臺灣博碩士論文加值系統

鐮孢菌屬(Fusarium)包含F. oxysporum、F. solani、F. graminearum與F. verticillioides等，且會在植物及人體造成嚴重的感染。臨床上，鐮孢菌感染發生的頻率在所有侵入性絲狀真菌的感染中排名第三，僅次於麴菌屬(Aspergillus)及毛黴目(Mucorales)，而感染的病例中以F. solani及F. oxysporum最多；然而，在植物及人類上，對於鐮孢菌感染的治療仍缺乏有效利器。陽離子態的抗菌胜肽多黏菌素B (polymyxin B) 具有抑制白色念珠菌與新型隱球菌的能力，但針對鐮孢菌的效果仍未知。本研究測試多黏菌素B對12株感染人類及植物(香蕉、番茄、瓜類、豆類、小麥及玉米)之鐮孢菌的抗菌能力。透過稀釋平板法、亞甲藍液染色及XTT還原法測試，此藥劑對所有受測菌株皆具殺真菌能力，其中對大部分菌株的最低殺菌濃度為32或64 μg/mL。多黏菌素B可降低鐮孢菌分生孢子的發芽率，且能造成膜系的缺陷，但厚膜孢子不受藥劑影響。多黏菌素B可與posaconazole產生協同作用(synergistic effect)，且具促進藥物fluconazole、voriconazole或amphotericin B對鐮孢菌抑制作用的潛力；多黏菌素B雖在抑制白色念珠菌及隱球菌的效果中與fluconazole有協同作用，然而在鐮孢菌屬中並未發現一樣的情形，顯示酵母菌及絲狀真菌如鐮孢菌在演化中的多樣性。

The genus Fusarium comprises many species, including F. oxysporum, F. solani, F. graminearum, and F. verticillioides, and causes severe infections in plants and humans. In clinical settings, Fusarium is the third most frequent mold to cause invasive fungal infections, after Aspergillus and the Mucorales. F. solani and F. oxysporum are the most prevalent Fusarium species to cause clinical disease. However, few effective antifungal drugs are available to treat both human and plant Fusarium infections. The cationic peptide antibiotic polymyxin B (PMB) exhibits antifungal activity against the human fungal pathogens Candida albicans and Cryptococcus neoformans, but its efficacy against Fusarium species is unknown. In this study, we tested the antifungal activity of PMB against 12 Fusarium strains that infect humans and plants (banana, tomato, melon, pea, wheat, and maize). PMB was fungicidal against all 12 Fusarium strains, with minimum fungicidal concentrations of 32 or 64 μg/mL for most strains tested, as evidenced by broth dilution, methylene blue staining, and XTT reduction assays. PMB can reduce the germination rates of conidia, but not chlamydospores, and can cause defects in cell membrane integrity in Fusarium strains. PMB exhibits synergistic activity with posaconazole, and can potentiate the effect of fluconazole, voriconazole, or amphotericin B against Fusarium species. However, PMB does not show synergistic effects with fluconazole against Fusarium species as it does against Candida glabrata and C. neoformans, indicating evolutionary divergence of mechanisms between yeast pathogens and the filamentous fungus Fusarium.

致謝 I
摘要 II
Abstract III
Table of Contents IV
1.Introduction 1
2.Materials and Methods
2.1.Strains, growth media, and chemicals 5
2.2.Conidia solution preparation 6
2.3.Determination of conidia germination rate 6
2.4.Determination of minimum inhibitory concentration 6
2.5.Determination of minimum fungicidal concentrations 7
2.6.Determination of fractional inhibitory concentration indices 8
2.7.Propidium iodide staining 9
2.8.RNA extraction and quantitative real-time RT-qPCR analysis 10
2.9.Induction of chlamydospore formation
and determination of germination rate 11
3.Results and Discussion
3.1.Polymyxin B exhibits novel antifungal activity against Fusarium species 12
3.2.Polymyxin B exhibits fungicidal activity against Fusarium species 14
3.3.Conidia, but not chlamydospore, germination of Fusarium strains
is inhibited by polymyxin B 15
3.4.Hypothetical mode of action of polymyxin B 16
3.5.Polymyxin B exhibits synergistic activity with posaconazole,
and potentiates the effect with fluconazole, voriconazole
or amphotericin B against Fusarium strains 17
4.Conclusions 19
5.Acknowledgement 20
 
6.Tables
Table 1.Fusarium strains used in this study 21
Table 2.MICs and MFCs of polymyxin B for 12 Fusarium strains 22
Table 3.Synergistic antifungal activity between polymyxin B (PMB) and
fluconazole (FLC), voriconazole (VRC), posaconazole (PSC) or
amphotericin B (AmB) against 12 Fusarium strains 23
Table 4.Primers used in RT-qPCR 24
7.References 25
8.Figures
Figure 1.Polymyxin B exhibits fungicidal activity against Fusarium strains
infecting humans or plants 30
Figure 2.Polymyxin B inhibits conidia germination of Fusarium strains 32
Figure 3.Polymyxin B damages cell membrane integrity of Fusarium strains 34
Figure 4.The expression of Fusarium CHS1 gene
is slightly affected by polymyxin B 36
Figure 5.Germination rate of chlamydospores produced by
Fusarium oxysporum f. sp. cubense or lycopersici
was not inhibited by polymyxin B 37
Figure S1.Polymyxin B exhibits novel antifungal activity against multiple
Fusarium species infecting humans and plants 39
Figure S2.Polymyxin B demonstrates antibacterial activity against
Pseudomonas aeruginosa that was frequently associated
with the keratitis in humans 40
Figure S3.XTT-based colormetric assays to determine
minimum fungicidal concentration of
polymyxin B against twelve Fusarium species 41
9.Appendix
Appendix A.Roles of PSD2 in Fusarium oxysporum f. sp. lycopersici 4287 43
Appendix B.A clinical case in stingrays infected with Fusarium species 52

[1]Dananche C, Cassier P, Sautour M, Gautheron N, Wegrzyn J, Perraud M, et al. Fungaemia caused by Fusarium proliferatum in a patient without definite immunodeficiency. Mycopathologia. 2015;179(1-2):135-40. doi: 10.1007/s11046-014-9817-6. PubMed PMID: 25253233.
[2]Nucci M, Anaissie E. Fusarium infections in immunocompromised patients. Clinical microbiology reviews. 2007;20(4):695-704. doi: 10.1128/CMR.00014-07. PubMed PMID: 17934079; PubMed Central PMCID: PMC2176050.
[3]Chang DC, Grant GB, O''Donnell K, Wannemuehler KA, Noble-Wang J, Rao CY, et al. Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. JAMA : the journal of the American Medical Association. 2006;296(8):953-63. doi: 10.1001/jama.296.8.953. PubMed PMID: 16926355.
[4]Broekaert N, Devreese M, De Baere S, De Backer P, Croubels S. Modified Fusarium mycotoxins unmasked: From occurrence in cereals to animal and human excretion. Food Chem Toxicol. 2015;80:17-31. doi: 10.1016/j.fct.2015.02.015. PubMed PMID: 25725190.
[5]Rajput AQ, Arain MH, Pathan MA, Jiskani MM, Lodhi AM. Efficacy of different fungicides against Fusarium wilt of cotton caused by Fusarium oxysporum f. sp vasinfectum. Pakistan J Bot. 2006;38(3):875-80. PubMed PMID: WOS:000242117000044.
[6]Avozani A, Tonin RB, Reis EM, Camera J, Ranzi C. In vitro sensitivity of Fusarium graminearum isolates to fungicides. Summa Phytopathologica. 2014;40:231-47.
[7]Guzman-Cottrill JA, Zheng X, Chadwick EG. Fusarium solani endocarditis successfully treated with liposomal amphotericin B and voriconazole. The Pediatric infectious disease journal. 2004;23(11):1059-61. PubMed PMID: 15545866.
[8]Speeleveld E, Gordts B, Van Landuyt HW, De Vroey C, Raes-Wuytack C. Susceptibility of clinical isolates of Fusarium to antifungal drugs. Mycoses. 1996;39(1-2):37-40. PubMed PMID: 8786755.
[9]Altun A, Kurna SA, Sengor T, Altun G, Olcaysu OO, Aki SF, et al. Effectiveness of posaconazole in recalcitrant fungal keratitis resistant to conventional antifungal drugs. Case reports in ophthalmological medicine. 2014;2014:701653. doi: 10.1155/2014/701653. PubMed PMID: 25184064; PubMed Central PMCID: PMC4144149.
[10]Ranawaka RR, Nagahawatte A, Gunasekara TA. Fusarium onychomycosis: prevalence, clinical presentations, response to itraconazole and terbinafine pulse therapy, and 1-year follow-up in nine cases. International journal of dermatology. 2015. doi: 10.1111/ijd.12906. PubMed PMID: 26223159.
[11]Chen KJ, Sun MH, Lai CC, Wu WC, Chen TL, Kuo YH, et al. Endophthalmitis caused by Pseudomonas aeruginosa in Taiwan. Retina. 2011;31(6):1193-8. doi: 10.1097/IAE.0b013e3181fbce5c. PubMed PMID: 21283056.
[12]Chuang CC, Hsiao CH, Tan HY, Ma DH, Lin KK, Chang CJ, et al. Staphylococcus aureus ocular infection: methicillin-resistance, clinical features, and antibiotic susceptibilities. PloS one. 2012;8(8):e42437. doi: 10.1371/journal.pone.0042437. PubMed PMID: 22880135; PubMed Central PMCID: PMC3413655.
[13]Stansly PG, Schlosser ME. Studies on Polymyxin: Isolation and Identification of Bacillus polymyxa and Differentiation of Polymyxin from Certain Known Antibiotics. Journal of bacteriology. 1947;54(5):549-56. PubMed PMID: 16561391; PubMed Central PMCID: PMC526590.
[14]Hancock RE. Peptide antibiotics. Lancet. 1997;349(9049):418-22. doi: 10.1016/S0140-6736(97)80051-7. PubMed PMID: 9033483.
[15]Nation RL, Li J, Cars O, Couet W, Dudley MN, Kaye KS, et al. Framework for optimisation of the clinical use of colistin and polymyxin B: the Prato polymyxin consensus. The Lancet Infectious diseases. 2014. doi: 10.1016/S1473-3099(14)70850-3. PubMed PMID: 25459221.
[16]Parchuri S, Mohan S, Cunha BA. Extended spectrum beta-lactamase-producing Klebsiella pneumoniae chronic ambulatory peritoneal dialysis peritonitis treated successfully with polymyxin B. Heart & lung : the journal of critical care. 2005;34(5):360-3. doi: 10.1016/j.hrtlng.2004.11.001. PubMed PMID: 16157192.
[17]Sarria JC, Angulo-Pernett F, Kimbrough RC, McVay CS, Vidal AM. Use of intravenous polymyxin B during continuous venovenous hemodialysis. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 2004;23(4):340-1. doi: 10.1007/s10096-004-1106-8. PubMed PMID: 15007705.
[18]Deris ZZ, Akter J, Sivanesan S, Roberts KD, Thompson PE, Nation RL, et al. A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity. J Antibiot (Tokyo). 2014;67(2):147-51. doi: 10.1038/ja.2013.111. PubMed PMID: 24169795; PubMed Central PMCID: PMCPMC3943757.
[19]Ogita A, Nagao Y, Fujita K, Tanaka T. Amplification of vacuole-targeting fungicidal activity of antibacterial antibiotic polymyxin B by allicin, an allyl sulfur compound from garlic. J Antibiot (Tokyo). 2007;60(8):511-8. doi: 10.1038/ja.2007.65. PubMed PMID: 17827662.
[20]Pankey G, Ashcraft D, Kahn H, Ismail A. Time-kill assay and Etest evaluation for synergy with polymyxin B and fluconazole against Candida glabrata. Antimicrobial agents and chemotherapy. 2014;58(10):5795-800. doi: 10.1128/AAC.03035-14. PubMed PMID: 25049251; PubMed Central PMCID: PMC4187950.
[21]Zhai B, Lin X. Evaluation of the anticryptococcal activity of the antibiotic polymyxin B in vitro and in vivo. International journal of antimicrobial agents. 2013;41(3):250-4. doi: 10.1016/j.ijantimicag.2012.11.006. PubMed PMID: 23313397.
[22]Zhai B, Zhou H, Yang L, Zhang J, Jung K, Giam CZ, et al. Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect. The Journal of antimicrobial chemotherapy. 2010;65(5):931-8. doi: 10.1093/jac/dkq046. PubMed PMID: 20167587; PubMed Central PMCID: PMC2851492.
[23]Ben-Ami R, Lewis RE, Tarrand J, Leventakos K, Kontoyiannis DP. Antifungal activity of colistin against mucorales species in vitro and in a murine model of Rhizopus oryzae pulmonary infection. Antimicrob Agents Chemother. 2010;54(1):484-90. doi: 10.1128/AAC.00956-09. PubMed PMID: 19858263; PubMed Central PMCID: PMCPMC2798530.
[24]White TJ, Bruns, T., Lee, S. and Talyor, J. PCR Protocols: a Guide to Methods and Applications. M. Innis DG, J. Sninsky, T. White, editor. Orlando, Florida: Academic Press; 1990.
[25]O''Donnell K, Kistler HC, Cigelnik E, Ploetz RC. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proc Natl Acad Sci U S A. 1998;95(5):2044-9. PubMed PMID: 9482835; PubMed Central PMCID: PMCPMC19243.
[26]CLSI. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard-second edition. CLSI document M38-A2. Wayne, PA: Clinical and Laboratory Standards Institute. 2008.
[27]Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother. 2003;52(1):1. doi: 10.1093/jac/dkg301. PubMed PMID: 12805255.
[28]Dijksterhuis J, Sanders M, Gorris LG, Smid EJ. Antibiosis plays a role in the context of direct interaction during antagonism of Paenibacillus polymyxa towards Fusarium oxysporum. J Appl Microbiol. 1999;86(1):13-21. PubMed PMID: 10030009.
[29]Bosscha MI, van Dissel JT, Kuijper EJ, Swart W, Jager MJ. The efficacy and safety of topical polymyxin B, neomycin and gramicidin for treatment of presumed bacterial corneal ulceration. Br J Ophthalmol. 2004;88(1):25-8. PubMed PMID: 14693766; PubMed Central PMCID: PMCPMC1771930.
[30]Tajima K, Miyake T, Koike N, Hattori T, Kumakura S, Yamaguchi T, et al. In vivo challenging of polymyxins and levofloxacin eye drop against multidrug-resistant Pseudomonas aeruginosa keratitis. J Infect Chemother. 2014;20(6):343-9. doi: 10.1016/j.jiac.2013.10.015. PubMed PMID: 24726376.
[31]Ly NS, Bulman ZP, Bulitta JB, Baron C, Rao GG, Holden PN, et al. Optimization of Polymyxin B in Combination with Doripenem To Combat Mutator Pseudomonas aeruginosa. Antimicrobial agents and chemotherapy. 2016;60(5):2870-80. doi: 10.1128/AAC.02377-15. PubMed PMID: 26926641.
[32]Pfaller MA, Sheehan DJ, Rex JH. Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization. Clinical microbiology reviews. 2004;17(2):268-80. PubMed PMID: 15084501; PubMed Central PMCID: PMCPMC387411.
[33]Justo JA, Bosso JA. Adverse reactions associated with systemic polymyxin therapy. Pharmacotherapy. 2015;35(1):28-33. doi: 10.1002/phar.1493. PubMed PMID: 25266910.
[34]Grau-Campistany A, Manresa A, Pujol M, Rabanal F, Cajal Y. Tryptophan-containing lipopeptide antibiotics derived from polymyxin B with activity against Gram positive and Gram negative bacteria. Biochim Biophys Acta. 2015. doi: 10.1016/j.bbamem.2015.11.011. PubMed PMID: 26607008.
[35]Rabanal F, Grau-Campistany A, Vila-Farres X, Gonzalez-Linares J, Borras M, Vila J, et al. A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity. Sci Rep. 2015;5:10558. doi: 10.1038/srep10558. PubMed PMID: 26024044; PubMed Central PMCID: PMCPMC4603705.
[36]Schafer K, Di Pietro A, Gow NA, MacCallum D. Murine model for Fusarium oxysporum invasive fusariosis reveals organ-specific structures for dissemination and long-term persistence. PLoS One. 2014;9(2):e89920. doi: 10.1371/journal.pone.0089920. PubMed PMID: 24587124; PubMed Central PMCID: PMCPMC3937399.
[37]Schroers HJ, O''Donnell K, Lamprecht SC, Kammeyer PL, Johnson S, Sutton DA, et al. Taxonomy and phylogeny of the Fusarium dimerum species group. Mycologia. 2009;101(1):44-70. PubMed PMID: 19271670.
[38]Cohen E. Chitin Biochemistry: Synthesis and Inhibition. Annual Review of Entomology. 1987;32(1):71-93. doi: doi:10.1146/annurev.en.32.010187.000443.
[39]Rogg LE, Fortwendel JR, Juvvadi PR, Steinbach WJ. Regulation of expression, activity and localization of fungal chitin synthases. Med Mycol. 2012;50(1):2-17. doi: 10.3109/13693786.2011.577104. PubMed PMID: 21526913; PubMed Central PMCID: PMCPMC3660733.
[40]Chang YL, Yu SJ, Heitman J, Wellington M, Chen YL. New facets of antifungal therapy. Virulence. 2016:1-15. doi: 10.1080/21505594.2016.1257457. PubMed PMID: 27820668.
[41]O''Donnell K, Sutton DA, Rinaldi MG, Magnon KC, Cox PA, Revankar SG, et al. Genetic diversity of human pathogenic members of the Fusarium oxysporum complex inferred from multilocus DNA sequence data and amplified fragment length polymorphism analyses: evidence for the recent dispersion of a geographically widespread clonal lineage and nosocomial origin. J Clin Microbiol. 2004;42(11):5109-20. doi: 10.1128/JCM.42.11.5109-5120.2004. PubMed PMID: 15528703; PubMed Central PMCID: PMCPMC525153.
[42]Lin Y-H, Chang J-Y, Liu E-T, Chao C-P, Huang J-W, Chang P-FL. Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4. Eur J Plant Pathol. 2009;123:353–65.
[43]Ma LJ, van der Does HC, Borkovich KA, Coleman JJ, Daboussi MJ, Di Pietro A, et al. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature. 2010;464(7287):367-73. doi: 10.1038/nature08850. PubMed PMID: 20237561; PubMed Central PMCID: PMCPMC3048781.
[44]Ma LJ, Shea T, Young S, Zeng Q, Kistler HC. Genome Sequence of Fusarium oxysporum f. sp. melonis Strain NRRL 26406, a Fungus Causing Wilt Disease on Melon. Genome Announc. 2014;2(4). doi: 10.1128/genomeA.00730-14. PubMed PMID: 25081257; PubMed Central PMCID: PMCPMC4118060.
[45]Coleman JJ, Rounsley SD, Rodriguez-Carres M, Kuo A, Wasmann CC, Grimwood J, et al. The genome of Nectria haematococca: contribution of supernumerary chromosomes to gene expansion. PLoS Genet. 2009;5(8):e1000618. doi: 10.1371/journal.pgen.1000618. PubMed PMID: 19714214; PubMed Central PMCID: PMCPMC2725324.
[46]Cuomo CA, Guldener U, Xu JR, Trail F, Turgeon BG, Di Pietro A, et al. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science (New York, NY. 2007;317(5843):1400-2. PubMed PMID: 17823352.