Automatic Discrimination of Species within the Enterobacter cloacae Complex Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Supervised Algorithms.

The Enterobacter cloacae complex (ECC) encompasses heterogeneous clusters of species that have been associated with nosocomial outbreaks. These species may have different acquired antimicrobial resistance and virulence mechanisms, and their identification is challenging. This study aims to develop predictive models based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles and machine learning for species-level identification. A total of 219 ECC and 118 Klebsiella aerogenes clinical isolates from three hospitals were included. The capability of the proposed method to differentiate the most common ECC species (Enterobacter asburiae, Enterobacter kobei, Enterobacter hormaechei, Enterobacter roggenkampii, Enterobacter ludwigii, and Enterobacter bugandensis) and K. aerogenes was demonstrated by applying unsupervised hierarchical clustering with principal-component analysis (PCA) preprocessing. We observed a distinctive clustering of E. hormaechei and K. aerogenes and a clear trend for the rest of the ECC species to be differentiated over the development data set. Thus, we developed supervised, nonlinear predictive models (support vector machine with radial basis function and random forest). The external validation of these models with protein spectra from two participating hospitals yielded 100% correct species-level assignment for E. asburiae, E. kobei, and E. roggenkampii and between 91.2% and 98.0% for the remaining ECC species; with data analyzed in the three participating centers, the accuracy was close to 100%. Similar results were obtained with the Mass Spectrometric Identification (MSI) database developed recently (https://msi.happy-dev.fr) except in the case of E. hormaechei, which was more accurately identified with the random forest algorithm. In short, MALDI-TOF MS combined with machine learning was demonstrated to be a rapid and accurate method for the differentiation of ECC species.

Microbiological diagnostic procedures for respiratory cystic fibrosis samples in Spain: towards standard of care practices.

BACKGROUND: The microbiological procedures for cystic fibrosis (CF) samples of 17 participating Spanish centers were examined to verify their compliance with current international and national guidelines and to implement the best standards of care for microbiology practices. A 47-item questionnaire covering different CF microbiology aspects was sent to participant laboratories. Telephone interviews were performed when necessary. Data about samples processing for bacteria, mycobacteria and fungi were collected. RESULTS: Gene sequencing (71%), MALDI-TOF (59%) or both (94%) were available for most laboratories. Susceptibility testing was performed by automated microdilution systems (94%) and manual diffusion methods (59%). However, a low use of selective media for Staphylococcus aureus (59%) and Burkholderia cepacia complex (71%), and of epidemiological typing methods (41%) was reported. CONCLUSIONS: Most Spanish laboratories are in agreement with consensus guidelines for the processing of CF respiratory samples, but need to improve in the use of specific selective media and typing methods for epidemiologic studies.

Statistical Evaluation of Metaproteomics and 16S rRNA Amplicon Sequencing Techniques for Study of Gut Microbiota Establishment in Infants with Cystic Fibrosis.

Newborn screening for cystic fibrosis (CF) can identify affected but asymptomatic infants. The selection of omic technique for gut microbiota study is crucial due to both the small amount of feces available and the low microorganism load. Our aims were to compare the agreement between 16S rRNA amplicon sequencing and metaproteomics by a robust statistical analysis, including both presence and abundance of taxa, to describe the sequential establishment of the gut microbiota during the first year of life in a small size sample (8 infants and 28 fecal samples). The taxonomic assignations by the two techniques were similar, whereas certain discrepancies were observed in the abundance detection, mostly the lower predicted relative abundance of Bifidobacterium and the higher predicted relative abundance of certain Firmicutes and Proteobacteria by amplicon sequencing. During the first months of life, the CF gut microbiota is characterized by a significant enrichment of Ruminococcus gnavus, the expression of certain virulent bacterial traits, and the detection of human inflammation-related proteins. Metaproteomics provides information on composition and functionality, as well as data on host-microbiome interactions. Its strength is the identification and quantification of Actinobacteria and certain classes of Firmicutes, but alpha diversity indices are not comparable to those of amplicon sequencing. Both techniques detected an aberrant microbiota in our small cohort of infants with CF during their first year of life, dominated by the enrichment of R. gnavus within a human inflammatory environment. IMPORTANCE In recent years, some techniques have been incorporated for the study of microbial ecosystems, being 16S rRNA gene sequencing being the most widely used. Metaproteomics provides the advantage of identifying the interaction between microorganisms and human cells, but the available databases are less extensive as well as imprecise. Few studies compare the statistical differences between the two techniques to define the composition of an ecosystem. Our work shows that the two methods are comparable in terms of microorganism identification but provide different results in alpha diversity analysis. On the other hand, we have studied newborns with cystic fibrosis, for whom we have described the establishment of an intestinal ecosystem marked by the inflammatory response of the host and the enrichment of Ruminococcus gnavus.

Multicenter Performance Evaluation of MALDI-TOF MS for Rapid Detection of Carbapenemase Activity in Enterobacterales: The Future of Networking Data Analysis With Online Software.

In this study, we evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories during a multicenter networking validation study. The study was divided into three different stages: "software design," "intercenter evaluation," and "clinical validation." First, a standardized procedure with an online software for data analysis was designed. Carbapenem resistance was detected by measuring imipenem hydrolysis and the results were automatically interpreted using the Clover MS data analysis software (Clover BioSoft, Spain). Second, a series of 74 genotypically characterized Enterobacterales (46 carbapenemase-producers and 28 non carbapenemase-producers) were analyzed in 8 international centers to ensure the reproducibility of the method. Finally, the methodology was evaluated independently in all centers during a 2-month period and results were compared with the reference standard for carbapenemase detection used in each center. The overall agreement rate relative to the reference method for carbapenemase resistance detection in clinical samples was 92.5%. The sensitivity was 93.9% and the specificity, 100%. Results were obtained within 60 min and accuracy ranged from 83.3 to 100% among the different centers. Further, our results demonstrate that MALDI-TOF MS is an outstanding tool for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories. The use of a simple in-house procedure with online software allows routine screening of carbapenemases in diagnostics, thereby facilitating early and appropriate antimicrobial therapy.

Good practice regarding smoking cessation management in Spain: Challenges and opportunities for primary care physicians and nurses.

INTRODUCTION: We analyze the activities carried out by primary care (PC) physicians and nurses with respect to smoking cessation and evaluate their self-reported training, knowledge, and behavior. METHODS: A cross-sectional study was conducted including 1514 PC physicians and nurses from June 2016 to March 2017, in Spain. The main variable was Good Practice (GP) in attention to smokers. To identify associated factors, a multilevel logistic regression model was used adjusted for sex, age, type of center, contract, years of employment, tobacco consumption, and self-reported training/knowledge. RESULTS: Of the 792 physicians and 722 nurses, 48.6% referred to GP in smoking cessation management. The finding related to: being a non-smoker (OR=1.8; 95% CI: 1.2-2.5) or ex-smoker (OR=1.4; 95% CI: 1.02-2.1), having a good level of knowledge (OR=1.8; 95% CI: 1.3-2.4) and training (OR=2.4; 95% CI: 1.8-3.2), and, to a lesser extent, being female (OR=1.3; 95% CI: 1.03-1.7), and work experience >10 years (OR=1.4; 95% CI: 1.03-1.9). The main GP barriers were: lack of time (45.5%), organizational problems (48.4%), and 35.4% lack of training. CONCLUSIONS: The GP of PC physicians and nurses regarding smoking cessation management is related to being non-smokers or ex-smokers, and having sufficient training and knowledge. Lack of time and organizational problems were considered to be the main barriers. The promotion of training activities in the Spanish National Health Service with the support of scientific societies is required.

Antibiotic prescription patterns in Spanish cystic fibrosis patients: results from a national multicenter study.

OBJECTIVE: Information about antibiotic prescription patterns for cystic fibrosis (CF) patients and, specifically, about inhaled treatment strategies for their management is lacking in Spain due to the absence of a national patient registry. In this study we present data about antibiotic prescription in the Spanish CF context that were obtained in a multicenter study, being inhaled treatment strategies the special focus of this work. METHODS: Twenty-four specialized CF units (12 adult, 12 pediatric) from 17 tertiary-care hospitals covering all Spanish Autonomous Communities provided sputa and clinical data from 15 consecutive patients. Data about antibiotic and non-antibiotic therapies prescribed to these patients during the year prior inclusion (2013) were retrospectively collected. RESULTS: The multicenter study included 341 CF patients from all age groups and clinical status. The prevalence of oral, inhaled and intravenous therapies was 89% (n = 302), 80% (n = 273) and 31% (n = 105), respectively. The most prevalent oral agents were ciprofloxacin (n = 177, 59%), cotrimoxazole (n = 109, 36%) and amoxicillin-clavulanate (n = 99, 33%), whereas ceftazidime (n = 53, 50%), tobramycin (n = 43, 41%) and meropenem (n = 41, 49%) were the most prevalent intravenous ones. Two or more different inhaled antibiotics were administered to 67 patients (24%), 51 of them receiving 2 drugs continuously in alternating schemes. Nebulization of intravenous specific antibiotics was common (n = 39) and, in some cases, was used for maintenance purposes. CONCLUSIONS: These results show that the treatment of CF patients is evolving more rapidly than clinical consensus guidelines. Clinical trials evaluating new specific inhaled combinations and new alternative treatment regimes of the existing ones are needed.

Objetivos: Existen actualmente pocos datos acerca de las pautas de tratamiento antimicrobiano administradas a los pacientes con fibrosis quística (FQ) en España, sobre todo en lo que se refiere a la antibioterapia inhalada. Esta escasez de conocimiento se debe principalmente a la ausencia de un registro nacional de datos de pacientes. En 2013 se llevó a cabo el primer estudio multicéntrico español focalizado en la microbiología de la FQ. En este trabajo presentamos los patrones de prescripción de antimicrobianos administrados durante un año a los pacientes incluidos en dicho estudio.Métodos: Se contó con la participación de 24 unidades de FQ (12 de adultos y 12 de pediatría) procedentes de 17 hospitales españoles. Cada unidad reclutó a 15 pacientes de manera consecutiva, que aportaron muestras respiratorias y datos clínicos. Se recogieron de manera retrospectiva los tratamientos antibióticos y no antibióticos administrados a estos pacientes durante el año previo a su inclusión en el estudio.Resultados: Se incluyeron 341 pacientes con FQ de todos los rangos de edad y de gravedad clínica. La prevalencia de antibioterapia oral, inhalada e intravenosa fue del 89% (n = 302), 80% (n = 273) y 31% (n = 105), respectivamente. Los fármacos administrados con mayor frecuencia por vía oral fueron ciprofloxacino (n = 177, 59%), cotrimoxazol (n = 109, 36%) y amoxicilina-clavulánico (n = 99, 33%), siendo ceftazidima (n = 53, 50%), tobramicina (n = 43, 41%) y meropenem (n = 41, 49%) los más frecuentes por vía intravenosa. Se administraron dos o más antibióticos por vía inhalada a 67 pacientes (24%), habiendo recibido 51 de ellos 2 antibióticos simultáneamente de manera rotatoria. La nebulización de antibióticos con formulación intravenosa fue una práctica común (n = 39) y, en algunos casos, se utilizó durante un tiempo prolongado como terapia de mantenimiento.Conclusiones: Los esquemas de tratamiento observados en este estudio demuestran que la terapia antibiótica de la FQ evoluciona más rápidamente que las recomendaciones reflejadas en las guías clínicas. Es necesario evaluar estos nuevos esquemas con estudios clínicos, así como con otros fármacos inhalados de reciente aparición y su papel en los esquemas existentes.