Carrera de Biotecnología
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Item Análisis genómico de Escherichia coli portadoras de genes de resistencia mcr-1 y blaOXA-48 de origen ambiental ecuatoriano utilizando secuenciación de tercera generación(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2024-02) Villacís López, Keyla Monserrath; Calero Cáceres, William RicardoThe present research emphasizes the importance of analyzing antimicrobial resistance (AMR) both globally and in Ecuador, focusing on the analysis of two complete genome sequences of E. coli strains (62A and 17A) of environmental origin, which harbor resistance genes. The relevance of this work in global public health is significant, highlighting the urgency to address this issue. Using the Oxford Nanopore Technologies (ONT) sequencing technique and bioinformatics tools, a detailed characterization of the chromosomes and plasmids of the strains was achieved. The analysis was carried out on the platform of the Center for Genomic Epidemiology (CGE-DTU), allowing the identification of resistance genes, virulence factors, plasmids, sequence types, including multilocus sequence typing (MLST). Additionally, the genetic environment was studied using DiGAling and phylogeny through the cgMLST method of the Galaxy platform, using the iTOL tool for visualization. The results revealed that both strains have an approximate genomic size of 4.8 Mb, and the resistance genes are located on IncFII type plasmids. They also share phylogenetic profiles and antibiotic resistance, particularly highlighting the E. coli 17A strain, being the first in Ecuador reported with the blaOXA-48 gene. These discoveries are vital for the surveillance and control of E. coli, underscoring the importance of an integrated One Health approach, which considers the relationship between human, animal, and environmental health; allowing for coordinated strategies against the challenges of antibiotic resistance.Item Análisis genómico de Escherichia coli portadoras de genes de resistencia mcr-1 y blaOXA-48 de origen ambiental ecuatoriano utilizando secuenciación de tercera generación(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2024-02) Jaramillo Mariño, Vivian Arlette; Calero Cáceres, William RicardoThe present research emphasizes the importance of analyzing antimicrobial resistance (AMR) both globally and in Ecuador, focusing on the analysis of two complete genome sequences of E. coli strains (62A and 17A) of environmental origin, which harbor resistance genes. The relevance of this work in global public health is significant, highlighting the urgency to address this issue. Using the Oxford Nanopore Technologies (ONT) sequencing technique and bioinformatics tools, a detailed characterization of the chromosomes and plasmids of the strains was achieved. The analysis was carried out on the platform of the Center for Genomic Epidemiology (CGE-DTU), allowing the identification of resistance genes, virulence factors, plasmids, sequence types, including multilocus sequence typing (MLST). Additionally, the genetic environment was studied using DiGAling and phylogeny through the cgMLST method of the Galaxy platform, using the iTOL tool for visualization. The results revealed that both strains have an approximate genomic size of 4.8 Mb, and the resistance genes are located on IncFII type plasmids. They also share phylogenetic profiles and antibiotic resistance, particularly highlighting the E. coli 17A strain, being the first in Ecuador reported with the blaOXA-48 gene. These discoveries are vital for the surveillance and control of E. coli, underscoring the importance of an integrated One Health approach, which considers the relationship between human, animal, and environmental health; allowing for coordinated strategies against the challenges of antibiotic resistance.Item Obtención del péptido antimicrobiano aedesina por medio de un sistema de autoescisión mediado por inteina, utilizando un sistema de expresión en Escherichia coli BL21 (DE3)(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2023-09) Mejía Bonilla, Dimitri Serge; García Solís, Mario DanielAntibiotics have significantly improved people's lifestyle since their implementation. However, at present, the indiscriminate use of these drugs has generated a health crisis due to antimicrobial resistance. The current trend shows an increase in the number of resistant strains that cannot be treated with conventional antibiotics, which has led to the need to develop a new generation of antibiotics. One of the most promising alternatives is the application of antimicrobial peptides, among which aedesin stands out. The present research project focuses on the implementation of a purification strategy for aedesin. This strategy consists of an intein-mediated self-cleavable system, which possesses a prohibition condition that disables self-cleavage in the presence of high salt concentrations. A heterologous system of synthesis in E. coli BL21 was used for expression. In conclusion, synthesis in bacteria offers several advantages compared to other methods of obtaining peptides. Furthermore, the strategy used for the purification of aedesin has proven to be effective. However, there are still challenges to be overcome, such as peptide toxicity and basal expression. Despite these drawbacks, the successful expression and purification of aedesin position it as an interesting candidate for analysis in future clinical studies.Item Evaluación de la presencia de genes de resistencia a carbapenémicos, tigeciclina y colistina mediada por plásmidos en aislados de Escherichia coli procedentes del río Cutuchi y del canal Latacunga-Salcedo-Ambato(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2023-09) Lagla Tipán, Nataly Gabriela; Calero Cáceres, William RicardoThe Cutuchi River and the Latacunga-Salcedo-Ambato irrigation channel exhibits significant contamination by antibiotic-resistant bacteria from wastewater discharges. This study analyzed E. coli isolates collected from April 2022 to March 2023 in both water bodies, focusing on the presence of last resort antibiotic resistance genes (carbapenems, tigecycline, colistin) transmitted by plasmids (LARGs). Under the research line of Microbiology and Biotechnology, the presence of LARGs in E. coli isolates was evaluated using conventional Polymerase Chain Reaction (PCR) and gel electrophoresis. Transconjugation tests were performed using the E. coli J53 as recipient strain. Phenotypic resistance profiles were determined by the Kirby-Bauer method and broth microdilution, and the genotypic profile was evaluated using conventional PCR, ERIC-PCR, and gel electrophoresis. Three isolates with mcr-1 colistin mobile resistance genes were detected, two carriers of carbapenem-hydrolyzing betalactamase SHV, and one carrier of carbapenemase OXA-48, which represents the first environmental isolation reported in Ecuador. Transconjugation determined the transmissibility of these genes by horizontal gene transfer (HGT). Phenotypic analysis revealed that the isolates carrying the evaluated genes are extremely drug-resistant (XDR). In addition, genotypic analysis determined that most of the studied genes are transferable by transconjugation and are associated with other antibiotic resistance genes.Item Evaluación de la presencia de genes de resistencia a carbapenémicos, tigeciclina y colistina mediada por plásmidos en aislados de Escherichia coli procedentes del río Cutuchi y del canal Latacunga-Salcedo-Ambato(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2023-09) Jerez Lluglla, Gabriela Estefania; Calero Cáceres, William RicardoThe Cutuchi River and the Latacunga-Salcedo-Ambato irrigation channel exhibits significant contamination by antibiotic-resistant bacteria from wastewater discharges. This study analyzed E. coli isolates collected from April 2022 to March 2023 in both water bodies, focusing on the presence of last resort antibiotic resistance genes (carbapenems, tigecycline, colistin) transmitted by plasmids (LARGs). Under the research line of Microbiology and Biotechnology, the presence of LARGs in E. coli isolates was evaluated using conventional Polymerase Chain Reaction (PCR) and gel electrophoresis. Transconjugation tests were performed using the E. coli J53 as recipient strain. Phenotypic resistance profiles were determined by the Kirby-Bauer method and broth microdilution, and the genotypic profile was evaluated using conventional PCR, ERIC-PCR, and gel electrophoresis. Three isolates with mcr-1 colistin mobile resistance genes were detected, two carriers of carbapenem-hydrolyzing betalactamase SHV, and one carrier of carbapenemase OXA-48, which represents the first environmental isolation reported in Ecuador. Transconjugation determined the transmissibility of these genes by horizontal gene transfer (HGT). Phenotypic analysis revealed that the isolates carrying the evaluated genes are extremely drug-resistant (XDR). In addition, genotypic analysis determined that most of the studied genes are transferable by transconjugation and are associated with other antibiotic resistance genes.Item Análisis de secuencias de genoma completo de Escherichia coli aisladas de vegetales y comida callejera de Ecuador(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2023-03) Sánchez Gavilanes, Lissette Katherine; Calero Cáceres, William RicardoWhole genome sequencing (WGS) is a fundamental tool for the analysis and detection of antimicrobial resistance genes (ARGs), virulence factors, and mobile genetic elements (MGEs) in bacterial genomes. Despite their advantages for epidemiological analysis, applying WGS in Ecuador is scarce and limited to Academia. The present study focused on in silico characterization of WGS of Escherichia coli isolated from vegetables and street food from markets in Ambato and Riobamba. The research line of this research is Microbiology and Biotechnology. For this study, paired-end read length sequences of 300 bp were used. In silico characterization was carried out through bioinformatics tools to detect ARGs, virulence, and MGE genes. Two isolates harbor the stx2 genes and various virulence factors. It was also identified that three strains carried the blaTEM and blaCMY genes, and different ADR determinants confer resistance to aminoglycosides, quinolones, tetracyclines, and sulfamethoxazole. Phylogenetic analysis indicated that the evolutionary interactions of resistance genes, virulence factors, and plasmid types did not correlate with the geographic origin and source of isolation, showing high diversity. These findings highlight the need to use WGS as a surveillance tool to analyze pathogenic E. coli in food matrices, taking into account a One Health perspective.Item Obtención de un péptido antimicrobiano autoescindible inducido por inteina utilizando un sistema de expresión en Escherichia coli(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2023-03) Cordones Chimborazo, Kerly Lissette; García Solís, Mario DanielNowadays, there is a high prevalence of antibiotic resistance, which poses a danger to public health. In response to this global problem, arises the creation of antimicrobial peptides (PAMs), due to the wide antibacterial spectrum they exhibit. Based on this, cecropins sequences were used to design a peptide through ancestral reconstruction, from which an ancestral peptide called CRP_N0 was obtained. The current methods for the production of PAMs are chemical synthesis, plant cell production and direct extraction from natural sources. However, these methods have a high economic value and a low efficiency. Therefore, the peptide CRP_N0 was expressed by an intein-induced self-splitting system linked by its N-terminal end to six histidines. The six-histidine label was used to purify the fusion protein intein-CRP_N0 and the removal of intein from the peptide CRP_N0. The construction was inserted into the plasmid pET-11b and expressed in Escherichia coli BL21 pG-Tf1. To maximize the expression of the fission protein, different induction times were tested with IPTG and the optimal expression time was determined to be 4 hours. In addition, an excision test was also conducted in which different temperature and pH conditions were evaluated to induce self-excision of inteine, where it was determined that at 25 degrees Celsius and a basic pH (10) a high yield is obtained in the cutting of intein.Item Caracterización de los perfiles de resistencia antimicrobiana de Escherichia coli aislada de mamíferos en cautiverio del EcoZoológico San Martín – Baños de Agua Santa(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2022-09) Medina Pujos, Anabell Yessenia; Calero Cáceres, William RicardoAntimicrobial resistance (AMR) has become a public health problem around the world; therefore, to prevent the emergence and spread of pathogenic bacteria, measures should be taken by adopting a One Health perspective. Around the world, one of the factors for the emergence of resistant pathogens has been the zoonotic exchange between animals and humans, and its association with the use of different antimicrobials in human and veterinary medicine. For this reason, the study of wild or captive species is essential. This research focuses on characterizing antimicrobial resistance profiles in E. coli isolates from captive mammals at the San Martín Eco Zoo in Baños de Agua Santa. From a total of 27 faecal samples collected, 90 strains were isolated. 32 strains presented resistance to more than three families of antibiotics; however, only strains E57 and E75 isolated from ocelots (Leopardus pardalis) showed resistance to six families of antibiotics. The antibiotics with the highest percentage of resistance were ampicillin (97.5), ceftriaxone (95), cefuroxime (95), trimethoprim/sulfamethoxazole (52.5), ceftazidime (47.5) and ciprofloxacin (37.5). The generated profiles showed the relationship between resistance to antimicrobials and the diet of the mammals studied, observing that those isolated from mammals with a meat-based diet were multi-resistant E. coli; In addition, the antibiotic treatment received helped with the presence of more resistant isolates. The results obtained indicate that captive species can be potential reservoirs of multi-resistant bacteria to antibiotics.