Revisiting the mercury cycle in marine sediments: A potential multifaceted role for Desulfobacterota.

Marine sediments impacted by urban and industrial pollutants are typically exposed to reducing conditions and represent major reservoirs of toxic mercury species. Mercury methylation mediated by anaerobic microorganisms is favored under such conditions, yet little is known about potential microbial mechanisms for mercury detoxification. We used culture-independent (metagenomics, metabarcoding) and culture-dependent approaches in anoxic marine sediments to identify microbial indicators of mercury pollution and analyze the distribution of genes involved in mercury reduction (merA) and demethylation (merB). While none of the isolates featured merB genes, 52 isolates, predominantly affiliated with Gammaproteobacteria, were merA positive. In contrast, merA genes detected in metagenomes were assigned to different phyla, including Desulfobacterota, Actinomycetota, Gemmatimonadota, Nitrospirota, and Pseudomonadota. This indicates a widespread capacity for mercury reduction in anoxic sediment microbiomes. Notably, merA genes were predominately identified in Desulfobacterota, a phylum previously associated only with mercury methylation. Marker genes involved in the latter process (hgcAB) were also mainly assigned to Desulfobacterota, implying a potential central and multifaceted role of this phylum in the mercury cycle. Network analysis revealed that Desulfobacterota were associated with anaerobic fermenters, methanogens and sulfur-oxidizers, indicating potential interactions between key players of the carbon, sulfur and mercury cycling in anoxic marine sediments.

Chitosan Films Incorporated with Exopolysaccharides from Deep Seawater Alteromonas Sp.

Two Alteromonas sp. strains isolated from deep seawater were grown to promote the production of exopolysaccharides (EPS, E611 and E805), which were incorporated into chitosan solutions to develop films. The combination of the major marine polysaccharides (chitosan and the isolated bacterial EPS) resulted in the formation of homogenous, transparent, colorless films, suggesting good compatibility between the two components of the film-forming formulation. With regards to optical properties, the films showed low values of gloss, in the range of 5-10 GU, indicating the formation of non-glossy and rough surfaces. In addition to the film surface, both showed hydrophobic character, with water contact angles higher than 100 º, regardless of EPS addition. Among the two EPS under analysis, chitosan films with E805 showed better mechanical performance, leading to resistant, flexible, easy to handle films.

Novel Vibrio spp. Strains Producing Omega-3 Fatty Acids Isolated from Coastal Seawater.

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA) (20:5n-3) and docosahexaenoic acid (DHA) (22:6n-3), are considered essential for human health. Microorganisms are the primary producers of omega-3 fatty acids in marine ecosystems, representing a sustainable source of these lipids, as an alternative to the fish industry. Some marine bacteria can produce LC-PUFAs de novo via the Polyunsaturated Fatty Acid (Pfa) synthase/ Polyketide Synthase (PKS) pathway, which does not require desaturation and elongation of saturated fatty acids. Cultivation-independent surveys have revealed that the diversity of microorganisms harboring a molecular marker of the pfa gene cluster (i.e., pfaA-KS domain) is high and their potential distribution in marine systems is widespread, from surface seawater to sediments. However, the isolation of PUFA producers from marine waters has been typically restricted to deep or cold environments. Here, we report a phenotypic and genotypic screening for the identification of omega-3 fatty acid producers in free-living bacterial strains isolated from 5, 500, and 1000 m deep coastal seawater from the Bay of Biscay (Spain). We further measured EPA production in pelagic Vibrio sp. strains collected at the three different depths. Vibrio sp. EPA-producers and non-producers were simultaneously isolated from the same water samples and shared a high percentage of identity in their 16S rRNA genes, supporting the view that the pfa gene cluster can be horizontally transferred. Within a cluster of EPA-producers, we found intraspecific variation in the levels of EPA synthesis for isolates harboring different genetic variants of the pfaA-KS domain. The maximum production of EPA was found in a Vibrio sp. strain isolated from a 1000 m depth (average 4.29% ± 1.07 of total fatty acids at 10 °C, without any optimization of culturing conditions).

Agentes etiológicos en pacientes con enfermedad diarreica aguda detectados por PCR en niños de 0 a 14 años de edad en el Hospital Metropolitano de Quito / Etiological agents in patients with acute diarrheal disease detected by PCR test in children from 0 to 14 years old in Hospital Metropolitano of Quito

Abstract: Objective: Determine the prevalence of gastrointestinal infections in children from 0 to 14 years old at Hospital Metropolitano of Quito from August 2017 to May 2018. Methods: A cross sectional study was carried out to determine the prevalence of gastrointestinal infections in children from 0 to 14 years old at Hospital Metropolitano of Quito. There were 58 patients with gastrointestinal infection from August 2017 to May 2018. We used the results from PCR gastrointestinal panel to determine the etiology of the disease. Results: We studied 58 patients with gastrointestinal infection between 0 to 14 years old. We found 79% of bacterial and 21% viral etiology. The most frequent agent was Clostridium difficile (15%) followed by enteropathogenic E. coli (14%), enteroagregative E. coli (12%). The most important virus was norovirus followed by rotavirus, and Giardia lamblia as a parasite. The most frequent coinfections were Clostridium difficile-Campylobacter, Clostridium difficile-enteropathogenic E. coli, enteropathogenic E. coli-enterotoxigenic E. coli. The months of the year where the greatest number of infections occurred were April and May. Conclusions: The etiology of gastrointestinal infections is a very important issue to study and manage because of its high incidence at the pediatric population, so a timely diagnosis and comprehensive management must be made.

Resumen: Objetivo: determinar la prevalencia de infecciones gastrointestinales en niños de 0 a 14 años que acudieron al Hospital Metropolitano de Quito durante el período comprendido entre agosto de 2017 y mayo de 2018. Método: estudio descriptivo transversal para determinar la prevalencia de infecciones gastrointestinales en niños de 0 a 14 años de edad en el Hospital Metropolitano de Quito. Se presentaron 58 casos de niños diagnosticados de infección gastrointestinal o gastroenteritis, durante un lapso de 10 meses desde agosto de 2017 hasta mayo de 2018. Para el diagnóstico etiológico se utilizaron los resultados del panel gastrointestinal por PCR. Resultados: se obtuvo 79% de infecciones bacterianas y 21% virales. La bacteria más frecuente fue el Clostridium difficile (15%) seguido por E. coli enteropatógena (14%) y E. coli enteroagregativa (12%). De los virus, el más frecuente fue el norovirus seguido por el rotavirus. De los parásitos, la Giardia lamblia. Las coinfecciones más frecuentes fueron causadas por Clostridium difficile-Campylobacter, Clostridium difficile-E. coli enteropatógena, E. coli enteropatógena-E. coli enterotoxigénica. La época del año de mayor incidencia de infecciones gastrointestinales abarcó abril y mayo. Conclusiones: la etiología de las infecciones gastrointestinales es un tema muy importante por su elevada incidencia en la población pediátrica, lo que motiva a realizar el diagnóstico y manejo integral oportunos

Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past.

A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain) allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100°C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30°C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions.

In Silico/In Vivo Insights into the Functional and Evolutionary Pathway of Pseudomonas aeruginosa Oleate-Diol Synthase. Discovery of a New Bacterial Di-Heme Cytochrome C Peroxidase Subfamily.

As previously reported, P. aeruginosa genes PA2077 and PA2078 code for 10S-DOX (10S-Dioxygenase) and 7,10-DS (7,10-Diol Synthase) enzymes involved in long-chain fatty acid oxygenation through the recently described oleate-diol synthase pathway. Analysis of the amino acid sequence of both enzymes revealed the presence of two heme-binding motifs (CXXCH) on each protein. Phylogenetic analysis showed the relation of both proteins to bacterial di-heme cytochrome c peroxidases (Ccps), similar to Xanthomonas sp. 35Y rubber oxidase RoxA. Structural homology modelling of PA2077 and PA2078 was achieved using RoxA (pdb 4b2n) as a template. From the 3D model obtained, presence of significant amino acid variations in the predicted heme-environment was found. Moreover, the presence of palindromic repeats located in enzyme-coding regions, acting as protein evolution elements, is reported here for the first time in P. aeruginosa genome. These observations and the constructed phylogenetic tree of the two proteins, allow the proposal of an evolutionary pathway for P. aeruginosa oleate-diol synthase operon. Taking together the in silico and in vivo results obtained we conclude that enzymes PA2077 and PA2078 are the first described members of a new subfamily of bacterial peroxidases, designated as Fatty acid-di-heme Cytochrome c peroxidases (FadCcp).

Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity.

Pseudomonas aeruginosa displays the ability to perform bioconversion of oleic acid into a class of hydroxylated fatty acids known as oxylipins. A diol synthase activity is responsible for such a conversion, which proceeds through the dioxygenation of oleic acid to release hydroperoxide 10-H(P)OME ((10S)-hydroxy-(8E)-octadecenoic acid), followed by conversion of the hydroperoxide intermediate into 7,10-DiHOME ((7S,10S)-dihydroxy-(8E)-octadecenoic acid), both of which accumulate in the culture supernatant. Several mutants of P. aeruginosa PAO1 were analyzed for the production of 10-H(P)OME and 7,10-DiHOME and two of them (ORFs PA2077 and PA2078), unable to release hydroxylated fatty acids, were detected and selected for further analysis. Involvement of ORFs PA2077 and PA2078 in oleate-diol synthase activity was confirmed, and their respective role in the conversion of oleic acid was analyzed by mutation complementation. Activity restoration revealed that gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction, whereas PA2078 encodes for the (7S,10S)-hydroperoxide diol synthase enzyme (7,10-DS) which allows the conversion of 10-H(P)OME into 7,10-DiHOME. Heterologous expression of both enzymes separately showed that no hetero-complex formation is required for enzymatic activity. Bioinformatics and RT-PCR analysis revealed that both genes constitute a new fine regulated oleate-diol synthase operon, originated by a gene duplication event followed by neofunctionalization for environmental adaptation, being unprecedented in prokaryotes.

Functional characterization of ExFadLO, an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa.

Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance.

Sensitization and exposure to house dust and storage mites in high-altitude areas of ecuador.

BACKGROUND: Few studies have addressed exposure and sensitization to mite allergens in Andean countries. OBJECTIVES: To identify the main mite species in 3 locations at different altitudes in Ecuador and to verify skin test reactivity to various mite species in allergic individuals in Quito, Ecuador. METHODS: Mattress dust samples were collected in Quito (2,800 m above sea level), Cuenca (2,500 m above sea level), and Guayaquil (sea level). Mite species present in the samples were isolated, identified, and counted. Der p 1 and Der f 1 levels were measured using monoclonal antibody-based enzyme immunoassays. Four hundred thirty-five patients in Quito diagnosed as having allergic rhinitis or asthma underwent skin testing with commercial extracts of Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Tyrophagus putrescentiae, and Lepidoglyphus destructor. In addition, Glycyphagus domesticus, Acarus siro, and Aleuroglyphus ovatus were tested in 362, 262, and 279 patients, respectively. RESULTS: Twenty-one mite species were identified. Large populations of mites were detected above 2,500 m of altitude. All the dust samples contained detectable levels of Der p 1 or Der f 1. Positive skin prick test reactions to D. pteronyssinus, D. farinae, B. tropicalis, L. destructor, T. putrescentiae, A. ovatus, A. siro, and G. domesticus were obtained in 60.9%, 56.8%, 17.0%, 19.3%, 10.6%, 15.8%, 8.8%, and 11.0% of the patients, respectively. CONCLUSIONS: Most analyzed mattresses contained several species of mites. Mite allergen levels were high. This study confirms the importance of house dust and storage mite allergens in Ecuador in areas above 2,500 m of altitude, where humidity remains high year round.

Owl monkey MHC-DRB exon 2 reveals high similarity with several HLA-DRB lineages.

One hundred and ten novel MHC-DRB gene exon 2 nucleotide sequences were sequenced in 96 monkeys from three owl monkey species (67 from Aotus nancymaae, 30 from Aotus nigriceps and 13 from Aotus vociferans). Owl monkeys, like humans, have high MHC-DRB allele polymorphism, revealing a striking similarity with several human allele lineages in the peptide binding region and presenting major convergence with DRB lineages from several Catarrhini (humans, apes and Old World monkeys) rather than with others New World monkeys (Platyrrhini). The parallelism between human and Aotus MHC-DRB reveals additional similarities regarding variability pattern, selection pressure and physicochemical constraints in amino acid replacements. These observations concerning previous findings of similarity between the Aotus immune system molecules and their human counterparts affirm this specie's usefulness as an excellent animal model in biomedical research.

Characterizing cellular immune response to kinetoplastid membrane protein-11 (KMP-11) during Leishmania (Viannia) panamensis infection using dendritic cells (DCs) as antigen presenting cells (APCs).

In vitro peptide binding assays and DCs pulsed with recombinant KMP-11 (rKMP-11) plus six 20-mer overlapping peptides covering the entire protein of Leishmania (Viannia) panamensis (L(V)p) promastigotes were used to identify T-cell epitopes in this protein. Such in vitro binding assays, using HLA DRB1* 0101, -0401, -0701 and -1101 alleles, demonstrated that two peptide sequences (DEEFNKKMQEQNAKFFADKP and FKHKFAELLEQQKAAQYPSK) exhibited high HLA DRB1* 0401 allele binding capacity. rKMP-11 specific T-cell proliferation and cytokine production, derived from 13 volunteers exposed to the parasite, suggested that using autologous DCs as APCs becomes advantageous in uncovering T-cell epitopes promoting proliferation and differences in IFN-gamma and IL-4 production in T-cells from volunteers with ACTIVE and CURED undetectable disease when other APCs were used. The two peptides which bound in vitro to the HLA DRB1* 0401 allele were immunogenic in HLA DRB1* 04 volunteers, thus validating the use of in vitro binding assays for predicting epitopes in this protein. The experimental approach used here may prove useful for characterizing T-cell epitopes in a protein useful in designing peptide-based vaccine candidates for Leishmania and other intracellular pathogens.

Asma en niños y adolescentes y su relación con aeroalergenos ambientales en Quito

En el presente estudio, se investigó la prevalencia de sensivilización a aeroalergenos en 473 niños y adolescentes asmáticos residentes en Quito, utilizando la técnica del prick test. Observamos un predominio de la sensibilización a ácaros del polvo. Dermatophagoides pteronyssinus y dermatophagoides farinae, sobre los otros aeroalergenos probados, tanto en el porcentaje de sensibilización (p<0.01), como en el tamaño de sus pápulas (p<0.01). El grupo con mayores sensibilizaciones fue el de asmáticos entre 11 y 15 años (D. pteronyssinus 86.6 por ciento, D. Farinae 85.8 por ciento, pelo de perro 40,2 por ciento, pelo de gato 35,4 por ciento. Los pólenes de gramíneas sensibilizaron más en el grupo de 16 a 20 años (27,8 por ciento). Los hombres estuvieron más sensibilizados (p<0.007) que las mujeres, a los ácaros del polvo (D. pteronyssinus 67.3 por ciento vs 55.2 por ciento y D. Farinae 65.5 por ciento vs 52.6 por ciento). En resumen, demostramos que los adolescentesasm'aticos son el grupo más predispuesto a presentar pruebas cutáneas positivas a aeroalergenos, en los asmáticos menores de 3 años ya encontramos sensibilizaciones y respuestas adecuadas a las pruebas cutáneas con aeroalergenos, y que los principales alergenos sinsibilizantes en el grupo de asmáticos niños y adolescentes, son los ácaros del polvo de casa D. pteronyssinus y D. farinae.