**1.3 Antibiotic resistance and further perspectives**

Today, antibiotic resistance is a serious problem and antibiotics are losing their effectiveness what is especially important and have serious threats for humans whose health is already compromised by stress in modern way of life or by illness (HIV patients, immnocompromised persons that are under chemotherapy). Along with the increasing use of antibiotics and antibiotic agents, the resistance of bacteria to common and more

Macrofungi or mushrooms are not taxonomic categories, being most frequently used as terms for fungi with distinctive fruiting bodies, which are usually fleshy and edible, hypogeous or epigeous, large enough to be seen with the naked eye, and picked by hand

Lignicolous (wood-decaying) macrofungi, mostly belonging to the *Polyporaceae* family, are easily noticed, collected and recognized in the field. Taxonomically, these fungi mainly belong to the phyla Basidiomycota and Ascomycota, including about 20,000 known species, widely distributed on Earth. Recent estimations suggest that even more than 1.5 million species of fungi exist on our planet and about 140,000 species belong to macrofungi. However, only 10% of them are explored and 16% are cultured (Chang & Miles, 2004;

From the beginning until now, the humankind has always been faced with a problem of spreading of infectious diseases. Today, more than 150 compounds make arsenal of antimicrobial substances used in the treatment of infectious diseases. Antibiotics are defined as low molecular weight organic natural products (secondary metabolites or idiolites) made by microorganisms, which are active at low concentrations against other microorganisms. There are estimations that among 12,000 antibiotics known, approximately 55% are produced by Streptomyces, 11% by other Actinomycetes, 12% from other bacteria and 22% from filamentous fungi (Inouye et al., 2004). In its broadest definition an antibacterial is an agent that interferes with the growth and reproduction of bacteria. Unlike antibiotics, antibacterials are not used as medicine for humans or animals, but are now most commonly described as agents used to disinfect surfaces and eliminate potentially harmful bacteria found in products

Since Alexander Fleming`s discovery, in 1928, of the first antibiotic, called penicillin, produced by the mold *Penicillium chrysogenum*, a real revolution in medicine with a new era of antibiotics have started. Later, the entire group of β-lactam antibiotics (penicillins and cephalosporins) was discovered, followed by the Waxman`s discovery of streptomycin derived from Streptomyces bacteria, used in a treatment of tuberculosis), and then tetracyclines, quinolones, antifungal metabolites, antiparasitic substances and more recently antiviral drugs such as acyclovir. In 1971, the second significant antibiotic cyclosporin A and C were isolated from fungal organism *Hypocladium inflatum gams* (*Tolypocladium inflatum*) which is the asexual state of the pathogen of beetles *Elaphocordyceps subsessilis* (Petch) G.H. Sung, J.M. Sung & Spatafora). Its immunosupresive activity was revealed in 1976 by J.F. Borel and was approved for use 1983 in order to reduce the risk of organ rejection in

Today, antibiotic resistance is a serious problem and antibiotics are losing their effectiveness what is especially important and have serious threats for humans whose health is already compromised by stress in modern way of life or by illness (HIV patients, immnocompromised persons that are under chemotherapy). Along with the increasing use of antibiotics and antibiotic agents, the resistance of bacteria to common and more

such as soaps, detergents, health and skincare products and household cleaners.

transplant surgery (Upton, 2001 as cited in Giovannini, 2006).

**1.3 Antibiotic resistance and further perspectives**

**1.1 Macrofungi** 

(Chang and Miles, 2002, Karaman et al., 2012).

**1.2 Antibiotics and antimicrobial agents** 

Mueller, Bills & Foster, 2004).

frequently used antibiotics increased, resulting in low respond to the antibiotic treatment. The existance of multidrug-resistant diseases, once felt to be under control, increased as well, tuberculosis, penicillin-resistant pneumonia, resistant malaria (the cause of death of 1.1 million people in 1998), resistant strains of gonorrhea or dysentery caused by Shigella and Salmonella (2.2 million deaths in 1998).

Public concern about infection has been expanded, resulting in a greater public use of a variety of antibacterial agents designed to remove disease-causing organisms from external surfaces before they can enter the body. Today, antibacterials may also be impregnated into sponges, cutting boards, carpeting, and children's toys. However, if used too frequently and indiscriminately, certain antibacterial agents, those that leave trace chemical residues and that target particular processes in the life cycle of bacteria, may select for resistant strains (http://www.tufts.edu/med/apua/about\_issue/agents.shtml).

Furthermore, no new class of antibacterial substances has been developed to combat infectious diseases since 1970 (WHO, 2000). It is therefore necessary to find some new compounds to fight against these resistant microorganisms. Then starts the parallel struggle against antibiotic resistance exhibited in the continuous screening of new natural resources of undiscovered antibiotics from the nature. In this manner, the potential of mushrooms have a great advantage, even in comparison to the bacteria. Nowadays it is much more complicated to find new pharmaceutical active substances by chemical synthesis than from the existing and unexplored natural resources. Screenings of biological activities have made great progress in exploring the rich unlimited and undiscovered natural products in order to use it for production of pharmaceutical and agrochemical products (Anke, 1989). Many organisms were studied as potentially new resources of undiscovered bioactive components, among which fungi from the phylum Basidiomycota gave the promising results. In the forties, the pioneers in such research were Anchel, Hervey, Wilkins et al. and Florey et al. 1949, who tested extracts derived from fruiting bodies and mycelia cultures of more then 2000 species, resulting in isolation of a tricyclic diterpene antibiotic (pleuromutilin from *Pleurotus mutilus*). During nineties of the last century many new structures and biological activities were detected (Anke, 1989). Since then, numerous studies have been performed. Today we are witnessing very important struggle not only against microorganisms but also against other human diseases such as cancer, viral and other diseases.

#### **1.4 Antimicrobial substances - Antibiotics from fungi and macrofungi**

Microbial metabolites and their derivatives play an important role in the development of medicines. The use of these metabolites has grown extensively over the past century, starting with the Fleming's discovery of penicillin (1924), originally from *Penicillium notatum* filamentous micro-fungus, via Brotzu's discovery of cephalosporins from another fungus, mold *Cephalosporium acremonium* (*Acremonium chrysogenum* now), until today when the Japanese clinics use 30 penicillin derivatives and about 49 derivatives of cephalosporin. Although the metabolites originating from fungi were the main targets of antimicrobial screening, these studies were interrupted for a short time by Waksman's discovery of streptomycin (1945) originating from Actinomycetes. It is believed that the cause of the break helped by the fact that fungi often produce mycotoxins with pronounced cytotoxicity in humans and animals, and one example is the aflatoxin from the mold *Aspergillus flavus*, the most prominent cause of chronic hepatitis that leads to tumor malignancy.

Antibacterial Agents from Lignicolous Macrofungi 365

Many studies have shown that macrofungi produce many interesting pharmacological substances. By comparing the number of studied fungi with those whose chemical and pharmacological effects are completely unknown, we realized that only a very small, even insignificant fraction of potentially active fungal substances are known. For instance, the illustrative example is the species *Ganoderma lucidum*, witnessing that each species contains many different active components. In addition, production of certain secondary metabolites may depend on the characteristics of the strains (isolates) or culture conditions. Therefore, many scientists coping with this problem are actually trying to find new active compounds to be used in the future. It is clear that only a small number of active compounds studied *in vitro* or *in vivo* on animals as biological models suits the needs of allopathic medicine, defined by chemical composition, precise dosing, toxicology, pharmacodynamics and clinical studies.

Macrofungi need antibacterial and antifungal compounds to survive in their natural environment. Since fungi and humans share common microbial pathogens (e.g. *E. coli*, *S. aureus* and *P. areuginosa)*, antimicrobial compounds that are produced by fungi against microorganisms, can benefit to humans (animals). Compounds of special interest are those that exhibit antibacterial activities against multiresistant bacterial strains (methicillin

According to a recent biological evaluation, more than 75% of screened polypores showed strong antimicrobial activity inhibiting mostly Gram-positive bacterial strains (*B. subtilis*, *S. aureus* and *M. flavus)*. It was reported that new sesquiterpenoid hydroquinones produced by some species of the European *Ganoderma* genus, named ganomycins, inhibit the growth of

Based on our results of antibacterial screening, 60% methanol and 55% chloroform extracts reached a significant antibacterial activity, giving the diameter of inhibitory zone (>15mmØ) against one or more target bacteria. Gram –negative bacteria were less sensitive to the applied extracts than Gram-positive ones, except *G. lucidum* ethanolic extract (25mg/ml) against *P. aeruginosa* (h) and *E. coli* (ATCC 25922). Three extracts of lignicolous macrofungi *P. betulinus*, *C. versicolor* and *G. lucidum* showed a wide range of activities against all tested Gram-positive and some of Gram-negative bacteria, reaching MIC values mainly at a concentration of 17.5 mg/ml. Unlike methanol, chloroform extracts did not show concentration dependence while the concept of a dose response phenomenon- hormesis (low dose stimulation and high dose inhibition) may be used for explanation of this phenomenon. The precise composition of examined extracts of fungi is unknown and can only be assumed that the effect of crude extracts, which are concentration dependent, is a consequence of complex interactions between

In a recent screening of antibacterial activity of water and methanol crude extracts of the species *Meripilus giganteus* against nine species of Gram-positive and four species of Gram- negative bacteria, the most active extract was methanolic extract, inhibiting all the Gram–positive bacteria (mostly *S. aureusan, Rh. equi, Bacillus*) and only two Gram-negative ones, *C. perfringens* and *P. aeruginosa,* ATCC strains (Karaman et al., 2009b) The animal strains showed to be the most susceptible analyzed strains, indicating a possible application of this fungus against Grampositive bacterial infections in animals. Since water extract exhibited only a narrow antibacterial effect, we assumed that the obtained results could not be attributed to the compounds like proteins or polysaccharides. These results are in agreement with the literature data for similar polypore fungi (Lindequist et al., 2005; Zjawioni, 2004), demonstrated sterols and lanostanoid

resistant *S. aureus* – MRSA or vancomycine resistant *Enterococcus* – VRE).

methicillin-resistant *S. aureus* and other bacteria (Mothana et al., 2000).

cells and mixtures of compounds in the extracts (Karaman et al., 2009a).

However, in recent years the trend has changed and fungal metabolites have again attracted the attention of pharmacological research. This can be seen from the statistics presenting fungal metabolites increasingly important as bioactive agents and showing that the percentage of medicines versus the metabolites originating from actinomycetes are as it follows (according to the Journal of Antibiotics (I), Tokyo): 13 versus 66% (1983), 16 versus 74% (1990), 38 versus 53% (1994) and 47 versus 44% (2000), while the percentage of metabolites originating from the bacteria remained at about 8%, except 1983 when it was 21%. A similar tendency was observed for metabolites that are registered as patents in Japan, showing that the products from the fungi grew intensively: 11% (1983), over 21% (1990) to 36% (2000), and for the products from actinomycetes decreased sharply from 74% (1983), over 66% (1990) to 48% (2000). According to Tanaka and Omura (1993), 43% of more than 8000 new microbial metabolites were discovered thanks to Japanese scientists. It is possible that the abundance of secondary metabolites of fungi and actinomycetes, compared with bacteria and yeasts, is associated with the characteristics of the environment poor in nutrients. Nutritional limitation further induces secondary metabolism and production of various compounds, in order to exploit scarce nutrients in the best extent possible (Aldered et al.*,* 1999). Taking into account the antibiotic screening, review of Inouye et al.*,* 2004 showed that the number of antifungal metabolites increased significantly, anticancer metabolites - moderately, while the number of antibacterial metabolites decreased in the last ten years. However, the most significant increase was observed in bioactive metabolites of non-antibiotic mode of action, especially regarding the screening of inhibitors of cholesterol synthesis, of which 93% originated from fungi (Yagisawa, 2000).

In this sense it is considered that the eukaryotic fungal metabolites in action in mammalian cells could have far fewer side-effects compared with prokaryotic metabolites. Cultures of micro-organisms usually contain complex mixtures of different compounds, small and large molecular weight, what makes a direct pharmacological screening more difficult, considering the fact that can easily be masked by the activity of other compounds in the mixture. Being sessile organisms, which are in their natural environment constantly exposed to the influence of different competitors (parasitic organisms), it is not surprising that many antibiotics are isolated from fungi (Lindequist et al., 2005). Although today, still only compounds originating from micro-fungi or synthetic medicines have been used, literature data pointing to higher fungi, macro-fungi, primarily Basidiomycetes as natural sources rich in new antimicrobial substances are infrequently found (Suay et al.*,* 2000).

As potential new sources of natural antibiotics, lignicolous mushrooms again become the subject of study (Smania et al., 2001). The fact that humans and animals share common microbial pathogens with fungi (*E. coli, S. aureus* and *P. areuginosa*) has prompted the thought that they produce compounds that may have similar effects in humans (Zjawioni, 2004). In Western Europe, the interest for this group of fungi start with the discovery of antibiotics (penicillin), when a group of scientists with their pioneering research of new antibiotics originating from macrofungi Basidiomycota, led by M. Anchel, A. Hervey, WH Wilkins and Kavanagh, started research of extracts and culture mycelia and fruit body of about 2000 species (Florey et al.*,* 1949). This research has resulted in isolation of antibiotics three-cyclic diterpene pleuromutilin (Kavanagh et al.*,* 1951) from *Pleurotus mutilus* species. Pleuromutilin has demonstrated its antibacterial activity by inhibiting bacterial protein synthesis by interacting with RNA (Lorenzen & Anke, 1998). After that, the first semisynthetic antibiotic tiamulin was produced together with valnemuline, used in veterinary medicine (Egger & Reinshagen, 1976) for the treatment of *Mycoplasma* infections in animals (Lorenzen & Anke, 1998).

However, in recent years the trend has changed and fungal metabolites have again attracted the attention of pharmacological research. This can be seen from the statistics presenting fungal metabolites increasingly important as bioactive agents and showing that the percentage of medicines versus the metabolites originating from actinomycetes are as it follows (according to the Journal of Antibiotics (I), Tokyo): 13 versus 66% (1983), 16 versus 74% (1990), 38 versus 53% (1994) and 47 versus 44% (2000), while the percentage of metabolites originating from the bacteria remained at about 8%, except 1983 when it was 21%. A similar tendency was observed for metabolites that are registered as patents in Japan, showing that the products from the fungi grew intensively: 11% (1983), over 21% (1990) to 36% (2000), and for the products from actinomycetes decreased sharply from 74% (1983), over 66% (1990) to 48% (2000). According to Tanaka and Omura (1993), 43% of more than 8000 new microbial metabolites were discovered thanks to Japanese scientists. It is possible that the abundance of secondary metabolites of fungi and actinomycetes, compared with bacteria and yeasts, is associated with the characteristics of the environment poor in nutrients. Nutritional limitation further induces secondary metabolism and production of various compounds, in order to exploit scarce nutrients in the best extent possible (Aldered et al.*,* 1999). Taking into account the antibiotic screening, review of Inouye et al.*,* 2004 showed that the number of antifungal metabolites increased significantly, anticancer metabolites - moderately, while the number of antibacterial metabolites decreased in the last ten years. However, the most significant increase was observed in bioactive metabolites of non-antibiotic mode of action, especially regarding the screening of inhibitors of cholesterol

In this sense it is considered that the eukaryotic fungal metabolites in action in mammalian cells could have far fewer side-effects compared with prokaryotic metabolites. Cultures of micro-organisms usually contain complex mixtures of different compounds, small and large molecular weight, what makes a direct pharmacological screening more difficult, considering the fact that can easily be masked by the activity of other compounds in the mixture. Being sessile organisms, which are in their natural environment constantly exposed to the influence of different competitors (parasitic organisms), it is not surprising that many antibiotics are isolated from fungi (Lindequist et al., 2005). Although today, still only compounds originating from micro-fungi or synthetic medicines have been used, literature data pointing to higher fungi, macro-fungi, primarily Basidiomycetes as natural sources rich

As potential new sources of natural antibiotics, lignicolous mushrooms again become the subject of study (Smania et al., 2001). The fact that humans and animals share common microbial pathogens with fungi (*E. coli, S. aureus* and *P. areuginosa*) has prompted the thought that they produce compounds that may have similar effects in humans (Zjawioni, 2004). In Western Europe, the interest for this group of fungi start with the discovery of antibiotics (penicillin), when a group of scientists with their pioneering research of new antibiotics originating from macrofungi Basidiomycota, led by M. Anchel, A. Hervey, WH Wilkins and Kavanagh, started research of extracts and culture mycelia and fruit body of about 2000 species (Florey et al.*,* 1949). This research has resulted in isolation of antibiotics three-cyclic diterpene pleuromutilin (Kavanagh et al.*,* 1951) from *Pleurotus mutilus* species. Pleuromutilin has demonstrated its antibacterial activity by inhibiting bacterial protein synthesis by interacting with RNA (Lorenzen & Anke, 1998). After that, the first semisynthetic antibiotic tiamulin was produced together with valnemuline, used in veterinary medicine (Egger & Reinshagen, 1976) for the treatment of

synthesis, of which 93% originated from fungi (Yagisawa, 2000).

in new antimicrobial substances are infrequently found (Suay et al.*,* 2000).

*Mycoplasma* infections in animals (Lorenzen & Anke, 1998).

Many studies have shown that macrofungi produce many interesting pharmacological substances. By comparing the number of studied fungi with those whose chemical and pharmacological effects are completely unknown, we realized that only a very small, even insignificant fraction of potentially active fungal substances are known. For instance, the illustrative example is the species *Ganoderma lucidum*, witnessing that each species contains many different active components. In addition, production of certain secondary metabolites may depend on the characteristics of the strains (isolates) or culture conditions. Therefore, many scientists coping with this problem are actually trying to find new active compounds to be used in the future. It is clear that only a small number of active compounds studied *in vitro* or *in vivo* on animals as biological models suits the needs of allopathic medicine, defined by chemical composition, precise dosing, toxicology, pharmacodynamics and clinical studies.

Macrofungi need antibacterial and antifungal compounds to survive in their natural environment. Since fungi and humans share common microbial pathogens (e.g. *E. coli*, *S. aureus* and *P. areuginosa)*, antimicrobial compounds that are produced by fungi against microorganisms, can benefit to humans (animals). Compounds of special interest are those that exhibit antibacterial activities against multiresistant bacterial strains (methicillin resistant *S. aureus* – MRSA or vancomycine resistant *Enterococcus* – VRE).

According to a recent biological evaluation, more than 75% of screened polypores showed strong antimicrobial activity inhibiting mostly Gram-positive bacterial strains (*B. subtilis*, *S. aureus* and *M. flavus)*. It was reported that new sesquiterpenoid hydroquinones produced by some species of the European *Ganoderma* genus, named ganomycins, inhibit the growth of methicillin-resistant *S. aureus* and other bacteria (Mothana et al., 2000).

Based on our results of antibacterial screening, 60% methanol and 55% chloroform extracts reached a significant antibacterial activity, giving the diameter of inhibitory zone (>15mmØ) against one or more target bacteria. Gram –negative bacteria were less sensitive to the applied extracts than Gram-positive ones, except *G. lucidum* ethanolic extract (25mg/ml) against *P. aeruginosa* (h) and *E. coli* (ATCC 25922). Three extracts of lignicolous macrofungi *P. betulinus*, *C. versicolor* and *G. lucidum* showed a wide range of activities against all tested Gram-positive and some of Gram-negative bacteria, reaching MIC values mainly at a concentration of 17.5 mg/ml. Unlike methanol, chloroform extracts did not show concentration dependence while the concept of a dose response phenomenon- hormesis (low dose stimulation and high dose inhibition) may be used for explanation of this phenomenon. The precise composition of examined extracts of fungi is unknown and can only be assumed that the effect of crude extracts, which are concentration dependent, is a consequence of complex interactions between cells and mixtures of compounds in the extracts (Karaman et al., 2009a).

In a recent screening of antibacterial activity of water and methanol crude extracts of the species *Meripilus giganteus* against nine species of Gram-positive and four species of Gram- negative bacteria, the most active extract was methanolic extract, inhibiting all the Gram–positive bacteria (mostly *S. aureusan, Rh. equi, Bacillus*) and only two Gram-negative ones, *C. perfringens* and *P. aeruginosa,* ATCC strains (Karaman et al., 2009b) The animal strains showed to be the most susceptible analyzed strains, indicating a possible application of this fungus against Grampositive bacterial infections in animals. Since water extract exhibited only a narrow antibacterial effect, we assumed that the obtained results could not be attributed to the compounds like proteins or polysaccharides. These results are in agreement with the literature data for similar polypore fungi (Lindequist et al., 2005; Zjawioni, 2004), demonstrated sterols and lanostanoid

Antibacterial Agents from Lignicolous Macrofungi 367

Several antifungal metabolites with steroid structure have been also isolated from fungi A25822 A and B from *Geotrichum* (Gordee and Butler, 1975 as cited in Inouye et al., 2004) and from *Wallemia sebi*; Mer-NF8054 A and X from the genus *Aspergillus*. The most famous triterpene, favonol isolated from basidiomycetous *Favolashia* sp. (Anke et al., 1995 as cited in Inouye et al., 2004) is a metabolite that exhibited antifungal activity against Ascomycetes, Basidiomycetes, Zygomycetes and Oomycetes, but did not show antibacterial activity. Researchers of Merck Group have discovered four acidic terpenoids from filamentous fungi: ergokonin A (from *Trichoderma koningii*), ascosteroid (from *Ascotricha amphitricha*) arundifungin (steroid from *Arthrinium arundinis*) and enfumafungin (pentacyclic terpenoid from from mould *Trichoderma koningii*), ascosteroid (from ascomycetous *Ascotricha amphitricha*), arundifungin (steroid from mould *Arthrinium arundinis*) and enfumafungin (pentacyclic terpenoid from *Aureobasidium*), which were found to affect the biosynthesis of β-D-glucan but not the biosynthesis of steroids. Among them the best antifungal activity on *Candida* species

A large number of pharmacologically active substances like sesquiterpenes (Abraham, 2001), hydroquinones (Mothana et al., 2000), polysaccharides and complexes of polysaccharide-peptide (Liu, 1999), lanostanoide triterpenoids (Shiao, 1992, Leon et al., 2004) steroids (Smania, 2003), nucleosides, alkaloids and vitamins (Paterson, 2006) from fruitbodies of polypore fungi have been detected. Recent studies pronounced phenolic compounds (Turkoglu et al., 2007, Paterson, 2006, Ribeiro et al., 2007) as the main active antioxidative components in fungal extracts (Kityberger et al., 2007; Barros et al., 2007). It is assumed that antibacterial effects exhibited by fungal extracts of different polarites could be related to an overall effect of phenolic compounds (e.g. phenolic acids: caffeic acid, ellagic acid; flavonoids, hydroquinones) detected in similar extracts of the species *G.lucidum*, *F.velutipes*, *P.ostreatus* or organic acids (oxalic, malic) previously detected in *L. sulphureus*

**Polysaccharides.** Polysaccharide molecules that form an integral part of the fungal cell wall also exhibit antimicrobial properties (Stamets, 2002). Polysaccharides are the most important components of fungal bioactive substances, proven to provide many medical and therapeutic possibilities (Fan et al., 2006) while their antibiotic effect is often specific to certain microorganisms (Stamets, 2002). Most of these compounds belong to glucans or heteroglycans (Fan et al., 2006). It is believed that the antibacterial and antifungal effects of β-glucan is based on the activation and strengthening of the immune response, and their use is recommended in combination with other antibiotics and immunostimulators in prevention and treatment of infectious diseases, especially immunocompromised individuals (Chen & Seviour, 2007).

**Proteins and polypeptides**. Proteins that act inhibitory on microorganisms are found frequently in organisms of plant and animal species, whereas their presence is rare in fungi (Wang & Ng, 2006). It is believed that these proteins are often positively charged, and that the mechanism of their action is realized by forming ion channels in cell membranes of microorganisms as well as by competitive binding to host cell polysaccharide receptors (Cowan, 1999). Proteins and peptides are isolated from macrofungi whose antimicrobial

effect is limited to a small number of mostly phytopathogenic species (Table 1).

and species of *Aspergillus* genera showed enfumafungin.

**1.5 Chemical nature of antibacterial agents** 

and *F. hepatica*, as well as terpenoids.

**1.5.1 Products of primary metabolism** 

terpenoids as well as phenolic compounds as the main active components responsible for the obtained activity (Turkoglu et al., 2007; Barros et al., 2007; Elmastas et al., 2007).
