Unique architecture of microbial snottites from a methane driven biofilm revealed by confocal microscopy.

Microbial biofilms occur in many shapes and different dimensions. In natural and semi-artificial caves they are forming pendulous structures of 10 cm and more. In this study a methane driven microbial community of a former medicinal spring was investigated. The habitat was completely covered by massive biofilms and snottites with a wobbly, gelatinous appearance. By using fluorescence techniques in combination with confocal laser scanning microscopy the architecture of these so far unknown snottites was examined. The imaging approaches applied comprised reflection of geogenic and cellular origin, possible autofluorescence, nucleic acid staining for bacterial cells, protein staining for bacteria and extracellular fine structures, calcofluor white for ß 1 → 3, ß 1 → 4 polysaccharide staining for possible fungi as well as lectin staining for the extracellular biofilm matrix glycoconjugates. The results showed a highly complex, intricate structure with voluminous, globular, and tube-like glycoconjugates of different dimensions and densities. In addition, filamentous bacteria seem to provide additional strength to the snottites. After screening with all commercially available lectins, by means of fluorescence lectin barcoding and subsequent fluorescence lectin binding analysis, the AAL, PNA, LEA, and Ban lectins identified α-Fuc, ß-Gal, ß-GlcNAc, and α-Man with α-Fuc as a major component. Examination of the outer boundary with fluorescent beads revealed a potential outer layer which could not be stained by any of the fluorescent probes applied. Finally, suggestions are made to further elucidate the characteristics of these unusual microbial biofilms in form of snottites. RESEARCH HIGHLIGHTS: The gelatinous snottites revealed at the microscale a highly complex structure not seen before. The extracellular matrix of the snottite biofilm was identified as clusters of different shape and density. The matrix of snottites was examined by taking advantage of 78 fluorescently-labeled lectins. The extracellular matrix glycoconjugates of snottites identified comprised: α-Fuc, ß-Gal, ß-GlcNAc, and α-Man. Probing the snottite outer surface indicated an additional unknown stratum.

High resolution functional analysis and community structure of photogranules.

Photogranules are spherical aggregates formed of complex phototrophic ecosystems with potential for "aeration-free" wastewater treatment. Photogranules from a sequencing batch reactor were investigated by fluorescence microscopy, 16S/18S rRNA gene amplicon sequencing, microsensors, and stable- and radioisotope incubations to determine the granules' composition, nutrient distribution, and light, carbon, and nitrogen budgets. The photogranules were biologically and chemically stratified, with filamentous cyanobacteria arranged in discrete layers and forming a scaffold to which other organisms were attached. Oxygen, nitrate, and light gradients were also detectable. Photosynthetic activity and nitrification were both predominantly restricted to the outer 500 µm, but while photosynthesis was relatively insensitive to the oxygen and nutrient (ammonium, phosphate, acetate) concentrations tested, nitrification was highly sensitive. Oxygen was cycled internally, with oxygen produced through photosynthesis rapidly consumed by aerobic respiration and nitrification. Oxygen production and consumption were well balanced. Similarly, nitrogen was cycled through paired nitrification and denitrification, and carbon was exchanged through photosynthesis and respiration. Our findings highlight that photogranules are complete, complex ecosystems with multiple linked nutrient cycles and will aid engineering decisions in photogranular wastewater treatment.

N2 -fixation can sustain wastewater treatment performance of photogranules under nitrogen-limiting conditions.

Wastewater characteristics can vary significantly, and in some municipal wastewaters the N:P ratio is as low as 5 resulting in nitrogen-limiting conditions. In this study, the microbial community, function, and morphology of photogranules under nitrogen-replete (N+) and limiting (N-) conditions was assessed in sequencing batch reactors. Photogranules under N- condition were nitrogen deprived 2/3 of a batch cycle duration. Surprisingly, this nitrogen limitation had no adverse effect on biomass productivity. Moreover, phosphorus and chemical oxygen demand removal were similar to their removal under N+ conditions. Although performance was similar, the difference in granule morphology was obvious. While N+ photogranules were dense and structurally confined, N- photogranules showed loose structures with occasional voids. Microbial community analysis revealed high abundance of cyanobacteria capable of N2 -fixation. These were higher at N- (38%) than N+ (29%) treatments, showing that photogranules could adjust and maintain treatment performance and high biomass productivity by means of N2 -fixation.

The biofilm matrix: multitasking in a shared space.

The biofilm matrix can be considered to be a shared space for the encased microbial cells, comprising a wide variety of extracellular polymeric substances (EPS), such as polysaccharides, proteins, amyloids, lipids and extracellular DNA (eDNA), as well as membrane vesicles and humic-like microbially derived refractory substances. EPS are dynamic in space and time and their components interact in complex ways, fulfilling various functions: to stabilize the matrix, acquire nutrients, retain and protect eDNA or exoenzymes, or offer sorption sites for ions and hydrophobic substances. The retention of exoenzymes effectively renders the biofilm matrix an external digestion system influencing the global turnover of biopolymers, considering the ubiquitous relevance of biofilms. Physico-chemical and biological interactions and environmental conditions enable biofilm systems to morph into films, microcolonies and macrocolonies, films, ridges, ripples, columns, pellicles, bubbles, mushrooms and suspended aggregates - in response to the very diverse conditions confronting a particular biofilm community. Assembly and dynamics of the matrix are mostly coordinated by secondary messengers, signalling molecules or small RNAs, in both medically relevant and environmental biofilms. Fully deciphering how bacteria provide structure to the matrix, and thus facilitate and benefit from extracellular reactions, remains the challenge for future biofilm research.

Distinct glycoconjugate cell surface structures make the pelagic diatom Thalassiosira rotula an attractive habitat for bacteria.

Interactions between marine diatoms and bacteria have been studied for decades. However, the visualization of physical interactions between these diatoms and their colonizers is still limited. To enhance our understanding of these specific interactions, a new Thalassiosira rotula isolate from the North Sea (strain 8673) was characterized by scanning electron microscopy and confocal laser scanning microscopy (CLSM) after staining with fluorescently labeled lectins targeting specific glycoconjugates. To investigate defined interactions of this strain with bacteria the new strain was made axenic and co-cultivated with a natural bacterial community and in two- or three-partner consortia with different bacteria of the Roseobacter group, Gammaproteobacteria and Bacteroidetes. The CLSM analysis of the consortia identified six out of 78 different lectins as very suitable to characterize glycoconjugates of T. rotula. The resulting images show that fucose-containing threads were the dominant glycoconjugates secreted by the T. rotula cells but chitin and to a lesser extent other glycoconjugates were also identified. Bacteria attached predominantly to the fucose glycoconjugates. The colonizing bacteria showed various attachment patterns such as adhering to the diatom threads in aggregates only or attaching to both the surfaces and the threads of the diatom. Interestingly the colonization patterns of single bacteria differed strikingly from those of bacterial co-cultures, indicating that interactions between two bacterial species impacted the colonization of the diatom. Our observations help to better understand physical interactions and specific colonization patterns of distinct bacterial mono- and co-cultures with an abundant diatom of costal seas.

Matrix glycoconjugate characterization in multispecies biofilms and bioaggregates from the environment by means of fluorescently-labeled lectins.

Environmental biofilms represent a complex mixture of different microorganisms. Their identity is usually analyzed by means of nucleic acid-based techniques. However, these biofilms are also composed of a highly complex extracellular matrix produced by the microbes within a particular biofilm system. The biochemical identity of this extracellular matrix remains in many cases an intractable part of biofilms and bioaggregates. Consequently, there is a need for an approach that will give access to the fully hydrated structure of the extracellular matrix or at least a major part of it. A crucial compound of the matrix identified as carbohydrate-based polymers represents major structural and functional constituents. These glycoconjugates can be characterized by using fluorescently-labeled lectins in combination with confocal laser scanning microscopy. The lectin approach is defined previously, as fluorescence lectin barcoding (FLBC) and fluorescence lectin-binding analysis (FLBA), where FLBC is equal to the screening of a particular sample with all the commercially available lectins and FLBA is the actual analysis of the matrix throughout an experiment with a selected panel of lectins. As the application of immune-based techniques in environmental biofilm systems is impossible, the lectin approach is currently the only option for probing lectin-specific glycoconjugates in complex biofilms and bioaggregates. From all the commercially available lectins tested, the lectins such as AAL, HAA, WGA, ConA, IAA, HPA, and LEA showed the highest binding efficiency. Furthermore, 20 of the overall lectins tested showed an intermediate signal intensity, nevertheless very useful for the assessment of matrix glycoconjugates. With the data compiled, we shall virtually shed more light on the dark matter of the extracellular matrix and their 3-dimensional distribution in environmental biofilm systems. The results will be helpful in future studies with a focus on the extracellular matrix glycoconjugates present in environmental microbial communities.

Biofilm forming rhizobacteria affect the physiological and biochemical responses of wheat to drought.

Plant growth promoting rhizobacteria (PGPR) can attenuate the adverse effects of water deficit on plant growth. Since drought stress tolerance of bacteria has earlier been associated to biofilm formation, we aimed to investigate the role of bacterial biofilm formation in their PGPR activity upon drought stress. To this end, a biofilm-forming bacterial collection was isolated from the rhizospheres of native arid grassland plants, and characterized by their drought tolerance and evaluated on their plant growth promoting properties. Most bacterial strains formed biofilm in vitro. Most isolates were drought tolerant, produced auxins, showed 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity and solubilized mineral phosphate and potassium, but at considerably different levels. Greenhouse experiments with the most promising isolates, B1, B2 and B3, under three levels of water deficit and two wheat varieties led to an increased relative water content and increased harvest index at both moderate and severe water deficit. However, the bacteria did not affect these plant parameters upon regular watering. In addition, decreased hydrogen peroxide levels and increased glutathione S-transferase activity occurred under water deficit. Based on these results, we conclude that by improving root traits and antioxidant defensive system of wheat, arid grassland rhizospheric biofilm forming bacilli may promote plant growth under water scarcity.

Catabolism of sialic acids in an environmental microbial community.

Sialic acids are a family of nine-carbon negatively charged carbohydrates. In animals, they are abundant on mucosa surfaces as terminal carbohydrates of mucin glycoproteins. Some commensal and pathogenic bacteria are able to release, take up and catabolize sialic acids. Recently, sialic acids have been discovered to be widespread among most microorganisms. Although the catabolism of sialic acids has been intensively investigated in the field of host-microbe interactions, very limited information is available on microbial degradation of sialic acids produced by environmental microorganisms. In this study, the catabolic pathways of sialic acids within a microbial community dominated by 'Candidatus Accumulibacter' were evaluated. Protein alignment tools were used to detect the presence of the different proteins involved in the utilization of sialic acids in the flanking populations detected by 16S rRNA gene amplicon sequencing. The results showed the ability of Clostridium to release sialic acids from the glycan chains by the action of a sialidase. Clostridium and Chryseobacterium can take up free sialic acids and utilize them as nutrient. Interestingly, these results display similarities with the catabolism of sialic acids by the gut microbiota. This study points at the importance of sialic acids in environmental communities in the absence of eukaryotic hosts.

The importance of biofilm formation for cultivation of a Micrarchaeon and its interactions with its Thermoplasmatales host.

Micrarchaeota is a distinctive lineage assigned to the DPANN archaea, which includes poorly characterised microorganisms with reduced genomes that likely depend on interactions with hosts for growth and survival. Here, we report the enrichment of a stable co-culture of a member of the Micrarchaeota (Ca. Micrarchaeum harzensis) together with its Thermoplasmatales host (Ca. Scheffleriplasma hospitalis), as well as the isolation of the latter. We show that symbiont-host interactions depend on biofilm formation as evidenced by growth experiments, comparative transcriptomic analyses and electron microscopy. In addition, genomic, metabolomic, extracellular polymeric substances and lipid content analyses indicate that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host. Our study of the cell biology and physiology of a Micrarchaeon and its host adds to our limited knowledge of archaeal symbioses.

Environmental conditions affect the food quality of plastic associated biofilms for the benthic grazer Physa fontinalis.

With an ever-increasing amount of plastic pollution in the various aquatic ecosystems around the world, the effects on organisms are still not fully understood. Most studies focus on direct effects posed by plastic intake or entanglement, but plastic debris can also affect primary production of biofilms and have an indirect impact on its consumers. This study investigates the primary production on three common plastic types in freshwater and its food quality for a benthic grazer. We hypothesized that different polymer types affect biofilm composition as well as the life parameters of its consumers. We incubated polyethylene (PE), polyethylene terephthalate (PET) and polystyrene (PS) as well as glass (control) in a productive freshwater creek for natural biofilm establishment. To account for changes in the environmental conditions, the experiment was conducted twice during winter and late spring, respectively. These biofilms were offered to the freshwater gastropod Physa fontinalis as sole food source. Growth and reproduction of the snails were measured to monitor sublethal effects. Additionally, biofilm composition was observed using confocal laser scanning microscopy (CLSM). In winter, snails feeding off PET and PE showed a significantly lower egg production and lower growth rates were observed on PET. No such effects occurred in spring. CLSM data revealed, that algal growth was significantly lower on PE and PET during the winter treatment compared to PS and glass. Since we could only find these effects during the colder and darker months (January-March), the microbial colonization on PE and PET was inhibited by the substrate under less favorable conditions of temperature and light. Hence, benign conditions may mask the adverse effects of microplastic on food webs. Our findings show that future studies on the plastisphere will need to consider such variations to further understand the influence of plastic pollution on primary production and higher trophic levels.

Production of nonulosonic acids in the extracellular polymeric substances of "Candidatus Accumulibacter phosphatis".

Nonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in "Candidatus Accumulibacter phosphatis", a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of "Ca. Accumulibacter" to produce different types of NulOs. Proteomic analysis showed the ability of "Ca. Accumulibacter" to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of "Ca. Accumulibacter" in particular, and biofilms in general. KEY POINTS: •"Ca. Accumulibacter" has the potential to produce a range of nonulosonic acids. •Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids. •The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

Who put the film in biofilm? The migration of a term from wastewater engineering to medicine and beyond.

Sessile microorganisms were described as early as the seventeenth century. However, the term biofilm arose only in the 1960s in wastewater treatment research and was adopted later in marine fouling and in medical and dental microbiology. The sessile mode of microbial life was gradually recognized to be predominant on Earth, and the term biofilm became established for the growth of microorganisms in aggregates, frequently associated with interfaces, although many, if not the majority, of them not being continuous "films" in the strict sense. In this sessile form of life, microorganisms live in close proximity in a matrix of extracellular polymeric substances (EPS). They share emerging properties, clearly distinct from solitary free floating planktonic microbial cells. Common characteristics include the formation of synergistic microconsortia, using the EPS matrix as an external digestion system, the formation of gradients and high biodiversity over microscopically small distances, resource capture and retention, facilitated gene exchange as well as intercellular communication, and enhanced tolerance to antimicrobials. Thus, biofilms belong to the class of collective systems in biology, like forests, beehives, or coral reefs, although the term film addresses only one form of the various manifestations of microbial aggregates. The uncertainty of this term is discussed, and it is acknowledged that it will not likely be replaced soon, but it is recommended to understand these communities in the broader sense of microbial aggregates.

Candidatus Sulfurimonas marisnigri sp. nov. and Candidatus Sulfurimonas baltica sp. nov., thiotrophic manganese oxide reducing chemolithoautotrophs of the class Campylobacteria isolated from the pelagic redoxclines of the Black Sea and the Baltic Sea.

Species of the genus Sulfurimonas are reported and isolated from terrestrial habitats and marine sediments and water columns with steep redox gradients. Here we report on the isolation of strains SoZ1 and GD2 from the pelagic redoxcline of the Black Sea and the Baltic Sea, respectively. Both strains are gram-stain-negative and appear as short and slightly curved motile rods. The autecological preferences for growth of strain SoZ1 were 0-25°C (optimum 20°C), pH 6.5-9.0 (optimum pH 7.5-8.0) and salinity 10-40gL-1 (optimum 25gL-1). Preferences for growth of strain GD2 were 0-20°C (optimum 15°C), pH 7.0-8.0 (optimum pH 7.0-7.5) and salinity 5-40gL-1 (optimum 21gL-1). Strain SoZ1 grew chemolithoautotrophically, while strain GD2 also showed heterotrophic growth with short chained fatty acids as carbon source. Both species utilized hydrogen (H2), sulfide (H2S here taken as the sum of H2S, HS- and S2-), elemental sulfur (S0) and thiosulfate (S2O32-) as electron donors and nitrate (NO3-), oxygen (O2) and particulate manganese oxide (MnO2) as electron acceptors. Based on 16S rRNA gene sequence similarity, both strains cluster within the genus Sulfurimonas with Sulfurimonas gotlandica GD1T as the closest cultured relative species with a sequence similarity of 96.74% and 96.41% for strain SoZ1 and strain GD2, respectively. Strains SoZ1 and GD2 share a ribosomal 16S sequence similarity of 99.27% and were demarcated based on average nucleotide identity and average amino acid identity of the whole genome sequence. These calculations have been applied to the whole genus. We propose the names Candidatus Sulfurimonas marisnigri sp. nov. and Candidatus Sulfurimonas baltica sp. nov. for the thiotrophic manganese reducing culture isolates from the Black Sea and Baltic Sea, respectively.

Interaction of cyanobacteria with calcium facilitates the sedimentation of microplastics in a eutrophic reservoir.

Low-density microplastics are frequently found in sediments of many lakes and reservoirs. The processes leading to sedimentation of initially buoyant polymers are poorly understood for inland waters. This study investigated the impact of biofilm formation and aggregation on the density of buoyant polyethylene microplastics. Biofilm formation on polyethylene films (4 × 4 × 0.15 mm) was studied in a eutrophic reservoir (Bautzen, Saxony, Germany). Additionally, aggregation dynamics of small PE microplastics (~85 µm) with cyanobacteria were investigated in laboratory experiments. During summer phototrophic sessile cyanobacteria (Chamaesiphon spp. and Leptolyngbya spp.) precipitated calcite while forming biofilms on microplastics incubated in Bautzen reservoir. Subsequently the density of the biofilms led to sinking of roughly 10% of the polyethylene particles within 29 days of incubation. In the laboratory experiments planktonic cyanobacteria (Microcystis spp.) formed large and dense cell aggregates under the influence of elevated Ca2+ concentrations. These aggregates enclosed microplastic particles and led to sinking of a small portion (~0.4 %) of polyethylene microplastics. This study showed that both sessile and planktonic phototrophic microorganisms mediate processes influenced by calcium which facilitates densification and sinking of microplastics in freshwater reservoirs. Loss of buoyancy leads to particle sedimentation and could be a prerequisite for the permanent burial of microplastics within reservoir sediments.

Decorating the Anammox House: Sialic Acids and Sulfated Glycosaminoglycans in the Extracellular Polymeric Substances of Anammox Granular Sludge.

Anammox (anaerobic ammonium oxidation) bacteria are important for the nitrogen cycle in both natural environments and wastewater treatment plants. These bacteria have a strong tendency to grow in aggregates like biofilms and granular sludge. To understand the formation of anammox aggregates, it is required to unravel the composition of the extracellular polymeric substances (EPS), which are produced by the bacteria to develop into aggregates and granules. Here, we investigated anionic polymers in anammox granular sludge, focussing on sialic acids and sulfated glycosaminoglycans. Quantification assays and fluorescent stains indicated that sialic acids and sulfated glycosaminoglycans were present in the anammox EPS (1.6% equivalents of sialic acids and 2.4% equivalents of sulfated glycosaminoglycans). Additionally, the potential genes for the biosynthesis of sialic acids and sulfated glycosaminoglycans were analyzed in the anammox draft genomes. The finding of these components in anammox granular sludge and previously in other nonpathogenic bacteria pointed out that sialic acids and sulfated glycosaminoglycans are worth investigating in the context of a broader function in microbial communities and biofilm systems in general.

Biofouling, metal sorption and aggregation are related to sinking of microplastics in a stratified reservoir.

Microplastic particles entering aquatic systems are rapidly colonized by microbial biofilms. The presence of microbial biomass may cause sinking of particles and as a consequence prevent their transport to the oceans. We studied microbial colonization of different polymer particles exposed in the epi-, meta- and hypolimnion of a freshwater reservoir during late summer for 47 days. Parameters measured included biofilm formation, metal sorption and sinking velocities. Microbial biofilms contained bacteria, cyanobacteria and algae as well as inorganic particles such as iron oxides. Regardless of biofilm thickness and biovolumes of different biofilm constituents, single polyethylene (PE) particles stayed buoyant, whereas the sinking velocity of single polystyrene (PS) and polyethylene terephthalate (PET) particles did not change significantly compared to initial values. During exposition, a mixing event occurred, by which anoxic, iron-rich water from the hypolimnion was mixed with water from upper layers. This induced aggregation and sinking of hypolimnetic PE particles together with organic matter, cyanobacteria colonies and iron minerals.

Aerobic granular sludge contains Hyaluronic acid-like and sulfated glycosaminoglycans-like polymers.

Glycosaminoglycans (GAGs) are linear heteropolysaccharides containing a derivative of an amino sugar. The possibility of the presence of GAGs in aerobic granular sludge was studied by combining SDS-PAGE with Alcian Blue staining (at pH 2.5 and 1), FTIR, mammalian Hyaluronic acid and sulfated GAG analysis kits, enzymatic digestion and specific in situ visualization by Heparin Red and lectin staining. GAGs, including Hyaluronic acid-like and sulfated GAGs-like polymers were found in aerobic granular sludge. The sulfated GAGs-like polymers contained Chondroitin sulfate and Heparan sulfate/Heparin based on their sensitivity to the digestion by Chondroitinase ABC and Heparinase I & III. Heparin Red and lectin staining demonstrated that, the sulfated GAGs-like polymers were not only present in the extracellular matrix, but also filled in the space between the cells inside the microcolonies. The GAGs-like polymers in aerobic granules were different from those produced by pathogenic bacteria but resemble those produced by vertebrates. Findings reported here and in previous studies on granular sludge described in literature indicate that GAGs-like polymers might be widespread in granular sludge/biofilm and contribute to the stability of these systems. The extracellular polymeric substances (EPS) in granular sludge/biofilm are far more complicated than they are currently appreciated. Integrated and multidisciplinary analyses are significantly required to study the EPS.

Biofilms facilitate cheating and social exploitation of ß-lactam resistance in Escherichia coli.

Gram-negative bacteria such as Escherichia coli commonly resist ß-lactam antibiotics using plasmid-encoded ß-lactamase enzymes. Bacterial strains that express ß-lactamases have been found to detoxify liquid cultures and thus to protect genetically susceptible strains, constituting a clear laboratory example of social protection. These results are not necessarily general; on solid media, for instance, the rapid bactericidal action of ß-lactams largely prevents social protection. Here, we tested the hypothesis that the greater tolerance of biofilm bacteria for ß-lactams would facilitate social interactions. We used a recently isolated E. coli strain, capable of strong biofilm formation, to compare how cooperation and exploitation in colony biofilms and broth culture drives the dynamics of a non-conjugative plasmid encoding a clinically important ß-lactamase. Susceptible cells in biofilms were tolerant of ampicillin-high doses and several days of exposure were required to kill them. In support of our hypothesis, we found robust social protection of susceptible E. coli in biofilms, despite fine-scale physical separation of resistant and susceptible cells and lower rates of production of extracellular ß-lactamase. In contrast, social interactions in broth were restricted to a relatively narrow range of ampicillin doses. Our results show that ß-lactam selection pressure on Gram-negative biofilms leads to cooperative resistance characterized by a low equilibrium frequency of resistance plasmids, sufficient to protect all cells.

Insight Into Interactions of Thermoacidophilic Archaea With Elemental Sulfur: Biofilm Dynamics and EPS Analysis.

Biooxidation of reduced inorganic sulfur compounds (RISCs) by thermoacidophiles is of particular interest for the biomining industry and for environmental issues, e.g., formation of acid mine drainage (AMD). Up to now, interfacial interactions of acidophiles with elemental sulfur as well as the mechanisms of sulfur oxidation by acidophiles, especially thermoacidophiles, are not yet fully clear. This work focused on how a crenarchaeal isolate Acidianus sp. DSM 29099 interacts with elemental sulfur. Analysis by Confocal laser scanning microscopy (CLSM) and Atomic force microscopy (AFM) in combination with Epifluorescence microscopy (EFM) shows that biofilms on elemental sulfur are characterized by single colonies and a monolayer in first stage and later on 3-D structures with a diameter of up to 100 µm. The analysis of extracellular polymeric substances (EPS) by a non-destructive lectin approach (fluorescence lectin-barcoding analysis) using several fluorochromes shows that intial attachment was featured by footprints rich in biofilm cells that were embedded in an EPS matrix consisting of various glycoconjugates. Wet chemistry data indicate that carbohydrates, proteins, lipids and uronic acids are the main components. Attenuated reflectance (ATR)-Fourier transformation infrared spectroscopy (FTIR) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) indicate glucose and mannose as the main monosaccharides in EPS polysaccharides. EPS composition as well as sugar types in EPS vary according to substrate (sulfur or tetrathionate) and lifestyle (biofilms and planktonic cells). This study provides information on the building blocks/make up as well as dynamics of biofilms of thermoacidophilic archaea in extremely acidic environments.

Encrustations on ureteral stents from patients without urinary tract infection reveal distinct urotypes and a low bacterial load.

BACKGROUND: Current knowledge of the urinary tract microbiome is limited to urine analysis and analysis of biofilms formed on Foley catheters. Bacterial biofilms on ureteral stents have rarely been investigated, and no cultivation-independent data are available on the microbiome of the encrustations on the stents. RESULTS: The typical encrustations of organic and inorganic urine-derived material, including microbial biofilms formed during 3-6 weeks on ureteral stents in patients treated for kidney and ureteral stones, and without reported urinary tract infection at the time of stent insertion, were analysed. Next-generation sequencing of the 16S rRNA gene V3-V4 region revealed presence of different urotypes, distinct bacterial communities. Analysis of bacterial load was performed by combining quantification of 16S rRNA gene copy numbers by qPCR with microscopy and cultivation-dependent analysis methods, which revealed that ureteral stent biofilms mostly contain low numbers of bacteria. Fluorescence microscopy indicates the presence of extracellular DNA. Bacteria identified in biofilms by microscopy had mostly morphogenic similarities to gram-positive bacteria, in few cases to Lactobacillus and Corynebacterium, while sequencing showed many additional bacterial genera. Weddellite crystals were absent in biofilms of patients with Enterobacterales and Corynebacterium-dominated microbiomes. CONCLUSIONS: This study provides novel insights into the bacterial burden in ureteral stent encrustations and the urinary tract microbiome. Short-term (3-6 weeks) ureteral stenting is associated with a low load of viable and visible bacteria in ureteral stent encrustations, which may be different from long-term stenting. Patients could be classified according to different urotypes, some of which were dominated by potentially pathogenic species. Facultative pathogens however appear to be a common feature in patients without clinically manifested urinary tract infection. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02845726 . Registered on 30 June 2016-retrospectively registered.