Análisis fenotípico, genómico y bioinformático de los elementos genéticos asociados a resistencia a antibióticos y biocidas en enterobacterias

Antibióticos y biocidas son compuestos ampliamente utilizados desde hace más de 50 años para inhibir o eliminar el crecimiento microbiano y se diferencian fundamentalmente en el espectro, mecanismo de acción y aplicaciones. Los antibióticos se emplean en el tratamiento de infecciones bacterianas, además de ser usados como profilácticos o promotores de crecimiento. Los biocidas se utilizan como antisépticos, conservantes o desinfectantes para controlar el crecimiento y proliferación de diferentes microorganismos (bacterias, hongos, virus) en el entorno hospitalario, doméstico, veterinario o industrial. El riesgo de corresistencia y/o resistencia cruzada a ambos compuestos constituye un problema de Salud Pública de primer orden. Los objetivos de esta tesis son: i) determinar la sensibilidad a biocidas ampliamente usados en el entorno hospitalario y comunitario (triclosan, TRI; cloruro de benzalconio, BKC; clorhexidina, CHX; hipoclorito de sodio, NaOCl) en los principales patógenos oportunistas de animales y humanos, ii) evaluar la aparición de resistencia a los biocidas y antibióticos tras exposición a estos dos grupos de compuestos, iii) analizar genotípica, fenotípica, y poblacionalmente los elementos genéticos transferibles descritos que confieren resistencia a biocidas (bombas de eflujo SMR que confieren resistencia a BKC, qac) y antibióticos (sulfamidas, genes sul). El análisis de la sensibilidad de una colección de aislados habitualmente patógenos oportunistas (Enterobacteriaceae, Enterococcus spp., Listeria spp. y Staphylococcus spp.) a los biocidas TRI, CHX, BKC y NaOCl reveló una baja prevalencia de microorganismos tolerantes a estos compuestos. Sin embargo, los estudios de exposición in vitro a biocidas y/o antibióticos de diferentes enterobacterias (Escherichia coli, Klebsiella pneumoniae y Salmonella enterica) permitieron identificar una diversidad de fenotipos de sensibilidad reducida (e.g. TRIR, BKCR, TRIR/BKCR, TRIR/BKCR/CHXR), sensibilidad aumentada (e.g. TRIHS, TRIHS/BKCHS, TRIHS/CHXHS) y combinaciones de ambas a diferentes biocidas (TRIHS/BKCR/CHXR, TRIR/CHXHS) y antibióticos que podrían reflejar la existencia de rutas compensatorias. Los mutantes con marcada reducción de sensibilidad presentaron un coste biológico elevado y sobreexpresaron los reguladores globales (marA o ramA). El metabolismo de fuentes de carbono y nitrógeno de estos mutantes sufrió alteraciones relacionadas con mayor patogenicidad en el caso de E. coli. El análisis transcriptómico de aislados y mutantes de S. enterica con sensibilidad disminuida a biocidas reveló un aumento de expresión de diversos genes implicados en la síntesis proteica, metabolismo, membrana, estrés y virulencia, indicando que la respuesta bacteriana a los biocidas es multifactorial. Finalmente, los estudios de caracterización genética y análisis poblacional de los elementos genéticos transferibles portadores de genes de resistencia a antibióticos (sul) y biocidas (qac) pusieron de manifiesto diferentes eventos de captura, selección y dispersión. Tanto los elementos que contienen sul2 (plataformas derivadas de plásmidos IncQ y asociadas a ISCR2) como sul3 y qacI (integrones inusuales de clase 1) se encuentran en un número limitado de transposones localizados en una variedad de plásmidos conjugativos de Enterobacteriaceae de origen clínico y comunitario. [ABSTRACT]Antibiotics and biocides are widely used for many purposes in daily life, in general for inhibiting or killing potentially pathogenic bacteria and they differ from each other in different aspects, including the spectrum of activity, the mechanism of action and the applications. First, antibiotics are mostly used as therapeutical agents or growth promoters, while biocides are used as antiseptics, disinfectants, preservatives and detergents. Second, most biocides do not act on a specific cell target and mechanisms responsible for their reduced susceptibility are poorly characterized in contrast to those associated with antibiotics. Most works on this topic have been performed using laboratory-generated mutants and have allowed to establish that the over-expression of efflux pumps AcrAB or AcrEF, which are controlled by global transcriptional regulators such as marAB, ramA and soxRS, constitutes the main cause of biocide tolerance and confers diverse low-level antibiotic resistance phenotypes. Adaptation to biocides may impair cellular homeostasis, increase the activity of efflux pumps, or change expression of proteins regulating invasiveness, virulence or stress response. Whether or not all of these changes are needed for adaptation to the presence of biocides or they just reflect fitness changes associated with such adaptation remains to be established. Finally, bacterial susceptibility based on either the likelihood of treatment failure (clinical breakpoints) or the upper limit of tolerance in the wild-type population (epidemiological cut-off value, ECOFF), are clearly defined for most antibiotics and species but fully unexplored for biocides. Resistance to antimicrobials is nowadays an outstanding problem in Public Health, because of the scarce options to treat and/or prevent infections caused by multiresistant pathogens. Since both antibiotics and biocides are widely used, often in combination, a potential risk to develop cross-resistance is of concern. Here, we focused on the association of reduced susceptibility to both antibiotics and biocides among Enterobacteriaceae, a bacterial family of opportunistic pathogens of humans and animals, and thus, very exposed to these two groups of antimicrobials. We especially analysed the link between sulphonamides and biocide resistance because the widespread of platforms containing genes putatively coding for resistance to such compounds since early 30´s in both hospital and community settings..

Circadian rhythm in critically ill patients: Insights from the eICU Database

OBJECTIVE: To investigate the circadian variation among critically ill patients and its association with clinical characteristics and survival to hospital discharge in a large population of patients in the intensive care unit (ICU). METHODS: Circadian variation was analyzed by fitting cosinor models to hourly blood pressure (BP) measurements in patients of the eICU Collaborative Research Database with an ICU length of stay of at least 3 days. We calculated the amplitude of the 24-hour circadian rhythm and time of the day when BP peaked. We determined the association between amplitude and time of peak BP and severity of illness, medications, mechanical intubation, and survival to hospital discharge. RESULTS: Among 23,355 patients (mean age 65 years, 55% male), the mean amplitude of the 24-hour rhythm was 4.5 ± 3.1 mm Hg. Higher APACHE-IV scores, sepsis, organ dysfunction, and mechanical ventilation were associated with a lower amplitude and a shifted circadian rhythm (P < .05 for all). The timing of the BP peak was associated with in-hospital mortality (P < .001). Higher BP amplitude was associated with shorter ICU (2 mm Hg amplitude: 7.0 days, 8 mm Hg amplitude: 6.7 days) and hospital (2 mm Hg amplitude: 11.8 days, 8 mm Hg amplitude: 11.3 days) lengths of stay and lower in-hospital mortality (2 mm Hg amplitude: 18.2%, 8 mm Hg amplitude: 15.2%) (P < .001 for all). CONCLUSION: The 24-hour rhythm is dampened and phase-shifted in sicker patients and those on mechanical ventilation, vasopressors, or inotropes. Dampening and phase shifting are associated with a longer length of stay and higher in-hospital mortality