Cyanobacterial extra-metabolites against some pathogenic bacteria

Ten cyanobacterial species (Nostoc calcicola, Nostoc commune, Nostoc entophytum, Nostoc minutum, Nostoc palndosum, Nostoc passerianum, Nostoc punctiforme, Anabaena ambigua, Anabaena amomala, and Anabaena doliolum) were isolated from the mangrove region of Ras Mohammed (Sinai, Egypt), and were tested for their allelopathic activities including inhibitory and/or promoting effects against two Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus) and two Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Data suggested two types of allelopathic effects: one type which always appeared in cyanobacterial medium as in the case with N. minutum (medium that inhibits the growth of all tested bacterial species). The other type is induced only when cyanobacteria are in contact with bacteria; this is the case when the growth of both B. subtilis and S. aureus were inhibited in co-culture with N. commune. On the other hand, promotion effects of bacterial growth were observed when grown in cyanobacterial metabolites in most of studied cyanobacterial species. The biological assays for aqueous and methanolic extracts of the two Nostoc species revealed that both extracts for each species were not toxic at concentrations of 0.52 and 0.59 g L -1 water extract for N. commune and N. minutum, respectively and 0.31 and 0.425 g L -1 for methanolic extract for N. commune and N. minutum, respectively. No mortality was observed in tested mice within 72 h.


INTRODUCTION
Research activities concerning the investigation of products of metabolism of plants and other groups of organisms were undertaken not only for a better understanding of nature but also to discover metabolites of possible use by humans in different fields of interest.Cyanobacteria are very old groups of prokaryotic organisms that produce a variety of secondary metabolites of allelopathic activity (Mundt et al., 2001).The screening of extracts or isolated compounds from different natural sources is a common way to discover biologically active metabolites.In such research activities, cyanobacteria were found to be a rich source for various products of commercial and pharmaceutical interest as primary metabolites such as carbohydrates, proteins, fatty acids, vitamins or pigments (Borowitzka, 1988a, b, *Corresponding author.E-mail: ibraheemborie@hotmail.com.1995) and/or various secondary metabolites such as phenolic compounds (De Cano et al., 1990;Pedersen and Da Silva, 1973;Volk, 2005), peptides, alkaloids or terpenoids and ne-glycosides (Ramamurthy, 1970;Bonjouklian et al., 1991), which showed some different bioactivities as antifungal, antiviral and antibacterial (Abdel-Raouf, 2004;Volk and Furkert, 2006;Hassan, 2007).
The aim of the present investigation was to quantify the allelopathic potential of the extra-cellular metabolites and/or the culture of ten selected local cyanobacterial species against two Gram positive and two Gram negative species.

Collection of cyanobacterial isolates
Ten cyanobacterial isolates were isolated from the mangrove regions at Ras Mohammed, Sinai, Egypt and re-cultured on artificial medium in laboratory.In this respect, Z-medium described by Staub (1961) and Allen's free nitrogen medium described by Hughes et al. (1958) modified by Allen (1968), using the techniques described by Esmarch (1914) and EL-Ayouty and Ayyad (1972), were used for isolation and culturing of the cyanobacterial isolates.
Unialgal and axenic cultures were obtained as described by Pringsheim (1949).These cultures were subjected to different trials to employ bacteria free cultures according to Felfoldy and Zsuzsa (1959) and Hoshaw and Rosewski (1973).
These purified cyanobacterial species were identified according to Smith (1950) and Deskichairy (1959).Logarithmic growth phase of each cyanobacterial species was detected to obtain healthy growth mass and vigorous extracellular products to be used in the allelopathic activity.All test organisms were kindly supplied by Biotechnological Research Center, Al-Azhar University (For Boys), Cairo, Egypt.These organisms were sub-cultured on specific culture media until used in the experiments.
After incubation at pH 7 for 48 h in the dark at 30°C, the number of viable bacteria was determined by a plate test by counting colony forming units per milliliter.Controls were done with bacteria in plain cyanobacterial media.Screening tests were run two times.Data for CFU are obtained from three parallel platings.

Detection of mixed cultures
Suspension of the four tested bacteria (10 µl, OD670 = 0.3 to 0.4) were added separately into the two Nostoc species cultures (50 ml, Ibraheem et al. 6721 exponential phase of growth).An aliquot of the mixture was taken for determination of CFU at the start of the experiment and every 48 h for 16 days.The pH was routinely checked.

Toxicity test
Mouse bioassay for toxicity test of both aqueous and methanolic extracts of the dry matter of the two Nostoc species to mice was evaluated by injecting mice with different concentrations for these extracts.It was conducted in Animal Laboratory, Faculty of Science, Beni-Suef University.

Statistical analysis
Analysis of variance (one-way ANOVA) was employed to determine if treatments were significantly different from each other (Zar, 1984).
Results were significantly different at the levels of 5 and 1%.All data were of three replicates.

RESULTS
The most common filamentous cyanobacteria inhabiting the mangrove samples have been detected either by direct observation or by the culture method.The greatest abundance percentage was recorded by two genera Nostoc and Anabaena; from which we isolated seven species belonging to Nostoc sp.When the supernatant of the ten cyanobacterial cultures were screened for allelopathic activity against bacteria in most assays, growth promoting effects were observed.At maximum, this led to a 20.7-fold increase of CFU of P. aeruginosa with supernatant of N. palndosum compared with control.On the other hand, supernatant of N. minutum did not show comparable growth promoting effects, but there were significant inhibitory effects on all studied bacterial species.
Concerning the effect of N. commune culture on the tested bacteria, there was slight inhibitory effect on E. coli and slight promoting effect on P. aeruginosa, S. aureus and B. subtilis.By a second series of experiments, we tested whether bacteria might induce the formation of bioactive compounds in Nostoc minutum and N. commune.Thus, we co-cultured the two cyanobacterial species with the four bacteria and monitored the amount of viable bacteria by the CFU assay.
In co-culture, N. minutum inhibited the growth of all tested bacterial species (Figure 1).This is in agreement with the effect of the cyanobacterial supernatant alone (Table 1).A most interesting resistance effect was observed with E. coli, after an initial decrease in count, it increased again and reached the original level (Fig. 1A 1 ).
N. commune inhibited the growth of both E. coli (Figure 2B 1 ) and P. aeruginosa (Figure 2B 2 ).This is in contrast to the effect of cyanobacterial supernatant alone which did not show any effect on growth of P. aeruginosa after 48 h incubation period (Table 1).
In the presence of N. commune, the growth of B. subtilis was enhanced (Figure 2B 3 ).This is in agreement with the effect of N. commune supernatant (Table 1).In a co-culture of N. commune and S. aureus, however, bacterial growth was inhibited (Figure 2B 4 ).This is in striking contrast to the slightly promoting effect of the cyanobacterial growth as shown in Table 1.
The biological assays for aqueous and methanolic extracts of the two Nostoc species revealed that both extracts for each species were not toxic at concentrations of 0.52 and 0.59 g L -1 water extract for N. commune and N. minutum, respectively and 0.31 and 0.425 g L -1 for methanolic extract for N. commune and N. minutum, respectively.No mortality was observed in tested mice within 72 h.

Interactions between microalgae and bacteria in aqueous
or co-cultures have studied repeatedly.In most cases, growth of microalgae is enhanced in the presence of bacteria (de Bashan et al., 2002), although the mechanism remain to be clarified.Mouget et al. (1995) suggested that growth promoting effect of bacteria was as a result of oxygen reduction of the surface of microalgae.Only few reports dealt with an impairment of microalgal growth by bacteria (Gozales-Bashan et al., 2000) or with growth promotion of bacteria by microalgal substances as shown by the present data of this investigation.Gaumann and Jaag (1950) reported on the production of vitamin A by various microalgae, which therefore might support the growth of microorganisms.The release of organic compounds is a well established feature of different kinds of microalgae and has been studied repeatedly (Hellebust, 1974).
The promoting effects of supernatants of cyanobacterial cultures on the growth of heterotrophic bacteria, as shown in Table 1, were most probably a result of a general accumulation of organic material that is derived from sporulation processes, dying cells, cell wall remnants and diffusing slime materials.However, it is   interesting that in some cases, promoting effects can act selectively on Gram negative or Gram positive bacteria or are even lacking (Safonova and Reisser, 2005).
Therefore, in our experiments, the release of substances that specifically promote the growth of bacteria cannot be ruled out.
Comparably few studies deal with antibacterial compounds that are released by living microalgae (Jones, 1988).For cyanobacteria, the release of antibacterial compounds was shown for Scytonema sp., N. muscorum and Chroococcus turgidus (Chetsumon et al., 1993).In the present study, we observed a slightly inhibiting effect of N. commune on E. coli, but no inhibitory effects were detected against the other tested bacteria.On the other hand, N. minutum showed significant inhibiting effects against all tested bacteria.
So, from the obtained results, we suggest that there exist two types of antibacterial effects of cyanobacteria: the constitutive type when antibacterial substances are generally released by cyanobacteria into their culture media, such as, substances released by N. minutum and acting on all tested bacteria (Table 1) and the induced type when antibacterial substances are only formed by cyanobacteria in the presence of bacteria, such as induced by N. commune against E. coli, P. aeruginosa and S. aureus.The chemical nature of those constitutive and induced substances is not clear.As has been shown here, constitutive substances might be water soluble molecules; whether synthesis of induced substances depends on special chemical signals produced by bacteria or requires cell-to-cell contact between bacteria and cyanobacteria as shown in the co-culture of N. minutum and tested bacteria (Figure 2).

Table 1 .
Effect of cyanobacterial supernatants on the growth of Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus.Data for CFU are obtained from three parallel platings after 48 h of incubation in dark at 30°C.