Regulación de la producción de DNA extracelular en Bacillus subtilis y estudio de su distribución en los cuerpos aéreos

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-06-2018Esta tesis tiene embargado el acceso al texto completo hasta el 27-12-2019El objeto de estudio de esta Tesis Doctoral fue la producción de DNA extracelular (eDNA) en la estirpe silvestre de Bacillus sutilis 3610. Con anterioridad en nuestro laboratorio se pudo demostrar que esta bacteria gram-positiva es capaz de producir elevadas cantidades de eDNA en respuesta a las señales de quorum sensing liberadas en fase exponencial tardía del crecimiento. Tras demostrar que la producción de eDNA y el desarrollo de la competencia temprana comparten la misma ruta de señalización celular, se planteó la hipótesis de que comS, un pequeño gen hasta ahora relacionado exclusivamente con el desarrollo de la competencia tardía, es el factor regulador responsable de la divergencia entre los fenómenos de competencia y producción de eDNA. En este trabajo hemos podido demostrar dicha hipótesis y describir por vez primera la regulación positiva que comS ejerce sobre los genes de motilidad. Al profundizar en el estudio de la relación existente entre los procesos de producción de eDNA, motilidad y competencia, nuestros resultados mostraron que la adquisición de motilidad en la población celular es necesaria tanto para la correcta producción de eDNA como para el desarrollo de la competencia. Finalmente, se investigó la producción de eDNA en el seno de las comunidades multicelulares producidas por Bacillus subtilis 3610. Nuestros resultados permiten descartar que el eDNA posea un papel estructural, a diferencia de lo que se ha descrito en otros microorganismos, al mismo tiempo que nos permitieron identificar por primera vez la presencia de una compleja estructura interna formada por micro-conductos. El estudio detallado de los mismos permitió comprobar la presencia de células con motilidad desplazándose en su interior. En conjunto, los resultados presentados en este trabajo de investigación han permitido describir nuevas e interesantes interacciones implicadas en los procesos de diferenciación celular, así como profundizar en la compleja estructura interna tridimensional desarrollada por las comunidades multicelulares de Bacillus subtilisExtracellular DNA production is a widespread feature present among many microorganisms. Particularly, the undomesticated strain B. subtilis 3610, has been shown to produce significant amounts of eDNA under very specific intracellular regulation and in response to quorum sensing signals at the end of the exponential phase. Previous work demonstrated that eDNA production and early competence share the same signaling pathway. That work led to the hypothesis that comS, a small gene exclusively described in relation to competence development, was the key factor regulating eDNA production. In this work, we demonstrate for the first time that comS is not only necessary for eDNA production, but it also regulates motility. Moreover, we studied the relationship between eDNA production, motility and competence and found out that motility is necessary for both eDNA production and competence development within the cell community. Finally, we studied the presence of eDNA in the multicellular structures created by B. subtilis communities and called aereal bodies or fruiting bodies, which are the living form they usually adopt in nature. Our results show that eDNA has not a structural role within aereal bodies formation. Moreover, we found a complex internal structure made up with micro-channels and filled with motile cells actively moving inside them. Overall, the results presented in this work describe new and interesting interactions related to cell differentiation processes, and contribute to the knowledge of the internal three-dimensional complex structure of the Bacillus subtilis multicellular communitie

Cost-effectiveness analysis of using innovative therapies for the management of moderate-to-severe ulcerative colitis in Spain

Background: To evaluate the cost-effectiveness of tofacitinib in comparison to vedolizumab for the treatment of moderate-to-severe ulcerative colitis (UC) after failure or intolerance to conventional therapy (bio-naive) or first-line biologic treatment (bio-experienced), from the Spanish National Health System (NHS) perspective. Methods: A lifetime Markov model with eight-week cycles was developed including five health states: remission, response, active UC, remission after surgery, and death. Response and remission probabilities (for induction and maintenance periods) were obtained from a multinomial network meta-analysis. Drug acquisition – biosimilar prices included – (ex-factory price with mandatory deductions), adminis- tration, surgery, patient management, and adverse event management costs (€, year 2019) were considered. A 3% discount rate (cost/outcomes) was applied. Probabilistic and deterministic sensitivity analyses (PSA) were conducted. Results: Tofacitinib was dominant versus vedolizumab (both in bio-naive and bio-experienced patients) entailing total cost savings of €23,816 (bio-naïve) and €11,438 (bio-experienced). Differences in quality- adjusted life-year (QALY) were smaller than 0.1 for both populations. PSA results showed that tofacitinib has a high probability of being cost-effective (bio-naïve: 82.5%; bio-experienced: 90.6%) versus vedolizumab. Conclusions: From the Spanish NHS perspective, tofacitinib could be a dominant treatment (less costly and more effective) in comparison to vedolizumab, with relevant cost savings and similar QALY gains

Health and economic impact of the correct diagnosis of transthyretin cardiac amyloidosis in Spain

Objective: to estimate the health and economic impact of the reduction in mortality and cardiovascular hospitalizations, associated with correct diagnosis of cardiac transthyretin amyloidosis (ATTR-CM), from the Spanish National Health System (NHS) perspective. Methods: a costs and effects analysis were performed (probabilistic Markov model) with time horizons between 1 and 15 years, comparing the correct diagnosis of ATTR-CM versus the non-diagnosis. Transition probabilities were obtained from the ATTR-ACT study (placebo arm) and from the literature. Costs and healthcare resources were obtained from Spanish sources ( 2019) and from a panel of Spanish clinical experts. Results: after 1, 5, 10 and 15 years, the diagnosis of ATTR-CM would generate a gain of 0.031 (95%CI 0.025; 0.038); 0.387 (95%CI 0.329; 0.435); 0.754 (95%CI 0.678; 0.781) and 0.944 (95%CI 0.905; 0.983) life years per patient, respectively, with savings of 212 (95%CI -632; 633), 2,289 (95%CI 2,250; 2,517), 2,859 (95%CI 2,584; 3,149) and 2,906 (95%CI 2,669; 3,450) per patient, respectively, versus the non-diagnosis. Conclusions: just by correctly diagnosing ATTR-CM, years of life would be gained, cardiovascular hospitalizations would be avoided, and savings would be generated for the NHS, compared to the non-diagnosis of the disease

The cost-effectiveness of isavuconazole compared to voriconazole, the standard of care in the treatment of patients with invasive mould diseases, prior to differential pathogen diagnosis in Spain

Background: Invasive mould diseases are associated with high morbidity, mortality and economic impact. Its treatment is often started prior to differential pathogen diagnosis. Isavuconazole is approved for treatment of invasive aspergillosis (IA) and invasive mucormycosis (IM) when amphotericin-B is not indicated. Objectives: To estimate the cost-effectiveness of isavuconazole vs voriconazole for the treatment of adult patients with possible IA prior to differential pathogen diagnosis, in Spain. Methods: A decision tree analysis was performed using the Spanish Healthcare System perspective. Among all patients with possible IA, it was considered that 7.81% actually had IM. Costs for laboratory analysis, management of adverse events, hospitalisation and drugs per patient, deaths and long-term effects in life years (LYs) and quality-adjusted LYs (QALYs) were considered. Efficacy data were obtained from clinical trials and utilities from the literature. Deterministic and probabilistic sensitivity analyses (PSA) were conducted. Results: In patients with possible IA and when compared to voricanozole, isavuconazole showed an incremental cost of 4758.53€, besides an incremental effectiveness of +0.49 LYs and +0.41 QALYs per patient. The Incremental Cost Effectiveness Ratio was 9622.52€ per LY gained and 11,734.79€ per QALY gained. The higher cost of isavuconazole was due to drug acquisition. Main parameters influencing results were mortality, treatment duration and hospitalisation days. The PSA results showed that isavuconazole has a probability of being cost-effective of 67.34%, being dominant in 24.00% of cases. Conclusions: Isavuconazole is a cost-effective treatment compared to voriconazole for patients with possible IA for a willingness to pay threshold of 25,000€ per additional QALY

Cost-utility analysis of the use of the 20-valent anti-pneumococcal vaccine (PCV20) in adults older than 60 years in Spain

Background and objectives: A cost-utility analysis was conducted to assess the efficiency of implementing a PCV20 vaccination strategy in the Spanish adult population older than 60 years, for the prevention of non-bacteremic pneumococcal pneumonia (NBP) and invasive pneumococcal disease (IPD). Methods: A Markov model, with annual cycles and a time horizon of 10 years was used. The analysis population was stratified by age and risk groups. The comparator was the sequential vaccination with the 15-valent pneumococcal conjugate vaccine (PCV15) followed by one dose of the pneumococcal polysaccharide vaccine (PPV23). The base case analysis was performed from the National Healthcare System (NHS) perspective including direct costs (€2018) and applying a discount of 3% to future costs and outcomes. Alternative scenarios explored a shorter time horizon (5 years), the societal perspective and other available vaccination strategies. All the parameters and assumptions were validated by a panel of experts. To evaluate the robustness of the model, deterministic and probabilistic sensitivity analyses (PSA) were carried out. Results: The results of the study showed that the vaccination strategy with PCV20 is a dominant option compared to the sequential regimen (PCV15 + PPSV23), resulting in direct cost savings of €85.7 M over 10 years, with a small increase in quality-adjusted life years (QALYs). PCV20 vaccination avoided 2,161 cases of IPD, 19,470 of NBP and 3,396 deaths and according to the PSA, the probability of PCV20 being cost-effective compared to a sequential regimen (PCV15 + PPSV23) was 100%. Conclusions/Recommendations: In the Spanish adult population older than 60 years, the vaccination strategy with one dose of PCV20 is more effective and less expensive (dominant) than vaccination with a sequential schedule with PCV15 and PPSV23