Identification and characterization of antimicrobial peptides from the larvae of black soldier fly, hermetia illucens (diptera: stratiomyidae) and blowfly, chrysomya megacephala (diptera: calliphoridae) as potential solution for antimicrobial resistance

Antimicrobial resistance (AMR) has caused million fatalities on a worldwide scale, originating from the emergence of resistance in bacteria and fungi against commonly employed antibiotics. The larvae of Hermetia illucens (Diptera: Stratiomyidae) and Chrysomya megacephala (Diptera: Calliphoridae) thrive in environments contaminated with bacteria, such as decaying waste and carrion. Therefore, the objective of our research was to identify potential antimicrobial compounds that can protect the larvae from bacterial infections. Haemolymph from approximately 200 larvae of C. megacephala and H. illucens, previously exposed to Methicillin Resistant Staphylococcus aureus (patient’s isolate) or Escherichia coli, was acquired, purified, and fractionated utilizing Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Fractions exhibiting antimicrobial properties were further examined through Quadrupole Time-of-Flight Liquid Chromatography Mass Spectrometry (QTOF-LCMS). The resultant mass spectra were converted into amino acid sequences through de novo algorithms. These sequences were subsequently analysed through bioinformatics using diverse protein databases, including the Antimicrobial Peptide Database version 3 (APD3), UniProt, and the National Centre for Biotechnology Information (NCBI), for comparative evaluation with previously reported peptides. Furthermore, antimicrobial prediction tools from databases such as APD3, Database of Antimicrobial Activity and Structure of Peptides (DBSAAP), Database of Antimicrobial Peptide (DBAMP), and Collection of Antimicrobial Peptide version 4 (CAMPR4) were utilized to anticipate the potential antimicrobial characteristics of the peptide sequences. The results indicated that a specific peptide (HGCGRLSKWFRQPGLLLSVKR), renamed as Chryso-AMP1 demonstrated significant similarity to C. megacephala’s heat shock protein 70 (hsp70), Meanwhile, for H. illucens, the peptide DPAVKPGRLPSAGLLAVLK (renamed as Hil-AMP1) displayed 100% identical identity to a beclin-1-like protein These peptides were synthesized and subjected to antimicrobial assessments against various pathogenic bacteria, including MRSA, Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 10240), Staphylococcus epidermidis (ATCC 12228), and Bacillus subtilis (ATCC 6633). The examined peptide Chryso-AMP1 exhibited efficacy against these bacteria, with a minimum inhibitory concentration (MIC) of 0.06 mg/ml and an inhibition zone ranging from 8 to 11 mm at a concentration of 1 mg/ml. Unfortunately, no antibacterial activity was observed for the peptide Hil-AMP1 from H. illucens. This highlights the significance of supporting the utilization of antimicrobial databases for predictive intents with laboratory experiments. The cytotoxicity of the peptide was gauged using immortalized keratinocyte (HaCat) and human corneal epithelial cell lines (HCEC), revealing an average cell viability exceeding 80% at concentration of 0.06 mg/ml. In conclusion, the peptide Chryso-AMP1 could serve as a promising candidate for future antimicrobial drug development as potential solution to antimicrobial resistance.