Preloading Long-Chain Quaternary Ammonium Groups to Synthesize a High-Efficient Anion-Exchange Resin for Eliminating Bacterial Contaminants in Drinking Water.

Bacterial contamination in drinking water is a global health concern, necessitating the development of highly efficient treatment techniques. Anion-exchange resins (AERs) have long been employed for removing anionic contaminants from drinking water, but their performance for bacterial contamination is poor. Here, we develop a novel AER (AER6-1) with exceptional bactericidal effects and ultrafast adsorption rates of extracellular DNA (eDNA) (2.2- and 11.5-fold compared to other AERs) achieved through preloading quaternary ammonium groups (QAGs) with hexyl chain (-C6-N+-) on the resin exterior and successively grafting QAGs with a methyl chain (-C1-N+-) inside a resin pore. The AER6-1 outperforms other commercial AERs and ultraviolet disinfection, exhibiting superior elimination of total bacteria, potential pathogens (Escherichia coli and Pseudomonas aeruginosa), eDNA, and antibiotic resistance genes (mexF, mexB, and bacA) in actual drinking water, while maintaining a comparable anion exchange capacity with other commercial AERs. Theoretical calculations of density functional theory and xDLVO combined with XPS elucidate the crucial roles of hydrogen bonding and hydrophobic force provided by the resin skeleton and -C6-N+- in cleaving the bacterial cell membrane and increasing the adsorption kinetics on eDNA. This study broadens the scope of AERs and highlights an effective way of simultaneously removing bacterial and anionic contaminants from drinking water.

Aggregatibacter actinomycetemcomitans Dispersin B: The Quintessential Antibiofilm Enzyme.

The extracellular matrix of most bacterial biofilms contains polysaccharides, proteins, and nucleic acids. These biopolymers have been shown to mediate fundamental biofilm-related phenotypes including surface attachment, intercellular adhesion, and biocide resistance. Enzymes that degrade polymeric biofilm matrix components, including glycoside hydrolases, proteases, and nucleases, are useful tools for studying the structure and function of biofilm matrix components and are also being investigated as potential antibiofilm agents for clinical use. Dispersin B is a well-studied, broad-spectrum antibiofilm glycoside hydrolase produced by Aggregatibacter actinomycetemcomitans. Dispersin B degrades poly-N-acetylglucosamine, a biofilm matrix polysaccharide that mediates biofilm formation, stress tolerance, and biocide resistance in numerous Gram-negative and Gram-positive pathogens. Dispersin B has been shown to inhibit biofilm and pellicle formation; detach preformed biofilms; disaggregate bacterial flocs; sensitize preformed biofilms to detachment by enzymes, detergents, and metal chelators; and sensitize preformed biofilms to killing by antiseptics, antibiotics, bacteriophages, macrophages, and predatory bacteria. This review summarizes the results of nearly 100 in vitro and in vivo studies that have been carried out on dispersin B since its discovery 20 years ago. These include investigations into the biological function of the enzyme, its structure and mechanism of action, and its in vitro and in vivo antibiofilm activities against numerous bacterial species. Also discussed are potential clinical applications of dispersin B.

Low concentrations of tetrabromobisphenol A promote the biofilm formation of methicillin-resistant Staphylococcus aureus.

The effect and underlying mechanism of tetrabromobisphenol A (TBBPA), a plastic additive, on biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA USA300) remain unknown. This study first investigated the impact of different concentrations of TBBPA on the growth and biofilm formation of USA300. The results indicated that a low concentration (0.5 mg/L) of TBBPA promoted the growth and biofilm formation of USA300, whereas high concentrations (5 mg/L and 10 mg/L) of TBBPA had inhibitory effects. Further exploration revealed that the low concentration of TBBPA enhance biofilm formation by promoting the synthesis of extracellular proteins, release of extracellular DNA (eDNA), and production of staphyloxanthin. RTqPCR analysis demonstrated that the low concentration of TBBPA upregulated genes associated with extracellular protein synthesis (sarA, fnbA, fnbB, aur) and eDNA formation (atlA) and increased the expression of genes involved in staphyloxanthin biosynthesis (crtM), suggesting a potential mechanism for enhanced resistance of USA300 to adverse conditions. These findings shed light on how low concentrations of TBBPA facilitate biofilm formation in USA300 and highlight the indirect impact of plastic additives on pathogenic bacteria in terms of human health. In the future, in-depth studies about effects of plastic additives on pathogenicity of pathogenic bacteria should be conducted. CAPSULE: The protein and eDNA contents in biofilms of methicillin-resistant Staphylococcus aureus are increased by low concentrations of TBBPA.

d-Methionine-induced DNases disperse established Burkholderia pseudomallei biofilms and promotes ceftazidime susceptibility.

Burkholderia pseudomallei biofilm is correlated with pathogenesis, antibiotic resistance, and relapsing cases of melioidosis, leading to challenges in clinical management. There is increasing interest in employing biofilm dispersal agents as adjunctive treatments for biofilm-associated infections. Methionine (Met) has shown promise as an anti-biofilm agent by inducing bacterial DNase production, resulting in the degradation of extracellular DNA (eDNA) and dispersion of bacterial biofilm. In this study, we investigated the impact of 0.05-50 µM D-Met and L-Met on the 24-h established biofilm of a clinical isolate, B. pseudomallei H777. Our findings revealed the ability of D-Met and L-Met to disperse the established biofilm in a non-dose-dependent manner accompanied by eDNA depletion. Real-time PCR analysis further identified an up-regulation of bacterial nuclease genes, including recJ, eddB, nth, xth, and recD, in the presence of 0.05 µM D-Met. Similarly, recJ and eddB in B. pseudomallei were up-regulated in response to the presence of 0.05 µM L-Met. Notably, D-Met enhanced the susceptibility of B. pseudomallei H777 biofilm cells to ceftazidime. Our findings indicate a correlation between methionine supplementation and the up-regulation of nuclease genes, leading to eDNA depletion and the dispersal of preformed B. pseudomallei H777 biofilm. This enhances the susceptibility of biofilm cells to ceftazidime, showing promise in combating biofilm-associated B. pseudomallei infections.

Viral and thermal lysis facilitates transmission of antibiotic resistance genes during composting.

While the distribution of extracellular ARGs (eARGs) in the environment has been widely reported, the factors governing their release remain poorly understood. Here, we combined multi-omics and direct experimentation to test whether the release and transmission of eARGs are associated with viral lysis and heat during cow manure composting. Our results reveal that the proportion of eARGs increased 2.7-fold during composting, despite a significant and concomitant reduction in intracellular ARG abundances. This relative increase of eARGs was driven by composting temperature and viral lysis of ARG-carrying bacteria based on metagenome-assembled genome (MAG) analysis. Notably, thermal lysis of mesophilic bacteria carrying ARGs was a key factor in releasing eARGs at the thermophilic phase, while viral lysis played a relatively stronger role during the non-thermal phase of composting. Furthermore, MAG-based tracking of ARGs in combination with direct transformation experiments demonstrated that eARGs released during composting pose a potential transmission risk. Our study provides bioinformatic and experimental evidence of the undiscovered role of temperature and viral lysis in co-driving the spread of ARGs in compost microbiomes via the horizontal transfer of environmentally released DNA. IMPORTANCE: The spread of antibiotic resistance genes (ARGs) is a critical global health concern. Understanding the factors influencing the release of extracellular ARGs (eARGs) is essential for developing effective strategies. In this study, we investigated the association between viral lysis, heat, and eARG release during composting. Our findings revealed a substantial increase in eARGs despite reduced intracellular ARG abundance. Composting temperature and viral lysis were identified as key drivers, with thermal lysis predominant during the thermophilic phase and viral lysis during non-thermal phases. Moreover, eARGs released during composting posed a transmission risk through horizontal gene transfer. This study highlights the significance of temperature and phage lysis in ARG spread, providing valuable insights for mitigating antibiotic resistance threats.

Nucleic Acids-Based Biomarkers for Alzheimer's Disease Diagnosis and Novel Molecules to Treat the Disease.

Alzheimer's disease (AD) represents the most common form of dementia and affects million people worldwide, with a high social burden and considerable economic costs. AD diagnosis benefits from a well-established panel of laboratory tests that allow ruling-in patients, along with FDG and amyloid PET imaging tools. The main laboratory tests used to identify AD patients are Aß40, Aß42, the Aß42/Aß40 ratio, phosphorylated Tau 181 (pTau181) and total Tau (tTau). Although they are measured preferentially in the cerebrospinal fluid (CSF), some evidence about the possibility for blood-based determination to enter clinical practice is growing up. Unfortunately, CSF biomarkers for AD and, even more, the blood-based ones, present a few flaws, and twenty years of research in this field did not overcome these pitfalls. The tale even worsens when the issue of treating AD is addressed due to the lack of effective strategies despite the many decades of attempts by pharmaceutic industries and scientists. Amyloid-based drugs failed to stop the disease, and no neuroinflammation-based drugs have been demonstrated to work so far. Hence, only symptomatic therapy is available, with no disease-modifying treatment on hand. Such a desolate situation fully justifies the active search for novel biomarkers to be used as reliable tests for AD diagnosis and molecular targets for treating patients. Recently, a novel group of molecules has been identified to be used for AD diagnosis and follow-up, the nuclei acid-based biomarkers. Nucleic acid-based biomarkers are a composite group of extracellular molecules consisting of DNA and RNA alone or in combination with other molecules, including proteins. This review article reports the main findings from the studies carried out on these biomarkers during AD, and highlights their advantages and limitations.

Anthropogenic pollution may enhance natural transformation in water, favouring the spread of antibiotic resistance genes.

Aquatic ecosystems are crucial in the antimicrobial resistance cycle. While intracellular DNA has been extensively studied to understand human activity's impact on antimicrobial resistance gene (ARG) dissemination, extracellular DNA is frequently overlooked. This study examines the effect of anthropogenic water pollution on microbial community diversity, the resistome, and ARG dissemination. We analyzed intracellular and extracellular DNA from wastewater treatment plant effluents and lake surface water by shotgun sequencing. We also conducted experiments to evaluate anthropogenic pollution's effect on transforming extracellular DNA (using Gfp-plasmids carrying ARGs) within a natural microbial community. Chemical analysis showed treated wastewater had higher anthropogenic pollution-related parameters than lake water. The richness of microbial community, antimicrobial resistome, and high-risk ARGs was greater in treated wastewaters than in lake waters both for intracellular and extracellular DNA. Except for the high-risk ARGs, richness was significantly higher in intracellular than in extracellular DNA. Several ARGs were associated with mobile genetic elements and located on plasmids. Furthermore, Gfp-plasmid transformation within a natural microbial community was enhanced by anthropogenic pollution levels. Our findings underscore anthropogenic pollution's pivotal role in shaping microbial communities and their antimicrobial resistome. Additionally, it may facilitate ARG dissemination through extracellular DNA plasmid uptake.

Beyond the double helix: the multifaceted landscape of extracellular DNA in Staphylococcus aureus biofilms.

Staphylococcus aureus forms biofilms consisting of cells embedded in a matrix made of proteins, polysaccharides, lipids, and extracellular DNA (eDNA). Biofilm-associated infections are difficult to treat and can promote antibiotic resistance, resulting in negative healthcare outcomes. eDNA within the matrix contributes to the stability, growth, and immune-evasive properties of S. aureus biofilms. eDNA is released by autolysis, which is mediated by murein hydrolases that access the cell wall via membrane pores formed by holin-like proteins. The eDNA content of S. aureus biofilms varies among individual strains and is influenced by environmental conditions, including the presence of antibiotics. eDNA plays an important role in biofilm development and structure by acting as an electrostatic net that facilitates protein-cell and cell-cell interactions. Because of eDNA's structural importance in biofilms and its ubiquitous presence among S. aureus isolates, it is a potential target for therapeutics. Treatment of biofilms with DNase can eradicate or drastically reduce them in size. Additionally, antibodies that target DNABII proteins, which bind to and stabilize eDNA, can also disperse biofilms. This review discusses the recent literature on the release, structure, and function of eDNA in S. aureus biofilms, in addition to a discussion of potential avenues for targeting eDNA for biofilm eradication.

Extracellular components in enteroaggregative Escherichia coli biofilm and impact of treatment with proteinase K, DNase or sodium metaperiodate.

Enteroaggregative E. coli (EAEC) is a major cause of diarrhea worldwide. EAEC are highly adherent to cultured epithelial cells and make biofilms. Both adherence and biofilm formation rely on the presence of aggregative adherence fimbriae (AAF). We compared biofilm formation from two EAEC strains of each of the five AAF types. We found that AAF type did not correlate with the level of biofilm produced. Because the composition of the EAEC biofilm has not been fully described, we stained EAEC biofilms to determine if they contained protein, carbohydrate glycoproteins, and/or eDNA and found that EAEC biofilms contained all three extracellular components. Next, we assessed the changes to the growing or mature EAEC biofilm mediated by treatment with proteinase K, DNase, or a carbohydrate cleavage agent to target the different components of the matrix. Growing biofilms treated with proteinase K had decreased biofilm staining for more than half of the strains tested. In contrast, although sodium metaperiodate only altered the biofilm in a quantitative way for two strains, images of biofilms treated with sodium metaperiodate showed that the EAEC were more spread out. Overall, we found variability in the response of the EAEC strains to the treatments, with no one treatment producing a biofilm change for all strains. Finally, once formed, mature EAEC biofilms were more resistant to treatment than biofilms grown in the presence of those same treatments.

The effects of climate and soil depth on living and dead bacterial communities along a longitudinal gradient in Chile.

Soil bacterial communities play a critical role in shaping soil stability and formation, exhibiting a dynamic interaction with local climate and soil depth. We employed an innovative DNA separation method to characterize microbial assemblages in low-biomass environments such as deserts and distinguish between intracellular DNA (iDNA) and extracellular DNA (eDNA) in soils. This approach, combined with analyses of physicochemical properties and co-occurrence networks, investigated soil bacterial communities across four sites representing diverse climatic gradients (i.e., arid, semi-arid, Mediterranean, and humid) along the Chilean Coastal Cordillera. The separation method yielded a distinctive unimodal pattern in the iDNA pool alpha diversity, increasing from arid to semi-arid climates and decreasing in humid environments, highlighting the rapid feedback of the iDNA community to increasing soil moisture. In the arid region, harsh surface conditions restrict bacterial growth, leading to peak iDNA abundance and diversity occurring in slightly deeper layers than the other sites. Our findings confirmed the association between specialist bacteria and ecosystem-functional traits. We observed transitions from Halomonas and Delftia, resistant to extreme arid environments, to Class AD3 and the genus Bradyrhizobium, associated with plants and organic matter in humid environments. The distance-based redundancy analysis (dbRDA) analysis revealed that soil pH and moisture were the key parameters that influenced bacterial community variation. The eDNA community correlated slightly better with the environment than the iDNA community. Soil depth was found to influence the iDNA community significantly but not the eDNA community, which might be related to depth-related metabolic activity. Our investigation into iDNA communities uncovered deterministic community assembly and distinct co-occurrence modules correlated with unique bacterial taxa, thereby showing connections with sites and key environmental factors. The study additionally revealed the effects of climatic gradients and soil depth on living and dead bacterial communities, emphasizing the need to distinguish between iDNA and eDNA pools.

Nanoscale surface defects of goethite governing DNA adsorption process and formation of the Goethite-DNA conjugates.

In urbanized areas, extracellular DNA (exDNA) is suspected of carrying genes with undesirable traits like virulence genes (VGs) or antibiotic resistance genes (ARGs), which can spread through horizontal gene transfer (HGT). Hence, it is crucial to develop novel approaches for the mitigation of exDNA in the environment. Our research explores the role of goethite, a common iron mineral with high adsorption capabilities, in exDNA adsorption processes. We compare well-crystalline, semi-crystalline, and nano goethites with varying particle sizes to achieve various specific surface areas (SSAs) (18.7-161.6 m2/g) and porosities. We conducted batch adsorption experiments using DNA molecules of varying chain lengths (DNA sizes: <11 Kb, <6 Kb, and <3 Kb) and assessed the impact of Ca2+ and biomacromolecules on the adsorption efficacy and mechanisms. Results show that porosity and pore structure significantly influence DNA adsorption capacity. Goethite with well-developed meso- and macroporosity demonstrated enhanced DNA adsorption. The accumulation of DNA on the goethite interface led to substantial aggregation in the system, thus the formation of DNA-goethite conjugates, indicating the bridging between mineral particles. DNA chain length, the presence of Ca2+, and the biomacromolecule matrix also affected the adsorption capacity and mechanism. Interactions between DNA and positively charged biomacromolecules or Ca2+ led to DNA compaction, allowing greater DNA accumulation in pores. However, a high concentration of biomacromolecules led to the saturation of the goethite surface, inhibiting DNA adsorption. AFM imaging of goethite particles after adsorption suggested the formation of the DNA multilayer. The study advances understanding of the environmental behavior of exDNA and its interaction with iron oxyhydroxides, offering insights into developing more effective methods for ARGs removal in wastewater treatment plants. By manipulating the textural properties of goethite, it's possible to enhance exDNA removal, potentially reducing the spread of biocontamination in urban and industrial environments.

Poly-ß-(1→6)-N-acetyl-D-glucosamine mediates surface attachment, biofilm formation, and biocide resistance in Cutibacterium acnes.

Background: The commensal skin bacterium Cutibacterium acnes plays a role in the pathogenesis of acne vulgaris and also causes opportunistic infections of implanted medical devices due to its ability to form biofilms on biomaterial surfaces. Poly-ß-(1→6)-N-acetyl-D-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm formation and biocide resistance in a wide range of bacterial pathogens. The objective of this study was to determine whether C. acnes produces PNAG, and whether PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro. Methods: PNAG was detected on the surface of C. acnes cells by fluorescence confocal microscopy using the antigen-specific human IgG1 monoclonal antibody F598. PNAG was detected in C. acnes biofilms by measuring the ability of the PNAG-specific glycosidase dispersin B to inhibit biofilm formation and sensitize biofilms to biocide killing. Results: Monoclonal antibody F598 bound to the surface of C. acnes cells. Dispersin B inhibited attachment of C. acnes cells to polystyrene rods, inhibited biofilm formation by C. acnes in glass and polypropylene tubes, and sensitized C. acnes biofilms to killing by benzoyl peroxide and tetracycline. Conclusion: C. acnes produces PNAG, and PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro. PNAG may play a role in C. acnes skin colonization, biocide resistance, and virulence in vivo.

A review of distribution and functions of extracellular DNA in the environment and wastewater treatment systems.

Extracellular DNA refers to DNA fragments existing outside the cell, originating from various cell release mechanisms, including active secretion, cell lysis, and phage-mediated processes. Extracellular DNA serves as a vital environmental biomarker, playing crucial ecological and environmental roles in water bodies. This review is summarized the mechanisms of extracellular DNA release, including pathways involving cell lysis, extracellular vesicles, and type IV secretion systems. Then, the extraction and detection methods of extracellular DNA from water, soil, and biofilm are described and analyzed. Finally, we emphasize the role of extracellular DNA in microbial community systems, including its significant contributions to biofilm formation, biodiversity through horizontal gene transfer, and electron transfer processes. This review offers a comprehensive insight into the sources, distribution, functions, and impacts of extracellular DNA within aquatic environments, aiming to foster further exploration and understanding of extracellular DNA dynamics in aquatic environments as well as other environments.

Stage-dependent immunity orchestrates AQP4 antibody-guided NMOSD pathology: a role for netting neutrophils with resident memory T cells in situ.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease of the CNS characterized by the production of disease-specific autoantibodies against aquaporin-4 (AQP4) water channels. Animal model studies suggest that anti-AQP4 antibodies cause a loss of AQP4-expressing astrocytes, primarily via complement-dependent cytotoxicity. Nonetheless, several aspects of the disease remain unclear, including: how anti-AQP4 antibodies cross the blood-brain barrier from the periphery to the CNS; how NMOSD expands into longitudinally extensive transverse myelitis or optic neuritis; how multiphasic courses occur; and how to prevent attacks without depleting circulating anti-AQP4 antibodies, especially when employing B-cell-depleting therapies. To address these knowledge gaps, we conducted a comprehensive 'stage-dependent' investigation of immune cell elements in situ in human NMOSD lesions, based on neuropathological techniques for autopsied/biopsied CNS materials. The present study provided three major findings. First, activated or netting neutrophils and melanoma cell adhesion molecule-positive (MCAM+) helper T (TH) 17/cytotoxic T (TC) 17 cells are prominent, and the numbers of these correlate with the size of NMOSD lesions in the initial or early-active stages. Second, forkhead box P3-positive (FOXP3+) regulatory T (Treg) cells are recruited to NMOSD lesions during the initial, early-active or late-active stages, suggesting rapid suppression of proinflammatory autoimmune events in the active stages of NMOSD. Third, compartmentalized resident memory immune cells, including CD103+ tissue-resident memory T (TRM) cells with long-lasting inflammatory potential, are detected under "standby" conditions in all stages. Furthermore, CD103+ TRM cells express high levels of granzyme B/perforin-1 in the initial or early-active stages of NMOSD in situ. We infer that stage-dependent compartmentalized immune traits orchestrate the pathology of anti-AQP4 antibody-guided NMOSD in situ. Our work further suggests that targeting activated/netting neutrophils, MCAM+ TH17/TC17 cells, and CD103+ TRM cells, as well as promoting the expansion of FOXP3+ Treg cells, may be effective in treating and preventing relapses of NMOSD.

Biofilm-disrupting effects of phage endolysins LysAm24, LysAp22, LysECD7, and LysSi3: breakdown the matrix.

The ability of most opportunistic bacteria to form biofilms, coupled with antimicrobial resistance, hinder the efforts to control widespread infections, resulting in high risks of negative outcomes and economic costs. Endolysins are promising compounds that efficiently combat bacteria, including multidrug-resistant strains and biofilms, without a low probability of subsequent emergence of stable endolysin-resistant phenotypes. However, the details of antibiofilm effects of these enzymes are poorly understood. To elucidate the interactions of bacteriophage endolysins LysAm24, LysAp22, LysECD7, and LysSi3 with bacterial films formed by Gram-negative species, we estimated their composition and assessed the endolysins' effects on the most abundant exopolymers in vitro. The obtained data suggests a pronounced efficiency of these lysins against biofilms with high (Klebsiella pneumoniae) and low (Acinetobacter baumannii) matrix contents, or dual-species biofilms, resulting in at least a twofold loss of the biomass. These peptidoglycan hydrolases interacted diversely with protective compounds of biofilms such as extracellular DNA and polyanionic carbohydrates, indicating a spectrum of biofilm-disrupting effects for bacteriolytic phage enzymes. Specifically, we detected disruption of acid exopolysaccharides by LysAp22, strong DNA-binding capacity of LysAm24, both of these interactions for LysECD7, and neither of them for LysSi3.

Multitasking functions of bacterial extracellular DNA in biofilms.

Bacterial biofilms are intricate ecosystems of microbial communities that adhere to various surfaces and are enveloped by an extracellular matrix composed of polymeric substances. Within the context of bacterial biofilms, extracellular DNA (eDNA) originates from cell lysis or is actively secreted, where it exerts a significant influence on the formation, stability, and resistance of biofilms to environmental stressors. The exploration of eDNA within bacterial biofilms holds paramount importance in research, with far-reaching implications for both human health and the environment. An enhanced understanding of the functions of eDNA in biofilm formation and antibiotic resistance could inspire the development of strategies to combat biofilm-related infections and improve the management of antibiotic resistance. This comprehensive review encapsulates the latest discoveries concerning eDNA, encompassing its origins, functions within bacterial biofilms, and significance in bacterial pathogenesis.

Spore-DNA localization and extraction efficiencies of Bacillus subtilis for accurate results in quantitative real-time polymerase chain reaction.

Mechanical bead disruption is an efficient DNA extraction method from spore cells for subsequent quantification of the spore population by quantitative polymerase chain reaction（qPCR）. In this study, to validate spore DNA localization and extraction efficiencies, the fractionated DNA included the total DNA（tDNA）extracted from spore cells and intracellular（iDNA）and extracellular DNA（eDNA）extracted from fractionated spores through chemical decoating and alkaline lysis buffers, each followed by bead disruption. Furthermore, alkaline lysis buffer-treated spore cells were intensively washed three and five times after each centrifugation to determine how the amount of DNA is affected by repeated centrifugation. This process was achieved through fractionated spore pellet and suspension treatments with propidium monoazide xx（PMAxx）before mechanical bead disruption. Three fractionated and extracted DNAs were assessed with qPCR. The amount of eDNA was higher than that of iDNA, and closer to tDNA levels in the qPCR assay. These results indicted the following: 1）amount of eDNA was more than iDNA and responsible for majority of amount of tDNA through the combination method involving alkaline lysis buffer and bead disruption, 2）lysis buffer partially eliminated the eDNA fragments through multiple washing steps, but it was not largely independent of the number of times centrifugation was performed.

Evaluating a polymicrobial biofilm model for structural components by co-culturing Komagataeibacter hansenii produced bacterial cellulose with Pseudomonas aeruginosa PAO1.

A polymicrobial biofilm model of Komagataeibacter hansenii and Pseudomonas aeruginosa was developed to understand whether a pre-existing matrix affects the ability of another species to build a biofilm. P. aeruginosa was inoculated onto the preformed K. hansenii biofilm consisting of a cellulose matrix. P. aeruginosa PAO1 colonized and infiltrated the K. hansenii bacterial cellulose biofilm (BC), as indicated by the presence of cells at 19 µm depth in the translucent hydrogel matrix. Bacterial cell density increased along the imaged depth of the biofilm (17-19 µm). On day 5, the average bacterial count across sections was 67 ± 4 % P. aeruginosa PAO1 and 33 ± 6 % K. hansenii. Biophysical characterization of the biofilm indicated that colonization by P. aeruginosa modified the biophysical properties of the BC matrix, which inlcuded increased density, heterogeneity, degradation temperature and thermal stability, and reduced crystallinity, swelling ability and moisture content. This further indicates colonization of the biofilm by P. aeruginosa. While eDNA fibres - a key viscoelastic component of P. aeruginosa biofilm - were present on the surface of the co-cultured biofilm on day 1, their abundance decreased over time, and by day 5, no eDNA was observed, either on the surface or within the matrix. P. aeruginosa-colonized biofilm devoid of eDNA retained its mechanical properties. The observations demonstrate that a pre-existing biofilm scaffold of K. hansenii inhibits P. aeruginosa PAO1 eDNA production and suggest that eDNA production is a response by P. aeruginosa to the viscoelastic properties of its environment.

Characterization of the Fusarium circinatum biofilm environmental response role.

The capacity to form biofilms is a common trait among many microorganisms present on Earth. In this study, we demonstrate for the first time that the fatal pine pitch canker agent, Fusarium circinatum, can lead a biofilm-like lifestyle with aggregated hyphal bundles wrapped in extracellular matrix (ECM). Our research shows F. circinatum's ability to adapt to environmental changes by assuming a biofilm-like lifestyle. This was demonstrated by varying metabolic activities exhibited by the biofilms in response to factors like temperature and pH. Further analysis revealed that while planktonic cells produced small amounts of ECM per unit of the biomass, heat- and azole-exposed biofilms produced significantly more ECM than nonexposed biofilms, further demonstrating the adaptability of F. circinatum to changing environments. The increased synthesis of ECM triggered by these abiotic factors highlights the link between ECM production in biofilm and resistance to abiotic stress. This suggests that ECM-mediated response may be one of the key survival strategies of F. circinatum biofilms in response to changing environments. Interestingly, azole exposure also led to biofilms that were resistant to DNase, which typically uncouples biofilms by penetrating the biofilm and degrading its extracellular DNA; we propose that DNases were likely hindered from reaching target cells by the ECM barricade. The interplay between antifungal treatment and DNase enzyme suggests a complex relationship between eDNA, ECM, and antifungal agents in F. circinatum biofilms. Therefore, our results show how a phytopathogen's sessile (biofilm) lifestyle could influence its response to the surrounding environment.

Synthetic Nuclease-Producing Microbiome Achieves Efficient Removal of Extracellular Antibiotic Resistance Genes from Wastewater Effluent.

Antibiotic resistance gene (ARG) transmission poses significant threats to human health. The effluent of wastewater treatment plants is demonstrated as a hotspot source of ARGs released into the environment. In this study, a synthetic microbiome containing nuclease-producing Deinococcus radiodurans was constructed to remove extracellular ARGs. Results of quantitative polymerase chain reaction (qPCR) showed significant reduction in plasmid RP4-associated ARGs (by more than 3 orders of magnitude) and reduction of indigenous ARG sul1 and mobile genetic element (MGE) intl1 (by more than 1 order of magnitude) in the synthetic microbiome compared to the control without D. radiodurans. Metagenomic analysis revealed a decrease in ARG and MGE diversity in extracellular DNA (eDNA) of the treated group. Notably, whereas eight antibiotic-resistant plasmids with mobility risk were detected in the control, only one was detected in the synthetic microbiome. The abundance of the nuclease encoding gene exeM, quantified by qPCR, indicated its enrichment in the synthetic microbiome, which ensures stable eDNA degradation even when D. radiodurans decreased. Moreover, intracellular ARGs and MGEs and pathogenic ARG hosts in the river receiving treated effluent were lower than those in the river receiving untreated effluent. Overall, this study presents a new approach for removing extracellular ARGs and further reducing the risk of ARG transmission in receiving rivers.