The Role of Seaweed Antimicrobials in Selection for Antibiotic Resistance

Antibiotic resistance is quickly becoming one of the biggest modern-day threats to human health. It has not only been observed in the clinic but in natural environments as well. Selection for antimicrobial resistant bacteria in the marine environment has been shown to be driven factors such as low concentrations of antibiotics entering the environment through discharge from wastewater treatment plants and run off from agricultural sites. However, antimicrobial resistance is likely to not be solely due to anthropogenic pollution, as it is an ancient mechanism and has been found in environments with minimal human exposure. Here we investigated whether natural antimicrobial producers, i.e. seaweeds, select for antibiotic resistant bacteria. We used both culture-based and molecular techniques to characterise the bacterial communities associated with different seaweed species, focusing on the human pathogens Vibrio, E. coli and S. aureus. Vibrio was harboured by all the seaweeds tested but E. coli and S. aureus were not. For the first time, we tested if Vibrio isolated from seaweed are locally adapted to their host macroalgae using a novel seaweed media assay. Our results showed Vibrio did not display local adaption. We tested the resistance profiles of bacteria isolated from seaweeds and found Vibrio showed cross-resistance to antibiotics and natural antimicrobial, in the form of methanolic seaweed extracts. We can conclude seaweeds harbour antibiotic resistant bacteria, but specific species of seaweeds do not select for specific antibiotic resistance. We quantified the prevalence of a biomarker for antibiotic resistance, the intI1 gene, and found seaweed select for antibiotic resistant bacteria independent of anthropogenic pollution, suggesting seaweed-associated bacterial resistance is an intrinsic mechanism. Using metagenomics, we characterised possible antimicrobial resistance genes associated with different seaweed species from which we were able identify eflamycin, aminocoumarin and fluoroquinolone resistance genes on all the seaweeds tested. Two of the antibiotic classes are produced by Streptomyces, which is present on seaweeds. Suggesting resistance on seaweeds is selected for by bacterial community or the genes characterised show cross-resistance to seaweed antimicrobials

Antifungal and antibacterial electrospun wound dressings for complex wounds

Introduction: Management of open fractures wounds, diabetic ulcers and military wounds frequently involve infections with gram-positive or gram-negative bacteria and in some cases invasive fungal infections which are linked to mycotic emboli and delays in reconstructive efforts or amputations. Topical antibiotics and antifungals are recommended and local delivery of antimicrobials through beads or bead pouches along with a water impermeable dressing has been shown to be beneficial 1 . Here we present a dressing prepared by alternating electrospun polymeric mats loaded with combination of antifungal (amphotericin B, AmB) and antibacterial (vancomycin) agents in clinically relevant concentrations that can be used for the treatment of complex wounds. Methods: Electrospun membranes were produced using a Spraybase 30kV electrospinning kit attached to a syringe pump (NE-1000). The collection distance was set to 12 cm and a voltage of 16.5 kV was utilised. Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) (8g) was dissolved in acidified methanol and AmB or vancomycin were dispersed prior to the addition of dichloromethane to elicit a suspension that was perfused at 8mL/h via the inner needle of a co-axial electrospinning needle (~900 µm, Rame-hart Instrument Co). Membranes were collected on foil after a stable “cone-jet” mode and a uniform fiber production process was achieved and stored under desiccated conditions in the fridge till further use. Release experiments were conducted in phosphate buffer (50mM, pH 7.4) and levels were quantified using a validated HPLC method 2 . Membranes were analysed using FT-IR, DSC and TGA and their morphology using SEM 3 . Disk diffusion inhibition halo assays against Candida albicans (strain) were performed as previously described 3 . Results: Amphotericin B membranes contained near 100% of AmB sprayed (1.05±0.96 mg/g) and demonstrated a fibrous morphology with higher curvature than Soluplus electrospun fibers (Figure 1). AmB electrospun dressings were amorphous and FT-IR indicated hydrogen bonding between the protonated amine of Amphotericin B and carbonyl groups of Soluplus. Near 30% release was achieved after 1 hour, while a controlled release profile was observed for the first 2 days. Released AmB was present in monomeric form (UV). Inhibition halo of AmB dressings or AmB DMSO impregnated filter papers (6mm, 10 µg) resulted in comparable halos against C. albicans (23 ± 1 mm and 24 ± 1mm respectively). Currently work is undertaken to characterise vancomycin dressings and combined dressings. Conclusions/Implications: Prepared dressings can be utilised for treatment of complex fungal infected wounds to avoid mycotic embolism