Detection of Shiga toxin-encoding genes in small community water supplies

Shiga toxin (Stx), one of the most potent bacterial toxins known, can cause bloody diarrhea, hemolytic uremic syndrome, kidney failure and death. The aim of this pilot was to investigate the occurrence of Shiga toxin-encoding genes, stx (stx1 and stx2) from total coliform (TC) and E. coli positive samples from small community water systems. After aliquots for TC and E. coli analyses were removed, the remnant volume of the samples was enriched, following a protocol developed for this study. Fifty-two per cent of the samples tested by multiplex PCR were positive for the presence of the stx genes; this percentage was higher in raw water samples. The stx2 gene was more abundant. Testing larger volumes of the samples increase the sensitivity of our assay, providing an alternative protocol for the detection of Shiga toxin-producing E. coli (STEC) that might be missed by the TC assay. This study confirms the presence of Stx encoding genes in source and distributed water for all systems sampled and suggests STEC as a potential health risk in small systems

Diversity of chemosynthetic thiosulfate oxidizing bacteria from diffuse flow hydrothermal vents and their role in mercury detoxification:

The mixing of hydrothermal fluids with seawater creates chemical, temperature, and pH gradients that support chemosynthetic primary production at deep-sea vents. These fluids are enriched with reduced sulfur compounds and their oxidation under aerobic conditions is considered the main chemosynthetic process at the vents. The main objective of my research was to gain a better understanding of the aerobic chemosynthetic thiosulfate oxidation processes at deep-sea hydrothermal vents, by studying the abundance and diversity of chemosynthetic thiosulfate oxidizing bacteria, and their role on the detoxification of heavy metals, with an emphasis on mercury detoxification. Fluids, sediments, and biomass from microbial colonization experiments were collected during several expeditions to the East Pacific Rise (EPR) at “9º50’N, 104º17’W” and to the Guaymas Basin, Gulf of California. Microbial isolations were carried out from diluted and undiluted samples. Isolates were identified by 16S rRNA gene analysis. The isolates obtained in pure cultures were related to the genera Thiomicrospira, Halothiobacillus, Hydrogenovibrio, Thioclava, Thalassospira, and Pelagibaca, as well as a new isolate EPR 70, which was described as a new species, Salinisphaera hydrothermalis. The isolates were further characterized, and their functional genes encoding enzymes for carbon fixation (RubisCO) and thiosulfate oxidation (SoxB) were analyzed. The Most Probable Number (MPN) technique was carried out in order to determine the abundance of chemosynthetic thiosulfate oxidizing bacteria, and the values obtained were compared with the total number of microorganisms per sample, estimated from microscopic direct counts. Our data show that this group of microorganisms represented from 103 to 107 cells per ml of sample, which accounts for about 0.002% to 14.1% of the total cell counts per sample. The chemical composition of the fluids was analyzed, and results indicated that hydrothermal fluids were enriched in mercury with concentrations comparable to the concentrations found in contaminated surface waters. MPN counts were done with the addition of mercury in order to determine the percentage of chemosynthetic thiosulfate oxidizing bacteria that were mercury resistant. Results indicated that from 0.2 to 24.6% of the chemosynthetic bacteria were resistant, suggesting an adaptation to life in the presence of this toxic metal.Ph.D.Includes bibliographical references (p. 89-98)by Melitza Crespo-Medin

The Family Salinisphaeraceae

The family Salinisphaeraceae (Class Gammaproteobacteria, Order Salinisphaerales) comprises a single genus, Salinisphaera, and six species: S. shabanensis, S. hydrothermalis, S. dokdonensis, S. orenii, S. halophila, and S. japonica. All members of the family Salinisphaeraceae were isolated from marine/oceanic and high-salinity environments. These bacteria have coccoid or short rod morphologies and are halophilic or halotolerant. All known members of the family Salinisphaeraceae are heterotrophic, mesophilic aerobes, although S. hydrothermalis was shown to be a facultative chemolithoautotroph. Isolation and characterization of new members of the Salinisphaeraceae, as well as in-depth studies of the currently known species, will allow for a better understanding of this family.(undefined

Methane Dynamics in a Tropical Serpentinizing Environment: The Santa Elena Ophiolite, Costa Rica

Uplifted ultramafic rocks represent an important vector for the transfer of carbon and reducing power from the deep subsurface into the biosphere and potentially support microbial life through serpentinization. This process has a strong influence upon the production of hydrogen and methane, which can be subsequently consumed by microbial communities. The Santa Elena Ophiolite (SEO) on the northwestern Pacific coast of Costa Rica comprises ∼250 km2 of ultramafic rocks and mafic associations. The climatic conditions, consisting of strongly contrasting wet and dry seasons, make the SEO a unique hydrogeological setting, where water-rock reactions are enhanced by large storm events (up to 200 mm in a single storm). Previous work on hyperalkaline spring fluids collected within the SEO has identified the presence of microorganisms potentially involved in hydrogen, methane, and methanol oxidation (such as Hydrogenophaga, Methylobacterium, and Methylibium spp., respectively), as well as the presence of methanogenic Archaea (such as Methanobacterium). Similar organisms have also been documented at other serpentinizing sites, however their functions have not been confirmed. SEO’s hyperalkaline springs have elevated methane concentrations, ranging from 145 to 900 µM, in comparison to the background concentrations (<0.3 µM). The presence and potential activity of microorganisms involved in methane cycling in serpentinization-influenced fluids from different sites within the SEO were investigated using molecular, geochemical, and modeling approaches. These results were combined to elucidate the bioenergetically favorable methane production and/or oxidation reactions in this tropical serpentinizing environment. The hyperalkaline springs at SEO contain a greater proportion of Archaea and methanogens than has been detected in any terrestrial serpentinizing system. Archaea involved in methanogenesis and anaerobic methane oxidation accounted from 40 to 90% of total archaeal sequences. Genes involved in methanogenic metabolisms were detected from the metagenome of one of the alkaline springs. Methanogenic activities are likely to be facilitated by the movement of nutrients, including dissolved inorganic carbon (DIC), from surface water and their infiltration into serpentinizing groundwater. These data provide new insight into methane cycle in tropical serpentinizing environments.Las rocas ultramáficas levantadas representan un vector importante para la transferencia de carbono y el poder reductor desde el subsuelo profundo hacia la biosfera y potencialmente sustentan la vida microbiana a través de la serpentinización. Este proceso tiene una fuerte influencia en la producción de hidrógeno y metano, que posteriormente pueden ser consumidos por las comunidades microbianas. La ofiolita de Santa Elena (SEO) en la costa noroeste del Pacífico de Costa Rica comprende ~250 km2 de rocas ultramáficas y asociaciones máficas. Las condiciones climáticas, consistentes en estaciones húmedas y secas fuertemente contrastadas, hacen del SEO un escenario hidrogeológico único, donde las reacciones agua-roca se ven potenciadas por grandes tormentas (hasta 200 mm en una sola tormenta). Trabajos previos sobre fluidos de manantiales hiperalcalinos recolectados dentro de la SEO han identificado la presencia de microorganismos potencialmente involucrados en la oxidación de hidrógeno, metano y metanol (como Hydrogenophaga, Methylobacterium y Mthylibium spp., respectivamente), así como la presencia de Archaea metanogénica ( como Methanobacterium). También se han documentado organismos similares en otros sitios de serpentinización, sin embargo, sus funciones no han sido confirmadas. Los manantiales hiperalcalinos de SEO tienen concentraciones elevadas de metano, que van desde 145 a 900 µM, en comparación con las concentraciones de fondo (<0,3 µM). Se investigó la presencia y la actividad potencial de los microorganismos involucrados en el ciclo del metano en fluidos influenciados por la serpentinización de diferentes sitios dentro del SEO utilizando enfoques moleculares, geoquímicos y de modelado. Estos resultados se combinaron para dilucidar la producción bioenergéticamente favorable de metano y/o las reacciones de oxidación en este ambiente serpentinizante tropical. Los manantiales hiperalcalinos en SEO contienen una mayor proporción de Archaea y metanógenos de lo que se ha detectado en cualquier sistema serpentinizante terrestre. Las arqueas involucradas en la metanogénesis y la oxidación anaeróbica de metano representaron del 40 al 90% del total de secuencias de arqueas. Se detectaron genes implicados en metabolismos metanogénicos a partir del metagenoma de uno de los manantiales alcalinos. Es probable que las actividades metanogénicas se vean facilitadas por el movimiento de nutrientes, incluido el carbono inorgánico disuelto (DIC), desde las aguas superficiales y su infiltración en las aguas subterráneas serpentinizantes. Estos datos brindan una nueva perspectiva del ciclo del metano en ambientes serpentinizantes tropicales.Universidad Nacional, Costa RicaEscuela de Químic

Characterization of Per- and Polyfluoroalkyl Substances (PFAS) and Other Constituents in MSW Landfill Leachate from Puerto Rico

Elevated per- and polyfluoroalkyl substance (PFAS) concentrations have been reported in municipal solid waste (MSW) landfill leachate with higher levels in wet and warmer subtropical climates. Information about landfill leachate characteristics is much more limited in tropical climates. In this study, 20 landfill leachate samples were collected from three MSW landfills on the tropical island of Puerto Rico and results were compared against landfills nationally and within Florida, USA. The samples collected in Puerto Rico underwent physical-chemical analysis, as well as a quantitative analysis of 92 PFAS. Samples described in this study include discrete leachate types, such as leachate, gas condensate, and leachate which has undergone on-site treatment (e.g., RO treatment, phytoremediation, lagoons). A total of 51 PFAS were detected above quantitation limits, including perfluorohexylphosphonic acid, a perfluoroalkyl acid (PFAA) which has not been reported previously in landfill leachate. ∑PFAS concentrations in this study (mean: 38,000 ng L ), as well as concentrations of individual PFAS, are significantly higher than other reported MSW landfill leachate concentrations. The profiles of leachates collected from on-site treatment systems indicate possible transformation of precursor PFAS as a result of treatment processes - oxidizing conditions, for example, may facilitate aerobic transformation, increase the concentrations of PFAAs, and possibly increase the apparent ∑PFAS concentration. Extreme climate events, including rising temperatures and more frequent hurricanes, have placed additional strain on the solid waste management infrastructure on the island - adding complexity to an already challenging PFAS management issue. As concern grows over PFAS contamination in drinking water, these findings should inform solid waste and leachate management decisions in order to minimize PFAS emissions in island environments

Deep-sea hydrothermal vent Epsilonproteobacteria encode a conserved and widespread nitrate reduction pathway (Nap)

Despite the frequent isolation of nitrate-respiring Epsilonproteobacteria from deep-sea hydrothermal vents, the genes coding for the nitrate reduction pathway in these organisms have not been investigated in depth. In this study we have shown that the gene cluster coding for the periplasmic nitrate reductase complex (nap) is highly conserved in chemolithoautotrophic, nitrate-reducing Epsilonproteobacteria from deep-sea hydrothermal vents. Furthermore, we have shown that the napA gene is expressed in pure cultures of vent Epsilonproteobacteria and it is highly conserved in microbial communities collected from deep-sea vents characterized by different temperature and redox regimes. The diversity of nitrate-reducing Epsilonproteobacteria was found to be higher in moderate temperature, diffuse flow vents than in high temperature black smokers or in low temperatures, substrate-associated communities. As NapA has a high affinity for nitrate compared with the membrane-bound enzyme, its occurrence in vent Epsilonproteobacteria may represent an adaptation of these organisms to the low nitrate concentrations typically found in vent fluids. Taken together, our findings indicate that nitrate reduction is widespread in vent Epsilonproteobacteria and provide insight on alternative energy metabolism in vent microorganisms. The occurrence of the nap cluster in vent, commensal and pathogenic Epsilonproteobacteria suggests that the ability of these bacteria to respire nitrate is important in habitats as different as the deep-sea vents and the human body