The efficacy and safety of an enzyme-containing lozenge for dental biofilm control - A randomized controlled pilot trial.

OBJECTIVES: To evaluate the effect of daily use of a multiple-enzyme lozenge on de novo plaque formation, on gingivitis development, and on the oral microbiome composition. METHODS: This trial with two parallel arms included 24 healthy adults allocated to the Active (n=12) or Placebo (n=12) group. Subjects consumed one lozenge three times daily for seven days, and no oral hygiene procedures were allowed. Differences in de novo plaque accumulation between a baseline period, and one and seven days of intervention were assessed by the Turesky-modification of the Quigley-and-Hein-Plaque-Index (TM-QHPI). The development of gingivitis after seven days of intervention was assessed by the Gingival Index (GI). Plaque and saliva samples were collected at baseline and after seven days of intervention, and evaluated by 16S rRNA gene sequencing. RESULTS: All subjects completed the study, and no adverse events were reported. After one day, the average TM-QHPI was significantly lower in the Active than in the Placebo group, as compared to baseline (p=0.012). After 7 days, average TM-QHPI values did not differ significantly between groups (p=0.37). GI values did not increase during the intervention period, with no difference between groups (p=0.62). Bacterial richness increased in both plaque and saliva samples over a seven-day oral hygiene-free period, with a statistically significant difference for the saliva samples (p=0.0495) between groups. CONCLUSIONS: A multiple-enzymes lozenge decreased the build-up of de novo plaque after one day and slowed down the process of species increment in saliva. The lozenge may be an adjunct to regular mechanical plaque removal. CLINICAL SIGNIFICANCE: Dental plaque is the main cause of caries, gingivitis, and periodontitis. The search for therapeutic adjuncts to mechanical plaque removal that have no harmful effects on the oral microbiome is important. Treatment with multiple plaque-matrix degrading enzymes is a promising non-biocidal approach to plaque control.

GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms.

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluorescent protein reporters in their native environment, because they must be exported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detected msfGFP fluorescence inside but not outside bacterial cells, indicating a failure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically integrated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.

Antibody-Drug Conjugates to Treat Bacterial Biofilms via Targeting and Extracellular Drug Release.

The treatment of implant-associated bacterial infections and biofilms is an urgent medical need and a grand challenge because biofilms protect bacteria from the immune system and harbor antibiotic-tolerant persister cells. This need is addressed herein through an engineering of antibody-drug conjugates (ADCs) that contain an anti-neoplastic drug mitomycin C, which is also a potent antimicrobial against biofilms. The ADCs designed herein release the conjugated drug without cell entry, via a novel mechanism of drug release which likely involves an interaction of ADC with the thiols on the bacterial cell surface. ADCs targeted toward bacteria are superior by the afforded antimicrobial effects compared to the non-specific counterpart, in suspension and within biofilms, in vitro, and in an implant-associated murine osteomyelitis model in vivo. The results are important in developing ADC for a new area of application with a significant translational potential, and in addressing an urgent medical need of designing a treatment of bacterial biofilms.

Polyether Ionophore Antibiotics Target Drug-Resistant Clinical Isolates, Persister Cells, and Biofilms.

Polyether ionophores are complex natural products known to transport various cations across biological membranes. While several members of this family are used in agriculture (e.g., as anti-coccidiostats) and have potent antibacterial activity, they are not currently being pursued as antibiotics for human use. Polyether ionophores are typically grouped as having similar functions, despite the fact that they significantly differ in structure; for this reason, how their structure and activity are related remains unclear. To determine whether certain members of the family constitute particularly interesting springboards for in-depth investigations and future synthetic optimization, we conducted a systematic comparative study of eight different polyether ionophores for their potential as antibiotics. This includes clinical isolates from bloodstream infections and studies of the compounds' effects on bacterial biofilms and persister cells. We uncover distinct differences within the compound class and identify the compounds lasalocid, calcimycin, and nanchangmycin as having particularly interesting activity profiles for further development. IMPORTANCE Polyether ionophores are complex natural products used in agriculture as anti-coccidiostats in poultry and as growth promoters in cattle, although their precise mechanism is not understood. They are widely regarded as antimicrobials against Gram-positive bacteria and protozoa, but fear of toxicity has so far prevented their use in humans. We show that ionophores generally have very different effects on Staphylococcus aureus, both in standard assays and in more complex systems such as bacterial biofilms and persister cell populations. This will allow us to focus on the most interesting compounds for future in-depth investigations and synthetic optimizations.

The Role of Nanoscale Distribution of Fibronectin in the Adhesion of Staphylococcus aureus Studied by Protein Patterning and DNA-PAINT.

Staphylococcus aureus is a widespread and highly virulent pathogen that can cause superficial and invasive infections. Interactions between S. aureus surface receptors and the extracellular matrix protein fibronectin mediate the bacterial invasion of host cells and is implicated in the colonization of medical implant surfaces. In this study, we investigate the role of distribution of both fibronectin and cellular receptors on the adhesion of S. aureus to interfaces as a model for primary adhesion at tissue interfaces or biomaterials. We present fibronectin in patches of systematically varied size (100-1000 nm) in a background of protein and bacteria rejecting chemistry based on PLL-g-PEG and studied S. aureus adhesion under flow. We developed a single molecule imaging assay for localizing fibronectin binding receptors on the surface of S. aureus via the super-resolution DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique. Our results indicate that S. aureus adhesion to fibronectin biointerfaces is regulated by the size of available ligand patterns, with an adhesion threshold of 300 nm and larger. DNA-PAINT was used to visualize fibronectin binding receptor organization in situ at ∼7 nm localization precision and with a surface density of 38-46 µm-2, revealing that the engagement of two or more receptors is required for strong S. aureus adhesion to fibronectin biointerfaces.

Aptamer-Targeted Drug Delivery for Staphylococcus aureus Biofilm.

Treatment of Staphylococcus aureus biofilm infections using conventional antibiotic therapy is challenging as only doses that are sublethal to the biofilm can be administered safely to patients. A potential solution to this challenge is targeted drug delivery. In this study, we tailored an aptamer-targeted liposomal drug delivery system for accumulation and delivery of antibiotics locally in S. aureus biofilm. In our search for a suitable targeting ligand, we identified six DNA aptamers that bound to S. aureus cells in biofilms, and we demonstrated that one of these aptamers could facilitate accumulation of liposomes around S. aureus cells inside the biofilm. Aptamer-targeted liposomes encapsulating a combination of vancomycin and rifampicin were able to eradicate S. aureus biofilm upon 24 h of treatment in vitro. Our results point to that aptamer-targeted drug delivery of antibiotics is a potential new strategy for treatment of S. aureus biofilm infections.

Host factors abolish the need for polysaccharides and extracellular matrix-binding protein in Staphylococcus epidermidis biofilm formation.

Introduction. Staphylococcus epidermidis is predominant in implant-associated infections due to its capability to form biofilms. It can deploy several strategies for biofilm development using either polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA) and/or proteins, such as the extracellular matrix-binding protein (Embp).Hypothesis/Gap Statement. We hypothesize that the dichotomic regulation of S. epidermidis adhesins is linked to whether it is inside a host or not, and that in vitro biofilm investigations in laboratory media may not reflect actual biofilms in vivo.Aim. We address the importance of PIA and Embp in biofilm grown in 'humanized' media to understand if these components play different roles in biofilm formation under conditions where bacteria can incorporate host proteins in the biofilm matrix.Methodology. S. epidermidis 1585 WT (deficient in icaADBC), and derivative strains that either lack embp, express embp from an inducible promotor, or express icaADBC from a plasmid, were cultivated in standard laboratory media, or in media with human plasma or serum. The amount, structure, elasticity and antimicrobial penetration of biofilms was quantified to describe structural differences caused by the different matrix components and growth conditions. Finally, we quantified the initiation of biofilms as suspended aggregates in response to host factors to determine how quickly the cells aggregate in response to the host environment and reach a size that protects them from phagocytosis.Results. S. epidermidis 1585 required polysaccharides to form biofilm in laboratory media. However, these observations were not representative of the biofilm phenotype in the presence of human plasma. If human plasma were present, polysaccharides and Embp were redundant for biofilm formation. Biofilms formed in human plasma were loosely attached and existed mostly as suspended aggregates. Aggregation occurred after 2 h of exposing cells to plasma or serum. Despite stark differences in the amount and composition of biofilms formed by polysaccharide-producing and Embp-producing strains in different media, there were no differences in vancomycin penetration or susceptibility.Conclusion. We suggest that the assumed importance of polysaccharides for biofilm formation is an artefact from studying biofilms in laboratory media void of human matrix components. The cell-cell aggregation of S. epidermidis can be activated by host factors without relying on either of the major adhesins, PIA and Embp, indicating a need to revisit the basic question of how S. epidermidis deploys self-produced and host-derived matrix components to form antibiotic-tolerant biofilms in vivo.

Combination of Rhamnolipid and Chitosan in Nanoparticles Boosts Their Antimicrobial Efficacy.

Nanomaterials have emerged as antimicrobial agents due to their unique physical and chemical properties. The development of nanoparticles (NPs) composed of natural biopolymers and biosurfactants have sparked interest, as they can be obtained without the use of complex chemical synthesis and toxic materials. In this study, we develop antimicrobial nanoparticles combining the biopolymer chitosan with the biosurfactant rhamnolipid. Addition of rhamnolipid reduced the size and polydispersity index of chitosan nanoparticles showing a more positive surface charge with improved stability, suggesting that chitosan-free amino groups are predominantly present on the surface of nanoparticles. Antimicrobial activity of chitosan/rhamnolipid nanoparticles (C/RL-NPs) against Staphylococcus strains surpassed that of either single rhamnolipid or chitosan, both in planktonic bacteria and biofilms. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of C/RL-NPs were determined considering the concentration of each individual molecule in NPs. MIC values of 14/19 µg mL-1 and MBC of 29/37 µg mL-1 were observed for S. aureus DSM 1104 and MIC and MBC of 29/37 and 58/75 µg mL-1 were observed against S. aureus ATCC 29213, respectively. For S. epidermidis, MIC and MBC of 7/9 and 14/19 µg mL-1 were noticed. Chitosan and chitosan nanoparticles eliminate the bacteria present in the upper parts of biofilms, while C/RL-NPs were more effective, eradicating most sessile bacteria and reducing the number of viable cells below the detection limit, when NPs concentration of 58/75 µg mL-1 was applied for both S. aureus DSM 1104 and S. epidermidis biofilms. The improved antibacterial efficacy of C/RL-NPs was linked to the increased local delivery of chitosan and rhamnolipid at the cell surface and, consequently, to their targets in Gram-positive bacteria. The combination of chitosan and rhamnolipid offers a promising strategy to the design of novel nanoparticles with low cytotoxicity, which can be exploited in pharmaceutical and food industries.

Development of a Label-Free LSPR-Apta Sensor for Staphylococcus aureus Detection.

The risk of foodborne diseases has increased over the last years. We have developed a simple, portable, and label-free optical sensor via aptamer recognition of Staphylococcus aureus at nanostructured plasmonic elements. The developed aptamers conjugated to a localized surface plasmon resonance (LSPR) sensing device were applied in both pure culture and artificially contaminated milk samples enabling a limit of detection of 103 CFU/mL for S. aureus in milk. There was no need for a pre-enrichment step, and the total analysis time decreased from 30 min to 120 s. Finite-difference time-domain was used to simulate the experimentally measured optical responses for a range of different sensor designs (100 and 200 nm disks), addressing the role of the near field and intrinsic refractive index sensitivity. A comparison of the aptamer to antibody-based recognition approaches showed that the thickness of the sensing layer was critical with a significantly larger response for the thinner aptamer layer. Comparison of differently sized metal nanostructures showed a significantly higher sensitivity for 200 nm diameter compared to 100 nm diameter disk structures resulting from both increases in bulk refractive index sensitivity and the extent to which the local field extends out from the metal surface. These findings confirmed that the developed gold nanodisk-based LSPR sensing chips could facilitate sensitive detection of S. aureus in food samples.

Innate glycosidic activity in metallic implants for localized synthesis of antibacterial drugs.

Iron-containing metallic implants are shown herein to mediate hydrolysis of glycosidic linkages. Using glucuronide prodrugs for broad-spectrum fluoroquinolone antibacterial agents, we capitalize on this behaviour and perform localized synthesis of antimicrobials which affords a significant zone of inhibition of bacterial growth around the metallic material.

Identification and Directed Development of Non-Organic Catalysts with Apparent Pan-Enzymatic Mimicry into Nanozymes for Efficient Prodrug Conversion.

Nanozymes, nanoparticles that mimic the natural activity of enzymes, are intriguing academically and are important in the context of the Origin of Life. However, current nanozymes offer mimicry of a narrow range of mammalian enzymes, near-exclusively performing redox reactions. We present an unexpected discovery of non-proteinaceous enzymes based on metals, metal oxides, 1D/2D-materials, and non-metallic nanomaterials. The specific novelty of these findings lies in the identification of nanozymes with apparent mimicry of diverse mammalian enzymes, including unique pan-glycosidases. Further novelty lies in the identification of the substrate scope for the lead candidates, specifically in the context of bioconversion of glucuronides, that is, human metabolites and privileged prodrugs in the field of enzyme-prodrug therapies. Lastly, nanozymes are employed for conversion of glucuronide prodrugs into marketed anti-inflammatory and antibacterial agents, as well as "nanozyme prodrug therapy" to mediate antibacterial measures.

Mesoporous silica nanoparticles carrying multiple antibiotics provide enhanced synergistic effect and improved biocompatibility.

Treatment of polymicrobial infections requires combination therapy with drugs that have different antimicrobial spectra and possibly work in synergy. However, the different pharmacokinetics and adverse side effects challenge the simultaneous delivery of multiple drugs at the appropriate concentrations to the site of infection. Formulation of multiple drugs in nano-carrier systems may improve therapeutic efficacy by increasing the local concentration and lowering the systemic concentration, leading to fewer side effects. In this study, we loaded polymyxin B and vancomycin on bare and carboxyl-modified mesoporous silica nanoparticles (B-MSNs and C-MSNs, respectively) to achieve simulataneous local delivery of antibiotics against Gram-positive and -negative bacteria. Polymyxin B adsorbed preferentially to nanoparticles compared to vancomycin. The total antibiotic loading was 563 µg and 453 µg per mg B-MSNs or C-MSNs, respectively. Both B-MSNs and C-MSNs loaded with antibiotics were effective against Gram-negative and Gram-positive bacteria. The antibiotics had synergistic interactions against Gram-negative bacteria, and the antimicrobial efficacy was higher for antibiotic-loaded C-MSNs compared to free antibiotics at the same concentration even though the cytotoxicity was lower. Our study shows that formulations of existing antibiotics in nanocarrier systems can improve their therapeutic efficiency, indicating that combination therapy with drug-loaded silica nanoparticles may provide a better treatment outcome for infections that require high concentrations of multiple drugs.

Ultra-dense polymer brush coating reduces Staphylococcus epidermidis biofilms on medical implants and improves antibiotic treatment outcome.

Staphylococcal biofilm formation is a severe complication of medical implants, leading to high antibiotic tolerance and treatment failure. Ultra-dense poly(ethylene glycol) (udPEG) coating resists adsorption of proteins, polysaccharides and extracellular DNA. It is therefore uniquely resistant to attachment by Staphylococcus epidermidis, which remains loosely adhered to the surface. Our aim was to determine if S. epidermidis remains susceptible to antibiotics when adhering to udPEG, and if udPEG coatings can improve the treatment outcome for implant-associated infections. We tested the in vitro efficacy of vancomycin treatment on recently adhered S. epidermidis AUH4567 on udPEG, conventional PEG or titanium surfaces using live/dead staining and microscopy. udPEG was then applied to titanium implants and inserted subcutaneously in mice and inoculated with S. epidermidis to induce infection. Mice were given antibiotic prophylaxis or a short antibiotic treatment. One group was given immunosuppressive therapy. After five days, implants and surrounding tissue were harvested for CFU enumeration. Only few S. epidermidis cells adhered to udPEG compared to conventional PEG and uncoated titanium, and a much lower fraction of cells on udPEG survived antibiotic treatment in vitro. In vivo, the bacterial load on implants in mice receiving vancomycin treatment was significantly lower on udPEG-coated compared to uncoated implants, also in neutropenic mice. Our results suggest that the improved outcome results from the coating's anti-adhesive properties that leads to less biofilm and increased efficacy of antibiotic treatment. Thus, the combination of udPEG with antibiotics is a promising strategy to prevent acute implant-associated infections that arise due to perioperative contaminations. STATEMENT OF SIGNIFICANCE: Infections of medical implants is an ever-present danger. Here, bacteria develop biofilms that cannot be eradicated with antibiotics. By using an ultra-dense polymer-brush coating (udPEG), bacterial attachment and the subsequent biofilm formation can be reduced, resulting in increased antibiotic susceptibility of bacteria surrounding the implant. udPEG combined with antibiotics proved to significantly reduce bacteria on implants inserted into mice, in our animal model. As the coating is not antibacterial per se, it does not induce antimicrobial resistance and its effect is independent of the bacterial species. Our results are encouraging for the prospect of preventing and treating implant-associated infections that arise due to perioperative contaminations.

Combatting implant-associated biofilms through localized drug synthesis.

Bacterial contamination of implantable biomaterials is a significant socioeconomic and healthcare burden. Indeed, bacterial colonization of implants after surgery has a high rate of incidence whereas concurrent prophylaxis using systemic antibiotics has limited clinical success. In this work, we develop enzyme-prodrug therapy (EPT) to prevent and to treat bacteria at interfaces. Towards the overall goal, novel prodrugs for fluoroquinolone antibiotics were developed on a privileged glucuronide scaffold. Whereas carbamoyl prodrugs were not stable and not suitable for EPT, glucuronides containing self-immolative linker between glucuronic acid masking group and the antibiotic were stable in solution and readily underwent bioconversion in the presence of ß-glucuronidase. Surface coatings for model biomaterials were engineered using sequential polymer deposition technique. Resulting coatings afforded fast prodrug conversion and mediated antibacterial measures against planktonic species as evidenced by pronounced zone of bacterial growth inhibition around the biomaterial surface. These biomaterials coupled with the glucuronide prodrugs also effectively combatted bacteria within established biofilms and also successfully prevented bacterial colonization of the surface. To our knowledge, this is the first report of EPT engineered to the surface of biomaterials to mediate antibacterial measures.

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms.

Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.

Preclinical evaluation of potential infection-imaging probe [68 Ga]Ga-DOTA-K-A9 in sterile and infectious inflammation.

The development of bacteria-specific infection radiotracers is of considerable interest to improve diagnostic accuracy and enabling therapy monitoring. The aim of this study was to determine if the previously reported radiolabelled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) conjugated peptide [68 Ga]Ga-DOTA-K-A9 could detect a staphylococcal infection in vivo and distinguish it from aseptic inflammation. An optimized [68 Ga]Ga-DOTA-K-A9 synthesis omitting the use of acetone was developed, yielding 93 ± 0.9% radiochemical purity. The in vivo infection binding specificity of [68 Ga]Ga-DOTA-K-A9 was evaluated by micro positron emission tomography/magnetic resonance imaging of 15 mice with either subcutaneous Staphylococcus aureus infection or turpentine-induced inflammation and compared with 2-deoxy-2-[18 F]fluoro-D-glucose ([18 F]FDG). The scans showed that [68 Ga]Ga-DOTA-K-A9 accumulated in all the infected mice at injected doses ≥3.6 MBq. However, the tracer was not found to be selective towards infection, since the [68 Ga]Ga-DOTA-K-A9 also accumulated in mice with inflammation. In a concurrent in vitro binding evaluation performed with a 5-carboxytetramethylrhodamine (TAMRA) fluorescence analogue of the peptide, TAMRA-K-A9, the microscopy results suggested that TAMRA-K-A9 bound to an intracellular epitope and therefore preferentially targeted dead bacteria. Thus, the [68 Ga]Ga-DOTA-K-A9 uptake observed in vivo is presumably a combination of local hyperemia, vascular leakiness and/or binding to an epitope present in dead bacteria.

Effect of DNase Treatment on Adhesion and Early Biofilm Formation of Enterococcus Faecalis.

OBJECTIVE: Extracellular DNA (eDNA) has been shown to be important for biofilm stability of the endodontic pathogen Enterococcus faecalis. In this study, we hypothesized that treatment with DNase prevents adhesion and disperses young E. faecalis biofilms in 96-well plates and root canals of extracted teeth. METHODS: E. faecalis eDNA in 96-well plates was visualized with TOTO-1®. The effect of DNase treatment was assessed in 96-well plates and in extracted single-rooted premolars (n=37) using a two-phase crossover design. E. faecalis was treated with DNase (50 Kunitz/mL) or heat-inactivated DNase for 1 h during adhesion or after 24 h of biofilm formation. In 96-well plates, adhering cells were quantified using confocal microscopy and digital image analysis. In root canals, the number of adhering cells was determined in dentine samples based on colony forming unit counts. Data from the 96-well plate were analyzed using one-tailed t-tests, and data from extracted teeth were analyzed using mixed-effect Poisson regressions. RESULTS: eDNA was present in wells colonized by E. faecalis after 1 h of adhesion and 24 h of biofilm formation; it was removed by DNase treatment, as evidenced by TOTO®-1 staining. DNase treatment reduced the area covered by cells in 96-well plates after 1 h (P<0.05), but not after 24 h (P=0.96). No significant differences in the number of adhering cells were observed in extracted teeth after 1 (P=0.14) and 24 h (P=0.98). CONCLUSION: DNase treatment does not disperse endodontic E. faecalis biofilms. The sole use of DNase as an anti-biofilm agent in root canal treatments is not recommendable.

Loading of polymyxin B onto anionic mesoporous silica nanoparticles retains antibacterial activity and enhances biocompatibility.

Polymyxin B is a polycationic antibiotic used as the last line treatment against antibiotic-resistant Gram negative bacteria. However, application of polymyxin B is limited because of its toxicity effects. Herein, we used bare and surface modified mesoporous silica nanoparticles (MSNs) with an average diameter of 72.29 ±â€¯8.17 nm as adsorbent for polymyxin B to improve its therapeutic properties. The polymyxin B adsorption onto MSN surfaces was explained as a function of pH, type of buffer and surface charge of nanoparticles, according to the ζ-potential of silica nanoparticles and adsorption kinetics results. The highest value of the adsorption capacity (about 401 ±â€¯15.38 mg polymyxin B/ g silica nanoparticles) was obtained for the bare nanoparticles in Tris buffer, pH 9. Release profiles of polymyxin B showed a sustained release pattern, fitting Power law and Hill models. The antibiotic molecules-loaded nanoparticles showed enhanced antibacterial activity compared to free antibiotic against different Gram negative bacteria. Biocompatibility evaluation results revealed that loading of polymyxin B onto MSNs can decrease the cytotoxicity effects of the drug by reducing ROS generation. Our results suggest that formulation of drugs by adsorption onto MSNs may offer a way forward to overcome the adverse effects of some antibiotics such as polymyxin B without compromising their antimicrobial properties.

Extracellular DNA Contributes to Dental Biofilm Stability.

Extracellular DNA (eDNA) is a major matrix component of many bacterial biofilms. While the presence of eDNA and its role in biofilm stability have been demonstrated for several laboratory biofilms of oral bacteria, there is no data available on the presence and function of eDNA in in vivo grown dental biofilms. This study aimed to determine whether eDNA was part of the matrix in biofilms grown in situ in the absence of sucrose and whether treatment with DNase dispersed biofilms grown for 2.5, 5, 7.5, 16.5, or 24 h. Three hundred biofilms from 10 study participants were collected and treated with either DNase or heat-inactivated DNase for 1 h. The bacterial biovolume was determined with digital image analysis. Staining with TOTO®-1 allowed visualization of eDNA both on bacterial cell surfaces and, with a cloud-like appearance, in the intercellular space. DNase treatment strongly reduced the amount of biofilm in very early stages of growth (up to 7.5 h), but the treatment effect decreased with increasing biofilm age. This study proves the involvement of eDNA in dental biofilm formation and its importance for biofilm stability in the earliest stages. Further research is required to uncover the interplay of eDNA and other matrix components and to explore the therapeutic potential of DNase treatment for biofilm control.

Differences in Gene Expression Profiles between Early and Late Isolates in Monospecies Achromobacter Biofilm.

Bacteria of genus Achromobacter are emerging pathogens in cystic fibrosis (CF) capable of biofilm formation and development of antimicrobial resistance. Evolutionary adaptions in the transition from primary to chronic infection were assessed by transcriptomic analysis of successive isolates of Achromobacter xylosoxidans from a single CF patient. Several efflux pump systems targeting antimicrobial agents were upregulated during the course of the disease, whereas all genes related to motility were downregulated. Genes annotated to subsystems of sulfur metabolism, protein metabolism and potassium metabolism exhibited the strongest upregulation. K+ channel genes were hyperexpressed, and a putative sulfite oxidase was more than 1500 times upregulated. The transcriptome patterns indicated a pivotal role of sulfur metabolism and electrical signalling in Achromobacter biofilms during late stage CF lung disease.