"Pseudomonas" spp. associated with coffee plants : comparative genomics, niche specificity and biotechnological potential

Orientador: Jorge Mauricio Costa MondegoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: "Pseudomonas" é um grupo bacteriano com capacidade de colonizar uma grande gama de ambientes. As do grupo fluorescente são conhecidas principalmente por serem benéficas aos vegetais, conferindo um ganho na produção e dando maior proteção contra possíveis patógenos. Durante as anotações do banco de ESTs de Coffee arabica detectou-se uma grande quantidade de sequências de "Pseudomonas" no banco. Através de isolamentos feitos nas folhas, rizosfera e das raízes de Coffea arabica constatou-se a presença destas bactérias e, posteriormente, foram selecionados três isolados distintos de cada ambiente mencionado; seus genomas foram sequenciados e em paralelo foram executados experimentos para verificar diferenças fisiológicas entre os isolados. Os genes identificados a partir do isolado de folha sugerem que ele está adaptado às tensões abióticas do ambiente da superfície foliar, o que confirmamos por experimentos de estresse osmótico e privação de ferro. O isolado a partir da região interna das raízes possui receptores sideróforos de acromobactina e aerobactina, mas não possui os genes responsáveis pela produção desses sideróforos, sugerindo uma interessante estratégia de competição baseada no furto de sideróforos de outras espécies. Todas as três bactérias foram capazes de aumentar o crescimento de plântulas de alface, principalmente o isolado de rizosfera. Curiosamente, o isolado de folha foi capaz de colonizar as folhas das plantas após a bacterização das sementes, sugerindo uma inespecificidade quanto ao hospedeiro. Além disso, os três isolados foram capazes de degradar compostos fenólicos, e em alguns casos utilizando-os como fonte metabólica. Entretanto, elas não foram capazes de utilizarem cafeína como fontes de nitrogênio e carbono. Em termos de análise de controle biológico, o isolado de folha foi capaz de diminuir o dano provocado pela ferrugem-do-café, podendo ser uma alternativa para o controle dessa praga. A partir dos nossos dados, discutimos a relação entre o conteúdo gênico e os dados experimentais indicando que existem adaptações dessas bactérias para viverem em associação com o cafeeiro em diferentes nichosAbstract: "Pseudomonas" is a bacterial group capable of colonizing a wide range of environments. The fluorescent group are known mainly for being beneficial to plants, providing a gain in production and providing great protection against possible pathogens. During the annotations of the EST bank of Coffee arabica, a large number of "Pseudomonas" sequences were detected in the bank. Through isolations made on the leaves, rhizosphere and roots of Coffea arabica, the presence of these bacteria was found and, subsequently, three distinct isolates from each mentioned environment were selected; their genomes were sequenced and in parallel experiments were performed to verify physiological differences between the isolates. The genes identified from the leaf isolate suggest that it is adapted to the abiotic stresses of the leaf surface environment, which is confirmed by experiments on osmotic stress and iron deprivation. The isolate from the inner region of the roots has siderophores of acromobactin and aerobactin receptors, but does not have the genes responsible for the production of these siderophores, suggesting an interesting competition strategy based on the theft of siderophores of other species. All three bacteria were able to increase the growth of lettuce seedlings, mainly the rhizosphere isolate. Interestingly, the leaf isolate was able to colonize the leaves of the plants after bacterization of the seeds, suggesting a non-specificity regarding the host. In addition, the three isolates were able to degrade phenolic compounds, and in some cases using them as a metabolic source. However, they were unable to use caffeine as sources of nitrogen and carbon. In terms of biological control analysis, the leaf isolate was able to reduce the damage caused by coffee rust, and could be an alternative for the control of this pathogen. From our data, we discussed the relationship between gene content and experimental data indicating that there are adaptations of these bacteria to live in association with coffee in different nichesDoutoradoMicrobiologiaDoutor em Genetica e Biologia Molecular2014/11544-4FAPES

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Differences between the Leaf Mycobiome of <i>Coffea arabica</i> and Wild Coffee Species and Their Modulation by Caffeine/Chlorogenic Acid Content

The study of microbes associated with the coffee tree has been gaining strength in recent years. In this work, we compared the leaf mycobiome of the traditional crop Coffea arabica with wild species Coffea racemosa and Coffea stenophylla using ITS sequencing for qualitative information and real-time PCR for quantitative information, seeking to relate the mycobiomes with the content of caffeine and chlorogenic acid in leaves. Dothideomycetes, Wallemiomycetes, and Tremellomycetes are the dominant classes of fungi. The core leaf mycobiome among the three Coffea species is formed by Hannaella, Cladosporium, Cryptococcus, Erythrobasidium, and Alternaria. A network analysis showed that Phoma, an important C. arabica pathogen, is negatively related to six fungal species present in C. racemosa and C. stenophylla and absent in C. arabica. Finally, C. arabica have more than 35 times the concentration of caffeine and 2.5 times the concentration of chlorogenic acid than C. stenophylla and C. racemosa. The relationship between caffeine/chlorogenic acid content, the leaf mycobiome, and genotype pathogen resistance is discussed

Leaf-associated bacterial microbiota of coffee and its correlation with manganese and calcium levels on leaves

Abstract Coffee is one of the most valuable agricultural commodities and the plants’ leaves are the primary site of infection for most coffee diseases, such as the devastating coffee leaf rust. Therefore, the use of bacterial microbiota that inhabits coffee leaves to fight infections could be an alternative agricultural method to protect against coffee diseases. Here, we report the leaf-associated bacteria in three coffee genotypes over the course of a year, with the aim to determine the diversity of bacterial microbiota. The results indicate a prevalence of Enterobacteriales in Coffea canephora, Pseudomonadales in C. arabica ‘Obatã’, and an intriguing lack of bacterial dominance in C. arabica ‘Catuaí’. Using PERMANOVA analyses, we assessed the association between bacterial abundance in the coffee genotypes and environmental parameters such as temperature, precipitation, and mineral nutrients in the leaves. We detected a close relationship between the amount of Mn and the abundance of Pseudomonadales in ‘Obatã’ and the amount of Ca and the abundance of Enterobacteriales in C. canephora. We suggest that mineral nutrients can be key drivers that shape leaf microbial communities

Additional file 1 of Functional genomics analysis of a phyllospheric Pseudomonas spp with potential for biological control against coffee rust

Additional file 1: Figure S1. Phylogenetic tree containing MN1F and other Pseudomonas species. Phylogenetic tree generated in TYGS using the FastME 2.1.6.1 program present on the website. 100 replications were used, with an average branch support of 89.6% according to the developer’s specifications. Figure S2. Measurement of caffeine incorporation as carbon and nitrogen source. Experiment to verify if MN1F was able to use caffeine as a source of carbon and nitrogen. Primary sources were removed and caffeine was offered as an alternative source. The intention was to induce cells to “replace” glucose and NH4Cl with caffeine as sources of carbon and nitrogen respectively, which did not occur. Figure S3. Sketch summarizing the set-up of leaf disc experiment of infection with H. vastatrix. Table S1. MN1F insertion sequences detected by the ISFinder web tool. Pseudomonas syringae was the main IS donor with 34 copies

Screening policies, preventive measures and in-hospital infection of COVID-19 in global surgical practices

Background In a surgical setting, COVID-19 patients may trigger in-hospital outbreaks and have worse postoperative outcomes. Despite these risks, there have been no consistent statements on surgical guidelines regarding the perioperative screening or management of COVID-19 patients, and we do not have objective global data that describe the current conditions surrounding this issue. This study aimed to clarify the current global surgical practice including COVID-19 screening, preventive measures and in-hospital infection under the COVID-19 pandemic, and to clarify the international gaps on infection control policies among countries worldwide.Methods During April 2-8, 2020, a cross-sectional online survey on surgical practice was distributed to surgeons worldwide through international surgical societies, social media and personal contacts. Main outcome and measures included preventive measures and screening policies of COVID-19 in surgical practice and centers' experiences of in-hospital COVID-19 infection. Data were analyzed by country's cumulative deaths number by April 8, 2020 (high risk, &gt;5000; intermediate risk, 100-5000; low risk, &lt;100).Results A total of 936 centers in 71 countries responded to the survey (high risk, 330 centers; intermediate risk, 242 centers; low risk, 364 centers). In the majority (71.9%) of the centers, local guidelines recommended preoperative testing based on symptoms or suspicious radiologic findings. Universal testing for every surgical patient was recommended in only 18.4% of the centers. In-hospital COVID-19 infection was reported from 31.5% of the centers, with higher rates in higher risk countries (high risk, 53.6%; intermediate risk, 26.4%; low risk, 14.8%; P&lt;0.001). Of the 295 centers that experienced in-hospital COVID-19 infection, 122 (41.4%) failed to trace it and 58 (19.7%) reported the infection originating from asymptomatic patients/staff members. Higher risk countries adopted more preventive measures including universal testing, routine testing of hospital staff and use of dedicated personal protective equipment in operation theatres, but there were remarkable discrepancies across the countries.Conclusions This large international survey captured the global surgical practice under the COVID-19 pandemic and highlighted the insufficient preoperative screening of COVID-19 in the current surgical practice. More intensive screening programs will be necessary particularly in severely affected countries/institutions