Tesis Medicina Veterinaria
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Item Identificación de genes de virulencia (ipaH) en Shigella spp aisladas de carne de pollo que se expende en el cantón Ambato(2025-02) Toapaxi Chiquito Elizabeth Fernanda; Cruz Quintana Sandra MargaritaShigella spp. infection is usually contracted by consuming contaminated food, with poor hygiene in handling it being a key risk. Likewise, unsanitary conditions in animal slaughter can be an important source of meat contamination. This study focused on the identification of a virulence gene in Shigella spp. isolated from chicken meat marketed in the Ambato canton. Of the 17 Shigella strains used, they were previously isolated and identified using molecular techniques in previous research and were kept in the Microbiology and Molecular Biology Laboratory of the Faculty of Agricultural Sciences of the Technical University of Ambato. In particular, the ipaH gene, a key virulence marker of this bacteria, was investigated. For the detection and analysis of this gene, advanced molecular techniques were used, including the Polymerase Chain Reaction (PCR) and agarose gel electrophoresis. First, pure and diluted Shigella spp DNA, previously extracted from chicken meat samples, was used for subsequent amplification. In the PCR phase, a specific primer designed to amplify the target sequence corresponding to the ipaH gene, with a size of 423 base pairs (bp), was used. In conclusion, the literature review revealed a remarkable diversity of virulence factors, and the presence of the ipaH gene is included in all the references consulted. However, the results obtained in the PCR did not show amplification of the gene, which reflected a 0% prevalence of the ipaH gene in Shigella isolates previously obtained from chicken meat. Finally, according to epidemiological data collected in the last five years, the presence of shigellosis has been recorded in Ecuador. In this regard, it is relevant to note that, both in 2023 and in the research carried out in 2024, the existence of this disease in the province of Tungurahua is not reported, nor is the presence of the related gene in the isolated strains used for the study. However, it is observed that these strains continue to circulate in neighboring provinces.Item Detección de genes de resistencia a antibióticos betalactámicos (blaTEM y blaCTX-M) en Shigella spp. aislada de carne de pollo que se expende en la ciudad de Ambato(2025-02) Rubio Obando Gregory Matheo; Cruz Quintana Sandra MargaritaChicken meat is a widely consumed food that can act as a vehicle for the transmission of antibiotic-resistant pathogenic microorganisms. In this context, the main objective of this research was to determine the presence of the beta-lactam resistance genes blaTEM and blaCTX-M in Shigella spp. strains isolated from chicken meat sold in the city of Ambato. 17 samples of chicken meat were collected from slaughterhouses (9 samples) and informal sales points (8 samples), and phenotypic and molecular identification of the strains was performed. The Shigella species identified were S. sonnei (58.82%), S. flexneri (29.41%) and S. dysenteriae (11.77%). The blaTEM gene was detected in 23.53% of the samples analyzed, while blaCTX-M was present in 88.23%. Both resistant strains were found in S. sonnei and S. dysenteriae, but not in S. flexneri. The highest presence of these genes was found in samples from unregistered sales points, suggesting that inadequate hygienic-sanitary conditions contribute to the spread of resistant bacteria. The results obtained partially agree with previous studies carried out in other regions of Ecuador and Latin America, where the S. flexneri species has historically been the most prevalent. However, a change in epidemiology is observed, with a predominance of S. sonnei in the samples analyzed, which could be explained by the capacity of this species to acquire resistance genes more easily. This study highlights the importance of implementing sanitary control and surveillance measures in the chicken meat production and marketing chain, in order to prevent the spread of bacteria resistant to beta-lactam antibiotics and reduce the associated risks to public health.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 Presencia de genes de resistencia a fluoroquinolonas y betalactámicos en Proteus mirabilis aislados en carne de pollo en el cantón Ambato(2024-08) Fiallos Barros, Lizbeth Estefanía; Cruz Quintana, Sandra MargaritaThis study focused on the identification and analysis of antimicrobial resistance genes in Proteus mirabilis strains isolated from chicken meat samples from the city of Ambato. In particular, two key genes were investigated: gyrA associated with resistance to fluoroquinolones, and blaTEM-1, related to beta-lactam resistance. For the detection and analysis of these genes, advanced molecular techniques were used, including Polymerase Chain Reaction (PCR) and gel electrophoresis. First, DNA extraction from the samples was performed using standardized methods that ensure purity and adequate concentration of DNA for subsequent amplification, in the PCR phase, specific primers were used to amplify the target sequences of the gyrA and blaTEM-1 genes. The results showed successful amplification of the gyrA gene in all samples analyzed, with a base size of 850 bp, confirming a 100% prevalence of this gene in Proteus mirabilis strains. On the other hand, the amplification of the blaTEM- 1 gene was not universal in all samples, it was detected in 58.3% of the strains analyzed. This finding suggests the presence of variability in beta-lactam resistance in the samples, indicating that although blaTEM-1 is a prevalent gene it is not the only resistance mechanism present. This result underscores the need to consider other betalactam resistance genes and other resistance mechanisms in future studies. This research provides detailed insight into antimicrobial resistance, the high prevalence of the gyrA gene and the considerable presence of the blaTEM-1 gene indicate significant resistance to fluoroquinolones and beta-lactams, respectively. These findings emphasize the importance of continuous and detailed monitoring of antimicrobial resistance in the food chain to implement effective control strategies and protect public health.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 Evaluación de la presencia de los genes de resistencia a antibióticos betalactámicos y glucopéptidos en Mammaliicoccus sciuri aislado de la carne de pollo que se expende en Ambato(2024-08) Barreno Mayorga, Jessica Fernanda; Cruz Quintana, Sandra MargaritaAntibiotic resistance represents a significant public health problem due to the ability of bacteria to cause difficult-to-treat infections. The objective of this research is to detect the presence of resistance genes to beta-lactam antibiotics (amoxicillin + clavulanic acid) and glycopeptides (vancomycin) in the bacteria Mammaliicoccus sciuri isolated from chicken meat sold in Ambato. The research was carried out by collecting Mamamlicocus samples isolated from chicken meat and analyzing them in the laboratory to determine the presence of the aforementioned resistance genes, using PCR, DNA extraction and electrophoresis. Obtaining as results the presence of resistance genes in beta-lactams with an amplification of the band of 162 bp but no amplification of glycopeptides was found, which means that there are different ways for the existence of resistance. The identification and monitoring of these genes are crucial to evaluate food safety and prevent the spread of resistant bacteria in the final food chain.Item Valoración de los perfiles antimicrobianos y genes de resistencia de Salmonella enterica aislada en carne de pollo expendida en Ambato”(2024-08) Aráuz Paredes, David Isaac; Cruz Quintana, Sandra MargaritaSalmonella enterica is a versatile bacteria that perceives very refined antimicrobial resistance mechanisms, causing a worldwide problem with regard to public health, which is why it should be investigated according to the “One Health” approach, which unites human, animal and environmental health professionals to mitigate the development of bacterial resistance, because the routes of contamination of these bacteria are through foods of animal origin, whose spread is more effective due to the poor hygiene standards implemented in slaughter sites. The objective of this research was to assess the antimicrobial profiles and resistance genes of Salmonella enterica isolated in chicken meat sold in Ambato. A Salmonella enterica strain was used, from which two samples were obtained (Salmonella enterica subespecie enterica serovar Enteritidis strain ATCC 13076 and Salmonella enterica strain FDAARGOS_768), where they were developed in two study phases. In the first phase, the use of the Kirby Bauer method was highlighted to evaluate the antibioresistance of the strain against a group of 5 antibiotics, these being Amoxicillin plus clavulanic acid (AMC), Ciprofloxacin (CIP), Gentamicin (GM), Oxytetracycline (OT) and Sulfamethoxazole plus trimethoprim (SXT), of which were classified according to the size of their inhibition zone into Resistant, Intermediate and Sensitive, following the regulations imposed by the CLSI. The results obtained were total resistance to OT, intermediate resistance to CIP and sensitivity to GM, AMC and SXT, the latter acting effectively against Salmonella enterica. In the second phase, only Oxytetracycline was taken to observe the presence of resistance genes, since it was the only antibiotic that was resistant. To visualize the resistance genes, the tet(4) gene primers were used, due to the high prevalence rate in previous research, and the same PCR conditions with which the gene was obtained were also applied. However, the results were negative without the presence of the chosen gene, for this reason it was considered pertinent to explain the molecular mechanisms that confer resistance to the bacteria against each group of antibiotics, starting with tetracyclines and ending with sulfonamides.Item Evaluación de antibiorresistencia en bacterias grampositivas aisladas en carne de pollo que se expende en el cantón Ambato, provincia de Tungurahua(2024-02) Velastegui Viteri, Cristian David; Cruz Quintana, Sandra MargaritaThe present investigation was carried out with the main purpose of evaluating the antibacterial resistance in Gram positive bacteria (Vagococcus lutrae, Mammaliicoccus sciuri and Kurthia gibsonii) isolated in chicken meat sold in Ambato Canton, province of Tungurahua, because bacterial resistance to antibiotics is a specific aspect of the natural evolution of bacteria, The study was carried out with strains of (Vagococcus lutrae, Mammaliicococcus sciuri and Kurthia gibsonii) that were isolated and molecularly identified from chicken meat sold in the canton of Ambato from previous investigations, which are kept in the Laboratory of Microbiology and Molecular Biology of the Faculty of Agricultural Sciences of the Technical University of Ambato. It was found that there is the presence of antibioresistance in gram-positive bacteria, specifically in strains of Vagococcus lutrae, Mammaliicoccus sciuri and Kurthia gibsonii, isolated from samples of chicken meat marketed in the Ambato Canton, province of Tungurahua. Likewise, the results obtained by the agar diffusion method revealed significant patterns of antibiotic resistance in the Gram-positive bacteria studied. Resistance profiles against various antibiotics used in both Veterinary and Human Medicine were identified, highlighting the importance of addressing resistance in an interdisciplinary context and the analysis of virulence factors in strains of Vagococcus lutrae, Mammaliicococcus sciuri and Kurthia gibsonii provided valuable information on the possible risks associated with these bacteria in chicken meat. Keywords: EVALUATION, ANTIBIORRESISTANCE, BACTERIA, GRAM POSITIVE, CHICKEN MEAT, AMBATO, TUNGURAHUA.Item Evaluación de antibiorresistencia de cepas de Shigella spp aisladas en carne de pollo en la ciudad de Ambato, provincia de Tungurahua(2024-02) Soto Villacis, Joselyn Anabel; Cruz Quintana, Sandra MargaritaShigella spp exhibits remarkably high and highly developed antibiotic resistance mechanisms, which constitutes a global challenge for public health. One of the main routes of transmission of this resistance is observed in foods of animal origin, highlighting that the lack of hygiene and sanitary control in slaughter centers contributes to its spread. The objective of this research was to evaluate the antibioresistance of Shigella spp strains isolated from chicken meat in the city of Ambato, Tungurahua Province. We worked with 17 strains of Shigella spp subjected to two phases of research. In the first phase, the Kirby Bauer phenotypic method (diskplate) was used to determine the resistance, intermediate or susceptibility of the strains to a group of drugs that includes Azithromycin (AZM), Gentamicin (CN), Ceftriaxone (CRO), Amoxicillin + clavulanic acid (AMC) and Ciprofloxacin (CIP). The results were analyzed using the Kruskal Wallis test, complemented by the Mann Whitney U test. A resistance of 82.4% was obtained for CIP, 64.7% for AMC, 29.4% for CRO, 23.5% for CN and 5.9% for AZM. In the second phase, the Minimum Inhibitory Concentration (MIC) of the strains that showed sensitivity in phase 1 to the aforementioned antibiotics was determined. The results were analyzed descriptively using curves, establishing the MICs for AZM 160 μg/ml, CN 10 μg/ml, AMC 160 μg/ml, CRO 1.25 μg/ml and CIP 20 μg/ml, following the CLSI cut-off points in 2016. It was considered essential to investigate the resistance mechanisms exhibited by Shigella spp. First, we focused on fluoroquinolones, as this group showed significant resistance due to the presence of chromosomal mutations. In addition, resistance related to the production of ESBL was observed for beta-lactams, as well as the alteration of the target site present in aminoglycosides, the existence of genes such as mphA or ermB in macrolides. Keywords: Shigella spp, antibioresistance, MIC, mechanisms, antibioticsItem Evaluación de la sensibilidad a los antibióticos y factores de virulencia de bacterias Gram negativas aisladas de la carne de pollo que se expenden en el cantón de Ambato (Comamonas kerstersii, Aeromonas veronii, Pseudomonas aeruginosa)(2024-02) Ramírez Pérez, Juan José; Cruz Quintana, Sandra MargaritaThe following research work was developed in the Laboratories of the Technical University of Ambato, with the objective of evaluating the sensitivity to antibiotics and virulence factors of the following bacteria: Comamonas kerstersii, Aeromonas veronii, and Pseudomonas aeruginosa, isolated from meat of chicken that is spent in the Ambato canton. We worked with 6 samples, carrying out the management of the experiment in three phases. The statistical method used was the Kolmogorov Smirov method to determine if there is a normal distribution. Demonstrating the results, the following data: Pseudomonas aeruginosa and Comamonas kerstersii have greater sensitivity to the antibiotics used, while Aeromonas veronii showed resistance. The molecular mechanisms of resistance vary according to the type of bacteria and the antibiotic. Finally, as a defense mechanism, bacteria develop virulence factors to evade the response of the body's immune system. Keywords: Comamonas kerstersii, Aeromonas veronii, and Pseudomonas aeruginosa, sensitivity, resistance, antibiotic, bacteria, virulence factors.