Antibiotic-resistant infection is a major health problem, and a limited number of drugs are currently approved as antibiotics. Soil bacteria are promising sources in the search for novel antibiotics. The aim of the present study is to isolate and assess soil bacteria with anti-MRSA activity and improve their capabilities by UV mutagenesis. Soil samples from the upper south of Thailand were screened for antibacterial activity using the cross-streak method. Agar well diffusion was used to examine the activity of isolates against a spectrum of human pathogens. The most active isolate was identified by 16S rRNA sequencing, and the production kinetics and stability were investigated. The most promising isolate was mutated by UV radiation, and the resulting activity and strain stability were studied. The results show that isolates from the cross-streak method could inhibit Staphylococcus aureus TISTR 517 (94 isolates) and Escherichia coli TISTR 887 (67 isolates). Nine isolates remained active against S. aureus TISTR 517 and MRSA, and eight isolates inhibited the growth of E. coli TISTR 887 as assessed using agar well diffusion. The most active strain was Brevibacillus sp. SPR-20, which had the highest activity at 24 h of incubation. The active substances in culture supernatants exhibited more than 90% activity when subjected to treatments involving various heat, enzymes, surfactants, and pH conditions. The mutant M201 showed significantly higher activity (109.88–120.22%) and strain stability compared to the wild-type strain. In conclusion, we demonstrate that soil Brevibacillus sp. is a potential resource that can be subjected to UV mutagenesis as a useful approach for improving the production of anti-MRSA in the era of antibiotic resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a high-priority pathogen because its infection is associated with a high mortality rate. It is urgent to search for new agents to treat such an infection. Our previous study isolated a soil bacterium (Brevibacillus sp. SPR-20), showing the highest antimicrobial activity against S. aureus TISTR 517 and MRSA strains. The present study aimed to purify and characterize anti-MRSA substances produced by SPR-20. The result showed that five active substances (P1–P5) were found, and they were identified by LC-MS/MS that provided the peptide sequences of 14–15 residues. Circular dichroism showed that all peptides contained β-strand and disordered conformations as the major secondary structures. Only P1–P4 adopted more α-helix conformations when incubated with 50 mM SDS. These anti-MRSA peptides could inhibit S. aureus and MRSA in concentrations of 2–32 μg/mL. P1 (NH2-VVVNVLVKVLPPPVV-COOH) had the highest activity and was identified as a novel antimicrobial peptide (AMP). The stability study revealed that P1 was stable in response to temperature, proteolytic enzymes, surfactant, and pH. The electron micrograph showed that P1 induced bacterial membrane damage when treated at 1× MIC in the first hour of incubation. The killing kinetics of P1 was dependent on concentration and time. Mechanisms of P1 on tested pathogens involved membrane permeability, leakage of genetic material, and cell lysis. The P1 peptide at a concentration up to 32 μg/mL showed hemolysis of less than 10%, supporting its safety for human erythrocytes. This study provides promising anti-MRSA peptides that might be developed for effective antibiotics in the post-antibiotic era.

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Antibiotic resistance is a major health concern worldwide. In our previous study, some bacterial isolates exhibited antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). However, the production of antibacterial substances by native microorganisms is limited by biosynthetic genes. This study aimed to improve the antibacterial activity of SPR19 using atmospheric and room temperature plasma mutagenesis (ARTP). The results showed that SPR19 belonged to the Brevibacillus genus. The growth curves and production kinetics of antibacterial substances were investigated. Argon-based ARTP was applied to SPR19, and the 469 mutants were preliminarily screened using agar overlay method. The remaining 25 mutants were confirmed by agar well diffusion assay against S. aureus TISTR 517 and MRSA isolates 142, 1096, and 2468. M285 exhibited the highest activity compared to the wild-type strain (10.34–13.59%) and this mutant was stable to produce the active substances throughout 15 generations consistently. The antibacterial substances from M285 were tolerant to various conditions (heat, enzyme, surfactant, and pH) while retaining more than 90% of their activities. Therefore, Brevibacillus sp. SPR19 is a potential source of antibacterial substances. ARTP mutagenesis is a powerful method for strain improvement that can be utilized to treat MRSA infection in the future.

Methicillin-resistant Staphylococcus aureus (MRSA) is a severe threat to public health globally. The development of novel agents has encountered the repeated mechanism of drug resistance. This study aimed to investigate an anti-MRSA substance isolated from a promising soil bacterium. The result showed that an isolate (WUL10) was in the Brevibacillus genus. The minimum inhibitory concentration (MIC) of the purified substance was 1 µg/mL against S. aureus TISTR 517 and MRSA strains. This substance showed the bactericidal effect at the concentration of 1–2 µg/mL against these bacterial indicators. The activity of the substance retained more than 95% when encountering high temperatures and a wide range of pH, but it was sensitive to proteolytic enzymes and SDS. It was identified as a novel antimicrobial peptide (KVLVKYLGGLLKLAALMV-COOH) with the predicted structure of α-helix. The substance could rupture the cell wall of the tested pathogen. MIC and MBC of the synthesized peptide were 16 and 64 µg/mL, respectively. The difference in the activity between the isolated and synthetic peptides might be from the synergistic effects of other AMPs in the purified substance. This novel AMP would provide an advantage for further development of anti-MRSA substances to manage the situation of antibiotic resistance.