effects. The extracts were most effective against S. epidermidis. PTCC 1114 and to a lesser extent S. typhi. PTCC 1108. Ten extracts inhibited E. coli. PTCC 1047. Most inhibition was produced by methanol extracts. These results disagreed with Ghasemi et al. (Citation2003) and Kellam and Walker (Citation1989).
The most potent species, Hapalosiphon hiberinicus. FS 33, was collected from different paddy fields of Fars Province. Interestingly, specimens of this species from paddy fields of Khoozestan Province showed no antibacterial production. The cyanobacteria of paddy fields of Iran showed different antimicrobial behaviors (Tables and ).
It was interesting that some species showed similar characteristics. For example, F. ambigua. FS18 that was collected from Gillan Province showed very strong and permanent antibacterial abilities, agreeing with reported activity by Ghasemi et al. (Citation2003) for the same species collected in Golestan Province.
It seems that among all of the species studied in the current work for antibacterial and antifungal activity, Fischerella. and Hapalosiphon. had most prominent effects and display a potential that warrants further investigation.
Data also indicated that the highest percentage of colonization and distribution of cyanobacteria in soil cultures and also the highest rate of antimicrobial activity belonged to northern provinces (70.5% in Golestan, Gillan, and Mazandaran Provinces). The lower colonization was seen in Fars (17.7%), Isfahan (5.9%), and Ilam (5.9%), respectively.
As results indicated (), none of isolated samples from Khoozestan Province (south of Iran) demonstrated antimicrobial activity. Also, the distribution and colonization of microalgae in these cultures were lower than other locations. Nostoc microscopicum., which was the dominant species in these paddy fields, exhibited no antimicrobial ability (data not shown). The current results show that most of the investigated cyanobacteria induced a response in at least one of the test systems applied. The most promising cyanobacterial strains for further studies appeared to be Fischerella ambigua. FS 18, Hapalosiphon hibernicus. FS 33, and Oscillatoria angusta. FS 19.
Acknowledgments
The authors wish to thank Dr. Y. Ghasemi and Mr. G. Zarrini for collection and spectral studies. Mrs. Ladan Baftehchi is also thanked for her participation in isolation of microalgae.
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