Semi-purified Antimicrobial Proteins from Oyster Hemolymph Inhibit Pneumococcal Infection.

Pneumococcal infections caused by Streptococcus pneumoniae are a leading cause of morbidity and mortality globally, particularly among children. The ability of S. pneumoniae to form enduring biofilms makes treatment inherently difficult, and options are further limited by emerging antibiotic resistance. The discovery of new antibiotics, particularly those with antibiofilm activity, is therefore increasingly important. Antimicrobial proteins and peptides (AMPs) from marine invertebrates are recognised as promising pharmacological leads. This study determined the in vitro antibacterial activity of hemolymph and unique protein fractions from an Australian oyster (Saccostrea glomerata) against multi-drug-resistant S. pneumoniae. We developed a successful method for hemolymph extraction and separation into 16 fractions by preparative HPLC. The strongest activity was observed in fraction 7: at 42 µg/mL protein, this fraction was bactericidal to S. pneumoniae and inhibited biofilm formation. Proteomic analysis showed that fraction 7 contained relatively high abundance of carbonic anhydrase, cofilin, cystatin B-like, and gelsolin-like proteins, while surrounding fractions, which showed lower or no antibacterial activity, contained these proteins in lower abundance or not at all. This work supports traditional medicinal uses of oysters and contributes to further research and development of novel hemolymph/AMP-based treatments for pneumococcal infections.

Out of control: The need for standardised solvent approaches and data reporting in antibiofilm assays incorporating dimethyl-sulfoxide (DMSO).

Bacteria in biofilm formations are up to 1000 times less susceptible to antibiotics than their planktonic counterparts. Recognition of the role of biofilms in ∼80% of chronic infections, their contribution to bacterial tolerance and development of antimicrobial resistance, and thus the search for compounds with antibiofilm properties, has increased greatly in recent years. The need for robust experimental methods is therefore critical but currently undermined by inappropriate controls when dimethyl-sulfoxide (DMSO) is used to enhance test compound solubility. DMSO is known to have a limited effect on planktonic growth, but emerging data indicates that the solvent can affect biofilm formation even at low concentrations. Here, we present both a literature review on the application of DMSO in in vitro antibiofilm studies, as well as a series of experiments and Bayesian hormetic dose-response modelling to define the effects of DMSO alone and in combination with standard antibiotics using two clinically important biofilm-forming bacteria. DMSO has been used in 76 published studies to solubilise a wide variety of synthesised and natural products, including plant extracts, isolated secondary metabolites, modified lead molecules and proteins, in in vitro antibiofilm assays. DMSO solvent concentrations to which biofilms were exposed ranged between <1 and 100% but unfortunately, 35% of articles did not specify the DMSO concentrations used, 50% of articles did not include solvent controls and, of those that did, 26% did not specify control concentrations, 47% did not report or discuss control data, and 53% omitted media controls. In a further 12 studies, DMSO is used as a biofilm treatment, demonstrating the antibiofilm properties of this solvent at higher concentrations. We provide evidence that DMSO (between 0.03 and 25%) significantly inhibits biofilm formation in Pseudomonas aeruginosa, but not Streptococcus pneumoniae, and acts synergistically with standard antibiotics at very low concentrations (<1%). Interestingly, intermediate concentrations of DMSO (∼6%) strongly promote the growth of P. aeruginosa biofilms. As the research community strives to identify bioactive antimicrobial compounds, there is a need for increased scientific rigour when using DMSO as a solvent in antibiofilm assays.

Molluscan Compounds Provide Drug Leads for the Treatment and Prevention of Respiratory Disease.

Respiratory diseases place an immense burden on global health and there is a compelling need for the discovery of new compounds for therapeutic development. Here, we identify research priorities by critically reviewing pre-clinical and clinical studies using extracts and compounds derived from molluscs, as well as traditional molluscan medicines, used in the treatment of respiratory diseases. We reviewed 97 biomedical articles demonstrating the anti-inflammatory, antimicrobial, anticancer, and immunomodulatory properties of >320 molluscan extracts/compounds with direct relevance to respiratory disease, in addition to others with promising bioactivities yet to be tested in the respiratory context. Of pertinent interest are compounds demonstrating biofilm inhibition/disruption and antiviral activity, as well as synergism with approved antimicrobial and chemotherapeutic agents. At least 100 traditional medicines, incorporating over 300 different mollusc species, have been used to treat respiratory-related illness in cultures worldwide for thousands of years. These medicines provide useful clues for the discovery of bioactive components that likely underpin their continued use. There is particular incentive for investigations into anti-inflammatory compounds, given the extensive application of molluscan traditional medicines for symptoms of inflammation, and shells, which are the principal molluscan product used in these preparations. Overall, there is a need to target research toward specific respiratory disease-related hypotheses, purify bioactive compounds and elucidate their chemical structures, and develop an evidence base for the integration of quality-controlled traditional medicines.

Toxicity of manganese to various life stages of selected marine cnidarian species.

Manganese (Mn) pollution in marine waters is increasing and sensitivities to this metal vary widely among marine species. The aims of this study were to characterise Mn chemistry in seawater, and evaluate the toxic effects of Mn on various life stages of two scleractinian corals - the branching sp. Acropora spathulata and massive sp. Platygyra daedalea, and the anemone Exaiptasia pallida. Analytical and theoretical characterisation experiments showed that 97-100% of Mn (II) additions ≤ 200 mg/L in seawater were soluble over 72 h and largely assumed labile complexes. Concentrations estimated to reduce coral fertilisation success by 50% (5.5-h EC50) were 237 mg/L for A. spathulata and 164 mg/L for P. daedalea. A relatively low 72-h LC50 of 7 mg/L was calculated for A. spathulata larvae. In a pilot test using fragments of adult A. spathulata, intact coral tissue rapidly sloughed away from the underlying skeleton at very low concentrations with a 48-h EC50 of just 0.7 mg/L. For E. pallida, survival, tentacle retraction and reproduction were unaffected by prolonged high exposures (12-d NOEC 54 mg/L). This study provides important data supporting the derivation of separate water quality guidelines for Mn in systems with and without coral - a decision recently considered by Australian and New Zealand authorities. It demonstrates the high sensitivity of coral larvae and adult colonies to Mn and the potential risks associated with relying on other early life stage tests and/or E. pallida as ecotoxicological representatives of critically important scleractinian corals.

Trace element contaminant uptake in phytocap vegetation and implications for koala habitat, Lismore, Australia.

Phytocapping is increasingly regarded as an economical and environmentally sustainable post-closure landfill management strategy. During 2013, a phytocap comprised of koala habitat trees was established on a historic landfill site in Lismore as part of an Australian trial program (A-ACAP). This case study was conducted to determine trace element contamination of the Lismore phytocap soil and foliage, and identify risks to grazing koalas. Foliage of Eucalyptus tereticornis, a key koala food tree, and Acacia melanoxylon, a reference native species, were assessed at the phytocap and an uncontaminated reference site. Concentrations of Ag, As, Hg and Pb were significantly higher in foliage from the phytocap compared to that from the reference site (p < 0.0001df 7, 52). Mean trace element concentrations in phytocap E. tereticornis foliage were compliant with state and international standards for contaminants in food and animal feed (NSW State Government, 2010; WHO and FAO, 2015) and soil was compliant with national health-based investigation levels for contaminated sites (NEPC, 2011). However, contaminant distribution was not homogenous, and As and Pb concentrations exceeded guidelines in some soil and foliage samples. Based on available guidelines and weekly dietary intake calculations, risks to koala health posed by trace element contamination of phytocap foliage are currently low, though should be managed by continued monitoring as the vegetation matures.