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[Resumen] La epidemiología basada en aguas residuales (EAR) es fundamental para la monitorización de patógenos responsables de enfermedades infecciosas. Esta Tesis Doctoral presenta en detalle el proyecto COVIDBENS, un programa multidisciplinar que integra las áreas de microbiología, estadística y bioinformática, implementado en el área metropolitana de A Coruña durante la pandemia de COVID-19, con el objetivo de monitorizar el virus SARS-CoV-2 en las aguas residuales de la depuradora de EDAR Bens, que da servicio a los municipios de A Coruña, Arteixo, Cambre, Culleredo y Oleiros. COVIDBENS operó como un sistema de alerta temprana de brotes de SARS-CoV-2 y de sus variantes emergentes, proporcionando información crucial para comprender la verdadera magnitud de la epidemia en la población y facilitando la toma de decisiones por parte de las autoridades. Además, se monitorizó la carga viral de SARS-CoV-2 (copias de ARN/L) a nivel local a partir de las aguas residuales de residencias de mayores ubicadas en el área metropolitana de A Coruña para prever nuevos brotes entre los residentes y evaluar la eficacia de la campaña de vacunación. Este trabajo demostró una vez más que la EAR es una estrategia rentable para prevenir brotes de enfermedades emergentes o futuras amenazas pandémicas., [Resumo] A epidemioloxía baseada en augas residuais (EAR) é fundamental para o rastrexo de patóxenos responsables de enfermidades infecciosas. Esta Tese Doutoral presenta en detalle o proxecto COVIDBENS, un programa multidisciplinar que integra as áreas de microbioloxía, estatística e bioinformática, implementado na área metropolitana da Coruña durante a pandemia de COVID-19, co obxectivo de rastrexar o virus SARS-CoV-2 nas augas residuais da depuradora de EDAR Bens, que serve aos municipios da Coruña, Arteixo, Cambre, Culleredo e Oleiros. COVIDBENS operou como un sistema de alerta temperá de gromos de SARS-CoV-2 e das súas variantes emerxentes, proporcionando información crucial para comprender a verdadeira magnitude da epidemia na poboación e facilitando a toma de decisións por parte das autoridades. Ademais, rastrexouse a carga viral de SARS-CoV-2 (copias de ARN /L) a nivel local a partir das augas residuais de residencias de maiores situadas na área metropolitana da Coruña para prever novos gromos entre os residentes e avaliar a eficacia da campaña de vacinación. Este traballo demostrou unha vez máis que a EAR é unha estratexia rendible para prever gromos de enfermidades emerxentes ou futuras ameazas pandémicas., [Abstract] Wastewater-based epidemiology (WBE) is essential for monitoring pathogens responsible for infectious diseases. This Doctoral Thesis presents in detail the COVIDBENS project, a multidisciplinary program that integrates the areas of microbiology, statistics and bioinformatics, implemented in the metropolitan area of A Coruña with the aim of monitoring SARS-CoV-2 in wastewater from the wastewater treatment plant of Bens, which serves to the municipalities of A Coruña, Arteixo, Cambre, Culleredo and Oleiros. COVIDBENS worked as an early warning system for SARS-CoV-2 outbreaks and its emerging variants, providing crucial information to understand the true magnitude of the epidemic in the population and facilitating decision-making by authorities. Furthermore, the viral load of SARS-CoV-2 (RNA copies/L) at local level was monitored from the wastewater of nursing homes located in the metropolitan area of A Coruña to predict new outbreaks among residents and to evaluate the effectiveness of the vaccination campaign. This work once again demonstrated that the WBE is a cost-effective strategy to prevent emerging diseases outbreaks or future pandemic threats.

[Abstract] The incidence of colorectal cancer (CRC) has increased worldwide, and early diagnosis is crucial to reduce mortality rates. Therefore, new noninvasive biomarkers for CRC are required. Recent studies have revealed an imbalance in the oral and gut microbiomes of patients with CRC, as well as impaired gut vascular barrier function. In the present study, the microbiomes of saliva, crevicular fluid, feces, and non-neoplastic and tumor intestinal tissue samples of 93 CRC patients and 30 healthy individuals without digestive disorders (non-CRC) were analyzed by 16S rRNA metabarcoding procedures. The data revealed that Parvimonas, Fusobacterium, and Bacteroides fragilis were significantly over-represented in stool samples of CRC patients, whereas Faecalibacterium and Blautia were significantly over-abundant in the non-CRC group. Moreover, the tumor samples were enriched in well-known periodontal anaerobes, including Fusobacterium, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Co-occurrence patterns of these oral microorganisms were observed in the subgingival pocket and in the tumor tissues of CRC patients, where they also correlated with other gut microbes, such as Hungatella. This study provides new evidence that oral pathobionts, normally located in subgingival pockets, can migrate to the colon and probably aggregate with aerobic bacteria, forming synergistic consortia. Furthermore, we suggest that the group composed of Fusobacterium, Parvimonas, Bacteroides, and Faecalibacterium could be used to design an excellent noninvasive fecal test for the early diagnosis of CRC. The combination of these four genera would significantly improve the reliability of a discriminatory test with respect to others that use a single species as a unique CRC biomarker.

[Resumo] A resistencia antibiótica, con Acinetobacter baumannii e Enterobacter cloacae complex (ECC) como microorganismos clave, constitúe unha das principais causas de morbimortalidade a nivel mundial. O estudo destas poboacións resulta esencial para desenvolver estratexias que mitiguen o impacto das infeccións na saúde pública. Nesta Tese Doutoral analízanse dúas coleccións multicéntricas nacionais de cepas clínicas, unha de Acinetobacter spp. e outra de ECC, co propósito de determinar a súa epidemioloxía, patróns e mecanismos de resistencia antibiótica, así como de explorar a actividade de novas alternativas terapéuticas. Deste xeito, cefiderocol posicionouse como o antibiótico máis activo contra A. baumannii, seguido pola colistina, a pesar do descenso significativo na eficacia desta na última década. Sublíñase tamén a problemática relacionada co ST2, con numerosos illamentos XDR, e cunha representación destacada da OXA-23, que evidencia un cambio paradigmático na epidemioloxía de A. baumannii resistente a carbapenémicos en España. Esta resistencia, mediada na súa maioría por carbapenemasas de clase D, queda aplacada polo inhibidor LN-1-255, o cal restaura o potencial de antibióticos como imipenem ou sulbactam. Doutra banda, nesta Tese caracterízase por primeira vez a diseminación plasmídica da carbapenemasa OXA-24/40 entre illamentos de Acinetobacter no-baumannii pertencentes á colección, así como a súa propagación internacional. Finalmente, en referencia a ECC, demostrouse a asociación de VIM-1 coa redución da sensibilidade ao cefiderocol, abordando aspectos microbiolóxicos, bioquímicos e moleculares. Ademais, destácase a influencia de factores adicionais na restrición da actividade desta cefalosporina de última xeración en ECC., [Resumen] La resistencia antibiótica, con Acinetobacter baumannii y Enterobacter cloacae complex (ECC) como microorganismos clave, constituye una causa importante de morbimortalidad a nivel global. El estudio de estas poblaciones es esencial para desarrollar estrategias que mitiguen el impacto de sus infecciones en la salud pública. En la presente Tesis Doctoral se analizan dos colecciones multicéntricas nacionales de cepas clínicas, una de Acinetobacter spp. y otra de ECC, con el propósito de determinar su epidemiología, sus patrones y mecanismos de resistencia antibiótica, así como de explorar la actividad de nuevas alternativas terapéuticas. En este sentido, cefiderocol se posiciona como el antibiótico más activo contra A. baumannii, seguido de colistina, a pesar de la disminución significativa en la eficacia de esta en la última década. Se subraya asimismo la problemática relacionada con el ST2, con numerosos aislados XDR, y una representación destacada de OXA-23, que evidencia un cambio paradigmático en la epidemiología de A. baumannii resistente a carbapenémicos en España. Esta resistencia, mediada en su mayoría por carbapenemasas de clase D, queda aplacada por el inhibidor LN-1-255, que recupera el potencial de compuestos como imipenem o sulbactam. Por otro lado, en esta Tesis se caracteriza por primera vez la diseminación plasmídica de la carbapenemasa OXA-24/40 entre aislados de Acinetobacter no-baumannii pertenecientes a la colección, así como su propagación internacional. Finalmente, en referencia a ECC, se demuestra la asociación de VIM-1 con la reducción de la sensibilidad a cefiderocol, abordando aspectos microbiológicos, bioquímicos y moleculares. Además, se destaca la influencia de factores adicionales en la restricción de la actividad de esta cefalosporina de última generación en ECC., [Abstract] Antibiotic resistance, involving Acinetobacter baumannii and the Enterobacter cloacae complex (ECC) as pivotal microorganisms, significantly contributes to global morbimortality. Studying these populations is crucial for developing effective strategies to lessen the impact of the resulting infections on public health. In this Doctoral Thesis two multicentric national collections of clinical strains, one comprising Acinetobacter spp. and the other ECC isolates, are examined to discern their epidemiology, resistance patterns and mechanisms, and to ultimately explore the activity of new therapeutic alternatives. Cefiderocol emerges as the most potent antibiotic against A. baumannii, followed by colistin, despite its notable decrease in efficacy over the past decade. Attention is drawn to the issue of ST2, characterized by numerous XDR isolates and a notable representation of OXA-23, signaling a paradigm shift in carbapenem-resistant A. baumannii epidemiology in Spain. This resistance, largely mediated by class D carbapenemases, is mitigated by the LN-1-255 inhibitor, restoring the potential of compounds like imipenem or sulbactam. On another note, in this Thesis, plasmid-mediated dissemination of the carbapenemase OXA-24/40 among isolates of Acinetobacter non-baumannii belonging to the collection is characterized for the first time, highlighting its international spread. Concerning ECC, the association of VIM-1 with diminished susceptibility to cefiderocol is demonstrated, addressing microbiological, biochemical, and molecular factors. Furthermore, emphasis is placed on the influence of additional factors restricting the activity of this latest generation cephalosporin in ECC.

The dynamics of SARS-CoV-2 transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the USA became increasingly significant. Notably, Galicia’s major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

[Resumen] El microbioma humano desempeña un papel esencial en el mantenimiento de las funciones fisiológicas normales. Estudios previos han demostrado la implicación del microbioma en el desarrollo de patologías, tales como el cáncer colorrectal (CCR). En el presente trabajo, se describió el bacterioma de muestras de saliva, fluído crevicular, heces y tejidos de tumores primarios y de metastásis, procedentes de una cohorte de 93 pacientes de CCR, mediante tecnologías de secuenciación de próxima generación. Los resultados obtenidos se compararon con el bacterioma de muestras de individuos sin CCR, con el objetivo principal de comprender la implicación de las bacterias en el desarrollo del CCR. Los resultados mostraron que el microbioma intestinal de pacientes de CCR estaba desequilibrado y enriquecido en patógenos periodontales. Una combinación específica de estos patógenos orales, ausentes en muestras de personas sin CCR, se propone para su uso como biomarcador para el diagnóstico de CCR empleando muestras no invasivas (heces). Uno de estos patógenos, Parvimonas micra, detectado en la cavidad oral y en carcinomas colorrectales, fue estudiado en profundidad, en una cohorte aleatoria de 20 pacientes, demostrándose mediante genómica comparativa, que esta bacteria es capaz de translocarse desde la cavidad subgingival hasta el colon, formando consorcios sinérgicos con otras bacterias., [Resumo] O microbioma humán desempeña un papel esencial no mantemento das funcións fisiolóxicas normais. Estudos previos demostraron a implicación do microbioma no desenvolvemento de diversas patoloxías, tales como o cancro colorrectal (CCR). No presente traballo, describiuse o bacterioma de mostras de saliva, fluído crevicular xinxival, feces e tecidos de tumores primarios e de metastásis, procedentes dunha cohorte de 93 pacientes de CCR, mediante tecnoloxías de secuenciación de próxima xeración. Os resultados obtidos comparáronse co bacterioma de mostras de individuos sen CCR, co obxectivo principal de comprender a implicación das bacterias no desenvolvemento do CCR. Os resultados mostraron que o microbioma intestinal de pacientes con CCR estaba desequilibrado e enriquecido en patóxenos periodontais. Unha combinación específica destes patóxenos orais, ausentes en mostras de persoas sen CCR, proponse para o seu emprego como biomarcador para o diagnóstico de CCR empregando mostras non invasivas (feces). Un destes patógenos, Parvimonas micra, detectado na cavidade oral e nos carcinomas colorrectais, foi estudado en profundidade nunha cohorte aleatoria de 20 pacientes, demostrándose, mediante xenómica comparativa, que esta bacteria é capaz de translocarse desde a cavidade subxinxival ata o colon, formando consorcios sinérxicos con outras bacterias., [Abstract] The human microbiome plays an essential role in maintaining normal physiological functions. Previous studies have demonstrated its involvement in the development of different diseases such as colorectal cancer (CCR). This study used next-generation sequencing technologies to characterize the bacteriome of saliva, gingival crevicular fluid, feces, primary tumor tissues, and metastatic tissues from a cohort of 93 CRC patients. The results obtained were compared with bacteriome profiles of samples from a group of individuals without CCR, with the main objective of understanding the role of oral and gut bacteria in the CCR development. The findings revealed an imbalanced and a periodontal pathogen-enrichment in the intestinal microbiome of CCR patients. Notably, a specific combination of oral pathogens, absent in no CCR individuals, was identified as a potential biomarker for non-invasive CCR diagnosis using fecal samples. Among these pathogens, Parvimonas micra, detected in the oral cavity and in colorectal carcinomas, was extensively studied in a randomized cohort of 20 CCR patients. Comparative genome analysis demonstrated that this bacterium was able to translocate from the subgingival cavity to the colon forming synergistic consortia with other oral bacteria.

[Abstract] Oral and intestinal samples from a cohort of 93 colorectal cancer (CRC) patients and 30 healthy controls (non-CRC) were collected for microbiome analysis. Saliva (28 non-CRC and 94 CRC), feces (30 non-CRC and 97 CRC), subgingival fluid (20 CRC), and tumor tissue samples (20 CRC) were used for 16S metabarcoding and/or RNA sequencing (RNAseq) approaches. A differential analysis of the abundance, performed with the ANCOM-BC package, adjusting the P-values by the Holm-Bonferroni method, revealed that Parvimonas was significantly over-represented in feces from CRC patients (P-value < 0.001) compared to healthy controls. A total of 11 Parvimonas micra isolates were obtained from the oral cavity and adenocarcinoma of CRC patients. Genome analysis identified a pair of isolates from the same patient that shared 99.2% identity, demonstrating that P. micra can translocate from the subgingival cavity to the gut. The data suggest that P. micra could migrate in a synergistic consortium with other periodontal bacteria. Metatranscriptomics confirmed that oral bacteria were more active in tumor than in non-neoplastic tissues. We suggest that P. micra could be considered as a CRC biomarker detected in non-invasive samples such as feces.

[Abstract] Wastewater-based epidemiology (WBE) has become an effective tool in the surveillance of infectious diseases such as COVID-19. In this work, we performed a brief study of monitoring the SARS-CoV-2 viral load in wastewater from six nursing homes located in the metropolitan area of A Coruña (Spain) between December 2020 and March 2021. The main objective was to detect SARSCoV- 2 outbreaks among residents and study the efficacy of the vaccination campaign. SARS-CoV-2 viral load (RNA copies per L of wastewater) was determined by reverse-transcription quantitative PCR (RT-qPCR) using the quantification cycle (Cq) values for the nucleocapsid (N) gene. Our results showed that the increase in viral load preceded the increase in clinical cases, favoring an early warning system that detects COVID-19 outbreaks in advance, making it possible to contain and stop the transmission of the virus among residents. In addition, the efficacy of the new COVID-19 vaccines was evidenced, since after the vaccination campaign in nursing homes in A Coruña, it was observed that many residents did not present any symptoms of the disease, although they excreted high amounts of virus in their feces. WBE is a cost-effective strategy that should be implemented in all cities to prevent new emerging diseases or future pandemic threats.

[Abstract] In the day-to-day work of bioinformatics, the use of integrated software packages, which encompass a wide range of tools, enables the development of pipelines for omics data analysis. Within the various existing pipelines, we focus on the analysis of the 16S rRNA gene as it allows for the study of diversity and taxonomy of prokaryotic microorganisms such as Bacteria and Archaea. However, these pipelines often involve a sequence of multiple tools that require intermediate steps before further processing can proceed, as in the case between Cutadapt and DADA2. In fact, in a typical pipeline, the values for DADA2 input arguments ’trunc-len-f’ and ’trunc-len-r’ are extracted from the output of Cutadapt. The best approach for selecting optimal values (aka the trimming positions) is graphically visualizing Cutadapt output and manually selecting the most accurate trimming position length. Therefore, we propose the automation of this specific intermediate step between Cutadapt and DADA2 tools, by selecting values displayed in the graphs that meet the filtering criteria. This automation has been incorporated into a custom pipeline for the analysis of the microbiome in 16S paired-end samples from colorectal cancer patients, and could potentially serve as a standardization approach in these processes

[Abstract] In the last decades, personalized medicine has been increasing its presence in different fields of medicine, including ophthalmology. A new factor that can help us direct medicine towards the challenge of personalized treatments is the microbiome. The gut microbiome plays an important role in controlling immune response, and dysbiosis has been associated with immune-mediated diseases such as non-infectious uveitis (NIU). In this review, we gather the published evidence, both in the pre-clinical and clinical studies, that support the possible role of intestinal dysbiosis in the pathogenesis of NIU, as well as the modulation of the gut microbiota as a new possible therapeutic target. We describe the different mechanisms that have been proposed to involve dysbiosis in the causality of NIU, as well as the potential pharmacological tools that could be used to modify the microbiome (dietary supplementation, antibiotics, fecal microbiota transplantation, immunomodulators, or biologic drugs) and, consequently, in the control of the NIU. Furthermore, there is increasing scientific evidence suggesting that the treatment with anti-TNF not only restores the composition of the gut microbiota but also that the study of the composition of the gut microbiome will help predict the response of each patient to anti-TNF treatment.

[Resumen]: El virus SARS-CoV-2 está compuesto por una nucleocápside que engloba su material genético y la proteína N, rodeada por una membrana bilipídica, que contiene las proteínas M y E, y una corona de espinas, que contienen la proteína S. La proteína S se encarga de unirse a los receptores ACE2 presentes en gran variedad de células del cuerpo humano, sobre todo en el epitelio alveolar pulmonar y en el epitelio intestinal, en donde se aloja de forma persistente. Cuando el virus se une a estos receptores, se puede desencadenar una cascada inflamatoria que origina la enfermedad COVID-19, que incluso puede llegar a desencadenar la muerte del individuo. Un porcentaje importante de infectados no desarrollan la enfermedad y permanecen asintomáticos aunque su cuerpo esté invadido por el virus. De hecho, SARS-CoV-2 se excreta en las heces de las personas infectadas, tengan o no síntomas de COVID-19, desde el inicio de la infección hasta mucho tiempo después de dar negativo en muestras nasofaríngeas1. Por eso, las estaciones depuradoras de aguas residuales (EDAR) sirven como sistemas de vigilancia epidemiológica y permiten conocer la verdadera magnitud de la pandemia de COVID-19 a partir del análisis de la cantidad de virus presente en sus aguas.

[Abstract] Background The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach. Results A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%. Conclusions Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.

[Abstract] The quantification of the SARS-CoV-2 RNA load in wastewater has emerged as a useful tool to monitor COVID–19 outbreaks in the community. This approach was implemented in the metropolitan area of A Coruña (NW Spain), where wastewater from a treatment plant was analyzed to track the epidemic dynamics in a population of 369,098 inhabitants. Viral load detected in the wastewater and the epidemiological data from A Coruña health system served as main sources for statistical models developing. Regression models described here allowed us to estimate the number of infected people (R2 = 0.9), including symptomatic and asymptomatic individuals. These models have helped to understand the real magnitude of the epidemic in a population at any given time and have been used as an effective early warning tool for predicting outbreaks in A Coruña municipality. The methodology of the present work could be used to develop a similar wastewater-based epidemiological model to track the evolution of the COVID–19 epidemic anywhere in the world where centralized water-based sanitation systems exist.

[Resumo] Acinetobacter baumannii é un patóxeno oportunista que xurdiu nos últimos anos como un dos microorganismos máis perigosos dentro do ambiente hospitalario. Na presente tese doutoral caracterizáronse dous novos factores de virulencia que poderían explicar parcialmente a habilidade que ten A. baumannii para causar unha infección. O xene LH92_11085 de A. baumannii MAR002, implicado na formación dun pilus tipo chaperona- usher, atopouse sobreexpresado en células do biofilm en comparación con células planctónicas. A inactivación deste xene resultou nunha redución na capacidade da cepa de formar biofilm e de adherirse a células epiteliais e nun descenso na virulencia. O xene A1S_0242 (feoA) da cepa A. baumannii ATCC 17978, que está implicado na captación de ferro e que se atopou sobreexpresado durante un proceso de pneumonía, xoga un papel na adhesión, na formación de biofilm, na resistencia ó estrés oxidativo e na virulencia in vivo. Todos os resultados obtidos durante esta tese doutoral demostraron a contribución destes xenes na patoxénese de A. baumannii., [Resumen] Acinetobacter baumannii es un patógeno oportunista que ha emergido en los últimos años como uno de los microorganismos más peligrosos dentro del ambiente hospitalario. En la presente tesis doctoral se caracterizaron dos nuevos factores de virulencia que podrían explicar parcialmente la habilidad que tiene A. baumannii para causar una infección. El gen LH92_11085 de A. baumannii MAR002, implicado en la formación de un pilus tipo chaperona-usher, se encontró sobreexpresado en células del biofilm en comparación con células planctónicas. La inactivación de este gen resultó en una reducción en la capacidad de la cepa de formar biofilm y de adherirse a células epiteliales y un descenso en la virulencia. El gen A1S_0242 (feoA) de la cepa A. baumannii ATCC 17978, que está implicado en la captación de hierro y que se encontró sobreexpresado durante un proceso de neumonía, juega un papel en la adhesión, la formación de biofilm, la resistencia al estrés oxidativo y en la virulencia in vivo. Todos los resultados obtenidos durante esta tesis doctoral demostraron la contribución de estos genes a la patogénesis de A. baumannii., [Abstract] Acinetobacter baumannii is an opportunist pathogen that has emerged in the last years as one of the most dangerous microorganisms living in hospital environments. In the present work two new virulence factors, that could partially explain the ability of A. baumannii to cause an infection, have been characterized. The LH92_11085 gene of the A. baumannii MAR002 strain, involved in the formation of a chaperone-usher pilus system, was found as over-expressed in biofilm cells compared to planktonic cells. The inactivation of this gene resulted in a reduction in the capacity of the MAR002 strain to form biofilm and to adhere to human epithelial cells and in a decrease in virulence. The A1S_0242 gene (feoA) from the A. baumannii ATCC 17978 strain, which is involved in iron uptake and that was found as over-expressed during the course of a pneumonia infection, plays a role in adhesion, biofilm formation, resistance to oxidative stress and in virulence. Data obtained in this work demostrate the contribution of both genes to the pathogenesis of A. baumannii.

[Abstract] The discovery of new antibiotics that are effective against Acinetobacter baumannii and Enterobacteralesis a research priority. Several essential oils (EOs) have displayed some antimicrobial activity and could potentially act as antibiotic adjuvants. Research in this area aims to develop new therapeutic alternatives to treat infections caused by these pathogens. MICs of different EOs were determined against A. baumannii and Klebsiella pneumoniae. Combined disk diffusion tests and checkerboard assays were used to study the synergy between the EOs and antibiotics. The fractional inhibitory concentration index (FICindex) was calculated in order to categorize the interaction. Time-kill assays were also performed. The EOs that displayed the highest levels of antimicrobial activity were clove (Syzygium aromaticum L.) and thyme (Thymus zygis L.). Combined disk diffusion tests and checkerboard assays revealed synergy between these EOs and colistin. Addition of either clove or thyme EO decreased the MIC of colistin by 8- to 64-fold and 8- to 128-fold in the colistin-resistant A. baumannii and K. pneumoniae strains, respectively (FICindex ≤ 0.5, synergy). MICs were also reduced in the colistin-susceptible strains. Time-kill assays also indicated the strong activity of the combined therapy. In summary, the use of clove or thyme EO in combination with colistin could improve the efficacy of the antibiotic and significantly reduce the concentrations needed to inhibit growth of A. baumannii and K. pneumoniae.

[Abstract] The important nosocomial pathogen Acinetobacter baumannii presents a quorum sensing (QS) system (abaI/abaR) mediated by acyl-homoserine-lactones (AHLs) and several quorum quenching (QQ) enzymes. However, the roles of this complex network in the control of the expression of important virulence-related phenotypes such as surface-associated motility and biofilm formation is not clear. Therefore, the effect of the mutation of the AHL synthase AbaI, and the exogenous addition of the QQ enzyme Aii20J on surface-associated motility and biofilm formation by A. baumannii ATCC® 17978TM was studied in detail. The effect of the enzyme on biofilm formation by several multidrug-resistant A. baumannii clinical isolates differing in their motility pattern was also tested. We provide evidence that a functional QS system is required for surface-associated motility and robust biofilm formation in A. baumannii ATCC® 17978TM. Important differences were found with the well-studied strain A. nosocomialis M2 regarding the relevance of the QS system depending on environmental conditions The in vitro biofilm-formation capacity of A. baumannii clinical strains was highly variable and was not related to the antibiotic resistance or surface-associated motility profiles. A high variability was also found in the sensitivity of the clinical strains to the action of the QQ enzyme, revealing important differences in virulence regulation between A. baumannii isolates and confirming that studies restricted to a single strain are not representative for the development of novel antimicrobial strategies. Extracellular DNA emerges as a key component of the extracellular matrix in A. baumannii biofilms since the combined action of the QQ enzyme Aii20J and DNase reduced biofilm formation in all tested strains. Results demonstrate that QQ strategies in combination with other enzymatic treatments such as DNase could represent an alternative approach for the prevention of A. baumannii colonization a

[Abstract] Acinetobacter baumannii is currently considered one of themost problematic nosocomial microorganisms. In the present work the hisF gene from the ATCC 17978 strain and the AbH12O-A2 clinical isolate of A. baumannii was found over-expressed during the course of murine pneumonia infections. The study demonstrated that the A. baumannii ATCC 17978 mutant strain lacking the hisF gene induces a sub-lethal pneumonia infection in mice, while the complemented mutant strain increased its virulence. This histidine auxotroph mutant showed an increase on IL-6 secretion and leukocytes recruitment during infections. Furthermore, data revealed that the hisF gene, implicated in the innate immunity and inflammation, is involved in virulence during a pneumonia infection, which may partly explain the ability of this strain to persist in the lung. We suggest that HisF, essential for full virulence in this pathogen, should be considered a potential target for developing new antimicrobial therapies against A. baumannii.