Instituto de Investigación do Medio Acuático para unha Saúde Global (iARCUS)

Permanent URI for this collectionhttps://hdl.handle.net/10347/42832

O Instituto de Investigación do Medio Acuático para unha Saúde Global (iARCUS) creouse en 1998 como Instituto de Acuicultura co obxectivo de ser un “centro de investigación, desenvolvemento, innovación, transferencia e formación en campos relacionados coa biotecnoloxía aplicada á acuicultura e á conservación dos recursos da acuicultura”. Os grupos que o compoñen abarcan aspectos de I+D relacionados coa Biotecnoloxía en Acuicultura, incluída a Xenética, a Microbioloxía, a Patoloxía, a Inmunoloxía, a Fisioloxía e a Bioquímica, aplicados ás dúas áreas principais da acuicultura: a piscicultura e o cultivo de invertebrados (moluscos e crustáceos), incluíndo as poboacións cultivadas e salvaxes.

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Hindered amine light stabilizers in indoor dust: method development and occurrence
(Springer, 2026-05-05) Castro Varela, Gabriela; Pereiro, Pablo; Santos Rodríguez, Ysabel; Perín, Mauricio; Rodríguez Pereiro, Isaac; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
Hindered amine light stabilizers (HALS) are polymer additives extensively used to improve the durability of plastic materials by inhibiting degradation induced by ultraviolet radiation. Due to their effectiveness, HALS are incorporated into a wide variety of polymeric products intended for both indoor and outdoor applications. However, because these compounds are not chemically bound to the polymer, they can be released into the environment through processes such as volatilization, abrasion, and dissolution. As a result, HALS may accumulate in dust and other environmental matrices. Their occurrence in indoor and outdoor dust raises concerns regarding environmental persistence and potential human exposure, underscoring the need for robust and sensitive analytical methods for their determination. In this study, a new analytical methodology for the determination of HALS in dust samples was developed and optimized. Different sample preparation techniques, including matrix solid-phase dispersion (MSPD), pressurized liquid extraction (PLE), and ultrasound-assisted extraction (UAE), were evaluated with the aim of improving extraction efficiency while minimizing matrix effects. Quantitative analysis was performed using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) equipped with a triple quadrupole (QqQ) mass analyzer, providing high sensitivity and selectivity. The optimized procedure was subsequently applied to dust samples collected from various indoor environments to investigate the occurrence and distribution of HALS. These findings contribute to a better understanding of HALS contamination in dust and associated human exposure pathways.
Optimization, validation and application of a novel multiresidue method for prioritization of contaminants of emerging concern in biosolids
(Elsevier, 2026-04-30) Barros Soto-Quiroga, Andrés; Castro Varela, Gabriela; Ramil Criado, María; Rodríguez Pereiro, Isaac; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
Biosolids generated during municipal wastewater treatment are a source of organic matter and essential macronutrients, making them valuable to increase the fertility of soils. However, they also contain variable concentrations of micropollutants, particularly pharmaceuticals, which can pose environmental risks. An analytical methodology based on QuEChERS extraction and LC-MS/MS analysis was optimized and applied to the determination of 59 micropollutants, with large variations in their polarities and different uses, in biosolids from sewage treatment plants (STPs). Extraction efficiencies above 80%, and moderate variations in the efficiency of electrospray ionization were achieved for most of the target analytes in biosolids from different STPs. The accuracy of the final method ranged from 80% to 120% for 57 out of 59 micropollutants, with limits of quantification between 1 and 5 ng g−1, except for chlorhexidine (50 ng g−1). Forty-nine pollutants showed detection frequencies above 70%, with thirty-six compounds quantified in 100% of biosolids from 27 STPs. Biocides and psychiatric drugs represented more than 75% of residues in biosolids, with average accumulated concentrations of 5149 and 2425 ng g−1 (dry weight, d.w.), respectively. Within the first group, the maximum concentrations of azithromycin and chlorhexidine stayed above 2300 ng g−1 and 11,000 ng g−1 d.w., respectively. Ecological risk assessment of biosolids-amended soils identified fifteen compounds whose potential leaching into aquatic environments warrants further investigation. Significative accumulation in biosolids of several pharmaceuticals listed in the Wastewater Directive must be considered together, with their residues in raw and treated wastewaters, to estimate their real removal at STPs.
Analytical challenges and occurrence of antibiotics in biosolids from municipal sewage treatment plants in North Spain
(Elsevier, 2026-04-24) Barros Soto-Quiroga, Andrés; Castro Varela, Gabriela; Ramil Criado, María; Rodríguez Pereiro, Isaac; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
Antibiotics are among the most concerning pharmaceutical contaminants released from municipal sewage treatment plants (STPs), occurring both in treated effluents and in dewatered biosolids. This study examines key analytical challenges during the determination of ten antibiotics in sewage wastes using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Furthermore, results of their distribution in dewatered biosolids from different STPs are presented. Fluoroquinolones were identified as the most problematic compounds due to sorption on glassware, strong interaction with sample matrix, and signal suppression effects during LC-MS/MS analysis. Optimized extraction, based on sonication of freeze-dried samples with a buffered acetonitrile–water solution (pH 4.4, 1:1), yielded average recoveries between 74% and 108%, with moderate variability across sludge types. During simultaneous quantification of multiclass antibiotics, fluoroquinolones exhibited moderate to high signal attenuation depending on the matrix. For this group of compounds, signal suppression could be mitigated by fractionating extracts using mixed-mode (reversed phase and cation exchange) sorbents. Combined with isotopically labelled surrogate standards, solvent based calibration enabled accurate quantification of all targeted compounds, achieving limits of quantification below 5 ng g⁻¹. Azithromycin, clarithromycin, norfloxacin, ciprofloxacin and ofloxacin were ubiquitous in dewatered biosolids, with median concentrations ranging from 7 ng g-1 (clarithromycin) to 1761 ng g-1 (ofloxacin). A mass balance assessment of emissions through treated wastewater and biosolids highlighted azithromycin, ciprofloxacin, norfloxacin, and ofloxacin as the priority antibiotics for monitoring in final dewatered solid waste streams (biosolids) from STPs.
Engineering of green sterilization technology to obtain biocompatible aerogels: Supercritical CO2 versus ethylene oxide and gamma radiation
(Elsevier, 2026-01-02) Carracedo Pérez, María; Boccia, Antonella Caterina; Ardao Palacios, Inés; Passos, Cláudia Pereira; Santos Rosales, Víctor; Santos Torres, Beatriz; Bernardo, Fábio Gabriel Pereira; Blanco Vales, María; Magariños Ferro, Beatriz; García González, Carlos A.; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Materiais (iMATUS); Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS); Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
The growing relevance of aerogels in biomedicine demands the choice of compatible sterilization techniques with these materials. Conventional methods, such as ethylene oxide (EO) and gamma radiation (γ-rays) sterilization, have significant drawbacks while facing important environmental restrictions. In this study, supercritical CO2 (scCO2) sterilization is tested for polysaccharide (starch and alginate) aerogels as an eco-friendly alternative to conventional procedures. Three post-processing treatments under different CO2 exposure regimes (static, dynamic and combined) and in the presence of H2O2 as additive were developed and assessed to reach sterility assurance levels (SAL) below 10−6. After sterilization, a vacuum treatment was implemented to ensure a low residual presence of H2O2 in the aerogels so that the material biocompatibility was not compromised according to in vitro cell tests with fibroblasts. The residual adsorbed H2O2 was quantified for the first time in aerogels by nuclear magnetic resonance spectroscopy. The effects of the supercritical sterilization treatments on the textural and chemical properties of the aerogels were evaluated and compared to those treated with EO and γ-rays. Results highlight the unique efficiency of scCO2 sterilization as a post-processing method that preserves the aerogel structure while offering an eco-sustainable potential for producing sterile and biocompatible materials.
Integrated computational fluid dynamics and experimental evaluation of a tubular membrane photoreactor for UVC-driven advanced oxidation at bench- and pilot-scale
(Elsevier, 2026-04-13) Santos, Carla S.; Marrocos, Paulo H.; Passalía, Claudio; Labas, Marisol D.; Brandi, Rodolfo J.; Montes Goyanes, Rosa; Quintana Álvarez, José Benito; Gomes, Ana I.; Vilar, Vítor J. P.; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía
This study combines computational fluid dynamics (CFD) simulations and experimental data to examine the hydrodynamic and photonic behavior of tubular membrane photoreactors operated under UVC and UVC/persulfate (UVC/PDS) conditions for the removal of organic micropollutants (OMPs). At bench-scale, CFD simulations revealed that the tangential inlet generated a helical flow pattern in the annular reaction zone (ARZ), promoting interaction between radial and axial velocity components. The effect of reflective surfaces on photon delivery was evaluated across three configurations: no reflector and one- and three-sided reflectors (N = 1 and N = 3). Reflective surfaces increased photon delivery to the ARZ, with the N = 3 configuration providing the highest optical efficiency and the best photolytic removal of the selected OMP in both demineralized water (DW) and conventional activated sludge (CAS) effluent. Based on these findings, a pilot-scale reactor preserving the same cross-sectional geometry and incorporating the N = 3 reflector was evaluated. Residence time distribution (RTD) experiments indicated a non-ideal plug flow with moderate axial dispersion, while CFD confirmed the persistence of the characteristic helical flow pattern. Under UVC alone, venlafaxine (VLX) removal remained limited, whereas under UVC/PDS operation removal exceeded 85% across all evaluated conditions. Simulations of the local volumetric rate of photon absorption (LVRPA) and spatial species distributions revealed a strong correlation between photon availability, oxidant distribution, and OMP degradation along the reactor length. These results provide valuable insights into the design of tubular membrane photoreactors and support their applicability for advanced urban wastewater treatment systems.
Tracing illicit and prescription drug use in a Spanish prison by combining wastewater analysis and pharmaceutical dispensing data
(Elsevier, 2026-04-12) Egaña, Iker; Nogales-García, Maite; Akhrimenko, Vladimir; González Gómez, Xiana; Rodil Rodríguez, María del Rosario; Montes Goyanes, Rosa; Quintana Álvarez, José Benito; Mestre-Pintó, Juan Ignacio; Orive, Gorka; Lertxundi, Unax; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía
Background and aim We combined wastewater-based epidemiology (WBE) and pharmaceutical dispensing records to distinguish between illicit and prescription drug use. Methods We collected 24-h composite wastewater samples during three one-week campaigns in a prison in northern Spain. Samples were analysed for pharmaceuticals, illicit drugs, nicotine and alcohol. Population-normalised daily loads (PNDLs) were converted to consumption estimates and compared with Spanish population WBE estimates and pharmacy dispensing data. Results Wastewater analysis showed high use of several pharmaceuticals, with the highest average PNDL for gabapentinoids. Pregabalin and gabapentin estimates from wastewater exceeded pharmacy dispensing, suggesting internal diversion. Cannabis consumption was about four times higher than community estimates and cocaine was also higher in all campaigns. Ketamine and MDMA use were above community medians during the February campaign and nicotine use was nearly ten times higher than community levels. Conclusion WBE identifies prescription drug diversion, illicit drug markets and quantifies licit substance use in prisons.
Associations between food group intake and serum levels of selenium and other essential and toxic trace elements in adults
(Springer, 2026-02-28) Rivas, Inés; Miranda Castañón, Marta Inés; Herrero Latorre, Carlos; Monte Secades, Rafael; López Alonso, María Marta; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Patoloxía Animal
Purpose: To examine how the habitual consumption of major food groups is related to serum concentrations of essential and toxic trace elements in adults and to identify key dietary predictors of adequate status. Methods: In this cross-sectional analysis, 465 healthy adults (Galicia, Spain; 2020–2022) completed a validated semi-quantitative food frequency questionnaire; foods were categorised in 13 groups. Fasting serum concentrations of 14 trace elements were measured by ICP-MS. Group differences across element tertiles (clinical categories for selenium) were compared using the Kruskal–Wallis test. Random Forest (RF) models were constructed to evaluate multivariate dietary predictors for each element. Age- and sex-adjusted logistic regression were used to identify food group predictors of adequate (> 90 µg/L) vs. non-adequate selenium. Results: Seafood intake was positively associated with serum selenium concentrations and strongly tracked serum arsenic and mercury concentrations. Dairy, fruit and meat were also included in the RF models. Seafood ranked among the top three RF predictors for 13/14 elements; dairy ranked in all models. Logistic regression indicated higher odds of adequate selenium with greater seafood intake (OR 1.009; 95% CI 1.003–1.015; p = 0.007) and inverse associations with oil (OR 0.947; p = 0.018) and grains (OR 0.992; p = 0.036); positive trends in legume and nut consumption were identified. Individuals with adequate selenium reported consumption of ~ 4 fish servings/week. Conclusion: Integrating dietary and biomarker data revealed selenium to be the most vulnerable micronutrient. Promoting regular consumption of fish (and possibly nuts) while moderating high-oil/high-grain intake may enhance selenium without exceeding toxic metal reference limits.
Evaluation of peroxymonosulfate addition as a strategy to enhance Cryptosporidium inactivation in solar water disinfection
(Elsevier, 2026-03-03) Martín García, Sandra; Couso Pérez, Seila; Gómez Couso, Hipólito; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
Solar disinfection (SODIS) is a low-cost water treatment used in areas without access to safe water, with well-established efficacy against bacteria, but limited for virus and protozoa. Cryptosporidium parvum is a waterborne protozoan resistant to conventional water disinfection methods, representing a major challenge in public health. This study evaluates for the first time the enhancement of SODIS against C. parvum using peroxymonosulfate (PMS). Distilled and well water with different PMS concentrations, in the absence or presence of Fe2+ , were spiked with C. parvum oocysts and exposed to simulated solar radiation at 40 °C for 6 h, directly and under a polyethylene terephthalate (PET), polypropylene (PP) and polymethylmethacrylate (PMMA) covers. Oocyst viability was evaluated by quantifying 70 kDa heat shock protein mRNA using reverse transcription qPCR. Results showed that PMS concentrations ≥ 0.5 mM, in the absence and presence of Fe2+, reached > 4 logarithmic reduction (LR) in the oocyst survival when water was exposed uncovered. Under all plastic covers, > 4 LR was achieved in the absence of Fe2+ in well water. Toxicity assays with Daphnia magna showed that, in the presence of Fe2+, 0.5 mM PMS-treated well water under PP and PMMA covers caused only 10% mortality after 24 and 48 h. Overall, results demonstrated that the incorporation of PMS in SODIS procedures can effectively inactivate C. parvum oocysts and reduces exposure time.
Regulatory effects on virulence and phage susceptibility revealed by sdiA mutation in Klebsiella pneumoniae
(Frontiers Media, 2025) Silva Bea, Sergio; Maseda, Pablo; Otero Casal, Ana María; Romero Bernárdez, Manuel; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
Introduction: The World Health Organization has identified multi-drug resistant Klebsiella pneumoniae strains as the highest priority in 2024. Understanding the regulatory routes of virulence features is crucial for the development of novel anti-virulence strategies. SdiA, a LuxR-like quorum sensing (QS) receptor that responds to N-acyl-homoserine lactones (AHLs), is involved in the regulation of virulence traits in some Gram-negative bacteria. The function of this receptor in the virulence of K. pneumoniae remains uncertain. The objective of the present study was to elucidate the function of SdiA in K. pneumoniae biofilm formation and virulence. Methods: To this end, a genetic knockout of sdiA was conducted, and virulence-related phenotypic studies were performed following AHL provision. Results and Discussion: The results demonstrate that sdiA deficiency increases susceptibility to phage infection and human serum resistance, and promotes biofilm maturation and cell filamentation, although no effect on virulence was observed in vivo in the Galleria mellonella infection model. On the other hand, C6-HSL promoted sdiA-dependent biofilm maturation, capsule production and serum resistance while reducing virulence against G. mellonella in the absence of sdiA. The addition of C6-HSL did not affect phage susceptibility. The results of this study demonstrate that AHLs and SdiA exert a dual influence on virulence phenotypes, operating both independently and hierarchically. These findings provide new insights into the virulence of K. pneumoniae and its regulation by SdiA.
¿A qué pregunta responde la educación para el desarrollo sostenible? Una respuesta inconveniente
(Sociedad Andaluza de Filosofía, 2026-01-15) Meira Cartea, Pablo; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
El artículo realiza un análisis crítico de las claves históricas, ideológicas, éticas y políticas que subyacen en la controversia entre Educación Ambiental (EA) y Educación para el Desarrollo Sostenible (EDS), una polémica que ha condicionado la evolución del campo de las respuestas de la crisis socio-ambiental desde la década de los años ochenta del siglo pasado. En esta aproximación se reivindica el carácter multiparadigmático y plural del campo de la EA frente a los sesgos que han pretendido convertir la EDS en un marco hegemónico para salvaguardar los principios de producción y reproducción cultural de la economía global de mercado, principalmente los referidos al mito del crecimiento como condición para el progreso humano y la sostenibilidad ambiental.
First Description and Characterisation of Lactococcus garvieae Strains Causing Septicaemic Disease in Farmed Sea Bass (Dicentrarchus labrax; Linnaeus) in Spain
(Wiley, 2025-11-27) Fouz Rodríguez, Belén; Carballeda Carrasco, Evelyn; Barriga Cuartero, Javier; Torres Corral, Yolanda; Robles, Álvaro; Zarza, Carlos; Santos Rodríguez, Ysabel; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
This study presents the first description and characterisation of Lactococcus garvieae isolated from diseased European sea bass (Dicentrarchus labrax) farmed in Spain. During autumn 2023, two fish farms were affected by infectious outbreaks causing moderate cumulative fish mortality (5%-10%). Diseased fish showed clinical signs of haemorrhagic septicaemia, compatible with lactococcosis. Standardised screening tests revealed the presence of Gram-positive cocci in the kidney, spleen and brain of the diseased fish, and negative results for parasites and viruses. Bacterial cultures recovered from the internal organs of all diseased fish were identified as Lactococcus garvieae by phenotypic, genetic, immunological and proteomic analyses. Strains were sensitive to oxytetracycline, florfenicol and erythromycin and resistant to trimethoprim sulfamethoxazole. In addition, representative isolates were virulent to juvenile sea bass and Senegalese sole (Solea senegalensis) after intracoelomic challenge with doses ranging from 106 to 107 CFU/fish. SDS-PAGE and immunoblotting analyses, using rabbit serum anti L. garvieae CECT 5274 and serum from trout immunised with a bivalent oil-based vaccine against L. garvieae-Yersinia ruckeri, showed that the current strains of sea bass shared some antigenic proteins with strains of L. garvieae from other hosts and with the reference strain of L. petauri DSM104842. Our overall results confirm the presence of this emerging pathogen in Spanish marine aquaculture and suggest that commercially available lactococcosis vaccines could confer some protection to sea bass, helping to prevent this new threat.
The Temperature-Dependent Expression of the High-Pathogenicity Island Encoding Piscibactin in Vibrionaceae Results From the Combined Effect of the AraC-Like Transcriptional Activator PbtA and Regulatory Factors From the Recipient Genome
(Frontiers, 2021-11-19) Afonso Lages, Marta Carolina; Lemos Ramos, Manuel Luis; Balado Dacosta, Miguel; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
The high-pathogenicity island irp-HPI is widespread among Vibrionaceae encoding the piscibactin siderophore system. The expression of piscibactin genes in the fish pathogen Vibrio anguillarum is favored by low temperatures. However, information about the regulatory mechanism behind irp-HPI gene expression is scarce. In this work, in-frame deletion mutants of V. anguillarum defective in the putative regulators AraC1 and AraC2, encoded by irp-HPI, and in the global regulators H-NS and ToxRS, were constructed and their effect on irp-HPI gene expression was analyzed at 15 and 25°C. The results proved that only AraC1 (renamed as PbtA) is required for the expression of piscibactin biosynthesis and transport genes. PbtA inactivation led to an inability to grow under iron restriction, a loss of the outer membrane piscibactin transporter FrpA, and a significant decrease in virulence for fish. Inactivation of the global repressor H-NS, which is involved in silencing of horizontally acquired genes, also resulted in a lower transcriptional activity of the frpA promoter. Deletion of toxR-S, however, did not have a relevant effect on the expression of the irp-HPI genes. Therefore, while irp-HPI would not be part of the ToxR regulon, H-NS must exert an indirect effect on piscibactin gene expression. Thus, the temperature-dependent expression of the piscibactin-encoding pathogenicity island described in V. anguillarum is the result of the combined effect of the AraC-like transcriptional activator PbtA, harbored in the island, and other not yet defined regulator(s) encoded by the genome. Furthermore, different expression patterns were detected within different irp-HPI evolutionary lineages, which supports a long-term evolution of the irp-HPI genomic island within Vibrionaceae. The mechanism that modulates piscibactin gene expression could also be involved in global regulation of virulence factors in response to temperature changes.
Structure and biosynthetic assembly of piscibactin, a siderophore from photobacterium damselae subsp. piscicida, predicted from genome analysis
(Wiley, 2012-09-05) Souto, Alba; Montaos, Marcos A.; Rivas Fontenla, Amable José; Balado Dacosta, Miguel; Rodríguez Osorio, Carlos; Rodríguez, Jaime; Lemos Ramos, Manuel Luis; Jiménez, Carlos; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
From the cultures of Photobacterium damselae subsp. piscicida, the aetiological agent of fish pasteurellosis, a new siderophore named piscibactin (1), was isolated as its gallium and iron(III) complexes along with a possible intermediate of its biosynthesis, prepiscibactin (2). Analysis of the gene cluster involved in the siderophore biosynthesis allowed the partial prediction of the structures. Thus, an NRPS-mediated mechanism similar to that for yersiniabactin was suggested by protein sequence comparisons. The final structures were solved by NMR and MS methods and by DFT molecular modeling. The results obtained in the structural and functional characterization of piscibactin enabled the proposal of a biosynthetic pathway.
Assessing the impact of viticulture plant protection products in the pollution of surface and ground waters
(Elsevier, 2025-12-03) Fernández Fernández, V.; Ramil Criado, María; Díaz Losada, E.; Loureiro Rodríguez, M.D.; Trigo Córdoba, E.; Andrades, M.S.; Marín Benito, J.M.; Rodríguez Cruz, M.S.; Rodríguez Pereiro, Isaac; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía
Plant protection products, particularly fungicides and insecticides, are intensively used in viticulture. Washing from leaves of vines, runoff transport, and soil penetration might lead to pollution of surface and ground waters. Herein, we present a systematic study on the occurrence and evolution of fungicides and insecticides in samples from two major wine production areas in Spain (La Rioja and Galicia). Above 400 water samples, corresponding to eight sub-zones, from two regions with different pedoclimatic conditions, were analyzed by liquid chromatography accurate mass spectrometry. Total residues of target compounds followed a homogeneous distribution in all sub-zones except one; nevertheless, their average values were slightly lower in La Rioja than in Galicia (181.1 ng L−1 and 260.8 ng L−1, respectively). The highest pollution levels in the complete set of samples were measured in summer campaigns, corresponding to the most intensive application season of fungicides and insecticides. Nonetheless, in some wells, residues increased from summer to autumn, confirming their leaching to ground water. Fungicides were prevalent versus insecticides with distribution patterns varying between regions. Tetraconazole, fluopyram and boscalid recorded higher detection frequencies in waters from La Rioja (from 16 % to 24 %) than in Galicia (between 4 % and 10 %). The opposite trend was observed for azoxystrobin (11 % vs 35 %), zoxamide (3 % vs 34 %), mandipropamide (1 % vs 27 %) and fluopicolide (1 % vs 23 %), with lower detection rates in La Rioja compared to Galicia. Attending to detection frequencies and ratios between measured and predicted non-effect concentrations, azoxystrobin, carbendazim, fluopicolid, tebuconazole and zoxamide were rated as the most environmental concerning pollutants. Suspect screening of accurate MS data revealed the presence of additional pesticides, including herbicides and their transformation products, in the monitored aquatic environments
FvtA Is the Receptor for the Siderophore Vanchrobactin in Vibrio anguillarum: Utility as a Route of Entry for Vanchrobactin Analogues
(American Society for Microbiology (ASM), 2009-05-01) Balado Dacosta, Miguel; Lemos Ramos, Manuel Luis; Rodríguez Osorio, Carlos; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
Some strains of Vibrio anguillarum, the causative agent of vibriosis in a variety of marine animals, produce a catechol-type siderophore named vanchrobactin. The biosynthetic pathway and regulation of vanchrobactin are quite well understood. However, aspects concerning its entry into the cell have remained uncharacterized. In the present study we characterized two genes, fvtA and orf13, encoding potential TonB-dependent ferric-vanchrobactin receptors in serotype O2 V. anguillarum strain RV22. We found that an fvtA mutant was defective for growth under iron limitation conditions and for utilization of vanchrobactin, suggesting that fvtA encodes the vanchrobactin receptor of V. anguillarum. Interestingly, an orf13 mutant was not significantly affected, and results of reverse transcriptase PCR, as well as analysis of outer membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggested that this gene is not expressed. Furthermore, fatA, a plasmid gene coding for the anguibactin receptor in plasmid pJM1-harboring strains, is also present in the chromosome of RV22, although it is inactivated by insertion of transposases. In addition, we found that FvtA is the route of entry for vanchrobactin analogues, and there is evidence that it recognizes primarily the catechol-iron center. These analogues are potential candidate vectors for a Trojan horse strategy aimed at generating antimicrobial compounds exploiting the same route of entry for native siderophores. We found that fvtA and vanchrobactin biosynthesis genes are ubiquitous in both vanchrobactin- and anguibactin-producing V. anguillarum strains, which reinforces the utility of the vanchrobactin route of entry for the design of future strategies for the control of vibriosis.
Two Catechol Siderophores, Acinetobactin and Amonabactin, Are Simultaneously Produced by Aeromonas salmonicida subsp. salmonicida Sharing Part of the Biosynthetic Pathway
(American Chemical Society, 2015-10-13) Balado Dacosta, Miguel; Souto, Alba; Vences, Ana; Careaga, Valeria P; Valderrama, Katherine; Segade, Yuri; Rodríguez, Jaime; Rodríguez Osorio, Carlos; Jiménez, Carlos; Lemos Ramos, Manuel Luis; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
The iron uptake mechanisms based on siderophore synthesis used by the fish pathogen Aeromonas salmonicida subsp. salmonicida are still not completely understood, and the precise structure of the siderophore(s) is unknown. The analysis of genome sequences revealed that this bacterium possesses two gene clusters putatively involved in the synthesis of siderophores. One cluster is a candidate to encode the synthesis of acinetobactin, the siderophore of the human pathogen Acinetobacter baumannii, while the second cluster shows high similarity to the genes encoding amonabactin synthesis in Aeromonas hydrophila. Using a combination of genomic analysis, mutagenesis, biological assays, chemical purification, and structural determination procedures, here we demonstrate that most A. salmonicida subsp. salmonicida strains produce simultaneously the two siderophores, acinetobactin and amonabactin. Interestingly, the synthesis of both siderophores relies on a single copy of the genes encoding the synthesis of the catechol moiety (2,3-dihydroxybenzoic acid) and on one encoding a phosphopantetheinyl transferase. These genes are present only in the amonabactin cluster, and a single mutation in any of them abolishes production of both siderophores. We could also demonstrate that some strains, isolated from fish raised in seawater, produce only acinetobactin since they present a deletion in the amonabactin biosynthesis gene amoG. Our study represents the first evidence of simultaneous production of acinetobactin and amonabactin by a bacterial pathogen and reveals the plasticity of bacterial genomes and biosynthetic pathways. The fact that the same siderophore is produced by unrelated pathogens highlights the importance of these systems and their interchangeability between different bacteria.
Genomic analysis of the marine fish pathogen Photobacterium damselae subsp. piscicida: Insertion sequences proliferation is associated with chromosomal reorganisations and rampant gene decay
(Elsevier, 2017-10) Balado Dacosta, Miguel; Benzekri, Hicham; Labella, Alejandro M.; Claros, M. Gonzalo; Manchado, Manuel; Borrego, Juan J.; Rodríguez Osorio, Carlos; Lemos Ramos, Manuel Luis; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
Photobacterium damselae subsp. piscicida (Pdp) is an intracellular fish pathogen that causes photobacteriosis, a disease proven deadly in farmed fish worldwide. This work focuses on the analysis of genome sequences, chromosomes structure and gene contents of two strains from Sparus aurata (DI21) and Solea senegalensis (L091106-03H), isolated on the Spanish Atlantic coast. The comparative genomic analysis revealed that DI21 and L091106-03H share 98% of their genomes, including two virulence plasmids: pPHDP70 encoding siderophore piscibactin synthesis and pPHDP10 encoding the apoptotic toxin AIP56. Both genomes harbour a surprisingly large number of IS elements accounting for 12–17% of the total genome, representing an IS density of 0.15 elements per kb, one of the highest IS density values in a bacterial pathogen. This massive proliferation of ISs is responsible for the generation of a high number of pseudogenes that caused extensive loss of biological functions. Pseudogene formation is one of the main features of Pdp genome that explains most of the ecological and phenotypic differences with respect to its sibling subspecies P. damselae subsp. damselae and to other Vibrionaceae. Evidence was also found proving the existence of two chromosomal configurations depending on the origin of the strains: an European and an Asian/American types of genome organisation, reinforcing the idea of the existence of two geographically-linked clonal lineages in Pdp. In short, our study suggests that the host-dependent lifestyle of Pdp allowed massive IS proliferation and gene decay processes, which are major evolutionary forces in the shaping of the Pdp genome.
Identification of the Ferric-Acinetobactin Outer Membrane Receptor in Aeromonas salmonicida subsp. salmonicida and Structure-Activity Relationships of Synthetic Acinetobactin Analogues
(American Chemical Society, 2016-12-12) Balado Dacosta, Miguel; Segade, Yuri; Rey Varela, Diego; Rodríguez Osorio, Carlos; Rodríguez, Jaime; Lemos Ramos, Manuel Luis; Jiménez, Carlos; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in several fish species, produces acinetobactin and amonabactin as siderophores. In a previous study, we chemically characterized these siderophores and proposed a biosynthetic pathway based on genetic analysis. However, the internalization mechanisms of ferric-acinetobactin and ferric-amonabactin remain largely unknown. In the present study, we demonstrate that the outer membrane protein FstB is the ferric-acinetobactin receptor in A. salmonicida since an fstB defective mutant is unable to grow under iron limitation and does not use acinetobactin as an iron source. In order to study the effect that structural changes in acinetobactin have on its siderophore activity, a collection of acinetobactin-based analogues was synthesized, including its enantiomer and four demethylated derivatives. The biological activity of these analogues on an fstB(+) strain compared to an fstB(−) strain allowed structure–activity relationships to be elucidated. We found a lack of enantiomer preference on the siderophore activity of acinetobactin over A. salmonicida or on the molecular recognition by FstB protein receptor. In addition, it was observed that A. salmonicida could not use acinetobactin analogues when imidazole or a similar heterocyclic ring was absent from the structure. Surprisingly, removal of the methyl group at the isoxazolidinone ring induced a higher biological activity, thus suggesting alternative route(s) of entry into the cell that must be further investigated. It is proposed that some of the synthetic acinetobactin analogues described here could be used as starting points in the development of novel drugs against A. salmonicida and probably against other acinetobactin producers like the human pathogen Acinetobacter baumannii.
A gene cluster involved in the biosynthesis of vanchrobactin, a chromosome-encoded siderophore produced by Vibrio anguillarum
(Microbiology Society, 2006-12-01) Balado Dacosta, Miguel; Rodríguez Osorio, Carlos; Lemos Ramos, Manuel Luis; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
Vibrio anguillarum serotype O2 strains produce a catechol siderophore named vanchrobactin, which has been identified as N-[N′-(2,3-dihydroxybenzoyl)-arginyl]-serine. This work describes a chromosomal region that harbours the genetic determinants necessary for the biosynthesis of vanchrobactin. The authors have identified the genes involved in 2,3-dihydroxybenzoic acid (DHBA) biosynthesis (vabA, vabB and vabC) and activation (vabE), and a gene (vabF) encoding a non-ribosomal peptide synthetase, which is putatively involved in the assembly of the siderophore components. Also described are the identification and characterization of genes encoding a putative vanchrobactin exporter (vabS) and a siderophore esterase (vabH). In-frame deletion mutants in vabA, vabB, vabC, vabE, vabF and vabH were impaired for growth under conditions of iron limitation, and the analysis of culture supernatants by chrome azurol-S and cross-feeding assays showed almost no production of siderophores in any of the vabABCEF mutants. In addition, deletion mutations of vabA, vabB and vabC abolished production of DHBA, as assessed by chemical and biological analyses. Complementation of each mutant with the corresponding gene provided in trans confirmed the involvement of this gene cluster in the biosynthesis of DHBA and vanchrobactin in V. anguillarum strain RV22. Based on chemical and genetic data, and on published models for other catechol siderophores, a model for vanchrobactin biosynthesis is proposed.
Biosynthetic and regulatory elements involved in the production of the siderophore vanchrobactin in Vibrio anguillarum
(Microbiology Society, 2008-05-01) Balado Dacosta, Miguel; Rodríguez Osorio, Carlos; Lemos Ramos, Manuel Luis; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
Some Vibrio anguillarum strains produce a catechol-type siderophore named vanchrobactin, whose biosynthetic pathway has not been completely elucidated. In addition to the previously described genes vabA, vabC, vabB, vabE, vabF, vabS and vabH, in the present study we have identified the genes encoding a DAHP (3-deoxy-d-arabino-heptulosonate-7-phosphate) synthetase (vabG), a phosphopantheteinyl transferase (vabD), a LysR-family transcriptional regulator (vabR) and a putative siderophore receptor (fvtA). A deletion affecting vabG or vabD greatly reduced growth under iron-limiting conditions, whereas deletion of vabR did not have significant effects. Vanchrobactin production was abolished in the vabD mutant, whereas the vabG mutant retained a residual vanchrobactin production ability. Reverse transcriptase-mediated PCR indicated that this 11-gene cluster is organized into six iron-regulated transcriptional units. Transcriptional lacZ fusions demonstrated that the ferric uptake regulator (Fur) protein is the main iron-responsive regulator of these genes. Interestingly, the vabG gene was strongly iron-repressed, but Fur was not essential for this repression. In addition, the maximal expression from the vabG promoter was achieved only in the presence of an intact copy of vabR. Analysis of the β-galactosidase activities of a fvtA : : lacZ fusion in a vabB mutant and in the presence of added vanchrobactin suggested that a ferric-vanchrobactin-dependent activator plays a positive regulatory role in transcription of the fvtA–vabD operon. This possibility is reinforced by the presence of a predicted AraC box upstream of fvtA. We propose that vanchrobactin biosynthesis is subjected to a complex regulatory circuitry aimed at adjusting vanchrobactin production for the maintenance of iron homeostasis in V. anguillarum.

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