UNASSIGNED: 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-β-(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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

To address the high tolerance of biofilms to antibiotics, it is urgent to develop new strategies to fight against these bacterial consortia. An innovative antibiofilm nanovector drug delivery system, consisting of Dispersin B-permethylated-β-cyclodextrin/ciprofloxacin adamantyl (DspB-β-CD/CIP-Ad), is described here. For this purpose, complexation assays between CIP-Ad and (i) unmodified β-CD and (ii) different derivatives of β-CD, which are 2,3-O-dimethyl-β-CD, 2,6-O-dimethyl-β-CD, and 2,3,6-O-trimethyl-β-CD, were tested. A stoichiometry of 1/1 was obtained for the β-CD/CIP-Ad complex by NMR analysis. Isothermal Titration Calorimetry (ITC) experiments were carried out to determine Ka, ΔH, and ΔS thermodynamic parameters of the complex between β-CD and its different derivatives in the presence of CIP-Ad. A stoichiometry of 1/1 for β-CD/CIP-Ad complexes was confirmed with variable affinity according to the type of methylation. A phase solubility study showed increased CIP-Ad solubility with CD concentration, pointing out complex formation. The evaluation of the antibacterial activity of CIP-Ad and the 2,3-O-dimethyl-β-CD/CIP-Ad or 2,3,6-O-trimethyl-β-CD/CIP-Ad complexes was performed on Staphylococcus epidermidis (S. epidermidis) strains. The Minimum Inhibitory Concentration (MIC) studies showed that the complex of CIP-Ad and 2,3-O-dimethyl-β-CD exhibited a similar antimicrobial activity to CIP-Ad alone, while the interaction with 2,3,6-O-trimethyl-β-CD increased MIC values. Antimicrobial assays on S. epidermidis biofilms demonstrated that the synergistic effect observed with the DspB/CIP association was partly maintained with the 2,3-O-dimethyl-β-CDs/CIP-Ad complex. To obtain this \"all-in-one\" drug delivery system, able to destroy the biofilm matrix and release the antibiotic simultaneously, we covalently grafted DspB on three carboxylic permethylated CD derivatives with different-length spacer arms. The strategy was validated by demonstrating that a DspB-permethylated-β-CD/ciprofloxacin-Ad system exhibited efficient antibiofilm activity.

BACKGROUND: A bacterial biofilm is a cluster of bacterial cells embedded in a self-produced matrix of extracellular polymeric substances such as DNA, proteins, and polysaccharides. Several diseases have been reported to cause by bacterial biofilms, and difficulties in treating these infections are of concern. This work aimed to identify the inhibitor with the highest binding affinity for the receptor protein by screening various inhibitors obtained from Azorella species for a potential target to inhibit dispersin B. This work shows that azorellolide has the highest binding affinity (- 8.2 kcal/mol) among the compounds tested, followed by dyhydroazorellolide, mulinone A, and 7-acetoxy-mulin-9,12-diene which all had a binding affinity of - 8.0 kcal/mol. To the best of our knowledge, this is the first study to evaluate and contrast several diterpene compounds as antibacterial biofilm chemicals.
METHODS: Here, molecular modelling techniques tested 49 diterpene compounds of Azorella and six FDA-approved antibiotics medicines for antibiofilm activity. Since protein-like interactions are crucial in drug discovery, AutoDock Vina was initially employed to carry out structure-based virtual screening. The drug-likeness and ADMET properties of the chosen compounds were examined to assess the antibiofilm activity further. Lipinski\'s rule of five was then applied to determine the antibiofilm activity. Then, molecular electrostatic potential was used to determine the relative polarity of a molecule using the Gaussian 09 package and GaussView 5.08. Following three replica molecular dynamic simulations (using the Schrodinger program, Desmond 2019-4 package) that each lasted 100 ns on the promising candidates, binding free energy was estimated using MM-GBSA. Structural visualisation was used to test the binding affinity of each compound to the crystal structure of dispersin B protein (PDB: 1YHT), a well-known antibiofilm compound.

Bacterial biofilms consist of bacterial cells embedded within a self-produced extracellular polymeric substance (EPS) composed of exopolysaccharides, extra cellular DNA, proteins and lipids. The enzyme Dispersin B (DspB) is a CAZy type 20 β-hexosaminidase enzyme that catalyses the hydrolysis of poly-N-acetylglucosamine (PNAG), a major biofilm polysaccharide produced by a wide variety of biofilm-forming bacteria. Native PNAG is partially de-N-acetylated, and the degree of deacetylation varies between species and dependent on the environment. We have previously shown that DspB is able to perform both endo- and exo-glycosidic bond cleavage of PNAG depending on the de-N-acetylation patterns present in the PNAG substrate. Here, we used a combination of synthetic PNAG substrate analogues, site-directed mutagenesis and in vitro biofilm dispersal assay to investigate the molecular basis for the endo-glycosidic cleavage activity of DspB and the importance of this activity for dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. We found that D242 contributes to the endoglycosidase activity of DspB through electrostatic interactions with cationic substrates in the -2 binding site. A DspBD242N mutant was highly deficient in endoglycosidase activity while maintaining exoglycosidase activity. When used to disperse S. epidermidis biofilms, this DspBD242N mutant resulted in an increase in residual biofilm biomass after treatment when compared to wild-type DspB. These results suggest that the de-N-acetylation of PNAG in S. epidermidis biofilms is not uniformly distributed and that the endoglycosidase activity of DspB is required for efficient biofilm dispersal.

Bacterial biofilms consist of surface-attached communities that secrete polymeric substances to form a biofilm matrix, generating a local microenvironment which helps protect from external factors. One such matrix component produced by a diverse list of microorganisms is the polysaccharide poly-β-1,6-N-acetylglucosamine (PNAG). Dispersin B is a PNAG-specific glycosyl hydrolase, which by leveraging its unique specificity, can be used to design a macromolecular fluorescent PNAG binding probe. An active site mutant of Dispersin B was fused to a fluorescent protein, to generate a probe that bound PNAG but did not hydrolyze its polysaccharide target. The ease and versatility of this strategy has made it possible to study PNAG in the context of maturing biofilms, as the probe tends to sequester in regions of high PNAG density. In this chapter, typical workflows from probe construction to cell-binding and imaging experiments are described.

Prosthetic joint infections (PJI) are frequent complications of arthroplasties. Their treatment is made complex by the rapid formation of bacterial biofilms, limiting the effectiveness of antibiotic therapy. In this study, we explore the effect of a tri-enzymatic cocktail (TEC) consisting of an endo-1,4-β-d-glucanase, a β-1,6-hexosaminidase, and an RNA/DNA nonspecific endonuclease combined with antibiotics of different classes against biofilms of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli grown on Ti-6Al-4V substrates. Biofilms were grown in Trypticase soy broth (TSB) with 10 g/liter glucose and 20 g/liter NaCl (TGN). Mature biofilms were assigned to a control group or treated with the TEC for 30 min and then either analyzed or reincubated for 24 h in TGN or TGN with antibiotics. The cytotoxicity of the TEC was assayed against MG-63 osteoblasts, primary murine fibroblasts, and J-774 macrophages using the lactate dehydrogenase (LDH) release test. The TEC dispersed 80.3 to 95.2% of the biofilms\' biomass after 30 min. The reincubation of the treated biofilms with antibiotics resulted in a synergistic reduction of the total culturable bacterial count (CFU) compared to that of biofilms treated with antibiotics alone in the three tested species (additional reduction from 2 to more than 3 log10 CFU). No toxicity of the TEC was observed against the tested cell lines after 24 h of incubation. The combination of pretreatment with TEC followed by 24 h of incubation with antibiotics had a synergistic effect against biofilms of S. aureus, S. epidermidis, and E. coli Further studies should assess the potential of the TEC as an adjuvant therapy in in vivo models of PJI.

One major factor inhibiting natural wound-healing processes is infection through bacterial biofilms, particularly in the case of chronic wounds. In this study, the micro/nanostructure of a wound dressing was optimized in order to obtain a more efficient antibiofilm protein-release profile for biofilm inhibition and/or detachment. A 3D substrate was developed with asymmetric polyhydroxyalkanoate (PHA) membranes to entrap Dispersin B (DB), the antibiofilm protein. The membranes were prepared using wet-induced phase separation (WIPS). By modulating the concentration and the molecular weight of the porogen polymer, polyvinylpyrrolidone (PVP), asymmetric membranes with controlled porosity were obtained. PVP was added at 10, 30, and 50% w/w, relative to the total polymer concentration. The physical and kinetic properties of the quaternary nonsolvent/solvent/PHA/PVP systems were studied and correlated with the membrane structures obtained. The results show that at high molecular weight (Mw = 360 kDa) and high PVP content (above 30%), pore size decreased and the membrane became extremely brittle with serious loss of physical integrity. This brittle effect was not observed for low molecular weight PVP (Mw = 40 kDa) at comparable contents. Whatever the molecular weight, porogen content up to 30% increased membrane surface porosity and consequently protein uptake. Above 30% porogen content, the pore size and the physical integrity/mechanical robustness both decreased. The PHA membranes were loaded with DB and their antibiofilm activity was evaluated against Staphylococcus epidermidis biofilms. When the bacterial biofilms were exposed to the DB-loaded PHA membrane, up to 33% of the S. epidermidis biofilm formation was inhibited, while 26% of the biofilm already formed was destroyed. These promising results validate our approach based on the development of bioactive-protein-loaded asymmetric membranes for antibiofilm strategies in situations where traditional antibiotic therapies are ineffective.

The exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm-dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity on de-N-acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site-directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the -1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

Regenerative medicine has become an extremely valuable tool offering an alternative to conventional therapies for the repair and regeneration of tissues. The re-establishment of tissue and organ functions can be carried out by tissue engineering strategies or by using medical devices such as implants. However, with any material being implanted inside the human body, one of the conundrums that remains is the ease with which these materials can get contaminated by bacteria. Bacterial adhesion leads to the formation of mature, alive and complex three-dimensional biofilm structures, further infection of surrounding tissues and consequent development of complicated chronic infections. Hence, novel tissue engineering strategies delivering biofilm-targeted therapies, while at the same time allowing tissue formation are highly relevant. In this study our aim was to develop surface modified polyhydroxyalkanoate-based fiber meshes with enhanced bacterial anti-adhesive and juvenile biofilm disrupting properties for tissue regeneration purposes. Using reactive and amphiphilic star-shaped macromolecules as an additive to a polyhydroxyalkanoate spinning solution, a synthetic antimicrobial peptide, Amhelin, with strong bactericidal and anti-biofilm properties, and Dispersin B, an enzyme promoting the disruption of exopolysaccharides found in the biofilm matrix, were covalently conjugated to the fibers by addition to the solution before the spinning process. Staphylococcus epidermidis is one of the most problematic pathogens responsible for tissue-related infections. The initial antibacterial screening showed that Amhelin proved to be strongly bactericidal at 12 μg/ml and caused >50% reductions of biofilm formation at 6 μg/ml, while Dispersin B was found to disperse >70% of pre-formed biofilms at 3 μg/ml. Regarding the cytotoxicity of the agents toward L929 murine fibroblasts, a CC50 of 140 and 115 μg/ml was measured for Amhelin and Dispersin B, respectively. Optimization of the electrospinning process resulted in aligned fibers. Surface activated fibers with Amhelin and Dispersin B resulted in 83% reduction of adhered bacteria on the surface of the fibers. Additionally, the materials developed were found to be cytocompatible toward L929 murine fibroblasts. The strategy reported in this preliminary study suggests an alternative approach to prevent bacterial adhesion and, in turn biofilm formation, in materials used in regenerative medicine applications such as tissue engineering.

Dispersin B (DspB) has shown a great potential for the hydrolysis of polymeric β-1,6-N-acetyl-d-glucosamine (PNAG) to disperse the biofilms formed by various bacteria but with no killing activity. Here we have investigated whether a silver-binding peptide (AgBP) fused to DspB can induce the in situ formation of silver nanoparticles (AgNP) and conjugated to the structure of DspB so that the bacteria cells released from the dispersed biofilm will be killed by the conjugated AgNP. However, the desired conjugate could be obtained because of the silver ions itself was found to precipitate DspB. But, the fusion of AgBP2 to DspB (AgBP2-DspB) could generate at least 2 fold higher activity against soluble substrate 4-nitrophenyl N-acetyl-β-D-glucosaminide (NP-GlcNAc). By applying to a preformed Staphylococcus epidermidis biofilm, AgBP2-DspB could clear 69% of the biofilm while only 37% could be cleared by DspB as observed by fluorescent microscope. As measured by crystal violet staining, biofilm could be eradicated to the same extent by loading AgBP2-DspB activity level approximately 20 fold lower than that of DspB. The biofilm formation could be prevented on a AgBP2-DspB immobilized surface as observed by confocal laser microscope.