17 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (supplement): 17–23, 2018, ISSN 2543-8832 DOI: 10.24917/25438832.3supp.2 Anna Čuvalová1*, Imrich Strapáč2, Lívia Handrová1, Vladimír Kmeť1 1Institute of Animal Physiology, Centre of Biosciences of the SAS, Soltesovej 4/6, 040 01 Kosice, Slovak Republic, *cuvalova@saske.sk 2Department of Chemistry, Biochemistry and Biophysics, Institute of Pharmaceutical Chemistry, University of Veterinary Medicine and Pharmacy, Komenskeho 73, 041 81 Kosice, Slovak Republic Antibiofilm activity of mushroom extracts against Staphylococcus aureus F. J. Rosen. Introduction In recent years, a growing interest has developed in the mechanisms of the action of natural products, because they are a major source of chemical diversity and have pro- vided important therapeutic agents for many bacterial diseases (Payne et al., 2007). Mushrooms have long been appreciated for their taste, �avour, desirable aroma, tex- ture, and nutraceutical and medicinal attributes (Strapáč et al., 2016). Moreover, they are a renowned source of products with an array of bioactivities, from antibacterial to antiviral, cytotoxic, anti-in�ammatory, anti-feeding, antifungal or antioxidant and might be a valuable resource in the search of new bioactive extracts to inhibit bio�lm production (Martín-Rodríguez et al., 2014; Alves et al., 2014). In this context, �avo- noids and phenolic compounds have been revealed as potential inhibitors of bio�lm formation and the production of virulence factors in the pathogenic bacteria by inter- fering with quorum sensing mechanisms (Nazzaro et al., 2013). �e main factors associated with bio�lm formation are the iron uptake system and adhesive matrix proteins. Adhesion is favoured by the presence of virulence factors known as adhesins, which are grouped in a family known as the microbial surface com- ponents recognising adhesive matrix molecules (MSCRAMM). Major proteins adhes- ins in this group include �bronectin binding proteins A and B (FnBpA, FnBpB), bone sialoprotein binding protein (Bbp), iron regulated surface determinants A and B (IsdA, IsdB), and serine aspartate repeat gene proteins D and E (SdrD, SdrE) (Rasmussen et al., 2013; Cucarella et al., 2001). Staphylococcus aureus F. J. Rosen also produces cyto- toxins and hemolysins (𝛼, 𝛽, 𝛾 and δ), which possess the ability to form pores in host cells enabling lysis. In staphylococci, the expression of a series of toxins and virulence factors are controlled by the accessory gene regulator (Agr) system (Jarraud et al., 2002). A nn a Č uv al ov á, Im ric h S tr ap áč L ív ia H an dr ov á V la di m ír K m eť 18 In the present study, a water extract obtained from a sample of Macrolepiota pro- cera (Scop.) Singer, Pleurotus ostreatus (Jacq.) P. Kumm., Auricularia auricula-judae (Bull.) Quél., Armillaria mellea (Vahl) P. Kumm. and Laetiporus sulphureus (Bull.) Murrill was explored for its antibio�lm activity against Staphylococcus aureus strains. Material and methods Bacterial strains �e following staphylococci strains were used in this study from our own laboratory: Staphylococcus aureus No. 5 and S. aureus No. 51, isolated from ixodid ticks (Acari); S. aureus No. 12, and S. aureus No. 14, isolated from ewe´s milk. All cultures were identi�ed by matrix-assisted laser desorption/ ionization (MALDI) biotyper (Bruker Daltonik, Leipzig, Germany). All staphylococci strains were cultured at 37°C on blood agar (Blood agar base No. 2, Oxoid, Basingstoke, United Kingdom and with 5% de�- brinated sheep blood). Mushroom extracts �e preparation of mushrooms water extracts were determined as described previ- ously (Strapáč et al., 2016). For our analysis, we used 1 kg of freshly harvested fruiting bodies of Macrolepiota procera, Armillaria mellea and Laetiporus sulphureus, collected in the autumn of 2014 in an area of Dargov, Bankov near Košice and Ižkovice, respec- tively, in the Slovak Republic. �e last two are commercially available mushrooms, Pleurotus ostreatus and Auricularia auricula-judae. Water extracts were prepared by the extraction of 100 mg samples in 2 cm3 of water for 24 h with occasional vigorous stirring at 8°C in a refrigerator. �en, the extracts were �ltered and stored at 4°C. Bio�lm production assay For the detection of bio�lm formation, the crystal violet method with Nunc Maxi- sorp plates were used (Nunc, Roskilde, Denmark) by a previously published method (O´Toole, 2011) with some modi�cations. Overnight cultures of Staphylococcus au- reus were removed from each well and 3 times washed with saline, �xed with metha- nol, and stained with 0.1% crystal violet. �e bound dye was released with 33% acetic acid, and the optical density (OD) at 570 nm was measured by using a Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, Vermont, USA). Anti-bio�lm activity of mushroom extracts To detect the e�ects of mushrooms water extracts of Macrolepiota procera, Pleurotus ostreatus, Auricularia auricula-judae, Armillaria mellea, and Laetiporus sulphureus on staphylococcal bio�lm formation, 10 µl of extracts were added to the each well and 19 A ntibiofilm activity of m ushroom extracts against Staphylococcus aureus F. J. R osen. the plates were incubated 24 h at 37°C. �e bio�lm quanti�cation has been described above. Wells containing medium were used as blank controls. �e percentage of the inhibition of biofilm formation was calculated according to the following equation: (1 – OD590 of test / OD590 of untreated control) × 100%. Polymerase chain reaction �e presence of virulence genes of staphylococci was carried out by polymerase chain reaction (PCR). Strains were screened for the following genes: α-hemolysin gene (hla) as described by (Jarraud et al., 2002), serine-aspartate repeat proteins E gene (sdrE) (Sabat et al., 2006), iron-regulated surface determinants A gene (isdA) (Verkaik et al., 2010) and B gene (isdB) (Waryah et al., 2016), bone sialoprotein – binding protein gene (bbp) (Tristan et al., 2003), �bronectin- binding protein A gene (fnbpA) (Booth et al., 2001), iron-siderophore transporter gene (sirB) (Dale et al., 2004), and accessory gene regulator (agr II) (Shopsin et al., 2003). Statistical analysis All assays were performed in eight replicates and the means as well as the standard deviations were calculated. A one-way analysis of variance (ANOVA) and Tukey’s test were used to compare data utilising Statistica 9.0 so�ware (StatSo�, Tulsa, Oklahoma, USA). Results In present study, genes hla and isdA were found to occur in all Staphylococci. �e presence of sdrE gene was detected in three out of four strains. Genes agrII, isdB, bbp and sirB were only detected in strains isolated from ixodid ticks. Gene fnbpA was detected in Staphylococcus aureus No. 51. �e bio�lm formation of S. aureus strains was reduced by all mushrooms extracts without a�ecting the bacterial growth. �e best results were observed for the Armillaria mellea (70.87%), Pleurotus ostreatus (67.00%), Laetiporus sulphureus (64.14%) and Auricularia auricula-judae (62.77%), while Macrolepiota procera showed the lowest reduction of bio�lm forma- tion (47.72%). �e extracts reduced bio�lm formation in the range of 47.72–70.87%, which means that the bio�lm was formed in the presence of extracts in the range of 29.13–52.28%. We showed that a more signi�cant anti-bio�lm e�ect of the ex- tracts, except for M. procera, was of Staphylococcus aureus isolated from ixodid ticks (82.00%) in comparison to S. aureus isolated from ewe’s milk (50.00%) (Tab. 1). �e extract from M. procera had a similar e�ect on strains isolated from ixodid ticks (46.50%) and ewe’s milk (49.00%). A nn a Č uv al ov á, Im ric h S tr ap áč L ív ia H an dr ov á V la di m ír K m eť 20 Tab. 1. �e e�ect of mushrooms extracts on bio�lm formation of Staphylococcus aureus F. J. Rosen. 5 isolated from ixodid ticks; signi�cant di�erences are indicated with asterisks (* – p < 0.05, ** – p < 0.01, *** – p < 0.001, Tukey’s test) Name of mushrooms Mean value n = 8 Standard deviation (±SD) Control 0.216 0.100 Armillaria mellea 0.037** 0.006 Laetiporus sulphureus 0.042** 0.014 Pleurotus ostreatus 0.025*** 0.009 Auricularia auricula-judae 0.039** 0.010 Macrolepiota procera 0.150 0.048 Discussion We investigated the main factors associated with bio�lm formation. �e results of our study are in agreement with Kateete et al. (2011) who reported that 100% isolates had hla gene and with Verkaik et al. (2010) who reported that 100% isolates had isdA gene. Tristan et al. (2003) reported positivity rates of fnbA and bbp among Staphylococcus aureus isolates 28.00% and 22.00%. Liu et al. (2015) showed that 68.10% of isolates contained the sdrE gene. Our study focused on the antibio�lm activity of mushrooms extracts. We have con�rmed here the great potential of mushrooms to produce antibio�lm compounds, and we showed good antibio�lm e�ects of aqueous extracts in terms of the reduction of bio�lm formation. Similar observations have been made previously by others with di�erent bacteria (Kostić et al., 2017), and showed the antibio�lm activity of Armillar- ia mellea extract against Pseudomonas aeruginosa. Antibio�lm activity was associated with content of phenolic compounds and organic acids. Another study that worked on the organic extracts of Macrolepiota procera and Laetiporus sulphureus showed anti- bio�lm activity against S. aureus (Carvalho et al., 2015). Li and Dong (2010) reported the inhibition of Escherichia coli T. Escher. bio�lm formation (73.00%) by Auricularia auricula-judae extract. �is is a pioneer study since, as far as we know, there are no reports on the antibio�lm activity by the mushroom extracts of Pleurotus ostreatus, against S. aureus; nevertheless, other studies are required to elucidate the mechanism of action. Conclusion Extracts from mushrooms are a complex of di�erent chemical compounds. An identi- �cation and understanding of the mechanisms of mushrooms extracts action will en- able their further application to new innovative strategies for the control of microbial contamination and infection via the food chain. 21 Acknowledgement �is study was supported by the Slovak projects APVV 14-0274 and VEGA 2/0085/18. References Alves, M.J., Ferreira, I.C.F.R., Lourenço, I., Costa, E., Martins, A., Pintado, M. (2014). Wild Mushroom Extracts as Inhibitors of Bacterial Bio�lm Formation. Pathogens, 3, 667–679. 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Heterogeneity of the humoral immune response following Staphylococcus aureus bacteremia. European Journal of Clinical Microbiology & Infectious Diseases, 29(5), 509–518. DOI: 10.1007/s10096-010-0888-0 Waryah, C.B., Gogoi-Tiwari, J., Wells, K., Eto, K.Y., Masoumi, E., Costantino, P., Kotiw, M., Muk- kur, T. (2016). Diversity of virulence factors associated with West Australian methicillin-sen- sitive Staphylococcus aureus isolates of human origin. BioMed Research International, 1–10. DOI: 10.1155/2016/8651918 Abstract Mushrooms are a renowned source of products with an array of bioactivities, from antibacterial to antiviral, cytotoxic, anti-feeding, antifungal, or antioxidant and might be a valuable resource in the search of new bioactive extracts to inhibit bio�lm production. We demonstrate the e�ect of �ve mushroom water extracts, Macrolepiota procera, Pleurotus ostreatus, Auricularia auricula-judae, Armillaria mellea, and Laetiporus sul- phurous on bio�lm formation of four Staphylococcus aureus strains isolated from ixodid ticks (Acari) and ewe´s milk. �e PCR was used for the detection of virulence genes (hla, isdA, B, bbp, sirB, fnbpA, sdrE, agr II). �e ability of bio�lm formation and anti-bio�lm activity of mushrooms extracts was assessed in a quantitative crystal violet assay. �e bio�lm formation of S. aureus strains was signi�cantly reduced by all mushrooms extracts (p < 0.001). We showed that a more signi�cant anti-bio�lm e�ect of the extracts was of Staphylococcus aureus isolated from ixodid ticks in comparison to Staphylococcus aureus isolated from ewe´s milk. In the present study, A. mellea, P. ostreatus, L. sulphurous, A. auricula-judae, and M. procera extracts inhibited bio�lm formation by 70.87%, 67.00%, 64.14%, 62.77% and 47.71%, respectively. �e results sug- gest that compounds in mushrooms extracts might be useful to control and handle detrimental infections caused by animal and human pathogens. Key words: bio�lm, fungi, ixodid ticks (Acari), milk, Staphylococcus aureus Received: [2018.05.30] Accepted: [2018.11.12] 23 Aktywność antybiofilmowa ekstraktów grzybowych przeciw Staphylococcus aureus F. J. Rosen. Streszczenie Grzyby są znanym źródłem produktów bioaktywnych – począwszy od środków przeciwbakteryjnych po przeciwwirusowe, cytotoksyczne, przeciwlękowe, przeciwgrzybicze lub przeciwutleniające. Mogą być cennym źródłem nowych bioaktywnych ekstraktów poszukiwanych w celu zahamowania produk- cji bio�lmu bakteryjnego. W pracy pokazano wpływ pięciu wodnych ekstraktów z Macrolepiota proce- ra, Pleurotus ostreatus, Auricularia auricula-judae, Armillaria mellea i Laetiporus sulphurous na tworzenie się bio�lmu czterech szczepów Staphylococcus aureus, izolowanych z kleszczy (Acari) i mleka owczego. Do wykrywania genów wirulencji (hla, isdA, B, bbp, sirB, fnbpA, sdrE, agr II) zastosowano metodę PCR. Zdolność tworzenia bio�lmów i aktywność anty-bio�lmową ekstraktów grzybów oceniano w analizie ilościowej �oletem krystalicznym. Tworzenie bio�lmu szczepów S. aureus było znacznie mniejsze w śro- dowisku ekstraktów z grzybów (p < 0,001). Wykazaliśmy, że bardziej wrażliwy na działanie anty-bio- �lmowe ekstraktów grzybów był S. aureus wyizolowany z kleszczy niż wyizolowany z mleka owczego. W  niniejszych badaniach, A. mellea, P. ostreatus, L. sulphurous, A. auricula-judae i  M. procera, ha- mowały tworzenie się bio�lmu (o odpowiednio 70,87%, 67,00%, 64,14%, 62,77% i 47,71%). Wyniki sugerują, że związki zawarte w wodnych ekstraktach z grzybów mogą być przydatne do kontrolowania i zwalczania szkodliwych infekcji powodowanych przez patogeny zwierzęce i ludzkie. Słowa kluczowe: bio�lm, grzyby, kleszcze (Acari), mleko, Staphylococcus aureus Information about authors Anna Čuvalová She is interested in anti-bio�lm activities of natural compounds using the static and dynamic bio�lm mo- dels with resistant staphylococci (MRSA and MRCoNS), Escherichia coli (ESBL and cefotaximases) and Pseudomonas aeruginosa on various surfaces (plastics, catheters and food grade stainless sheet). Imrich Strapáč He is interested in bio�lm and anti-bio�lm activities of natural compounds using the static and dynamic bio�lm models with Escherichia coli, and resistant staphylococci (MRSA and MRCoNS). �e area of his interest is genetic ecology and genes encoding factors of virulence, metabolism, and the spreading of these genes. He studies the resistance occurrence in animal, which could serve as a reservoir of antibiotic resistance in indicator bacteria. Lívia Handrová http://orcid.org/0000-0002-0985-1771 �e main area of her interest is genetic ecology and the spread of antibiotic resistance genes. She studies the resistance occurrence in small mammals, which could serve as a reservoir of antibiotic resistance (ESBL, plasmid encoded chinolone resistance, carbapenemases) in indicator bacteria Escherichia coli, Pseudomonas aeruginosa and Staphylococcus spp. Vladimír Kmeť http://orcid.org/0000-0002-8081-8579 He is interested in bio�lm and anti-bio�lm activities of natural compounds using the static and dyna- mic bio�lm models with Escherichia coli, resistant staphylococci (MRSA and MRCoNS). �e area of his interest is genetic ecology and gene encoding factors of virulence, metabolism, and the spreading of these genes. He studies the resistance occurrence in animal, which could serve as a reservoir of antibiotic resistance in indicator bacteria. A ntibiofilm activity of m ushroom extracts against Staphylococcus aureus F. J. R osen.