Antimicrobial peptides from amphibian skin secretion are a promising source for the development of alternative antibiotics against the urgent antibiotic resistance. Methicillin-resistant S. aureus (MRSA) has been found to persist in both early and late disease course of cystic fibrosis (CF). Japonicin-2LF was isolated from the skin secretion of Fujian Large-headed Frog (Limnonectes fujianensis) via the combination of cDNA cloning and MS/MS sequencing. The antimicrobial and anti-biofilm activities of Japonicin-2LF were evaluated using both reference and clinic isolated strains. The permeability of the cell membrane treated by the peptide was revealed by fluorescent staining. The cytotoxicity was examined by haemolysis, MTT and LDH assays. Wax moth larvae (Galleria mellonella) infection model was applied to assess the efficacy of Japonicin-2LF against the reference and clinic MRSA isolates in vivo. Japonicin-2LF exhibited potent antimicrobial activity, particularly against Gram-positive bacteria Staphylococcus aureus and MRSA, killing the bacteria via membrane permeabilisation. Additionally, Japonicin-2LF demonstrated the inhibition and eradication of biofilms, particularly against the biofilm of MRSA by eradicating the biofilm matrix as well as killing all the sessile bacteria. In the in vivo assay, Japonicin-2LF significantly decreased the mortality of MRSA acute infected larvae. In conclusion, it is a novel antimicrobial peptide discovered from the skin secretion of Limnonectes fujianensis, and particularly effective against both planktonic and sessile MRSA. The further in vivo study suggests that Japonicin-2LF could be a potential drug candidate to control the MRSA infection in cystic fibrosis patients.

DMPC-10A (ALWKKLLKK-Cha-NH₂) is a 10-mer peptide derivative from the N-terminal domain of Dermaseptin-PC which has shown broad-spectrum antimicrobial activity as well as a considerable hemolytic effect. In order to reduce hemolytic activity and improve stability to endogenous enzymes, a D-amino acid enantiomer (DMPC-10B) was designed by substituting all L-Lys and L-Leu with their respective D-form amino acid residues, while the Ala¹ and Trp³ remained unchanged. The D-amino acid enantiomer exhibited similar antimicrobial potency to the parent peptide but exerted lower cytotoxicity and hemolytic activity. Meanwhile, DMPC-10B exhibited remarkable resistance to hydrolysis by trypsin and chymotrypsin. In addition to these advantages, DMPC-10B exhibited an outstanding antibacterial effect against Methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae using the Galleria mellonella larva model and displayed synergistic activities with gentamicin against carbapenem-resistant K. pneumoniae strains. This indicates that DMPC-10B would be a promising alternative for treating antibiotic-resistant pathogens.

Amphibian skin secretion is an ideal source of antimicrobial peptides that are difficult to induce drug resistance to due to their membrane-targeting mechanism as a new treatment scheme. In this study, a natural antimicrobial peptide Temporin-1CEh was identified by molecular cloning and mass spectrometry from the skin secretions of the Chinese forest frog (Rana chensinensis). Through the study of the structure and biological activity, it was found that Temporin-1CEh was a helical peptide from the Temporin family, and possessed good anti-Gram-positive bacteria activity through the mechanism of membrane destruction. Seven analogues were further designed to obtain broad-spectrum antimicrobial activity and higher stability in different physiological conditions. The results showed that T1CEh-KKPWW showed potent antibacterial activity with significantly increasing the activity against Gram-negative bacteria in vitro and in vivo with low haemolysis. In addition, T1CEh-KKPWW2 showed high sensitivity to the pH, serum or salts conditions, which applied a branched structure to allow the active units of the peptide to accumulate. Even though the haemolytic activity was increased, the stable antibacterial activity made this novel analogue meet the conditions to become a potential candidate in future antimicrobial and antibiofilm applications.

Summary The objective of this work was to study the effects of inoculation with an arbuscular mycorrhizal fungus (AMF), Glomus mosseae, and/or a phosphate-solubilising fungus (PSF), Mortierella sp. SM-1, on the rhizospheric niche of beach plum (Prunus maritima) when grown under 1% (w/v) NaCl stress. Salinisation adversely affected colonisation by AMF, microorganism populations, available-P, and hydrolysable-N concentrations in the soil, as well as phosphatase, urease, and proteinase activities in the rhizospheric niche of beach plum, resulting in a significant reduction (P 0.05) in the dry weight (DW) of the plants. Plants inoculated with AMF and/or PSF avoided these adverse changes. Combined inoculation with AMF and PSF promoted AMF colonisation, increased plant DWs, and significantly increased bacterial and actinomycete numbers on the root surface and in roots, and nitrogen-fixing bacterial numbers on the root surface of beach plum plants. Dual inoculation with AMF plus PSF also resulted in significantly higher phosphatase, urease, and protease activities, and higher concentrations of available-P and hydrolysable-N in rhizosphere soil than following a single inoculation with AMF or PSF. After 90 d of 1% (w/v) NaCl stress, soil pH values in the rhizosphere of beach plum plants co-inoculated with AMF plus PSF were significantly lower than in the rhizosphere of plants inoculated with AMF or PSF alone.We conclude that inoculation with AMF plus the application PSF synergistically improved the micro-ecological environment and increased soil fertility in the rhizosphere of beach plum, which improved the tolerance of these plants to saline stress.