ANTIBACTERIAL ACTIVITY OF FOMITOPSIS BETULINA CULTURAL LIQUID

The antibacterial activity of Fomitopsis betulina cultural liquid (native, native concentrated, lyophilized, dried) against standard bacteria (Escherichia coli АТСС 25922, Pseudomonas aeruginosa АТСС 27853, Staphylococcus aureus АТСС 25923), and clinical isolates (Acinetobacter baumannii 50/1496 MBL, A. baumannii 88/2995 MBL, E. coli 116/3196 KPC, Klebsiella pneumoniae 6/509 ESBL, AmpC, KPC, P. aeruginosa 99/3066 MBL, P. aeruginosa 125/3343 MBL, S. haemoliticus 22/824 MRSA, S. aureus 134/3569 MRCNS) has been evaluated by the serial dilutions method. The antibacterial activity of F. betulina against S. haemoliticus and A. baumannii has been found for the first time. All samples of F. betulina cultural liquid demonstrated the inhibitory effect against standard bacterial strains at the minimum bactericidal concentration (MBC) ranging from >2.0 up to 18.75 mg/ml, and against multidrug-resistant clinical isolates with MBC from 7.8 up to 48.42 mg/ml. The dried F. betulina cultural liquid showed the highest antimicrobial activity against standard bacteria and clinical isolates, except A. baumannii 50/1496 MBL, while native concentrated cultural liquid was the most effective against this pathogen. The study showed that the antibacterial activity of the cultural liquid of F. betulina was improved by concentration and drying. The results obtained indicate that F. betulina cultural liquid contains alternative antimicrobial agents, useful for the treatment of bacterial diseases and might be a perspective substance for the pharmaceutical industries.

Thus, some research has been done to investigate the antibacterial potential of birch polypore, using different methods, test bacteria, samples of mushroom, fractions or single compounds. There are still many ways to explore this relevant and important problem. The culture liquid of the fungus contains a full complex of biologically active substances, which is impossible in the case of extraction by one or another solvent. It is not excluded that a number of such substances potentiate the action of each other. The aim of this research was to evaluate the antibacterial activity of Fomitopsis betulina (Bull.) B.K. Cui, M.L. Han and Y.C. Dai, strain 327 cultural liquid against some standard bacteria and clinical isolates.

Material and Methods
Fomitopsis betulina was supplied by the IBK [14] and were maintained in necessary conditions [15] The bacterial strains were selected from standard ones as much as used for similar studies and because of their pathogenic characteristics. Tested microorganisms were prepared as in the study [15].
Culture media and conditions are consistent with those in a previous study [15]. The dry weight was determined as follows. After preparation of mycelium and cultural liquid [15] culture liquid was frozen (-80 °C) and lyophilized (Telstar Cryodos-80, Terrassa, Barcelona, Spain) or dried at 60 °C (Snol-58/350, UMEGA, Republic of Lithuania) to a constant weight.
The antibacterial activity of F. betulina cultural liquid (native, native concentrated, lyophilized, and dried) against bacterial standard strains and clinical isolates was determined by the series of the twofold dilutions method [16]. Initial solutions of the F. betulina culture liquid were obtained by measuring a certain amount (in terms of dry matter): native cultural liquid (its concen-Immunology and microbiology tration ranged from 0.146 up to 18.750 mg/ml), concentrated native cultural liquid (with concentration between 0.75 and 96.85 mg/ml), lyophilized, and dried samples of F. betulina culture liquid were dissolved in pure Dimethyl sulfoxide (DMSO) in concentration from 2.0 up to 250 mg/ml. DMSO was chosen as a non-toxic solvent and due to its very good permeability through biological membranes. A test bacterial culture (0.5 ml) in Mueller-Hinton broth (MHB) was added to the sterile tubes already containing the 0.5 ml of twofold serially diluted samples of F. betulina cultural liquid in MHB. Positive controls were tubes with a bacterial suspension in 0.5 ml of MHB, sterility controls -tubes with MHB. Negative controls were tubes with bacterial suspension in MHB with pure DMSO.
The minimum concentration (MIC) is the lowest concentration of the sample that will inhibit the visible growth of test-bacteria. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of a sample that gave the complete absence of growth on the agar surface. Incubation was carried out at 37 °C for 24 hours.
All the assays were carried out in triplicate. For the antimicrobial assay, the MBC which killed all the organisms was recorded.

Results and Discussion
In respect that using of different toxic solvents for extraction is often of no practical use in therapy we focused our study on different aggregate forms of the mushroom cultural liquid (native, native concentrated, lyophilized, and dried). Due to the color of the F. betulina cultural liquid samples in almost all test tubes, the determination of the MIC (the lowest concentration of the tested sample, able to inhibit the growth of bacteria) was complicated and, unfortunately, the saturated brown color of the serial dilutions of culture liquid blocked the establishing of MIC (Fig. 1).

Fig. 1. Serial dilutions of dried F. betulina cultural liquid dissolved in DMSO
The MBC values against standard bacteria were determined as an evaluation of the antibacterial activity of the F. betulina samples ( Table 1). The test bacteria possess varied susceptibility to the different samples of F. betulina cultural liquid. The native cultural liquid and lyophilized cultural liquid of F. betulina showed an exclusively static activity against test bacteria, presenting MBC of 18.75 and 7.8 mg/ml, respectively. It was established a better efficiency of the native concentrated F. betulina cultural liquid, compared to its native form. These results are in agreement with a previous study [16] where the extracts from the liquid media showed antibacterial effects only when they were concentrated. The antibacterial activity of F. betulina cultural liquid also has been increased due to drying, which was expected. It was established, that pure DMSO (Negative control) possessed some antibacterial activity against test bacteria ( Table 1), but in our experiments with the culture liquid, the presence of DMSO was less, than MBC of pure DMSO. Thus, the inhibition effect of our samples, dissolved in DMSO, on the bacterial growth, can be explained only by the antibacterial activity of F. betulina culture liquid. Our investigations confirm the results of previous studies that F. betulina possesses the antibacterial activity against similar bacteria [2,[5][6][7][8][9][10][11][12][13]. Samples of F. betulina cultural liquid, studied by us, possessed weaker activity against S. aureus ATCC 25923, but were active against P. aeruginosa АТСС 27853, compared to the ethanolic extracts of P. betulinus fruit bodies [12]. Different species of macrofungi extracts were also tested against these standard bacteria [18][19][20][21][22][23] [21]; ethyl acetate (3.125 mg/ml) and acetone (12.5 mg/ml) extracts from Cordyceps taii mycelia [22]; methanol extract of Coriolus versicolor carpophores (5.0 mg/ml) [20]; methanol (62.5 mg/ml), ethanol and aqueous extracts (31.25 mg/ml) of Pluerotus squarrosulus carpophores [18]; methanolic extract of Cantharellus cibarius carpophores (20.0 mg/ml) [19]. At the same time, the antimicrobial activity of F. betulina culture liquid against S. aureus ATCC 25923 was inferior to MBC of the chloroform extract (1.56 mg/ml) of Cordyceps taii mycelia [22], chloroform extract of Hygrophorus agathosmus (125 µg/ml), dichloromethane extract (>1000 µg/ml) of Suillus collitinus carpophores [23], and ethanol extract (0.625 mg/ml) of Hyphodontia paradoxa fruit body [21]. Gram-negative bacteria E. coli АТСС 25922 showed less sensitivity to tested samples of F. betulina cultural liquid than other mushroom extracts, reported in similar studies: MBC of the dichloromethane extract of Suillus collitinus sporokarps was >1000 μg/ml [23], MBC of the ethanol extracts of fruiting bodies of 31 mushrooms like Amanita citrina, Am. muscaria, Am. pantherina, Am. porphyria, Bjerkandera adusta, Clavicorona pyxidata, Cortinarius armillatus, C. sanguineus, Daedaleopsis confragosa, Fomes fomentarius, Fomitopsis pinicola, Gymnopilus penetrans, Heterobasidion annosum, Hygrophoropsis aurantiaca, Hyphodontia paradoxa, Lactarius aurantiacus, L. helvus, L. vellereus, Lenzites betulinus, Panellus stypticus, Pseudoclitocybe cyanthiformis, Psilocybe fascicularis, Ps. lateritia, Rhodocollybia maculate, Russula fragilis, Scleroderma citrinum, Stereum hirsutum, Thelephora terrestris, Trametes hirsuta, Trichaptum fuscoviolaceum, Tubaria furfuracea were determined at concentration between 0.625 and 5 mg/ml [21]. In contrast, the tested samples of F. betulina cultural liquid inhibited the growth of P. aeruginosa АТСС 27853 significantly higher, than the methanol and ethanol extracts of Pluerotus squarrosulus fruit bodies with MBC at 62.5 mg/ml and 31.25 mg/ml, respectively [18]. We noted that results of the studies, available in the literature, are entirely focused on evaluation of the antibacterial properties of mushroom extracts in contrast to this work.
In the last few decades the problem of resistance to antibacterial drugs is greatly global and has a great value for public health. Therefore, the next step was to study the antibiotic activity of F. betulina culture liquid against the most important drug-resistant strains that produce various types of betalactamase. Considering that the tested bacteria were clinical isolates with high antibi-otic resistance profile, the promising results have been established in our study (Table 2, Fig. 2, 3)

Conclusion
Thus, the analysis of literature data indicates a significant interest of researchers to the studying of the antibacterial activity of higher fungi and their potential for the production of antibacterial compounds. Based on the obtained results, Fomitopsis betulina occupies a certain niche among the studied mushrooms. All forms of F. betulina cultural liquid (native, native concentrated, lyophilized, and dried), used in this study, were found to have the antibacterial activity in varying degrees against tested microorganisms: standard bacterial strains S. aureus АТСС 25923, E. coli АТСС 25922, P. aeruginosa АТСС 27853 at concentration from >2.0 up to 18 The study showed that the antibacterial activity of F. betulina cultural liquid improved when it was concentrated and dried. The highest antibacterial activity was established in dried F. betulina cultural liquid and might be a perspective antibacterial substance in the pharmaceutical industries. Further studies are needed for isolation and determination of biologically active agents, responsible for the antimicrobial properties of F. betulina cultural liquid, which could be used in the treatment or prevention of many human diseases.