Influencia de la resistencia a imipenen y de diferentes mecanismos de virulencia en acinetobacter baumannii en la colonización o infección de vías respiratorias y la mortalidad en pacientes con ventilación mecánica en cuidados intensivos

Falta palabras clavesAcinetobacter baumannii es un bacilo gramnegativo no fermentador con gran importancia clínica por su capacidad para colonizar superficies y pacientes hospitalizados, especialmente a aquellos ingresados en unidades de cuidados intensivos, y su habilidad para desarrollar resistencias microbianas. La infección que causa con mayor frecuencia es la neumonía asociada a ventilación mecánica (NAVM). En los últimos años, se han venido expandiendo cepas resistentes a carbapenemas, que habían sido el tratamiento de elección para estas infecciones. Los factores de virulencia mediante los que actúa A. baumannii no son bien conocidos. Este estudio tenía como objetivos: 1. Evaluar la mortalidad asociada a la colonización de vías respiratorias por A. baumannii vs. la de las infecciones respiratorias por esta bacteria, con o sin bacteriemia, en los pacientes críticos con ventilación mecánica. 2. Comparar la presencia de determinados factores de virulencia entre las cepas clínicas colonizantes y productoras de infección: formación de biofilm, motilidad, expresión de OmpA, adquisición de hierro y presencia de hemolisinas. 3. Evaluar la presencia de infección respiratoria, el desarrollo de bacteriemia y la mortalidad en los pacientes críticos con ventilación mecánica relacionados con la adquisición de cepas con distintos factores de virulencia bacteriana, anteriormente caracterizados. 4. Estudiar la asociación entre la presencia de mecanismos de resistencia a imipenem (baja expresión de porinas y presencia de carbapenemasas) y los factores de virulencia en la colección de cepas caracterizadas a partir del objetivo primero. Se realizó un estudio de cohortes prospectivo en el que se reclutó a 100 adultos con ventilación mecánica durante más de 48 horas en la UCI del Hospital Virgen del Rocío y aislamiento de A. baumannii en muestra de aspirado traqueobronquial. La variable resultado principal analizada fue la mortalidad a 30 días desde la colonización o el debut de la NAVM (según el caso), detectando que esta fue similar en los 57 pacientes con NAVM que en los 43 pacientes colonizados (27,9% vs. 24,6%). La bacteriemia por A. baumannii fue un factor de riesgo de menor supervivencia y el tratamiento con colistina, que fue muy prevalente, se comportó como factor protector. Se seleccionaron 127 aislados de A. baumannii procedentes de los 100 pacientes (113 aislados procedían de muestras respiratorias y 15 de hemocultivos). Se realizó un estudio de clonalidad mediante PFGE, que reveló la existencia de 7 pulsotipos diferentes con dos clones predominantes. Se estudió la producción de biofilm mediante estudio fenotípico, encontrando que diecisiete aislados eran hiperproductores de biofilm, con una distribución clonal. Se estudió la motilidad en superficie; solamente 14 aislados no eran motiles, sin relación clonal. Ningún aislado presentó hemólisis en medio con eritrocitos humanos ni tampoco había diferencias entre ellos en la capacidad de adquisición de hierro. Se seleccionaron 29 cepas para estudiar la producción de OmpA. La expresión de OmpA fue superior en los aislados procedentes de pacientes con NAVM bacteriémica, seguida por la de los pacientes con NAVM sin bacteriemia y, finalmente, la expresión más baja la producían los aislados procedentes de pacientes colonizados. La expresión de OmpA también se relacionó con la mortalidad en los pacientes con NAVM. El resto de factores de virulencia estudiados no mostraron asociación con la mortalidad ni la producción de infección. Todos los aislados fueron sensibles a colistina con una CMI90 muy baja, de 0,12 mg/l. Todos menos uno fueron resistentes a carbapenemas. Todos los aislados resistentes a carbapenemas portaban una carbapenemasa tipo oxacilinasa, siendo la más frecuente OXA-58 (104 casos) seguida de OXA-40. Ningún aislado producía OXA-23 o metalobetalactamasas. Aunque 7 aislados presentaban ausencia de la proteína de membrana externa de 29 KDa CarO, esto no se relacionó con un incremento de la resistencia a carbapenemas.Premio Extraordinario de Doctorado U

Inference on filtered and smoothed probabilities in Markov-switching autoregressive models

We derive a statistical theory that provides useful asymptotic approximations to the distributions of the single inferences of filtered and smoothed probabilities, derived from time series characterized by Markov-switching dynamics. We show that the uncertainty in these probabilities diminishes when the states are separated, the variance of the shocks is low, and the time series or the regimes are persistent. As empirical illustrations of our approach, we analyze the U.S. GDP growth rates and the U.S. real interest rates. For both models, we illustrate the usefulness of the confidence intervals when identifying the business cycle phases and the interest rate regimes.M. Camacho and M. Ruiz acknowledge the financial support from projects ECO2016-76178-P and ECO2015-65637-P, respectively. Rocio Alvarez acknowledges the financial support from project CIP16013 (Universidad Central de Chile). This study is the result of the activity carried out under the program Groups of Excellence of the region of Murcia, the Fundación Séneca, Science and Technology Agency of the region of Murcia project 19884/GERM/15

Avoiding sexual interference: herkogamy and dichogamy in style dimorphic flowers of Narcissus broussonetii (Amaryllidaceae)

Spatial (herkogamy) or temporal (dichogamy) separation of sex organs are mechanisms considered to restrict self-pollination and promote outcrossing. Additionally, avoidance of self-interference is proposed to be the driving force for the evolution of these mechanisms, particularly in self-incompatible species. However, species with anthers and stigmas at different levels may increase the rate of imprecise pollen transfer, resulting in pollen discounting. Non-reciprocal stylar dimorphism has been considered a transitional, unstable stage towards the evolution of reciprocal style dimorphism (distyly), to simultaneously avoid interference and lack of precision. In this study we investigate the spatial and temporal separation of sex organs in a population of the style dimorphic and self-incompatible Narcissus broussonetii and their consequences in the reciprocity between the sex organs of morphs and their fecundity. First, we evaluated the relative growth of sex organs after anthesis. Then, we studied the stigma receptivity along the flower lifespan including its effect on seed production in both morphs. Finally, given the weak reciprocity between the sex organs of morphs of this species, we estimated population genetic diversity parameters in Long- and Short-styled plants to explore differences between them as a result of rates of inbreeding due to different mating strategies. We observed that Long-styled plants and Short-styled plants present different strategies to avoid sexual interference and both of them had negative consequences in the reciprocity between the sex organs of morphs. Long-styled plants exhibited a delay in stigma receptivity and a higher growth rate of the style after anthesis, while Short-styled plants presented higher herkogamy and no delay in stigma receptivity. These findings suggest that the avoidance of self-interference, in stylar dimorphic Narcissus species, seems to be more critical than improving of reciprocity between the sex organs of morphs. This might explain why reciprocal herkogamy (distyly) is rare in the genus.España, Ministerio de Economía y Competitividad (CGL2009-12565 and CGL2013-45037 PGC2018-099608-B-I00)ESpaña, Plan Andaluz de Investigación (RNM-210)España, Ministerio de Economía y Competitividad (BES-2014-067795 and EEBB-I-16-10587

In vitro Activity of Pentamidine Alone and in Combination With Aminoglycosides, Tigecycline, Rifampicin, and Doripenem Against Clinical Strains of Carbapenemase-Producing and/or Colistin-Resistant Enterobacteriaceae

Enterobacteriaceae cause different types of community- and hospital-acquired infections. Moreover, the spread of multidrug-resistant Enterobacteriaceae is a public health problem and the World Health Organization pointed them among the pathogens in which the search of new antibiotics is critical. The objective of this study was to analyze the in vitro activity of pentamidine alone and in combination with gentamicin, tobramycin, amikacin, tigecycline, rifampicin, or doripenem against eight clinical strains of carbapenemase-producing and/or colistin-resistant Enterobacteriaceae: five carbapenemase-producing Klebsiella pneumoniae, one carbapenemase-producing Escherichia coli, and two colistin-resistant Enterobacter cloacae. MIC and MBC were determined following standard protocols. MIC results were interpreted for all the antibiotics according to the EUCAST breakpoints but for rifampicin in which the French FSM breakpoint was used. Bactericidal and synergistic activity of pentamidine alone and in combination with antibiotics at concentrations of 1xMIC was measured by time-kill curves. For one selected strain, K. pneumoniae OXA-48/CTX-M-15 time-kill curves were performed also at 1/2xMIC of pentamidine. All studies were performed in triplicate. Pentamidine MIC range was 200-800 μg/mL. The 50, 12.5, 62.5, 87.5, and 62.5% of the strains were susceptible to gentamicin, tobramycin, amikacin, tigecycline, and doripenem, respectively. Only the two E. cloacae strains were susceptible to rifampicin. Pentamidine alone at 1xMIC showed bactericidal activity against all strains, except for the E. cloacae 32 strain. The bactericidal activity of pentamidine alone was also observed in combination. The combinations of pentamidine were synergistic against E. cloacae 32 with amikacin and tobramycin at 24 h and with tigecycline at 8 h. Pentamidine plus rifampicin was the combination that showed synergistic activity against more strains (five out of eight). Pentamidine plus doripenem did not show synergy against any strain. At 1/2xMIC, pentamidine was synergistic with all the studied combinations against the K. pneumoniae OXA-48/CTX-M-15 strain. In summary, pentamidine alone and in combination shows in vitro activity against carbapenemase-producing and/or colistin-resistant Enterobacteriaceae. Pentamidine appears to be a promising option to treat infections caused by these pathogens.Plan Nacional de I+D+I 2013-2016 REIPI RD16/0016/0009Instituto de Salud Carlos III. Subdirección General de Redes y Centros de Investigación Cooperativa REIPI RD16/0016/0009Ministerio de Economía, Industria y Competitividad REIPI RD16/0016/0009Spanish Network for Research in Infectious Diseases REIPI RD16/0016/0009European Development Regional Fun

Phylogeny, Resistome, and Virulome of Escherichia coli Causing Biliary Tract Infections

Escherichia coli is the most frequent Gram-negative bacilli involved in intra-abdominal infections. However, despite high mortality rates associated with biliary tract infections due to E. coli, there is no study focusing on this pathogen. In this study, we have characterized a group of 15 E. coli isolates obtained from 12 patients with biliary tract infections. Demographic and clinical data of the patients were recovered. Phylogeny, resistome, and virulome analysis through whole genome sequencing and biofilm formation were investigated. Among the 15 E. coli isolates, no predominant sequence type (ST) was identified, although 3 of them belonged to unknown STs (20%). Resistance to ampicillin, amoxicillin/clavulanic acid, cotrimoxazole, and quinolones was more present in these isolates; whereas, third and fourth generation cephalosporins, carbapenems, amikacin, tigecycline, and colistin were highly active. Moreover, high diversity of virulence factors has been found, with sfa, fimH, and gad the most frequently detected genes. Interestingly, 26.6% of the E. coli isolates were high biofilm-producers. Altogether, our data characterized for the first time E. coli isolates associated with biliary tract infections in terms of genomic relationship, resistome, and virulome.España, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades (CP15/00132)España, Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (RD16/0016/0009

Clinical efficacy of β-lactam/β-lactamase inhibitor combinations for the treatment of bloodstream infection due to extended-spectrum β-lactamase- producing Enterobacteriaceae in haematological patients with neutropaenia: a study protocol for a retrospect observational study (BICAR).

Introduction: Bloodstream infection (BSI) due to extended-spectrum β-lactamase-producing Gram- negative bacilli (ESBL-GNB) is increasing at an alarming pace worldwide. Although β-lactam/β- lactamase inhibitor (BLBLI) combinations have been suggested as an alternative to carbapenems for the treatment of BSI due to these resistant organisms in the general population, their usefulness for the treatment of BSI due to ESBL-GNB in haematological patients with neutropaenia is yet to be elucidated. The aim of the BICAR study is to compare the efficacy of BLBLI combinations with that of carbapenems for the treatment of BSI due to an ESBL-GNB in this population. Methods and analysis: A multinational, multicentre, observational retrospective study. Episodes of BSI due to ESBL-GNB occurring in haematological patients and haematopoietic stem cell transplant recipients with neutropaenia from 1 January 2006 to 31 March 2015 will be analysed. The primary end point will be case- fatality rate within 30 days of onset of BSI. The secondary end points will be 7-day and 14-day case- fatality rates, microbiological failure, colonisation/ infection by resistant bacteria, superinfection, intensive care unit admission and development of adverse events. Sample size: The number of expected episodes of BSI due to ESBL-GNB in the participant centres will be 260 with a ratio of control to experimental participants of 2. Ethics and dissemination: The protocol of the study was approved at the first site by the Research Ethics Committee (REC) of Hospital Universitari de Bellvitge. Approval will be also sought from all relevant RECs. Any formal presentation or publication of data from this study will be considered as a joint publication by the participating investigators and will follow the recommendations of the International Committee of Medical Journal Editors (ICMJE). The study has been endorsed by the European Study Group for Bloodstream Infection and Sepsis (ESGBIS) and the European Study Group for Infections in Compromised Hosts (ESGICH).Ministerio de Economía y Competitividad REIPI RD12/001

In vitro activity of pentamidine alone and in combination with antibiotics against multidrug-resistant clinical Pseudomonas aeruginosa strains

Multidrug-resistant (MDR) Pseudomonas aeruginosa is a public health problem causing both community and hospital-acquired infections, and thus the development of new therapies for these infections is critical. The objective of this study was to analyze in vitro the activity of pentamidine as adjuvant in combinations to antibiotics against seven clinical P. aeruginosa strains. The Minimum Inhibitory Concentration (MIC) was determined following standard protocols, and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints; however, the gentamicin activity was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. The bactericidal in vitro activity was studied at 1×MIC concentrations by time–kill curves, and also performed in three selected strains at 1/2×MIC of pentamidine. All studies were performed in triplicate. The pentamidine MIC range was 400–1600 µg/mL. Four of the strains were MDR, and the other three were resistant to two antibiotic families. The combinations of pentamidine at 1×MIC showed synergistic activity against all the tested strains, except for pentamidine plus colistin. Pentamidine plus imipenem and meropenem were the combinations that showed synergistic activity against the most strains. At 1/2×MIC, pentamidine plus antibiotics were synergistic with all three analyzed strains. In summary, pentamidine in combination with antibiotics showed in vitro synergy against multidrug-resistant P. aeruginosa clinical strains, which suggests its possible use as adjuvant to antibiotics for the therapy of infections from MDR P. aeruginosa.Instituto de Salud Carlos III Proyectos de Investigación en Salud PI18-01842Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Red Española de Investigación en Enfermedades Infecciosas REIPI RD16 / 0016/0009Fondo Regional de Desarrollo Europeo Una forma de lograr Europa, Operativa programa Crecimiento inteligente 2014-2020. T.CUniversidad de Sevilla. Servicio Andaluz de Salud, Junta de Andalucía C1-0038-2019Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanciado por la Unión Fondo Regional de Desarrollo RD16 / 0016/0009Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanciado por European Development Regional Fund (A Way to Achieve Europe) y por la Red Española de Investigación en Enfermedades Infecciosas JR17 / 0002

Colistin Dosage without Loading Dose Is Efficacious when Treating Carbapenem- Resistant Acinetobacter baumannii Ventilator- Associated Pneumonia Caused by Strains with High Susceptibility to Colistin.

Objectives This study aims to analyze the mortality and the length of ICU stay (LOS) of A. baumannii VAP compared to respiratory colonization in patients with mechanical ventilation (MV). Methods A prospective cohort study was performed in an ICU of adult patients (February 2010–June 2011). One hundred patients on MV with A. baumannii in lower respiratory airways were recruited, and classified as VAP or airways colonization according to CPIS criteria, with a punctuation 6. LOS, 30-days mortality, A. baumannii bacteremia, and clinical features including antibiotic therapy were recorded. Multivariate analysis (linear and Cox regression) and survival analysis (Kaplan-Meier curves) were performed. Results Fifty-seven VAP and 43 colonized A. baumannii patients were analyzed. Among the A. bau- mannii strains, 99% were non-susceptible to carbapenems and the MIC 90 of colistin was 0.12 mg/l. Therapy was appropriate in 94.6% of VAP patients, most of them with colistin 6 MIU/day, although in 13 (23.6%) cases colistin was started 48 hours after the onset of VAP. Mortality was similar in both groups (VAP 24.6% vs. colonized 27.9%, p = 0.7). Bacteremia and acute kidney insufficiency were associated with decreased survival (p = 0.02 and p = 0.04, respectively) in VAP patients. LOS was 21.5 (11.5–42.75) vs. 9 (6–22) days for VAP and colonized patients (p = 0.004). VAP (p = 0.003) and age (p = 0.01) were independently related to a longer LOS. Conclusions Multidrug-resistant A. baumannii VAP treated with colistin does not have a different mortality compared to lower airways colonization, among patients on mechanical-ventilation, in a set- ting of high susceptibility to colistin of A. baumannii.Ministerio de Economía y Competitividad REIPI RD12/ 0015/000

Role of blaTEM and OmpC in the piperacillin-tazobactam resistance evolution by E. coli in patients with complicated intra-abdominal infection

Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that bla gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of bla or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4 g/0.5 g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of bla and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and bla and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of bla gene in P/T-R isolates, generated by three different genetic events: (1) IS26-mediated duplication of the bla gene, (2) generation of a small multicopy plasmid (ColE-like) carrying bla, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms.This work was funded by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad (grant PI19/01009), by Plan Nacional de I+D+i 2013–2016, and by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (grant RD16/0016/0009), cofinanced by the European Development Regional Fund “A way to make Europe”/”Investing in your future”. A.R.V, R.A-M, J.A.L. and J.M.C. were supported (grant CB21/13/00006) by CIBERINFEC - Consorcio Centro de Investigación Biomédica en Red, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea – NextGenerationEU. L.G.B. was partly financed by a grant from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) and is supported by the Subprograma Rio Hortega, Instituto Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (CM22/00196). J.M.O.R. is supported by the Subprograma Sara Borrell, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (CD21/00098). A.R.V. is supported by the Subprograma Juan Rodés, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (JR20/00023), and Y.S. has received a Miguel Servet Tipo II contract from the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spain (grant CPII20/00018). Funding for open access publishing: Universidad Pablo de OlavidePeer reviewe

Bases genéticas de la adquisición de la resistencia in vivo a piperacilina-tazobactam en Escherichia coli

Trabajo presentado en el XXVI Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica, celebrado en Santiago de Compostela (España) del 01 al 03 de junio de 2023.Peer reviewe