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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. Instituto de Salud Carlos III (ISCIII)

Programa Oficial de Doutoramento en Bioloxía Celular e Molecular . 5004V01 [Resumo] Klebsiella pneumoniae ST15 é un clon de alto risco con unha gran plasticidade xenética e portador dunha gran diversidade de xenes de resistencia a antimicrobianos. Está implicado na produción de gromos hospitalarios en todo o mundo e posúe unha gran capacidade para colonizar o tracto gastrointestinal, o que supón un factor de risco importante para o posterior desenvolvemento dunha infección. Esta Tesis Doctoral aborda o estudo dos mecanismos implicados na patoxénesis de K. pneumoniae ST15 co obxectivo de buscar novas dianas terapéuticas e estratexias alternativas para o manexo das infeccións causadas por este clon de alto risco. Para iso utilizamos como modelo un clon epidémico de K. pneumoniae ST15 illado durante un gran gromo producido no Hospital Universitario de A Coruña. A análise comparativa do seu xenoma fronte a outros clons pertencentes a diferentes STs de K. pneumoniae permitiunos identificar as características xenómicas específicas deste clon que poderían estar implicadas no seu éxito como patóxeno. Encontramos que o clon ST15 posúe dous factores de virulencia relacionados coa adherencia que non están presentes no resto de STs analizados. Identificáronse e caracterizáronse por primeira vez en K. pneumoniae un sistema de tipo chaperona-usher pili, o sistema Kpi, e un sistema de secreción de dous compoñentes, o sistema FhaB/FhaC. Estes sistemas están directamente relacionados co fenotipo altamente adherente mostrado polo clon epidémico ST15, xa que están implicados na capacidade de este clon para adherirse a diferentes células eucariotas e formar biofilm. Ademais, os sistemas Kpi e FhaB/FhaC están estreitamente relacionados con este ST e confírenlle unha maior eficacia biolóxica. 20 Por outro lado, demostrouse que o sistema Kpi ten un papel determinante tanto na habilidade mostrada por este clon epidémico para colonizar o tracto gastrointestinal nun modelo murino de colonización, como no seu potencial patoxénico nun modelo de infección en Galleria mellonella. Tendo en conta que os dous sistemas caracterizados neste estudio están claramente asociados co clon de alto risco ST15, estes poden considerarse biomarcadores óptimos para identificar este clon de maneira específica. Neste contexto, ambos sistemas poderían ser usados como diana para detectar K. pneumoniae ST15 con unha alta sensibilidade e especificidade. Finalmente, obtivemos e caracterizamos un extracto salino de Vaccinium corymbosum con un alto contido en polifenois. Este extracto inhibe a adhesión a células eucariotas intestinais e a formación de biofilm na cepa Kp3380. O efecto antiadherente producido por este extracto reduce significativamente a colonización intestinal producida por K. pneumoniae nun modelo murino. [Resumen] Klebsiella pneumoniae ST15 es un clon de alto riesgo con una gran plasticidad genética y portador de una gran diversidad de genes de resistencia a antimicrobianos. Está implicado en la producción de brotes hospitalarios en todo el mundo y posee una gran capacidad para colonizar el tracto gastrointestinal, lo que supone un factor de riesgo importante para el posterior desarrollo de una infección. Esta Tesis Doctoral aborda el estudio de los mecanismos implicados en la patogénesis de K. pneumoniae ST15 con el objetivo de buscar nuevas dianas terapéuticas y estrategias alternativas para el manejo de las infecciones causadas por este clon de alto riesgo. Para ello utilizamos como modelo un clon epidémico de K. pneumoniae ST15 aislado durante un gran brote producido en el Hospital Universitario de A Coruña. El análisis comparativo de su genoma frente a otros clones pertenecientes a diferentes STs de K. pneumoniae nos permitió identificar las características genómicas específicas de este clon que podrían estar implicadas en su éxito como patógeno. Encontramos que el clon ST15 posee dos factores de virulencia relacionados con la adherencia que no están presentes en el resto de STs analizados. Se han identificado y caracterizado por primera vez en K. pneumoniae un sistema tipo chaperona-usher pili, el sistema Kpi, y un sistema de secreción de dos componentes, el sistema FhaB/FhaC. Estos sistemas están directamente relacionados con el fenotipo altamente adherente mostrado por el clon epidémico ST15, ya que están implicados en la capacidad de este clon para adherirse a diferentes células eucariotas y formar biofilm. Además, los sistemas Kpi y FhaB/FhaC están estrechamente relacionados con este ST y le confieren una mayor eficacia biológica. 22 Por otra parte, se ha demostrado que el sistema Kpi tiene un papel determinante tanto en la habilidad mostrada por este clon epidémico para colonizar el tracto gastrointestinal en un modelo murino de colonización, como en su potencial patogénico en un modelo de infección en Galleria mellonella. Teniendo en cuenta que los dos sistemas caracterizados en este estudio están claramente asociados con el clon de alto riesgo ST15, estos pueden considerarse biomarcadores óptimos para identificar este clon de manera específica. En este contexto, ambos sistemas podrían ser usados como diana para detectar K. pneumoniae ST15 con una alta sensibilidad y especificidad. Finalmente, hemos obtenido y caracterizado un extracto salino de Vaccinium corymbosum con un alto contenido en polifenoles. Este extracto inhibe la adhesión a células eucariotas intestinales y la formación de biofilm en la cepa Kp3380. El efecto anti-adherente producido por este extracto reduce significativamente la colonización intestinal producida por K. pneumoniae en un modelo murino. [Abstract] Klebsiella pneumoniae ST15 is a high-risk clone with high genetic plasticity carrying a wide diversity of antimicrobial resistance genes. It is implicated in the production of hospital outbreaks worldwide and it has a high capacity to colonize the gastrointestinal tract, which is a major risk factor for the subsequent development of an infection. This Doctoral Thesis deals with the study of the mechanisms involved in the pathogenesis of K. pneumoniae ST15 with the aim of searching for new therapeutic targets and alternative strategies for the management of infections caused by this highrisk clone. For this purpose, we used as a model an epidemic clone of K. pneumoniae ST15 isolated during a large outbreak at the University Hospital of A Coruña. Comparative analysis of its genome against other clones belonging to different K. pneumoniae STs allowed us to identify specific genomic features of this clone that could be involved in its success as a pathogen. We found that clone ST15 possesses two virulence factors related to adherence that are not present in the rest of the STs analysed. A chaperone-usher pili system, the Kpi system, and a two-component secretion system, the FhaB/FhaC system, have been identified and characterized for the first time in K. pneumoniae. These systems are directly related to the highly adherent phenotype displayed by the epidemic clone ST15, as they are involved in the ability of this clone to adhere to different eukaryotic cells and form biofilm. In addition, the Kpi and FhaB/FhaC systems are closely related to this ST and confer greater fitness. On the other hand, it has been demonstrated that the Kpi system has a determining role both in the ability shown by this epidemic clone to colonize the 24 gastrointestinal tract in a murine model of colonization, and in its pathogenic potential in a model of infection in Galleria mellonella. Considering that the two systems characterized in this study are clearly associated with the high-risk clone ST15, they can be considered optimal biomarkers to identify this clone specifically. In this context, both systems could be used as targets to detect K. pneumoniae ST15 with high sensitivity and specificity. Finally, we have obtained and characterized a saline extract of Vaccinium corymbosum with a high polyphenol content. This extract inhibits intestinal eukaryotic cell adhesion and biofilm formation in the strain Kp3380. The antiadhesive effect produced by this extract significantly reduces intestinal colonization by K. pneumoniae in a murine model.

[Introduction] The main aim of this study was to assess changes in the epidemiology and clinical presentation of Acinetobacter baumannii over a 10-year period, as well as risk factors of mortality in infected patients. [Method] Prospective, multicentre, hospital-based cohort studies including critically ill patients with A. baumannii isolated from any clinical sample were included. These were divided into a first period (“2000 study”) (one month), and a second period (“2010 study”) (two months). Molecular typing was performed by REP-PCR, PFGE and MSLT. The primary endpoint was 30-day mortality. [Results] In 2000 and 2010, 103 and 108 patients were included, and the incidence of A. baumannii colonization/infection in the ICU decreased in 2010 (1.23 vs. 4.35 cases/1000 patient-days; p < 0.0001). No differences were found in the colonization rates (44.3 vs. 38.6%) or infected patients (55.7 vs. 61.4%) in both periods. Overall, 30-day mortality was similar in both periods (29.1 vs. 27.8%). The rate of pneumonia increased from 46.2 in 2000 to 64.8% in 2010 (p < 0.001). Performing MSLT, 18 different sequence types (ST) were identified (18 in 2000, 8 in 2010), but ST2 and ST79 were the predominant clones. ST2 isolates in the ICU increased from 53.4% in the year 2000 to 73.8% in 2010 (p = 0.002). In patients with A. baumannii infection, the multivariate analysis identified appropriate antimicrobial therapy and ST79 clonal group as protective factors for mortality. [Conclusions] At 10 years of the first analysis, some variations have been observed in the epidemiology of A. baumannii in the ICU, with no changes in mortality. Epidemic ST79 clone seems to be associated with a better prognosis and adequate treatment is crucial in terms of survival. [Introducción] El principal objetivo fue evaluar los cambios en la epidemiología a lo largo de un periodo de 10 años, así como la presentación clínica y los factores predictores de mortalidad en los pacientes críticos infectados por Acinetobacter baumannii. [Método] Estudio de cohortes prospectivo y multicéntrico en el que se incluyeron pacientes críticos con A. baumannii aislado de cualquier muestra clínica. Se consideró un primer período («estudio de 2000») (un mes) y un segundo («estudio de 2010») (2 meses). La tipificación molecular se realizó mediante REP-PCR, PFGE y MSLT. La variable resultado primaria fue la mortalidad a los 30 días. [Resultados] En 2000 y 2010 se incluyeron 103 y 108 pacientes, respectivamente, y la incidencia de colonización/infección por A. baumannii en la UCI disminuyó en 2010 respecto al 2000 (1,23 vs. 4,35 casos/1.000 pacientes-días; p < 0,0001). No se encontraron diferencias en la tasa de colonización (44,3 vs. 38,6%) o infección (55,7 vs. 61,4%) en ambos periodos. En general, la mortalidad a los 30 días fue similar en ambos periodos (29,1 vs. 27,8%). La tasa de neumonía aumentó desde el 46,2% en 2000 al 64,8% en 2010 (p < 0,001). Mediante MSLT, se identificaron 18 tipos de secuencias diferentes (ST) (18 en 2000, 8 en 2010), pero ST2 y ST79 fueron los clones predominantes. La identificación de ST2 aumentó en la UCI desde el 53,4% en 2000 al 73,8% en 2010 (p = 0,002). En los pacientes infectados, el tratamiento antimicrobiano apropiado y el grupo clonal ST79 fueron factores protectores de mortalidad en el análisis multivariante. [Conclusiones] A los 10 años del primer análisis se han observado algunos cambios en la epidemiología de A. baumannii en la UCI, sin cambios en la mortalidad. El clon ST79 epidémico parece estar asociado con un mejor pronóstico, y el tratamiento adecuado es crucial en términos de supervivencia.

Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans.The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However,the mechanisms promoting the dissemination of such highrisk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the littlecharacterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K.pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella. The findings provide information about the pathobiology and epidemicity of Kpi+ K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization., This research was supported by Projects p-01216A and IJCI-2016-29524 (to A.P.), funded by the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) and the Minestry of Economy and Competetiveness(MINECO), respectively. It was also supported by Projects PI11/01034 (to M.P.), PI14/00059 and PI17/1482 (to M.P. and A.B.), and PI18/00501 (to G.B.),included in the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 and funded by the Instituto de Salud Carlos III (ISCIII) and Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/006) cofinanced by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014-2020. Grant BFU2016-77835-R of the MINECO (to A.R.) also supported this research. E.G. was financially supported by the SEIMC project. J.C.V.-U. was financially supported by the PFIS (Contratos Predoctorales de Formación en Investigación en Salud) program (F18/00315);J.A.V. was financially supported by IN607A 2016/22; M.M.-G. was financially supported by a Clara Roy grant (SEIMC); A.B. was financially supported by the Miguel Servet program (ISCIII, Spain); B.K.R.-J. was financially supported by Marie S. Curie Action SaPhaDe project (MSCA-IF-GF-836754); and A.P. was financially supported by the Juan de la Cierva program (MINECO, IJCI-2016-29524).

Acinetobacter spp. are found in 53% of air colonization samples from the hospital environment. In this work, we sequenced all the genome of airborne Acinetobacter sp. strain 5-2Ac02. We found important features at the genomic level in regards to the rhizome. By phylogenetic analysis, A. towneri was the species most closely related to Acinetobacter sp. 5-2Ac02.