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Background: Peri-implantitis has become an inexorable clinical challenge in implantology. Topical immunomodulatory epoxy-tiglianes (EBCs), derived from the Queensland blushwood tree, which induce remodeling and resolve dermal infection via induction of the inflammasome and biofilm disruption, may offer a novel therapeutic approach., Design: In vitro antimicrobial activity of EBC structures (EBC-46, EBC-1013 and EBC-147) against Streptococcus mutans , Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in minimum inhibitory concentration, growth curve and permeabilization assays were determined. Antibiofilm activity was assessed using minimum biofilm eradication concentration (MBEC) experiments. Biofilm formation and disruption assays were analyzed using confocal laser scanning microscopy, scanning electron microscopy and direct plate counting., Results: The observed antimicrobial efficacy of the tested compounds (EBC-1013 > EBC-46 > EBC-147) was directly related to significant membrane permeabilization and growth inhibition ( p  < 0.05) against planktonic S. mutans and P. gingivalis . Antibiofilm activity was evident in MBEC assays, with S. mutans biofilm formation assays revealing significantly lower biomass volume and increased DEAD:LIVE cell ratio observed for EBC-1013 ( p  < 0.05). Furthermore, biofilm disruption assays on titanium discs induced significant biofilm disruption in S. mutans and P. gingivalis ( p  < 0.05)., Conclusions: EBC-1013 is a safe, semi-synthetic, compound, demonstrating clear antimicrobial biofilm disruption potential in peri-implantitis., Competing Interests: This work was supported by QBiotics Group Limited, Yungaburra, Australia, who also supplied the EBC compounds used in this study. DWT. KEH, MFP and LCP have received research funding from QBiotics Group. JS is an employee of QBiotics Group. PR is the Executive Director and Chief Scientific Officer of QBiotics Group. The other authors have no conflicts of interest to disclose., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The management of antibiotic-resistant, bacterial biofilm infections in chronic skin wounds is an increasing clinical challenge. Despite advances in diagnosis, many patients do not derive benefit from current anti-infective/antibiotic therapies. Here, we report a novel class of naturally occurring and semisynthetic epoxy-tiglianes, derived from the Queensland blushwood tree ( Fontainea picrosperma) , and demonstrate their antimicrobial activity (modifying bacterial growth and inducing biofilm disruption), with structure/activity relationships established against important human pathogens. In vitro, the lead candidate EBC-1013 stimulated protein kinase C (PKC)-dependent neutrophil reactive oxygen species (ROS) induction and NETosis and increased expression of wound healing-associated cytokines, chemokines, and antimicrobial peptides in keratinocytes and fibroblasts. In vivo, topical EBC-1013 induced rapid resolution of infection with increased matrix remodeling in acute thermal injuries in calves. In chronically infected diabetic mouse wounds, treatment induced cytokine/chemokine production, inflammatory cell recruitment, and complete healing (in six of seven wounds) with ordered keratinocyte differentiation. These results highlight a nonantibiotic approach involving contrasting, orthogonal mechanisms of action combining targeted biofilm disruption and innate immune induction in the treatment of chronic wounds.

In a number of chronic respiratory diseases e.g. cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), the production of viscous mucin reduces pulmonary function and represents an effective barrier to diffusion of inhaled therapies e.g. antibiotics. Here, a 2-compartment Transwell model was developed to study impaired diffusion of the antibiotic colistin across an artificial sputum (AS) matrix/medium and to quantify its antimicrobial activity against Pseudomonas aeruginosa NH57388A biofilms (alone and in combination with mucolytic therapy). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) revealed that the presence of AS medium significantly reduced the rate of colistin diffusion (> 85% at 48 h; p < 0.05). Addition of alginate oligosaccharide (OligoG CF-5/20) significantly improved colistin diffusion by 3.7 times through mucin-rich AS medium (at 48 h; p < 0.05). Increased diffusion of colistin with OligoG CF-5/20 was shown (using confocal laser scanning microscopy and COMSTAT image analysis) to be associated with significantly increased bacterial killing (p < 0.05). These data support the use of this model to study drug and small molecule delivery across clinically-relevant diffusion barriers. The findings indicate the significant loss of colistin and reduced effectiveness that occurs with mucin binding, and support the use of mucolytics to improve antimicrobial efficacy and lower antibiotic exposure., (© 2022. The Author(s).)

Attachment of polysaccharide carriers is increasingly being used to achieve precision delivery and improved effectiveness of protein and peptide drugs. Although it is clear that their clinical effectiveness relies on the purity and integrity of the conjugate in storage, as well as following administration, instability of polysaccharide-based conjugates can reduce the protective efficacy of the polymer, which may adversely affect the bioactive's potency. As a model, these studies used dextrin-colistin conjugates, with varying degrees of polymer modification (1, 2.5 and 7.5 mol% succinoylation) to assess the effect of storage temperature (- 20, 4, 21 and 37 °C) and duration (up to 12 months) on saccharide and colistin release and antimicrobial activity. Estimation of the proportion of saccharide release (by comparison of area under the curve from size exclusion chromatograms) was more pronounced at higher temperatures (up to 3 and 35% at - 20 °C and 37 °C, respectively after 12 months), however, repeated freeze-thaw did not produce any measurable release of saccharides, while addition of amylase (20, 100, 500 IU/L) caused rapid release of saccharides (> 70% total within 24 h). At all temperatures, conjugates containing the lowest degree of succinoylation released the highest proportion of free colistin, which increased with storage temperature, however no trend in saccharide release was observed. Despite the clear physical effects of prolonged storage, antimicrobial activity of all samples was only altered after storage at 37 °C for 12 months (> threefold decreased activity). These results demonstrate significant release of saccharides from dextrin-colistin conjugates during prolonged storage in buffered solution, especially at elevated temperature, which, in most cases, did not affect antimicrobial activity. These findings provide vital information about the structure-activity relationship of dextrin-colistin conjugates, prior to full-scale commercial development, which can subsequently be applied to other polysaccharide-protein and -peptide conjugates.

Novel therapeutics designed to target the polymeric matrix of biofilms requires innovative techniques to accurately assess their efficacy. Here, multiple particle tracking (MPT) was developed to characterize the physical and mechanical properties of antimicrobial resistant (AMR) bacterial biofilms and to quantify the effects of antibiotic treatment. Studies employed nanoparticles (NPs) of varying charge and size (40-500 nm) in Pseudomonas aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus (MRSA) biofilms and also in polymyxin B (PMB) treated Escherichia coli biofilms of PMB-sensitive (PMB Sens ) IR57 and PMB-resistant (PMB R ) PN47 strains. NP size-dependent and strain-related differences in the diffusion coefficient values of biofilms were evident between PAO1 and MRSA. Dose-dependent treatment effects induced by PMB in PMB Sens E. coli biofilms included increases in diffusion and creep compliance (P < 0.05), not evident in PMB treatment of PMB R E. coli biofilms. Our results highlight the ability of MPT to quantify the diffusion and mechanical effects of antibiotic therapies within the AMR biofilm matrix, offering a valuable tool for the pre-clinical screening of anti-biofilm therapies.

The recent emergence of resistance to colistin, an antibiotic of last resort with dose-limiting toxicity, has highlighted the need for alternative approaches to combat infection. This study aimed to generate and characterise alginate oligosaccharide ("OligoG")-polymyxin (polymyxin B and E (colistin)) conjugates to improve the effectiveness of these antibiotics. OligoG-polymyxin conjugates (amide- or ester-linked), with molecular weights of 5200-12,800 g/mol and antibiotic loading of 6.1-12.9% w / w , were reproducibly synthesised. In vitro inflammatory cytokine production (tumour necrosis factor alpha (TNFα) ELISA) and cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) of colistin (2.2-9.3-fold) and polymyxin B (2.9-27.2-fold) were significantly decreased by OligoG conjugation. Antimicrobial susceptibility tests (minimum inhibitory concentration (MIC), growth curves) demonstrated similar antimicrobial efficacy of ester- and amide-linked conjugates to that of the parent antibiotic but with more sustained inhibition of bacterial growth. OligoG-polymyxin conjugates exhibited improved selectivity for Gram-negative bacteria in comparison to mammalian cells (approximately 2-4-fold). Both OligoG-colistin conjugates caused significant disruption of Pseudomonas aeruginosa biofilm formation and induced bacterial death (confocal laser scanning microscopy). When conjugates were tested in an in vitro "time-to-kill" (TTK) model using Acinetobacter baumannii , only ester-linked conjugates reduced viable bacterial counts (~2-fold) after 4 h. Bi-functional OligoG-polymyxin conjugates have potential therapeutic benefits in the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, directly reducing toxicity whilst retaining antimicrobial and antibiofilm activities.