Quorum Quenching Strains Isolated from the Microbiota of Sea Anemones and Holothurians Attenuate Vibrio corallilyticus Virulence Factors and Reduce Mortality in Artemia salina

The English text was corrected by Michael O'Shea. This research was funded by the Spanish Ministry of the Economy and Competitiveness (grant number PID2019-106704RB-100/AEI/10.13039/501100011033). Jose Carlos Reina is supported by an FPU fellowship from the Spanish Ministry of Education, Culture and Sport (fellowship number FPU15/01717).Interference with quorum-sensing (QS) intercellular communication systems by the enzymatic disruption of N-acylhomoserine lactones (AHLs) in Gram-negative bacteria has become a promising strategy to fight bacterial infections. In this study, seven strains previously isolated from marine invertebrates and selected for their ability to degrade C6 and C10-HSL, were identified as Acinetobacter junii, Ruegeria atlantica, Microbulbifer echini, Reinheimera aquimaris, and Pseudomonas sihuiensis. AHL-degrading activity against a wide range of synthetic AHLs were identified by using an agar well diffusion assay and Agrobacterium tumefaciens NTL4 and Chromobacterium violaceum CV026 and VIR07 as biosensors. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis indicated that this activity was not due to an AHL lactonase. All the strains degraded Vibrio coralliilyticus AHLs in coculture experiments, while some strains reduced or abolished the production of virulence factors. In vivo assays showed that strains M3-111 and M3-127 reduced this pathogen’s virulence and increased the survival rate of Artemia salina up to 3-fold, indicating its potential use for biotechnological purposes. To our knowledge, this is the first study to describe AHLdegrading activities in some of these marine species. These findings highlight that the microbiota associated with marine invertebrates constitute an important underexplored source of biological valuable compounds.Spanish Government PID2019-106704RB-100/AEI/10.13039/501100011033 FPU15/0171

Silencing of Phytopathogen Communication by the Halotolerant PGPR Staphylococcus Equorum Strain EN21

I.S. wishes to thank MINECO for her Ramón and Cajal contract, M.R. wishes to thank MITRAMISS for his “Joven Investigador” contract, Michael O´Shea corrected the English version of the manuscript and Clementina Pozo bioassayed the toxicity of EN21. The authors thank Juan Sanjuan Pinilla research group (EEZ, CSIC) for kindly supply Pseudomonas syringae pv. tomato DC3000 strain.Supplementary materials can be found at http://www.mdpi.com/2076-2607/8/1/42/s1.Increasing world food demand together with soil erosion and indiscriminate use of chemical fertilization highlight the need to adopt sustainable crop production strategies. In this context, a combination of plant growth-promoting rhizobacteria (PGPR) and pathogen management represents a sustainable and effcient alternative. Though little studied, halophilic and halotolerant PGPR could be a beneficial plant growth promotion strategy for saline and non-saline soils. The virulence of many bacterial phytopathogens is regulated by quorum sensing (QS) systems. Quorum quenching (QQ) involves the enzymatic degradation of phytopathogen-generated signal molecules, mainly N-acyl homoserine lactones (AHLs). In this study, we investigate plant growth-promoting (PGP) activity and the capacity of the halotolerant bacterium Staphylococcus equorum strain EN21 to attenuate phytopathogens virulence through QQ.We used biopriming and in vivo tomato plant experiments to analyse the PGP activity of strain EN21. AHLinactivation was observed to reduce Pseudomonas syringae pv. tomato infections in tomato and Arabidopsis plants. Our study of Dickeya solani, Pectobacterium carotovorum subsp. carotovorum and Erwinia amylovora bacteria in potato tubers, carrots and pears, respectively, also demonstrated the effectiveness of QS interruption by EN21. Overall, this study highlights the potential of strain S. equorum EN21 in plant growth promotion and QQ-driven bacterial phytopathogen biocontrol.The study was funded by the Ramón y Cajal MINECO program (RYC-2014-15532) in Spai

Plant growth-promoting activity and quorum quenchingmediated biocontrol of bacterial phytopathogens by Pseudomonas segetis strain P6

This research was funded by grants from the Spanish Ministry of the Economy and Competitiveness (AGL2015-68806-R) and the Ramon y Cajal program (RYC-2014-15532). The authors wish to thank Michael O'Shea for proofreading the manuscript and Juan Sanjuan Pinilla's research group (EEZ-CSIC) for kindly supplying the Pseudomonas syringae pv. tomato strain DC3000.Given the major threat of phytopathogenic bacteria to food production and ecosystem stability worldwide, novel alternatives to conventional chemicals-based agricultural practices are needed to combat these bacteria. The objective of this study is to evaluate the ability of Pseudomonas segetis strain P6, which was isolated from the Salicornia europaea rhizosphere, to act as a potential biocontrol agent given its plant growth-promoting (PGP) and quorum quenching (QQ) activities. Seed biopriming and in vivo assays of tomato plants inoculated with strain P6 resulted in an increase in seedling height and weight. We detected QQ activity, involving enzymatic degradation of signal molecules in quorum sensing communication systems, against a broad range of N-acylhomoserine lactones (AHLs). HPLC-MRM data and phylogenetic analysis indicated that the QQ enzyme was an acylase. The QQ activity of strain P6 reduced soft rot symptoms caused by Dickeya solani, Pectobacterium atrosepticum and P. carotovorum on potato and carrot. In vivo assays showed that the PGP and QQ activities of strain P6 protect tomato plants against Pseudomonas syringae pv. tomato, indicating that strain P6 could have biotechnological applications. To our knowledge, this is the first report to show PGP and QQ activities in an indigenous Pseudomonas strain from Salicornia plants.Spanish Ministry of the Economy and Competitiveness AGL2015-68806-RSpanish Government RYC-2014-1553

Peribacillus castrilensis sp. nov.: A Plant-Growth-Promoting and Biocontrol Species Isolated From a River Otter in Castril, Granada, Southern Spain

A strictly aerobic, chemoheterotrophic, endospore-forming, Gram-positive, rod-shaped bacterial strain N3T was isolated from the feces of a river otter in Castril (Granada, southern Spain). It is halotolerant, motile, and catalase-, oxidase-, ACC deaminase-, and C4- and C8-lipase-positive. It promotes tomato plant growth and can reduce virulence in Erwinia amylovora CECT 222T and Dickeya solani LMG 25993T through interference in their quorum-sensing systems, although other antagonistic mechanisms could also occur. A phylogenetic analysis of the 16S rRNA gene sequence as well as the phenotypic and phylogenomic analyses indicated that the strain N3T is a novel species of the genus Peribacillus, with the highest 16S rRNA sequence similar to that of Bacillus frigoritolerans DSM 8801T (99.93%) and Peribacillus simplex DSM 1321T (99.80%). Genomic digital DNA–DNA hybridization (dDDH) between the strain N3T and Bacillus frigoritolerans DSM 8801T and Peribacillus simplex was 12.8 and 69.1%, respectively, and the average nucleotide identity (ANIb) of strain N3T and Bacillus frigoritolerans DSM 8801T and Peribacillus simplex was 67.84 and 93.21%, respectively. The genomic G + C content was 40.3 mol%. Its main cellular fatty acids were anteiso-C15 : 0 and iso-C15 : 0. Using 16S rRNA phylogenetic and in silico phylogenomic analyses, together with the chemotaxonomic and phenotypic data, we demonstrated that the type strain N3T (=CECT 30509T = LMG 32505T) is a novel species of the genus Peribacillus and the name Peribacillus castrilensis sp. nov. is proposed.Spanish Ministerio de Educación y Ciencia (AGL-2015-68806-R)Spanish Ministry of the Economy and Competitiveness (PID2019-106704RB-100/AEI/10.13039/501100011033)Plan Andaluz de Investigación (Research Group BIO 188)University of Granada Programme (Empleo Garantía Juvenil)Spanish Ministerio de Educación, Cultura y Deporte (FPU15/01717

Study of Bacterial Community Composition and Correlation of Environmental Variables in Rambla Salada, a Hypersaline Environment in South-Eastern Spain

We studied the bacterial community in Rambla Salada in three different sampling sites and in three different seasons and the effect of salinity, oxygen, and pH. All sites samples had high diversity and richness (Rr > 30). The diversity indexes and the analysis of dendrograms obtained by DGGE fingerprint after applying Pearson’s and Dice’s coefficient showed a strong influence of sampling season. The Pareto-Lorenz (PL) curves and Fo analysis indicated that the microbial communities were balanced and despite the changing environmental conditions, they can preserve their functionality. The main phyla detected by DGGE were Bacteroidetes (39.73%), Proteobacteria (28.43%), Firmicutes (8.23%), and Cyanobacteria (5.14%). The majority of the sequences corresponding to uncultured bacteria belonged to Bacteroidetes phylum. Within Proteobacteria, the main genera detected were Halothiobacillus and Roseovarius. The environmental factors which influenced the community in a higher degree were the salinity and oxygen. The bacteria belonging to Bacteroidetes and Proteobacteria were positively influenced by salinity. Nevertheless, bacteria related to Alpha- and Betaproteobacteria classes and phylum Firmicutes showed a positive correlation with oxygen and pH but negative with salinity. The phylumCyanobacteria were less influenced by the environmental variables. The bacterial community composition of Rambla Salada was also studied by dilution-to-extinction technique. Using this method, 354 microorganisms were isolated. The 16S sequences of 61 isolates showed that the diversity was very different to those obtained by DGGE and with those obtained previously by using classic culture techniques. The taxa identified by dilution-to-extinction were Proteobacteria (81.92%), Firmicutes (11.30%), Actinobacteria (4.52%), and Bacteroidetes (2.26%) phyla with Gammaproteobacteria as predominant class (65.7%). The main genera were: Marinobacter (38.85%), Halomonas (20.2%), and Bacillus (11.2%). Nine of the 61 identified bacteria showed less than 97% sequence identity with validly described species and may well represent new taxa. The number of bacteria in different samples, locations, and seasons were calculated by CARD-FISH, ranging from 54.3 to 78.9% of the total prokaryotic population. Inconclusion, the dilution-to-extinction technique could be a complementary method to classical culture based method, but neither gets to cultivate the major taxa detected by DGGE. The bacterial community was influenced significantly by the physico-chemical parameters (specially the salinity and oxygen), the location and the season of sampling.This research was supported by grants from the Dirección General de Investigación Científica y Técnica (CGL2005-05947; CGL2008-02399; CGL2011-25748), Ministerio de Economía y Competitividad and from the Plan Andaluz de Invesigacion (P07-CVI-03150; CVI06226), Spain

Potential of the quorum-quenching and plant-growth promoting halotolerant Bacillus toyonensis AA1EC1 as biocontrol agent

The use of fertilizers and pesticides to control plant diseases is widespread in intensive farming causing adverse effects together with the development of antimicrobial resistance pathogens. As the virulence of many Gram-negative phytopathogens is controlled by N-acyl- homoserine lactones (AHLs), the enzymatic disruption of this type of quorum-sensing (QS) signal molecules, mechanism known as quorum quenching (QQ), has been proposed as a promising alternative antivirulence therapy. In this study, a novel strain of Bacillus toyonensis isolated from the halophyte plant Arthrocaulon sp. exhibited numerous traits associated with plant growth promotion (PGP) and degraded a broad range of AHLs. Three lactonases and an acylase enzymes were identified in the bacterial genome and verified in vitro. The AHL-degrading activity of strain AA1EC1 significantly attenuated the virulence of relevant phytopathogens causing reduction of soft rot symptoms on potato and carrots. In vivo assays showed that strain AA1EC1 significantly increased plant length, stem width, root and aerial dry weights and total weight of tomato and protected plants against Pseudomonas syringae pv. tomato. To our knowledge, this is the first report to demonstrate PGP and QQ activities in the species B. toyonensis that make this strain as a promising phytostimulant and biocontrol agent.Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033ESF Investing in your future (Grant: RYC2019- 026481-I)Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 (Grant: PID2019-106704RB-100)Consejería de Universidad, Investigación e Innovación de la Junta de AndalucíaERDF A way of making Europe (Grant: B-AGR-222-UGR20

Diversity and antimicrobial potential in sea anemone and holothurian microbiomes

Marine invertebrates, as holobionts, contain symbiotic bacteria that coevolve and develop antimicrobial substances. These symbiotic bacteria are an underexplored source of new bioactive molecules to face the emerging antibiotic resistance in pathogens. Here, we explored the antimicrobial activity of bacteria retrieved from the microbiota of two sea anemones (Anemonia sulcata, Actinia equina) and two holothurians (Holothuria tubulosa, Holothuria forskali). We tested the antimicrobial activity of the isolated bacteria against pathogens with interest for human health, agriculture and aquaculture. We isolated 27 strains with antibacterial activity and 12 of these isolates also showed antifungal activity. We taxonomically identified these strains being Bacillus and Vibrio species the most representative producers of antimicrobial substances. Microbiome species composition of the two sea anemones was similar between them but differed substantially of seawater bacteria. In contrast, microbiome species composition of the two holothurian species was different between them and in comparison with the bacteria in holothurian feces and seawater. In all the holobiont microbiomes Bacteroidetes was the predominant phylum. For each microbiome, we determined diversity and the rank-abundance dominance using five fitted models (null, preemption, log-Normal, Zipf and Zipf-Mandelbrot). The models with less evenness (i.e. Zipf and Zipf-Mandelblot) showed the best fits in all the microbiomes. Finally, we tracked (using the V4 hypervariable region of 16S rRNA gene) the relative abundance of these 27 isolates with antibacterial activity in the total pool of sequences obtained for the microbiome of each holobiont. Coincidences, although with extremely low frequencies, were detected only in the microbiome of H. forskali. This fact suggests that these isolated bacteria belong to the long tail of rare symbiotic bacteria. Therefore, more and more sophisticated culture techniques are necessary to explore this apparently vast pool of rare symbiontic bacteria and to determine their biotechnological potentiality.This work was supported by Campus de Excelencia Internacional BIOTIC (CEI Biotic) Universidad de Granada, http://biotic.ugr.es, Grant PBS46. iMare Natural S.L. provided support in the form of salaries for author PAA, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the `author contributions' section

Governance Review Commission General Rationale Rev 2

Bacterial infectious diseases produced by Vibrio are the main cause of economic losses in aquaculture. During recent years it has been shown that the expression of virulence genes in some Vibrio species is controlled by a population-density dependent gene-expression mechanism known as quorum sensing (QS), which is mediated by the diffusion of signal molecules such as N-acylhomoserine lactones (AHLs). QS disruption, especially the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), is one of the novel therapeutic strategies for the treatment of bacterial infections. In this study, we present the detection of AHLs in 34 marine Vibrionaceae strains. Three aquaculture-related pathogenic Vibrio strains, V. mediterranei VibC-Oc-097, V. owensii VibC-Oc-106 and V. coralliilyticus VibC-Oc-193 were selected for further studies based on their virulence and high production of AHLs. This is the first report where the signal molecules have been characterized in these emerging marine pathogens and correlated to the expression of virulence factors. Moreover, the results of AHL inactivation in the three selected strains have been confirmed in vivo against brine shrimps (Artemia salina) and Manila clams (Venerupis philippinarum). This research contributes to the development of future therapies based on AHL disruption, the most promising alternatives for fighting infectious diseases in aquaculture.This research was supported in part by grants from the Ministerio de Economía y Competitividad (Spain) (http://www.mineco.gob. es/portal/site/mineco/idi) (AGL2012-39274-C02- 02; AGL2015-68806-R; AGL2015-63740-C2-2-R). José Carlos Reina is supported by a FPU fellowship rom the Ministerio de Educación, Cultura y Deporte (Spain) (https://www.mecd.gob.es/ portada-mecd/) (FPU15-01717) and a Initiation to Research fellowship for Master Students (2016) from the University of Granada (Spain

Effective Tetradentate Compound Complexes against Leishmania spp. that Act on Critical Enzymatic Pathways of These Parasites

The spectrum and efficacy of available antileishmanial drugs is limited. In the present work we evaluated in vitro the antiproliferative activity of 11 compounds based on tetradentate polyamines compounds against three Leishmania species (L. braziliensis, L. donovani and L. infantum) and the possible mechanism of action. We identified six compounds (3, 5, 6, 7, 8 and 10) effective against all three Leishmania spp both on extracellular and intracellular forms. These six most active leishmanicidal compounds also prevent the infection of host cells. Nevertheless, only compound 7 is targeted against the Leishmania SOD. Meanwhile, on the glucose metabolism the tested compounds have a species-specific effect on Leishmania spp.: L. braziliensis was affected mainly by 10 and 8, L. donovani by 7, and L. infantum by 5 and 3. Finally, the cellular ultrastructure was mainly damaged by 11 in the three Leishmania spp. studied. These identified antileishmania candidates constitute a good alternative treatment and will be further studied.This research was funded by THE SPANISH MINISTRY OF ECONOMY AND COMPETITIVENESS (MINECO), CONSOLIDER-INGENIO 2010, grant number CDS2010-00065; THE EUROPEAN RESEARCH FOUNDATION, grant number ERC-2009-StG-239910; THE MICINN, grant number CTQ2009-08464; and INNPLANTA, grant number INP-2011-0059-PCT-420000-ACT1

A new teaching strategy to teach microbiology through its history

Ante el nuevo reto de la implantación del Espacio Europeo de Educación Superior los métodos de estudio deben adaptarse y servirse de herramientas innovadoras. Una buena estrategia para comprender una ciencia, su metodología y objetivos, es conocer su historia. Esta estrategia se hace especialmente útil en el caso de la Microbiología por ser una disciplina joven, que se ha cimentado precisamente en la metodología que sus creadores han ido elaborando. El objetivo de este trabajo ha sido crear una herramienta de utilidad en el aprendizaje de la Microbiología. Para ello hemos tomado como punto de apoyo su historia, es decir, explicar la Microbiología mediante la descripción de los descubrimientos y hechos que contribuyeron al desarrollo de esta ciencia. Para ello se ha creado una página web con dos aplicaciones independientes que operan sobre una base de datos común. 1. La primera de carácter público para los alumnos aunque con posibilidad de acceso restringido; se pueden consultar los contenidos sin posibilidad de alterarlos. 2. La otra aplicación es privada, los profesores pueden administrar los contenidos, y hacer uso de diferentes herramientas que facilitan la gestión de los mismos.In the light of the challenge presented by the European Higher Education Area, study strategies will have to adapt themselves and take advantage of innovative tools provided by modern information technology. One good way of understanding a science, its methodology and objectives is by taking an interest in its history. This approach is especially useful with regard to Microbiology, which is a fairly young discipline founded upon and made cohesive by the methods devised and constantly elaborated on by its designers. The aim of this work has been to offer guidance towards learning Microbiology. To this end we have chosen as our starting point, and indeed one of the keystones of our approach, the history of the subject itself; that is to say, an explanation of Microbiology via a description of the discoveries and milestones that have contributed to the development of this science. Thus we have set up a web page with two separate applications operating on one common database. 1. The first is openly available to the students, although allowing the possibility of restricted access. Its contents may be consulted but not altered. 2. The second is accessible only by lecturers, who may administer its contents and resort to different tools to facilitate the management of the information available on the site