Phenology and ecological role of aerobic anoxygenic phototrophs in freshwaters.

BACKGROUND: Aerobic anoxygenic phototrophic (AAP) bacteria are heterotrophic bacteria that supply their metabolism with light energy harvested by bacteriochlorophyll-a-containing reaction centers. Despite their substantial contribution to bacterial biomass, microbial food webs, and carbon cycle, their phenology in freshwater lakes remains unknown. Hence, we investigated seasonal variations of AAP abundance and community composition biweekly across 3 years in a temperate, meso-oligotrophic freshwater lake. RESULTS: AAP bacteria displayed a clear seasonal trend with a spring maximum following the bloom of phytoplankton and a secondary maximum in autumn. As the AAP bacteria represent a highly diverse assemblage of species, we followed their seasonal succession using the amplicon sequencing of the pufM marker gene. To enhance the accuracy of the taxonomic assignment, we developed new pufM primers that generate longer amplicons and compiled the currently largest database of pufM genes, comprising 3633 reference sequences spanning all phyla known to contain AAP species. With this novel resource, we demonstrated that the majority of the species appeared during specific phases of the seasonal cycle, with less than 2% of AAP species detected during the whole year. AAP community presented an indigenous freshwater nature characterized by high resilience and heterogenic adaptations to varying conditions of the freshwater environment. CONCLUSIONS: Our findings highlight the substantial contribution of AAP bacteria to the carbon flow and ecological dynamics of lakes and unveil a recurrent and dynamic seasonal succession of the AAP community. By integrating this information with the indicator of primary production (Chlorophyll-a) and existing ecological models, we show that AAP bacteria play a pivotal role in the recycling of dissolved organic matter released during spring phytoplankton bloom. We suggest a potential role of AAP bacteria within the context of the PEG model and their consideration in further ecological models.

Plasma-derived extracellular vesicle surface markers CD45, CD326 and CD56 correlate with the stage of osteoarthritis: a primary study of a novel and promising diagnostic tool of the disease.

Recently, there is a growing interest in the research based on extracellular vesicles (EVs) which represent paracrine factors secreted by almost all cell types. Both, normal and pathological cells are able to release various types of EVs with different physiological properties, functions and compositions. EVs play an important role in intercellular communication, mechanism and tissue repair. Moreover, EVs could help not only in the treatment of diseases but also in their diagnostics. This work focused on the evaluation of the potential of EVs being used as biomarkers for the diagnosis of osteoarthritis (OA) based on a comparison of the composition of EVs separated from platelet-poor plasma (PPP) of healthy donors and OA patients at different stages of OA. OA is established as a complex syndrome with extensive impact on multiple tissues within the synovial joint. It is a chronic disease of musculoskeletal system that mainly affects the elderly. Depending on the use of the Kellgren-Lawrence classification system, there are four grades of OA which have a negative impact on patients' quality of life. It is very difficult to detect OA in its early stages, so it is necessary to find a new diagnostic method for its timely detection. PPP samples were prepared from whole blood. PPP-EVs were separated from 3 groups of donors-healthy control, early stage OA, end-stage OA, and their content was compared and correlated. EVs from PPP were separated by size exclusion chromatography and characterized in terms of their size, yield and purity by NTA, western blotting, ELISA and flow cytometry. Detection of surface markers expression in EVs was performed using MACSPlex approach. Inflammatory and growth factors in EVs were analysed using MAGPix technology. Our study confirmed significant differences between EVs surface markers of patients and healthy controls correlating with the age of donor (CD63, CD31 and ROR1) and stage of OA (CD45, CD326 and CD56), respectively. Circulating EVs have been under extensive investigation for their capability to predict OA pathology diagnosis as potential targets for biomarker discovery. Taken together, obtained results indicated that PPP-EVs surface markers could be used as potential biomarkers in the early diagnosis of OA.

Bacterial Community Structure of Two Cold Sulfur Springs in Slovakia (Central Europe).

Sulfur-oxidizing bacteria, especially those from hot springs, have attracted the attention of microbiologists for more than 150 years. In contrast, the microbial diversity of cold sulfur springs remains largely unrecognized. Culture-dependent and culture-independent approaches were used to study the diversity of sulfur-oxidizing bacterial communities in two cold sulfur springs in Slovakia. Geological conditions and resulting spring water chemistry appear to be major factors influencing the composition of the sulfur-oxidizing bacterial community. Bacterial communities in both springs were found to be dominated by Proteobacteria with Epsilonproteobacteria being prevalent in the high-salinity Stankovany spring and Alpha- and Gammaproteobacteria in the low-salinity Jovsa spring. Limited overlap was found between culture-dependent and culture-independent approaches with multiple taxa of cultivated sulfur-oxidizing bacteria not being detected by the culture-independent metagenomics approach. Moreover, four cultivated bacterial isolates could represent novel taxa based on the low similarity of their 16S rRNA gene sequence (similarity lower than 98%) to sequences of known bacteria. Our study supports the current view that multiple approaches are required to assess the bacterial diversity in natural habitats and indicates that sulfur springs in Slovakia harbor unique, yet-undescribed microorganisms.

Beneficial Effect of Faecal Microbiota Transplantation on Mild, Moderate and Severe Dextran Sodium Sulphate-Induced Ulcerative Colitis in a Pseudo Germ-Free Animal Model.

Transplantation of faecal microbiota (FMT) is generally considered a safe therapeutic procedure with few adverse effects. The main factors that limit the spread of the use of FMT therapy for idiopathic inflammatory bowel disease (IBD) are the necessity of minimising the risk of infection and transfer of another disease. Obtaining the animal model of UC (ulcerative colitis) by exposure to DSS (dextran sodium sulphate) depends on many factors that significantly affect the result. Per os intake of DSS with water is individual for each animal and results in the development of a range of various forms of induced UC. For this reason, the aim of our study was to evaluate the modulation and regenerative effects of FMT on the clinical and histopathological responses and the changes in the bowel microenvironment in pseudo germ-free (PGF) mice of the BALB/c line subjected to chemical induction of mild, moderate and serious forms of UC. The goal was to obtain new data related to the safety and effectiveness of FMT that can contribute to its improved and optimised use. The animals with mild and moderate forms of UC subjected to FMT treatment exhibited lower severity of the disease and markedly lower damage to the colon, including reduced clinical and histological disease index and decreased inflammatory response of colon mucosa. However, FMT treatment failed to achieve the expected therapeutic effect in animals with the serious form of UC activity. The results of our study indicated a potential safety risk involving development of bacteraemia and also translocation of non-pathogenic representatives of bowel microbiota associated with FMT treatment of animals with a diagnosed serious form of UC.

The Role of Synovial Membrane in the Development of a Potential In Vitro Model of Osteoarthritis.

There is a lack of in vitro models able to plausibly represent the inflammation microenvironment of knee osteoarthritis (OA). We analyzed the molecules released from OA tissues (synovial membrane, cartilage, infrapatellar fat pad) and investigated whether the stimulation of human synovial fibroblasts (SFs), with synthetic cytokines (IL-1ß and TNF-α or IFN-γ) or conditioned media (CM) from OA tissues, influence the SFs' response, in the sense of pro-inflammatory cytokines, chemokines, growth factors, and degradative enzymes modulation. Human SFs were obtained from OA synovial membranes. SFs and their CM were analyzed for biomarkers, proliferation rate, protein profile and gene expression, before and after stimulation. Real-time PCR and multiplex assays quantified OA-related gene expression and biomolecule production. Unlike other activators, CM from OA synovial membrane (CM-SM), significantly up-regulated all genes of interest (IL-6, IL-8, MMP-1, MMP-3, RANTES, MCP-1, TSG-6, YKL-40) in SFs. Multiplex immunoassay analysis showed that levels of OA-related cytokines (IL-6, IL-8, MCP 1, IL-1Ra), chemokine (RANTES) and growth factor (VEGF), produced by CM-SM stimulated SFs, increased significantly compared to non-stimulated SFs. Molecules released from the SM from OA patients induces OA-like changes in vitro, in specific OA synovial populations (SFs). These findings promote the use and establish a compelling in vitro model that simulates the versatility and complexity of the OA disease. This model, in the future, will allow us to study new cell therapies or test drugs by reducing or avoiding animal models.

Study of microbiocenosis of canine dental biofilms.

Dental biofilm is a complex microbial community influenced by many exogenous and endogenous factors. Despite long-term studies, its bacterial composition is still not clearly understood. While most of the research on dental biofilms was conducted in humans, much less information is available from companion animals. In this study, we analyzed the composition of canine dental biofilms using both standard cultivation on solid media and amplicon sequencing, and compared the two approaches. The 16S rRNA gene sequences were used to define the bacterial community of canine dental biofilm with both, culture-dependent and culture-independent methods. After DNA extraction from each sample, the V3-V4 region of the 16S rRNA gene was amplified and sequenced via Illumina MiSeq platform. Isolated bacteria were identified using universal primers and Sanger sequencing. Representatives of 18 bacterial genera belonging to 5 phyla were isolated from solid media. Amplicon sequencing largely expanded this information identifying in total 284 operational taxonomic units belonging to 10 bacterial phyla. Amplicon sequencing revealed much higher diversity of bacteria in the canine dental biofilms, when compared to standard cultivation approach. In contrast, cultured representatives of several bacterial families were not identified by amplicon sequencing.

Lineage-Specific Growth Curves Document Large Differences in Response of Individual Groups of Marine Bacteria to the Top-Down and Bottom-Up Controls.

Marine bacterioplankton represent a diverse assembly of species differing largely in their abundance, physiology, metabolic activity, and role in microbial food webs. To analyze their sensitivity to bottom-up and top-down controls, we performed a manipulation experiment where grazers were removed, with or without the addition of phosphate. Using amplicon-reads normalization by internal standard (ARNIS), we reconstructed growth curves for almost 300 individual phylotypes. Grazer removal caused a rapid growth of most bacterial groups, which grew at rates of 0.6 to 3.5 day-1, with the highest rates (>4 day-1) recorded among Rhodobacteraceae, Oceanospirillales, Alteromonadaceae, and Arcobacteraceae. Based on their growth response, the phylotypes were divided into three basic groups. Most of the phylotypes responded positively to both grazer removal as well as phosphate addition. The second group (containing, e.g., Rhodobacterales and Rhizobiales) responded to the grazer removal but not to the phosphate addition. Finally, some clades, such as SAR11 and Flavobacteriaceae, responded only to phosphate amendment but not to grazer removal. Our results show large differences in bacterial responses to experimental manipulations at the phylotype level and document different life strategies of marine bacterioplankton. In addition, growth curves of 130 phylogroups of aerobic anoxygenic phototrophs were reconstructed based on changes of the functional pufM gene. The use of functional genes together with rRNA genes may significantly expand the scientific potential of the ARNIS technique. IMPORTANCE Growth is one of the main manifestations of life. It is assumed generally that bacterial growth is constrained mostly by nutrient availability (bottom-up control) and grazing (top-down control). Since marine bacteria represent a very diverse assembly of species with different metabolic properties, their growth characteristics also largely differ accordingly. Currently, the growth of marine microorganisms is typically evaluated using microscopy in combination with fluorescence in situ hybridization (FISH). However, these laborious techniques are limited in their throughput and taxonomical resolution. Therefore, we combined a classical manipulation experiment with next-generation sequencing to resolve the growth dynamics of almost 300 bacterial phylogroups in the coastal Adriatic Sea. The analysis documented that most of the phylogroups responded positively to both grazer removal and phosphate addition. We observed significant differences in growth kinetics among closely related species, which could not be distinguished by the classical FISH technique.

Common Presence of Phototrophic Gemmatimonadota in Temperate Freshwater Lakes.

Members of the bacterial phylum Gemmatimonadota are ubiquitous in most natural environments and represent one of the top 10 most abundant bacterial phyla in soil. Sequences affiliated with Gemmatimonadota were also reported from diverse aquatic habitats; however, it remains unknown whether they are native organisms or represent bacteria passively transported from sediment or soil. To address this question, we analyzed metagenomes constructed from five freshwater lakes in central Europe. Based on the 16S rRNA gene frequency, Gemmatimonadota represented from 0.02 to 0.6% of all bacteria in the epilimnion and between 0.1 and 1% in the hypolimnion. These proportions were independently confirmed using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Some cells in the epilimnion were attached to diatoms (Fragilaria sp.) or cyanobacteria (Microcystis sp.), which suggests a close association with phytoplankton. In addition, we reconstructed 45 metagenome-assembled genomes (MAGs) related to Gemmatimonadota They represent several novel lineages, which persist in the studied lakes during the seasons. Three lineages contained photosynthesis gene clusters. One of these lineages was related to Gemmatimonas phototrophica and represented the majority of Gemmatimonadota retrieved from the lakes' epilimnion. The other two lineages came from hypolimnion and probably represented novel photoheterotrophic genera. None of these phototrophic MAGs contained genes for carbon fixation. Since most of the identified MAGs were present during the whole year and cells associated with phytoplankton were observed, we conclude that they represent truly limnic Gemmatimonadota distinct from the previously described species isolated from soils or sediments.IMPORTANCE Photoheterotrophic bacterial phyla such as Gemmatimonadota are key components of many natural environments. Its first photoheterotrophic cultured member, Gemmatimonas phototrophica, was isolated in 2014 from a shallow lake in the Gobi Desert. It contains a unique type of photosynthetic complex encoded by a set of genes which were likely received via horizontal transfer from Proteobacteria We were intrigued to discover how widespread this group is in the natural environment. In the presented study, we analyzed 45 metagenome-assembled genomes (MAGs) that were obtained from five freshwater lakes in Switzerland and Czechia. Interestingly, it was found that phototrophic Gemmatimonadota are relatively common in euphotic zones of the studied lakes, whereas heterotrophic Gemmatimonadota prevail in deeper waters. Moreover, our analysis of the MAGs documented that these freshwater species contain almost the same set of photosynthesis genes identified before in Gemmatimonas phototrophica originating from the Gobi Desert.

Innovative Animal Model of DSS-Induced Ulcerative Colitis in Pseudo Germ-Free Mice.

The aim of this study was to investigate the use of a standardized animal model subjected to antibiotic treatment, and the effects of this treatment on the course of dextran sodium sulphate (DSS)-induced colitis in mice. By decontamination with selective antibiotics and observation of pathogenesis of ulcerative colitis (UC) induced chemically by exposure of mice to various concentrations of DSS, we obtained an optimum animal PGF model of acute UC manifested by mucin depletion, epithelial degeneration and necrosis, leading to the disappearance of epithelial cells, infiltration of lamina propria and submucosa with neutrophils, cryptitis, and accompanied by decreased viability of intestinal microbiota, loss of body weight, dehydration, moderate rectal bleeding, and a decrease in the selected markers of cellular proliferation and apoptosis. The obtained PGF model did not exhibit changes that could contribute to inflammation by means of alteration of the metabolic status and the induced dysbiosis did not serve as a bearer of pathogenic microorganisms participating in development of ulcerative colitis. The inflammatory process was induced particularly by exposure to DSS and its toxic action on compactness and integrity of mucosal barrier in the large intestine. This offers new possibilities of the use of this animal model in studies with or without participation of pathogenic microbiota in IBD pathogenesis.

Light and Primary Production Shape Bacterial Activity and Community Composition of Aerobic Anoxygenic Phototrophic Bacteria in a Microcosm Experiment.

Phytoplankton is a key component of aquatic microbial communities, and metabolic coupling between phytoplankton and bacteria determines the fate of dissolved organic carbon (DOC). Yet, the impact of primary production on bacterial activity and community composition remains largely unknown, as, for example, in the case of aerobic anoxygenic phototrophic (AAP) bacteria that utilize both phytoplankton-derived DOC and light as energy sources. Here, we studied how reduction of primary production in a natural freshwater community affects the bacterial community composition and its activity, focusing primarily on AAP bacteria. The bacterial respiration rate was the lowest when photosynthesis was reduced by direct inhibition of photosystem II and the highest in ambient light condition with no photosynthesis inhibition, suggesting that it was limited by carbon availability. However, bacterial assimilation rates of leucine and glucose were unaffected, indicating that increased bacterial growth efficiency (e.g., due to photoheterotrophy) can help to maintain overall bacterial production when low primary production limits DOC availability. Bacterial community composition was tightly linked to light intensity, mainly due to the increased relative abundance of light-dependent AAP bacteria. This notion shows that changes in bacterial community composition are not necessarily reflected by changes in bacterial production or growth and vice versa. Moreover, we demonstrated for the first time that light can directly affect bacterial community composition, a topic which has been neglected in studies of phytoplankton-bacteria interactions.IMPORTANCE Metabolic coupling between phytoplankton and bacteria determines the fate of dissolved organic carbon in aquatic environments, and yet how changes in the rate of primary production affect the bacterial activity and community composition remains understudied. Here, we experimentally limited the rate of primary production either by lowering light intensity or by adding a photosynthesis inhibitor. The induced decrease had a greater influence on bacterial respiration than on bacterial production and growth rate, especially at an optimal light intensity. This suggests that changes in primary production drive bacterial activity, but the effect on carbon flow may be mitigated by increased bacterial growth efficiencies, especially of light-dependent AAP bacteria. Bacterial activities were independent of changes in bacterial community composition, which were driven by light availability and AAP bacteria. This direct effect of light on composition of bacterial communities has not been documented previously.

Diel changes and diversity of pufM expression in freshwater communities of anoxygenic phototrophic bacteria.

The anoxygenic phototrophic bacteria (APB) are an active component of aquatic microbial communities. While DNA-based studies have delivered a detailed picture of APB diversity, they cannot provide any information on the activity of individual species. Therefore, we focused on the expression of a photosynthetic gene by APB communities in two freshwater lakes (Cep lake and the Rímov Reservoir) in the Czech Republic. First, we analyzed expression levels of pufM during the diel cycle using RT-qPCR. The transcription underwent a strong diel cycle and was inhibited during the day in both lakes. Then, we compared DNA- (total) and RNA-based (active) community composition by sequencing pufM amplicon libraries. We observed large differences in expression activity among different APB phylogroups. While the total APB community in the Rímov Reservoir was dominated by Betaproteobacteria, Alphaproteobacteria prevailed in the active library. A different situation was encountered in the oligotrophic lake Cep where Betaproteobacteria (order Burkholderiales) dominated both the DNA and RNA libraries. Interestingly, in Cep lake we found smaller amounts of highly active uncultured phototrophic Chloroflexi, as well as phototrophic Gemmatimonadetes. Despite the large diversity of APB communities, light repression of pufM expression seems to be a common feature of all aerobic APB present in the studied lakes.

Two rep genes in small cryptic plasmid pKST21 of Escherichia coli.

The complete nucleotide sequence of a small cryptic plasmid pKST21 from Escherichia coli was determined. This plasmid is 1,460 bp long with an overall GC content of 51 %. Based on sequence analysis, the presence of two segments with different average GC density was observed. The segment with higher GC content revealed 98-90 % similarity to several small plasmids of E. coli and to pCR1 from Gram-positive Corynebacterium renale. Plasmid pKST21 possesses two conversely oriented open reading frames encoding proteins with a high degree of amino acid identity to Rep proteins involved in replication. ORF1 encodes replication protein similar to RepA protein of Bartonella tribocorum or Bacillus cereus plasmids or to the putative plasmid Rep protein from ecologically close Selenomonas ruminantium. ORF2 similarly encodes a replication protein, which shares 97 % homology with Rep protein from C. renale. Genetic diversity observed in plasmid pKST21 indicates a mosaic structure of the plasmid with different segments acquired from different sources. Deletion analysis showed that both fragments carrying the repA and repB genes are necessary for the replication of pKST21 in E. coli. The presence of plasmid with the same gene composition was revealed in 14 % of tested E. coli isolates from the rumen of sheep. All these strains produced identical ERIC-PCR profiles indicating isogenic origin of the strain and lack of horizontal gene transfer of pKST21 plasmid.

Variability of putative rep gene cassettes in Selenomonas ruminantium plasmids.

Characteristic feature of the most of Selenomonas ruminantium cryptic plasmids is the presence of short, conserved sequences encompassing the gene for replication protein creating a potential rep gene cassette. PCR-based experiment was designed to analyse the genetic organization of putative plasmid rep modules and to assess S. ruminantium plasmid biodiversity. Analysed PCR amplicons contained single open reading frames encoding for putative replication proteins. While most of the derived protein sequences were often found to be conserved among putative plasmid molecules, at noncoding regions, genetic variability was observed to various extents. Complete nucleotide sequence of a plasmid was determined that contained probably a new rep gene only distantly related to known selenomonas Rep proteins but at noncoding regions shared high homology with already known plasmids. Our results document considerable structural instability and sequence variability of analysed rep gene cassettes and suggest a modular structure of S. ruminantium plasmids potentially accessible for rep gene module exchanges.