Development of a reporter system for screening anti-biofilm activities

Biofilm formation is responsible for increasing antibiotic tolerance in pathogenic bacteria. It is estimated that approximately 80% of chronic infections are associated with this phenomenon. Therefore, the search for therapeutic agents with specific biofilm targets has become of vital importance. One of the main strategies is the search for enzymes that degrade the different components of the extracellular matrix. However, since the composition of the matrix varies among the different microorganisms, an alternative would be to interfere with the signaling cascades that lead to the formation of the biofilm or stimulate its dispersion

FleN and FlhF as new regulatory elements in the lifestyle switch in Pseudomonas putida

Motivation:Pseudomonas putida is a soil bacterium that can be found in nature as individual motile cells or as part of sessile communities called biofilms (1). Biofilm formation could be considered an adaptive strategy as it provide higher resistance against adverse agents, antimicrobial treatments, ultraviolet radiation or dehydration, increasing bacteria survival (2). The switch from a planktonic lifestyle to biofilm formation in P.putida is regulated by FleQ and the intracellular levels of c-di-GMP (3). Isolation of insertion mutants in flhF gene, defective in biofilm formation, suggests the involvement of additional elements in the regulation of this process. In this work, we have characterized the transcriptional organization of flhA, flhF, fleN and fliA genes and the role of FlhF and FleN in biofilm development in P.putida. Methods: Planktonic growth and biofilm formation curves, adhesion and swimming-motility assays. Gene expression analysis: β-galactosidase assays and RT-PCR. Electrophoretic mobility shift assays (EMSA).Results and Conclusions: To test the role of FlhF and FleN in biofilm development, we carried out a phenotypic characterization of ΔflhF and ΔfleN mutants. Experiments shown that ΔfleN mutant is not able to form biofilm whereas ΔflhF mutant exhibits a wild-type phenotype. Adhesion assays indicate that ΔfleN mutant has a reduced adhesion, whereas ΔflhF can properly adhere to the surface. On the other hand, both mutant show reduced swimming motility. These resuts suggests that both, FleN and FlhF, are involved in swimming motility, but only FleN is necessary for biofilm formation, probably by altering adhesion capacity of the bacteria. Bioinformatic tools predict that flhF and fleN form an operon with the upstream gene flhA and the downstream gene fliA. In order to corroborate this hypothesis, we have done RT-PCR using RNA from the mutants. Results shown that these genes are structured in a single operon: flhAFfleNfliA.To determine a possible regulatory role of these elements in biofilm formation and motility, β-galactosidase assays were performed to analyse the expression of biofilm and flagellum related promoters. Results shown that FleN downregulates both types of promoters whereas FlhF is only involved in the regulation of one flagellar gene. The regulatory role of FleN has been further studied in vitro by EMSA with the regulator FleQ. Results suggest that FleN is required for FleQ to bind to its target promoters

FleQ, FleN and c-di-GMP coordinately regulate cellulose production in Pseudomonas syringae pv. tomato DC3000

The second messenger cyclic di-GMP (c-di-GMP) controls the transition between motility and sessility in many bacterial species by a variety of mechanisms, including the production of multiple exopolysaccharides. Pseudomonas syringae pv. tomato (Pto) DC3000 is a plant pathogenic bacteria able to synthesize acetylated cellulose under high c-di-GMP levels thanks to the expression of the wssABCDEFGHI operon. Increased cellulose production enhances air-liquid biofilm formation and generates a wrinkled colony phenotype on solid media. We previously showed that under low levels of c-di-GMP, the regulators FleQ and AmrZ bound to adjacent sequences at the wss promoter inhibiting its expression, but only FleQ responded to the presence of c-di-GMP by activating cellulose production. In the present work, we advance in the knowledge of this complex regulation in Pto DC3000 by shedding light over the role of FleN in this process. The distinctive features of this system are that FleN and FleQ are both required for repression and activation of the wss operon under low and high c-di-GMP levels, respectively. We have also identified three putative FleQ binding sites at the wss promoter and show that FleQ/FleN-ATP binds at those sites under low c-di-GMP levels, inducing a distortion of DNA, impairing RNA polymerase binding, and repressing wss transcription. However, binding of c-di-GMP induces a conformational change in the FleQ/FleN-ATP complex, which relieves the DNA distortion, allows promoter access to the RNA polymerase, and leads to activation of wss transcription. On the other hand, AmrZ is always bound at the wss promoter limiting its expression independently of FleQ, FleN and c-di-GMP levels

Functional dissection of the large adhesion protein

There are numerous microorganisms which have the ability to switch between being in planktonic stateor form biofilms, these biofilms are complex communities of microorganisms attached to surfaces orassociated with interfaces. This concept has a lot of relevance in ecology due to the fact that thesemicrobial communities are often composed of multiple species that interact with each other and theirenvironment. The longest gene in the Gram-negative bacterium Pseudomonas putida genome encodesLapA, a >9000 amino-acid surface adhesin essential to surface adhesion and biofilm formation. LapA isa complex protein, containing numerous functional domains and a large array of repeated sequences.However, the exact function of any of these elements in LapA is unknown.The strategy of our project is the construction of different lapA variants containing internal deletions ofthe putative functional domains, and study the role of each of these in LapA-dependent phenotypes .Each version will be inserted in the chromosome of a Δ lapA mutant using a Tn 7 -based delivery system.An Initial synthetic construct containing the complete N-terminal and C-terminal domains and a 3xHAtag for immunodetection, but lacking all repeated sequences is already available, and constructsbearing progressively shorter N-terminal domains are underway. Addition of different numbers ofrepeats will be tested afterwards.Phenotypic assays will include swimming and adhesion assays using different surfaces, biofilmformation curves and microscopic assessment of biofilm morphology under different conditions. Weexpect that this approach will provide useful insight into the functions of the different domains of LapAand the dynamics of biofilm development in P. putida

Development of tools for the widespread implementation of CRISPR-Cas technologies in Gram-negative bacteria

CRISPR-Cas technology has made a huge impact due to its potential for editing, regulating and targeting genomes overthe past years. It has been proven a very powerful and useful tool with lot of potential and a wide range of applications within the biotechnological, pharmaceutical and food industry, among others. Nevertheless, one of the biggestshortcomings of this technology is that, despite the multiple of applications already available, there is potential for many moreuses yet to be discovered.According to that, the Bacteria domain holds the promise of an entire world of opportunities and possibilities, as thistechnology has not been implemented, with the exception of Escherichia coli and a few other species. Many examples ofhighly interesting organisms are Gram-negative bacteria, such as the symbiotic nitrogen-fixing Rhizobium and Shinorizobium spp. Developing the CRISPR-Cas system to be able to modify genetically these organisms, establishingspecific protocols for directed genome or gene expression manipulation, would be a game changer and set a baseline forfurther research. The aim of our project is to develop CRISPR-Cas-based genome editing tools of widespread use in Gram-negative bacteria and test their fucntion in three members of the Rhizobial grou, namely Rhizobium legumirosarum, Shinorhizobium melilotiand Sinorhizobium fredii. The strategy designed consists on the construction two broad host-range plasmids: one expressing the nuclease Cas12a, and a second one expressing a Cas12a gRNA, and containing the gRNA target sequence and GFP (Green Fluorescent Protein) as a reproter gene. The ability of Cas12a to induce loss of this reporter plasmid (and the GFP reporter encoded therein) will be assessed, and the efficiency by which expression of different repair systems enable persistance of the plasmid by introducing mutations at the cleaved target calculated. The combination of efficient target cleavage and efficient mutation-inducing repair will provide a suitable toolkit for genome engineering in these organisms. As we progress with this project we expect to be able to offer the scientific community a complete toolbox of Cas nucleases, repair systems and testing procedures that will allow the identification of the optimal tool combination for each organism

Transformation of process water in the agri-food industry for its use in the agriculture in order to improve the agricultural processes.

The agricultural sector is facing a challenge because they need to increase the productivity of the agricultural crops due to the growing global population. Moreover, another problem is the negative impact that the chemical fertilizersand pesticides have in the ecosystems and human health. For this reason, investigation is needed to find other type of fertilizers to avoid this detrimental impact. Nowadays, biostimulants are playing an important role, they are considered as plant growth regulators because they enhance flowering, germination, plant growth and crop productivity and areenvironmental-friendly [1].In this study, the efficiency of the microalgaes and their use as biostimulants have been put to test. In order to prove the effects in the plant growth and crop productivity using lettuce as test crop, seven solutions have been prepared with different concentrations of natural fertilizer, microorganisms and microalgae. After that, these solutions weretested to prove if microalgae promoted seed germination and plant development in radishes' seed. Finally, cut flowers (daisies) were used to prove the efficiency of microalgaes in the maintenance of post-harvey, so four solutions which had water, aspirin, microalgae culture (with the microalgae) and seaweed liquid extract (without the microalgae) wereprepared.The results showed improve in the test crops as well as in the cut flowers. This can be explained because microalgae produce phytohormones like auxins, gibberelins, cytokinins and abscisc acid and other exogenous molecules as lipids, aminoacids, polysaccharide, etc. That can cause this beneficial effect [2].In conclusion, the microalgae seems to be a perfect substitute for the chemical fertilizers because of its benefits and non-harmful effects

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Prospective individual patient data meta-analysis of two randomized trials on convalescent plasma for COVID-19 outpatients

Data on convalescent plasma (CP) treatment in COVID-19 outpatients are scarce. We aimed to assess whether CP administered during the first week of symptoms reduced the disease progression or risk of hospitalization of outpatients. Two multicenter, double-blind randomized trials (NCT04621123, NCT04589949) were merged with data pooling starting when = 50 years and symptomatic for <= 7days were included. The intervention consisted of 200-300mL of CP with a predefined minimum level of antibodies. Primary endpoints were a 5-point disease severity scale and a composite of hospitalization or death by 28 days. Amongst the 797 patients included, 390 received CP and 392 placebo; they had a median age of 58 years, 1 comorbidity, 5 days symptoms and 93% had negative IgG antibody-test. Seventy-four patients were hospitalized, 6 required mechanical ventilation and 3 died. The odds ratio (OR) of CP for improved disease severity scale was 0.936 (credible interval (CI) 0.667-1.311); OR for hospitalization or death was 0.919 (CI 0.592-1.416). CP effect on hospital admission or death was largest in patients with <= 5 days of symptoms (OR 0.658, 95%CI 0.394-1.085). CP did not decrease the time to full symptom resolution

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe