Estudio de nuevas dianas terapéuticas frente a Acinetobacter baumannii mediante análisis transcriptómico in vivo y evaluación de nuevas estrategias antimicrobianas

Programa Oficial de Doctorado en Ciencias de la Salud. 5007V01[Resumen] Acinetobacter baumannii es un patógeno nosocomial con gran plasticidad genética y resistencia antimicrobiana, implicado en brotes hospitalarios difíciles de controlar. El estudio del genoma de este patógeno nos permite seleccionar aquellos genes más interesantes como posibles dianas terapéuticas. En esta Tesis se presenta un análisis transcriptómico global del ARN bacteriano aislado de los pulmones de ratones infectados con A. baumannii, con la finalidad de identificar aquellos genes implicados en el desarrollo de la infección por neumonía. Los genes hisF y lpxB se encontraron sobreexpresados durante la infección. HisF está implicado en la persistencia en pulmón debido a la inhibición del reclutamiento de macrófagos y la producción de IL-6 por parte del huésped. La traducción de LpxB, proteína esencial implicada en la síntesis de LPS, ha sido inhibida empleando tecnología antisentido. La colistina, antibiótico con elevada toxicidad, se utiliza en el tratamiento de cepas multirresistentes y es considerado de última opción terapéutica. El inhibidor de SPasas MD3 presenta sinergia con la colistina, siendo así un candidato prometedor en el tratamiento de infecciones causadas por A. baumannii, incluyendo cepas resistentes a colistina. El análisis transcriptómico in vivo nos permitió identificar una serie de genes como dianas terapéuticas potenciales y se evaluaron nuevas estrategias y compuestos antimicrobianos para el tratamiento de A.[Resumo] Acinetobacter baumannii é un patóxeno nosocomial que presenta unha gran plasticidade xenética e resistencia antimicrobiana frecuentemente implicado en brotes hospitalarios difíciles de controlar. O estudo do xenoma deste patóxeno permítenos seleccionar aqueles xenes máis interesantes como posibles dianas terapéuticas. Nesta Tese preséntase unha análise transcriptómica global do ARN bacteriano illado directamente dos pulmóns de ratos infectados con A. baumannii, coa finalidade de identifica-los xenes implicados no desenvolvemento da infección. Os xenes hisF e lpxB atopáronse sobreexpresados durante a infección. HisF atópase implicado na persistencia en pulmón pola inhibición do reclutamento de macrófagos e da producción de IL-6 por parte do hospedador. A traducción de LpxB, proteína esencial implicada na síntese de LPS, foi inhibida empregando tecnoloxía antisentido A colistina, antibiótico cunha elevada toxicidade, emprégase principalmente no tratamento de cepas multirresistentes e é considerado de última opción terapéutica. O MD3 é un inhibidor de SPasas que presenta sinerxia coa colistina, sendo así un candidato prometedor no tratamento de infeccións causadas por A. baumannii, incluindo cepas resistentes a colistina. A análise transcriptómica in vivo permitiunos identificar unha serie de xenes como dianas terapéuticas potenciais e avaliáronse novas estratexias e compostos antimicrobianos para o tratamento de A. baumannii.[Abstract] Acinetobacter baumannii is a nosocomial pathogen that presents great genetic plasticity and antimicrobial resistance frequently implied hospital outbreaks difficult to control. The study of the genome of this pathogen allows us to select the most interesting genes as possible therapeutic targets. This Thesis presents a global transcriptomic analysis of bacterial RNA isolated directly from the lungs of mice infected with A. baumannii, in order to identify those genes involved in the development of pneumonia infection. hisF and lpxB genes were found to be overexpressed during infection. HisF is involved in lung persistence due to inhibition of macrophage recruitment and IL-6 production by the host. The translation of LpxB, the essential protein involved in the synthesis of LPS, has been inhibited using antisense technology. Colistin, an antibiotic with a high toxicity, is mainly used in the treatment of multi-resistant strains and is considered the last therapeutic option. MD3 is a SPasas inhibitor that presents synergy with colistin, thus being a promising candidate in the treatment of infections caused by A. baumannii, including colistin-resistant strains. In vivo transcriptomic analysis allowed us to identify several genes as potential therapeutic targets and evaluated new strategies and antimicrobial compounds for the treatment of A. baumannii

Pneumonia infection in mice reveals the involvement of the feoA gene in the pathogenesis of Acinetobacter baumannii

[Abstract] Acinetobacter baumannii has emerged in the last decade as an important nosocomial pathogen. To identify genes involved in the course of a pneumonia infection, gene expression profiles were obtained from A. baumannii ATCC 17978 grown in mouse infected lungs and in culture medium. Gene expression analysis allowed us to determine a gene, the A1S_0242 gene (feoA), over-expressed during the pneumonia infection. In the present work, we evaluate the role of this gene, involved in iron uptake. The inactivation of the A1S_0242 gene resulted in an increase susceptibility to oxidative stress and a decrease in biofilm formation, in adherence to A549 cells and in fitness. In addition, infection of G. mellonella and pneumonia in mice showed that the virulence of the Δ0242 mutant was significantly attenuated. Data presented in this work indicated that the A1S_0242 gene from A. baumannii ATCC 17978 strain plays a role in fitness, adhesion, biofilm formation, growth, and, definitively, in virulence. Taken together, these observations show the implication of the feoA gene plays in the pathogenesis of A. baumannii and highlight its value as a potential therapeutic target.This work has been funded by Projects PI15/00860 to GB, CP13/00226 to AB, PI11/01034 to MP and P14/00059 and PI17/01482 to MP and AB, all integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the ISCIII – General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe”. The study was also funded by the project IN607A 2016/22 (Consellería de Cultura, Educación e Ordenación Universitaria) to G.B. Also supported by Planes Nacionales de I+D+i 2008–2011 / 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 and REIPI RD16/0016/006) co-financed by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014–2020. J. A. Vallejo was financially supported by the Sara Borrell Programme (ISCIII, Spain CD13/00373), J.C. Vázquez-Ucha was financially supported by the Miguel Servet Programme (ISCIII, Spain CP13/00226) and M. Martínez-Guitián was financially supported by the grant Clara Roy (Spanish Society of Clinical Microbiology and Infectious Diseases). We thank M. I. Voskuil (Dept. of Immunology and Microbiology, University of Colorado Medical School, CO, USA) for providing pMo130.Xunta de Galicia. Consellería de Cultura, Educación e Ordenación Universitaria; IN607A 2016/2

Determinants of Eurasian Otter (Lutra Lutra) Diet in a Seasonally Changing Reservoir

This is an accepted version of the published document. This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: https://doi.org/10.1007/s10750-020-04208-y[Abstract] Otter diet in reservoirs is known to experience seasonal changes. We selected a reservoir with a large population of exclusively wintering great cormorants and seasonal changes in stored water volume to test the relative influence of abiotic and biotic factors on otter foraging ecology. DNA metabarcoding of otter spraints revealed a dietary change from autumn to winter. Otters had a diet dominated by the exotic goldfish in autumn, but predated intensively on the native northern straight-mouth nase in winter. This change was likely caused by predation of cormorants on goldfish and to fish biology. Secondly, macroscopic analysis of spraints revealed that otters shifted from a diet dominated by fish (in terms of biomass) to a diet dominated by red swamp crayfish during spring–summer, when the latter became overabundant. As revealed by modelling, this second shift was most likely influenced by the sudden increase in stored water volume in spring, but also by the cumulative effect of cormorant predation on fish during autumn–winter. Macroscopic analyses of otter spraints collected in a second reservoir with no cormorants revealed a lack of seasonality. Hence, the combined influence of both biotic and abiotic factors explained otter diet seasonality in a lentic-water novel ecosystem.This work received funding from Xunta de Galicia (Grants GRC2014/050 and ED431C 2018/57) and Universidade da Coruña. A Martínez-Abraín was supported by an Isidro Parga Pondal research contract by Xunta de Galicia during the period 2011–2016Xunta de Galicia; GRC2014/050Xunta de Galicia; ED431C 2018/5

Draft Genome Sequences of Two Epidemic OXA-48-Producing Klebsiella pneumoniae Clinical Strains Isolated during a Large Outbreak in Spain

[Abstract] We report here the draft genome sequences of Klebsiella pneumoniae strains Kp1803 and Kp3380 isolated during a large outbreak at A Coruña Hospital in Spain. The final genome assemblies for Kp1803 and Kp3380 comprise approximately 6.6 and 6.1 Mb, respectively, and both strains have G+C contents of 57.2%.This work was supported by grants from the Spanish Society of Infectious Diseases and Clinical Microbiology to A.P. and by projects PI11/01034 and P14/00059 to M.P., integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the Instituto de Salud Carlos III (ISCII), General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A Way of Making Europe.” We also thank the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 to G.B.), cofinanced by the European Development Regional Fund (EDRF) and by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad

In Vitro and In Vivo Assessment of the Efficacy of Bromoageliferin, an Alkaloid Isolated from the Sponge Agelas dilatata, against Pseudomonas aeruginosa

[Abstract] The pyrrole-imidazoles, a group of alkaloids commonly found in marine sponges belonging to the genus Agelas, display a wide range of biological activities. Herein, we report the first chemical study of the secondary metabolites of the sponge A. dilatata from the coastal area of the Yucatan Peninsula (Mexico). In this study, we isolated eight known alkaloids from an organic extract of the sponge. We used NMR and MS analysis and comparison with existing databases to characterize the alkaloids: ageliferin (1), bromoageliferin (2), dibromoageliferin (3), sceptrin (4), nakamuric acid (5), 4-bromo-1H-pyrrole-2-carboxylic acid (6), 4,5-dibromopyrrole-2-carboxylic acid (7) and 3,7-dimethylisoguanine (8). We also evaluated, for the first time, the activity of these alkaloids against the most problematic multidrug-resistant (MDR) pathogens, i.e., the Gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Bromoageliferin (2) displayed significant activity against P. aeruginosa. Comparison of the antibacterial activity of ageliferins 1–3 (of similar structure) against P. aeruginosa revealed some relationship between structure and activity. Furthermore, in in vitro assays, 2 inhibited growth and biofilm production in clinical strains of P. aeruginosa. Moreover, 2 increased the survival time in an in vivo Galleria mellonella model of infection. The findings confirm bromoageliferin (2) as a potential lead for designing new antibacterial drugs.This work was supported by Grants RTI2018-093634-B-C22 (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, both co-funded by the FEDER Programme from the European Union and BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP). The study was also funded by projects GRC2018/039 and Agrupación Estratégica CICA-INIBIC ED431E 2018/03 (Consellería de Educación, Universidades e Formación Profesional) from the Xunta de Galicia (autonomous government of the region). This work was also funded by Projects PI17/01482 awarded to AB and PI15/00860 to GB, all within in the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 and funded by the ISCIII-General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe” and operative program Intelligent Growth 2014-2020. The study was also funded by project IN607A 2016/22 (GAIN- Agencia Gallega de Innovación - Consellería de Economía, Emprego e Industria) awarded to GB. D.P.-P. received a fellowship from the program National Council of Science and Technology (CONACYT) of Mexico and the Secretariat of Research, Innovation and Higher Education (SIIES) of Yucatan. Mexico and the Secretariat of Research, Innovation and Higher Education (SIIES) of Yucatan. JCVU was financially supported by the pFIS Program (FI18/00315), MMG by a Clara Roy grant (SEIMC) and CLM by IN606A-2019/029Xunta de Galicia; 0474_BLUEBIOLAB_1_EXunta de Galicia; GRC2018/039Xunta de Galicia; ED431E 2018/03Xunta de Galicia; IN607A 2016/22Xunta de Galicia; IN606A-2019/02

Involvement of HisF in the persistence of Acinetobacter baumannii during a pneumonia infection

[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.Instituto de Salud Carlos III; PI15/00860Instituto de Salud Carlos III; PI14/00059Instituto de Salud Carlos III; PI17/01482Axencia Galega de Innovación; IN607A 2016/22Spanish Network for Research in Infectious Diseases; REIPI RD12/0015/0014Spanish Network for Research in Infectious Diseases; REIPI RD16/0016/006Instituto de Salud Carlos III; FI18/00315Xunta de Galicia; IN606A-2019/029Xunta de Galicia; IN607A 2016/2

Activity of the β-Lactamase inhibitor LN-1-255 against carbapenem-hydrolyzing class D β-lactamases from Acinetobacter baumannii

The number of infections caused by Gram-negative pathogens carrying carbapenemases is increasing, and the group of carbapenem-hydrolyzing class D β-lactamases (CHDLs) is especially problematic. Several clinically important CHDLs have been identified in A. baumannii, including OXA-23, OXA-24/40, OXA-58, OXA-143, OXA-235, and the chromosomally encoded OXA-51. The selection and dissemination of carbapenem-resistant A. baumannii strains constitutes a serious global threat. Carbapenems have been successfully utilized as last resort antibiotics for the treatment of multi-drug-resistant A. baumannii infections. However, the spread of OXA carbapenemases is compromising the continued use of these antimicrobials. In response to this clinical issue, it is necessary and urgent to design and develop new specific inhibitors with efficacy against these enzymes. The aim of this work is to characterize the inhibitory activity of LN-1-255 (a 6-alkylidene-2-substituted penicillin sulfone) and compare it to that of two established inhibitors (avibactam and tazobactam) against the most relevant enzymes of each group of class D carbapenemases in A. baumannii. The β-lactamase inhibitor LN-1-255 demonstrated excellent microbiological synergy and inhibition kinetics parameters against all tested CHDLs, and a significantly higher activity than tazobactam and avibactam. A combination of carbapenems and LN-1-255 was effective against A. baumannii class D carbapenemases. Docking assays confirmed the affinity of LN-1-255 for the active site of these enzymes. LN-1-255 represents a potential new β-lactamase inhibitor, which may have a significant role in eradicating infections caused by A. baumannii isolates carrying CHDLsThis work was supported by the Spanish National Plans for Scientific Research, Development and Technological Innovation 2008-2011 and 2013-2016 and funded by the ISCIII- General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (ERDF) “A way of making Europe”: PI12/00552 to G.B. and PI14/00059 to M.P. and A.B. Also, this study was supported in part by funds from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (USA) under award numbers R01AI063517 and R01AI100560, by funds and/or facilities provided by the Cleveland Department of Veterans Affairs, the Veterans Affairs Merit Review Program Award 1I01BX001974 and the Geriatric Research Education and Clinical Center VISN 10 to R.A.B., and by the Spanish Ministry of Economy and Competiveness (SAF2013-42899-R), Xunta de Galicia (GRC2013-041) and the European Regional Development Fund (ERDF) to C.GB. J.V. was financially supported by the Sara Borrell Programme ISCIII-FEDER (CD13/00373). J.V.H. and A.B. were financially supported by the Miguel Servet Programme ISCIII-FEDER (CP13/00226)S

Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent Adduct

The first example of an ammonium derivative that causes a specific modification of the active site of type I dehydroquinase (DHQ1), a dehydratase enzyme that is a promising target for antivirulence drug discovery, is described. The resolution at 1.35 Å of the crystal structure of DHQ1 from Salmonella typhi chemically modified by this ammonium derivative revealed that the ligand is covalently attached to the essential Lys170 through the formation of an amine. The detection by mass spectroscopy of the reaction intermediates, in conjunction with the results of molecular dynamics simulations, allowed us to explain the inhibition mechanism and the experimentally observed differences between S. typhi and Staphylococcus aureus enzymes. The results presented here reveal that the replacement of Phe225 in St-DHQ1 by Tyr214 in Sa-DHQ1 and its hydrogen bonding interaction with the conserved water molecule observed in several crystal structures protects the amino adduct against further dehydration/aromatization reactions. In contrast, for the St-DHQ1 enzyme, the carboxylate group of Asp114, with the assistance of this water molecule, would trigger the formation of a Schiff base that can undergo further dehydration reactions until full aromatization of the cyclohexane ring is achieved. Moreover, in vitro antivirulence studies showed that the reported compound is able to reduce the ability of Salmonella Enteritidis to kill A459 respiratory cells. These studies have identified a good scaffold for the design of irreversible inhibitors that can be used as drugs and has opened up new opportunities for the development of novel antivirulence agents by targeting the DHQ1 enzymeFinancial support from the Spanish Ministry of Science and Innovation (SAF2013-42899-R), Xunta de Galicia (GRC2013-041), and the European Regional Development Fund (ERDF) is gratefully acknowledged. E.L. thanks the Xunta de Galicia for his postdoctoral fellowship. A.B. thanks the Miguel Servet Programme ISCIII-FEDER (CP13/00226) and the ISCIIIGeneral Subdirection of Assesment and Promotion of the Research (PI14/00059) for financial supportS

LN-1-255, a penicillanic acid sulfone able to inhibit the class D carbapenemase OXA-48

This is a pre-copyedited, author-produced version of an article accepted for publication in Journal of Antimicrobial Chemotherapy following peer review. The version of record Juan A. Vallejo, Marta Martínez-Guitián, Juan C. Vázquez-Ucha, Concepción González-Bello, Margarita Poza, John D. Buynak, Christopher R. Bethel, Robert A. Bonomo, German Bou, Alejandro Beceiro; LN-1-255, a penicillanic acid sulfone able to inhibit the class D carbapenemase OXA-48, Journal of Antimicrobial Chemotherapy, Volume 71, Issue 8, 1 August 2016, Pages 2171–2180 is available online at: https://doi.org/10.1093/jac/dkw105Objectives Carbapenemases are the most important mechanism responsible for carbapenem resistance in Enterobacteriaceae. Among carbapenemases, OXA-48 presents unique challenges as it is resistant to β-lactam inhibitors. Here, we test the capacity of the compound LN-1-255, a 6-alkylidene-2′-substituted penicillanic acid sulfone, to inhibit the activity of the carbapenemase OXA-48. Methods The OXA-48 gene was cloned and expressed in Klebsiella pneumoniae and Escherichia coli in order to obtain MICs in the presence of inhibitors (clavulanic acid, tazobactam and sulbactam) and LN-1-255. OXA-48 was purified and steady-state kinetics was performed with LN-1-255 and tazobactam. The covalent binding mode of LN-1-255 with OXA-48 was studied by docking assays. Results Both OXA-48-producing clinical and transformant strains displayed increased susceptibility to carbapenem antibiotics in the presence of 4 mg/L LN-1-255 (2–32-fold increased susceptibility) and 16 mg/L LN-1-255 (4–64-fold increased susceptibility). Kinetic assays demonstrated that LN-1-255 is able to inhibit OXA-48 with an acylation efficiency (k2/K) of 10 ± 1 × 104 M−1 s−1 and a slow deacylation rate (koff) of 7 ± 1 × 10−4 s−1. IC50 was 3 nM for LN-1-255 and 1.5 μM for tazobactam. Lastly, kcat/kinact was 500-fold lower for LN-1-255 than for tazobactam. Conclusions In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases.This work was supported by the Spanish National Plans for Scientific Research, Development and Technological Innovation 2013-16 and funded by the ISCIII-General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (ERDF) ‘A way of making Europe’: PI12/00552 to G. B. and PI14/00059 to M. P. and A. B. Also, research reported in this publication was supported in part by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (USA) under Award Numbers R01AI100560, R01AI063517 and R01AI072219 to R. A. B. This study was supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs (USA), Award Number 1I01BX001974 to R. A. B. from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development and the Geriatric Research Education and Clinical Center VISN 10 (USA) to R. A. B. This study was also supported by the Spanish Ministry of Economy and Competiveness (SAF2013-42899-R), Xunta de Galicia (Spain) (GRC2013-041) and the European Regional Development Fund (ERDF) to C. G.-B, and supported by National Institutes of Health (USA) to J. D. B. (1R15AI109624). J. V. A. was financially supported by the Sara Borrell Programme ISCIII-FEDER (CD13/00373). J. V. H. and A. B. were financially supported by the Miguel Servet Programme ISCIII-FEDER (CP13/00226)S

In-Depth Analysis of the Role of the Acinetobactin Cluster in the Virulence of Acinetobacter baumannii

[Abstract] Acinetobacter baumannii is a multidrug-resistant pathogen that represents a serious threat to global health. A. baumannii possesses a wide range of virulence factors that contribute to the bacterial pathogenicity. Among them, the siderophore acinetobactin is one of the most important, being essential for the development of the infection. In this study we performed an in-depth analysis of the acinetobactin cluster in the strain A. baumannii ATCC 17978. For this purpose, nineteen individual isogenic mutant strains were generated, and further phenotypical analysis were performed. Individual mutants lacking the biosynthetic genes entA, basG, basC, basD, and basB showed a significant loss in virulence, due to the disruption in the acinetobactin production. Similarly, the gene bauA, coding for the acinetobactin receptor, was also found to be crucial for the bacterial pathogenesis. In addition, the analysis of the ΔbasJ/ΔfbsB double mutant strain demonstrated the high level of genetic redundancy between siderophores where the role of specific genes of the acinetobactin cluster can be fulfilled by their fimsbactin redundant genes. Overall, this study highlights the essential role of entA, basG, basC, basD, basB and bauA in the pathogenicity of A. baumannii and provides potential therapeutic targets for the design of new antivirulence agents against this microorganism.This work was funded by Projects PI15/00860 awarded to GB and PI17/01482 to AB and MP, all within in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the ISCIII – General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe.” The study was also funded by project IN607A 2016/22 (GAIN- Agencia Gallega de Innovación – Consellería de Economía, Emprego e Industria) awarded to GB. This work was also supported by Planes Nacionales de I + D + i 2008–2011/2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/006) co-financed by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014–2020. This work was also supported by Grant RTI2018-093634-B-C22 (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, co-funded by the FEDER Programme from the European Union and Xunta de Galicia for the support of Grant ED431E 2018/03 for CICA-INIBIC strategic and the initiative “Seed Projects 2019–2020.” JV-U was financially supported by the ISCIII project FI18/00315, LÁ-F by the ISCIII project PI14/00059 and the IN606B-2018/011, MM-G was financially supported by the Grant Clara Roy (SEIMC, Spanish Society of Clinical Microbiology and Infectious Diseases), KC-P by IN607A 2016/22 and AECC (Asociación Española Contra el Cáncer) predoctoral fellowship and LA by Xunta de Galicia co-funded with the European Social Fund (FSE) of the European Union (ED481A-2019/081)Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED431E 2018/03Xunta de Galicia; IN606B-2018/011Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED481A-2019/08