Ultrastructural changes in methicillin-resistant Staphylococcus aureus (MRSA) induced by a novel cyclic peptide ASP-1 from Bacillus subtilis: A scanning electron microscopy (SEM) study / Cambios ultraestructurales en Staphylococcusaureus resistente a meticilina (SARM) inducidos por un nuevo péptido cíclico ASP-1 de Bacillussubtilis: un estudio con microscopio electrónico de barrido (MEB)

ABSTRACT Increasing antimicrobial resistance amongStaphylococcus aureusnecessitates a new antimicrobial with a different site of action. We have isolated a novel cyclic peptide-1 (ASP-1) fromBacillussubtiliswith potent activity against methicillin-resistantS. aureus(MRSA) at a minimum inhibitory concentration (MIC) of 8-64μg/ml. Scanning electron micrographs demonstrated drastic changes in the cellular architecture of ASP-1 treated cells ofS. aureusATCC 29213 and an MRSA clinical isolate at MICs, with damages to the cell wall, membrane lysis and probable leakage of cytoplasmic contents at minimum bactericidal concentrations. The ultrastructure alterations induced by ASP-1 have also been compared with those of oxacillin-treated MRSA cells at its MIC using scanning electron microscopy.

RESUMEN El incremento de la resistencia antimicrobiana entre los tipos deS. aureusexige un nuevo agente antimicrobiano con un sitio de acción diferente. Aislamos un nuevo péptido cíclico (ASP-1) deBacillussubtiliscon potente actividad frente aS. aureusresistente a meticilina (SARM) en una concentración inhibitoria mínima (CIM) de 8-64μg/ml. Las micrografías obtenidas con microscopio electrónico de barrido mostraron cambios drásticos en la arquitectura celular de las células deS. aureusATCC 29213 tratadas con ASP-1, y un aislamiento clínico de SARM a la CIM, con daños a la pared celular, lisis de la membrana y probable fuga de contenido citoplasmático a concentraciones bactericidas mínimas. Comparamos también, las alteraciones de la ultraestructura inducidas por ASP-1 con las de células de SARM tratadas con oxacilina a su CIM, utilizando microscopio electrónico de barrido.

Ultrastructural changes in methicillin-resistant Staphylococcus aureus (MRSA) induced by a novel cyclic peptide ASP-1 from Bacillus subtilis: A scanning electron microscopy (SEM) study.

Increasing antimicrobial resistance among Staphylococcus aureus necessitates a new antimicrobial with a different site of action. We have isolated a novel cyclic peptide-1 (ASP-1) from Bacillussubtilis with potent activity against methicillin-resistant S. aureus (MRSA) at a minimum inhibitory concentration (MIC) of 8-64µg/ml. Scanning electron micrographs demonstrated drastic changes in the cellular architecture of ASP-1 treated cells of S. aureus ATCC 29213 and an MRSA clinical isolate at MICs, with damages to the cell wall, membrane lysis and probable leakage of cytoplasmic contents at minimum bactericidal concentrations. The ultrastructure alterations induced by ASP-1 have also been compared with those of oxacillin-treated MRSA cells at its MIC using scanning electron microscopy.

Purification and characterisation of a novel antistaphylococcal peptide (ASP-1) from Bacillus sp. URID 12.1.

A strong antistaphylococcal peptide (ASP-1) from Bacillus subtilis URID 12.1 strain that is active against cefoxitin- and methicillin-resistant Staphylococcus aureus clinical isolates was purified to homogeneity by solvent extraction, silica gel-based adsorption chromatography and reversed-phase high-performance liquid chromatography. The peptide sequence of ASP-1 as determined by MALDI-TOF/MS and ESI-FTICR-MS was acetylated Phe-Thr-Ala-Val-Dhb-Phe-Ile/Leu. The peptide was further analysed by alkaline hydrolysis, ESI-Q-TOF-MS and an ion mobility assay, which detected the presence of a lactone ring in the intact peptide and a cyclic nature, subsequently revealing the linearised peptide sequence as acPhe-Leu-Phe-Thr-Val-Ala-Dhb. Based on the molecular mass (804.5 Da), peptide sequence and amino acid composition, ASP-1 was identified as a lactone ring-containing peptide similar to TL-119, a poorly studied cyclic depsipeptide. Circular dichroism spectroscopy revealed its predominantly random structure in aqueous solution and its ß-sheet conformation in methanol. Minimum inhibitory concentrations (MICs) of the purified peptide against S. aureus and methicillin-resistant S. aureus (MRSA) ranged from 2 µg/mL to 64 µg/mL. At sub-MICs and 1× MIC, ASP-1 showed a strong antibiofilm characteristic. ASP-1 at a concentration of 128 µg/mL did not show haemolytic activity, and no cytotoxicity was observed against hepatic carcinoma and breast carcinoma cell lines at the same concentration. Peptide ASP-1 with anti-MRSA and antibiofilm abilities and non-haemolytic and non-cytotoxic properties has not been reported previously. These findings suggest that it may serve as a lead molecule for developing alternative topical antibacterial agents.

A broad-spectrum antimicrobial activity of Bacillus subtilis RLID 12.1.

In the present study, an attempt was made to biochemically characterize the antimicrobial substance from the soil isolate designated as RLID 12.1 and explore its potential applications in biocontrol of drug-resistant pathogens. The antimicrobial potential of the wild-type isolate belonging to the genus Bacillus was determined by the cut-well agar assay. The production of antimicrobial compound was recorded maximum at late exponential growth phase. The ultrafiltered concentrate was insensitive to organic solvents, metal salts, surfactants, and proteolytic and nonproteolytic enzymes. The concentrate was highly heat stable and active over a wide range of pH values. Partial purification, zymogram analysis, and TLC were performed to determine the preliminary biochemical nature. The molecular weight of the antimicrobial peptide was determined to be less than 2.5 kDa in 15% SDS-PAGE and in zymogram analysis against Streptococcus pyogenes. The N-terminal amino acid sequence by Edman degradation was partially determined to be T-P-P-Q-S-X-L-X-X-G, which shows very insignificant identity to other antimicrobial peptides from bacteria. The minimum inhibitory concentrations of dialysed and partially purified ion exchange fractions were determined against some selected gram-positive and gram-negative bacteria and some pathogenic yeasts. The presence of three important antimicrobial peptide biosynthesis genes ituc, fend, and bmyb was determined by PCR.