Quantification of Extracellular DNA Network Abundance and Architecture within Streptococcus gordonii Biofilms Reveals Modulatory Factors.

Extracellular DNA (eDNA) is an important component of biofilm matrix that serves to maintain biofilm structural integrity, promotes genetic exchange within the biofilm, and provides protection against antimicrobial compounds. Advances in microscopy techniques have provided evidence of the cobweb- or lattice-like structures of eDNA within biofilms from a range of environmental niches. However, methods to reliably assess the abundance and architecture of eDNA remain lacking. This study aimed to address this gap by development of a novel, high-throughput image acquisition and analysis platform for assessment of eDNA networks in situ within biofilms. Utilizing Streptococcus gordonii as the model, the capacity for this imaging system to reliably detect eDNA networks and monitor changes in abundance and architecture (e.g., strand length and branch number) was verified. Evidence was provided of a synergy between glucans and eDNA matrices, while it was revealed that surface-bound nuclease SsnA could modify these eDNA structures under conditions permissive for enzymatic activity. Moreover, cross talk between the competence and hexaheptapeptide permease systems was shown to regulate eDNA release by S. gordonii. This novel imaging system can be applied across the wider field of biofilm research, with potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit. IMPORTANCE Extracellular DNA (eDNA) is critical for maintaining the structural integrity of many microbial biofilms, making it an attractive target for the management of biofilms. However, our knowledge and targeting of eDNA are currently hindered by a lack of tools for the quantitative assessment of eDNA networks within biofilms. Here, we demonstrate use of a novel image acquisition and analysis platform with the capacity to reliably monitor the abundance and architecture of eDNA networks. Application of this tool to Streptococcus gordonii biofilms has provided new insights into how eDNA networks are stabilized within the biofilm and the pathways that can regulate eDNA release. This highlights how exploitation of this novel imaging system across the wider field of biofilm research has potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit.

Cellulose Nanofibril Formulations Incorporating a Low-Molecular-Weight Alginate Oligosaccharide Modify Bacterial Biofilm Development.

Cellulose nanofibrils (CNFs) from wood pulp are a renewable material possessing advantages for biomedical applications because of their customizable porosity, mechanical strength, translucency, and environmental biodegradability. Here, we investigated the growth of multispecies wound biofilms on CNF formulated as aerogels and films incorporating the low-molecular-weight alginate oligosaccharide OligoG CF-5/20 to evaluate their structural and antimicrobial properties. Overnight microbial cultures were adjusted to 2.8 × 109 colony-forming units (cfu) mL-1 in Mueller Hinton broth and growth rates of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus 1061A monitored for 24 h in CNF dispersions sterilized by γ-irradiation. Two CNF formulations were prepared (20 g m-2) with CNF as air-dried films or freeze-dried aerogels, with or without incorporation of an antimicrobial alginate oligosaccharide (OligoG CF-5/20) as a surface coating or bionanocomposite, respectively. The materials were structurally characterized by scanning electron microscopy (SEM) and laser profilometry (LP). The antimicrobial properties of the formulations were assessed using single- and mixed-species biofilms grown on the materials and analyzed using LIVE/DEAD staining with confocal laser scanning microscopy (CLSM) and COMSTAT software. OligoG-CNF suspensions significantly decreased the growth of both bacterial strains at OligoG concentrations >2.58% (P < 0.05). SEM showed that aerogel-OligoG bionanocomposite formulations had a more open three-dimensional structure, whereas LP showed that film formulations coated with OligoG were significantly smoother than untreated films or films incorporating PEG400 as a plasticizer (P < 0.05). CLSM of biofilms grown on films incorporating OligoG demonstrated altered biofilm architecture, with reduced biomass and decreased cell viability. The OligoG-CNF formulations as aerogels or films both inhibited pyocyanin production (P < 0.05). These novel CNF formulations or bionanocomposites were able to modify bacterial growth, biofilm development, and virulence factor production in vitro. These data support the potential of OligoG and CNF bionanocomposites for use in biomedical applications where prevention of infection or biofilm growth is required.

Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa.

Pseudomonas aeruginosa plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in P. aeruginosa biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in P. aeruginosa QS regulation was studied by using Chromobacterium violaceum CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; P < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C4-AHL and 3-oxo-C12-AHL production (≥2%; P < 0.05). Moreover, quantitative PCR showed that reduced expression of both the las and rhl systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; P < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; P < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease (P < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in P. aeruginosa PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation.

A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness.

In chronic respiratory disease, the formation of dense, 3-dimensional "microcolonies" by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates (n = 3) and reference nonmucoid and mucoid multidrug-resistant (MDR) CF isolates (n = 7). Bacterial growth and biofilm development and disruption were studied using cell viability assays and image analysis with scanning electron and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production (P < 0.05) and the formation (at 48 h) of discrete (>10-µm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 µg/ml) in AS medium compared to Mueller-Hinton (MH) medium. In contrast, in an established-biofilm model in AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment, however, induced dose-dependent biofilm disruption (P < 0.05) and led to colistin retaining its antimicrobial activity (P < 0.05). While circular dichroism indicated that OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, mass spectrometry demonstrated that the oligomers induced dose-dependent (>0.2%; P < 0.05) reductions in pseudomonal quorum-sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung and highlight a novel approach to treat MDR pseudomonal infections.

Streptococcus gordonii comCDE (competence) operon modulates biofilm formation with Candida albicans.

Candida albicans is a pleiomorphic fungus that forms mixed species biofilms with Streptococcus gordonii, an early colonizer of oral cavity surfaces. Activation of quorum sensing (QS; intercellular signalling) promotes monospecies biofilm development by these micro-organisms, but the role of QS in mixed species communities is not understood. The comCDE genes in S. gordonii encode a sensor-regulator system (ComDE), which is activated by the comC gene product (CSP, competence stimulating peptide) and modulates expression of QS-regulated genes. Dual species biofilms of S. gordonii ΔcomCDE or ΔcomC mutants with C. albicans showed increased biomass compared to biofilms of S. gordonii DL1 wild-type with C. albicans. The ΔcomCDE mutant dual species biofilms in particular contained more extracellular DNA (eDNA), and could be dispersed with DNase I or protease treatment. Exogenous CSP complemented the S. gordonii ΔcomC transformation deficiency, as well as the ΔcomC-C. albicans biofilm phenotype. Purified CSP did not affect C. albicans hyphal filament formation but inhibited monospecies biofilm formation by C. albicans. The results suggest that the S. gordonii comCDE QS-system modulates the production of eDNA and the incorporation of C. albicans into dual species biofilms.

The Impact of Probiotic Supplementation on Cognitive, Pathological and Metabolic Markers in a Transgenic Mouse Model of Alzheimer's Disease.

Brain degenerative disorders such as Alzheimer's disease (AD) can be exacerbated by aberrant metabolism. Supplementation with probiotic bacteria is emerging as a promising preventative strategy for both neurodegeneration and metabolic syndrome. In this study, we assess the impact of the Lab4b probiotic consortium on (i) cognitive and pathological markers of AD progression and (ii) metabolic status in 3xTg-AD mice subjected to metabolic challenge with a high fat diet. The group receiving the probiotic performed better in the novel object recognition test and displayed higher hippocampal neuronal spine density than the control group at the end of the 12 weeks intervention period. These changes were accompanied by differences in localised (brain) and systemic anti-inflammatory responses that favoured the Probiotic group together with the prevention of diet induced weight gain and hypercholesterolaemia and the modulation of liver function. Compositional differences between the faecal microbiotas of the study groups included a lower Firmicutes:Bacteroidetes ratio and less numbers of viable yeast in the Probiotic group compared to the Control. The results illustrate the potential of the Lab4b probiotic as a neuroprotective agent and encourage further studies with human participants.

Probiotics reduce self-reported symptoms of upper respiratory tract infection in overweight and obese adults: should we be considering probiotics during viral pandemics?

Gut microbiome manipulation to alter the gut-lung axis may potentially protect humans against respiratory infections, and clinical trials of probiotics show promise in this regard in healthy adults and children. However, comparable studies are lacking in overweight/obese people, who have increased risks in particular of viral upper respiratory tract infections (URTI). This Addendum further analyses our recent placebo-controlled trial of probiotics in overweight/obese people (focused initially on weight loss) to investigate the impact of probiotics upon the occurrence of URTI symptoms. As well as undergoing loss of weight and improvement in certain metabolic parameters, study participants taking probiotics experienced a 27% reduction in URTI symptoms versus control, with those ≥45 years or BMI ≥30 kg/m2 experiencing greater reductions. This symptom reduction is apparent within 2 weeks of probiotic use. Gut microbiome diversity remained stable throughout the study in probiotic-treated participants. Our data provide support for further trials to assess the potential role of probiotics in preventing viral URTI (and possibly also COVID-19), particularly in overweight/obese people.

The Impact of Lab4 Probiotic Supplementation in a 90-Day Study in Wistar Rats.

The anti-inflammatory and cholesterol lowering capabilities of probiotic bacteria highlight them as potential prophylactics against chronic inflammatory diseases, particularly cardiovascular disease. Previous studies in silico, in vitro, and in vivo suggest that the Lab4 probiotic consortium may harbour such capabilities and in the current study, we assessed plasma levels of cytokines/chemokines, short chain fatty acids and lipids and faecal levels of bile acids in a subpopulation of healthy Wistar rats included in 90-day repeat dose oral toxicity study. In the rats receiving Lab4, circulating levels of pro-inflammatory interleukin-6, tumour necrosis factor-α and keratinocyte chemoattractant/growth regulated oncogene were significantly lower compared to the control group demonstrating a systemic anti-inflammatory effect. These changes occurred alongside significant reductions in plasma low density lipoprotein cholesterol and increases in faecal bile acid excretion implying the ability to lower circulating cholesterol via the deconjugation of intestinal bile acids. Correlative analysis identified significant associations between plasma tumour necrosis factor-α and the plasma total cholesterol:high density lipoprotein cholesterol ratio and faecal levels of bifidobacteria in the Lab4 rats. Together, these data highlight Lab4 supplementation as a holistic approach to CVD prevention and encourages further studies in humans.

The interaction of wood nanocellulose dressings and the wound pathogen P. aeruginosa.

Chronic wounds pose an increasingly significant worldwide economic burden (over £1 billion per annum in the UK alone). With the escalation in global obesity and diabetes, chronic wounds will increasingly be a significant cause of morbidity and mortality. Cellulose nanofibrils (CNF) are highly versatile and can be tailored with specific physical properties to produce an assortment of three-dimensional structures (hydrogels, aerogels or films), for subsequent utilization as wound dressing materials. Growth curves using CNF (diameter <20nm) in suspension demonstrated an interesting dose-dependent inhibition of bacterial growth. In addition, analysis of biofilm formation (Pseudomonas aeruginosa PAO1) on nanocellulose aerogels (20g/m2) revealed significantly less biofilm biomass with decreasing aerogel porosity and surface roughness. Importantly, virulence factor production by P. aeruginosa in the presence of nanocellulose materials, quantified for the first time, was unaffected (p>0.05) over 24h. These data demonstrate the potential of nanocellulose materials in the development of novel dressings that may afford significant clinical potential.