Antibiotic Resistomes and Microbiomes in the Surface Water along the Code River in Indonesia Reflect Drainage Basin Anthropogenic Activities.

Water and sanitation are important factors in the emergence of antimicrobial resistance in low- and middle-income countries. Drug residues, metals, and various wastes foster the spread of antibiotic resistance genes (ARGs) with the help of mobile genetic elements (MGEs), and therefore, rivers receiving contaminants and effluents from multiple sources are of special interest. We followed both the microbiome and resistome of the Code River in Indonesia from its pristine origin at the Merapi volcano through rural and then city areas to the coast of the Indian Ocean. We used a SmartChip quantitative PCR with 382 primer pairs for profiling the resistome and MGEs and 16S rRNA gene amplicon sequencing to analyze the bacterial communities. The community structure explained the resistome composition in rural areas, while the city sampling sites had lower bacterial diversity and more ARGs, which correlated with MGEs, suggesting increased mobility potential in response to pressures from human activities. Importantly, the vast majority of ARGs and MGEs were no longer detectable in marine waters at the ocean entrance. Our work provides information on the impact of different influents on river health as well as sheds light on how land use contributes to the river resistome and microbiome.

Fertilizing with Animal Manure Disseminates Antibiotic Resistance Genes to the Farm Environment.

The dissemination of antibiotic resistance genes to the environment is an important factor causing increased prevalence of resistant pathogens. Manure is an important fertilizer, but it contains diverse resistance genes. Therefore, its application to fields may lead to increased abundance of resistance genes in the environment. Farming environments exposed to animal manure have not been studied extensively in countries with comparably low antibiotic use, such as Finland. The effects of manure storage and application to fields on the abundance of resistance genes were studied on two dairy cattle farms and two swine farms in southern Finland. Samples were taken from farms during the 2013 cropping season. Copy numbers of carbapenem (), sulfonamide (), and tetracycline () resistance genes were measured with quantitative polymerase chain reaction, and the data were analyzed using linear mixed models. The relative abundance of antibiotic resistance genes increased about fourfold in soil after manure application. Carbapenemase encoding was detected on all of the studied farms, which indicated that the gene is dispersed in the farm environment. The relative abundance of antibiotic resistance genes increased in stored manure compared with fresh manure roughly fivefold. This study shows that antibiotic resistance genes are disseminated on Finnish production animal farms. The spreading of resistance genes in farm-associated environments could possibly be limited by experimenting with new manure handling methods that could reduce the abundance of the genes in manure used for land application.

Genome-derived insights into the biology of the hepatotoxic bloom-forming cyanobacterium Anabaena sp. strain 90.

BACKGROUND: Cyanobacteria can form massive toxic blooms in fresh and brackish bodies of water and are frequently responsible for the poisoning of animals and pose a health risk for humans. Anabaena is a genus of filamentous diazotrophic cyanobacteria commonly implicated as a toxin producer in blooms in aquatic ecosystems throughout the world. The biology of bloom-forming cyanobacteria is poorly understood at the genome level. RESULTS: Here, we report the complete sequence and comprehensive annotation of the bloom-forming Anabaena sp. strain 90 genome. It comprises two circular chromosomes and three plasmids with a total size of 5.3 Mb, encoding a total of 4,738 genes. The genome is replete with mobile genetic elements. Detailed manual annotation demonstrated that almost 5% of the gene repertoire consists of pseudogenes. A further 5% of the genome is dedicated to the synthesis of small peptides that are the products of both ribosomal and nonribosomal biosynthetic pathways. Inactivation of the hassallidin (an antifungal cyclic peptide) biosynthetic gene cluster through a deletion event and a natural mutation of the buoyancy-permitting gvpG gas vesicle gene were documented. The genome contains a large number of genes encoding restriction-modification systems. Two novel excision elements were found in the nifH gene that is required for nitrogen fixation. CONCLUSIONS: Genome analysis demonstrated that this strain invests heavily in the production of bioactive compounds and restriction-modification systems. This well-annotated genome provides a platform for future studies on the ecology and biology of these important bloom-forming cyanobacteria.

Virulence genes of Aeromonas isolates, bacterial endotoxins and cyanobacterial toxins from recreational water samples associated with human health symptoms.

Exposure to cyanobacterial water blooms has been associated with various kinds of adverse health effects. In addition to cyanobacteria and their toxins, the bacteria associated with cyanobacteria could also be the etiological agents. We isolated Aeromonas strains (n = 176) from water samples (n = 38) taken from sites where cyanobacteria were suspected to have caused human health symptoms, of which fever and gastrointestinal symptoms were the most common. The isolates were screened by PCR for six virulence gene types (12 genes). The majority (90%) of the strains contained at least one of the virulence genes. Most common amplification products were those of genes (act/aerA/hlyA) that encode cytotoxic enterotoxin and haemolytic products. The genes encoding cytotonic enterotoxins (ast and alt), phospholipase (lip/pla/lipH3/alp-1), elastase (ahyB) and flagellin subunits (flaA/flaB) were also present in 5-37% of the Aeromonas strains. Analysed toxins (cyanobacterial hepatotoxins and neurotoxins, and bacterial endotoxins) were not detectable or were present in only low concentrations in the majority of the samples. The results indicated that the toxins were unlikely to be the main cause of the reported adverse health effects, whereas more attention should be paid to bacteria associated with cyanobacteria as a source of health effects.

Depolymerization of biorefinery lignin by improved laccases of the white-rot fungus Obba rivulosa.

Fungal laccases are attracting enzymes for sustainable valorization of biorefinery lignins. To improve the lignin oxidation capacity of two previously characterized laccase isoenzymes from the white-rot fungus Obba rivulosa, we mutated their substrate-binding site at T1. As a result, the pH optimum of the recombinantly produced laccase variant rOrLcc2-D206N shifted by three units towards neutral pH. O. rivulosa laccase variants with redox mediators oxidized both the dimeric lignin model compound and biorefinery poplar lignin. Significant structural changes, such as selective benzylic α-oxidation, were detected by nuclear magnetic resonance analysis, although no polymerization of lignin was observed by gel permeation chromatography. This suggests that especially rOrLcc2-D206N is a promising candidate for lignin-related applications.

Production of Recombinant Laccase From Coprinopsis cinerea and Its Effect in Mediator Promoted Lignin Oxidation at Neutral pH.

Laccases are multi-copper oxidases that use molecular oxygen as the electron acceptor to oxidize phenolic and indirectly also non-phenolic substrates by mechanisms involving radicals. Due to their eco-friendliness and broad substrate specificity, laccases span a wide range of biotechnological applications. We have heterologously expressed a laccase from the coprophilic basidiomycete Coprinopsis cinerea (CcLcc9) in the methylotrophic yeast Pichia pastoris. The recombinant CcLcc9 (rCcLcc9) oxidized 2,6-dimethoxyphenol in the neutral pH range, and showed thermostability up to 70°C. The rCcLcc9 efficiently oxidized veratryl alcohol to veratraldehyde in the presence of low molecular weight mediators syringyl nitrile, methyl syringate and violuric acid, which are syringyl-type plant phenolics that have shown potential as natural co-oxidants for lignocellulosic materials. In addition, rCcLcc9 is able to depolymerize biorefinery hardwood lignin in the presence of methyl syringate and syringyl nitrile as indicated by gel permeation chromatography, and infrared spectral and nucleic magnetic resonance analyses. Furthermore, we showed that several added-value aromatic compounds, such as vanillin, vanillic acid, syringaldehyde, syringic acid and p-hydroxybenzoic acid, were formed during sequential biocatalytic chemical degradation of biorefinery lignin, indicating that rCcLcc9 harbors a great potential for sustainable processes of circular economy and modern biorefineries.

Culture-independent evidence for the persistent presence and genetic diversity of microcystin-producing Anabaena (Cyanobacteria) in the Gulf of Finland.

The late summer mass occurrences of cyanobacteria in the Baltic Sea are among the largest in the world. These blooms are rarely monotypic and are often composed of a diverse assemblage of cyanobacteria. The toxicity of the blooms is attributed to Nodularia spumigena through the production of the hepatotoxic nodularin. However, the microcystin hepatotoxins have also been reported from the Baltic Sea on a number of occasions. Recent evidence links microcystin production in the Gulf of Finland directly to the genus Anabaena. Here we developed a denaturing gradient gel electrophoresis (DGGE) method based on the mcyE microcystin synthetase gene and ndaF nodularin synthetase gene that allows the culture-independent discrimination of microcystin- and nodularin-producing cyanobacteria directly from environmental samples. We PCR-amplified microcystin and nodularin synthetase genes from environmental samples taken from the Gulf of Finland and separated them on a denaturing gradient gel using optimized conditions. Sequence analyses demonstrate that uncultured microcystin-producing Anabaena strains are genetically more diverse than previously demonstrated from cultured strains. Furthermore, our data show that microcystin-producing Anabaena are widespread in the open Gulf of Finland. Non-parametric statistical analysis suggested that salinity plays an important role in defining the distribution of microcystin-producing Anabaena. Our results indicate that microcystin-producing blooms are a persistent phenomenon in the Gulf of Finland.

Increasing oxygen deficiency changes rare and moderately abundant bacterial communities in coastal soft sediments.

Coastal hypoxia is a major environmental problem worldwide. Hypoxia-induced changes in sediment bacterial communities harm marine ecosystems and alter biogeochemical cycles. Nevertheless, the resistance of sediment bacterial communities to hypoxic stress is unknown. We investigated changes in bacterial communities during hypoxic-anoxic disturbance by artificially inducing oxygen deficiency to the seafloor for 0, 3, 7, and 48 days, with subsequent molecular biological analyses. We further investigated relationships between bacterial communities, benthic macrofauna and nutrient effluxes across the sediment-water-interface during hypoxic-anoxic stress, considering differentially abundant operational taxonomic units (OTUs). The composition of the moderately abundant OTUs changed significantly after seven days of oxygen deficiency, while the abundant and rare OTUs first changed after 48 days. High bacterial diversity maintained the resistance of the communities during oxygen deficiency until it dropped after 48 days, likely due to anoxia-induced loss of macrofaunal diversity and bioturbation. Nutrient fluxes, especially ammonium, correlated positively with the moderate and rare OTUs, including potential sulfate reducers. Correlations may reflect bacteria-mediated nutrient effluxes that accelerate eutrophication. The study suggests that even slightly higher bottom-water oxygen concentrations, which could sustain macrofaunal bioturbation, enable bacterial communities to resist large compositional changes and decrease the harmful consequences of hypoxia in marine ecosystems.

Genetic diversity in strains of the genus Anabaena isolated from planktonic and benthic habitats of the Gulf of Finland (Baltic Sea).

Late summer cyanobacterial blooms in the Baltic Sea contain Anabaena sp. together with Nodularia spumigena and Aphanizomenon flos-aquae. Although Anabaena is common especially in the Gulf of Finland, very little is known about its genetic diversity. Here we undertook a molecular phylogenetic study of 68 Anabaena strains isolated from the brackish Gulf of Finland. We sequenced the 16S rRNA genes from 54 planktonic and 14 benthic Anabaena strains, and rbcL and rpoC1 genes from a subset of these strains. Phylogenetic trees showed that Anabaena strains, from both planktonic and benthic habitats, were genetically diverse. Although the Anabaena strains were morphologically diverse, in our study only one genetically valid species was found to exist in the plankton. Evolutionary distances between benthic Anabaena strains were greater than between planktonic strains, suggesting that benthic habitats allow for the maintenance of greater genetic diversity than planktonic habitats. A number of novel lineages containing only sequences obtained in this study were compiled in the phylogenetical analyses. Thus, it seemed that novel lineages of the genus Anabaena may be present in the Baltic Sea. Our results demonstrate that the Baltic Sea Anabaena strains show surprisingly high genetic diversity.

Maternal gut and breast milk microbiota affect infant gut antibiotic resistome and mobile genetic elements.

The infant gut microbiota has a high abundance of antibiotic resistance genes (ARGs) compared to adults, even in the absence of antibiotic exposure. Here we study potential sources of infant gut ARGs by performing metagenomic sequencing of breast milk, as well as infant and maternal gut microbiomes. We find that fecal ARG and mobile genetic element (MGE) profiles of infants are more similar to those of their own mothers than to those of unrelated mothers. MGEs in mothers' breast milk are also shared with their own infants. Termination of breastfeeding and intrapartum antibiotic prophylaxis of mothers, which have the potential to affect microbial community composition, are associated with higher abundances of specific ARGs, the composition of which is largely shaped by bacterial phylogeny in the infant gut. Our results suggest that infants inherit the legacy of past antibiotic consumption of their mothers via transmission of genes, but microbiota composition still strongly impacts the overall resistance load.

Strains of the cyanobacterial genera Calothrix and Rivularia isolated from the Baltic Sea display cryptic diversity and are distantly related to Gloeotrichia and Tolypothrix.

Strains of the cyanobacterial genus Calothrix display pronounced tapering filaments. These cyanobacteria are benthic, have a worldwide distribution and are among the most easily recognizable cyanobacterial genera. However, it is not clear whether cyanobacterial strains assigned to the genus Calothrix constitute a natural monophyletic group. We sequenced 16S rRNA genes from 42 cyanobacterial cultures and environmental samples belonging to the genus Calothrix, and the morphologically similar genera Rivularia, Gloeotrichia and Tolypothrix. Phylogenetic analysis of the 16S rRNA gene identified large sequence diversity among the Calothrix morphotype strains. Our results demonstrate that Calothrix, Gloeotrichia and Tolypothrix do not form a monophyletic group but instead display a high level of genetic diversity. The evolutionary distances between cyanobacteria, morphologically identified as Calothrix, suggest that they belong to at least five different genera. Our results also suggest that the genus Gloeotrichia is distantly related to the genus Calothrix. We found correlations between genetic grouping and morphology in redundancy analysis. However, morphology alone was not sufficiently reliable to distinguish strains from different 16S rRNA gene clusters. The high level of diversity that we observed confirms the hypothesis that the Rivulariaceae are species rich.

Bacterial diversity and function in the Baltic Sea with an emphasis on cyanobacteria.

In this article we summarize the current knowledge of Baltic Sea cyanobacteria, focusing on diversity, toxicity, and nitrogen fixation in the filamentous heterocystous taxa. We also review the recent results of our microbial diversity studies in planktonic and benthic habitats in the Baltic Sea. Based on molecular analyses, we have improved the understanding of cyanobacterial population structure by assessing genetic diversity within species that are morphologically inseparable. Moreover, we have studied microbial functions such as toxin production and nitrogen fixation in situ under different environmental conditions. Phosphorus limitation of bloom-forming, nitrogen-fixing cyanobacteria was clearly verified, emphasizing the importance of continuous efforts to reduce this element in the Baltic Sea. We have designed a rapid and reliable detection method for the toxic cyanobacterium Nodularia spumigena, which can be used to study bloom formation of this important toxin producer.

Evaluating the mobility potential of antibiotic resistance genes in environmental resistomes without metagenomics.

Antibiotic resistance genes are ubiquitous in the environment. However, only a fraction of them are mobile and able to spread to pathogenic bacteria. Until now, studying the mobility of antibiotic resistance genes in environmental resistomes has been challenging due to inadequate sensitivity and difficulties in contig assembly of metagenome based methods. We developed a new cost and labor efficient method based on Inverse PCR and long read sequencing for studying mobility potential of environmental resistance genes. We applied Inverse PCR on sediment samples and identified 79 different MGE clusters associated with the studied resistance genes, including novel mobile genetic elements, co-selected resistance genes and a new putative antibiotic resistance gene. The results show that the method can be used in antibiotic resistance early warning systems. In comparison to metagenomics, Inverse PCR was markedly more sensitive and provided more data on resistance gene mobility and co-selected resistances.

High-throughput quantification of antibiotic resistance genes from an urban wastewater treatment plant.

Antibiotic resistance among bacteria is a growing problem worldwide, and wastewater treatment plants have been considered as one of the major contributors to the dissemination of antibiotic resistance to the environment. There is a lack of comprehensive quantitative molecular data on extensive numbers of antibiotic resistance genes (ARGs) in different seasons with a sampling strategy that would cover both incoming and outgoing water together with the excess sludge that is removed from the process. In order to fill that gap we present a highly parallel quantitative analysis of ARGs and horizontal gene transfer potential over four seasons at an urban wastewater treatment plant using a high-throughput qPCR array. All analysed transposases and two-thirds of primer sets targeting ARGs were detected in the wastewater. The relative abundance of most of the genes was highest in influent and lower in effluent water and sludge. The resistance profiles of the samples cluster by sample location with a shift from raw influent through the final effluents and dried sludge to the sediments. Wastewater discharge enriched only a few genes, namely Tn25 type transposase gene and clinical class 1 integrons, in the sediment near the discharge pipe, but those enriched genes may indicate a potential for horizontal gene transfer.

Bacterial communities in Arctic first-year drift ice during the winter/spring transition.

Horizontal and vertical variability of first-year drift-ice bacterial communities was investigated along a North-South transect in the Fram Strait during the winter/spring transition. Two different developmental stages were captured along the transect based on the prevailing environmental conditions and the differences in bacterial community composition. The differences in the bacterial communities were likely driven by the changes in sea-ice algal biomass (2.6-5.6 fold differences in chl-a concentrations). Copiotrophic genera common in late spring/summer sea ice, such as Polaribacter, Octadecabacter and Glaciecola, dominated the bacterial communities, supporting the conclusion that the increase in the sea-ice algal biomass was possibly reflected in the sea-ice bacterial communities. Of the dominating bacterial genera, Polaribacter seemed to benefit the most from the increase in algal biomass, since they covered approximately 39% of the total community at the southernmost stations with higher (>6 µg l(-1) ) chl-a concentrations and only 9% at the northernmost station with lower chl-a concentrations (<6 µg l(-1) ). The sea-ice bacterial communities also varied between the ice horizons at all three stations and thus we recommend that for future studies multiple ice horizons be sampled to cover the variability in sea-ice bacterial communities in spring.

Aquaculture changes the profile of antibiotic resistance and mobile genetic element associated genes in Baltic Sea sediments.

Antibiotics are commonly used in aquaculture and they can change the environmental resistome by increasing antibiotic resistance genes (ARGs). Sediment samples were collected from two fish farms located in the Northern Baltic Sea, Finland, and from a site outside the farms (control). The sediment resistome was assessed by using a highly parallel qPCR array containing 295 primer sets to detect ARGs, mobile genetic elements and the 16S rRNA gene. The fish farm resistomes were enriched in transposon and integron associated genes and in ARGs encoding resistance to antibiotics which had been used to treat fish at the farms. Aminoglycoside resistance genes were also enriched in the farm sediments despite the farms not having used aminoglycosides. In contrast, the total relative abundance values of ARGs were higher in the control sediment resistome and they were mainly genes encoding efflux pumps followed by beta-lactam resistance genes, which are found intrinsically in many bacteria. This suggests that there is a natural Baltic sediment resistome. The resistome associated with fish farms can be from native ARGs enriched by antibiotic use at the farms and/or from ARGs and mobile elements that have been introduced by fish farming.

Ice formation and growth shape bacterial community structure in Baltic Sea drift ice.

Drift ice, open water and under-ice water bacterial communities covering several developmental stages from open water to thick ice were studied in the northern Baltic Sea. The bacterial communities were assessed with 16S rRNA gene terminal-restriction fragment length polymorphism and cloning, together with bacterial abundance and production measurements. In the early stages, open water and pancake ice were dominated by Alphaproteobacteria and Actinobacteria, which are common bacterial groups in Baltic Sea wintertime surface waters. The pancake ice bacterial communities were similar to the open-water communities, suggesting that the parent water determines the sea-ice bacterial community in the early stages of sea-ice formation. In consolidated young and thick ice, the bacterial communities were significantly different from water bacterial communities as well as from each other, indicating community development in Baltic Sea drift ice along with ice-type changes. The thick ice was dominated by typical sea-ice genera from classes Flavobacteria and Gammaproteobacteria, similar to those in polar sea-ice bacterial communities. Since the thick ice bacterial community was remarkably different from that of the parent seawater, results indicate that thick ice bacterial communities were recruited from the rarer members of the seawater bacterial community.

Bacterial community dynamics and activity in relation to dissolved organic matter availability during sea-ice formation in a mesocosm experiment.

The structure of sea-ice bacterial communities is frequently different from that in seawater. Bacterial entrainment in sea ice has been studied with traditional microbiological, bacterial abundance, and bacterial production methods. However, the dynamics of the changes in bacterial communities during the transition from open water to frozen sea ice is largely unknown. Given previous evidence that the nutritional status of the parent water may affect bacterial communities during ice formation, bacterial succession was studied in under ice water and sea ice in two series of mesocosms: the first containing seawater from the North Sea and the second containing seawater enriched with algal-derived dissolved organic matter (DOM). The composition and dynamics of bacterial communities were investigated with terminal restriction fragment length polymorphism (T-RFLP), and cloning alongside bacterial production (thymidine and leucine uptake) and abundance measurements (measured by flow cytometry). Enriched and active sea-ice bacterial communities developed in ice formed in both unenriched and DOM-enriched seawater (0-6 days). γ-Proteobacteria dominated in the DOM-enriched samples, indicative of their capability for opportunistic growth in sea ice. The bacterial communities in the unenriched waters and ice consisted of the classes Flavobacteria, α- and γ-Proteobacteria, which are frequently found in natural sea ice in polar regions. Furthermore, the results indicate that seawater bacterial communities are able to adapt rapidly to sudden environmental changes when facing considerable physicochemical stress such as the changes in temperature, salinity, nutrient status, and organic matter supply during ice formation.

Sulphonamide and trimethoprim resistance genes persist in sediments at Baltic Sea aquaculture farms but are not detected in the surrounding environment.

Persistence and dispersal of antibiotic resistance genes (ARGs) are important factors for assessing ARG risk in aquaculture environments. Here, we quantitatively detected ARGs for sulphonamides (sul1 and sul2) and trimethoprim (dfrA1) and an integrase gene for a class 1 integron (intI1) at aquaculture facilities in the northern Baltic Sea, Finland. The ARGs persisted in sediments below fish farms at very low antibiotic concentrations during the 6-year observation period from 2006 to 2012. Although the ARGs persisted in the farm sediments, they were less prevalent in the surrounding sediments. The copy numbers between the sul1 and intI1 genes were significantly correlated suggesting that class 1 integrons may play a role in the prevalence of sul1 in the farm sediments through horizontal gene transfer. In conclusion, the presence of ARGs may limit the effectiveness of antibiotics in treating fish illnesses, thereby causing a potential risk to the aquaculture industry. However, the restricted presence of ARGs at the farms is unlikely to cause serious effects in the northern Baltic Sea sediment environments around the farms.

Sediment bacterial communities reflect the history of a sea basin.

How entire microbial communities are structured across stratified sediments from the historical standpoint is unknown. The Baltic Sea is an ideal research object for historical reconstruction, since it has experienced many fresh- and brackish water periods and is depleted of dissolved oxygen, which increases the sediment's preservation potential. We investigated the bacterial communities, chemical elements (e.g. Cr, Pb Na, P, Sr and U) and sediment composition in a stratified sediment core dated by radiocarbon and spanning 8000 years of Baltic Sea history, using up-to-date multivariate statistics. The communities were analysed by 16S rRNA gene terminal restriction fragment length polymorphism. The communities of the deep Early Litorina and surface Late Litorina Sea laminae were separated from the communities of the middle Litorina Sea laminae, which were associated with elevated concentrations of U and Sr trace elements, palaeo-oxygen and palaeosalinity proxies. Thus, the Litorina Sea laminae were characterized by past oxygen deficiency and salinity increase. The communities of the laminae, bioturbated and homogeneous sediments were differentiated, based on the same historical sea phases, with correct classifications of 90%. Palaeosalinity was one of the major parameters that separated the bacterial communities of the stratified sediments. A discontinuous spatial structure with a surprising increase in community heterogeneity was detected in Litorina Sea sediments from 388 to 422 cm deep, which suggests that a salinity maximum occurred in the central Gulf of Finland app. 6200-6600 years ago. The community heterogeneity decreased from the surface down to 306 cm, which reflected downcore mineralization. The plateau of the decrease was in the app. 2000-year-old sediment layers. Bacterial community data may be used as an additional tool in ocean-drilling projects, in which it is important to detect mineralization plateaus both to determine historically comparable portions of sediment samples and historical events, such as sea-level rise culminations.