Actividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género Candida
La candidiasis es una de las infecciones más frecuentes en pacientes hospitalizados o con alteraciones en su sistema inmunológico, actualmente se ha convertido en una problemática creciente debido a la resistencia adquirida por las levaduras del género Candida a diferentes antifúngicos utilizados en...
- Autores:
-
Chaves Alfonso, Gabriel German
Coronado Ladino, Yenifer Tatiana
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2019
- Institución:
- Colegio Mayor de Cundinamarca
- Repositorio:
- Repositorio Colegio Mayor de Cundinamarca
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unicolmayor.edu.co:unicolmayor/3714
- Acceso en línea:
- https://repositorio.unicolmayor.edu.co/handle/unicolmayor/3714
- Palabra clave:
- Microorganismos
Dominio C-terminal antimicrobiano
Cepas
Candida albicans
Péptido antimicrobiano
Curvas de crecimiento
Catelicidina humana LL37
- Rights
- closedAccess
- License
- Derechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2018
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dc.title.spa.fl_str_mv |
Actividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género Candida |
title |
Actividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género Candida |
spellingShingle |
Actividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género Candida Microorganismos Dominio C-terminal antimicrobiano Cepas Candida albicans Péptido antimicrobiano Curvas de crecimiento Catelicidina humana LL37 |
title_short |
Actividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género Candida |
title_full |
Actividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género Candida |
title_fullStr |
Actividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género Candida |
title_full_unstemmed |
Actividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género Candida |
title_sort |
Actividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género Candida |
dc.creator.fl_str_mv |
Chaves Alfonso, Gabriel German Coronado Ladino, Yenifer Tatiana |
dc.contributor.advisor.none.fl_str_mv |
Muñoz Henao, Julián Esteban Pinilla Bermúdez, Gladys |
dc.contributor.author.none.fl_str_mv |
Chaves Alfonso, Gabriel German Coronado Ladino, Yenifer Tatiana |
dc.subject.lemb.none.fl_str_mv |
Microorganismos Dominio C-terminal antimicrobiano Cepas |
topic |
Microorganismos Dominio C-terminal antimicrobiano Cepas Candida albicans Péptido antimicrobiano Curvas de crecimiento Catelicidina humana LL37 |
dc.subject.proposal.spa.fl_str_mv |
Candida albicans Péptido antimicrobiano Curvas de crecimiento Catelicidina humana LL37 |
description |
La candidiasis es una de las infecciones más frecuentes en pacientes hospitalizados o con alteraciones en su sistema inmunológico, actualmente se ha convertido en una problemática creciente debido a la resistencia adquirida por las levaduras del género Candida a diferentes antifúngicos utilizados en el tratamiento de esta micosis. Como alternativa terapéutica han sido estudiados en las últimas décadas varios péptidos antimicrobianos con el fin de investigar nuevos tratamientos de amplio espectro, especialmente para levaduras productoras de biopelículas. Este es el caso de Candida albicans, una levadura comensal que hace parte del microbiota del ser humano, pero que en pacientes inmunosuprimidos puede causar infecciones cutáneas, subcutáneas y sistémicas, con el agravante de presentar resistencia en algunos casos a la terapia convencionalmente usada para su tratamiento. Los péptidos antimicrobianos están presentes en animales, plantas y otros microorganismos, haciendo parte del sistema inmune innato. La catelicidina humana LL37, péptido antimicrobiano catiónico, es una molécula que contiene tanto un dominio catelina como un dominio C-terminal antimicrobiano. Basados en lo anterior, se realizó un estudio de tipo cuantitativo; estudios descriptivos y explicativos en donde se utilizaron tres cepas, dos de ellas correspondientes a Candida albicans y una correspondiente a una cepa proveniente de candidiasis recurrente, probando en cada una de ellas cinco concentraciones distintas de péptidos análogos de LL-37 como: AC-1, LL-37,1, AC-2 y D, evidenciando su poder inhibitorio mediante las curvas de crecimiento obtenidas en el equipo BioScreen C. Teniendo en cuenta los cuatro péptidos utilizados, se demostró que los péptidos AC-1 y D evidenciaron una actividad inhibitoria significativa en las tres cepas incluidas en el estudio; con respecto a los otros péptidos, cabe resaltar que LL-37,1 tuvo una excelente capacidad inhibitoria en la cepa Candida albicans 256. |
publishDate |
2019 |
dc.date.issued.none.fl_str_mv |
2019-01 |
dc.date.accessioned.none.fl_str_mv |
2021-11-23T19:59:21Z |
dc.date.available.none.fl_str_mv |
2021-11-23T19:59:21Z |
dc.type.spa.fl_str_mv |
Trabajo de grado - Pregrado |
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http://purl.org/coar/resource_type/c_7a1f |
dc.type.coarversion.spa.fl_str_mv |
http://purl.org/coar/version/c_970fb48d4fbd8a85 |
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info:eu-repo/semantics/bachelorThesis |
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https://purl.org/redcol/resource_type/TP |
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58675 |
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dc.relation.references.spa.fl_str_mv |
Alvarez-moreno CA. Burden of Fungal Infections in Colombia. 2018;1–13. Merseguel KB, Nishikaku AS, Rodrigues AM, Padovan AC, Carmona R, Salles A, et al. Genetic diversity of medically important and emerging Candida species causing invasive infection. 2015;1–11. Papon N, Courdavault V, Clastre M, Bennett RJ, Nac N-C. Emerging and Emerged Pathogenic Candida Species : Beyond the Candida albicans Paradigm. 2013;9(9). Whibley N, Gaffen SL. Beyond Candida albicans: Mechanisms of immunity to non-albicans Candida species. Cytokine. 2015;76(1):42–52. Pfaller MA, Messer SA, Jones RN, Castanheira M. Antifungal susceptibilities of Candida , Cryptococcus neoformans and Aspergillus fumigatus from the Asia and Western Paci fi c region : data from the SENTRY antifungal surveillance program ( 2010 – 2012 ). 2015;(November 2014):1–6. Available from: http://dx.doi.org/10.1038/ja.2015.29 Pfaller MA. Antifungal Drug Resistance : Mechanisms , Epidemiology , and Consequences for Treatment. AJM [Internet]. 2012;125(1):S3–13. Available from: http://dx.doi.org/10.1016/j.amjmed.2011.11.001 Pfaller MA, Moet GJ, Messer SA, Jones RN, Castanheira M. Geographic Variations in Species Distribution and Echinocandin and Azole Antifungal Resistance Rates among Candida Bloodstream Infection Isolates : Report from the SENTRY Antimicrobial Surveillance Program ( 2008 to 2009 ) ᰔ. 2011;49(1):396–9. Tema DE. Péptidos antimicrobianos. 2010;14(1):55–67. de la Fuente-Núñez C, Silva ON, Lu TK, Franco OL. Antimicrobial peptides: Role in human disease and potential as immunotherapies. Pharmacol Ther. 2017; Tsai PW, Yang CY, Chang HT, Lan CY. Human antimicrobial peptide LL-37 inhibits adhesion of Candida albicans by interacting with yeast cell-wall carbohydrates. PLoS One. 2011;6(3). Carrascosa JM. Mecanismo De acción De ustekinumab y su relevancia en la patogénesis De la psoriasis. Impacto en el sistema inmune. Actas Dermosifiliogr. 2012;103(SUPPL. 2):7–15. Antinori S, Milazzo L, Sollima S, Galli M, Corbellino M. European Journal of Internal Medicine Candidemia and invasive candidiasis in adults : A narrative review. Eur J Intern Med [Internet]. 2016; Available from: http://dx.doi.org/10.1016/j.ejim.2016.06.029 Blumberg HM, Jarvis WR, Soucie JM, Edwards JE, Patterson JE, Pfaller MA, et al. Risk Factors for Candidal Bloodstream Infections in Surgical Intensive Care Unit Patients : The NEMIS Prospective Multicenter Study. 2001;6:177– 86. Giongo JL, de Almeida Vaucher R, Fausto VP, Quatrin PM, Lopes LQS, Santos RCV, et al. Anti-Candida activity assessment of Pelargonium graveolens oil free and nanoemulsion in biofilm formation in hospital medical supplies. Microb Pathog. 2016;100:170–8. Kodedová M, Sychrová H. Synthetic antimicrobial peptides of the halictines family disturb the membrane integrity of Candida cells. Biochim Biophys Acta - Biomembr. 2017;1859(10):1851–8. Scarsini M, Tomasinsig L, Arzese A, D’Este F, Oro D, Skerlavaj B. Antifungal activity of cathelicidin peptides against planktonic and biofilm cultures of Candida species isolated from vaginal infections. Peptides. 2015;71:211–21. Maurya IK, Thota CK, Sharma J, Tupe SG, Chaudhary P, Singh MK, et al. Mechanism of action of novel synthetic dodecapeptides against Candida albicans. Biochim Biophys Acta - Gen Subj. 2013;1830(11):5193–203. Chang H-T, Tsai P-W, Huang H-H, Liu Y-S, Chien T-S, Lan C-Y. LL37 and hBD-3 elevate the β-1,3-exoglucanase activity of Candida albicans Xog1p, resulting in reduced fungal adhesion to plastic. Biochem J. 2012;441(3):963– 70. Kabir MA, Hussain MA, Ahmad Z. Candida albicans : A Model Organism for Studying Fungal Pathogens. 2012;2012. Cassone A. Vulvovaginal Candida albicans infections: Pathogenesis, immunity and vaccine prospects. BJOG An Int J Obstet Gynaecol. 2015;122(6):785–94. Netea MG, Brown GD, Kullberg BJ, Gow NAR. An integrated model of the recognition of Candida albicans by the innate immune system. Nat Rev Microbiol. 2008;6(1):67–78. Shibata N, Kobayashi H, Suzuki S. Immunochemistry of pathogenic yeast, Candida species, focusing on mannan. Proc Jpn Acad Ser B Phys Biol Sci. 2012;88(6):250–65. Mothibe J V., Patel M. Pathogenic characteristics of Candida albicans isolated from oral cavities of denture wearers and cancer patients wearing oral prostheses. Microb Pathog. 2017;110:128–34. Luis J, Pozo D. Revista Iberoamericana de Micología Candidiasis asociada a biopelículas. 2016;33(3):176–83. Nobile CJ, Fox EP, Nett JE, Sorrells TR, Mitrovich QM, Hernday AD, et al. A recently evolved transcriptional network controls biofilm development in Candida albicans. Cell. 2012;148(1–2):126–38. Rajendran R, Sherry L, Nile CJ, Sherriff A, Johnson EM, Hanson MF, et al. Biofilm formation is a risk factor for mortality in patients with Candida albicans bloodstream infection-Scotland, 2012-2013. Clin Microbiol Infect. 2016;22(1):87–93. Horn DL, Neofytos D, Anaissie EJ, Fishman JA, Steinbach WJ, Olyaei AJ, et al. Epidemiology and Outcomes of Candidemia in 2019 Patients : Data from the Prospective Antifungal Therapy Alliance Registry. 2019;48(August 2018):1695–703. McCarty TP, Pappas PG. Invasive Candidiasis. Infect Dis Clin North Am. 2016;30(1):103–24. Clancy CJ, Nguyen MH. Finding the missing 50% of invasive candidiasis: How nonculture diagnostics will improve understanding of disease spectrum and transform patient care. Clin Infect Dis. 2013;56(9):1284–92. Kaaniche FM, Allela R, Cherif S, Algia N ben. Invasive candidiasis in critically ill patients. Trends Anaesth Crit Care. 2016;11:1–5. Valdivielso-Ramos M, Mauleón C. Diagnóstico diferencial de la candidiasis sistémica. Piel. 2012;27(10):581–90. Sauget M, Valot B, Bertrand X, Hocquet D. Can MALDI-TOF Mass Spectrometry Reasonably Type Bacteria ? Trends Microbiol [Internet]. 2016;xx:1–9. Available from: http://dx.doi.org/10.1016/j.tim.2016.12.006 Iavazzo C, Gkegkes ID, Zarkada IM, Falagas ME. Boric Acid for Recurrent Vulvovaginal Candidiasis : 2011;20(8). Tardif KD, Schlaberg R. Development of a real-time PCR assay for the direct detection of Candida species causing Vulvovaginal candidiasis. Diagn Microbiol Infect Dis. 2017;88(1):39–40. Sun M-G, Huang Y, Xu Y-H, Cao Y-X. Efficacy of vitamin B complex as an adjuvant therapy for the treatment of complicated vulvovaginal candidiasis: An in vivo and in vitro study. Biomed Pharmacother. 2017;88:770–7. Blostein F, Levin E, Wagner J, Foxman B. Recurrent Vulvovaginal Candidiasis,. Ann Epidemiol. 2017; Authors T, Munksgaard B. Oral mucosal fungal infections. 2009;49:39–59. Millsop JW, Fazel N. Oral candidiasis. Vol. 34, Clinics in Dermatology. Elsevier B.V.; 2016. 487-494 p. Khullar G, Vignesh P, Lau YL, Rudramurthy SM, Rawat A, De D, et al. Chronic Mucocutaneous Candidiasis. J Allergy Clin Immunol Pract. 2017;5(4):1119–21. Moriyama B, Gordon LA, Mccarthy M, Stacey A, Walsh TJ, Penzak SR. NIH Public Access. 2015;57(12):718–33. Warrilow AG, Parker JE, Kelly DE, Kelly SL. Candida albicans and Homo sapiens. 2013;57(3):1352–60. Emami S, Tavangar P, Keighobadi M. An overview of azoles targeting sterol 14α-demethylase for antileishmanial therapy. Eur J Med Chem. 2017;135:241–59. Azanza JR, Montejo M. Farmacocinética y farmacodinamia. Interacciones y efectos secundarios. Comparación con otras equinocandinas. Enferm Infecc Microbiol Clin. 2008;26(SUPPL. 14):14–20. Kinoshita H, Yoshioka M, Ihara F, Nihira T. Cryptic antifungal compounds active by synergism with polyene antibiotics. J Biosci Bioeng. 2016;121(4):394–8. Perea A. Anfotericina B forma liposómica : un perfil farmacocinético exclusivo . Una historia inacabada . 2012;25(1):17–24. Candida D, De E, Porte L, León P, Gárate C, Guzmán AM, et al. Susceptibilidad a azoles y anfotericina B de aislados de. 2010;149–55. Brilhante RSN, Paiva MAN, Sampaio MS, Teixeira CEC. Macrobrachium amazonicum: 2011;76:268–77. Brilhante RSN, Paiva MAN, Sampaio CMS, Castelo-Branco DSCM, Teixeira CEC, de Alencar LP, et al. Azole resistance in Candida spp. isolated from Catú Lake, Ceará, Brazil: An efflux-pump-mediated mechanism. Brazilian J Microbiol. 2016;47(1):33–8. Morace G, Perdoni F, Borghi E. Antifungal drug resistance in Candida species. J Glob Antimicrob Resist. 2014;2(4):254–9. Fernandes T, Silva S, Henriques M. Candida tropicalis biofilm’s matrix- involvement on its resistance to amphotericin B. Diagn Microbiol Infect Dis. 2015;83(2):165–9. Téllez GA, Castaño JC. Péptidos antimicrobianos. Grup Inmunol Mol. 2010;14(1):55–67. Rossi DC, Munoz JE, Carvalho DD, Belmonte R, Faintuch B, Borelli P, et al. Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis. BMC Microbiol. 2012;12(1):28. Zhu M, Liu P, Niu ZW. A perspective on general direction and challenges facing antimicrobial peptides. Chinese Chem Lett. 2016; Ageitos JM, S??nchez-P??rez A, Calo-Mata P, Villa TG. Antimicrobial peptides (AMPs): Ancient compounds that represent novel weapons in the fight against bacteria. Biochem Pharmacol. 2016; Fabisiak A, Murawska N, Fichna J. LL-37: Cathelicidin-related antimicrobial peptide with pleiotropic activity. Pharmacol Reports. 2016;68(4):802–8. Vandamme D, Landuyt B, Luyten W, Schoofs L. A comprehensive summary of LL-37, the factoctum human cathelicidin peptide. Cell Immunol. 2012;280(1):22–35. Wang G. Structures of Human Host Defense Cathelicidin LL-37 and Its Smallest Antimicrobial Peptide KR-12 in Lipid Micelles * □. 2008; Malmsten M. Interactions of Antimicrobial Peptides with Bacterial Membranes and Membrane Components. 2016;16–24. Wong JH, Ng TB, Legowska A, Rolka K, Hui M, Cho CH. Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans. Peptides. 2011;32(10):1996–2002. Nobile CJ, Johnsonb AD. Candida albicans biofilms and human disease. Annu Rev Microbiol. 2015;69:71–92. Hamid S, Zainab S, Faryal R, Ali N, Sharafat I. Inhibition of secreted aspartyl proteinase activity in biofilms of Candida species by mycogenic silver nanoparticles. Artif Cells, Nanomedicine Biotechnol. 2017;(2016):1–7. Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Adherence and biofilm formation of non-Candida albicans Candida species. Trends Microbiol. 2011;19(5):241–7. Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, Cormick TMC, Ghannoum MA. Biofilm Formation by the Fungal Pathogen Candida albicans : Development , Architecture , and Drug Resistance. 2001;183(18):5385–94. Tanaka M, Takamura Y, Kawakami T, Aimoto S, Saito H. Effect of amino acid distribution of amphipathic helical peptide derived from human apolipoprotein A-I on membrane curvature sensing. FEBS Lett [Internet]. 2013;587(5):510–5. Hollmann A, Martínez M, Noguera ME, Augusto MT, Disalvo A, Santos NC, et al. Colloids and Surfaces B : Biointerfaces Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide – membrane interactions of three related antimicrobial peptides. Colloids Surfaces B Biointerfaces [Internet]. 2016;141:528–36. Nuijens T, Piva E, Kruijtzer JAW, Rijkers DTS, Liskamp RMJ, Quaedflieg PJLM. Enzymatic C-terminal amidation of amino acids and peptides. Tetrahedron Lett [Internet]. 2012;53(29):3777–9. Alvares DS, Wilke N, Ruggiero J. BBA - Biomembranes E ff ect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide. BBA - Biomembr [Internet]. 2018;1860(3):737–48. Aminoácidos y péptidos 1 . :49–62. Tsai P, Cheng Y, Hsieh W, Lan C. Responses of Candida albicans to the Human Antimicrobial Peptide LL-37. 2014;52(8). Guirao-abad JP, Sánchez-fresneda R, Alburquerque B, Hernández JA, Argüelles J. Ac ce p cr t. Int J Med Microbiol [Internet]. 2017; Touil HFZ, Boucherit-otmani Z, Boucherit K. ScienceDirect In vitro activity of antifungal combinations against planktonic and sessile cells of Candida albicans isolated from medical devices in an intensive care department. J Mycol Med [Internet]. 2018;6–10. C LM, Novoa C, Carlos M. Uso de anfotericina B deoxicolato y sus reacciones adversas en un hospital universitario en Chile. 2007; |
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Derechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2018 |
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https://creativecommons.org/licenses/by-nc-sa/4.0/ |
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Atribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0) |
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Derechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2018 https://creativecommons.org/licenses/by-nc-sa/4.0/ Atribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0) http://purl.org/coar/access_right/c_14cb |
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Facultad de Ciencias de la Salud |
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Bogotá D.C |
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Bacteriología y Laboratorio Clínico |
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Muñoz Henao, Julián Esteban8c3a047deeb142fcc831a877d6096885Pinilla Bermúdez, Gladys428561c7b44bd4a7c33f25d8f79d473cChaves Alfonso, Gabriel German14d8b07be89c1bbe1a22d1264e42af0eCoronado Ladino, Yenifer Tatiana6b471d61abd250de4d725113be53b4d02021-11-23T19:59:21Z2021-11-23T19:59:21Z2019-01https://repositorio.unicolmayor.edu.co/handle/unicolmayor/371458675La candidiasis es una de las infecciones más frecuentes en pacientes hospitalizados o con alteraciones en su sistema inmunológico, actualmente se ha convertido en una problemática creciente debido a la resistencia adquirida por las levaduras del género Candida a diferentes antifúngicos utilizados en el tratamiento de esta micosis. Como alternativa terapéutica han sido estudiados en las últimas décadas varios péptidos antimicrobianos con el fin de investigar nuevos tratamientos de amplio espectro, especialmente para levaduras productoras de biopelículas. Este es el caso de Candida albicans, una levadura comensal que hace parte del microbiota del ser humano, pero que en pacientes inmunosuprimidos puede causar infecciones cutáneas, subcutáneas y sistémicas, con el agravante de presentar resistencia en algunos casos a la terapia convencionalmente usada para su tratamiento. Los péptidos antimicrobianos están presentes en animales, plantas y otros microorganismos, haciendo parte del sistema inmune innato. La catelicidina humana LL37, péptido antimicrobiano catiónico, es una molécula que contiene tanto un dominio catelina como un dominio C-terminal antimicrobiano. Basados en lo anterior, se realizó un estudio de tipo cuantitativo; estudios descriptivos y explicativos en donde se utilizaron tres cepas, dos de ellas correspondientes a Candida albicans y una correspondiente a una cepa proveniente de candidiasis recurrente, probando en cada una de ellas cinco concentraciones distintas de péptidos análogos de LL-37 como: AC-1, LL-37,1, AC-2 y D, evidenciando su poder inhibitorio mediante las curvas de crecimiento obtenidas en el equipo BioScreen C. Teniendo en cuenta los cuatro péptidos utilizados, se demostró que los péptidos AC-1 y D evidenciaron una actividad inhibitoria significativa en las tres cepas incluidas en el estudio; con respecto a los otros péptidos, cabe resaltar que LL-37,1 tuvo una excelente capacidad inhibitoria en la cepa Candida albicans 256.1. INTRODUCCIÓN 18 2. OBJETIVOS 22 2.1) OBJETIVO GENERAL 22 2.2) OBJETIVOS ESPECÍFICOS 22 3. JUSTIFICACIÓN 23 4. ANTECEDENTES 25 4.1) ESTADO DEL ARTE 25 5. MARCO CONCEPTUAL 27 5.1) GENERALIDADES DE LEVADURAS DEL GÉNERO Candida 27 PATOLOGÍAS OCASIONADAS POR Candida spp 28 5.2) OTROS TIPOS DE CANDIDIASIS 30 5.3) CANDIDIASIS VULVOVAGINAL 30 5.4) CANDIDIASIS ORAL Y MUCOCUTANEA CRONICA 31 5.5) ANTIFÚNGICOS CONTRA LAS CANDIDIASIS 32 5.5.1) Azoles 32 5.5.2) Equinocandinas 33 5.5.3) Polienos 33 5.6) RESISTENCIA A LOS ANTIFÚNGICOS DE Candida spp 34 5.7) PÉPTIDOS ANTIMICROBIANOS 35 5.8) BIOPELÍCULAS EN LEVADURAS DEL GÉNERO Candida 40 6. DISEÑO METODOLÓGICO 43 6.1) UNIVERSO, POBLACIÓN Y MUESTRA 43 6.2) MATERIALES Y MÉTODOS 43 6.3) ANÁLISIS ESTADÍSTICO 43 6.4) DETERMINACIÓN DE FASES DE CRECIMIENTO PARA C. albicans 256, Candida FVF, C. albicans SC5314. 44 6.5) CURVAS DE CRECIMIENTO UTILIZANDO LOS PÉPTIDOS (AC-1, LL-37-1, AC-2 Y D). 44 6.6) CONTROL PARA C. albicans 256, C. albicans SC5314 y Candida FVF. 45 7. RESULTADOS 47 7.1) FASES DE CRECIMIENTO 47 7.2) CURVAS DE CRECIMIENTO UTILIZANDO LOS PÉPTIDOS ANTIMICROBIANOS 49 8. DISCUSIÓN 57 9. CONCLUSIONES 62 10. REFERENCIASPregradoBacteriólogo(a) y Laboratorista Clínico75p.application/pdfspaUniversidad Colegio Mayor de CundinamarcaFacultad de Ciencias de la SaludBogotá D.CBacteriología y Laboratorio ClínicoDerechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2018https://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/closedAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_14cbActividad antifúngica in Vitro de péptidos análogos derivados de LL-37 contra levaduras del género CandidaTrabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fhttp://purl.org/coar/version/c_970fb48d4fbd8a85Textinfo:eu-repo/semantics/bachelorThesishttps://purl.org/redcol/resource_type/TPinfo:eu-repo/semantics/publishedVersionAlvarez-moreno CA. Burden of Fungal Infections in Colombia. 2018;1–13.Merseguel KB, Nishikaku AS, Rodrigues AM, Padovan AC, Carmona R, Salles A, et al. Genetic diversity of medically important and emerging Candida species causing invasive infection. 2015;1–11.Papon N, Courdavault V, Clastre M, Bennett RJ, Nac N-C. Emerging and Emerged Pathogenic Candida Species : Beyond the Candida albicans Paradigm. 2013;9(9).Whibley N, Gaffen SL. Beyond Candida albicans: Mechanisms of immunity to non-albicans Candida species. Cytokine. 2015;76(1):42–52.Pfaller MA, Messer SA, Jones RN, Castanheira M. Antifungal susceptibilities of Candida , Cryptococcus neoformans and Aspergillus fumigatus from the Asia and Western Paci fi c region : data from the SENTRY antifungal surveillance program ( 2010 – 2012 ). 2015;(November 2014):1–6. Available from: http://dx.doi.org/10.1038/ja.2015.29Pfaller MA. Antifungal Drug Resistance : Mechanisms , Epidemiology , and Consequences for Treatment. AJM [Internet]. 2012;125(1):S3–13. Available from: http://dx.doi.org/10.1016/j.amjmed.2011.11.001Pfaller MA, Moet GJ, Messer SA, Jones RN, Castanheira M. Geographic Variations in Species Distribution and Echinocandin and Azole Antifungal Resistance Rates among Candida Bloodstream Infection Isolates : Report from the SENTRY Antimicrobial Surveillance Program ( 2008 to 2009 ) ᰔ. 2011;49(1):396–9.Tema DE. Péptidos antimicrobianos. 2010;14(1):55–67.de la Fuente-Núñez C, Silva ON, Lu TK, Franco OL. Antimicrobial peptides: Role in human disease and potential as immunotherapies. Pharmacol Ther. 2017;Tsai PW, Yang CY, Chang HT, Lan CY. Human antimicrobial peptide LL-37 inhibits adhesion of Candida albicans by interacting with yeast cell-wall carbohydrates. PLoS One. 2011;6(3).Carrascosa JM. Mecanismo De acción De ustekinumab y su relevancia en la patogénesis De la psoriasis. Impacto en el sistema inmune. Actas Dermosifiliogr. 2012;103(SUPPL. 2):7–15.Antinori S, Milazzo L, Sollima S, Galli M, Corbellino M. European Journal of Internal Medicine Candidemia and invasive candidiasis in adults : A narrative review. Eur J Intern Med [Internet]. 2016; Available from: http://dx.doi.org/10.1016/j.ejim.2016.06.029Blumberg HM, Jarvis WR, Soucie JM, Edwards JE, Patterson JE, Pfaller MA, et al. Risk Factors for Candidal Bloodstream Infections in Surgical Intensive Care Unit Patients : The NEMIS Prospective Multicenter Study. 2001;6:177– 86.Giongo JL, de Almeida Vaucher R, Fausto VP, Quatrin PM, Lopes LQS, Santos RCV, et al. Anti-Candida activity assessment of Pelargonium graveolens oil free and nanoemulsion in biofilm formation in hospital medical supplies. Microb Pathog. 2016;100:170–8.Kodedová M, Sychrová H. Synthetic antimicrobial peptides of the halictines family disturb the membrane integrity of Candida cells. Biochim Biophys Acta - Biomembr. 2017;1859(10):1851–8.Scarsini M, Tomasinsig L, Arzese A, D’Este F, Oro D, Skerlavaj B. Antifungal activity of cathelicidin peptides against planktonic and biofilm cultures of Candida species isolated from vaginal infections. Peptides. 2015;71:211–21.Maurya IK, Thota CK, Sharma J, Tupe SG, Chaudhary P, Singh MK, et al. Mechanism of action of novel synthetic dodecapeptides against Candida albicans. Biochim Biophys Acta - Gen Subj. 2013;1830(11):5193–203.Chang H-T, Tsai P-W, Huang H-H, Liu Y-S, Chien T-S, Lan C-Y. LL37 and hBD-3 elevate the β-1,3-exoglucanase activity of Candida albicans Xog1p, resulting in reduced fungal adhesion to plastic. Biochem J. 2012;441(3):963– 70.Kabir MA, Hussain MA, Ahmad Z. Candida albicans : A Model Organism for Studying Fungal Pathogens. 2012;2012.Cassone A. Vulvovaginal Candida albicans infections: Pathogenesis, immunity and vaccine prospects. BJOG An Int J Obstet Gynaecol. 2015;122(6):785–94.Netea MG, Brown GD, Kullberg BJ, Gow NAR. An integrated model of the recognition of Candida albicans by the innate immune system. Nat Rev Microbiol. 2008;6(1):67–78.Shibata N, Kobayashi H, Suzuki S. Immunochemistry of pathogenic yeast, Candida species, focusing on mannan. Proc Jpn Acad Ser B Phys Biol Sci. 2012;88(6):250–65.Mothibe J V., Patel M. Pathogenic characteristics of Candida albicans isolated from oral cavities of denture wearers and cancer patients wearing oral prostheses. Microb Pathog. 2017;110:128–34.Luis J, Pozo D. Revista Iberoamericana de Micología Candidiasis asociada a biopelículas. 2016;33(3):176–83.Nobile CJ, Fox EP, Nett JE, Sorrells TR, Mitrovich QM, Hernday AD, et al. A recently evolved transcriptional network controls biofilm development in Candida albicans. Cell. 2012;148(1–2):126–38.Rajendran R, Sherry L, Nile CJ, Sherriff A, Johnson EM, Hanson MF, et al. Biofilm formation is a risk factor for mortality in patients with Candida albicans bloodstream infection-Scotland, 2012-2013. Clin Microbiol Infect. 2016;22(1):87–93.Horn DL, Neofytos D, Anaissie EJ, Fishman JA, Steinbach WJ, Olyaei AJ, et al. Epidemiology and Outcomes of Candidemia in 2019 Patients : Data from the Prospective Antifungal Therapy Alliance Registry. 2019;48(August 2018):1695–703.McCarty TP, Pappas PG. Invasive Candidiasis. Infect Dis Clin North Am. 2016;30(1):103–24.Clancy CJ, Nguyen MH. Finding the missing 50% of invasive candidiasis: How nonculture diagnostics will improve understanding of disease spectrum and transform patient care. Clin Infect Dis. 2013;56(9):1284–92.Kaaniche FM, Allela R, Cherif S, Algia N ben. Invasive candidiasis in critically ill patients. Trends Anaesth Crit Care. 2016;11:1–5.Valdivielso-Ramos M, Mauleón C. Diagnóstico diferencial de la candidiasis sistémica. Piel. 2012;27(10):581–90.Sauget M, Valot B, Bertrand X, Hocquet D. Can MALDI-TOF Mass Spectrometry Reasonably Type Bacteria ? Trends Microbiol [Internet]. 2016;xx:1–9. Available from: http://dx.doi.org/10.1016/j.tim.2016.12.006Iavazzo C, Gkegkes ID, Zarkada IM, Falagas ME. Boric Acid for Recurrent Vulvovaginal Candidiasis : 2011;20(8).Tardif KD, Schlaberg R. Development of a real-time PCR assay for the direct detection of Candida species causing Vulvovaginal candidiasis. Diagn Microbiol Infect Dis. 2017;88(1):39–40.Sun M-G, Huang Y, Xu Y-H, Cao Y-X. Efficacy of vitamin B complex as an adjuvant therapy for the treatment of complicated vulvovaginal candidiasis: An in vivo and in vitro study. Biomed Pharmacother. 2017;88:770–7.Blostein F, Levin E, Wagner J, Foxman B. Recurrent Vulvovaginal Candidiasis,. Ann Epidemiol. 2017;Authors T, Munksgaard B. Oral mucosal fungal infections. 2009;49:39–59.Millsop JW, Fazel N. Oral candidiasis. Vol. 34, Clinics in Dermatology. Elsevier B.V.; 2016. 487-494 p.Khullar G, Vignesh P, Lau YL, Rudramurthy SM, Rawat A, De D, et al. Chronic Mucocutaneous Candidiasis. J Allergy Clin Immunol Pract. 2017;5(4):1119–21.Moriyama B, Gordon LA, Mccarthy M, Stacey A, Walsh TJ, Penzak SR. NIH Public Access. 2015;57(12):718–33.Warrilow AG, Parker JE, Kelly DE, Kelly SL. Candida albicans and Homo sapiens. 2013;57(3):1352–60.Emami S, Tavangar P, Keighobadi M. An overview of azoles targeting sterol 14α-demethylase for antileishmanial therapy. Eur J Med Chem. 2017;135:241–59.Azanza JR, Montejo M. Farmacocinética y farmacodinamia. Interacciones y efectos secundarios. Comparación con otras equinocandinas. Enferm Infecc Microbiol Clin. 2008;26(SUPPL. 14):14–20.Kinoshita H, Yoshioka M, Ihara F, Nihira T. Cryptic antifungal compounds active by synergism with polyene antibiotics. J Biosci Bioeng. 2016;121(4):394–8.Perea A. Anfotericina B forma liposómica : un perfil farmacocinético exclusivo . Una historia inacabada . 2012;25(1):17–24.Candida D, De E, Porte L, León P, Gárate C, Guzmán AM, et al. Susceptibilidad a azoles y anfotericina B de aislados de. 2010;149–55.Brilhante RSN, Paiva MAN, Sampaio MS, Teixeira CEC. Macrobrachium amazonicum: 2011;76:268–77.Brilhante RSN, Paiva MAN, Sampaio CMS, Castelo-Branco DSCM, Teixeira CEC, de Alencar LP, et al. Azole resistance in Candida spp. isolated from Catú Lake, Ceará, Brazil: An efflux-pump-mediated mechanism. Brazilian J Microbiol. 2016;47(1):33–8.Morace G, Perdoni F, Borghi E. Antifungal drug resistance in Candida species. J Glob Antimicrob Resist. 2014;2(4):254–9.Fernandes T, Silva S, Henriques M. Candida tropicalis biofilm’s matrix- involvement on its resistance to amphotericin B. Diagn Microbiol Infect Dis. 2015;83(2):165–9.Téllez GA, Castaño JC. Péptidos antimicrobianos. Grup Inmunol Mol. 2010;14(1):55–67.Rossi DC, Munoz JE, Carvalho DD, Belmonte R, Faintuch B, Borelli P, et al. Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis. BMC Microbiol. 2012;12(1):28.Zhu M, Liu P, Niu ZW. A perspective on general direction and challenges facing antimicrobial peptides. Chinese Chem Lett. 2016;Ageitos JM, S??nchez-P??rez A, Calo-Mata P, Villa TG. Antimicrobial peptides (AMPs): Ancient compounds that represent novel weapons in the fight against bacteria. Biochem Pharmacol. 2016;Fabisiak A, Murawska N, Fichna J. LL-37: Cathelicidin-related antimicrobial peptide with pleiotropic activity. Pharmacol Reports. 2016;68(4):802–8.Vandamme D, Landuyt B, Luyten W, Schoofs L. A comprehensive summary of LL-37, the factoctum human cathelicidin peptide. Cell Immunol. 2012;280(1):22–35.Wang G. Structures of Human Host Defense Cathelicidin LL-37 and Its Smallest Antimicrobial Peptide KR-12 in Lipid Micelles * □. 2008;Malmsten M. Interactions of Antimicrobial Peptides with Bacterial Membranes and Membrane Components. 2016;16–24.Wong JH, Ng TB, Legowska A, Rolka K, Hui M, Cho CH. Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans. Peptides. 2011;32(10):1996–2002.Nobile CJ, Johnsonb AD. Candida albicans biofilms and human disease. Annu Rev Microbiol. 2015;69:71–92.Hamid S, Zainab S, Faryal R, Ali N, Sharafat I. Inhibition of secreted aspartyl proteinase activity in biofilms of Candida species by mycogenic silver nanoparticles. Artif Cells, Nanomedicine Biotechnol. 2017;(2016):1–7.Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Adherence and biofilm formation of non-Candida albicans Candida species. Trends Microbiol. 2011;19(5):241–7.Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, Cormick TMC, Ghannoum MA. Biofilm Formation by the Fungal Pathogen Candida albicans : Development , Architecture , and Drug Resistance. 2001;183(18):5385–94.Tanaka M, Takamura Y, Kawakami T, Aimoto S, Saito H. Effect of amino acid distribution of amphipathic helical peptide derived from human apolipoprotein A-I on membrane curvature sensing. FEBS Lett [Internet]. 2013;587(5):510–5.Hollmann A, Martínez M, Noguera ME, Augusto MT, Disalvo A, Santos NC, et al. Colloids and Surfaces B : Biointerfaces Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide – membrane interactions of three related antimicrobial peptides. Colloids Surfaces B Biointerfaces [Internet]. 2016;141:528–36.Nuijens T, Piva E, Kruijtzer JAW, Rijkers DTS, Liskamp RMJ, Quaedflieg PJLM. Enzymatic C-terminal amidation of amino acids and peptides. Tetrahedron Lett [Internet]. 2012;53(29):3777–9.Alvares DS, Wilke N, Ruggiero J. BBA - Biomembranes E ff ect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide. BBA - Biomembr [Internet]. 2018;1860(3):737–48.Aminoácidos y péptidos 1 . :49–62.Tsai P, Cheng Y, Hsieh W, Lan C. Responses of Candida albicans to the Human Antimicrobial Peptide LL-37. 2014;52(8).Guirao-abad JP, Sánchez-fresneda R, Alburquerque B, Hernández JA, Argüelles J. Ac ce p cr t. Int J Med Microbiol [Internet]. 2017;Touil HFZ, Boucherit-otmani Z, Boucherit K. ScienceDirect In vitro activity of antifungal combinations against planktonic and sessile cells of Candida albicans isolated from medical devices in an intensive care department. J Mycol Med [Internet]. 2018;6–10.C LM, Novoa C, Carlos M. Uso de anfotericina B deoxicolato y sus reacciones adversas en un hospital universitario en Chile. 2007;MicroorganismosDominio C-terminal antimicrobianoCepasCandida albicansPéptido antimicrobianoCurvas de crecimientoCatelicidina humana LL37ORIGINALTesis Final 1.0.pdfTesis Final 1.0.pdfapplication/pdf1293031https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3714/1/Tesis%20Final%201.0.pdf2788c3fd6c6bcb24d63c2310792e5dadMD51open accessTesis Final 1.0 Cartas.pdfTesis Final 1.0 Cartas.pdfapplication/pdf165241https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3714/2/Tesis%20Final%201.0%20Cartas.pdf6447d839328fdd4255c60d8cc7ce184fMD52metadata only accessDIAPOSITIVAS TESIS (1).pdfDIAPOSITIVAS TESIS (1).pdfapplication/pdf2036100https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3714/3/DIAPOSITIVAS%20TESIS%20%281%29.pdfe36d52e335446df725672b08a1d2e35bMD53open accessLICENSElicense.txtlicense.txttext/plain; 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Unicolmayorrepositorio@unicolmayor.edu.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