Actividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogos
ilustraciones, diagramas
- Autores:
-
Botina Rodríguez, Anyelly Tatiana
- Tipo de recurso:
- Fecha de publicación:
- 2023
- Institución:
- Universidad Nacional de Colombia
- Repositorio:
- Universidad Nacional de Colombia
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unal.edu.co:unal/85602
- Palabra clave:
- 570 - Biología::572 - Bioquímica
LfcinB
Actividad antimicrobiana
Escherichia coli
Candida tropicalis
Péptidos antimicrobianos
LfcinB
Antimicrobial peptides
Antimicrobial activity
Escherichia coli
Candida tropicalis
Microbiología
Bacteria
Tratamiento médico
Microbiology
Bacteria
Medical treatment
- Rights
- openAccess
- License
- Atribución-NoComercial 4.0 Internacional
| id |
UNACIONAL2_c593ac1db9cd9348fc603e886aafd58e |
|---|---|
| oai_identifier_str |
oai:repositorio.unal.edu.co:unal/85602 |
| network_acronym_str |
UNACIONAL2 |
| network_name_str |
Universidad Nacional de Colombia |
| repository_id_str |
|
| dc.title.spa.fl_str_mv |
Actividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogos |
| dc.title.translated.eng.fl_str_mv |
Antimicrobian activity of LfcinB (21-25)Pal RWQWRWQWR palindromic peptid and it analogues |
| title |
Actividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogos |
| spellingShingle |
Actividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogos 570 - Biología::572 - Bioquímica LfcinB Actividad antimicrobiana Escherichia coli Candida tropicalis Péptidos antimicrobianos LfcinB Antimicrobial peptides Antimicrobial activity Escherichia coli Candida tropicalis Microbiología Bacteria Tratamiento médico Microbiology Bacteria Medical treatment |
| title_short |
Actividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogos |
| title_full |
Actividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogos |
| title_fullStr |
Actividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogos |
| title_full_unstemmed |
Actividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogos |
| title_sort |
Actividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogos |
| dc.creator.fl_str_mv |
Botina Rodríguez, Anyelly Tatiana |
| dc.contributor.advisor.spa.fl_str_mv |
Rivera Monroy, Zuly Jenny Parra Giraldo, Claudia Marcela |
| dc.contributor.author.spa.fl_str_mv |
Botina Rodríguez, Anyelly Tatiana |
| dc.subject.ddc.spa.fl_str_mv |
570 - Biología::572 - Bioquímica |
| topic |
570 - Biología::572 - Bioquímica LfcinB Actividad antimicrobiana Escherichia coli Candida tropicalis Péptidos antimicrobianos LfcinB Antimicrobial peptides Antimicrobial activity Escherichia coli Candida tropicalis Microbiología Bacteria Tratamiento médico Microbiology Bacteria Medical treatment |
| dc.subject.proposal.spa.fl_str_mv |
LfcinB Actividad antimicrobiana Escherichia coli Candida tropicalis Péptidos antimicrobianos |
| dc.subject.proposal.eng.fl_str_mv |
LfcinB Antimicrobial peptides Antimicrobial activity Escherichia coli Candida tropicalis |
| dc.subject.unesco.spa.fl_str_mv |
Microbiología Bacteria Tratamiento médico |
| dc.subject.unesco.eng.fl_str_mv |
Microbiology Bacteria Medical treatment |
| description |
ilustraciones, diagramas |
| publishDate |
2023 |
| dc.date.issued.none.fl_str_mv |
2023 |
| dc.date.accessioned.none.fl_str_mv |
2024-02-02T15:43:43Z |
| dc.date.available.none.fl_str_mv |
2024-02-02T15:43:43Z |
| dc.type.spa.fl_str_mv |
Trabajo de grado - Maestría |
| dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/masterThesis |
| dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/acceptedVersion |
| dc.type.content.spa.fl_str_mv |
Text |
| dc.type.redcol.spa.fl_str_mv |
http://purl.org/redcol/resource_type/TM |
| status_str |
acceptedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://repositorio.unal.edu.co/handle/unal/85602 |
| dc.identifier.instname.spa.fl_str_mv |
Universidad Nacional de Colombia |
| dc.identifier.reponame.spa.fl_str_mv |
Repositorio Institucional Universidad Nacional de Colombia |
| dc.identifier.repourl.spa.fl_str_mv |
https://repositorio.unal.edu.co/ |
| url |
https://repositorio.unal.edu.co/handle/unal/85602 https://repositorio.unal.edu.co/ |
| identifier_str_mv |
Universidad Nacional de Colombia Repositorio Institucional Universidad Nacional de Colombia |
| dc.language.iso.spa.fl_str_mv |
spa |
| language |
spa |
| dc.relation.indexed.spa.fl_str_mv |
Bireme |
| dc.relation.references.spa.fl_str_mv |
Ángel Serra Valdés M, Cabrera E, Habana Cuba maserra L. Ciencias epidemioló gicas y salubristas. La resistencia microbiana en el contexto actual y la importancia del conocimiento y aplicación en la política antimicrobiana Microbial resistance in the current context and the importance of knowledge and application in antimicrobial policy [Internet]. Available from: http://www.revhabanera.sld.cu/index.php/rhab/article/view/2013 Vivas R, Barbosa AAT, Dolabela SS, Jain S. Multidrug-Resistant Bacteria and Alternative Methods to Control Them: An Overview. Microbial Drug Resistance. 2019 Jul 1;25(6):890–908. OMS, PLAN DE ACCIÓN MUNDIAL SOBRE LA RESISTENCIA A LOS ANTIMICROBIANOS. Luo Y, Song Y. Mechanism of antimicrobial peptides: Antimicrobial, antiinflammatory and antibiofilm activities. Vol. 22, International Journal of Molecular Sciences. MDPI; 2021. Freitas ED, Bataglioli RA, Oshodi J, Beppu MM. Antimicrobial peptides and their potential application in antiviral coating agents. Vol. 217, Colloids and Surfaces B: Biointerfaces. Elsevier B.V.; 2022. Tomita M, Wakabayashi H, Yamauchi K, Teraguchi S, Hayasawa H. Bovine lactoferrin and lactoferricin derived from milk: Production and applications. In: Biochemistry and Cell Biology. 2002. p. 109–12. Muñoz A, Marcos JF. Activity and mode of action against fungal phytopathogens of bovine lactoferricin-derived peptides. J Appl Microbiol. 2006 Dec;101(6):1199–207. Sun C, Li Y, Cao S, Wang H, Jiang C, Pang S, et al. Antibacterial activity and mechanism of action of bovine lactoferricin derivatives with symmetrical amino acid sequences. Int J Mol Sci. 2018 Oct 1;19(10). León-Calvijo MA, Leal-Castro AL, Almanzar-Reina GA, Rosas-Pérez JE, García-Castañeda JE, Rivera-Monroy ZJ. Antibacterial activity of synthetic peptides derived from lactoferricin against Escherichia coli ATCC 25922 and Enterococcus Faecalis ATCC 29212. Biomed Res Int. 2015;2015. Vargas-Casanova Y, Rodríguez-Mayor AV, Cardenas KJ, Leal-Castro AL, Muñoz-Molina LC, Fierro-Medina R, et al. Synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the RWQWR motif against Gram-positive and Gram-negative strains. RSC Adv. 2019;9(13):7239–45. Ardila-Chantré N, Hernández-Cardona AK, Pineda-Castañeda HM, Estupiñan-Torres SM, Leal-Castro AL, Fierro-Medina R, et al. Short peptides conjugated to non-peptidic motifs exhibit antibacterial activity. RSC Adv. 2020 Aug 10;10(49):29580–6. Wang B, Timilsena YP, Blanch E, Adhikari B. Lactoferrin: Structure, function, denaturation and digestion. Vol. 59, Critical Reviews in Food Science and Nutrition. Taylor and Francis Inc.; 2019. p. 580–96. Karav S, German JB, Rouquié C, le Parc A, Barile D. Studying lactoferrin N-glycosylation. Vol. 18, International Journal of Molecular Sciences.MDPI AG; 2017. Antoshin AA, Shpichka AI, Huang G, Chen K, Lu P, Svistunov AA, et al. Lactoferrin as a regenerative agent: The old-new panacea? Vol. 167, Pharmacological Research. Academic Press; 2021. Fernandes KE, Carter DA. The antifungal activity of lactoferrin and its derived peptides: Mechanisms of action and synergy with drugs against fungal pathogens. Vol. 8, Frontiers in Microbiology. Frontiers Research Foundation; 2017. Tomita M, Wakabayashi H, Yamauchi K, Teraguchi S, Hayasawa H. Bovine lactoferrin and lactoferricin derived from milk: Production and applications. In: Biochemistry and Cell Biology. 2002. p. 109–12. Ardila-Chantré N, Hernández-Cardona AK, Pineda-Castañeda HM, Estupiñan-Torres SM, Leal-Castro AL, Fierro-Medina R, et al. Short peptides conjugated to non-peptidic motifs exhibit antibacterial activity. RSC Adv. 2020 Aug 10;10(49):29580–6. León-Calvijo MA, Leal-Castro AL, Almanzar-Reina GA, Rosas-Pérez JE, García-Castañeda JE, Rivera-Monroy ZJ. Antibacterial activity of synthetic peptides derived from lactoferricin against Escherichia coli ATCC 25922 and Enterococcus Faecalis ATCC 29212. Biomed Res Int. 2015;2015. Vargas-Casanova Y, Rodríguez-Mayor AV, Cardenas KJ, Leal-Castro AL, Muñoz-Molina LC, Fierro-Medina R, et al. Synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the RWQWR motif against Gram-positive and Gram-negative strains. RSC Adv. 2019;9(13):7239–45. Vargas-Casanova Y, Carlos Villamil Poveda J, Jenny Rivera-Monroy Z, Ceballos Garzón A, Fierro-Medina R, Le Pape P, et al. PalindromicPeptide LfcinB (21-25)Pal Exhibited Antifungal Activity against Multidrug- Resistant Candida. ChemistrySelect. 2020 Jun 30;5(24):7236–42. Barragán-Cárdenas AC, Insuasty-Cepeda DS, Cárdenas-Martínez KJ, López-Meza J, Ochoa-Zarzosa A, Umaña-Pérez A, et al. LfcinB-Derived Peptides: Specific and punctual change of an amino acid in monomeric and dimeric sequences increase selective cytotoxicity in colon cancer cell lines. Arabian Journal of Chemistry. 2022 Aug 1;15(8). Barragán-Cárdenas A, Urrea-Pelayo M, Niño-Ramírez VA, Umaña-Pérez A, Vernot JP, Parra-Giraldo CM, et al. Selective cytotoxic effect against the MDA-MB-468 breast cancer cell line of the antibacterial palindromic peptide derived from bovine lactoferricin. RSC Adv. 2020 May 6;10(30):17593–601. Andrey K, Ortiz H. Caracterización fisicoquímica de un péptido polivalente, derivado de la Lactoferricina Bovina, candidato a fármaco para el tratamiento del Cáncer de Mama. 2021. Tornesello AL, Borrelli A, Buonaguro L, Buonaguro FM, Tornesello ML. Antimicrobial Peptides as Anticancer Agents: Functional Properties and Biological Activities. Vol. 25, Molecules. MDPI AG; 2020. Luo Y, Song Y. Mechanism of antimicrobial peptides: Antimicrobial, antiinflammatory and antibiofilm activities. Vol. 22, International Journal of Molecular Sciences. MDPI; 2021. Bin Hafeez A, Jiang X, Bergen PJ, Zhu Y. Antimicrobial peptides: An update on classifications and databases. Vol. 22, International Journal of Molecular Sciences. MDPI; 2021. Huan Y, Kong Q, Mou H, Yi H. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Vol. 11, Frontiers in Microbiology. Frontiers Media S.A.; 2020. Vila J, Sáez-López E, Johnson JR, Römling U, Dobrindt U, Cantón R, et al. Escherichia coli: An old friend with new tidings. Vol. 40, FEMS Microbiology Reviews. Oxford University Press; 2016. p. 437–63. Yu D, Banting G, Neumann NF. A review of the taxonomy, genetics, and biology of the genus escherichia and the type species E. coli. Can J Microbiol. 2021;67(8):553–71. Poirel L, Madec JY, Lupo A, Schink AK, Kieffer N, Nordmann P, et al. Antimicrobial Resistance in E. coli . Microbiol Spectr. 2018 Jul 27;6(4). Jang J, Hur HG, Sadowsky MJ, Byappanahalli MN, Yan T, Ishii S. Environmental E. coli: ecology and public health implications—a review. Vol. 123, Journal of Applied Microbiology. Blackwell Publishing Ltd; 2017. p. 570–81. Paitan Y. Current trends in antimicrobial resistance of E. coli. In: Current Topics in Microbiology and Immunology. Springer Verlag; 2018. p. 181– 211. Chong Y, Shimoda S, Shimono N. Current epidemiology, genetic evolution and clinical impact of extended-spectrum β-lactamase-producing E. coli and Klebsiella pneumoniae. Vol. 61, Infection, Genetics and Evolution. Elsevier B.V.; 2018. p. 185–8. Rapoport M, Faccone D, Pasteran F, Ceriana P, Albornoz E, Petroni A, et al. First description of mcr-1-mediated colistin resistance in human infections caused by E. coli in Latin America. Vol. 60, Antimicrobial Agents and Chemotherapy. American Society for Microbiology; 2016. p. 4412–3. Choi U, Lee CR. Distinct Roles of Outer Membrane Porins in Antibiotic Resistance and Membrane Integrity in E. coli. Front Microbiol. 2019;10(APR). Zuza-Alves DL, Silva-Rocha WP, Chaves GM. An update on C. tropicalisbased on basic and clinical approaches. Vol. 8, Frontiers in Microbiology. Frontiers Media S.A.; 2017. Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Candida glabrata, Candida parapsilosis and Candida tropicalis: Biology, epidemiology, pathogenicity and antifungal resistance. Vol. 36, FEMS Microbiology Reviews. 2012. p. 288–305. Heaney H, Laing J, Paterson L, Walker AW, Gow NAR, Johnson EM, et al. The environmental stress sensitivities of pathogenic candida species, including candida auris, and implications for their spread in the hospital setting. Med Mycol. 2020;58(6):744–55. Xin H. Effects of immune suppression in murine models of disseminated Candida glabrata and C. tropicalisinfection and utility of a synthetic peptide vaccine. Med Mycol. 2019 Aug 1;57(6):745–56. Kothavade RJ, Kura MM, Valand AG, Panthaki MH. Candida tropicalis: Its prevalence, pathogenicity and increasing resistance to fluconazole. Vol. 59, Journal of Medical Microbiology. 2010. p. 873–80. Pappas PG, Lionakis MS, Arendrup MC, Ostrosky-Zeichner L, Kullberg BJ. Invasive candidiasis. Nat Rev Dis Primers. 2018 May 11;4. de Oliveira JS, Pereira VS, Castelo-Branco D de SCM, Cordeiro R de A, Sidrim JJC, Brilhante RSN, et al. The yeast, the antifungal, and the wardrobe: A journey into antifungal resistance mechanisms of Candida tropicalis. Vol. 66, Canadian Journal of Microbiology. Canadian Science Publishing; 2020. p. 377–88. Pathakumari B, Liang G, Liu W. Immune defence to invasive fungal infections: A comprehensive review. Vol. 130, Biomedicine and Pharmacotherapy. Elsevier Masson SAS; 2020. Pemán J, Quindós G. Aspectos actuales de las enfermedades invasoras causadas por Candida y otros hongos levaduriformes. Vol. 33, Revista Iberoamericana de Micologia. Asociacion Espanola de Micologia; 2016. p. 133–9. Ann Chai LY, Denning DW, Warn P. C. tropicalisin human disease. Vol. 36, Critical Reviews in Microbiology. 2010. p. 282–98. von Lilienfeld-Toal M, Wagener J, Einsele H, Cornely OA, Kurzai O. Invasive Pilzinfektionen. Vol. 116, Deutsches Arzteblatt International. Deutscher Arzte-Verlag GmbH; 2019. p. 271–8. Vargas Casanova Y. Evaluación de la actividad antibacteriana de péptidos diméricos y tetraméricos derivados de lactoferricina bovina contra bacterias gram positivas y gram negativas. 2018. Khatri B, Nuthakki VR, Chatterjee J. Strategies to Enhance Metabolic Stabilities. In: Methods in Molecular Biology. Humana Press Inc.; 2019. p. 17–40. Barragán-Cárdenas AC, Insuasty-Cepeda DS, Vargas-Casanova Y, López-Meza JE, Parra-Giraldo CM, Fierro-Medina R, et al. Changes in Length and Positive Charge of Palindromic Sequence RWQWRWQWR Enhance Cytotoxic Activity against Breast Cancer Cell Lines. ACS Omega. 2022 National Committee for Clinical Laboratory Standards., Barry AL. Methods for determining bactericidal activity of antimicrobial agents : approved guideline. National Committee for Clinical Laboratory Standards; 1999. Pei J, Xiong L, Bao P, Chu M, Yan P, Guo X. Secondary Structural Transformation of Bovine Lactoferricin Affects Its Antibacterial Activity. Probiotics Antimicrob Proteins. 2021 Jun 1;13(3):873–84. Huertas Méndez NDJ, Vargas Casanova Y, Gómez Chimbi AK, Hernández E, Leal Castro AL, Melo Diaz JM, et al. Synthetic Peptides Derived from Bovine Lactoferricin Exhibit Antimicrobial Activity against E. coli ATCC 11775, S. maltophilia ATCC 13636 and S. enteritidis ATCC 13076. Molecules. 2017 Mar 12;22(3). Fernández de Ullivarri M, Arbulu S, Garcia-Gutierrez E, Cotter PD. Antifungal Peptides as Therapeutic Agents. Vol. 10, Frontiers in Cellular and Infection Microbiology. Frontiers Media S.A.; 2020. Carvajal SK, Vargas-Casanova Y, Pineda-Castañeda HM, GarcíaCastañeda JE, Rivera-Monroy ZJ, Parra-Giraldo CM. In Vitro Antifungal Activity of Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II against Cryptococcus neoformans var. grubii. Antibiotics. 2022 Dec 1;11(12). Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Vol. 58, Pharmacological Reviews. 2006. p. 621–81. Zhang J, Yu L, Fu Y, Zhao Y, Wang Y, Zhao J, et al. Tigecycline in combination with other antibiotics against clinical isolates of carbapenemresistant klebsiella pneumoniae in vitro. Ann Palliat Med. 2019 Nov 1;8(5):522–631. De M, Coli E, Aureus Laura YS, Bonilla Velásquez D. ACTIVIDAD ANTIBACTERIANA DEL PÉPTIDO LfcinB (20-25)4 CONTRA AISLADOS CLÍNICOS. 2021. Goel A, Gupta V, Singhal L, Palta S, Chander J. In vitro evaluation of antibiotic synergy for carbapenem-resistant Klebsiella pneumoniae clinical isolates. Indian Journal of Medical Research. 2021 Sep 1;154(3):520–6. Luterbach CL, Qiu H, Hanafin PO, Sharma R, Piscitelli J, Lin FC, et al. A Systems-Based Analysis of Mono- and Combination Therapy for Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections. Antimicrob Agents Chemother. 2022 Oct 1;66(10). Guía de práctica clínica [Internet]. Available from: https://orcid.org/0000- 0003-1898-9067 Jesús Fortún. Antifúngicos En El Tratamiento, Actualización en terapia antifúngica: nuevos fármacos e indicaciones_2010. Lazo W. Accion antifungica de las asociaciones sulfa-metoxazol fluconazol y sulfametoxazol flucitosina. Vol. 4. 1989. Médicas C, Dias Castro C, Leite Cavalcanti R;, De A;, Lima O. Revista Cubana de Estomatología Combined effect of Cinnamomum zeylanicum blume essential oil and nystatin on strains of non-albicans Candida Revista Cubana de Estomatología. 2013;50:192–200. Available from: http://www.redalyc.org/articulo.oa?id=378661952008 Milagros Esther Luyo Hermosa, Facultad de ciencias médicas escuela académico profesional de medicina, Efecto sinérgico antifúngico de la combinación de fluconazol y aceite esencial de Syzygium aromaticum sobre Candida albicans, in vitro, 2020. |
| dc.rights.coar.fl_str_mv |
http://purl.org/coar/access_right/c_abf2 |
| dc.rights.license.spa.fl_str_mv |
Atribución-NoComercial 4.0 Internacional |
| dc.rights.uri.spa.fl_str_mv |
http://creativecommons.org/licenses/by-nc/4.0/ |
| dc.rights.accessrights.spa.fl_str_mv |
info:eu-repo/semantics/openAccess |
| rights_invalid_str_mv |
Atribución-NoComercial 4.0 Internacional http://creativecommons.org/licenses/by-nc/4.0/ http://purl.org/coar/access_right/c_abf2 |
| eu_rights_str_mv |
openAccess |
| dc.format.extent.spa.fl_str_mv |
70 páginas |
| dc.format.mimetype.spa.fl_str_mv |
application/pdf |
| dc.publisher.spa.fl_str_mv |
Universidad Nacional de Colombia |
| dc.publisher.program.spa.fl_str_mv |
Bogotá - Ciencias - Maestría en Ciencias - Microbiología |
| dc.publisher.faculty.spa.fl_str_mv |
Facultad de Ciencias |
| dc.publisher.place.spa.fl_str_mv |
Bogotá, Colombia |
| dc.publisher.branch.spa.fl_str_mv |
Universidad Nacional de Colombia - Sede Bogotá |
| institution |
Universidad Nacional de Colombia |
| bitstream.url.fl_str_mv |
https://repositorio.unal.edu.co/bitstream/unal/85602/1/license.txt https://repositorio.unal.edu.co/bitstream/unal/85602/2/1032464270.2023.pdf https://repositorio.unal.edu.co/bitstream/unal/85602/3/1032464270.2023.pdf.jpg |
| bitstream.checksum.fl_str_mv |
eb34b1cf90b7e1103fc9dfd26be24b4a cac7f3045859005fe92c2cead2edcc0a 68861e32d1bd7c87e133f1569635bcdf |
| bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 |
| repository.name.fl_str_mv |
Repositorio Institucional Universidad Nacional de Colombia |
| repository.mail.fl_str_mv |
repositorio_nal@unal.edu.co |
| _version_ |
1806886098732843008 |
| spelling |
Atribución-NoComercial 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Rivera Monroy, Zuly Jenny4d01a798fcb62ec5f5a8b8214fe50c7c600Parra Giraldo, Claudia Marcelaffcf6fc0b59f6bfe28c5a794cb7e41d1600Botina Rodríguez, Anyelly Tatiana6b7c3ee49d9f1aae1181ac792fa64f212024-02-02T15:43:43Z2024-02-02T15:43:43Z2023https://repositorio.unal.edu.co/handle/unal/85602Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/ilustraciones, diagramasEl incremento en la resistencia de los microorganismos a los tratamientos convencionales, debido al uso indiscriminado de los antibióticos en humanos, animales y plantas, es un problema de salud pública a nivel mundial. En las últimas décadas se han venido realizando esfuerzos para la búsqueda de soluciones terapéuticas que permitan tratar las infecciones causadas por patógenos resistentes. Una de las alternativas más estudiadas son los péptidos antimicrobianos (PAMs), como la Lactoferricina bovina (LfcinB), que ha demostrado tener actividad antibacteriana, antifúngica, anticancerígena, entre otras. Se identificaron péptidos derivados de la LfcinB con actividad antibacteriana, dentro de los cuales uno de los más promisorios es el péptido palindrómico LfcinB (21-25)Pal: RWQWRWQWR. En este trabajo se evaluó la actividad antimicrobiana de 38 péptidos derivados de la secuencia palindrómica LfcinB (21-25)Pal en los cuales se realizaron modificaciones como: reemplazó de arginina por lisina, cambios puntuales de L-aminoácidos por D-aminoácidos, péptidos con la secuencia completa con D-aminoácidos y péptidos quiméricos que incluyen el motivo mínimo de la LfcinB. La actividad antibacteriana y antifúngica de estos péptidos fue evaluada en Escherichia coli ATCC 25922, C. tropicalis ATCC 1369 y en aislados clínicos, con fenotipos sensibles y resistentes, de Escherichia coli 1004, Escherichia coli 301755, C. tropicalis1018 y C. tropicalis883. Se realizaron curvas de letalidad para determinar el tipo de actividad antimicrobiana: mortalidad (bactericida, fungicida) o inhibición del desarrollo y crecimiento (bacteriostática, fungistática) y se realizaron curvas de sinergia e interacción con ciprofloxacina y fluconazol. El péptido palindrómico LfcinB (21-25)Pal presentó actividad en aislados clínicos sensibles y resistentes de E. coli y C. tropicalis, confirmando que la actividad antimicrobiana observada en cepas de referencia también se presenta en aislados clínicos. La secuencia derivada del palíndromo, H2N-RRWQWRRWQWRR-CONH2 presentó la mejor actividad frente a la cepa de referencia, el aislado clínico sensible y el aislado clínico resistente E. coli 301755. Este péptido presentó actividad bactericida y un efecto de indiferencia en combinación con ciprofloxacina. El péptido con mejor actividad en los aislados clínicos de C. tropicalis fue el péptido original LfcinB (21-25)Pal H2N-RWQWRWQWR-CONH2 que presentó actividad fungistática en la cepa de referencia y aislados clínicos e igualmente un efecto de sinergia en combinación con Fluconazol. En este trabajo se identificaron péptidos que presentaron actividad antimicrobiana contra cepas de referencia y aislados clínicos sensibles y resistentes, sugiriendo que estas moléculas pueden ser candidatas para estudios de desarrollo de agentes terapéuticos contra infecciones bacterianas y/o fúngicas. (Texto tomado de la fuente).The increase in the resistance of microrganisms to conventional treatments, due to the indiscriminate use of antibiotics in humans, animals, and plants, is a public health problem worldwide. In recent decades, efforts have been made to search for therapeutic solutions to treat infections caused by resistant pathogens. One of the most studied alternatives are antimicrobial peptides (PAMs), such as bovine lactoferricin (LfcinB), which has been shown to have antibacterial, antifungal, and anticancer activity, among others. Peptides derived from LfcinB with antibacterial activity were identified, among which one of the most promising is the palindromic peptide LfcinB (21-25)Pal: RWQWRWQWR. In this investigation, we evaluated the antimicrobial activity of 38 peptides derived from the palindromic sequence LfcinB (21-25)Pal in which modifications were made, such as: replacement of arginine by lysine, punctual changes of L-amino acids by D-amino acids, peptides with the complete sequence with D-amino acids, and chimeric peptides that include the minimal motif of LfcinB. The antibacterial and antifungal activity of these peptides was evaluated in Escherichia coli ATCC 25922, C. tropicalisATCC 1369 and in clinical isolates with sensitive and resistant phenotypes of Escherichia coli 1004, Escherichia coli 301755, C. tropicalis1018 and C. tropicalis883. Killing rate curves were performed to determine the type of antimicrobial activity: mortality (bactericidal, fungicidal) or growth inhibition (bacteriostatic, fungistatic) and synergy and interaction curves with ciprofloxacin and fluconazole were performed. The palindromic peptide LfcinB (21-25)Pal showed activity in sensitive and resistant clinical isolates of E. coli and C. tropicalis, confirming that the antimicrobial activity observed in reference strains is also present in clinical isolates. The sequence derived from the palindrome H2N-RRWQWRRWQWRRCONH2 showed the best activity against the reference strain, the sensitive clinical isolate and the resistant clinical isolate E. coli 301755. This peptide al showed bactericidal activity and an indifference effect in combination with ciprofloxacin. The peptide with the best activity in the clinical isolates of C. tropicalis was the original peptide LfcinB (21-25)Pal H2N-RWQWRWQWR- CONH2, which presented fungistatic activity in the reference strain and clinical isolates, as well as a synergistic effect in combination with Fluconazole. In this work, peptides that presented antimicrobial activity against reference strains and sensitive and resistant clinical isolates were identified, suggesting that these molecules may be candidates for development studies of therapeutic agents against bacterial and/or fungal infections.MaestríaMagíster en Ciencias - Microbiología70 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Ciencias - Maestría en Ciencias - MicrobiologíaFacultad de CienciasBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá570 - Biología::572 - BioquímicaLfcinBActividad antimicrobianaEscherichia coliCandida tropicalisPéptidos antimicrobianosLfcinBAntimicrobial peptidesAntimicrobial activityEscherichia coliCandida tropicalisMicrobiologíaBacteriaTratamiento médicoMicrobiologyBacteriaMedical treatmentActividad antimicrobiana del péptido palindrómico LfcinB (21-25)Pal RWQWRWQWR y sus análogosAntimicrobian activity of LfcinB (21-25)Pal RWQWRWQWR palindromic peptid and it analoguesTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMBiremeÁngel Serra Valdés M, Cabrera E, Habana Cuba maserra L. Ciencias epidemioló gicas y salubristas. La resistencia microbiana en el contexto actual y la importancia del conocimiento y aplicación en la política antimicrobiana Microbial resistance in the current context and the importance of knowledge and application in antimicrobial policy [Internet]. Available from: http://www.revhabanera.sld.cu/index.php/rhab/article/view/2013Vivas R, Barbosa AAT, Dolabela SS, Jain S. Multidrug-Resistant Bacteria and Alternative Methods to Control Them: An Overview. Microbial Drug Resistance. 2019 Jul 1;25(6):890–908.OMS, PLAN DE ACCIÓN MUNDIAL SOBRE LA RESISTENCIA A LOS ANTIMICROBIANOS.Luo Y, Song Y. Mechanism of antimicrobial peptides: Antimicrobial, antiinflammatory and antibiofilm activities. Vol. 22, International Journal of Molecular Sciences. MDPI; 2021.Freitas ED, Bataglioli RA, Oshodi J, Beppu MM. Antimicrobial peptides and their potential application in antiviral coating agents. Vol. 217, Colloids and Surfaces B: Biointerfaces. Elsevier B.V.; 2022.Tomita M, Wakabayashi H, Yamauchi K, Teraguchi S, Hayasawa H. Bovine lactoferrin and lactoferricin derived from milk: Production and applications. In: Biochemistry and Cell Biology. 2002. p. 109–12.Muñoz A, Marcos JF. Activity and mode of action against fungal phytopathogens of bovine lactoferricin-derived peptides. J Appl Microbiol. 2006 Dec;101(6):1199–207.Sun C, Li Y, Cao S, Wang H, Jiang C, Pang S, et al. Antibacterial activity and mechanism of action of bovine lactoferricin derivatives with symmetrical amino acid sequences. Int J Mol Sci. 2018 Oct 1;19(10).León-Calvijo MA, Leal-Castro AL, Almanzar-Reina GA, Rosas-Pérez JE, García-Castañeda JE, Rivera-Monroy ZJ. Antibacterial activity of synthetic peptides derived from lactoferricin against Escherichia coli ATCC 25922 and Enterococcus Faecalis ATCC 29212. Biomed Res Int. 2015;2015.Vargas-Casanova Y, Rodríguez-Mayor AV, Cardenas KJ, Leal-Castro AL, Muñoz-Molina LC, Fierro-Medina R, et al. Synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the RWQWR motif against Gram-positive and Gram-negative strains. RSC Adv. 2019;9(13):7239–45.Ardila-Chantré N, Hernández-Cardona AK, Pineda-Castañeda HM, Estupiñan-Torres SM, Leal-Castro AL, Fierro-Medina R, et al. Short peptides conjugated to non-peptidic motifs exhibit antibacterial activity. RSC Adv. 2020 Aug 10;10(49):29580–6.Wang B, Timilsena YP, Blanch E, Adhikari B. Lactoferrin: Structure, function, denaturation and digestion. Vol. 59, Critical Reviews in Food Science and Nutrition. Taylor and Francis Inc.; 2019. p. 580–96.Karav S, German JB, Rouquié C, le Parc A, Barile D. Studying lactoferrin N-glycosylation. Vol. 18, International Journal of Molecular Sciences.MDPI AG; 2017.Antoshin AA, Shpichka AI, Huang G, Chen K, Lu P, Svistunov AA, et al. Lactoferrin as a regenerative agent: The old-new panacea? Vol. 167, Pharmacological Research. Academic Press; 2021.Fernandes KE, Carter DA. The antifungal activity of lactoferrin and its derived peptides: Mechanisms of action and synergy with drugs against fungal pathogens. Vol. 8, Frontiers in Microbiology. Frontiers Research Foundation; 2017.Tomita M, Wakabayashi H, Yamauchi K, Teraguchi S, Hayasawa H. Bovine lactoferrin and lactoferricin derived from milk: Production and applications. In: Biochemistry and Cell Biology. 2002. p. 109–12.Ardila-Chantré N, Hernández-Cardona AK, Pineda-Castañeda HM, Estupiñan-Torres SM, Leal-Castro AL, Fierro-Medina R, et al. Short peptides conjugated to non-peptidic motifs exhibit antibacterial activity. RSC Adv. 2020 Aug 10;10(49):29580–6.León-Calvijo MA, Leal-Castro AL, Almanzar-Reina GA, Rosas-Pérez JE, García-Castañeda JE, Rivera-Monroy ZJ. Antibacterial activity of synthetic peptides derived from lactoferricin against Escherichia coli ATCC 25922 and Enterococcus Faecalis ATCC 29212. Biomed Res Int. 2015;2015.Vargas-Casanova Y, Rodríguez-Mayor AV, Cardenas KJ, Leal-Castro AL, Muñoz-Molina LC, Fierro-Medina R, et al. Synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the RWQWR motif against Gram-positive and Gram-negative strains. RSC Adv. 2019;9(13):7239–45.Vargas-Casanova Y, Carlos Villamil Poveda J, Jenny Rivera-Monroy Z, Ceballos Garzón A, Fierro-Medina R, Le Pape P, et al. PalindromicPeptide LfcinB (21-25)Pal Exhibited Antifungal Activity against Multidrug- Resistant Candida. ChemistrySelect. 2020 Jun 30;5(24):7236–42.Barragán-Cárdenas AC, Insuasty-Cepeda DS, Cárdenas-Martínez KJ, López-Meza J, Ochoa-Zarzosa A, Umaña-Pérez A, et al. LfcinB-Derived Peptides: Specific and punctual change of an amino acid in monomeric and dimeric sequences increase selective cytotoxicity in colon cancer cell lines. Arabian Journal of Chemistry. 2022 Aug 1;15(8).Barragán-Cárdenas A, Urrea-Pelayo M, Niño-Ramírez VA, Umaña-Pérez A, Vernot JP, Parra-Giraldo CM, et al. Selective cytotoxic effect against the MDA-MB-468 breast cancer cell line of the antibacterial palindromic peptide derived from bovine lactoferricin. RSC Adv. 2020 May 6;10(30):17593–601.Andrey K, Ortiz H. Caracterización fisicoquímica de un péptido polivalente, derivado de la Lactoferricina Bovina, candidato a fármaco para el tratamiento del Cáncer de Mama. 2021.Tornesello AL, Borrelli A, Buonaguro L, Buonaguro FM, Tornesello ML. Antimicrobial Peptides as Anticancer Agents: Functional Properties and Biological Activities. Vol. 25, Molecules. MDPI AG; 2020.Luo Y, Song Y. Mechanism of antimicrobial peptides: Antimicrobial, antiinflammatory and antibiofilm activities. Vol. 22, International Journal of Molecular Sciences. MDPI; 2021.Bin Hafeez A, Jiang X, Bergen PJ, Zhu Y. Antimicrobial peptides: An update on classifications and databases. Vol. 22, International Journal of Molecular Sciences. MDPI; 2021.Huan Y, Kong Q, Mou H, Yi H. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Vol. 11, Frontiers in Microbiology. Frontiers Media S.A.; 2020.Vila J, Sáez-López E, Johnson JR, Römling U, Dobrindt U, Cantón R, et al. Escherichia coli: An old friend with new tidings. Vol. 40, FEMS Microbiology Reviews. Oxford University Press; 2016. p. 437–63.Yu D, Banting G, Neumann NF. A review of the taxonomy, genetics, and biology of the genus escherichia and the type species E. coli. Can J Microbiol. 2021;67(8):553–71.Poirel L, Madec JY, Lupo A, Schink AK, Kieffer N, Nordmann P, et al. Antimicrobial Resistance in E. coli . Microbiol Spectr. 2018 Jul 27;6(4).Jang J, Hur HG, Sadowsky MJ, Byappanahalli MN, Yan T, Ishii S. Environmental E. coli: ecology and public health implications—a review. Vol. 123, Journal of Applied Microbiology. Blackwell Publishing Ltd; 2017. p. 570–81.Paitan Y. Current trends in antimicrobial resistance of E. coli. In: Current Topics in Microbiology and Immunology. Springer Verlag; 2018. p. 181– 211.Chong Y, Shimoda S, Shimono N. Current epidemiology, genetic evolution and clinical impact of extended-spectrum β-lactamase-producing E. coli and Klebsiella pneumoniae. Vol. 61, Infection, Genetics and Evolution. Elsevier B.V.; 2018. p. 185–8.Rapoport M, Faccone D, Pasteran F, Ceriana P, Albornoz E, Petroni A, et al. First description of mcr-1-mediated colistin resistance in human infections caused by E. coli in Latin America. Vol. 60, Antimicrobial Agents and Chemotherapy. American Society for Microbiology; 2016. p. 4412–3.Choi U, Lee CR. Distinct Roles of Outer Membrane Porins in Antibiotic Resistance and Membrane Integrity in E. coli. Front Microbiol. 2019;10(APR).Zuza-Alves DL, Silva-Rocha WP, Chaves GM. An update on C. tropicalisbased on basic and clinical approaches. Vol. 8, Frontiers in Microbiology. Frontiers Media S.A.; 2017.Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Candida glabrata, Candida parapsilosis and Candida tropicalis: Biology, epidemiology, pathogenicity and antifungal resistance. Vol. 36, FEMS Microbiology Reviews. 2012. p. 288–305.Heaney H, Laing J, Paterson L, Walker AW, Gow NAR, Johnson EM, et al. The environmental stress sensitivities of pathogenic candida species, including candida auris, and implications for their spread in the hospital setting. Med Mycol. 2020;58(6):744–55.Xin H. Effects of immune suppression in murine models of disseminated Candida glabrata and C. tropicalisinfection and utility of a synthetic peptide vaccine. Med Mycol. 2019 Aug 1;57(6):745–56.Kothavade RJ, Kura MM, Valand AG, Panthaki MH. Candida tropicalis: Its prevalence, pathogenicity and increasing resistance to fluconazole. Vol. 59, Journal of Medical Microbiology. 2010. p. 873–80.Pappas PG, Lionakis MS, Arendrup MC, Ostrosky-Zeichner L, Kullberg BJ. Invasive candidiasis. Nat Rev Dis Primers. 2018 May 11;4.de Oliveira JS, Pereira VS, Castelo-Branco D de SCM, Cordeiro R de A, Sidrim JJC, Brilhante RSN, et al. The yeast, the antifungal, and the wardrobe: A journey into antifungal resistance mechanisms of Candida tropicalis. Vol. 66, Canadian Journal of Microbiology. Canadian Science Publishing; 2020. p. 377–88.Pathakumari B, Liang G, Liu W. Immune defence to invasive fungal infections: A comprehensive review. Vol. 130, Biomedicine and Pharmacotherapy. Elsevier Masson SAS; 2020.Pemán J, Quindós G. Aspectos actuales de las enfermedades invasoras causadas por Candida y otros hongos levaduriformes. Vol. 33, Revista Iberoamericana de Micologia. Asociacion Espanola de Micologia; 2016. p. 133–9.Ann Chai LY, Denning DW, Warn P. C. tropicalisin human disease. Vol. 36, Critical Reviews in Microbiology. 2010. p. 282–98.von Lilienfeld-Toal M, Wagener J, Einsele H, Cornely OA, Kurzai O. Invasive Pilzinfektionen. Vol. 116, Deutsches Arzteblatt International. Deutscher Arzte-Verlag GmbH; 2019. p. 271–8.Vargas Casanova Y. Evaluación de la actividad antibacteriana de péptidos diméricos y tetraméricos derivados de lactoferricina bovina contra bacterias gram positivas y gram negativas. 2018.Khatri B, Nuthakki VR, Chatterjee J. Strategies to Enhance Metabolic Stabilities. In: Methods in Molecular Biology. Humana Press Inc.; 2019. p. 17–40.Barragán-Cárdenas AC, Insuasty-Cepeda DS, Vargas-Casanova Y, López-Meza JE, Parra-Giraldo CM, Fierro-Medina R, et al. Changes in Length and Positive Charge of Palindromic Sequence RWQWRWQWR Enhance Cytotoxic Activity against Breast Cancer Cell Lines. ACS Omega. 2022National Committee for Clinical Laboratory Standards., Barry AL. Methods for determining bactericidal activity of antimicrobial agents : approved guideline. National Committee for Clinical Laboratory Standards; 1999.Pei J, Xiong L, Bao P, Chu M, Yan P, Guo X. Secondary Structural Transformation of Bovine Lactoferricin Affects Its Antibacterial Activity. Probiotics Antimicrob Proteins. 2021 Jun 1;13(3):873–84.Huertas Méndez NDJ, Vargas Casanova Y, Gómez Chimbi AK, Hernández E, Leal Castro AL, Melo Diaz JM, et al. Synthetic Peptides Derived from Bovine Lactoferricin Exhibit Antimicrobial Activity against E. coli ATCC 11775, S. maltophilia ATCC 13636 and S. enteritidis ATCC 13076. Molecules. 2017 Mar 12;22(3).Fernández de Ullivarri M, Arbulu S, Garcia-Gutierrez E, Cotter PD. Antifungal Peptides as Therapeutic Agents. Vol. 10, Frontiers in Cellular and Infection Microbiology. Frontiers Media S.A.; 2020.Carvajal SK, Vargas-Casanova Y, Pineda-Castañeda HM, GarcíaCastañeda JE, Rivera-Monroy ZJ, Parra-Giraldo CM. In Vitro Antifungal Activity of Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II against Cryptococcus neoformans var. grubii. Antibiotics. 2022 Dec 1;11(12).Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Vol. 58, Pharmacological Reviews. 2006. p. 621–81.Zhang J, Yu L, Fu Y, Zhao Y, Wang Y, Zhao J, et al. Tigecycline in combination with other antibiotics against clinical isolates of carbapenemresistant klebsiella pneumoniae in vitro. Ann Palliat Med. 2019 Nov 1;8(5):522–631.De M, Coli E, Aureus Laura YS, Bonilla Velásquez D. ACTIVIDAD ANTIBACTERIANA DEL PÉPTIDO LfcinB (20-25)4 CONTRA AISLADOS CLÍNICOS. 2021.Goel A, Gupta V, Singhal L, Palta S, Chander J. In vitro evaluation of antibiotic synergy for carbapenem-resistant Klebsiella pneumoniae clinical isolates. Indian Journal of Medical Research. 2021 Sep 1;154(3):520–6.Luterbach CL, Qiu H, Hanafin PO, Sharma R, Piscitelli J, Lin FC, et al. A Systems-Based Analysis of Mono- and Combination Therapy for Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections. Antimicrob Agents Chemother. 2022 Oct 1;66(10).Guía de práctica clínica [Internet]. Available from: https://orcid.org/0000- 0003-1898-9067Jesús Fortún. Antifúngicos En El Tratamiento, Actualización en terapia antifúngica: nuevos fármacos e indicaciones_2010.Lazo W. Accion antifungica de las asociaciones sulfa-metoxazol fluconazol y sulfametoxazol flucitosina. Vol. 4. 1989.Médicas C, Dias Castro C, Leite Cavalcanti R;, De A;, Lima O. Revista Cubana de Estomatología Combined effect of Cinnamomum zeylanicum blume essential oil and nystatin on strains of non-albicans Candida Revista Cubana de Estomatología. 2013;50:192–200. Available from: http://www.redalyc.org/articulo.oa?id=378661952008Milagros Esther Luyo Hermosa, Facultad de ciencias médicas escuela académico profesional de medicina, Efecto sinérgico antifúngico de la combinación de fluconazol y aceite esencial de Syzygium aromaticum sobre Candida albicans, in vitro, 2020.LICENSElicense.txtlicense.txttext/plain; charset=utf-85879https://repositorio.unal.edu.co/bitstream/unal/85602/1/license.txteb34b1cf90b7e1103fc9dfd26be24b4aMD51ORIGINAL1032464270.2023.pdf1032464270.2023.pdfTesis de Maestría en Ciencias - Microbiologíaapplication/pdf2265614https://repositorio.unal.edu.co/bitstream/unal/85602/2/1032464270.2023.pdfcac7f3045859005fe92c2cead2edcc0aMD52THUMBNAIL1032464270.2023.pdf.jpg1032464270.2023.pdf.jpgGenerated Thumbnailimage/jpeg4697https://repositorio.unal.edu.co/bitstream/unal/85602/3/1032464270.2023.pdf.jpg68861e32d1bd7c87e133f1569635bcdfMD53unal/85602oai:repositorio.unal.edu.co:unal/856022024-02-02 23:03:40.658Repositorio Institucional Universidad Nacional de Colombiarepositorio_nal@unal.edu.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 |