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Universidad Autónoma Del Estado De México Maestría Y UNIVERSIDAD AUTÓNOMA DEL ESTADO DE MÉXICO MAESTRÍA Y DOCTORADO EN CIENCIAS AGROPECUARIAS Y RECURSOS NATURALES Evaluación del efecto bactericida in vitro de las nanopartículas de plata en cepas de Moraxella spp. multirresistentes aisladas en ovinos en el Estado de México. TESIS QUE PARA OBTENER EL GRADO DE MAESTRO EN CIENCIAS AGROPECUARIAS Y RECURSOS NATURALES. PRESENTA: MVZ. Giovany Ortiz Arana El Cerrillo Piedras Blancas, Toluca, Estado de México, abril de 2019. UNIVERSIDAD AUTÓNOMA DEL ESTADO DE MÉXICO MAESTRÍA Y DOCTORADO EN CIENCIAS AGROPECUARIAS Y RECURSOS NATURALES Evaluación del efecto bactericida in vitro de las nanopartículas de plata en cepas de Moraxella spp. multirresistentes aisladas en ovinos en el Estado de México. TESIS QUE PARA OBTENER EL GRADO DE MAESTRO EN CIENCIAS AGROPECUARIAS Y RECURSOS NATURALES. PRESENTA: MVZ. Giovany Ortiz Arana. COMITÉ DE TUTORES DR. MARTÍN TALAVERA ROJAS DR. JORGE PABLO ACOSTA DIBARRAT DR. EDGARDO SORIANO VARGAS El Cerrillo Piedras Blancas, Toluca, Estado de México, abril de 2019. Dedicatorias. A mis padres. Que gracias a consejos y palabras de aliento me han ayudado a crecer como persona y a luchar por lo que quiero, gracias por enseñarme valores que me han llevado a alcanzar mis metas. De antemano les agradezco su amor, cariño y cuidado. Este logró también es de ustedes. Los quiero mucho. A mis hermanos. Gracias por su apoyo, cariño, por los ratos de diversión en todo momento y por estar en los momentos más importantes de mi vida. A mis asesores. Agradezco su tiempo dedicado, paciencia y enseñanzas para la elaboración de este documento. A mis amigos. Gracias por su amistad y lealtad en todo momento, agradezco los momentos de risa que los conmemoró en mis recuerdos. A ti: Alicia gracias por tu cariño, apoyo, comprensión y confianza que has dado en todo este trayecto. I Resumen Evaluación del efecto bactericida in vitro de las nanopartículas de plata en cepas de Moraxella spp. multirresistentes aisladas en ovinos en el Estado de México. La queratoconjuntivitis contagiosa ovina (QCO) es una enfermedad que produce ceguera temporal o permanente en ovinos y caprinos, está asociada a un conjunto de diversos géneros bacterianos y virales y el tratamiento se basa en el uso de compuestos antimicrobianos, ya se reportado la aparición de cepas de Moraxella spp. con resistencia antimicrobiana asociados a la QCO, por esta razón se está en la búsqueda de nuevas alternativas al uso de antibióticos y actualmente se está empleando nuevas herramientas basadas en nanotecnología contra bacterias multirresistentes, en particular, las nanopartículas de plata (AgNPs) que han atraído mucha atención en el campo científico, debido a que tienen un alto potencial para resolver el problema de la aparición de bacterias multirresistentes y sobrepasar los mecanismos de resistencia. En este estudio se evaluó el efecto bactericida in vitro de las nanopartículas de plata contra aislamientos de Moraxella spp. multirresistentes. Para este estudio se partió de un muestreo probabilístico, utilizando la fórmula de poblaciones finitas, obteniéndose un total 209 muestras de casos clínicos con queratoconjuntivitis contagiosa ovina en unidades producción ovina del Estado de México, se realizó el aislamiento e identificación de Moraxella spp. mediante técnicas bacteriológicas y la amplificación mediante PCR de los genes 16s rRNA y rtxA y posteriormente se determinó su perfil de sensibilidad antimicrobiana a diversos antimicrobianos mediante el método de difusión en disco y se amplificaron siete genes de resistencia antimicrobiana. Finalmente se evaluó la eficacia del efecto bactericida de las nanopartículas de plata contra los aislamientos de Moraxella spp. multirresistentes. Se obtuvieron 60 aislamientos de Moraxella spp., en el 90% (54/60) solo se amplificó el gen 16S rRNA y el 95.0% (57/60) el gen rtxA, identificando (58/60) de los aislados como M. ovis y (2/60) como M. bovoculi. El 8.33% (5/60) resultaron aislamientos multirresistentes, 23.33% (14/60) aislamientos resistentes y 68.33% (41/60) aislamientos sensibles a todos los antibióticos. Se logró la amplificación de los genes sul1, sul2, tetB, qnrA, qnrB, BlaTEM, no se logró amplificar el gen floR en los aislados. Con respecto a la actividad bactericida de las AgNPs sobre los aislamientos de Moraxella ovis multirresistentes, resistentes y sensibles (MDR, R y S) y Moraxella bovoculi resistentes (R) se observó inhibición del crecimiento a partir de la concentración de 10.78 µg/L (100mM), mientras que la inhibición del crecimiento de las cepas controles de E. coli y S. aureus se obtuvo a partir de concentraciones de 5.39 µg (50mM) y 8.09 µg/L (75mM) respectivamente. Los casos de queratoconjuntivitis contagiosa ovina en México están relacionados con la presencia de Moraxella ovis y M bovoculi. El uso de las nanopartículas de plata son una alternativa para el tratamiento de la queratoconjuntivitis ovina provocada por bacterias del género Moraxella. Palabras Claves: Nanopartículas plata, Moraxella spp., resistencia bacteriana, PCR, genes de resistencia antimicrobiana. II Abstract. Evaluation of the bactericidal effect in vitro of the silver nanoparticles in multiresistant Moraxella spp. strains isolated in sheep in the State of Mexico. Contagious ovine keratoconjunctivitis (QCO) is a disease that produces temporary or permanent blindness in sheep and goats, is associated with a set of different bacterial and viral genera and the treatment is based on the use of antimicrobial compounds, the appearance of Moraxella spp strains with antimicrobial resistance associated with the QCO was reported. With resistance to these drugs, for this reason we are searching for new alternatives to the use of antibiotics and currently we are using new tools based on nanotechnology against multiresistant bacteria, in particular, the silver nanoparticles (AgNPs) that have attracted a lot of attention in the scientific field, because they have a high potential to solve the problem of the appearance of multiresistant bacteria and surpass the mechanisms of resistance. In this study, the in vitro bactericidal effect of silver nanoparticles against isolates of Moraxella spp. multi-resistant. For this study we started with a probabilistic sampling, using the formula of finite populations, obtaining a total of 209 samples of clinical cases with contagious ovine keratoconjunctivitis in sheep production units of the State of Mexico, isolating and identifying Moraxella spp. by means of bacteriological techniques and PCR amplification of the 16s rRNA and rtxA genes and subsequently their antimicrobial susceptibility profile was determined to several antimicrobials by the disk diffusion method and seven antimicrobial resistance genes were amplified. Finally, the effectiveness of the bactericidal effect of the silver nanoparticles against the isolates of Moraxella spp. multi-resistant. We obtained 60 isolates of Moraxella spp., in 90% (54/60) only the 16S rRNA gene was amplified and 95.0% (57/60) the rtxA gene, identifying (58/60) of the isolates as M. ovis and (2/60) as M. bovoculi. 8.33% (5/60) resulted in multiresistant isolates, 23.33% (14/60) resistant isolates and 68.33% (41/60) sensitive isolates to all antibiotics. The amplification of the sul1, sul2, tetB, qnrA, qnrB, BlaTEM genes was achieved; the floR gene could not be amplified in the isolates. With regard to the bactericidal activity of the AgNPs on the multirresistant, resistant and sensitive Moraxella ovis (MDR, R and S) and resistant Moraxella bovoculi (R) isolates, growth inhibition was observed from the concentration of 10.78 μg / L ( 100mM), while growth inhibition of the control strains of E. coli and S. aureus was obtained from concentrations of 5.39 μg (50mM) and 8.09μg / L (75mM) respectively. Cases of contagious ovine keratoconjunctivitis in México are related with the presence of Moraxella ovis and Moraxella bovoculi. The use of silver nanoparticles are an alternative for the treatment contagious ovine keratoconjunctivitis caused by bacterias of the genus Moraxella. Keywords; silver nanoparticles, Moraxella spp., resistance bacteria, PCR, genes resistance antimicrobial. III Agradecimientos. A la Universidad Autónoma del Estado de México por favorecer mi crecimiento como profesionista y persona. A la Facultad de Medicina Veterinaria y Zootecnia, Centro de Investigación y Estudios Avanzados en Salud Animal (CIESA), por el conocimiento adquirido con su cuerpo Académico e Instalaciones. Al CONACYT, por el apoyo como becario dentro de Programa de Maestría en Ciencias Agropecuarias y Recursos Naturales con número de registro REF 001553. El presente trabajo conto con el financiamiento del proyecto: “Nanotecnología como herramienta para el diagnóstico y tratamiento de enfermedades infecciosas” con clave 3998/2016RED financiado por la Secretaría de Educación Pública, registrado como “Biotecnología aplicada, enfermedades emergentes y zoonosis con la clave UAEM-CA-215 en la Universidad Autónoma del Estado de México”. El presente trabajo forma parte del proyecto: “Caracterización, fenotípica y genotípica y resistencia antimicrobiana de aislados de Moraxella spp. obtenidos de casos clínicos de queratoconjuntivitis ovina” con clave de registro 4629/2019SF, UAEM. IV ÍNDICE. Dedicatorias. ...................................................................................................................................... I Resumen...........................................................................................................................................
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