Nuevos antipatogénicos: identificación de nuevas bacterias marinas con actividad quorum quenching y aplicación en salud bucodental

En la formación de la biopelícula oral se necesitan diferentes genes, entre los que se encuentran los relacionados con la comunicación bacteriana, denominada QUORUM SENSING. Sin embargo, poco se conoce del papel del quorum sensing en la microbiota oral. La interceptación de esta comunicación, conocida como QUORUM QUENCHING, constituye una interesante alternativa al uso de antibióticos pues impide la producción de factores de virulencia. En este trabajo, se aislaron bacterias marinas con actividad quorum quenching para uso biotecnológico y se estudio el papel de las señales N-acil-homoserin-lactonas dentro de las biopelícula orales

The effect of bacteria on planula-larvae settlement and metamorphosis in the octocoral Rhytisma fulvum fulvum

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Freire, I., Gutner-Hoch, E., Muras, A., Benayahu, Y., & Otero, A. The effect of bacteria on planula-larvae settlement and metamorphosis in the octocoral Rhytisma fulvum fulvum. Plos One, 14(9), (2019): e0223214, doi:10.1371/journal.pone.0223214.While increasing evidence supports a key role of bacteria in coral larvae settlement and development, the relative importance of environmentally-acquired versus vertically-transferred bacterial population is not clear. Here we have attempted to elucidate the role of post-brooding-acquired bacteria on the development of planula-larvae of the octocoral Rhytisma f. fulvum, in an in vitro cultivation system employing different types of filtered (FSW) and autoclaved (ASW) seawater and with the addition of native bacteria. A good development of larvae was obtained in polystyrene 6-well cell culture plates in the absence of natural reef substrata, achieving a 60–80% of larvae entering metamorphosis after 32 days, even in bacteria-free seawater, indicating that the bacteria acquired during the brooding period are sufficient to support planulae development. No significant difference in planulae attachment and development was observed when using 0.45 μm or 0.22 μm FSW, although autoclaving the 0.45 μm FSW negatively affected larval development, indicating the presence of beneficial bacteria. Autoclaving the different FSW homogenized the development of the larvae among the different treatments. The addition of bacterial strains isolated from the different FSW did not cause any significant effect on planulae development, although some specific strains of the genus Alteromonas seem to be beneficial for larvae development. Light was beneficial for planulae development after day 20, although no Symbiodinium cells could be observed, indicating either that light acts as a positive cue for larval development or the presence of beneficial phototrophic bacteria in the coral microbiome. The feasibility of obtaining advanced metamorphosed larvae in sterilized water provides an invaluable tool for studying the physiological role of the bacterial symbionts in the coral holobiont and the specificity of bacteria-coral interactions.This work was supported by: EU FP7-Research Infrastructure Initiative Assemble (Association of European marine biological laboratories); EU FP7 Project Byefouling (grant agreement no 612717); Xunta de Galicia, Consellería de Cultura, Educación e Ordenación Universitaria (grant number ED431D 2017/22). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Use of Quorum Sensing Inhibition Strategies to Control Microfouling

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purposeThis work was supported by the European project BYEFOULING “Low-toxic cost-efficient environment-friendly antifouling materials” (FP7-OCEAN-2013 612717) and the project PID2019-104439RB-C21/AEI/10.13039/501100011033 of the Agencia Estatal de Investigación (AEI, Spain), and co-funded by the European Regional Development Fund of the European Union: A Way to Making Europe (FEDER). A.M. and A.P. were supported by predoctoral fellowships from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (ED481A-2015/311 and ED481A-2019/194). C.M. was supported by a postdoctoral fellowship from Xunta de Galicia (IN606B-2019/010)S

Acyl homoserine lactone-mediated quorum sensing in the oral cavity: a paradigm revisited

Acyl homoserine lactones (AHLs), the quorum sensing (QS) signals produced by Gram-negative bacteria, are currently considered to play a minor role in the development of oral biofilm since their production by oral pathogens has not been ascertained thus far. However, we report the presence of AHLs in different oral samples and their production by the oral pathogen Porphyromonas gingivalis. The importance of AHLs is further supported by a very high prevalence of AHL-degradation capability, up to 60%, among bacteria isolated from dental plaque and saliva samples. Furthermore, the wide-spectrum AHL-lactonase Aii20J significantly inhibited oral biofilm formation in different in vitro biofilm models and caused important changes in bacterial composition. Besides, the inhibitory effect of Aii20J on a mixed biofilm of 6 oral pathogens was verified using confocal microscopy. Much more research is needed in order to be able to associate specific AHLs with oral pathologies and to individuate the key actors in AHL-mediated QS processes in dental plaque formation. However, these results indicate a higher relevance of the AHLs in the oral cavity than generally accepted thus far and suggest the potential use of inhibitory strategies against these signals for the prevention and treatment of oral diseasesThis work was supported by the grant “Axudas do Programa de Consolidación e Estructuración de Unidades de Investigación Competitivas (GPC)” from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (ED431B2017/53) and by the project INTERREG-POCTEP-0227-CODIGOMAIS-1-E. A.M. was supported by a predoctoral fellowship from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (ED481A-2015/311)S

Multiple quorum quenching enzymes are active in the nosocomial pathogen acinetobacter baumannii ATCC17978

Acinetobacter baumannii presents a typical luxI/luxR quorum sensing (QS) system (abaI/abaR) but the acyl-homoserine lactone (AHL) signal profile and factors controlling the production of QS signals in this species have not been determined yet. A very complex AHL profile was identified for A. baumannii ATCC17978 as well as for A. nosocomialis M2, but only when cultivated under static conditions, suggesting that surface or cell-to-cell contact is involved in the activation of the QS genes. The analysis of A. baumanni clinical isolates revealed a strain-specific AHL profile that was also affected by nutrient availability. The concentration of OHC12-HSL, the major AHL found in A. baumannii ATCC17978, peaked upon stationary-phase establishment and decreases steeply afterwards. Quorum quenching (QQ) activity was found in the cell extracts of A. baumannii ATCC17978, correlating with the disappearance of the AHLs from the culture media, indicating that AHL concentration may be self-regulated in this pathogen. Since QQ activity was observed in strains in which AidA, a novel α/β-hydrolase recently identified in A. baumannii, is not present, we have searched for additional QQ enzymes in A. baumannii ATCC17978. Seven putative AHL-lactonase sequences could be identified in the genome and the QQ activity of 3 of them could be confirmed. At least six of these lactonase sequences are also present in all clinical isolates as well as in A. nosocomialis M2. Surface-associated motility and biofilm formation could be blocked by the exogenous addition of the wide spectrum QQ enzyme Aii20J. The differential regulation of the QQ enzymes in A. baumannii ATCC17978 and the full dependence of important virulence factors on the QS system provides a strong evidence of the importance of the AHL-mediated QS/QQ network in this speciesS

Evaluation of the anti-fouling efficacy of bacillus licheniformis extracts under environmental and natural conditions

There is an increasing interest in developing innovative coatings and testing natural products with anti-fouling activity to substitute current highly toxic biocides that have a harmful impact on marine organisms. Bacillus licheniformis species have shown different anti-biofilm and anti-fouling activities in vitro, but so far, its efficacy in field trials has not been tested. For this purpose, the capacity of different extracts of B. licheniformis NCTC 10341T to prevent micro and macro-fouling was first tested in vitro. The methanol cell extract (MCE) inhibited bacterial biofilm formation without significantly affecting planktonic growth and displayed a significant efficacy to prevent larval settlement of the macro-fouler Bugula neritina in vitro without inducing lethality. Additionally, the MCE presented low toxicity against the non-target species Artemia salina. The B. licheniformis MCE was then incorporated in a self-polishing paint at 2 and 5% w/w and tested in a static immersion experiment in the Gulf of Aqaba (northern Red Sea) for 180 days. Fouling coverage decreased by 30% in the 5% MCE-treated panels in comparison with the control panels. Differences in the anti-biofilm activity of the extracts depending on the culture medium highlight the importance of the strict control of culture conditions for the production of biomass with stable bioactive activity. The results indicate the potential of B. licheniformis NCTC 10341T crude extracts for environmentally friendly anti-fouling applications, although a deeper characterization of the bioactive compounds present in the B. licheniformis MCE and its mode of action is required to allow strict control of the activity of the extracts to achieve large-scale industrial productionThis work was supported by the European Union under Grant FP7-OCEAN-2013 612717 (Low-toxic cost-efficient environment-friendly anti-fouling materials). AM was supported by a predoctoral fellowship from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (ED481A-2015/311). CM was supported by a post-doctoral fellowship from Xunta de Galicia (IN606B-2019/010)S

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

Resveratrol-Loaded Hydrogel Contact Lenses with Antioxidant and Antibiofilm Performance

Contact lenses (CLs) are prone to biofilm formation, which may cause severe ocular infections. Since the use of antibiotics is associated with resistance concerns, here, two alternative strategies were evaluated to endow CLs with antibiofilm features: copolymerization with the antifouling monomer 2-methacryloyloxyethyl phosphorylcholine (MPC) and loading of the antioxidant resveratrol with known antibacterial activity. MPC has, so far, been used to increase water retention on the CL surface (Proclear® 1 day CLs). Both poly(hydroxyethyl methacrylate) (HEMA) and silicone hydrogels were prepared with MPC covering a wide range of concentrations (from 0 to 101 mM). All hydrogels showed physical properties adequate for CLs and successfully passed the hen’s egg-chorioallantoic membrane (HET-CAM) test. Silicone hydrogels had stronger affinity for resveratrol, with higher loading and a slower release rate. Ex vivo cornea and sclera permeability tests revealed that resveratrol released from the hydrogels readily accumulated in both tissues but did not cross through. The antibiofilm tests against Pseudomonas aeruginosa and Staphylococcus aureus evidenced that, in general, resveratrol decreased biofilm formation, which correlated with its concentration-dependent antibacterial capability. Preferential adsorption of lysozyme, compared to albumin, might also contribute to the antimicrobial activity. In addition, importantly, the loading of resveratrol in the hydrogels preserved the antioxidant activity, even against photodegradation. Overall, the designed hydrogels can host therapeutically relevant amounts of resveratrol to be sustainedly released on the eye, providing antibiofilm and antioxidant performanceThis research was funded by MINECO (SAF2017-83118-R), Agencia Estatal de Investigación (AEI) Spain, Xunta de Galicia (ED431C 2020/17), FEDER, and Fundação para a Ciência e Tecnologia (FCT) Portugal (UIDB/00100/2020 and UIDB/04585/2020). M. Vivero-Lopez acknowledges Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria) for a predoctoral research fellowship (ED481A-2019/120)S

Quorum sensing network in clinical strains of A. baumannii: AidA is a new quorum quenching enzyme

18 p.-4 fig.-6 tab. López, María et al.Acinetobacter baumannii is an important pathogen that causes nosocomial infections generally associated with high mortality and morbidity in Intensive Care Units (ICUs). Currently, little is known about the Quorum Sensing (QS)/Quorum Quenching (QQ) systems of this pathogen. We analyzed these mechanisms in seven clinical isolates of A. baumannii. Microarray analysis of one of these clinical isolates, Ab1 (A. baumannii ST-2_clon_2010), previously cultured in the presence of 3-oxo-C12-HSL (a QS signalling molecule) revealed a putative QQ enzyme (α/ß hydrolase gene, AidA). This QQ enzyme was present in all non-motile clinical isolates (67% of which were isolated from the respiratory tract) cultured in nutrient depleted LB medium. Interestingly, this gene was not located in the genome of the only motile clinical strain growing in this medium (A. baumannii strain Ab421_GEIH-2010 [Ab7], isolated from a blood sample). The AidA protein expressed in E. coli showed QQ activity. Finally, we observed downregulation of the AidA protein (QQ system attenuation) in the presence of H2O2 (ROS stress). In conclusion, most of the A. baumannii clinical strains were not surface motile (84%) and were of respiratory origin (67%). Only the pilT gene was involved in surface motility and related to the QS system. Finally, a new QQ enzyme (α/ß hydrolase gene, AidA protein) was detected in these strains.This study was funded by grant PI13/02390 awarded to MT within the State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General Directorate of evaluation and Promotion of Research - European Regional Development Fund "A way of Making Europe" and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015).Peer reviewe

Isolasi Bakteri Pendegradasi Senyawa Persisten Organic Pollutants Asal Tanah Inceptisol Karawang

Penggunaan pestisida yang tidak terkontrol akan mengganggu agroekosistem pertanian dan mencemari lingkungan. Jumlah pestisida yang beredar di Indonesia tahun 2006 terdaftar sebanyak 1336 formulasi, 2008 sebanyak 1702 formulasi, 2010 sebanyak 2048 formulasi, 2011 sebanyak 2247 formulasi. Di dalam tanah, karbon aktif peranan sebagai shelter atau rumah untuk mikroorganisme. Pori-pori kecil pada karbon aktif digunakan sebagai tempat tinggal bakteri, sedangkan pori besar dan retakan (cracks) digunakan sebagai tempat berkumpul. Penelitian dilakukan di Laboratorium Mikrobiologi Pusat Penelitian Biologi LIPI Cibinong Mei 2011 s/d Agustus 2011.Tujuan penelitian adalah menyeleksi bakteri dalam tanah yang berpotensi mendegradasi residu insektisida yang bersifat persistent organic poluttants (POPs). Isolasi dan karakterisasi mikroba pendegradasi POPs dilakukan dengan tiga tahapan, yaitu (1) Isolasi dan identifikasi mikroba pendegradasi POPs, (2) Uji karakteristik pertumbuhan isolat pada berbagai jenis POPs, dan (3) Penetapan residu insektisida POPs hasil kultur. Hasil terdapat 7 (tujuh) isolat yang mampu mendegradasi senyawa POPs, Lima isolat bersifat gram positif (BOB1, BOB2, BOB3, BOB4, BOB5) efektif untuk mendegradasi POPs berbahan aktif: lindan, heptaklor, DDT, dan dieldrin, sedangkan dua isolat bersifat gram negatif (BOB6 dan BOB7) efektif untuk mendegradasi POPs berbahan aktif aldrin