New antibiotic leads from marine molluscs for treating respiratory infection

Kate Summer

doi:10.25918/thesis.421

Respiratory infections place a significant burden on global health, while the rise of antimicrobial resistance is reducing the efficacy of available treatments. New antibiotics with novel mechanisms of action targeting respiratory pathogens and biofilms are therefore needed. Natural products have long served as original sources of most pharmaceuticals, and molluscs and other marine invertebrates are recognised as particularly productive leads for drug discovery and development. Antimicrobial proteins and peptides (AMPPs) are also increasingly targeted. The aim of this thesis was to assess the pharmacological potential of molluscan natural products, specifically AMPPs, from hemolymph of the Sydney Rock Oyster (SRO), Saccostrea glomerata, for further research and development as new antibiotic treatments for respiratory infection. The work presented in this thesis comprised firstly, a critical review of traditional molluscan medicines and biomedical evidence, demonstrating the medicinal relevance of bioactive products from this phylum to respiratory disease and highlighting the most promising leads, including hemolymph/AMPPs. Subsequently, we undertook broad in-vitro antibacterial-antibiofilm screening, during which the need for guidance on the use of dimethyl sulfoxide (DMSO) as a delivery solvent was realised. The work showed that, even at low concentrations (<2%), DMSO can have confounding effects on biofilm formation and dispersion, with variable effects between species and within species, which ultimately interferes with interpretation of data for the product of interest. It therefore highlighted the importance of appropriate, clearly defined DMSO controls and data calculations moving forward. The research then focused on SRO as a model species for drug discovery, and a method for reproducible hemolymph extraction and fractionation by preparative high-performance liquid chromatography (HPLC) was developed. This was followed by a series of bioassay-guided experiments to narrow down the active hemolymph protein fraction (named HPE) showing antibacterial/antibiofilm activity. Streptococcus pneumoniae and Streptococcus pyogenes were most susceptible to HPE: minimum bactericidal concentrations (MBCs) of HPE ranged between 4.4 to 24.1 μg/mL and concentrations between 4.8 to 19.3 µg/mL inhibited biofilm formation; 9.6 µg/mL HPE also killed all viable cells in pre-formed S. pneumoniae biofilms. In combination assays, the effectiveness of conventional antibiotics (ampicillin, gentamicin, trimethoprim and ciprofloxacin) was improved between 2 to 32-fold in the presence of HPE (1-12 µg/mL) against a range of clinically important bacteria including Streptococcus sp., Pseudomonas aeruginosa, Moraxella catarrhalis, Klebsiella pneumoniae and Staphylococcus aureus. HPE also had a good stability and safety profile with no evidence of cytotoxicity toward the A549 human respiratory cell line up to 205 µg/mL, which far exceeds effective antimicrobial concentrations. Proteomics analysis of HPE identified a number of proteins including abundant cystatin B-like protein and carbonic anhydrase. Overall, this work has scientifically substantiated the antimicrobial properties of medicinal products derived from SRO hemolymph. It highlights the potential utility of AMPPs in HPE as novel antibiotic treatments for respiratory infections, including those caused by Streptococcus spp. The work warrants further research and development of HPE, and presents great opportunities to value-add to the aquaculture industry and develop collaborations.

New antibiotic leads from marine molluscs for treating respiratory infection

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New antibiotic leads from marine molluscs for treating respiratory infection

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