A Mouthwash with Cetylpyridinium Chlorideis reducing salivary SARS-CoV-2 viral load in +COVID-19 / Un enjuague con Cloruro de Cetilpiridinio redujo la carga salival del virus SARSCoV-2 en pacientes con Covid-19

Aim: The aim of this randomised, double-blind, placebo-controlled pilot clinical trial is to evaluate the capacity of a mouthwash to reduce SARS-CoV-2 viral load in the saliva of patients with COVID-19. Methods: Twenty-three symptomatic SARS-CoV-2-positive outpatients were selected andrandomised into two groups and registered at NTC 04563689. Both groups rinsed and gargled for one minute with either distilled water (Placebo) or with 0.05% Cetylpyridinium chloride (CPC) plus 0.12% Chlorhexidine (CHX) mouthwash (PERIOAID Intensive Care). Saliva samples were collected before the use of placebo or mouthwash and after 15 minutes and 1 and 2 hours of either of the above treatment. A saliva sample was also taken five days after regular use of placebo or mouthwash twice daily. The virus was detected by qRT-PCR. Results: A great heterogeneity in the viral load values was observed at baseline in both groups for nasopharyngeal and saliva samples. Most of the patients who used the mouthwash (8/12) had a significant decrease in baseline viral load after 15 min (greater than 99% reduction). This inhibitory effect was maintained for up to two hours in 10 of the 12 patients. At five days, SARS-CoV-2 RNA was detectedin only 1 patient from the mouthwash group and in 5 from the placebo group. Conclusions: This study points out that a CPC mouthwash can reduce the viral load in saliva of COVID-positive patients. This finding may be important in transmission control of SARS-CoV-2. Nevertheless, the clinical relevance of CPC mouthwash-reduction on SARS-CoV-2 shedding in saliva requires further study.

Objetivo: El objetivo de este ensayo clínico piloto aleatorizado, doble ciego y controlado con placebo es evaluar la capacidad de un enjuague bucal para reducir la carga viral del SARS-CoV-2 en la saliva de pacientes con COVID-19. Materiales y métodos:Veintitrés pacientes ambulatorios positivos para SARS-CoV-2 sintomáticos fueron seleccionados y aleatorizados en dos grupos y registrados en el NTC 04563689. Ambos grupos se enjuagaron y hicieron gárgaras durante un minuto con agua destilada (placebo) o con cloruro decetilpiridinio al 0 ,05 % (CPC). ) más enjuague bucal con Clorhexidina (CHX) al 0,12% (PERIOAID Intensive Care). Se recolectaron muestras de saliva antes del uso de placebo o enjuague bucal y después de 15 minutos y 1 y 2 horas de cualquiera de los tratamientos anteriores. También se tomó una muestra de saliva cinco días después del uso regular de placebo o enjuague bucal dos veces al día. El virus fue detectado por qRT-PCR. Resultados:Se demostró una gran heterogeneidad en los valores de carga viral al inicio del estudio en grupos ambos para muestras de nasofaringe y saliva. La mayoría de los pacientes que usaron el enjuague bucal (8/12) tuvieron una disminución significativa en la carga viral inicial después de 15 minutos (reducción superior al 99 %). Este efecto inhibidor se mantuvo hasta dos horas en 10 de los 12 pacientes. A los cinco días, se detectó ARN del SARS-CoV-2 en solo 1 paciente del grupo de enjuague bucal y en 5 del grupo de placebo. Conclusiones:Este señala que un enjuague bucal CPC puedereducir la carga viral en saliva de pacientes COVID positivos. Este hallazgo puede ser importante en el control de la transmisión del SARS-CoV-2. Sin embargo, la relevancia clínica de la reducción del enjuague bucal con CPC en la excreción de SARS-CoV-2 en la saliva requiere más estudios.

Crystal structures of the closed form of Mycobacterium tuberculosis dihydrofolate reductase in complex with dihydrofolate and antifolates.

Tuberculosis is a disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious pathogen, with a high prevalence in developing countries in Africa and Asia. There still is a need for the development or repurposing of novel therapies to combat this disease owing to the long-term nature of current therapies and because of the number of reported resistant strains. Here, structures of dihydrofolate reductase from M. tuberculosis (MtDHFR), which is a key target of the folate pathway, are reported in complex with four antifolates, pyrimethamine, cycloguanil, diaverdine and pemetrexed, and its substrate dihydrofolate in order to understand their binding modes. The structures of all of these complexes were obtained in the closed-conformation state of the enzyme and a fine structural analysis indicated motion in key regions of the substrate-binding site and different binding modes of the ligands. In addition, the affinities, through Kd measurement, of diaverdine and methotrexate have been determined; MtDHFR has a lower affinity (highest Kd) for diaverdine than pyrimethamine and trimethoprim, and a very high affinity for methotrexate, as expected. The structural comparisons and analysis described in this work provide new information about the plasticity of MtDHFR and the binding effects of different antifolates.

Structural Basis for the Interaction and Processing of ß-Lactam Antibiotics by l,d-Transpeptidase 3 (LdtMt3) from Mycobacterium tuberculosis.

Targeting Mycobacterium tuberculosis peptidoglycans with ß-lactam antibiotics represents a strategy to address increasing resistance to antitubercular drugs. ß-Lactams inhibit peptidoglycan synthases such as l,d-transpeptidases, a group of carbapenem-sensitive enzymes that stabilize peptidoglycans through 3 → 3 cross-links. M. tuberculosis encodes five l,d-transpeptidases (LdtMt1-5), of which LdtMt3 is one of the less understood. Herein, we structurally characterized the apo and faropenem-acylated forms of LdtMt3 at 1.3 and 1.8 Å resolution, respectively. These structures revealed a fold and catalytic diad similar to those of other LdtsMt enzymes, supporting its involvement in transpeptidation reactions despite divergences in active site size and charges. The LdtMt3-faropenem structure indicated that faropenem is degraded after Cys-246 acylation, and possibly only a ß-OH-butyrate or an acetyl group (C2H3O) covalently attached to the enzyme remains, an observation that strongly supports the notion that LdtMt3 is inactivated by ß-lactams. Docking simulations with intact ß-lactams predicted key LdtMt3 residues that interact with these antibiotics. We also characterized the heat of acylation involved in the binding and reaction of LdtMt3 for ten ß-lactams belonging to four different classes, and imipenem had the highest inactivation constant. This work provides key insights into the structure, binding mechanisms, and degradation of ß-lactams by LdtMt3, which may be useful for the development of additional ß-lactams with potential antitubercular activity.

Effects of biocide exposure on P. Aeruginosa, E. coli and A. Baumannii complex isolates from hospital and household environments / Efectos de la exposición a biocidas en aislados ambientales de P. Aeruginosa, E. coli y del complejo A. Baumannii hospitalarios y de la comunidad

Background: Bacterial responses to biocide exposure and its effects on survival and persistence remain to be studied in greater detail. Aim: To analyse the viability and survival of environmental isolates from household and hospital settings after biocide exposure. Methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of chlorhexidine (CHxG), benzalkonium chloride (BAC) and triclosan (TC) were determined in isolates of Pseudomonas aeruginosa, Acinetobacter baumannii complex and Escherichia coli collected from hospital and house- holds environments. Viability was monitored after exposure and removal of biocides using agar cultures and flow cytometry. Findings: P. aeruginosa isolates showed greater tolerance for all biocides tested whereas A. baumannii complex and E. coli were less tolerant. When compared with reference strains, biocide tolerance was up to 8 to 13-fold higher for TC and BAC respectively. Flow cytometry showed that biocide exposure may induce viable but non-growing states in P. aeruginosa and E. coli isolates before becoming fully replicative. Changes in the susceptibility profile in one isolate of A. baumannii complex were observed after biocide exposure. Discussion: Bacteria isolates from hospital and households were able to recover after biocide exposure at bactericidal concentrations favouring persistence and spread of biocide-tolerant strains. This study reinforces that cleaning compliance should be monitored by non-culture based tests. Novel formulations in cleaning and disinfection protocols should be revisited in hospitals harbouring P. aeruginosa and A. baumannii multidrug resistant isolates.

Introducción: El efecto de la exposición a biocidas en las poblaciones bacterianas, su viabilidad y persistencia requieren de estudios detallados. Objetivo: analizar la viabilidad y persistencia de bacterias de ambientes hospitalarios y domésticos posterior a la exposición a biocidas. Materiales y Métodos: En un estudio experimental in vitro se determinó la concentración inhibitoria mínima (CIM) y la concentración bactericida (CBM) para chlorhexidina (CHxG), cloruro de benzalconio (BAC) y triclcosan (TC) en aislados de Pseudomonas aeruginosa (10), el complejo Acinetobacter baumannii (5) y Escherichia coli (5) obtenidos de ambientes hospitalarios y domésticos. La viabilidad y susceptibilidad bacteriana después de la exposición y remoción del biocida fue evaluada por citometria de flujo y cultivo. Resultados: Independiente de su procedencia P. aeruginosa presentó mayor tolerancia a todos los biocidas. El complejo A. baumannii y E. coli fueron hasta 8 a 13 veces más tolerantes a BAC y TC que las cepas de referencia. Se observó que la exposición a biocidas altamente efectivos induce formas viables no replicativas en P. aeruginosa y E. coli. Un aislado del complejo A baumannii presentó cambios en el perfil de susceptibilidad posterior a la exposición. Discusión: Aislados tanto de ambiente hospitalario como de la comunidad pueden recuperarse después de la exposición a concentraciones bactericidas de los biocidas favoreciendo la persistencia y diseminación de bacterias no replicativas. Por lo anterior métodos alternativos al cultivo deben utilizarse en el seguimiento de protocolos de limpieza y desinfección. Los tiempos de recuperación de la viabilidad bacteriana deben tenerse en cuenta en la formulación de protocolos para erradicar y/o controlar cepas hospitalarias de P. aeruginosa o A. baumannii multirresistentes.