ANTAGONISTIC PROPERTIES OF SOME MICROORGANISMS ISOLATED FROM BRAZILIAN TROPICAL SAVANNAH PLANTS AGAINST Staphylococcus COAGULASE-POSITIVE STRAIN

Endophytic microorganisms are relatively unstudied as potential sources of novel natural products for medical and commercial exploitation. The aim of this work was to investigate some Brazilian tropical savannah trees Cassia leptophylla and Prunus spp. in order to isolate the endophytic microorganisms associated with these plants. The samples were disinfected to eliminate the epiphytic population. Colonies were diluted and displayed as drops in media and growing colonies were inactivated. Staphylococcus coagulase-positive strain was used as indicator microorganism and subjected to the antibioses test. Data showed that the microorganisms isolated from Cassia leptophylla had no inhibition against Staphylococcus. On the other hand, microorganisms isolated from Prunus spp. leaves showed antibacterial activity and inhibited Staphylococcus when cultivated in peptone agar as well as in yeast extract agar. Investigation proceeds in order to classify the isolated microorganisms presenting bioactive substance and exploit the potential of the compounds produced to inhibit the indicator bacteria. Other bioactive properties will be investigated.


INTRODUCTION
The need for new and useful compounds to provide assistance and relief in all aspects of human condition is ever-growing.Drug resistance in bacteria, the appearance of life-threatening viruses, and the increase in the incidence of fungal infections in the world's population all underscore our inadequacy to cope with these medical problems.Endophytic microorganisms that colonize internal tissues of plants without causing any negative effects are relatively unstudied as potential sources of novel natural products for medical and commercial exploitation.Some endemic plants having an unusual longevity can generate bioactive natural products associated with endophytic microorganisms that produce the same natural products (1).Presumably, the simplest biological arrangement between these organisms is that the plant provides nutrition for the microbe and this one provides some form of protection for the plant.Endophytes promote plant growth and yield, suppress pathogens, may help to remove contaminants, solubilize phosphate, or contribute to assimilate nitrogen to plants (12).Brazilian tropical savannah plants are likely to be excellent specimens to begin a search for endophytic microorganisms (11,15).In Brazilian tropical savannah soils with native plants, the natural populations of actinomycetes in the microbiota can be superior to 75%, with predominance of the genera Streptomyces (6,8,16).As a group, the Streptomycetes provide nearly 80% of all of the world's antibiotics produced (3,4).A search reveals that most of these organisms have their origins in soil (10), and another biologically important niche that has been overlooked as a source of novel are the tissues of higher plants.This specialized and unique biological niche that supports the growth of microbes is the intracellular space between cells of higher plants.Novel endophytes usually have associated with them novel secondary natural products (17).
The aim of this work was to investigate the Brazilian tropical savannah trees Cassia leptophylla and Prunus spp. in order to isolate the associated endophytic microorganisms with potential for producing antibacterial substances.To eliminate the epiphytic population, the plants (leaves and caulis) were washed with distilled sterile water and neutral detergent and its surface disinfected with ethanol (70%/1min), sodium hypochlorite (2%/6min) and ethanol (70%/30s) and consecutively rinsed with distilled sterile water (10).The surface-disinfected plants were then aseptically sectioned into 0.5+/-0.7cmfragments, distributed onto the isolation media, yeast extract agar (YE) and peptone agar (PA), and incubated at room temperature for 4 days.At the same time, the disinfected plants were homogenized with peptone water 0.1% (1:9).Decimal dilutions were made and 100µl aliquots were displayed on the surface of agar medium and incubated (room temperature/4 days).From each of these plates, some colonies representing different morphologies were then picked.Preliminary microbial identification using Gram and Ziehl-Neelsen staining, melanin production and catalase activity were done.

The
Selected colonies were diluted in peptone water (0.1%) and displayed as drops (Pasteur pipette) in PA and YE as an additional part.Petri dishes were incubated (room temperature, and 37°C/48h).The bioassays were conducted using growing colonies in PA and YE and inactivating them by chloroform (20min).Plates were opened (30min) to evaporate the substance.At the same time, the reactivation of Staphylococcus coagulase-positive strain (BHI broth 24h/37°C) was made and one aliquot was used as the indicator microorganism.200µl of the culture properly reactivated was transferred to 10ml of semi solid BHI medium and shaken.This mixture was deposited onto the surface of plates containing the inactivated microorganisms and incubated (37°C/48h) for the observation of inhibition halos.

RESULTS
Ten colonies, chosen among those isolated from Cassia leptophylla and Prunus spp., were selected.Data showed that the microorganisms isolated from Cassia leptophylla had no antibiosis against Staphylococcus coagulase-positive strain when cultivated in PA and YE (Figure 1).
The macroscopic evaluations of one isolate from Prunus spp.were made.The observation of growing colonies in PA and YE were made by visual inspection of the morphological characteristics.The isolated microorganism showed different growing aspects when cultivated in these media (room temperature/48h).In YE, the colonies were yellowish, big, glowing and mucous, and in PA, white, small and with irregular borders (Figure 2).Gram-positive bacilli and spores as well as bacilli with one or two apical spores in Gram staining were observed, suggesting that only one propagule was isolated.No alcohol-acid cells in Ziehl-Neelsen staining were observed.It was not observed the melanoid pigment production.The catalase activity was positive.The investigation continues in order to identify the isolated endophytic microorganism.
The microorganisms isolated from Prunus spp.leaves showed distinct inhibition zones against Staphylococcus coagulase-positive strain.The white colony showed antibacterial activity against Staphylococcus, with inhibition zones of 1.6cm and 2.1cm diameter in media when cultivated in PA and YE, respectively (Figure 3).
Similarly, the inhibition of Staphylococcus by the other isolated microorganism (yellowish colony) presented inhibition zone of 2.0cm and 1.4cm diameter in media when cultivated in YE and PA, respectively (Figure 4).

DISCUSSION
In Pernambuco state, Brazil, Brito (2) isolated actinomycetes from beans (Phaseolus vulgaris).They showed that 6% of the isolated microorganisms had antibiotic activity against Staphylococcus aureus and Bacillus subtilis.In this present study, the isolated microorganisms from Prunus spp.showed activity against the tested Staphylococcus coagulase-positive strain.Data exhibited here suggests that Brazilian tropical savannah plants are good source to search endophytic microorganisms, emphasizing the potential of natural compounds that can be used in agriculture, clinics and pharmaceutical industry.
The high commercial potential of natural substances and the upcoming threat of multidrug resistant pathogens have brought research on actinomycetes into focus again during the last years.
In conclusion, endophytic microorganisms are a very promising source for production of bioactive compounds.Investigation proceeds in order to classify the microorganisms and exploit the potential of the substance produced to inhibit pathogenic bacteria.
Brazilian tropical savannah plants Cassia leptophylla and Prunus spp.were collected in an ecological reserve at São Carlos, São Paulo State, Brazil, and analyzed at the Microbiology Laboratory, at Federal University of São Carlos.R. P. Ratti et al.ANTAGONISTIC PROPERTIES OF SOME MICROORGANISMS ISOLATED FROM BRAZILIAN TROPICAL SAVANNAH PLANTS AGAINST Staphylococcus COAGULASE-POSITIVE STRAIN.J. Venom.Anim.Toxins incl.Trop.Dis., 2008, 14, 2, p. 296 R. P. Ratti et al.ANTAGONISTIC PROPERTIES OF SOME MICROORGANISMS ISOLATED FROM BRAZILIAN TROPICAL SAVANNAH PLANTS AGAINST Staphylococcus COAGULASE-POSITIVE STRAIN.J. Venom.Anim.Toxins incl.Trop.Dis., 2008, 14, 2, p. 297 R. P. Ratti et al.ANTAGONISTIC PROPERTIES OF SOME MICROORGANISMS ISOLATED FROM BRAZILIAN TROPICAL SAVANNAH PLANTS AGAINST Staphylococcus COAGULASE-POSITIVE STRAIN.J. Venom.Anim.Toxins incl.Trop.Dis., 2008, 14, 2, p. 299 Sardi et al. (14), working in Italy with about 500 endophytic actinomycetes isolated from roots from 28 different plants, showed that the Streptomyces were the predominant isolated genera.Yoo et al. (18) analyzed the capability of the Streptomyces spp.CS684 in order to inactivate methicillin-resistant Staphylococcus aureus (MRSA) and vancomycinresistant enterococci (VRE).Data showed that the Streptomyces spp.CS684 presented antibacterial activity against MRSA and VRE.Gu et al. (5) analyzed the capability of Streptomyces roseosporus strain in the inhibition of Staphylococcus aureus and showed that the S. roseosporus strain exhibited potent antibacterial activity against S. aureus. .P. Ratti et al.ANTAGONISTIC PROPERTIES OF SOME MICROORGANISMS ISOLATED FROM BRAZILIAN TROPICAL SAVANNAH PLANTS AGAINST Staphylococcus COAGULASE-POSITIVE STRAIN.J. Venom.Anim.Toxins incl.Trop.Dis., 2008, 14, 2, p. 300 R