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Cell-penetrating peptides (CPPs) are highly promising transfection agents that can deliver various compounds into living cells, including nucleic acids (NAs). Positively charged CPPs can form non-covalent complexes with negatively charged NAs, enabling simple and time-efficient nanoparticle preparation. However, as CPPs have substantially different chemical and physical properties, their complexation with the cargo and characteristics of the resulting nanoparticles largely depends on the properties of the surrounding environment, i.e., solution. Here, we show that the solvent used for the initial dissolving of a CPP determines the properties of the resulting CPP particles formed in an aqueous solution, including the activity and toxicity of the CPP-NA complexes. Using different biophysical methods such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission and scanning electron microscopy (TEM and SEM), we show that PepFect14 (PF14), a cationic amphipathic CPP, forms spherical particles of uniform size when dissolved in organic solvents, such as ethanol and DMSO. Water-dissolved PF14, however, tends to form micelles and non-uniform aggregates. When dissolved in organic solvents, PF14 retains its α-helical conformation and biological activity in cell culture conditions without any increase in cytotoxicity. Altogether, our results indicate that by using a solvent that matches the chemical nature of the CPP, the properties of the peptide-cargo particles can be tuned in the desired way. This can be of critical importance for in vivo applications, where CPP particles that are too large, non-uniform, or prone to aggregation may induce severe consequences.

Ballast water is a main vector of introduction of potentially harmful or pathogenic aquatic organisms. The development of genetic tools for ballast water monitoring has been underway and highlighted as a source for accurate and reliable data for decision making. We used 16S rRNA gene amplicon sequencing to analyze the microbial communities found in the ballast water of fifteen commercial ships routed through two Estonian ports. In parallel, samples from the port area were collected at the same time each ship visited. Fluorescence microscopy was utilized to assess the effectiveness of the treatment applied to ballast water. In addition, supplemental samples were collected from Hamburg Port (Germany) and a ballast tank decontamination system used at this port. The composition and diversity of bacterial communities varied greatly between obtained samples. The application of UV treatment did not demonstrate significant reduction in species richness estimates. The composition of microbial communities was significantly influenced by salinity, treatment (mainly untreated or UV treated) and the point of origin of the ballast water. Over a hundred potentially pathogenic bacterial taxa were found in relatively high abundance, including in ballast water that had received UV treatment. These shortcomings of stand-alone UV treatment of ballast water, especially when weak treatment is applied insufficiently, highlight the danger of possible harmful effects arising over time and the need for genetic tools for ballast water monitoring and management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Nanomaterials are prospective candidates for the elimination of viruses due to their multimodal mechanisms of action. Here, we tested the antiviral potential of a largely unexplored nanoparticle of cerium dioxide (CeO 2 ). Two nano-CeO 2 with opposing surface charge, (+) and (-), were assessed for their capability to decrease the plaque forming units (PFU) of four enveloped and two non-enveloped viruses during 1-h exposure. Statistically significant antiviral activity towards enveloped coronavirus SARS-CoV-2 and influenza virus was registered already at 20 mg Ce/l. For other two enveloped viruses, transmissible gastroenteritis virus and bacteriophage φ6, antiviral activity was evidenced at 200 mg Ce/l. As expected, the sensitivity of non-enveloped viruses towards nano-CeO 2 was significantly lower. EMCV picornavirus showed no decrease in PFU until the highest tested concentration, 2000 mg Ce/l and MS2 bacteriophage showed slight non-monotonic response to high concentrations of nano-CeO 2 (-). Parallel testing of antiviral activity of Ce 3+ ions and SiO 2 nanoparticles allows to conclude that nano-CeO 2 activity was neither due to released Ce-ions nor nonspecific effects of nanoparticulates. Moreover, we evidenced higher antiviral efficacy of nano-CeO 2 compared with Ag nanoparticles. This result along with low antibacterial activity and non-existent cytotoxicity of nano-CeO 2 allow us to propose CeO 2 nanoparticles for specific antiviral applications., (© 2022. The Author(s).)

Environmental disturbances influence bacterial community structure and functioning. To investigate the effect of environmental disturbance caused by changes in salinity on host-protected bacterial communities, we analyzed the microbiome within the gastrointestinal tract of Ampullaceana balthica in different salinities. A. balthica is a benthic gastropod found in fresh- and mesohaline waters. Whereas the total energy reserves of A. balthica were unaffected by an increase of salinity to 3, a high mortality rate was detected after a shift from freshwater to salinity 6 suggesting a major disruption of energy homeostasis. The shift to salinity 6 also caused a change in the gastrointestinal bacterial community composition. At salinity 3, the bacterial community composition of different host individuals was related either to the freshwater or salinity 6 gastrointestinal bacterial community, indicating an ambivalent nature of salinity 3. Since salinity 3 represents the range where aquatic gastropods are able to regulate their osmolarity, this may be an important tipping point during salinization. The change in the intestinal microbiome was uncoupled from the change in the water bacterial community and unrelated to the food source microbiome. Our study shows that environmental disturbance caused by salinity acts also on the host-protected microbiome. In light of the sea-level rise, our findings indicate that salinization of the near-shore freshwater bodies will cause changes in organisms' intestinal microbiomes if a critical salinity threshold (presumably ∼3) is exceeded., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kivistik, Käiro, Tammert, Sokolova, Kisand and Herlemann.)

Poultry meat is considered the most important source of Campylobacter spp. Because of rising antimicrobial resistance in Campylobacter spp., this study investigated the antimicrobial resistance of Campylobacter isolates from fresh broiler chicken meat originating from the Baltic countries sold in Estonian retail settings. Additionally, human clinical isolates obtained from patients with Campylobacter enteritis in Estonia were analysed. The aim of this study was to investigate the susceptibility of Campylobacter spp. to nalidixic acid, ciprofloxacin, tetracycline, streptomycin, erythromycin and gentamicin. The broth microdilution method with the EUCAMP2 panel was used for MIC determination and antimicrobial mechanisms were analysed using WGS data. A total of 46 Campylobacter strains were analysed, of which 26 (42.6%) originated from Lithuanian, 16 (26.2%) from Latvian, and 4 (6.6%) from Estonian fresh broiler chicken meat. In addition, 15 (24.6%) Campylobacter strains of patients with campylobacteriosis were tested. The antimicrobial resistance patterns of Campylobacter spp. isolated from fresh broiler chicken meat samples of Estonian, Latvian and Lithuanian origin collected in Estonian retail, and from patients with Campylobacter enteric infections, were determined. A total of 46 (75%) of the isolates tested were C. jejuni and 15 (25%) were C. coli . Campylobacter resistance was highest to nalidixic acid (90.2% of strains) and ciprofloxacin (90.2%), followed by tetracycline (57.4%), streptomycin (42.6%) and erythromycin (6.6%). All strains were sensitive to gentamicin. Additionally, antimicrobial resistance genes and point mutations were detected in 27 C. jejuni and 8 C. coli isolates previously assigned as resistant with the phenotypic method. A high antibiotic resistance of Campylobacter spp. in Lithuanian- and Latvian-origin broiler chicken meat and Estonian clinical isolates was found. Similar antibiotic resistance patterns were found for broiler chicken meat and human Campylobacter isolates.

Since 2005 campylobacteriosis has been the most commonly reported gastrointestinal infection in humans in the European Union with more than 200,000 cases annually. Also Campylobacter is one of the most frequent cause of food-borne outbreaks with 319 outbreaks reported to EFSA, involving 1,254 cases of disease and 125 hospitalizations in EU in 2019. Importantly poultry meat is one of the most common source for the sporadic Campylobacter infections and for strong-evidence campylobacteriosis food-borne outbreaks in EU. In present study, 429 fresh broiler chicken meat samples of Estonian, Latvian, and Lithuanian origin were collected from Estonian retail level and analyzed on a monthly basis between September 2018 and October 2019. Campylobacter spp. were isolated in 141 (32.9%) of 429 broiler chicken meat samples. Altogether 3 (1.8%), 49 (36.8%), and 89 (66.9%) of Estonian, Latvian, and Lithuanian origin broiler chicken meat samples were positive for Campylobacter spp. Among Campylobacter-positive samples, 62 (14.5%) contained Campylobacter spp. below 100 CFU/g and in 28 (6.5%) samples the count of Campylobacter spp. exceeded 1,000 CFU/g. A high prevalence of Campylobacter spp. in fresh broiler chicken meat of Lithuanian and Latvian origin in Estonian retail was observed. Additionally, 22 different multilocus sequence types were identified among 55 genotyped isolates of broiler chicken meat and human origin, of which 45 were Campylobacter jejuni (C. jejuni) and 10 were Campylobacter coli (C. coli). The most prevalent multilocus sequence types among C. jejuni was ST2229 and among C. coli ST832, ST872. C. jejuni genotypes found in both broiler chicken meat and human origin samples were ST122, ST464, ST7355, and ST9882, which indicates that imported fresh broiler chicken meat is likely the cause of human campylobacteriosis in Estonia., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In the context of healthcare-acquired infections, microbial cross-contamination and the spread of antibiotic resistance, additional passive measures to prevent pathogen carryover are urgently needed. Antimicrobial high-touch surfaces that kill microbes on contact or prevent their adhesion could be considered to mitigate the spread. Here, we demonstrate that photocatalytic nano-ZnO- and nano-ZnO/Ag-based antibacterial surfaces with efficacy of at least a 2.7-log reduction in Escherichia coli and Staphylococcus aureus viability in 2 h can be produced by simple measures using a commercial acrylic topcoat for wood surfaces. We characterize the surfaces taking into account cyclic wear and variable environmental conditions. The light-induced antibacterial and photocatalytic activities of the surfaces are enhanced by short-term cyclic wear, indicating their potential for prolonged effectivity in long-term use. As the produced surfaces are generally more effective at higher relative air humidity and silver-containing surfaces lost their contact-killing properties in dry conditions, it is important to critically evaluate the end-use conditions of materials and surfaces to be tested and select application-appropriate methods for their efficacy assessment.

The hydration of phospholipids, electrospun into polymeric nanofibers and used as templates for liposome formation, offers pharmaceutical advantages as it avoids the storage of liposomes as aqueous dispersions. The objective of the present study was to electrospin and characterize amphiphilic nanofibers as templates for the preparation of antibiotic-loaded liposomes and compare this method with the conventional film-hydration method followed by extrusion. The comparison was based on particle size, encapsulation efficiency and drug-release behavior. Chloramphenicol (CAM) was used at different concentrations as a model antibacterial drug. Phosphatidylcoline (PC) with polyvinylpyrrolidone (PVP), using ethanol as a solvent, was found to be successful in fabricating the amphiphilic composite drug-loaded nanofibers as well as liposomes with both methods. The characterization of the nanofiber templates revealed that fiber diameter did not affect the liposome size. According to the optical microscopy results, the immediate hydration of phospholipids deposited on the amphiphilic nanofibers occurred within a few seconds, resulting in the formation of liposomes in water dispersions. The liposomes appeared to aggregate more readily in the concentrated than in the diluted solutions. The drug encapsulation efficiency for the fiber-hydrated liposomes varied between 14.9 and 28.1% and, for film-hydrated liposomes, between 22.0 and 77.1%, depending on the CAM concentrations and additional extrusion steps. The nanofiber hydration method was faster, as less steps were required for the in-situ liposome preparation than in the film-hydration method. The liposomes obtained using nanofiber hydration were smaller and more homogeneous than the conventional liposomes, but less drug was encapsulated.

Non-precious-metal catalysts are promising alternatives for Pt-based cathode materials in low-temperature fuel cells, which is of great environmental importance. Here, we have investigated the bifunctional electrocatalytic activity toward the oxygen reduction reaction (ORR) and the oxygen evolution reaction (OER) of mixed metal (FeNi; FeMn; FeCo) phthalocyanine-modified multiwalled carbon nanotubes (MWCNTs) prepared by a simple pyrolysis method. Among the bimetallic catalysts containing nitrogen derived from corresponding metal phthalocyanines, we report the excellent ORR activity of FeCoN-MWCNT and FeMnN-MWCNT catalysts with the ORR onset potential of 0.93 V and FeNiN-MWCNT catalyst for the OER having E = 1.58 V at 10 mA cm OER = 1.58 V at 10 mA cm -2 . The surface morphology, structure, and elemental composition of the prepared catalysts were examined with scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The FeCoN-MWCNT and FeMnN-MWCNT catalysts were prepared as cathodes and tested in anion-exchange membrane fuel cells (AEMFCs). Both catalysts displayed remarkable AEMFC performance with a peak power density as high as 692 mW cm -2 for FeCoN-MWCNT.

Phage predation constitutes a major mortality factor for bacteria in aquatic ecosystems, and thus, directly impacts nutrient cycling and microbial community dynamics. Yet, the population dynamics of specific phages across time scales from days to months remain largely unexplored, which limits our understanding of their influence on microbial succession. To investigate temporal changes in diversity and abundance of phages infecting particular host strains, we isolated 121 phage strains that infected three bacterial hosts during a Baltic Sea mesocosm experiment. Genome analysis revealed a novel Flavobacterium phage genus harboring gene sets putatively coding for synthesis of modified nucleotides and glycosylation of bacterial cell surface components. Another novel phage genus revealed a microdiversity of phage species that was largely maintained during the experiment and across mesocosms amended with different nutrients. In contrast to the newly described Flavobacterium phages, phages isolated from a Rheinheimera strain were highly similar to previously isolated genotypes, pointing to genomic consistency in this population. In the mesocosm experiment, the investigated phages were mainly detected after a phytoplankton bloom peak. This concurred with recurrent detection of the phages in the Baltic Proper during summer months, suggesting an influence on the succession of heterotrophic bacteria associated with phytoplankton blooms., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Microplastic (MPL) contamination in the marine environment is extensively studied yet little is known about the extent of MPL abundance in seagrass beds. The aim of this study was to evaluate MPL accumulation in coastal seagrass (Zostera marina) beds in the Baltic Sea, Estonia. Surface water was sampled by pumping using 40 μm plankton net, and sediments by trowel. MPL was extracted with NaCl, identified by microscopy and ATR-FTIR on selected samples. Surface water in the seagrass beds had 0.04-1.2 (median 0.14) MPL/L, similar to other areas of the Baltic Sea. Sediments had 0-1817 (median 208) MPL/kg (dwt), much higher than previously recorded from adjacent unvegetated and offshore sediments, thereby suggesting a strong ability of the sediments in seagrass beds to retain MPL. Of identified MPL, blue fibres were dominant in both the sampled media. Sediment characterization showed a correlation between MPL counts with poorly sorted sediments., (Copyright © 2021. Published by Elsevier Ltd.)

Fungi are ecologically important in several ecosystem processes, yet their community composition, ecophysiological roles, and responses to changing environmental factors in historical sediments are rarely studied. Here we explored ancient fungal DNA from lake Lielais Svētiņu sediment throughout the Holocene (10.5 kyr) using the ITS metabarcoding approach. Our data revealed diverse fungal taxa and smooth community changes during most of the Holocene with rapid changes occurring in the last few millennia. More precisely, plankton parasitic fungi became more diverse from the Late Holocene (2-4 kyr) which could be related to a shift towards a cooler climate. The Latest Holocene (~2 kyr) showed a distinct increase in the richness of plankton parasites, mycorrhizal, and plant pathogenic fungi which can be associated with an increased transfer rate of plant material into the lake and blooms of planktonic organisms influenced by increased, yet moderate, human impact. Thus, major community shifts in plankton parasites and mycorrhizal fungi could be utilized as potential paleo-variables that accompany host-substrate dynamics. Our work demonstrates that fungal aDNA with predicted ecophysiology and host specificity can be employed to reconstruct both aquatic and surrounding terrestrial ecosystems and to estimate the influence of environmental change.

In this study, we aimed to characterize the population structure, drug resistance mechanisms, and virulence genes of Enterococcus isolates in Estonia. Sixty-one Enterococcus faecalis and 34 Enterococcus faecium isolates were collected between 2012 and 2014 across the country from various sites and sources, including farm animals and poultry ( n = 53), humans ( n = 12), environment ( n = 24), and wild birds ( n = 44). Clonal relationships of the strains were determined by whole-genome sequencing and analyzed by multi-locus sequence typing. We determined the presence of acquired antimicrobial resistance genes and 23S rRNA mutations, virulence genes, and also the plasmid or chromosomal origin of the genes using dedicated DNA sequence analysis tools available and/or homology search against an ad hoc compiled database of relevant sequences. Two E. faecalis isolates from human with vanB genes were highly resistant to vancomycin. Closely related E. faecalis strains were isolated from different host species. This indicates interspecies spread of strains and potential transfer of antibiotic resistance. Genomic context analysis of the resistance genes indicated frequent association with plasmids and mobile genetic elements. Resistance genes are often present in the identical genetic context in strains with diverse origins, suggesting the occurrence of transfer events., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Aun, Kisand, Laht, Telling, Kalmus, Väli, Brauer, Remm and Tenson.)

The effects of microplastics (MP) are extensively studied, yet hazard data from long-term exposure studies are scarce. Moreover, for sustainable circular use in the future, knowledge on the biological impact of recycled plastics is essential. The aim of this study was to provide long-term toxicity data of virgin vs recycled (mechanical recycling) low density polyethylene (LDPE) for two commonly used ecotoxicity models, the freshwater crustacean Daphnia magna and the terrestrial crustacean Porcellio scaber . LDPE MP was tested as fragments of 39.8 ± 8.82 µm (virgin) and 205 ± 144 µm (recycled) at chronic exposure levels of 1-100 mg LDPE/L ( D. magna ) and 0.2-15 g LDPE/kg soil ( P. scaber ). Mortality, reproduction, body length, total lipid content, feeding and immune response were evaluated. With the exception of very low inconsistent offspring mortality at 10 mg/L and 100 mg/L of recycled LDPE, no MP exposure-related adverse effects were recorded for D. magna . For P. scaber , increased feeding on non-contaminated leaves was observed for virgin LDPE at 5 g/kg and 15 g/kg. In addition, both LDPE induced a slight immune response at 5 g/kg and 15 g/kg with more parameters altered for virgin LDPE. Our results indicated different sublethal responses upon exposure to recycled compared to virgin LDPE MP.

Ultraviolet photoelectron spectroscopy (UPS) investigations of several gas-phase ionic liquid (IL) ion pairs have been conducted. [EMIM][OTF], [PYR 14 ][OTF], [EMIM][DCA], [PYR 14 ][DCA], [PYR 14 ][TCM], [PYR 14 ][FSI], [PYR 14 ][PF 6 ], [S 222 ][TFSI], [P 4441 ][TFSI], and [EMMIM][TFSI] vapor UPS spectra are presented for the first time. The experimental low-binding-energy cutoff value (highest occupied molecular orbital, HOMO energy) of the ionic liquid ion pairs, which is of great interest, has been measured. Many studies use calculated gas-phase electronic properties to estimate the liquid-phase electrochemical stability. Hybrid density functional theory (DFT) calculations have been used to interpret the experimental data. The gas-phase photoelectron spectra in conjunction with the theoretical calculations are able to verify most HOMO energies and assign them to the cation or anion. The hybrid M06 functional is shown to offer a very good description of the ionic liquid electronic structure. In some cases, the excellent agreement between the UPS spectra and the M06 calculation validates the conformer found and constitutes as a first indirect experimental determination of ionic liquid ion-pair structure. Comparisons with recent theoretical studies are made, and implications for electrochemical applications are discussed. The new data provide a much-needed reference for future ab initio calculations and support the argument that modeling of IL cations and anions separately is incorrect., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Highly active electrocatalysts for electrochemical oxygen reduction reaction (ORR) were prepared by high-temperature pyrolysis from 5-methylresorcinol, Co and/or Fe salts and dicyandiamide, which acts simultaneously as a precursor for reactive carbonitride template and a nitrogen source. The electrocatalytic activity of the catalysts for ORR in alkaline solution was studied using the rotating disc electrode (RDE) method. The bimetallic catalyst containing iron and cobalt (FeCoNC-at) showed excellent stability and remarkable ORR performance, comparable to that of commercial Pt/C (20 wt%). The superior activity was attributed to high surface metal and nitrogen contents. The FeCoNC-at catalyst was further tested in anion exchange membrane fuel cell (AEMFC) with poly-(hexamethyl-p-terphenylbenzimidazolium) (HMT-PMBI) membrane, where a high value of peak power density (P max  = 415 mW cm -2 ) was achieved., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In this work He/H 2 plasma jet treatment was used to reduce Pd ions in the aqueous solution with simultaneous deposition of created Pd nanoparticles to support materials. Graphene oxide (GO) and nitrogen-doped graphene oxide (NrGO) were both co-reduced with the Pd ions to formulate catalyst materials. Pd catalyst was also deposited on the surface of carbon black. The prepared catalyst materials were physically characterized using transmission electron microscopy, scanning electron microscopy and x-ray photoelectron spectroscopy. The plasma jet method yielded good dispersion of small Pd particles with average sizes of particles being: Pd/rGO 2.9 ± 0.6 nm, Pd/NrGO 2.3 ± 0.5 nm and Pd/Vulcan 2.8 ± 0.6 nm. The electrochemical oxygen reduction reaction (ORR) kinetics was explored using the rotating disk electrode method. Pd catalyst deposited on nitrogen-doped graphene material showed slightly improved ORR activity as compared to that on the nondoped substrate, however Vulcan carbon-supported Pd catalyst exhibited a higher specific activity for oxygen electroreduction.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum β-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 10 3 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gluten is a cereal protein that is incompletely digested by human proteolytic enzymes that create immunogenic peptides that accumulate in the gastrointestinal tract (GIT). Although both environmental and human bacteria have been shown to expedite gluten hydrolysis, gluten intolerance is a growing concern. Here we hypothesize that together with food, we acquire environmental bacteria that could impact our GIT with gluten-degrading bacteria. Using in vitro gastrointestinal simulation conditions, we evaluated the capacity of endophytic bacteria that inhabit root vegetables, potato (Solanum tuberosum), carrot (Daucus sativus), beet (Beta vulgaris), and topinambur (Jerusalem artichoke) (Helianthus tuberosus), to resist these conditions and degrade gluten. By 16S rDNA sequencing, we discovered that bacteria from the families Enterobacteriaceae, Bacillaceae, and Clostridiaceae most effectively multiply in conditions similar to the human GIT (microoxic conditions, 37 °C) while utilizing vegetable material and gluten as nutrients. Additionally, we used stomach simulation (1 h, pH 3) and intestinal simulation (1 h, bile salts 0.4%) treatments. The bacteria that survived this treatment retained the ability to degrade gluten epitopes but at lower levels. Four bacterial strains belonging to species Bacillus pumilus, Clostridium subterminale, and Clostridium sporogenes isolated from vegetable roots produced proteases with postproline cleaving activity that successfully neutralized the toxic immunogenic epitopes. KEY POINTS: • Bacteria from root vegetables can degrade gluten. • Some of these bacteria can resist conditions mimicking gastrointestinal tract.

Background: Next generation sequencing (NGS) technologies are extensively used to dissect the molecular mechanisms of host-parasite interactions in human pathogens. However, ecological studies have yet to fully exploit the power of NGS as a rich source for formulating and testing new hypotheses., Methods: We studied Eurasian perch (Perca fluviatilis) and its eye parasite (Trematoda, Diplostomidae) communities in 14 lakes that differed in humic content in order to explore host-parasite-environment interactions. We hypothesised that high humic content along with low pH would decrease the abundance of the intermediate hosts (gastropods), thus limiting the occurrence of diplostomid parasites in humic lakes. This hypothesis was initially invoked by whole eye RNA-seq data analysis and subsequently tested using PCR-based detection and a novel targeted metabarcoding approach., Results: Whole eye transcriptome results revealed overexpression of immune-related genes and the presence of eye parasite sequences in RNA-seq data obtained from perch living in clear-water lakes. Both PCR-based and targeted-metabarcoding approach showed that perch from humic lakes were completely free from diplostomid parasites, while the prevalence of eye flukes in clear-water lakes that contain low amounts of humic substances was close to 100%, with the majority of NGS reads assigned to Tylodelphys clavata., Conclusions: High intraspecific diversity of T. clavata indicates that massively parallel sequencing of naturally pooled samples represents an efficient and powerful strategy for shedding light on cryptic diversity of eye parasites. Our results demonstrate that perch populations in clear-water lakes experience contrasting eye parasite pressure compared to those from humic lakes, which is reflected by prevalent differences in the expression of immune-related genes in the eye. This study highlights the utility of NGS to discover novel host-parasite-environment interactions and provide unprecedented power to characterize the molecular diversity of cryptic parasites.

We have attempted to define the prevalence and risk factors of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-Enterobacteriaceae) carriage, and to characterize antimicrobial susceptibility, beta-lactamase genes, and major types of isolated strains in volunteers, with a specific focus on humans in contact with animals. Samples were collected from 207 volunteers (veterinarians, pig farmers, dog owners, etc.) and cultured on selective agar. Clonal relationships of the isolated ESBL-Enterobacteriaceae were determined by whole genome sequencing and multi-locus sequence typing. Beta-lactamases were detected using a homology search. Subjects filled in questionnaires analyzed by univariate and multiple logistic regression. Colonization with ESBL-Enterobacteriaceae was found in fecal samples of 14 individuals (6.8%; 95%CI: 3.75-11.09%). In multiple regression analysis, working as a pig farmer was a significant risk factor for ESBL-Enterobacteriaceae carriage (OR 4.8; 95%CI 1.2-19.1). The only species isolated was Escherichia coli that distributed into 11 sequence types. All ESBL-Enterobacteriaceae isolates were of CTX-M genotype, with the bla CTX-M-1 being the most prevalent and more common in pig farmers than in other groups. Despite the generally low prevalence of ESBL-Enterobacteriaceae in Estonia, the pig farmers may still pose a threat to transfer resistant microorganisms. The clinical relevance of predominant bla CTX-M-1 carrying E. coli is still unclear and needs further studies.

Differences in salinity are boundaries that act as barriers for the dispersal of most aquatic organisms. This creates distinctive biota in freshwater and brackish water (mesohaline) environments. To test how saline boundaries influence the diversity and composition of host-associated microbiota, we analyzed the microbiome within the digestive tract of Theodoxus fluviatilis , an organism able to cross the freshwater and mesohaline boundary. Alpha-diversity measures of the microbiome in freshwater and brackish water were not significantly different. However, the composition of the bacterial community within freshwater T. fluviatilis differed significantly compared with mesohaline T. fluviatilis and typical bacteria could be determined for the freshwater and the mesohaline digestive tract microbiome. An artificial increase in salinity surrounding these freshwater snails resulted in a strong change in the bacterial community and typical marine bacteria became more pronounced in the digestive tract microbiome of freshwater T. fluviatilis . However, the composition of the digestive tract microbiome in freshwater snails did not converge to that found within mesohaline snails. Within mesohaline snails, no cardinal change was found after either an increase or decrease in salinity. In all samples, Pseudomonas , Pirellula , Flavobacterium , Limnohabitans , and Acinetobacter were among the most abundant bacteria. These bacterial genera were largely unaffected by changes in environmental conditions. As permanent residents in T. fluviatilis , they may support the digestion of the algal food in the digestive tract. Our results show that freshwater and mesohaline water host-associated microbiomes respond differently to changes in salinity. Therefore, the salinization of coastal freshwater environments due to a rise in sea level can influence the gut microbiome and its functions with currently unknown consequences for, e.g., nutritional physiology of the host., (Copyright © 2020 Kivistik, Knobloch, Käiro, Tammert, Kisand, Hildebrandt and Herlemann.)

Clinical use of CuO nanoparticles (NPs) as antibacterials can be hampered by their toxicity to human cells. We hypothesized that certain surface functionalizations of CuO NPs may render NPs toxic to bacteria, but still be relatively harmless to human cells. To control this hypothesis, the toxicity of differently functionalized CuO NPs to bacteria Escherichia coli vs human cells (THP-1 macrophages and HACAT keratinocytes) was compared using similar conditions and end points. CuO NPs functionalized with polyethylene glycol (CuO-PEG), carboxyl (CuO-COOH, anionic), ammonium (CuO-NH 4 + , cationic) and unfunctionalized CuO NPs and CuSO 4 (controls) were tested. In general, the toxicity of Cu compounds decreased in the following order: CuO-NH 4 +  > unfunctionalized CuO > CuSO 4  > CuO-COOH > CuO-PEG. Positively charged unfunctionalized CuO and especially CuO-NH 4 + proved most toxic (24-h EC 50 = 21.7-47 mg/l) and had comparable toxicity to bacterial and mammalian cells. The multivariate analysis revealed that toxicity of these NPs was mostly attributed to their positive zeta potential, small hydrodynamic size, high Cu dissolution, and induction of reactive oxygen species (ROS) and TNF-α. In contrast, CuO-COOH and CuO-PEG NPs had lower toxicity to human cells compared to bacteria despite efficient uptake of these NPs by human cells. In addition, these NPs did not induce TNF-α and ROS. Thus, by varying the NP functionalization and Cu form (soluble salt vs NPs), it was possible to "target" the toxicity of Cu compounds, whereas carboxylation and PEGylation rendered CuO NPs that were more toxic to bacteria than to human cells envisaging their use in medical antibacterial products.

The groundwater biome is a poorly characterized habitat hypothesized to harbor uniquely diverse bacterial communities; the degree to which these communities differ from associated soils is a central question in environmental microbiology. We characterized the Bacterial community composition in 37 aquifer and 32 surface soil samples across the island of O'ahu, Hawai'i. Several bacterial phyla (Acetothermia, Omnitrophica, Parcubacteria, Peregrinibacteria) relatively abundant in the aquifer samples were rare to absent in the soils. Immense bacterial diversity detected in the deep aquifers indicates that these environments are not as homogenous as expected, but provide various niches and energy sources for wide variety of bacteria. A small proportion of OTUs were widespread in all the basal (0.63%) and all the dike aquifer (0.31%) samples. However, these core bacteria comprised an average of 31.8% (ranging 16.2%-62.0%) and 15.4% (0.1%-31.5%) of all sequences isolated from the basal and dike aquifers respectively. Bacterial community composition correlated significantly with the sodium, sulfate, potassium, total dissolved solids, nitrate, conductivity, and pH in the basal aquifers, while phosphate and bicarbonate levels were also highly important when dike water samples were included in the analyses. This was consistent with high relative abundance of putative chemolithoautoroph taxa in the aquifer communities relative to soils. Targeted molecular and culture-based fecal indicator microbial analyses indicated good water quality of aquifers. The dominance of unique, deeply branching lineages in tropical aquifers emphasizes a large adaptive potential in O'ahu's aquifers; variability among groundwater samples suggests that aquifer habitats are surprisingly variable potentially harboring a variety of chemolithotrophic energy sources. Although parallel analyses of conventional and alternative indicators indicated good groundwater quality, this study calls for groundwater monitoring programs which would consider public as well as ecosystem health., Competing Interests: The authors have declared that no competing interests exist.

Global warming, paired with eutrophication processes, is shifting phytoplankton communities towards the dominance of bloom-forming and potentially toxic cyanobacteria. The ecosystems of shallow lakes are especially vulnerable to these changes. Traditional monitoring via microscopy is not able to quantify the dynamics of toxin-producing cyanobacteria on a proper spatio-temporal scale. Molecular tools are highly sensitive and can be useful as an early warning tool for lake managers. We quantified the potential microcystin (MC) producers in Lake Peipsi using microscopy and quantitative polymerase chain reaction (qPCR) and analysed the relationship between the abundance of the mcyE genes, MC concentration, MC variants and toxin quota per mcyE gene. We also linked environmental factors to the cyanobacteria community composition. In Lake Peipsi, we found rather moderate MC concentrations, but microcystins and microcystin-producing cyanobacteria were widespread across the lake. Nitrate (NO 3 - ) was a main driver behind the cyanobacterial community at the beginning of the growing season, while in late summer it was primarily associated with the soluble reactive phosphorus (SRP) concentration. A positive relationship was found between the MC quota per mcyE gene and water temperature. The most abundant variant-MC-RR-was associated with MC quota per mcyE gene, while other MC variants did not show any significant impact.

The ultraviolet photoelectron spectrum of the [EMIM][B(CN) 4 ] ionic liquid was recorded and analyzed. Together with different ab initio calculation methods, detailed insight into the electronic structure of this simple room temperature ionic liquid is possible. The ion-pair approximation to the liquid electronic structure was not sufficient. Therefore bulk ab initio calculations were performed on a proposed crystal structure. The modelling of bulk electronic spectra is able to explain the experimental electronic structure of the ionic liquid. Most notably, the dispersion corrected PBE calculation (PBE-D3BJ) showed good agreement with the experimental UPS spectrum. The spectra simulated by the B97-D and the BLYP-D3(BJ) functionals were also in agreement with the experimental data. The LDA approximation only provided qualitative agreement while the optB88-vdW and CX-vdW functionals were not good. However, it will be shown that many requirements have to be met in order to accurately describe the electronic structure of this ionic liquid., Competing Interests: There are no conflicts of interests to declare., (This journal is © The Royal Society of Chemistry.)

Heterotrophic bacteria are important drivers of nitrogen (N) cycling and the processing of dissolved organic matter (DOM). Projected increases in precipitation will potentially cause increased loads of riverine DOM to the Baltic Sea and likely affect the composition and function of bacterioplankton communities. To investigate this, the effects of riverine DOM from two different catchment areas (agricultural and forest) on natural bacterioplankton assemblages from two contrasting sites in the Baltic Sea were examined. Two microcosm experiments were carried out, where the community composition (16S rRNA gene sequencing), the composition of a suite of N-cycling genes (metagenomics) and the abundance and transcription of ammonia monooxygenase (amoA) genes involved in nitrification (quantitative PCR) were investigated. The river water treatments evoked a significant response in bacterial growth, but the effects on overall community composition and the representation of N-cycling genes were limited. Instead, treatment effects were reflected in the prevalence of specific taxonomic families, specific N-related functions and in the transcription of amoA genes. The study suggests that bacterioplankton responses to changes in the DOM pool are constrained to part of the bacterial community, whereas most taxa remain relatively unaffected., (© FEMS 2019.)

Plants contain endophytic bacteria, whose communities both influence plant growth and can be an important source of probiotics. Here we used deep sequencing of a 16S rRNA gene fragment and bacterial cultivation to independently characterize the microbiomes of five plant species from divergent taxonomic orders-potato (Solanum tuberosum), carrot (Daucus sativus), beet (Beta vulgaris), neep (Brassica napus spp. napobrassica), and topinambur (Helianthus tuberosus). We found that both species richness and diversity tend to be higher in the peel, where Alphaproteobacteria and Actinobacteria dominate, while Gammaproteobacteria and Firmicutes dominate in the pulp. A statistical analysis revealed that the main characteristic features of the microbiomes of plant species originate from the peel microbiomes. Topinambur pulp displayed an interesting characteristic feature: it contained up to 108 CFUs of lactic acid bacteria, suggesting its use as a source of probiotic bacteria. We also detected Listeria sp., in topinambur pulps, however, the 16S rRNA gene fragment is unable to distinguish between pathogenic versus non-pathogenic species, so the evaluation of this potential health risk is left to a future study., Competing Interests: The authors have declared that no competing interests exist.

The Eurasian perch ( Perca fluviatilis ) is the most common fish of the Percidae family and is widely distributed across Eurasia. Perch is a popular target for professional and recreational fisheries, and a promising freshwater aquaculture species in Europe. However, despite its high ecological, economical and societal importance, the available genomic resources for P. fluviatilis are rather limited. In this work, we report de novo assembly and annotation of the whole genome sequence of perch. The linked-read based technology with 10X Genomics Chromium chemistry and Supernova assembler produced a draft perch genome ∼1.0 Gbp assembly (scaffold N 50 = 6.3 Mb; the longest individual scaffold of 29.3 Mb; BUSCO completeness of 88.0%), which included 281.6 Mb of putative repeated sequences. The perch genome assembly presented here, generated from small amount of starting material (0.75 ng) and a single linked-read library, is highly continuous and considerably more complete than the currently available draft of P. fluviatilis genome. A total of 23,397 protein-coding genes were predicted, 23,171 (99%) of which were annotated functionally from either sequence homology or protein signature searches. Linked-read technology enables fast, accurate and cost-effective de novo assembly of large non-model eukaryote genomes. The highly continuous assembly of the Eurasian perch genome presented in this study will be an invaluable resource for a range of genetic, ecological, physiological, ecotoxicological, functional and comparative genomic studies in perch and other fish species of the Percidae family., (Copyright © 2018 Ozerov et al.)

Most studies that utilize ancient DNA have focused on specific groups of organisms or even single species. Instead, the whole biodiversity of eukaryotes can be described using universal phylogenetic marker genes found within well-preserved sediment cores that cover the post-glacial period. Sedimentary ancient DNA samples from Lake Lielais Svētiņu, eastern Latvia, at a core depth of 1,050 cm in ~150 year intervals were used to determine phylotaxonomy in domain Eukaryota. Phylotaxonomic affiliation of >1,200 eukaryotic phylotypes revealed high richness in all major eukaryotic groups-Alveolata, Stramenopiles, Cercozoa, Chlorophyta, Charophyta, Nucletmycea, and Holozoa. The share of organisms that originate from terrestrial ecosystems was about one third, of which the most abundant molecular operational taxonomic units were Fungi and tracheal/vascular plants, which demonstrates the usefulness of using lake sediments to reconstruct the terrestrial paleoecosystems that surround them. Phylotypes that originate from the lake ecosystem belonged to various planktonic organisms; phyto-, proto,- and macrozooplankton, and vascular aquatic plants. We observed greater richness of several planktonic organisms that can be associated with higher trophic status during the warm climate period between 4,000 and 8,000 years ago and an increase in eukaryotic richness possibly associated with moderate human impact over the last 2,000 years., (© 2018 John Wiley & Sons Ltd.)

We have developed an easy-to-use and memory-efficient method called PhenotypeSeeker that (a) identifies phenotype-specific k-mers, (b) generates a k-mer-based statistical model for predicting a given phenotype and (c) predicts the phenotype from the sequencing data of a given bacterial isolate. The method was validated on 167 Klebsiella pneumoniae isolates (virulence), 200 Pseudomonas aeruginosa isolates (ciprofloxacin resistance) and 459 Clostridium difficile isolates (azithromycin resistance). The phenotype prediction models trained from these datasets obtained the F1-measure of 0.88 on the K. pneumoniae test set, 0.88 on the P. aeruginosa test set and 0.97 on the C. difficile test set. The F1-measures were the same for assembled sequences and raw sequencing data; however, building the model from assembled genomes is significantly faster. On these datasets, the model building on a mid-range Linux server takes approximately 3 to 5 hours per phenotype if assembled genomes are used and 10 hours per phenotype if raw sequencing data are used. The phenotype prediction from assembled genomes takes less than one second per isolate. Thus, PhenotypeSeeker should be well-suited for predicting phenotypes from large sequencing datasets. PhenotypeSeeker is implemented in Python programming language, is open-source software and is available at GitHub (https://github.com/bioinfo-ut/PhenotypeSeeker/)., Competing Interests: The authors have declared that no competing interests exist.

Background: We aimed to identify the main spreading clones, describe the resistance mechanisms associated with carbapenem- and/or multidrug-resistant P. aeruginosa and characterize patients at risk of acquiring these strains in Estonian hospitals., Methods: Ninety-two non-duplicated carbapenem- and/or multidrug-resistant P. aeruginosa strains were collected between 27th March 2012 and 30th April 2013. Clinical data of the patients was obtained retrospectively from the medical charts. Clonal relationships of the strains were determined by whole genome sequencing and analyzed by multi-locus sequence typing. The presence of resistance genes and beta-lactamases and their origin was determined. Combined-disk method and PCR was used to evaluate carbapenemase and metallo-beta-lactamase production., Results: Forty-three strains were carbapenem-resistant, 11 were multidrug-resistant and 38 were both carbapenem- and multidrug-resistant. Most strains (54%) were isolated from respiratory secretions and caused an infection (74%). Over half of the patients (57%) were ≥ 65 years old and 85% had ≥1 co-morbidity; 96% had contacts with healthcare and/or had received antimicrobial treatment in the previous 90 days. Clinically relevant beta-lactamases (OXA-101, OXA-2 and GES-5) were found in 12% of strains, 27% of which were located in plasmids. No Ambler class B beta-lactamases were detected. Aminoglycoside modifying enzymes were found in 15% of the strains. OprD was defective in 13% of the strains (all with CR phenotype); carbapenem resistance triggering mutations (F170 L, W277X, S403P) were present in 29% of the strains. Ciprofloxacin resistance correlated well with mutations in topoisomerase genes gyrA (T83I, D87N) and parC (S87 L). Almost all strains (97%) with these mutations showed ciprofloxacin-resistant phenotype. Multi-locus sequence type analysis indicated high diversity at the strain level - 36 different sequence types being detected. Two sequence types (ST108 (n = 23) and ST260 (n = 18)) predominated. Whereas ST108 was associated with localized spread in one hospital and mostly carbapenem-resistant phenotype, ST260 strains occurred in all hospitals, mostly with multi-resistant phenotype and carried different resistance genotype/machinery., Conclusions: Diverse spread of local rather than international P. aeruginosa strains harboring multiple chromosomal mutations, but not plasmid-mediated Ambler class B beta-lactamases, were found in Estonian hospitals., Trial Registration: This trial was registered retrospectively in ClinicalTrials.gov ( NCT03343119 ).

Application of efficient antimicrobial surfaces has been estimated to decrease both, the healthcare-associated infections and the spread of antibiotic-resistant bacteria. In this paper, we prepared ZnO and ZnO/Ag nanoparticle covered surfaces and evaluated their antimicrobial efficacy towards a Gram-negative bacterial model (Escherichia coli), a Gram-positive bacterial model (Staphylococcus aureus) and a fungal model (Candida albicans) in the dark and under UVA illumination. The surfaces were prepared by spin coating aliquots of ZnO and ZnO/Ag nanoparticle suspensions onto glass substrates. Surfaces contained 2 or 20 μg Zn/cm 2 and 0-0.02 μg Ag/cm 2 . No significant antimicrobial activity of the surfaces, except of those with the highest Ag or Zn content was observed in the dark. On the other hand, UVA illuminated surfaces containing 20 μg Zn/cm 2 and 2 μg Zn plus 0.02 μg Ag/cm 2 caused >3 log decrease in the viable counts of E. coli and S. aureus in 30 min. As proven by brilliant blue FCF dye degradation and elemental analysis of the surfaces, this remarkable antimicrobial activity was a combined result of photocatalytic effect and release of Zn and Ag ions from surfaces. Surfaces retained significant antibacterial and photocatalytic properties after several usage cycles. Compared to bacteria, yeast C. albicans was significantly less sensitive to the prepared surfaces and only about 1 log reduction of viable count was observed after 60 min UVA illumination. In conclusion, the developed ZnO/Ag surfaces exhibit not only high antibacterial activity but also some antifungal activity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The ultraviolet photoelectron spectrum (UPS) of the [EMIM][BF 4 ] ionic liquid was recorded and analyzed. Together with the gas-phase UPS spectrum of the [EMIM][BF 4 ] vapor and ab initio calculation methods, detailed insight into the electronic structure of this simple ionic liquid is possible. The low binding energy tail in the UPS spectrum is about 7.4 eV, in agreement with previous estimations of the HOMO-LUMO gap of the [EMIM][BF 4 ] ion-pair. The bulk ab initio calculations are able to explain most of the features in the spectrum. However, DFT consistently lacks accuracy in the description of the top of the valence band. The dispersion corrected PBE calculation (PBE-D3) did offer very good agreement with the experimental structure, but the recently-developed vdW-DF functionals C09, optPBE, optB88 and CX were found to offer the best agreement in terms of the electronic structure., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Bacteria are major drivers of biogeochemical nutrient cycles and energy fluxes in marine environments, yet how bacterial communities respond to environmental change is not well known. Metagenomes allow examination of genetic responses of the entire microbial community to environmental change. However, it is challenging to link metagenomes directly to biogeochemical process rates. Here, we investigate metagenomic responses in natural bacterioplankton communities to simulated environmental stressors in the Baltic Sea, including increased river water input, increased nutrient concentration, and reduced oxygen level. This allowed us to identify informative prokaryotic gene markers, responding to environmental perturbation. Our results demonstrate that metagenomic and metabolic changes in bacterial communities in response to environmental stressors are influenced both by the initial community composition and by the biogeochemical factors shaping the functional response. Furthermore, the different sources of dissolved organic matter (DOM) had the largest impact on metagenomic blueprint. Most prominently, changes in DOM loads influenced specific transporter types reflecting the substrate availability and DOC assimilation and consumption pathways. The results provide new knowledge for developing models of ecosystem structure and biogeochemical cycling in future climate change scenarios and advance our exploration of the potential use of marine microorganisms as markers for environmental conditions., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The Baltic Sea is one of the world's largest brackish water bodies and is characterised by pronounced physicochemical gradients where microbes are the main biogeochemical catalysts. Meta-omic methods provide rich information on the composition of, and activities within, microbial ecosystems, but are computationally heavy to perform. We here present the Baltic Sea Reference Metagenome (BARM), complete with annotated genes to facilitate further studies with much less computational effort. The assembly is constructed using 2.6 billion metagenomic reads from 81 water samples, spanning both spatial and temporal dimensions, and contains 6.8 million genes that have been annotated for function and taxonomy. The assembly is useful as a reference, facilitating taxonomic and functional annotation of additional samples by simply mapping their reads against the assembly. This capability is demonstrated by the successful mapping and annotation of 24 external samples. In addition, we present a public web interface, BalticMicrobeDB, for interactive exploratory analysis of the dataset.

The gastrointestinal tract (GIT) of fish supports a dynamic microbial ecosystem that is intimately linked to host nutrient acquisition, epithelial development, immune system priming, and disease prevention, and we are far from understanding the complex interactions among parasites, symbiotic gut bacteria, and host fitness. Here, we analyzed the effects of environmental factors and parasitic burdens on the microbial composition and diversity within the GIT of the brown trout ( Salmo trutta ). We focused on the emerging dangerous salmonid myxozoan parasite Tetracapsuloides bryosalmonae , which causes proliferative kidney disease in salmonid fish, to demonstrate the potential role of GIT micobiomes in the modulation of host-parasite relationships. The microbial diversity in the GIT displayed clear clustering according to the river of origin, while considerable variation was also found among fish from the same river. Environmental variables such as oxygen concentration, water temperature, and river morphometry strongly associated with both the river microbial community and the GIT microbiome, supporting the role of the environment in microbial assemblage and the relative insignificance of the host genotype and gender. Contrary to expectations, the parasite load exhibited a significant positive relationship with the richness of the GIT microbiome. Many operational taxonomic units (OTUs; n = 202) are more abundant in T. bryosalmonae -infected fish, suggesting that brown trout with large parasite burdens are prone to lose their GIT microbiome homeostasis. The OTUs with the strongest increase in infected trout are mostly nonpathogenic aquatic, anaerobic sediment/sludge, or ruminant bacteria. Our results underscore the significance of the interactions among parasitic disease, abiotic factors, and the GIT microbiome in disease etiology. IMPORTANCE Cohabiting microorganisms play diverse and important roles in the biology of multicellular hosts, but their diversity and interactions with abiotic and biotic factors remain largely unsurveyed. Nevertheless, it is becoming increasingly clear that many properties of host phenotypes reflect contributions from the associated microbiome. We focus on a question of how parasites, the host genetic background, and abiotic factors influence the microbiome in salmonid hosts by using a host-parasite model consisting of wild brown trout ( Salmo trutta ) and the myxozoan Tetracapsuloides bryosalmonae , which causes widely distributed proliferative kidney disease. We show that parasite infection increases the frequency of bacteria from the surrounding river water community, reflecting impaired homeostasis in the fish gut. Our results also demonstrate the importance of abiotic environmental factors and host size in the assemblage of the gut microbiome of fish and the relative insignificance of the host genotype and gender.

Indicator bacteria, which are conventionally used to evaluate recreational water quality, can originate from various non-human enteric and extra-enteric sources, hence they may not be indicative of human health risk nor do they provide information on the sources of contamination. In this study we utilized traditional (enterococci and Escherichia coli) and alternative (Clostridium perfringens) indicator bacteria, F + -specific coliphage, molecular markers for microorganisms associated with human sewage (human-associated Bacteroides and polyomaviruses), and microbial community analysis tools (16S rRNA gene fragment amplicon sequencing), to identify and evaluate human sewage-related impact in the Manoa watershed in Honolulu, Hawaii. Elevated concentrations of enterococci (geometric mean ranging from 1604 to 2575 CFU 100 mL -1 ) and C. perfringens (45-77 CFU 100 mL -1 ) indicated impairment of the urbanized section of the stream, while indicator bacteria concentrations decreased downstream in the tidally influenced Ala Wai Canal. The threshold values triggering water quality violation notifications in Hawaii were exceeded in 33.3-75.0% of samples collected at sites in the urbanized section of Manoa Stream, but were not exceeded in any of the samples collected at an upstream site located in a forested area. Correlation between indicator bacteria concentrations and rainfall amounts was weak to moderate but significant (E. coli R = 0.251, P = 0.009; enterococci R = 0.369, P < 0.001; C. perfringens R = 0.343, P < 0.001), while concentrations of human fecal-associated molecular markers were not significantly correlated with rainfall (human-associated Bacteroides, R = 0.131, P = 0.256; human-associated polyomaviruses, R = 0.213, P = 0.464). Presence of human sewage was confirmed by detection of human-associated Bacteroides and human polyomavirus in the urbanized section of Manoa Stream (83.3-100% and 41.7-66.7% positive samples respectively). It was further confirmed by microbial community analyses which suggested that an average 2.4-3.4% of the total bacterial population in this section was associated with sewage. Microbial community profiles were significantly influenced by rainfall (R 2  = 0.4390, P < 0.001), pH (R 2  = 0.3077, P = 0.006), salinity (R 2  = 0.2614, P = 0.038), and conductivity (R 2  = 0.2676, P = 0.031). Although microbial diversity fluctuated throughout the watershed, it was lower in the impaired section. Leaking sewer systems and illegal cross-connections are implicated in the impairment of the watershed, hence both the sewer and the storm water lines should be routinely inspected. Collectively, our data suggest that information derived from the analysis of microbial communities complements current marker-based microbial source tracking techniques and environmental monitoring programs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Roof-harvested rainwater (RHRW) is an important alternative source of water that many island communities can use for drinking and other domestic purposes when groundwater and/or surface water sources are contaminated, limited, or simply not available. The aim of this pilot-scale study was to investigate current RHRW practices in American Samoa (AS) and to evaluate and compare the quality of water from common potable water sources including RHRW stored in tanks, untreated stream water, untreated municipal well water, and treated municipal tap water samples. Samples were analyzed using culture-based methods, quantitative polymerase chain reaction (qPCR), and 16S amplicon sequencing-based methods. Based on indicator bacteria (total coliform and Escherichia coli) concentrations, the quality of RHRW was slightly lower than well and chlorinated tap water but exceeded that of untreated stream water. Although no Giardia or Leptospira spp. were detected in any of the RHRW samples, 86% of the samples were positive for Cryptosporidium spp. All stream water samples tested positive for Cryptosporidium spp. Opportunistic pathogens (Pseudomonas aeruginosa and Mycobacterium intracellulare) were also detected in the RHRW samples (71 and 21% positive samples, respectively). Several potentially pathogenic genera of bacteria were also detected in RHRW by amplicon sequencing. Each RHRW system was characterized by distinct microbial communities, 77% of operational taxonomic units (OTUs) were detected only in a single tank, and no OTU was shared by all the tanks. Risk of water-borne illness increased in the following order: chlorinated tap water/well water < RHRW < stream water. Frequent detection of opportunistic pathogens indicates that RHRW should be treated before use. Stakeholder education on RHRW system design options as well as on importance of regular cleaning and proper management techniques could improve the quality of the RHRW in AS.

Colorimetric gas sensing is demonstrated by thin films based on ultrasmall TiO 2 nanoparticles (NPs) on Si substrates. The NPs are bound into the film by p -toluenesulfonic acid (PTSA) and the film is made to absorb volatile organic compounds (VOCs). Since the color of the sensing element depends on the interference of reflected light from the surface of the film and from the film/silicon substrate interface, colorimetric detection is possible by the varying thickness of the NP-based film. Indeed, VOC absorption causes significant swelling of the film. Thus, the optical path length is increased, interference wavelengths are shifted and the refractive index of the film is decreased. This causes a change of color of the sensor element visible by the naked eye. The color response is rapid and changes reversibly within seconds of exposure. The sensing element is extremely simple and cheap, and can be fabricated by common coating processes.

A plasmid carrying the colistin resistance gene mcr-1 was isolated from a pig slurry sample in Estonia. The gene was present on a 33,311-bp plasmid of the IncX4 group. mcr-1 is the only antibiotic resistance gene on the plasmid, with the other genes mainly coding for proteins involved in conjugative DNA transfer (taxA, taxB, taxC, trbM, and the pilX operon). The plasmid pESTMCR was present in three phylogenetically very different Escherichia coli strains, suggesting that it has high potential for horizontal transfer., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Over the past century the spread of hypoxia in the Baltic Sea has been drastic, reaching its 'arm' into the easternmost sub-basin, the Gulf of Finland. The hydrographic and climatological properties of the gulf offer a broad suite of discrete niches for microbial communities. The current study explores spatiotemporal dynamics of bacterioplankton community in the Gulf of Finland using massively parallel sequencing of 16S rRNA fragments obtained by amplifying community DNA from spring to autumn period. The presence of redoxcline and drastic seasonal changes make spatiotemporal dynamics of bacterioplankton community composition (BCC) and abundances in such estuary remarkably complex. To the best of our knowledge, this is the first study that analyses spatiotemporal dynamics of BCC in relation to phytoplankton bloom throughout the water column (and redoxcline), not only at the surface layer. We conclude that capability to survive (or benefit from) shifts between oxic and hypoxic conditions is vital adaptation for bacteria to thrive in such environments. Our results contribute to the understanding of emerging patterns in BCCs that occupy hydrographically similar estuaries dispersed all over the world, and we suggest the presence of a global redox- and salinity-driven metacommunity. These results have important implications for understanding long-term ecological and biogeochemical impacts of hypoxia expansion in the Baltic Sea (and similar ecosystems), as well as global biogeography of bacteria specialized inhabiting similar ecosystems.

Massively parallel sequencing a small proportion of the whole genome at high coverage enables answering a wide range of questions from molecular evolution and evolutionary biology to animal and plant breeding and forensics. In this study, we describe the development of restriction-site associated DNA (RAD) sequencing approach for Ion Torrent PGM platform. Our protocol results in extreme genome complexity reduction using two rare-cutting restriction enzymes and strict size selection of the library allowing sequencing of a relatively small number of genomic fragments with high sequencing depth. We applied this approach to a common freshwater fish species, the Eurasian perch (Perca fluviatilis L.), and generated over 2.2 MB of novel sequence data consisting of ~17,000 contigs, identified 1259 single nucleotide polymorphisms (SNPs). We also estimated genetic differentiation between the DNA pools from freshwater (Lake Peipus) and brackish water (the Baltic Sea) populations and identified SNPs with the strongest signal of differentiation that could be used for robust individual assignment in the future. This work represents an important step towards developing genomic resources and genetic tools for the Eurasian perch. We expect that our ddRAD sequencing protocol for semiconductor sequencing technology will be useful alternative for currently available RAD protocols., (© 2015 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Slow sand filtration (SSF) has been widely used as a means of providing potable water due to its efficacy, low cost, and minimal maintenance. Advances in analytical instrumentation have revealed the occurrence of pharmaceutically active compounds (PhACs) in surface water as well as in groundwater. It is unclear if the presence of these compounds in the feed water can interfere with the performances of an SSF unit. The aim of this work was to examine i) the ability of two SSF units to remove six PhACs (caffeine, carbamazepine, 17-β estradiol [E2], estrone [E1], gemfibrozil, and phenazone), and ii) the impact of these PhACs on the removal of bacteria by two SSF units. The presence of PhACs in feed water for SSF can occur in surface waters impacted by wastewater or leakage from sewers and septic tanks, as well as in developing countries where unregulated use and improper disposal are prevalent. Two pilot-scale SSF units were used during the study. Unit B1 was fed with stream water with 1% of primary effluent added, while unit B2 was fed with stream water alone. Although limited removal (<10%) of carbamazepine, gemfibrozil, and phenazone occurred, the complete removal of caffeine, and the partial removal (11-92%) of E2 and E1 were observed in the two SSF units. The results of this study suggest that the occurrence of the selected PhACs, probably estrogens and caffeine, in the feed water at 50 μg L(-1) affected the ability of the schmutzdecke to remove total coliform and Escherichia coli. The bacterial removal achieved within the schmutzdecke dropped from 95% to less than 20% by the end of the study. This decrease in removal may be related to the change in the microbial community within the schmutzdecke. A diverse microbial community, including Bacteroidetes and several classes of Proteobacteria, was replaced by a microbial community in which Gammaproteobacteria was the predominant phylum (99%). Despite the low removal achieved within the schmutzdecke, removal of total coliform and E. coli greater than 99% occurred after both SSF units throughout the study. Bacterial removal occurred in the upper half of the sand filter. This was probably due to a diverse microbial community established in the packing material, in which Bacteroidetes (13-25%), Acidobacteria (7-17%) and several classes of Proteobacteria (35-52%) (Alpha-, Beta-, Delta-, and Gammaproteobacteria) were the predominant phyla., (Copyright © 2015 Elsevier B.V. All rights reserved.)

It has been suggested that plumage microorganisms play an important role in shaping the life histories of wild birds. Some bacteria may act as pathogens or cause damage to feathers, and thereby reduce individual fitness. Intense parental care in birds can result in a reduction of self-maintenance and preening behavior in parents and therefore might affect the dynamics of microbiota living on their feathers. However, experimental evidence of this relationship is virtually absent. We manipulated the parental effort of wild breeding pied flycatcher (Ficedula hypoleuca) females by modifying their brood size or temporarily removing male partners. We expected that experimentally decreasing or increasing parental effort would affect feather sanitation in females and therefore also bacterial density on their plumage. In accordance with this hypothesis, manipulation affected the density of free-living bacteria: females with reduced broods had the lowest number of free-living bacteria on their feathers, while females left without male partners had the highest. However, manipulation did not have a significant effect on the densities of attached bacteria. Our results provide experimental evidence that a trade-off between self-maintenance and parental effort affects plumage bacterial densities in birds.

This study explored the spatiotemporal dynamics of the bacterioplankton community composition in the Gulf of Finland (easternmost sub-basin of the Baltic Sea) based on phylogenetic analysis of 16S rDNA sequences acquired from community samples via pyrosequencing. Investigations of bacterioplankton in hydrographically complex systems provide good insight into the strategies by which microbes deal with spatiotemporal hydrographic gradients, as demonstrated by our research. Many ribotypes were closely affiliated with sequences isolated from environments with similar steep physiochemical gradients and/or seasonal changes, including seasonally anoxic estuaries. Hence, one of the main conclusions of this study is that marine ecosystems where oxygen and salinity gradients co-occur can be considered a habitat for a cosmopolitan metacommunity consisting of specialized groups occupying niches universal to such environments throughout the world. These niches revolve around functional capabilities to utilize different electron receptors and donors (including trace metal and single carbon compounds). On the other hand, temporal shifts in the bacterioplankton community composition at the surface layer were mainly connected to the seasonal succession of phytoplankton and the inflow of freshwater species. We also conclude that many relatively abundant populations are indigenous and well-established in the area.

Titanium dioxide is a photocatalyst with well-known ability to oxidise a wide range of organic contaminants as well as to destroy microbial cells. In the present work TiO2 nanoparticles with high specific surface area (150m(2)/g) were used to prepare nanostructured films. The TiO2 nanoparticle-based film in combination with UV-A illumination with intensity (22W/m(2)) comparable to that of the sunlight in the UV-A region was used to demonstrate light-induced antibacterial effects. Fast and effective inactivation of Escherichia coli cells on the prepared thin films was observed. Visualization of bacterial cells under scanning electron microscopy (SEM) showed enlargement of the cells, distortion of cellular membrane and possible leakage of cytoplasm after 10min of exposure to photoactivated TiO2. According to the plate counts there were no viable cells as early as after 20min of exposure to UV-A activated TiO2. In parallel to effects on bacterial cell viability and morphology, changes in saturated and unsaturated fatty acids - important components of bacterial cell membrane-were studied. Fast decomposition of saturated fatty acids and changes in chemical structure of unsaturated fatty acids were detected. Thus, we suggest that peroxidation and decomposition of membrane fatty acids could be one of the factors contributing to the morphological changes of bacteria observed under SEM, and ultimately, cell death., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The knowledge on potential harmful effects of metallic nanomaterials lags behind their increased use in consumer products and therefore, the safety data on various nanomaterials applicable for risk assessment are urgently needed. In this study, 11 metal oxide nanoparticles (MeOx NPs) prepared using flame pyrolysis method were analyzed for their toxicity against human alveolar epithelial cells A549, human epithelial colorectal cells Caco2 and murine fibroblast cell line Balb/c 3T3. The cell lines were exposed for 24 h to suspensions of 3-100 μg/mL MeOx NPs and cellular viability was evaluated using. Neutral Red Uptake (NRU) assay. In parallel to NPs, toxicity of soluble salts of respective metals was analyzed, to reveal the possible cellular effects of metal ions shedding from the NPs. The potency of MeOx to produce reactive oxygen species was evaluated in the cell-free assay. The used three cell lines showed comparable toxicity responses to NPs and their metal ion counterparts in the current test setting. Six MeOx NPs (Al2O3, Fe3O4, MgO, SiO2, TiO2, WO3) did not show toxic effects below 100 µg/mL. For five MeOx NPs, the averaged 24 h IC50 values for the three mammalian cell lines were 16.4 µg/mL for CuO, 22.4 µg/mL for ZnO, 57.3 µg/mL for Sb2O3, 132.3 µg/mL for Mn3O4 and 129 µg/mL for Co3O4. Comparison of the dissolution level of MeOx and the toxicity of soluble salts allowed to conclude that the toxicity of CuO, ZnO and Sb2O3 NPs was driven by release of metal ions. The toxic effects of Mn3O4 and Co3O4 could be attributed to the ROS-inducing ability of these NPs. All the NPs were internalized by the cells according to light microscopy studies but also proven by TEM, and internalization of Co3O4 NPs seemed to be most prominent in this aspect. In conclusion, this work provides valuable toxicological data for a library of 11 MeOx NPs. Combining the knowledge on toxic or non-toxic nature of nanomaterials may be used for safe-by-design approach.

The changes in optical properties during TiO₂ nanowire orientation in polydimethylsiloxane (PDMS) matrix under the influence of an electric field are strongly influenced by nanowire (NW) diameter. It was demonstrated for the first time that either positive or negative change in transmittance can be induced by NW alignment parallel to the electric field depending on the NW diameter. These effects can be explained by the interplay between scattering and reflectance. Experimental findings reported could be important for smart window applications for the regulation of visible or even infrared transparency, thus reducing the energy consumption by air conditioning systems in buildings and automobiles in the future.