Estudio descriptivo observacional retrospectivo desde Mayo de 2012 a Abril de 2014 en pacientes pediátricos con sospecha de enfermedad celíaca en el Área Sanitaria de A Coruña

La enfermedad celiaca ha ganado gran popularidad en los últimos años estando presente en la humanidad desde hace siglos. El conocimiento de la enfermedad celiaca (EC) ha evolucionado mucho en las últimas décadas, pudiendo saber cómo se produce, qué factores afectan a su aparición y qué síntomas y signos se manifiestan. Esto ha permitido diferenciar varios tipos de EC según el cuadro clínico, así como identificar grupos de riesgo que pueden ser susceptibles a la enfermedad, consiguiendo con la aparición de nuevas pruebas una gran rapidez en el diagnóstico. El objetivo de este trabajo es conocer la incidencia de la EC en individuos pediátricos en el Área Sanitaria de A Coruña, así como obtener diversos datos de la enfermedad en cuanto al sexo, grupos de edad, presentación clínica… que nos acerquen al perfil más común. Los resultados obtenidos muestran una incidencia elevada de la EC en A Coruña, 34,92 casos por cada 100.000 personas-año, siendo la enfermedad más común en mujeres, existiendo diferencias en la presentación clínica atendiendo a la edad, apareciendo síntomas gastrointestinales en niños de temprana edad, y desarrollándose enfermedades asociadas en un pequeño número de pacientes. Aparecen diferencias en la genética de los distintos pacientes, así como en el resultado de las pruebas histológicas, no obstante muchos aspectos de la EC se nos escapan debido al número reducido de casos encontrados así como al reducido tiempo del estudio (2 años) por lo que a pesar de mostrársenos una visión cercana de la enfermedad un conocimiento completo requeriría un esfuerzo mayor.Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2013/201

Nuevos inhibidores de β-lactamasas frente a patógenos gram negativos multirresistentes

Programa Oficial de Doutoramento en Ciencias da Saúde. 5007V01[Resumo] As especies Acinetobacter baumannii, Pseudomonas aeruginosa e a orde Enterobacterales son categorizados como patóxenos de prioridade crítica para o desenvolvemento de novos antimicrobianos. O grupo de antibióticos máis utilizado é o dos antibióticos β-lactámicos. Sendo as cefalosporinas e os carbapenems os de maior espectro de actividade. O uso destes antibióticos vese comprometido pola aparición e diseminación de β-lactamasas de espectro ampliado (BLEAs) e carbapenemasas. Unha opción para manter a actividade destes β-lactámicos é a utilización de inhibidores de β-lactamasas. Nesta Tese Doutoral determinouse a boa actividade do LN-1-255, unha penicilín sulfona en fase preclínica, fronte ás carbapenemasas OXA-48 de Enterobacterales e as principais β-lactamasas de clase D que hidrolizan carbapenems (CHDLs) de A. baumannii. Isto último tamén se demostrou en ensaios in vivo, onde a combinación de imipenem e LN-1- 255 diminuíu considerablemente a carga bacteriana. Por outra banda, tanto o LN-1-255 como dous derivados do mesmo demostraron ser capaces de inhibir a PDC-1 de P. aeruginosa, permitindo recuperar a actividade da ceftazidima. Finalmente demostrouse a eficacia das combinacións imipenem/relebactam, recentemente aprobada, cefepima/zidebactam e cefepima/taniborbactam, en fase clínica III, fronte a un estudo multicéntrico nacional de Enterobacterales produtores de carbapenemasas.[Resumen] Las especies Acinetobacter baumannii, Pseudomonas aeruginosa y el orden Enterobacterales son categorizados como patógenos de prioridad crítica para el desarrollo de nuevos antimicrobianos. El grupo de antibióticos más utilizado es el de los antibióticos β-lactámicos. Siendo las cefalosporinas y los carbapenems los de mayor espectro de actividad. El uso de estos antibióticos se ve comprometido por la aparición y diseminación de β-lactamasas de espectro ampliado (BLEAs) y carbapenemasas. Una opción para mantener la actividad de estos β-lactámicos es la utilización de inhibidores de β-lactamasas. En esta Tesis Doctoral se determinó la buena actividad del LN-1-255, una penicilín sulfona en fase preclínica, frente a las carbapenemasas OXA-48 de Enterobacterales y las principales β-lactamasas de clase D que hidrolizan carbapenems (CHDLs) de A. baumannii. Esto último también se demostró en ensayos in vivo, donde la combinación de imipenem y LN-1-255 disminuyó considerablemente la carga bacteriana. Por otra parte, tanto el LN-1-255 como dos derivados del mismo demostraron ser capaces de inhibir la PDC-1 de P. aeruginosa, permitiendo recuperar la actividad de la ceftazidima. Finalmente se demostró la eficacia de las combinaciones imipenem/relebactam, recientemente aprobada, cefepima/zidebactam y cefepima/taniborbactam, en fase clínica III, frente a un estudio multicéntrico nacional de Enterobacterales productores de carbapenemasas.[Abstract] Acinetobacter baumannii, Pseudomonas aeruginosa and the order Enterobacterales are categorised as critical priority pathogens for the development of new antimicrobials. The most widely used group of antibiotics is the β-lactam group. Cephalosporins and carbapenems have the highest spectrum of activity. The use of these antibiotics is compromised by the emergence and spread of extended-spectrum β-lactamases (ESBLs) and carbapenemases. One option to maintain the activity of these β-lactams is the use of β-lactamase inhibitors. In this Thesis, the good activity of LN-1-255, a penicillin sulphone in the preclinical phase, was determined against the OXA-48 carbapenemase of Enterobacterales and the carbapenem-hydrolysing class D β-lactamases (CHDLs) of A. baumannii. The latter was also demonstrated in in vivo trials, where the combination of imipenem and LN-1-255 significantly decreased the bacterial load. Moreover, both LN-1-255 and two derivatives of LN-1-255 were shown to be able to inhibit PDC-1 of P. aeruginosa, allowing the activity of ceftazidime to be restored. Finally, the efficacy of the recently approved imipenem/relebactam, and cefepime/zidebactam and cefepime/taniborbactam combinations, in clinical phase III, was demonstrated in a national multicentre study of carbapenemase-producing Enterobacterales

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

Carbapenem Resistance in Acinetobacter nosocomialis and Acinetobacter junii Conferred by Acquisition of blaOXA-24/40 and Genetic Characterization of the Transmission Mechanism between Acinetobacter Genomic Species

[Abstract] Carbapenem resistance is increasing among Gram-negative bacteria, including the genus Acinetobacter. This study aimed to characterize, for the first time, the development of carbapenem resistance in clinical isolates of Acinetobacter junii and Acinetobacter nosocomialis conferred by the acquisition of a plasmid-borne blaOXA-24/40 gene and also to characterize the dissemination of this gene between species of Acinetobacter. Carbapenem-resistant A. nosocomialis HUAV-AN66 and A. junii HUAV-AJ77 strains were isolated in the Arnau de Vilanova Hospital (Spain). The genomes were sequenced, and in silico analysis were performed to characterize the genetic environment and the OXA-24/40 transmission mechanism. Antibiotic MICs were determined, and horizontal transfer assays were conducted to evaluate interspecies transmission of OXA-24/40. Carbapenems MICs obtained were ≥64 mg/L for HUAV-AN66 and HUAV-AJ77. Genome analysis revealed the presence in both strains of a new plasmid, designated pHUAV/OXA-24/40, harboring the carbapenem-resistance gene blaOXA-24/40 and flanked by sequences XerC/XerD. pHUAV/OXA-24/40 was successfully transferred from A. nosocomialis and A. junii to a carbapenem-susceptible A. baumannii strain, thus conferring carbapenem resistance. A second plasmid (pHUAV/AMG-R) was identified in both clinical isolates for the successful horizontal transfer of pHUAV/OXA-24/40. blaOXA-24/40-carrying plasmids of the GR12 group and showing high identity with pHUAV/OXA-24/40 were identified in at least 8 Acinetobacter species. In conclusion the carbapenemase OXA-24/40 is described for the first time in A. nosocomialis and A. junii. In both isolates the blaOXA-24/40 gene was located in the GR12 pHUAV/OXA-24/40 plasmid. GR12 plasmids are implicated in the dissemination and spread of carbapenem resistance among Acinetobacter species. IMPORTANCE Acinetobacter baumannii is one of the most relevant pathogens in terms of antibiotic resistance. The main resistance mechanisms are the carbapenem-hydrolyzing class D β-lactamases (CHDLs), especially OXA-23 and OXA-24/40. In addition to A. baumannii, there are other species within the genus Acinetobacter, which in general exhibit much lower resistance rates. In this work we characterize for the first time two clinical isolates of Acinetobacter nosocomialis and Acinetobacter junii, isolated in the same hospital, carrying the carbapenemase OXA-24/40 and displaying high resistance rates to carbapenems. By means of bioinformatics analysis we have also been able to characterize the mechanism by which this carbapenemase is horizontally transferred interspecies of Acinetobacter spp. The dissemination of carbapenemase OXA-24/40 between non-baumannii Acinetobacter species is concerning since it prevents the use of most β-lactam antibiotics in the fight against these resistant isolates.This work was supported by Projects PI17/01482 and PI20/01212 awarded to A.B. and PI18/00501 to G.B., 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 work was also supported by CIBERINF (CIBER de Enfermedades Infecciosas). The study was also funded by project IN607A 2020/05 (GAIN- Agencia Gallega de Innovación - Consellería de Economía, Emprego e Industria) awarded to G.B. and IN607D 2021/12 awarded to A.B. This work was also supported by Planes Nacionales de I+D+i2008 to 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 Infectiosus Diseases (REIPI RD16/0016/006) cofinanced by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014–2020. J.A.-S.was financially supported by the Rio Hortega program (ISCIII, CM19/00219), J.C.V.-U. was financially supported by the pFIS program (ISCIII, PI17/01482), C.L.-M. was financially supported by IN606A-2019/029 Grant (Xunta de Galicia) and P.G.-S. was financially supported by IN607A 2020/05 Grant (Xunta de Galicia)Xunta de Galicia; IN607A 2020/05Xunta de Galicia; IN607D 2021/12Xunta 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

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

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

Epidemiology, resistance genomics and susceptibility of Acinetobacter species: results from the 2020 Spanish nationwide surveillance study

[Abstract]: Background: As increasing antibiotic resistance in Acinetobacter baumannii poses a global healthcare challenge, understanding its evolution is crucial for effective control strategies. Aim: We aimed to evaluate the epidemiology, antimicrobial susceptibility and main resistance mechanisms of Acinetobacter spp. in Spain in 2020, and to explore temporal trends of A. baumannii. Methods: We collected 199 single-patient Acinetobacter spp. clinical isolates in 2020 from 18 Spanish tertiary hospitals. Minimum inhibitory concentrations (MICs) for nine antimicrobials were determined. Short-read sequencing was performed for all isolates, and targeted long-read sequencing for A. baumannii. Resistance mechanisms, phylogenetics and clonality were assessed. Findings on resistance rates and infection types were compared with data from 2000 and 2010. Results: Cefiderocol and colistin exhibited the highest activity against A. baumannii, although colistin susceptibility has significantly declined over 2 decades. A. non-baumannii strains were highly susceptible to most tested antibiotics. Of the A. baumannii isolates, 47.5% (56/118) were multidrug-resistant (MDR). Phylogeny and clonal relationship analysis of A. baumannii revealed five prevalent international clones, notably IC2 (ST2, n = 52; ST745, n = 4) and IC1 (ST1, n = 14), and some episodes of clonal dissemination. Genes blaOXA-23, blaOXA-58 and blaOXA-24/40 were identified in 49 (41.5%), eight (6.8%) and one (0.8%) A. baumannii isolates, respectively. ISAba1 was found upstream of the gene (a blaOXA-51-like) in 10 isolates. Conclusions: The emergence of OXA-23-producing ST1 and ST2, the predominant MDR lineages, shows a pivotal shift in carbapenem-resistant A. baumannii (CRAB) epidemiology in Spain. Coupled with increased colistin resistance, these changes underscore notable alterations in regional antimicrobial resistance dynamics.This work was supported by Projects PI20/01212 and PI23/00851 awarded to AB, Project PI21/00704 awarded to GB, and Project PI22/01212 awarded to JA-S, funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union. The work was also supported by CIBER-Consorcio Centro de Investigación Biomédica en Red- de Enfermedades Infecciosas, Instituto de Salud Carlos III (CB21/13/00055), Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU and by project IN607A 2020/05 (GAIN-Agencia Gallega de InnovaciónConsellería de Economía, Emprego e Industria) awarded to GB and project IN607D 2021/12 awarded to AB. JA-S was financially supported by the Juan Rodés program (ISCIII, JR21/00026), JCV-U was financially supported by IN606B-2022/009 (Xunta de Galicia), CL-M was financially supported by PI20/01212 (ISCIII), PG-S was financially supported by IN606A 2021/021 Grant (Xunta de Galicia). MO was financially supported by IN607D 2021/12. IA-G was financially supported by the Rio Hortega program (ISCIII, CM21/00076). MM-G was financially supported by RSU.UDC.M505 (Ministerio de Universidades).Xunta de Galicia; IN607A 2020/05Xunta de Galicia; IN607D 2021/12Xunta de Galicia; IN606B-2022/009Xunta de Galicia; IN606A 2021/021Xunta de Galicia; IN607D 2021/12Universidade da Coruña; RSU.UDC.M50

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