Transcriptional analysis reveals new factors involved in the biofilm formation ability of "Acinetobacter baumannii"

Programa Oficial de Doutoramento en Ciencias da Saúde. 5007V01[Resumo] Acinetobacter baumannii é un patóxeno nosocomial que posúe unha enorme capacidade de adaptarse a condicións desfavorables, o que o converte nun importante problema de saúde pública. Os perfís de expresión xénica durante a formación de biopelículas en A. baumannii resultaron diferentes en comparación con células libres planctónicas en fase exponencial e estacionaria. Confirmouse que os xenes A1S_1507, A1S_3168, A1S_2042, A1S_0302 y A1S_0114 que codifican unha proteína de fimbria, una proteína dun pilus, un regulador transcripcional, unha proteína hipotética e un transportador de grupos acilo, respectivamente, están implicados na capacidade de formación de biopelículas en A. baumannii. Ademais, demostrouse que o xene A1S_0114 participa na adherencia a superficies bióticas e abióticas, en virulencia e na síntese dun metabolito denominado acinetin 505. Así mesmo, atopáronse moléculas de sRNAs que se expresan diferencialmente nas biopelículas con respecto ás células planctónicas. Entre eles, destacou o sRNA 13573, altamente expresado en células sésiles, que resultou estar implicado na formación de biopelículas e na adherencia a células eucariotas. Neste traballo, descríbense, dúas novas dianas terapéuticas que participan na patoxénese de A. baumannii: o xene A1S_0114 e o sRNA 13573.[Resumen] Acinetobacter baumannii es un patógeno nosocomial que posee una enorme capacidad de adaptarse a condiciones desfavorables, lo que lo convierte en un importante problema de salud pública. Los perfiles de expresión génica durante la formación de biopelículas en A. baumannii resultaron diferentes en comparación con células libres planctónicas en fase exponencial y estacionaria. Se confirmó que los genes A1S_1507, A1S_3168, A1S_2042, A1S_0302 y A1S_0114 que codifican una proteína de fimbria, una proteína de un pilus, un regulador transcripcional, una proteína hipotética y un transportador de grupos acilo, respectivamente, están implicados en la capacidad de formación de biopelículas en A. baumannii. Además, se demostró que el gen A1S_0114 participa en la adherencia a superficies bióticas y abióticas, en virulencia y en la síntesis de un metabolito denominado acinetin 505. Asimismo, se encontraron moléculas de sRNAs que se expresan diferencialmente en las biopelículas con respecto a las células planctónicas. Entre ellos, destacó el sRNA 13573, altamente expresado en células sésiles, que resultó estar implicado en formación de biopelículas y en la adherencia a células eucariotas. En este trabajo, se describen, dos nuevas dianas terapéuticas que participan en la patogénesis de A. baumannii: el gen A1S_0114 y el sRNA 13573.[Abstract] Acinetobacter baumannii is a nosocomial pathogen with a notable ability to adapt to stress conditions and develop resistance to multiple antimicrobial compounds, becoming a remarkable public health problem. Gene expression during biofilm formation in A. baumannii showed different profiles compared to planktonic cells in exponential and stationary phase of growth. We could confirm that genes A1S_1507, A1S_3168, A1S_2042, A1S_0302 y A1S_0114 coding a fimbrial protein, a pilus assembly protein, a transcriptional regulator, a hypothetical protein and an acyl-carrier protein, respectively, are involved in biofilm formation ability of A. baumannii. Furthermore, the A1S_0114 gene showed to play a role in attachment to biotic and abiotic surfaces, in virulence, and in the biosynthesis of a metabolite named as acinetin 505. Moreover, an important number of sRNAs differentially expressed in biofilm associated cells compared to planktonic cells were determined. Among them, the 13575 sRNA, highly expressed in biofilm, resulted to be involved in biofilm formation and in adherence to eukaryotic cells. In the present work, two new therapeutic targets involved in the pathogenesis of A. baumannii are described: the A1S_0114 gen and the 13573 sRNA

A New and Efficient Enrichment Method for Metagenomic Sequencing of Monkeypox Virus

[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.This work was supported by a grant from the SERGAS-Galician Healthcare Service (Program “Innova Saúde”) to GB, by CIBERINFEC and also by Instituto de Salud Carlos III (ISCIII) through the projects PI20/00413 to MP and PI21/00704 to G

Quorum Sensing as a Target for Controlling Surface Associated Motility and Biofilm Formation in Acinetobacter Baumannii ATCC® 17978TM

[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 and survival on surfaces and the prevention and treatment of infections caused by this pathogen.Xunta de Galicia; ED481A-2015/311Xunta de Galicia; IN606B-2019/010Biotechnology and Biological Sciences Research Council (United Kingdom); BB/R012415/1Xunta de Galicia; ED481A-2019/19

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

Contribution of the a-baumannii A1S_0114 gene to the interaction with eukaryotic cells and virulence

Genetic and functional studies showed that some components of the Acinetobacter baumannii ATCC 17978 A1S_0112-A1S_0119 gene cluster are critical for biofilm biogenesis and surface motility. Recently, our group has shown that the A1S_0114 gene was involved in biofilm formation, a process related with pathogenesis. Confirming our previous results, microscopy images revealed that the ATCC 17978 10114 derivative lacking this gene was unable to form a mature biofilm structure. Therefore, other bacterial phenotypes were analyzed to determine the role of this gene in the pathogenicity of A. baumannii ATCC 17978. The interaction of the ATCC 17978 parental strain and the 10114 mutant with A549 human alveolar epithelial cells was quantified revealing that the A1S_0114 gene was necessary for proper attachment to A549 cells. This dependency correlates with the negative effect of the A1S_0114 deletion on the expression of genes coding for surface proteins and pili-assembly systems, which are known to play a role in adhesion. Three different experimental animal models, including vertebrate and invertebrate hosts, confirmed the role of the A1S_0114 gene in virulence. All of the experimental infection assays indicated that the virulence of the ATCC 17978 was significantly reduced when this gene was inactivated. Finally, we discovered that the A1S_0114 gene was involved in the production of a small lipopeptide-like compound herein referred to as acinetin 505 (Ac-505). Ac-505 was isolated from ATCC 17978 spent media and its chemical structure was interpreted by mass spectrometry. Overall, our observations provide novel information on the role of the A1S_0114 gene in A. baumannii’s pathobiology and lay the foundation for future work to determine the mechanisms by which Ac-505, or possibly an Ac-505 precursor, could execute critical functions as a secondary metaboliteS

Modeling the Number of People Infected With SARS-COV-2 From Wastewater Viral Load in Northwest Spain

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[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.This work was supported by EDAR Bens S.A., A Coruña, Spain [grant references INV04020, INV12120 and INV05921 to MP], the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 funded by the ISCIII, Spain - General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe” [grant numbers PI15/00860 to GB, PI17/01482 and PI20/00413 to MP], the GAIN, Xunta de Galicia, Spain [grant number IN607A 2016/22 to GB, ED431C-2016/015 and ED431C-2020/14 to RC, ED431C 2017/58 to SL, ED431G 2019/01 to RC and SL, and ED431C 2017/66 to MCV], MINECO, Spain [grant number MTM2017-82724-R to RC], Ministerio de Ciencia e Innovación, Spain [grant number PID2020-113578RB-100 to RC], and the Spanish Network for Research in Infectious Diseases [REIPI RD16/0016/006 to GB]. The work was also supported by the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 - Research and Innovation Framework Programme under grant agreement no 871029. SR-F was financially supported by REIPI RD16/0016/006, KC-P by IN607A 2016/22 and the Spanish Association against Cancer (AECC) and JAV by IN607A 2016/22. Funding for open access charge: Universidade da Coruña/CISUGEDAR Bens S.A.; INV04020EDAR Bens S.A.; INV12120EDAR Bens S.A.; INV05921Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED431C-2016/015Xunta de Galicia; ED431C-2020/14Xunta de Galicia; ED431C 2017/58Xunta de Galicia; ED431G 2019/01Xunta de Galicia; ED431C 2017/6

Wastewater early warning system for SARS-CoV-2 outbreaks and variants in a Coruña, Spain

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract]: Wastewater-based epidemiology has been widely used as a cost-effective method for tracking the COVID-19 pandemic at the community level. Here we describe COVIDBENS, a wastewater surveillance program running from June 2020 to March 2022 in the wastewater treatment plant of Bens in A Coruña (Spain). The main goal of this work was to provide an effective early warning tool based in wastewater epidemiology to help in decision-making at both the social and public health levels. RT-qPCR procedures and Illumina sequencing were used to weekly monitor the viral load and to detect SARS-CoV-2 mutations in wastewater, respectively. In addition, own statistical models were applied to estimate the real number of infected people and the frequency of each emerging variant circulating in the community, which considerable improved the surveillance strategy. Our analysis detected 6 viral load waves in A Coruña with concentrations between 103 and 106 SARS-CoV-2 RNA copies/L. Our system was able to anticipate community outbreaks during the pandemic with 8-36 days in advance with respect to clinical reports and, to detect the emergence of new SARS-CoV-2 variants in A Coruña such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529 and BA.2) in wastewater with 42, 30, and 27 days, respectively, before the health system did. Data generated here helped local authorities and health managers to give a faster and more efficient response to the pandemic situation, and also allowed important industrial companies to adapt their production to each situation. The wastewater-based epidemiology program developed in our metropolitan area of A Coruña (Spain) during the SARS-CoV-2 pandemic served as a powerful early warning system combining statistical models with mutations and viral load monitoring in wastewater over time.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for open access charge: Universidade da Coruña/CISUG. This work was supported by EDAR Bens S.A., A Coruña, Spain [grant references INV04020, INV12120, INV05921, and INV148721 to MP], by the National Plan for Scientific Research, Development and Technological Innovation funded by the Institute of Health Carlos III (ISCIII), Spain—General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe” [grant references PI15/00860 to GB, PI17/01482, and PI20/00413 to MP], by the Galician Innovation Agency (GAIN) (Xunta de Galicia, Spain) [grant references IN607A 2016/22 to GB, ED431C-2016/015 and ED431C-2020/14 to RC, ED431C 2021/53 to SL and ED431G 2019/01 and COV20/00604 to RC and SL, by Ministry of Economic Affairs and Digital Transformation (MINECO), Spain [grant references MTM2017-82724-R to RC], by the Spanish Network for Research in Infectious Diseases [REIPI RD16/0016/0006 to GB], by the “Innova Saúde” Program, (INNOVAMICROLAB project) co-founded by the Galician Healthcare Service (SERGAS) and the Spanish Ministry of Science and Innovation, and by the Spanish Network of Research in Infectious Diseases (CIBERINFEC, ISCIII), and by the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 871029. SR-F was financially supported by REIPI RD16/0016/006, KC-P by IN607A 2016/22 and the Spanish Association against Cancer (AECC) and JAV by IN607A 2016/22. DP was funded by grant EPICOVIGAL FONDO SUPERA-COVID19 from Banco Santander-CSIC-CRUE, Spain, and grant CT850A-2 from (Health Knowledge Agency) ACIS SERGAS from the Consellería de Sanidade of Xunta de Galicia, Spain.EDAR Bens S.A.; INV04020EDAR Bens S.A.; INV12120EDAR Bens S.A.; INV05921EDAR Bens S.A.; INV148721Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED431C-2016/015Xunta de Galicia; ED431C-2020/14Xunta de Galicia; ED431C 2021/53Xunta de Galicia; ED431G 2019/01Xunta de Galicia; COV20/0060