Vigilancia epidemiológica y caracterización de mecanismos de resitencia a carbapenems en enterobacterias de origen clínico y de portadores

Las especies comensales del orden Enterobacterales pueden actuar como patógenos potenciales dando lugar a infecciones oportunistas, siendo uno de los principales agentes etiológicos en las infecciones tanto nosocomiales como comunitarias. La aparición de enterobacterias resistentes a carbapenems ha aumentado en los últimos años, debido no solo a la producción de carbapenemasas, sino también a otros mecanismos como la alteración de la permeabilidad de la membrana, como es el caso de la alteración en las porinas, que en combinación con la producción de otras beta-lactamasas conlleva a una resistencia a carbapenems, especialmente ertapenem. En Europa la propagación de enterobacterias productoras de carbapenemasa se ha visto incrementada en los últimos años. En nuestro país, destaca K. pneumoniae productora de OXA-48. En K. pneumoniae se han descrito ciertos clones epidémicos de alto riesgo, como ST11, ST15, ST340, ST258 y ST512, los cuales se asocian a cepas multirresistentes. En España, los clones más extendidos son ST405, ST11 y ST15.El primer objetivo de la tesis fue el estudio de enterobacterias con sensibilidad disminuida carbapenems en aislados de origen clínico recogidos entre 2012 y 2016 en el Servicio de Microbiología del Hospital Clínico Universitario Lozano Blesa de Zaragoza. Se estudiaron 40.557 cepas y se seleccionaron 51 con sensibilidad disminuida a carbapenems. Se detectó una baja prevalencia de enterobacterias productoras de carbapenemasas (0,02%). Estas cepas correspondían a las especies de C. freundii y K. aerogenes. A pesar de que en España la carbapenemasa más frecuentemente descrita es OXA-48, el único gen codificante de carbapenemasas detectado fue blaVIM. Este gen se encontró asociado a integrones de clase 1, lo que facilita su diseminación. Uno de los aislados fue productor de VIM-1. Esta variante es la más descrita en enterobacterias. El resto, presentaron el gen blaVIM-2 dentro de un nuevo integrón cuya estructura no había sido descrito hasta ahora (In1470).El segundo objetivo de esta tesis fue estudiar la prevalencia de pacientes ingresados en la UCI colonizados por enterobacterias con sensibilidad disminuida a carbapenems, así como el estudio de estas cepas. Para ello, durante 18 meses se obtuvieron frotis nasales, faríngeos y perineales, y se seleccionaron los aislados crecidos en un medio cromogénico para enterobacterias productoras de carbapenemasas. Hemos detectado una baja prevalencia (6,35%) de pacientes colonizados por enterobacterias con sensibilidad disminuida a carbapenems. La mayor parte de los aislados (78,94%) fueron cepas de K. pneumoniae multirresistentes, las cuales presentaron alta resistencia a beta-lactámicos, incluyendo ertapenem. Uno de esto aislados fue productor de VIM-2 y, además, todas ellas presentaron diferentes beta-lactamasas de espectro extendido y/o beta-lactamasas de tipo AmpC plasmídicas (CTX-M-15, SHV-2, SHV-76, DHA-1). La alteración de la estructura de las porinas OmpK35, OmpK36 y OmpK37 en combinación con las beta-lactamasas detectadas fue el principal mecanismo de resistencia a ertapenem detectado en nuestros aislados. La mayor parte de los aislados de K. pneumoniae multirresistentes pertenecieron al clon ST405, pero también encontramos clones de alto riesgo como ST340, ST15 y ST11. Además, también se aislaron cepas de C. freundii productoras de VIM-2 entre los aislados de portadores. Todos los aislados de C. freundii productores de carbapenemasas, tanto clínicos como de portadores, presentaron el mismo perfil de PFGE y, por tanto, asumimos que pertenecen al mismo clon, aunque no presentaron ninguna relación espacio- temporal ni originó brotes. Todos los aislados de enterobacterias productoras de VIM-2, presentaron este gen dentro del integrón no descrito hasta ahora In1470. Este integrón parece haber actuado como vehículo de transmisión de carbapenemasas entre enterobacterias en el entorno de nuestro hospital.<br /

Epidemiología molecular de enterobacterias productoras de carbapenemasas

En los últimos años se está observando un incremento de bacterias multirresistentes gramnegativas portadoras de genes codificantes de carbapenemasas. La emergencia de enterobacterias productoras de carbapenemasas como causa de infecciones es un asunto de gran preocupación, y la evolución futura indica un aumento de estas infecciones, tanto nosocomiales como adquiridas en la comunidad. Además, la inclusión de estos genes en elementos genéticos móviles (integrones, plásmidos) puede estar favoreciendo su diseminación entre diferentes géneros y especies bacterianas. El objetivo principal de este estudio es conocer cuál es la situación en el Sector Sanitario de Zaragoza III en cuanto a la prevalencia en muestras clínicas y caracterizar los mecanismos de resistencia a β-lactámicos, con especial atención a la producción de carbapenemasas, así como su posible asociación con mecanismos de resistencia a otras familias de antimicrobianos como aminoglucósidos y quinolonas. Para ello, se ha realizado un estudio entre Abril de 2012 y Agosto de 2014 en el Servicio de Microbiología del Hospital Clínico Universitario Lozano Blesa en el que se han seleccionado cepas de enterobacterias procedentes de muestras clínicas con un perfil de sensibilidad compatible de ser portadoras de carbapenemasas. En estos aislados se ha estudiado la sensibilidad a diferentes antibióticos para orientar los posibles mecanismos de resistencia responsables de dicho patrón de sensibilidad. A su vez, se han realizado diferentes test fenotípìcos para clasificar las enterobacterias como posibles cepas portadoras de β-lactamasas tipo BLEEs, AmpC y carbapenemasas. Posteriormente, se han caracterizado los mecanismos implicados en la resistencia a distintos antimicrobianos mediante métodos moleculares, principalmente los asociados con el fenotipo BLEE, AmpC y carbapenemasa. En las cepas portadoras de carbapenemasas se determinó la presencia de los integrones tipo 1, 2 y 3, así como las resistencias asociadas a antibióticos no β-lactámicos (quinolonas y aminoglucósidos)

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Design, synthèse et test d'efficacité des inhibiteurs de flavodoxine, Nitroethylene- et 7-Nitrobenzoxadiazole contre les souches clinniques antibio-resistantes d'Helicobacter pylori et dans les souris infectées

International audienceHelicobacter pylori (Hp) infection is the main cause of peptic ulcer and gastric cancer. Hp eradication rates have fallen due to increasing bacterial resistance to currently used broad-spectrum antimicrobials. We have designed, synthesized, and tested redox variants of nitroethylene- and 7-nitrobenzoxadiazole-based inhibitors of the essential Hp protein flavodoxin. Derivatives of the 7-nitrobenzoxadiazole lead, carrying reduced forms of the nitro group and/or oxidized forms of a sulfur atom, display high therapeutic indexes against several reference Hp strains. These inhibitors are effective against metronidazole-, clarithromycin-, and rifampicin-resistant Hp clinical isolates. Their toxicity for mice after oral administration is low, and, when administered individually at single daily doses for 8 days in a mice model of Hp infection, they decrease significantly Hp gastric colonization rates and are able to eradicate the infection in up to 60% of the mice. These flavodoxin inhibitors constitute a novel family of Hp-specific antimicrobials that may help fight the constant increase of Hp antimicrobial-resistant strains

Brazilian Flora 2020: Leveraging the power of a collaborative scientific network

International audienceThe shortage of reliable primary taxonomic data limits the description of biological taxa and the understanding of biodiversity patterns and processes, complicating biogeographical, ecological, and evolutionary studies. This deficit creates a significant taxonomic impediment to biodiversity research and conservation planning. The taxonomic impediment and the biodiversity crisis are widely recognized, highlighting the urgent need for reliable taxonomic data. Over the past decade, numerous countries worldwide have devoted considerable effort to Target 1 of the Global Strategy for Plant Conservation (GSPC), which called for the preparation of a working list of all known plant species by 2010 and an online world Flora by 2020. Brazil is a megadiverse country, home to more of the world's known plant species than any other country. Despite that, Flora Brasiliensis, concluded in 1906, was the last comprehensive treatment of the Brazilian flora. The lack of accurate estimates of the number of species of algae, fungi, and plants occurring in Brazil contributes to the prevailing taxonomic impediment and delays progress towards the GSPC targets. Over the past 12 years, a legion of taxonomists motivated to meet Target 1 of the GSPC, worked together to gather and integrate knowledge on the algal, plant, and fungal diversity of Brazil. Overall, a team of about 980 taxonomists joined efforts in a highly collaborative project that used cybertaxonomy to prepare an updated Flora of Brazil, showing the power of scientific collaboration to reach ambitious goals. This paper presents an overview of the Brazilian Flora 2020 and provides taxonomic and spatial updates on the algae, fungi, and plants found in one of the world's most biodiverse countries. We further identify collection gaps and summarize future goals that extend beyond 2020. Our results show that Brazil is home to 46,975 native species of algae, fungi, and plants, of which 19,669 are endemic to the country. The data compiled to date suggests that the Atlantic Rainforest might be the most diverse Brazilian domain for all plant groups except gymnosperms, which are most diverse in the Amazon. However, scientific knowledge of Brazilian diversity is still unequally distributed, with the Atlantic Rainforest and the Cerrado being the most intensively sampled and studied biomes in the country. In times of “scientific reductionism”, with botanical and mycological sciences suffering pervasive depreciation in recent decades, the first online Flora of Brazil 2020 significantly enhanced the quality and quantity of taxonomic data available for algae, fungi, and plants from Brazil. This project also made all the information freely available online, providing a firm foundation for future research and for the management, conservation, and sustainable use of the Brazilian funga and flora

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field