Biofilm Inhibition: A Concept for Many Applications

GEOMAR: 17 November 2025 Sherif S. Ebada, PhD Microbial Drugs Dept., HZI Braunschweig, Germany; [email protected]; [email protected] Biofilm Inhibition: A Concept for Many Applications Blackwellomyces roseostromatus Samsoniella aurantia Cordyceps javanica Laburnicola nematophila Beauveria neobassiana Cordyceps blackwelliae Agenda 1. Resume 2. Background in NPC. 3. Biofilm Inhibitors from Saprotrophic/Entomopathogens: R. Toshe, K. Phutthacharoen, S. S. Ebada, M. Stadler, et al. 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens: J.-P. Wennrich, C. Holzenkamp, E. Sepanian, S. S. Ebada et al. 5. Neurotrophic NPs from Basidiomycota: W. C. Sum, S. S. Ebada, J. C. Matasyoh, M. Stadler, et al. 1. Resume: Name: Sherif S. Ebada Academic Degree/Profession: - 2022-2024: GF Fellowship for Experienced Researcher, AvH. - 2021: Professor in Pharmacognosy and Nat. Prod. Chem., Faculty of Pharmacy, ASU, Egypt. - 2010: PhD in Natural Sciences (Mar. Nat. Prod. Chem.), Heinrich Heine University, Germany. - 2006: Msc. in Pharmacognosy, ASU, Egypt. - 2000: Bsc. in Pharmacy, Ain Shams Univerity, Egypt. Current: Erasmus Staff Mobility Program (KA171), Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University, Düsseldorf, Germany Permanent: Professor Department of Pharmacognosy Faculty of Pharmacy Ain-Shams University, Egypt. 2. Background in NPC 2. Background in NPC NHO NCH3 O O H N NH R3 O R1 1 3 4 6 8 1012 14 17 18 18' 17' 20 24 29 30 31 32 33 34 OH O R1= = R2 R2 (+)-Jaspamide 3 17 18 18' 17' OH R1= H = R2 (+)-Jaspamide Z6 HO 1 3 17 18 18' 17' OH O R1= (+)-Jaspamide Z5 HO OR2= H 19 19 OHO 1 19 1 3 17 18 18' 17' OH O R1= = R2 (+)-Jaspamide Q 19 1 3 17 18 18' 17' OH O R1= = R2 (+)-Jaspamide R 19 R3= Br R4= H R4 R3= Br R4= H R3= Br R4= H R3= Br R4= H R3= Br R4= H OH OO H HO OH OOH H HO OCH3 Arthrinin D Arthrinin A OH OO H HO Arthrinin B OH OO H HO Arthrinin C 2: R = H 4: R = CH3 OH O O OR 1: R = H 3: R = CH3 OH O O OR H H OOH R2O O OR4 R1O R3 6: R1=R3= OCH3, R2=R4= H 7: R1=R3=R4= H, R2= CH3 8: R1= OCH3, R2=R3= H, R4= CH3 9: R1=R4= H, R2= CH3, R3= OCH3 10: R1=R3= OCH3, R2= CH3, R4= H O OOH OH 1 O OH O O O O HO O O OH Agathisflavone-4',7,7"-trimethyl ether GF-AvH at HZI-BS (09.2022-06.2024) Exploring Novel Fungal Species for Metabolites Interfering with Biofilms of Human Pathogenic Microbes Microbial Drugs Department (MWIS) Prof. Dr. M. Stadler Mrs. C. Fritz-Braun Dr. H. Schrey Dr. M. Große Dr. Y. Marin-FelixDr. F. Surup Dr. B. Sandargo Dr. B. M. Kemkuignou 3. Biofilm Inhibitors from Saprotrophic/Entomopathogens https://www.pureline.com/biofilm-threats-and-the-chlorine-dioxide-solution/ Adhesion EPS matrix Biofilm Maturation Balance Biofilm: What, Why, When, Where? https://www.researchgate.net/publication/239735127 Biofilm: What, Why, When, Where? Surface Biofilm – Close up of a Streambed Biofilm Forming Microorganism – Electron Micrograph Cordyceps javanica Beauveria neobassiana Samsoniella aurantia Cordyceps blackwelliae Blackwellomyces roseostromatus b. Biofilm Inhibitors from Entomopathogenic fungi PDB Beauveria neobassiana Compounds 2-5: anti S. aureus biofilm of ca. 80% at 31 µg/mL Bioprospection of Tenellins Produced by the Entomopathogenic Fungus Beauveria neobassiana b. Biofilm Inhibitors from Entomopathogenic fungi b. Biofilm Inhibitors from Entomopathogenic Fungi Bioprospection of Tenellins Produced by the Entomopathogenic Fungus Beauveria neobassiana Cytotoxicity criteria: • 15-Hydroxylation. •N-Hydroxylation • 15-Hydroxylation and N-Hydroxylation Only tenellin exhibited slight antibacterial activity against Bacillus subtilis and Staphylococcus aureus. Halo et al. (2008) Journal of the American Chemical Society Cytotoxic activity (4) Positive Control IC50 (µM) Test Cell Line Epothilone B (nM)654321 0.65 5.76.80.7948.270.1n.a. L929 (fibroblast) 0.17 4.96.00.7920.264.7n.a.KB3.1 (ovary) 0.05 24.12.60.24n.d.n.d.n.d.A549 (lung) 0.07 7.38.12.0n.d.n.d.n.d.MCF-7 (breast) b. Biofilm Inhibitors from Entomopathogenic Fungi Bioprospection of Tenellins Produced by the Entomopathogenic Fungus Beauveria neobassiana •Tenellin derivatives (2-4) revealed promising activities in a dose-dependent manner. •In 2-pyridone derivatives, the aliphatic side chain at C-3 potentiates antibiofilm activity. •Hydroxylation at C-15 on of the side chain as in 15hydroxytenellin (5) negatively affected the biofilm inhibitory activity. b. Biofilm Inhibitors from Entomopathogenic Fungi Blackwellomyces roseostromatus b. Biofilm Inhibitors from Entomopathogenic Fungi. ES-242-9 (1) 1 3 4 5 6 7 89 10 4a 10a 8a 9a 11 1' 3' 4' 5' 6' 7' 8' 9' 10' 9a' 8a' 4a' 10a' 11' (S) O OHO O HO O OH O (S) (R) OH L-Trp L-Ala L-Phe O HN NH H N O O O O HN 2 3 4 1 11 6810 HA 1' 2' 3' 4' 5' 6' 7' 8' 10' 1'' 2'' 3'' 1''' 2''' 3''' 4''' 5''' 7''' 9''' L-Trp1 L-Trp2 L-Phe O HN NH H N O O O O HN 2 3 4 1 11 6810 H A 1' 2' 3' 4' 5 '6' 7' 8' 10' 1'' 2'' 3'' 1''' 2''' 3''' 4''' 5''' 7''' 9''' 10'' 8'' 7'' 6'' 5'' 4'' HN Beauverolide T (2)Beauverolide U (3) N O OHN O O NH O NH O O NL-Leu1 L-NMe-Ala1 L-NMe-Ala2 L-Pla L-Phe L-Leu2 1 2 3 4 5 9 6 78 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Cardinalisamide D (4) N O OHN O O O O NH O O N R R= H: Cardinalisamide B (5) R= CH3: Cardinalisamide C (6) Positive Control IC50 (µM) Epothilone B (nM) 65 4321Test Cell Line 0.65 26.0 ********L929 (murine) 0.17 8.42.236.2 ** 29.2 KB3.1 (cervix) 0.09 7.52.5 n.t.n.t.n.t.n.t.PC-3 (prostate) 0.07 13.96.9 n.t.n.t.n.t.n.t.MCF-7 (breast) 0.09 12.325.0 n.t.n.t.n.t.n.t.SKOV-3 (ovary) 0.06 8.54.3 n.t.n.t.n.t.n.t.A431 (skin) 0.05 11.111.5 n.t.n.t.n.t.n.t.A549 (lung) Cytotoxic (IC50 in µM) activity results of 1-6against mammalian cells. (*): Slight inhibition of cell proliferation, (**): no cytotoxic activity observed, n.t.: not tested. N O OHN O O O O NH O O N Cardinalisamide C (6) b. Biofilm Inhibitors from Entomopathogenic Fungi. Nematicidal assay of 1–6against Caenorhabditis elegans. Corrected mortality rate of compounds 1–6(10 µg mL-1, 50 µg mL-1 and 100 µg mL-1) against C. elegans after 18 h of treatment. A solution of ivermectin (1 µg mL-1) was used as a positive control. N O OHN O O O O NH O O N Cardinalisamide C (6) b. Biofilm Inhibitors from Entomopathogenic Fungi. II. Biofilm Inhibitors from Entomopathogenic Fungi. N OOH O HO Farinosone D (1) O N O NN OO O O O O O Beauvericin B (5) O N O NN OO O O O O O Beauvericin A (4) 1 HN HO O O OH 3 57911 13 14 15 16 1' 2' 3' 4' 5' 6' 7' R R = H: Farinosone A (2) R = OH: Farinosone B (3) O N O NN OO O O O O O Beauvericin C (6) HO Antibiofilm and Cytotoxic Metabolites from the Entomopathogenic Fungus Samsoniella aurantia b. Biofilm Inhibitors from Entomopathogenic Fungi Pathogenicity of Polyphilus sieberi towards Heterodera filipjevi.1 A-C. Fungus-infected eggs isolated from field-collected symptomatic cysts. D. Symptomatic cyst obtained from in vitro tests. E. Runner hyphae of P. sieberi growing in cyst mucilage. F–H. In vitro infection process of P. sieberi in nematode eggs. 1Ashrafi et al. (2018). Mycologia, 110(2), 286-299. Dr. W. Maier Dr. S. Ashrafi 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Cultivation Polyphilus sieberi Heterodera filipjevi infected by: Polyphilus sieberi LCMS analysis Activity-guided fractionation Antimicrobial testing Purification & structure elucidation Assessment of Pure Compounds Bacteria B. subtilis E. coli Fungi M. tenuis C. albicans Qualitative screening (Prescreening) Collaborations Plant parasitic nematodes (JKI) Human parasitic nematodes Screening Libraries (HIPS) Inhibtion of biofilm formation (H. Zeng) Quantitative testing Effects on C. elegans populations (J. Wennrich) Minimal Inhibitory concentration (MIC) (W. Collisi) Cytotoxicity (W. Collisi) RegionIsolation sourceStrain no.SpeciesGenus Villerupt, FrancePinus sylvetris roots a V16frankeniiPolyphilus Yozgat, TurkeyEggs of Heterodera filipyevi b 17Asieberi 17C Bugac, HungaryAsclepias syriaca roots c REF052 Fungal strains investigated in this study a Isolated by Damien Blaudez and Michel Chalot. b Isolated by Samad Ashrafi. c Isolated by Dániel G. Knapp and Gabor M. Kováks 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Control [O] MIC [µg/mL] 2 MIC [µg/mL] Organism 16.60.52 B. subtilis 0.2166.6 S. aureus 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens N (S) (R)(S) H N OH H O H N O OH HO O NH R HO Cl H N O 123 3a 3b 4 6 5 7 7a 8a HPI D-Tyr2 L-Tyr1 1'' 2'' 3'' 4'' 5'' 6'' 7'' 8'' 9'' 1: R= Cl 2: R= H HO OH OO (R) O 12 3 4 4a 5 5a 6 7 8 99a10 10a 11 13 15 12 14 O3 O (R) (R) O OH HO OH OOH OH O HO (R) 1' 2' 3' 4' 4'a 5' 5'a 6' 7' 8' 9' 9'a 10' 10'a 11' 13' 15' 12' 14' 1 3 4 4a 5 5a 6 8 99a10 10a 11 13 15 12 14 R 4: R= OH 5: R= H N N O O OO HO O OH OH OH H Sn 6: n= 1 7: n= 3 O O O O N O ON OH OH OH H O O O O N O ONH OH OH OH H S 89 O OH NH O HO O OH HO HO HO 10 O R OH O O HO 11: R= 12: R= O N H NH N N O O 13 L-diCl/ClTyr 1''' 2''' 3''' 4''' 5''' 6''' 7''' 8''' 9''' 1' 2' 3' 4' 5' 6' 7' 8' 9' 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens O (R) (R) O OH HO OH OOH OH O HO (R) 1' 2' 3' 4' 4'a 5' 5'a 6' 7' 8' 9' 9'a 10' 10'a 11' 13' 15' 12' 14' 1 3 4 4a 5 5a 6 8 99a10 10a 11 13 15 12 14 R R= OH: Talaroderxine D (4) R= H: Talaroderxine C (5) O HO OH O O HO Fusidic acid (11) Cytotoxic (IC50 in µM) activity results. Antimicrobial (MIC in µg/mL) activity results. 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Page 37 | 5: R= H 6: R= Cl 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Laburnicotides A–F: Acyclic N-acetyl Oligopeptides from the NematodeCyst Associated Fungus Laburnicola nematophila 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens (R) OH O O HO OH 98a 8 7 6 5 4a 4 3 2 1 1' 3' 4' 4a' 5' 6' 7' 8' 8a' 9' 1 2 3 4 5 6 7 8 98a 4a (R) OH O O OH O 10 11 12 OO OH O OH OH HO OH OH 2 3 4 5 6 1' 2' 3' 4' 5' 6' 1" 3" 5" 7" 9" 11" 13" 15" 17" 19" 20" 21" 22" 23" 24" 25" 26" R2 R1 3: R1, R2= H 2: R1= CH3, R2= H 1: R1= CH3, R2= OH 4 5 6 HO O OH O HO OH O OH O (R) 27" (S*) (S*) (S*)(S*) (S*) (S*) (S*) (R*) (S*) (R*) (R*) OO OH O OH OH HO OH 2 3 4 5 6 1' 2' 3' 4' 5' 6' 1" 3" 5" 7" 9" 11" 13" 15" 17" 19" 20" 21" 24" 25" 26" 28" 29" 22" 23" OH 27" 30" Dactylfungin A16,20 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Strain number IC 50 (µM) Positive Control Test Cell Line 1 2 3 4 5 6 Epothilone B (nM) Mouse fibroblast L929 * * n.d. 49.02 ** * 0.65 Human endocervival adenocarcinoma KB3.1 * 33.31 n.d. 63.02 76.01 * 0.17 Test Microorganism MIC (µg/mL) Positive Control (µg/mL) Staphylococcus aureus DSM 346 - 66.6 n.d. - - - 0.21 G Aspergillus fumigatus ATCC 204305 0.26 0.52 8.325 n.d. n.d. n.d. 0.312 A A. fumigatus CCF 3522 n.d. 0.52 n.d. n.d. n.d. n.d. 0.312 A A. fumigatus (azole-resistant) CCF 6651 - 2.08 0.52 n.d. n.d. n.d. 0.156 A A. fumigatus (azole-resistant) CCF 6674 16.6 0.52 0.52 n.d. n.d. n.d. 2.5 A C. albicans CCM 8215 - - 8.3 n.d. n.d. n.d. 1.25 A Cryptococcus neoformans CCF 1081 - 4.15 4.15 n.d. n.d. n.d. 2.5 A Mucor plumbeus CCF 2612 - - 1.04 n.d. n.d. n.d. 0.312 A Rhodotorula glutinis DSM 10134 - 8.3 n.d. - - - 4.20 N 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens 4. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Cordypyridones E–J (1–6), C (7) and D (8). Acknowledgement •In-house collaboration at HZI: T. Stradal, K. Rottner, M. Brönstrup and co-workers. •Co-authors of papers on Thai fungi; above all J. J. Luangsa-ard, K. D. Hyde and coworkers. •P. Crous, J. Collemare (WFBI) and coworkers. •Collaborators in the ERAFRICA project: J. C. Matasyoh (Kenya), C. Decock (MUCL Belgium). •I. Krisai-Greilhuber/H. Voglmayr (Vienna). •Expert technical assistance: W. Collisi, A. Skiba, E. Surges, A. Gollasch (HZI) and many others. •All coauthors of all publications. •Various funding agencies. This research benefitted from funding by the European Union’s Horizon 2020 research and innovation program (RISE) under the Marie Skłodowska-Curie grant agreement No. 101008129, project acronym “Mycobiomics”. https://cordis.europa.eu/project/id/101008129 https://zenodo.org/communities/mycobiomicsh2020-mscarise/records?q=&l=list&p=1&s=10&sort=newest Acknowledgement Thank You!