Caracterización de cepas clínicas de Staphylococcus Aereus aisladas en el Hospital Rural de Gambo (Etiopía). Efecto in vitro de compuestos dendríticos
- Autores: Cristina Verdú Expósito
- Directores de la Tesis: Juan Soliveri de Carranza (dir. tes.), Jorge Pérez Serrano (codir. tes.)
- Lectura: En la Universidad de Alcalá ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: José Luis Copa Patiño (presid.), Mercedes Marin Arriaza (secret.), Silvia Moreno Pinilla (voc.)
- Programa de doctorado: Programa de Doctorado en Ciencias de la Salud por la Universidad de Alcalá
- Materias:
- Enlaces
- Resumen
- español
Staphylococcus aureus es una bacteria ampliamente distribuida por todo el mundo, causante de un gran número de infecciones, que en ocasiones producen brotes, tanto de infecciones adquiridas en la comunidad como nosocomiales. Se trata además de un microorganismo con gran capacidad de adquirir nuevas características que lo hacen más adaptado y versátil, incluyendo resistencias a antimicrobianos y genes de patogenicidad. Lo anteriormente descrito, junto con el aumento de movimiento poblacional por todo el mundo, generan la necesidad de vigilancia de estas infecciones, para prevenir o superar problemas de salud pública a nivel global.Entre los años 2014 y 2018 se aislaron 80 cepas de S. aureus de pacientes de un hospital rural de Etiopía. Dichas cepas causaban infecciones, principalmente adquiridas en la comunidad, que presentaban una elevada virulencia y en ocasiones recurrencia, siendo en su mayor parte infecciones de piel y partes blandas.Los principales objetivos de esta tesis, fueron en primer lugar caracterizar genotípica y fenotípicamente dichas cepas, e intentar revelar la relación entre estas y su virulencia, así como sus manifestaciones clínicas y la evolución o procedencia de dichas cepas. También tratamos de estudiar el efecto de estas cepas sobre cultivos celulares humanos para poner de manifiesto la virulencia previamente determinada. Por otro lado, se pretendía desarrollar tratamientos alternativos a antibióticos, o limitar el uso de estos, debido a la facilidad de este microorganismo de manifestar resistencias.Para el estudio fenotípico, se estudiaron algunos factores de virulencia mediante técnicas bioquímicas o de cultivo, como son la formación de biopelículas, la sensibilidad antimicrobiana, la actividad DNasa y lecitinasa, y el tipo de hemólisis. Los resultados demostraron una baja prevalencia de resistencias a antibióticos y sólo una cepa SARM, justificado por el uso limitado de estos fármacos en la zona rural de estudio debido a la dificultad de adquisición de estos. Sin embargo, el análisis de factores de virulencia debidos a enzimas determinó una elevada actividad DNasa y un alto porcentaje de cepas que expresaron actividad lecitinasa. También se identificó una elevada proporción de cepas que forman biopelícula, en algunos casos muy altos.El estudio de caracterización molecular, realizado mediante análisis por MLST, estudio filogenético y detección de genes de virulencia, confirmó la identificación de cepas como S. aureus y determinó una elevada diversidad de STs, la mayor parte de ellos descritos por primera vez en Etiopía o en todo el mundo. Sin embargo, los estudios de filogenia establecieron relaciones cercanas entre muchos de las cepas. La detección de genes de virulencia demostró una elevada prevalencia de cepas que presentaban los genes pvl, hla and fnba. Además, se corroboró la relación entre el ST152 y el gen pvl, previamente descrita en otras zonas de África.Para estudiar el efecto de algunas de estas cepas seleccionadas sobre células humanas, se midieron los niveles producidos de la enzima lactato deshidrogenasa (LDH), para determinar la citotoxicidad, y se cuantificaron los niveles de TNF¿, IL-1ß y proteínas totales, así como de expresión de los genes TNF-¿, IL-1ß e IL-6, sobre co-cultivos de células similares a macrófagos y 11 cepas de S. aureus de Etiopía seleccionadas en función de su caracterización previa. Los resultados identificaron la cepa más inocua que coincidía con los resultados previos obtenidos de caracterización, pero no determinaron una relación clara entre los factores de patogenicidad encontrados en cada una de las cepas y la virulencia producida sobre células similares a macrófagos.
- English
Staphylococcus aureus is a bacterium widely distributed throughout the world, that causes many infections, which sometimes produce outbreaks of both community-acquired and nosocomial infections, being the latter more prevalent in developed countries. It is in these countries where their study and knowledge are extensive, opposite to what happens in developing countries, where the lack of resources and the presence of other endemic etiological agents displace the attention that it requires. It is also a microorganism very capable of acquiring new features that make it more adapted and versatile, including resistance to antimicrobials as well as pathogenicity determinants. This scenario together with the increase in the population movement worldwide generates the need for surveillance of infections caused by S. aureus, to prevent or overcome public health problems globally. Eighty strains of S. aureus were isolated from patients in a rural hospital in Ethiopia between 2014 and 2018. These strains caused highly virulent and sometimes recurrent infections mainly acquired in the community affecting infections mostly skin and soft tissue. The work conducted for this thesis has been divided into chapters as follows: Chapter 1 describes feature of S. aureus such as the taxonomic context, most important structural features, metabolism, mechanism of pathogenesis, main manifestations and diseases it produces, some of the main diagnostic identification techniques, antimicrobial agents most frequently used for treatment, and general data on epidemiology, as well as the geographical framing of the study, due to the source of the strains. This chapter also includes the main aims of the thesis, the first of which was to characterize these strains genotypically and phenotypically, trying to reveal the relationship between them and their virulence, as well as their clinical manifestations and their evolution or source. We also try to study the effect of some of these strains on human cell cultures to highlight previously determined virulence. Finally, it was intended to develop alternatives to antibiotic treatments, or limit the use of these, due to the ease of this microorganism to show resistance. In Chapter 2 the phenotypic characterization of these strains is performed, thus describing some of the main virulence factors revealed by biochemical or culture techniques, such as the formation of biofilms, antimicrobial sensitivity, DNase and lecithinase activity and the type of hemolysis. The methodology used to isolate and preserve the strains for further study is described, as well as the first step to identify them as S. aureus and the procedures used to study each of the factors named above. The antimicrobial sensitivity study showed a low prevalence of antibiotic resistance and the presence of a single MRSA strain (S. aureus resistant to methicillin), a fact justified by the limited use of these drugs in the rural study area due to the difficulty in acquiring them. However, the analysis of virulence factors due to enzymes determined a high DNase activity and a high percentage of strains that express lecithinase activity. A high proportion of strains that form biofilm was also identified, in some cases with very high values. Chapter 3 refers to the molecular characterization of the strains above mentioned, emphasizing on the molecular techniques as highly valuable tools for studying infections. It also describes the concept of molecular typification and phylogeny, as well as their application to the study of bacterial strains. The main virulence factors due to extracellular toxins and the genes that encode them are also described. Different techniques were used for these molecular studies requiring genomic DNA extraction of each strain and subsequent PCR amplification of diverse genes, some of which were also sequenced. Molecular typification was accomplished by multilocus sequence typing (MLST) and data were used for phylogenetic studies. Moreover, molecular evidence of the presence of the virulence gene hla (α hemolysin), tst (toxic shock syndrome toxin), pvl (Panton-Valentine Leukocidin), fnbA (fibronectin A adhesion protein) and mecA (resistance to methicillin) were also molecularly detected by PCR. Results confirmed the identification of the strains as S. aureus strains showing the MLST analysis a high diversity of STs, most of them described for the first time in Ethiopia or worldwide. However, phylogenetic studies established close relationships between many of these STs. The detection of virulence genes revealed a high prevalence of strains presenting genes pvl, hla and fnbA. In addition, the relationship between ST152 and the pvl gene, previously described in other areas of Africa, was corroborated. Chapter 4 studies the effect of some of the selected strains from S. aureus isolates on human cells. In this sense, bacterial pathogenesis and cellular immune response are described, as well as some of the main cytokines involved in it, such as tumor necrosis factor α (TNFα), interleukin 1β, and IL-6. It also explains the techniques of cell cultures and their use to study the cytotoxic effect and cellular response against infection causing bacterial strains, the aim of this chapter. Cytotoxicity was assessed by quantifying both the levels of lactate dehydrogenase (LDH), TNFα and IL-1β referred to total proteins produced and the levels of expression of TNF-α, IL genes -1β and IL-6 on co-cultures of macrophage-like cells and 11 strains of S. aureus from Ethiopia previously selected for their features. The results identified the most innocuous strain coinciding with the previous results of characterization but did not point to a clear relationship between the pathogenicity factors found in each of the strains and the virulence produced on macrophage-like cells. In Chapter 5, due to the increase in antimicrobial resistance over recent decades, as well as the ease of S. aureus in acquiring such resistance, the in vitro effect of dendritic systems on this bacterium was studied, as well as its combination with the cloxacillin antibiotic. These studies were conducted in a methicillin-sensitive (SASM) and a methicillin-resistant (SARM) collection strain, as well as in a methicillin-resistant strain of an isolate from Ethiopia. The minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) of five dendritic compounds and cloxacillin were analyzed by the plate dilution method. Based on the individual results, the MIC and MBC of the combination of each dendritic compound with cloxacillin against the SASM strain were analyzed. From these results the best combinations were selected to test them with the resistant strain. The results indicated that compound BDTR003 was the most effective individually showing bactericidal activity against the three S. aureus strains tested. However, the combination of cloxacillin and the BDTL050 dendrimer presented a synergistic effect on the SARM strain. This result opens up a possibility for new treatments against infections caused by resistant bacteria, which will be confirm in future studies elucidating the cause of such effectiveness. Finally, some general conclusions defined by the whole of the work carried out during the thesis are drawn, in which it is emphasized that, despite the low prevalence of antimicrobial resistance, strains of S. aureusisolated in a rural area of Ethiopia present pathogenicity determinants that, together with the socioeconomic and hygienic scenario of the population studied, could explain its high virulence.
- español
