Biocatalytic quantification of α-glucan in particulate marine organic matter

Marine algae drive the marine carbon cycle, converting carbon dioxide into organic material. A major component of this produced biomass is a variety of glycans; and yet their chemical composition and individual involvement in production, sedimentation and bacterial uptake remain largely unknown due to a lack of analytical tools for glycan-specific quantification. Marine α-glucans include a range of storage glycans from red and green algae, bacteria, fungi and animals. Although these compounds are likely to account for a high amount of the carbon stored in the oceans they have not been quantified in marine samples so far. Here we present a method to extract and quantify α-glucans (and compare it with the β-glucan laminarin) in particulate organic matter from algal cultures and environmental samples using a sequential physicochemical extraction and enzymes as α-glucan-specific probes. This enzymatic assay is more specific and less susceptible to side reactions than chemical hydrolysis. Using HPAEC-PAD to detect the hydrolysis products allows for a glycan quantification in particulate marine samples down to a concentration of ≈ 2 μg/L. We measured glucans in three cultured microalgae as well as in marine particulate organic matter from the North Sea and western North Atlantic Ocean. While the β-glucan laminarin from diatoms and brown algae is an essential component of marine carbon turnover, our results further indicate the significant contribution of starch-like α-glucans to marine particulate organic matter. Henceforth, the combination of glycan-linkage-specific enzymes and chromatographic hydrolysis product detection can provide a powerful tool in the exploration of marine glycans and their role in the global carbon cycle.

Species-specific reservoir effect estimates: A case study of archaeological marine samples from the Bering Strait

Due to the marine reservoir effect, radiocarbon dates of marine samples require a correction. Marine reservoir effects, however, may vary among different marine species within a given body of water. Factors such as diet, feeding depth and migratory behaviour all affect the 14C date of a marine organism. Moreover, there is often significant variation within single marine species. Whilst the careful consideration of the Δ R values of a single marine species in a given location is important, so too is the full range of Δ R values within an ecosystem. This paper illustrates this point, using a sample pairing method to estimate the reservoir effects in 17 marine samples, of eight different species, from the archaeological site of Ekven (Eastern Chukotka, Siberia). An OxCal model is used to assess the strength of these estimates. The marine reservoir effects of samples passing the model range from Δ R (Marine20) = 136 ± 41–Δ R = 460 ± 40. Marine reservoir effect estimates of these samples and other published samples are used to explore variability in the wider Bering Strait region. The archaeological implications of this variability are also discussed. The calibrating of 14C dates from human bone collagen, for example, could be improved by applying a dietary relevant marine reservoir effect correction. For humans from the site of Ekven, a Δ R (Marine20) correction of 289 ± 124 years or reservoir age correction of 842 ± 123 years is suggested.

A Toolbox for the Determination of Nitroaromatic Explosives in Marine Water, Sediment, and Biota Samples on Femtogram Levels by GC-MS/MS

To determine the amount of the explosives 1,3-dinitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and its metabolites in marine samples, a toolbox of methods was developed to enhance sample preparation and analysis of various types of marine samples, such as water, sediment, and different kinds of biota. To achieve this, established methods were adapted, improved, and combined. As a result, if explosive concentrations in sediment or mussel samples are greater than 10 ng per g, direct extraction allows for time-saving sample preparation; if concentrations are below 10 ng per g, techniques such as freeze-drying, ultrasonic, and solid-phase extraction can help to detect even picogram amounts. Two different GC-MS/MS methods were developed to enable the detection of these explosives in femtogram per microliter. With a splitless injector, limits of detection (LODs) between 77 and 333 fg/µL could be achieved in only 6.25 min. With the 5 µL programmable temperature vaporization—large volume method (PTV-LVI), LODs between 8 and 47 fg/µL could be achieved in less than 7 min. The detection limits achieved by these methods are among the lowest published to date. Their reliability has been tested and confirmed by measuring large and diverse sample sets.

Linezolid resistance genes in enterococci isolated from sediment and zooplankton in two Italian coastal areas

Linezolid is a last resort antibiotic for the treatment of severe infections caused by multi-resistant Gram-positives; although linezolid resistance remains uncommon, the number of linezolid-resistant enterococci has increased during recent years due to worldwide spread of acquired resistance genes (cfr, optrA and poxtA) in clinical, animal and environmental setting. In this study we investigated the occurrence of linezolid-resistant enterococci in marine samples from two coastal areas in Italy. Isolates grown on florfenicol-supplemented Slanetz-Bartley were investigated for their carriage of optrA, poxtA and cfr genes: optrA was found in one E. faecalis, poxtA in three E. faecium and two E. hirae and cfr was not found. Two of the three poxtA-carrying E. faecium and the two E. hirae showed related PFGE profiles. Two E. faecium belonged to the new ST1710, which clustered in the clonal complex CC94, encompassing nosocomial strains. S1-PFGE/hybridization assays showed a double (chromosome and plasmid) location of poxtA and plasmid location of optrA. WGS revealed that poxtA was contained in a Tn6657-like element carried by two plasmids (pEfm-EF3 and pEh-GE2) of similar size, found in different species, and that poxtA were flanked by two copies of IS1216 in both plasmids. In mating experiments all but one (E. faecalis EN3) strains were able to transfer the poxtA gene to E. faecium 64/3. The occurrence of linezolid resistance genes in enterococci from marine samples is of great concern and highlights the need to improve practices aimed at limiting the transmission of linezolid resistant strains to humans from the environmental reservoirs. Importance Linezolid is one of the few antimicrobials available to treat severe infections due to drug-resistant Gram-positive bacteria, thus the emergence of linezolid-resistant enterococci carrying transferable resistance determinants is of great concern for public health. Linezolid resistance genes (cfr, optrA and poxtA), often plasmid located, can be transmitted via horizontal gene transfer and have the potential to spread globally. This study highlights the first detection of enterococci carrying linezolid resistance genes from sediment and zooplankton samples in two coastal urban areas in Italy. The presence of clinically relevant resistant bacteria, such as linezolid-resistant enterococci, in marine environment could reflect their spillover from human and/or animal reservoirs and could indicate that also coastal seawaters could represent a source of these resistance genes.