**1.7.1 Techniques used in research of new substances**

Basidiomycota and fungi in general, represent an inexhaustible source of new substances, even though each species contains hundreds of active metabolites. Therefore, test systems for research of new substances must be fully simplified, fast, efficient and as cheap as possible (Hostettmann et al.*,* 1997, as cited in Giovaninni, 2006). In addition, biotests (bioassays) must be sufficiently sensitive to detect the activity of substances in low concentrations, in the so-called solid (crude) extracts.

Crude products can be used in antimicrobial testing disc-diffusion and broth-dilution assays to test for antibacterial properties including bioautography according to standard procedures (NCCLS or CLSI procedures). The use of standard cultures of familiar characteristics is recommended though several precautions have to be taken into account. In a recent study the differences between two screening methods applied were not statistically significant (t-test at level p< 0.05). Both *Meripilus* extracts analyzed (water and methanol) showed wider inhibition zones in disc-diffusion method, indicating that it is more appropriate for the testing of polar extracts (Karaman et al., 2009b). Similar results were confirmed for extracts of the genus *Fomes* although showed broader inhibitory zones using the method of "wells", compared with inhibitory zones obtained by disc-diffusion method. For other extracts, however, the disk-diffusion method could be recommended, indicating that polarity of active substances in extract influence on results obtained in particular method applied.

MIC and MBC determination is used to quantify antimicrobial activity using the two-fold dilution method according to CLSI guidelines. The MIC is defined as the lowest concentration preventing visible growth while complete absence of growth is considered as the MBC. The lower MIC or MBC values with respect to the extract concentration indicate a higher activity, implying better quality of the extract. To confirm MBCs, aliquots of the experimental suspensions (100μl) could be sub-cultured on Müeller Hinton agar plates incubated overnight.

Potent source of antibacterial agents is the species *M. giganteus* (50mg/ml), showing high activity against both groups of bacteria reaching MIC values in a wide range of concentrations (<17.5 -1125μg/ml). Various activities have been detected among different strains of *S. aureus*, indicating that fungal extracts are target specific on intraspecific level (strain specific).

Antibacterial assay may be performed in 96-well micro-plates instead of tubes. If 5% DMSO is applied for dissolving a negative control with 0.5% DMSO must be used to ensure that DMSO did not affect bacterial growth. Results are recorded after incubation at 35-37ºC for 18-24h and all the samples should be tested in triplicate.

Bioautography is one of the most effective tests for detection of antimicrobial metabolites, considering the fact that it localizes the place of the active component, therefore enabling the isolation of the active component precisely. Bioautography may be the direct, when microorganisms grow directly on the TLC plate, then contact, when the active compound is transferred from the TLC plates to inoculated agar and agar-spill-over (so-called immersion bioautography), when the inoculated agar medium is spilled over the TLC plate (Rahalison

Basidiomycota and fungi in general, represent an inexhaustible source of new substances, even though each species contains hundreds of active metabolites. Therefore, test systems for research of new substances must be fully simplified, fast, efficient and as cheap as possible (Hostettmann et al.*,* 1997, as cited in Giovaninni, 2006). In addition, biotests (bioassays) must be sufficiently sensitive to detect the activity of substances in low

Crude products can be used in antimicrobial testing disc-diffusion and broth-dilution assays to test for antibacterial properties including bioautography according to standard procedures (NCCLS or CLSI procedures). The use of standard cultures of familiar characteristics is recommended though several precautions have to be taken into account. In a recent study the differences between two screening methods applied were not statistically significant (t-test at level p< 0.05). Both *Meripilus* extracts analyzed (water and methanol) showed wider inhibition zones in disc-diffusion method, indicating that it is more appropriate for the testing of polar extracts (Karaman et al., 2009b). Similar results were confirmed for extracts of the genus *Fomes* although showed broader inhibitory zones using the method of "wells", compared with inhibitory zones obtained by disc-diffusion method. For other extracts, however, the disk-diffusion method could be recommended, indicating that polarity of active substances in extract influence on results obtained in particular

MIC and MBC determination is used to quantify antimicrobial activity using the two-fold dilution method according to CLSI guidelines. The MIC is defined as the lowest concentration preventing visible growth while complete absence of growth is considered as the MBC. The lower MIC or MBC values with respect to the extract concentration indicate a higher activity, implying better quality of the extract. To confirm MBCs, aliquots of the experimental suspensions (100μl) could be sub-cultured on Müeller Hinton agar plates

Potent source of antibacterial agents is the species *M. giganteus* (50mg/ml), showing high activity against both groups of bacteria reaching MIC values in a wide range of concentrations (<17.5 -1125μg/ml). Various activities have been detected among different strains of *S. aureus*, indicating that fungal extracts are target specific on intraspecific level

Antibacterial assay may be performed in 96-well micro-plates instead of tubes. If 5% DMSO is applied for dissolving a negative control with 0.5% DMSO must be used to ensure that DMSO did not affect bacterial growth. Results are recorded after incubation at 35-37ºC for

Bioautography is one of the most effective tests for detection of antimicrobial metabolites, considering the fact that it localizes the place of the active component, therefore enabling the isolation of the active component precisely. Bioautography may be the direct, when microorganisms grow directly on the TLC plate, then contact, when the active compound is transferred from the TLC plates to inoculated agar and agar-spill-over (so-called immersion bioautography), when the inoculated agar medium is spilled over the TLC plate (Rahalison

**1.7 Evaluation of antibacterial activity** 

method applied.

incubated overnight.

(strain specific).

**1.7.1 Techniques used in research of new substances** 

concentrations, in the so-called solid (crude) extracts.

18-24h and all the samples should be tested in triplicate.

et al., 1991). In the bioautography agar overlay method, the drug to be evaluated is adsorbed onto the TLC plate and the inoculum is laid onto the plate as a very thin layer (1 mm). The advantage of this method is that the amount of sample being used is very small and that the fractionalisation of the crude extracts on its different components simplifies the identification of active compound.26

In our recent work, the TLC chemical profile of the analyzed species of lignicolous macrofungi showed that they are rich in phenols, although the differences in the number and quality of the extracted compounds have been noticed. Comparing the TLC profiles, fungi can be classified into three groups according to the obtained retention factor e.g. Rf values representing the distance traveled by the compound divided by the distance traveled by the solvent: 1) three species: *C. versicolor, G. lucidum* and *G. applanatum* contain compounds with similar (Rf = 0.68, Rf = 0.69, Rf = 0.70, respectively), 2) five species *M. giganteus, L. sulphureus, F. velutipes, F. hepatica* and *P. ostreatus* showed a small amount of eluated compounds and intense fluorescence at the start line after the spraying, 3) the species *P. betulinus* expressed with three spots in the MeOH extracts (Rf = 0.62, Rf = 0.65, Rf = 0.68), which extinguished fluorescence in the UV 254th (Karaman, 2009c).

Furthermore we made slight modifications of the standard procedure of bioautography in the same study using the following: soft (top) agar (0.7% Nutrient agar) which was mixed with freshly prepared inoculum of bacteria (0.5Mac Farland optical density) and with the aqueous solution of tetrazolium red dye 0.1% w/v (1mg/ml)- 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) (3:1:0.1). The strain S*. aureusan* was used as the indicator organism. Amoxicillin (64µg/ml) was used as positive control. Approximately 10µl of the solution of each extract was applied on a TLC plates (silica gel 60, F 254, DC-Plastikfolien, 0.2 mm thick, Merck, Germany) for about 2h, equally prepared as a reference plate for chemical analysis. Bioautography test plate was developed in the same tank using the pre-determined mobile phase which was removed from the plate by drying with a stream of cool air from a heating gun. Separated spots were visualised under UV light and marked by pencil (Figure 2A). Developed plates were placed upside-down in the petri dishes containing bottom agar (nutrient agar, Torlak, Belgrade). Soft agar (07% Nutrient agar) was melted and poured into sterile tubes (100 ml) in which the dye and bacteria were added quickly. That mixture was flowed over the chromatograms in the petri dishes. After the agar has solidified, the plates were inverted and incubated at 35ºC for 24h. The clear zones on the chromatogram indicate areas of inhibition zones on the red background where bacteria are present. Comparing clearing zones with reference TLC plate according to Rf values the most active components of crude fungal extracts could be approximately detected (Fig. 1B).

Bioautography results showed many antibacterial compounds against animal strain of *S. aureus* that were mostly present in the polar region of the bioautogram. According to detected clearing zones, chloroform extracts were more active corresponding to more detected UV absorptive substances along the chromatogram. However, these substances were not active in methanolic extracts on bioautogram for *C. versicolor* and *P. betulinus.*

**Developing system:** toluene-ethyl acetate – 90% formic acid (5:4:1 v/v/v). **Detection:** 366 nm UV light without spraying. **Extracts:** lane 1- *M. giganteus* (MeOH), lane 2- *L. sulphureus*  (MeOH), lane 3- *C. versicolor* (MeOH), lane 4- *F. velutipes* (MeOH), lane 5- *G. lucidum* (EtOH), lane 6- *G. applanatum* (MeOH)*,* lane 7- *P. tigrinus* (MeOH)*,* lane 8- *P. betulinus* (MeOH)*,* lane 9- *P. ostreatus* (MeOH), lane 10- *F. hepatica* (MeOH), lane 2´- *L. sulphureus* (CHCl3),

Antibacterial Agents from Lignicolous Macrofungi 379

*Escherichia coli. E. coli*, Gram - bacteria, inhabits the gastro-intestinal tract of humans and warm-blooded animals, making their normal indigenous microflora. In immuno-suppressed patients, however, it can cause infections, sometimes fatal (Giovannini, 2006). Gram bacteria cause more problems than Gram +, as a result of their different cell wall structure. Since penicillin and cephalosporin antibiotics belong to the group that act at the level of cell wall synthesis, the exploration of new types of antibiotics is very important for group of Gr-

*C. albicans* belongs to Deuteromycota, representing yeasts forming pseudo-mycelia. It lives as a part of the normal human microflora, especially in the mucosa of the mouth and vagina. In immuno-suppressed individuals (AIDS, chemotherapy, inadequate nutrition and poor hygiene), or after prolonged use of antibiotics, it can cause disease called candidiasis, which is the most common caused by *C. albicans* as the most widespread species. It may affect almost any tissue, starting with simple children's thrush, and ending as the systemic infections. Most commonly it is manifested in the form of slimy mucus. *C. albicans*, is very

In the last decades of the 20th century, the study of macrofungi was intensified, including the research of structurally different metabolites (polysaccharides, glycoproteins, proteoglucans, terpenoids, fatty acids, proteins, lectins, etc..) originating from the primary or secondary metabolism of fungi, as well as different biological activities that they express. Metabolites from fungal fruit bodies or spores themselves are substantially different from those that come from extracellular liquid of the medium in which submerged mycelium was grown or from cells of the culture. Since the phenomenon of multidrug-resistance of microorganisms is on the rise, the studies of macrofungi increased in range, in spite of the fact that they are very slow growing organisms. The value of macrofungi and the dietary supplements, originating from these organisms, grows each year on the world market. They are very safe and considered as the factors useful in the daily diet, especially for people

Natural-products chemists further purify active chemicals from crude extracts by a variety of methods. The chemical structures of the puried material can then be analyzed. Techniques for further chemical analysis include chromatography, bioautography, radioimmunoassay, various methods of structure identication, or modern techniques such as atom bombardment mass spectrometry, Gas chromatography–mass spectrometry, highperformance liquid chromatography, capillary zone electrophoresis, nuclear magnetic reso-

The presented results indicate that extracts from lignicolous macrofungi could be used in the prevention and treatment of Gram-positive bacterial infections resistant to antibiotics in animals (humans), although further toxicity assays (*in vivo*) must be performed before its application. The fact that fungi can have bactericidal properties with low cytotoxicity to the animal host underscores their usefulness as natural sources of human or veterinary

convenient target organism in the detection of new antifungal drugs.

**2. Determination of active substances** 

suffering from various diseases.

**3. Conclusion** 

medicines.

nance spectroscopy, and X-ray crystallography.

organisms.

lane 3´- *C. versicolor* (CHCl3), lane 4´- *F. velutipes* (CHCl3), lane 6´- *G. applanatum* (CHCl3)*,* lane 7´- *P. tigrinus* (CHCl3)*,* lane 8´- *P. betulinus* (CHCl3) **B: Bioautogram of extracts for** *S. aureusa***. Extracts:** lane 1- *M. giganteus* (MeOH), lane 2- *L. sulphureus* (MeOH), lane 4- *F. velutipes* (MeOH), lane 3- *C. versicolor* (MeOH), lane 6- *G. applanatum* (MeOH)*,* lane 5- *G. lucidum* (EtOH), lane 7- *P. tigrinus* (MeOH)*,* lane 8- *P. betulinus* (MeOH)*,* lane 9- *P. ostreatus*  (MeOH), lane 10- *F. hepatica* (MeOH), lane 2´- *L. sulphureus* (CHCl3), lane 3´- *C. versicolor*  (CHCl3), lane 4´- *F. velutipes* (CHCl3), lane 6´- *G. applanatum* (CHCl3)*,* lane 7´- *P. tigrinus*  (CHCl3)*,* lane 8´- *P. betulinus* (CHCl3).

Fig. 1. **A:** TLC separation of crude extracts (methanol - MeOH and chloroform - CHCl3) of selected lignicolous species prepared for bioautography assay and **B:** bioautogram of extracts for Gram- positive bacteria *S. aureus,* animal strain

#### **1.8 Target organisms**

*Bacillus subtilis* is a Gram + bacteria, non-pathogenic to humans and can be used as a model organism in similar tests, since the representative of the same genus, bacteria *B. anthracis* is responsible for the disease anthrax, which is characterized by the appearance of edema, hemorrhage and tissue necrosis. It is common in some animals, often used as a biological weapon in bioterrorism. If an extract shows activity against *B.subtilis*, it is possible to be active against *B. anthracis* and possibly against other pathogenic Gram + bacteria such as specieses of the genera *Staphylococcus* and *Streptococcus.*

lane 3´- *C. versicolor* (CHCl3), lane 4´- *F. velutipes* (CHCl3), lane 6´- *G. applanatum* (CHCl3)*,* lane 7´- *P. tigrinus* (CHCl3)*,* lane 8´- *P. betulinus* (CHCl3) **B: Bioautogram of extracts for** *S. aureusa***. Extracts:** lane 1- *M. giganteus* (MeOH), lane 2- *L. sulphureus* (MeOH), lane 4- *F. velutipes* (MeOH), lane 3- *C. versicolor* (MeOH), lane 6- *G. applanatum* (MeOH)*,* lane 5- *G. lucidum* (EtOH), lane 7- *P. tigrinus* (MeOH)*,* lane 8- *P. betulinus* (MeOH)*,* lane 9- *P. ostreatus*  (MeOH), lane 10- *F. hepatica* (MeOH), lane 2´- *L. sulphureus* (CHCl3), lane 3´- *C. versicolor*  (CHCl3), lane 4´- *F. velutipes* (CHCl3), lane 6´- *G. applanatum* (CHCl3)*,* lane 7´- *P. tigrinus* 

Fig. 1. **A:** TLC separation of crude extracts (methanol - MeOH and chloroform - CHCl3) of selected lignicolous species prepared for bioautography assay and **B:** bioautogram of

*Bacillus subtilis* is a Gram + bacteria, non-pathogenic to humans and can be used as a model organism in similar tests, since the representative of the same genus, bacteria *B. anthracis* is responsible for the disease anthrax, which is characterized by the appearance of edema, hemorrhage and tissue necrosis. It is common in some animals, often used as a biological weapon in bioterrorism. If an extract shows activity against *B.subtilis*, it is possible to be active against *B. anthracis* and possibly against other pathogenic Gram + bacteria such

extracts for Gram- positive bacteria *S. aureus,* animal strain

as specieses of the genera *Staphylococcus* and *Streptococcus.*

**1.8 Target organisms** 

(CHCl3)*,* lane 8´- *P. betulinus* (CHCl3).

*Escherichia coli. E. coli*, Gram - bacteria, inhabits the gastro-intestinal tract of humans and warm-blooded animals, making their normal indigenous microflora. In immuno-suppressed patients, however, it can cause infections, sometimes fatal (Giovannini, 2006). Gram bacteria cause more problems than Gram +, as a result of their different cell wall structure. Since penicillin and cephalosporin antibiotics belong to the group that act at the level of cell wall synthesis, the exploration of new types of antibiotics is very important for group of Grorganisms.

*C. albicans* belongs to Deuteromycota, representing yeasts forming pseudo-mycelia. It lives as a part of the normal human microflora, especially in the mucosa of the mouth and vagina. In immuno-suppressed individuals (AIDS, chemotherapy, inadequate nutrition and poor hygiene), or after prolonged use of antibiotics, it can cause disease called candidiasis, which is the most common caused by *C. albicans* as the most widespread species. It may affect almost any tissue, starting with simple children's thrush, and ending as the systemic infections. Most commonly it is manifested in the form of slimy mucus. *C. albicans*, is very convenient target organism in the detection of new antifungal drugs.
