Purification and Identification of Antimicrobial Peptides Produced by Thermophilic Bacteria Isolated from Hot Springs

Every year, bacterial infections lead to over 7.7 million deaths, with 4.95 million of these caused by drug-resistant organisms. This growing resistance emphasizes the urgent need for a different approach to combat bacterial infections. Antimicrobial peptides (AMPs) offer a promising solution due to their unique mechanisms of action. However, their clinical application is often limited by their instability under extreme physiological conditions like high temperatures. To address this limitation, this study focuses on extracting and characterizing AMPs from thermophilic bacteria, specifically Bacillus licheniformis, Geobacillus stearothermophilus, Brevibacillus borstelensis AK1, and Anoxybacillus flavithermus AK1. These strains exhibited strong antimicrobial activity against Staphylococcus aureus and Escherichia coli. The antimicrobial peptides (AMPs) were purified using C18 reverse-phase column chromatography and characterized through multiple spectrometric techniques. UPLC revealed strong absorption peaks between 210 and 298 nm, while FTIR spectra showed characteristic peptide bands, including Amide A and B (3100-3500 cm^-1), Amide I (1600-1700 cm^-1), Amide II (1480-1600 cm^-1), and Amide III-VI (500-1300 cm^-1), confirming their proteinaceous nature. Complementary NMR analysis further supported these findings, with ¹H signals for alkyl amines (3-4 ppm), aliphatic groups (1-2 ppm), and amide protons (7-10 ppm), as well as ¹³C resonances characteristic of amide compounds. Bioactive fractions displayed molecular masses ranging from 500 to 2000 Da. In silico genome mining of Brevibacillus borstelensis AK1 and Anoxybacillus flavithermus AK1 revealed gene clusters encoding ribosomally synthesized and post-translationally modified peptides (RiPPs), nonribosomal peptides (NRPs), and a terpene. Despite some thermal sensitivity, the compounds retained substantial activity at elevated temperatures; notably, AMPs from B. borstelensis AK1 preserved over 84% inhibition even after exposure to 70 °C. These results highlight thermophilic bacteria from Saudi Arabian hot springs as a promising source of AMPs with potential applications in medicine, food preservation, and agriculture.