Ciencias Agropecuarias
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Item Presencia de genes de resistencia a los antibióticos en Shigella spp. aislada de carne de pollo que se expende en el cantón de Ambato.(2024-11) Pazmiño Orozco Andrés Sebastián; Cruz Quintana Sandra MargaritaShigella spp. is a common causal agent of diarrheal diseases around the world. The treatment of choice for shigellosis is the administration of antibiotics. However, the resistance that Shigella spp. has developed to these molecules poses a major challenge in the need to find effective treatment alternatives. Transmission of Shigella spp. is via the fecal-oral route, however, infection can result from the ingestion of contaminated food where poor hygiene during handling can be considered a risk factor for the occurrence of disease. Likewise, unhygienic conditions during the slaughter of animals could constitute one of the main sources of contamination of meat for human consumption. In this research, we start from previous studies where, from samples of chicken meat taken from points of sale and slaughterhouses in Ambato, 17 strains of Shigella spp. were isolated and molecularly identified and some of them, showed marked resitance mainly to two antibiotics: amoxicillin-clavulanate (AMC) and ciprofloxacin (CIP). The aim of this investigation was to verify the presence of a resistance gene for each of the antibiotics described above. For AMC, the blaTEM gene was searched for, which encodes for class A ESBL (extended spectrum β-lactamases), finding a prevalence of 23.53%, while for CIP, the gyrA gene was searched for, which encodes for the A subunits of DNA gyrase, finding a prevalence of 100%. One of the resistance mechanisms for AMC would be the production of TEM-like ESBLs, while specific mutations in a small region near the start of the gyrA gene, called the quinolone resistance determining region (QRDR), have been widely linked to CIP resistance in Enterobacteriaceae.Item Evaluación de la sensibilidad antibiótica y presencia de genes de resistencia en Enterococcus gallinarum aislado en carne de pollo en Ambato(2024-08) Villacis Gamboa, David Alexander; Cruz Quintana, Sandra MargaritaEnterococcus gallinarum is a Gram-positive bacterium that grows in short chains and can be found in the gastrointestinal tract of animals and humans at temperatures ranging from 10 - 37ºC. The aim of the study was to evaluate the antibiotic sensitivity and presence of resistance genes in Enterococcus gallinarum isolated from chicken meat in Ambato. The Kirby Bauer method with antibiotic discs was used to evaluate the sensitivity and resistance to vancomycin, erythromycin, ciprofloxacin, amoxicillin + clavulanic acid and trimethopin + sulfamethasone. The antibiotic chosen to detect resistance genes was vancomycin by DNA extraction, PCR and electrophoresis. PCR primers VanA F and VanA R were used to amplify a band of 732 base pairs. Resistance to vancomycin, erythromycin, ciprofloxacin and sensitivity to amoxicillin + clavulanic acid and trimethopin + sulfamethasone was shown. No vancomycin resistance genes were detected and the main molecular mechanisms of resistance of Enterococcus gallinarum to vancomycin were investigated as efflux pumps, resistance genes and target site modification. It was concluded that Enterococcus gallinarum from chicken meat sold in the canton of Ambato is sensitive to clavulanic acid and trimethopin + sulfamethasone and resistant to vancomycin, erythromycin and ciprofloxacin. To vancomycin it does not present resistance genes, its main resistance mechanisms are resistance genes, efflux pumps, modification of the target point.Item Evaluación de la sensibilidad a los antibióticos y presencia de genes de resistencia en Lactococcus garvieae aislado en carne de pollo que se expende en Ambato(2024-08) Chávez Romero, Anderson Xavier; Cruz Quintana, Sandra MargaritaLactococcus garvieae is a Gram-positive pathogenic bacterium, considered an emerging zoonotic pathogen, as reports of human infection are increasing. In the present research work, the antibioresistance of the L. garvieae strain and the presence of resistance genes in L. garvieae isolated from chicken meat sold in Ambato were evaluated. Sensitivity was identified using the Kirby Bauer method for the following antibiotics: Linezolid, Ceftriaxone, Amoxicillin+clavulanic acid, Lincomycin and Oxytetracycline. After obtaining results where four of the five antibiotics were resistant: Linezolid, Ceftriaxone Lincomycin and Oxytetracycline where we chose oxytetracycline, which showed the highest resistance and is the most widely used antibiotic in livestock farming. An arduous investigation was carried out to identify resistance genes, tetD and tetA, then the DNA of L. garvieae was extracted by the method of Aljanabi & Martinez modified, the PCR technique was carried out with the corresponding primers of the genes and ended with an electrophoresis where it was not identified that it has tetD and tetA genes of resistance to oxytetracycline and finally it was investigated on the molecular mechanisms of resistance of L. garvieae to the different resistant antibiotics.Item Aislamiento, caracterización cultural, morfológica, patogénica e identificación de genes AVR en Cladosporium fulvum Cooke.(2023-12) Sánchez Ortiz, Aldo Martin; Leiva Mora, MichelGray mold caused by Cladosporium fulvum was described by Cooke in 1883, which develops in nightshades, specifically in tomato crops, causing damage from the youngest leaves to the top of the plant. The objective of this research was to "Isolate and characterize culturally, morphologically and pathogenically monosporic isolates of Cladosporium fulvum" obtained from signs of gray mold on Solanum lycopersicum leaves to identify the presence or absence of Avr genes in the cantons of the province of Tungurahua (Ambato, Baños, Cevallos, Mocha, Patate, Pelileo, Píllaro, Tisaleo). For isolation, the leaf printing method was applied in potato dextrose (PDA) culture medium with gentamicin sulfate, to describe the cultural and morphological characteristics, the microculture method was used accompanied by the observation of fungal structures under an optical microscope. of transmitted light. In the pathogenic characterization, the isolates of C. fulvum were activated The conidia were counted in the Neubauer chamber of the differential cultivars previously sown on substrates and the inoculum was applied with a manual sprayer. For amplification, DNA extraction and lyophilization of each of the isolates was carried out prior to development. of conventional PCR with two denaturations at 94°C with different cycles and an annealing at 55°C and 34 cycles with a final extension of 72°C. Obtaining that in the 8 cantons of the province of Tungurahua they all showed olive green colors on the obverse, while on the back it was black, with a superficial elevation, plush texture and irregular shape, lobed edges and in some isolates there was pigmentation and perspiration liquid, the mycelial growth in the colonies was obtained with a length of 5.93 µm, width 2.8 µm, hyphae 5.37 µm and conidiophores 32.97 µm average values. In the pathogenic characterization it was evident that the degree of affectation 1 represented between 1 to 5% of the surface of leaves with signs of C. fulvum, while 2 varied from 6 to 20% in the differential cultivars (Cf0-Cf2- Cf4- Cf5-Cf6), finally in the detection of the presence or absence of genes, the presence of the genes Avr2, Avr4e, Ecp2, Ecp4 and Ecp5 and total absence of the genes Avr4, Avr9 and Ecp1 were observed. Based on the results and the tests carried out, it was possible to create bases for the selection of resistance genes for the production of S. lycopersicum hybrids in the province of Tungurahua.Item Caracterización molecular de genes de avirulencia del agente causal del moho gris de la hoja de Solanum lycopersicum L en la provincia de Tungurahua(2022-08) Flores Yanchaliquin, Diana Carolina; León Gordón, Olguer AlfredoCladosporium fulvum or leaf mold is a pathogen described by Cooke in 1883 that develops easily in tomato crops due to favorable temperature and humidity conditions. For the present investigation, the objective was to "Molecularly characterize avirulence genes of the causal agent of the mold of the leaves of Solanumlycopersicum L in the province of Tungurahua". For the molecular characterization of genes, fungal DNA was extracted from each canton (Ambato, Baños, Cevallos, Mocha, Patate, Pelileo, Píllaro, Tisaleo), which consisted of lyophilizing the mycelium, crushing and placing the respective buffers (Buffer A Naoh + Tween 20 and Buffer B Tris Hcl + Edta) and the extracted DNA was stored at -20°C. For amplification using conventional PCR, the amplification conditions for each Avr andEcp and their process were standardized, using dNTPs, Taq DNA Polymerase,forwrad-reverse primers, and ultrapure water with 25 μl of reaction. For visualization,1% agarose gels were made with the running buffer TAE 1X and a 100bp molecular marker (Invitogen) was used. Having as results the presence of the Avr2 and Avr4E avirulence gene, thus also verifying the absence of Avr4 and Avr9, while for extracellular proteins Ecp2, Ecp4 and Ecp5 are presented so that for Ecp1 it does notrecognize the presence of the geneItem Presencia de enterobacterias portadoras de genes de resistencia a antibióticos emergentes procedentes de aguas de riego y superficiales del Ecuador, año 2019.(2020-09) Mendoza León, David Israel; Calero Cáceres, William RicardoIn order to assess the degree of biological pollution of water in the country, the aim of this researching was the identification of bacteria resistant to emerging antibiotics from surface waters of 5 provinces of Ecuador. The sampling points for the analysis were determined based on a space-temporal study, based on historical data by the National Institute for Public Health Research (INSPI) based on to the presence of population, hospitals, crops, animal production and industries. The collection of samples was carried out from rivers, irrigation canals, collection points. About 71 samples were taken at strategic points based on US EPA-600 / 4- 79-0120 "Methods for the Collection and Analysis of Water and Waste", of which 40 (24 from the river and 16 from the irrigation canal ) corresponded to water and 31 samples (16 from the river and 15 from the irrigation canal) corresponded to sediments. Seeds were performed on Chromocult® and MacConkey agar enriched with 2 µg of Ceftriaxone. From the samples, the indicators of faecal contamination were E. coli and total coliforms, thus E. coli and ceftriaxone resistant total coliforms (2 µg / mL). 10 genes (mcr-1 mcr-2 mcr-3 mcr-4 mcr-5, blaKPC, blaNDM, blaIMP and blaVIM) were detected for antibiotic resistance in the PCR process, byconfirming the amplification they were identified and identified 9 positive isolates to emerging antibiotic resistance genes (8 for mcr-1 and, 1 for blaKPC).Item Efecto de la aplicación de gallinaza de compostaje tradicional y comercial sobre las densidades de bacterias resistentes y genes de resistencia a antibióticos en cultivo de rábano (Raphanus sativus) y lechuga (Lactuca sativa)(2020-09) Cruz Morales, Carla Marisol; Calero Cáceres, William RicardoPoultry manure is traditionally used as a good source of nutrients and organic matter in agricultural soils, especially in organic-farming systems. However, these materials can cause adverse effects on general soil conditions, since they contain high densities of potentially pathogenic enteric bacteria; such as antibiotic resistant bacteria (ARBs) and their corresponding antibiotic resistance genes (ARGs). The use of these materials may represent a probable route of dissemination of pathogenic microorganisms and their determinants through the food chain. This thesis is part of the project "EVALUACIÓN DE LA DISEMINACIÓN AMBIENTAL DE GENES DE RESISTENCIA A ANTIBIÓTICOS POR MEDIO DEL ESTIÉRCOL DE GALLINA" (HCU 1752-CU-P-2019). The aim of this thesis is the evaluation of the effect of the addition of traditional and commercial composting on the densities of ARBs and ARGs in vegetables (lettuce and radish). The virulence and resistance mechanisms of the detected E. coli isolates were evaluated. The results suggest that traditional composting chicken manure influences to a greater extent the spread of ARBs and ARGs in relation to commercial chicken manure. The E. coli isolates presented an antibiotic multiresistance profile. The transconjugation experiments suggest the mechanisms of antibiotic resistance of these bacteria were found encoded in chromosomes and not in plasmids. On the other hand, the qPCR technique was used to detect ARGs, which showed very high levels of detection for sul1 and blaTEM and low levels of detection to qnrS, tetW, ermB and 16S rDNA. The dissemination of ARBs and ARGs from traditional and commercial poultry manure to food is plausible, representing this a potential route of dissemination of antibiotic resistance determinants through the food chain.Item Determinación de la presencia de genes de resistencia a betalactámicos y evaluación de diversidad clonal en aislados de Escherichia coli de origen canino de la ciudad de Ambato(2020-03-19) Garcés Arias, Xiomara Sthefanny; Calero Cáceres, William RicardoThe evolution and spread of multiresistant bacteria is considered prioritary, considering their impact in public health and animal safety worldwide. Therefore, the detection of reservoirs, routes of dissemination and evolution of these bacteria is aimed in order to limit their negative effects on public and animal health. Extended-spectrum-betalactamase Escherichia coli producer is considered an emerging health problem. In Ecuador, there are few available data on the molecular epidemiology of those of ESBL- E. coli producing bacteria in animals and environment. In the present study, the presence of beta-lactam resistance genes was evaluated as well as the clonal diversity of E. coli isolates of canine origin from the city of Ambato. The highest presence of beta-lactam resistance genes occurred in dogs with owners, in contrast to stray dogs. The most frequently detected genes were blaCTX-M and blaTEM. The blaSHV and blaCMY genes had a lower detection rate, however, these were found in combination with the blaCTX-M and blaTEM genes, indicating that they are isolates with considerable virulence. The analysis of clonal relatedness through the amplification of palindromic repetitive regions (GTG5) detected that the isolates comes from different lineages, discarding the hypothesis that suggest the presence of multi-resistant clones with a common ancestor, suggesting that their genotypes were acquired through recent horizontal genetic transmission. Dogs can act as a silent reservoir of microorganisms with antibiotic multiresistance genotypes. It is necessary an extended review of waste management procedures is necessary, as well as interactions with humans and animals under certain circumstances like immunological disorders.Item Análisis bioinformático de cuatro secuencias de genoma completo de Salmonella entérica de origen avícola(2020-03-19) Villacis Grijalva, Joyce Elizabeth; Calero Cáceres, William RicardoThe application of molecular epidemiological tools applied to microbiology provides valuable evidence to elucidate the origin of clinical outbreaks. Additionally, provides information that supports the opportune intervention and takes of control measures. Nowadays, the raising of new omics technologies, as new generation sequencing (NGS) of complete genomes and the development of bioinformatics tools are improving the knowledge and the understanding of the microbiology. The implementation of these technological tools in microbiological studies is allowing to study in real-time and low cost the evolution and dissemination of new pathogens that are circulating across our geographical area. Salmonella enterica represents one of the most important pathogens that deserves special attention, considering its implications in clinical and productive sectors. Considering these reasons, it is important the realization of epidemiological studies using bioinformatic tools that allow analyze the origin, dissemination, virulence mechanisms and resistance genotypes. In this study, four S. enterica serovar Infantis and Kentucky from layer poultry origin obtained in Tungurahua and Cotopaxi were analyzed through WGS and the bioinformatic tools of Center of Genomic Epidemiology (University of Denmark) and BaseSpace (Illumina Inc). All the strains were categorized as human pathogens. According to their sequence-type, it belongs to the ST32 (Infantis) and ST152 (Kentucky). The strains harbor a plethora of antibiotic resistance genes. It is worth noting the presence of the beta-lactamases blaCTX-M-148 and blaCTX-M-65, which show an emerging raising in clinical and animal origin isolates around the world. Finally, our sequences were compared with sequences of analogous bacteria from different origins and geographical locations through phylogenetic trees. Our sequences are related to clinical isolates from the United States, suggesting that their share a common ancestor.Item Presencia de genes de resistencia a antibióticos emergentes en aislados de Escherichia coli procedentes de aguas superficiales y sedimentos del Río Cutuchi y del Canal Latacunga-Salcedo-Ambato(2018) Mero García, Manuel Efraín; Calero Cáceres, WilliamDirección de Innovación y Emprendimiento según el CÓDIGO ORGÁNICO DE LA ECONOMÍA SOCIAL DE LOS CONOCIMIENTOS, CREATIVIDAD E INNOVACIÓN, Aprobado Registro Oficial Nº 899 diciembre de 2016 Artículo 11.- “Atribuciones de la entidad encargada de la gestión de la propiedad intelectual y de la protección de conocimientos tradicionales”, se encuentra gestionando, el proceso de trámite del Registro como Derecho de Autor, y reserva de tesis relevante, en el Servicio Nacional de Derechos Intelectuales (SENADI).