Presentation Information
[P02-275]Evaluation of a novel biotechnological produced antibiotic containing Gramicidin S analogs against Staphylococcus aureus and Staphylococcus epidermidis
○Ana B. Moldes1, Ksenia Lvova1, Sara Silvério2, Ana Pereira2, Cátia Pereira2, Nuno da Silva2, Ligia R. Rodrigues2, Jose M. Cruz1 (1. Chemical Engineering Department, School of Industrial Engineering – CINTECX, University of Vigo. (Spain), 2. Center of Biological Engineering, University of Minho (Portugal))
Keywords:
Antimicrobial resistance,antibiotics,Gramicidin,bacteria,Aneurinibacillus
Antimicrobial resistance (AMR) is recognized by the World Health Organization (WHO) as a critical global health threat, increasingly aggravated by the massive use of conventional antibiotics. Thus, the discovery of new antibiotics and the development of structural analogs is essential. Gramicidin S (GrS) is a potent antimicrobial peptide; identifying new GrS analogs could provide distinct activity profiles with reduced cytotoxicity, offering new tools to respond to AMR. This study evaluates a novel biotechnological extract of GrS analogs, produced by fermentation followed by a single-step, room-temperature extraction of Aneurinibacillus aneurinilyticus (A. aneurinilyticus) biomass. The aim of this work is to compare the antimicrobial efficacy of this GrS extract against commercial GrS containing trifluoracetic acid (TFA). The antimicrobial study includes a relevant Gram-positive pathogen (Staphylococcus aureus) and a commensal of the skin microbiota (Staphylococcus epidermidis).
Antimicrobial activity was assessed via broth microdilution using dilutions of commercial GrS and biotechnological GrS extracts obtained in ethanol or PBS. Minimum Inhibitory Concentrations (MICs) were determined after 24 hours of incubation. To monitor population dynamics, optical density (OD) and colony-forming units (CFUs) were measured over 48–72 hours. Aliquots from wells with no visible growth were streaked onto Mueller-Hinton agar (MHA) plates to elucidate the mechanism of action (bacteriostatic vs. bactericidal).
Inhibition assays indicated that the biotechnologically produced ethanolic extract has efficacy comparable to commercial GrS against Staphylococcus aureus ATCC6538, achieving 100% inhibition at concentrations of 5 µg/mL and above. Conversely, a differential inhibitory profile was observed against Staphylococcus epidermidis ATCC1457. The biotechnological GrS extract exhibited higher inhibitory power against this commensal microorganism than the commercial standard. Kinetic analyses revealed that at inhibitory concentrations (5 µg/mL) against S. aureus, growth is suppressed during the initial 24–48 hours; nevertheless, after 48 hours the pathogen initiates an exponential growth phase. This was confirmed by plate streaking from inhibited wells, which demonstrated microbial recovery for both commercial and biotechnologically produced GrS.
The biotechnological extract of GrS analogs demonstrates highly satisfactory antimicrobial activity against S. aureus, paralleling the commercial standard containing TFA. Moreover, the biotechnologically produced GrS (free of TFA) showed higher inhibition of S. epidermidis, suggesting that easily extractable GrS analogs could inhibit a broader spectrum of Gram-positive microorganisms. Therefore, GrS analogs produced biotechnologically by A. aneurinilyticus can compete with commercial GrS containing TFA against Gram-positive bacteria. However, more assays are needed to determine the bactericidal dose.
Antimicrobial activity was assessed via broth microdilution using dilutions of commercial GrS and biotechnological GrS extracts obtained in ethanol or PBS. Minimum Inhibitory Concentrations (MICs) were determined after 24 hours of incubation. To monitor population dynamics, optical density (OD) and colony-forming units (CFUs) were measured over 48–72 hours. Aliquots from wells with no visible growth were streaked onto Mueller-Hinton agar (MHA) plates to elucidate the mechanism of action (bacteriostatic vs. bactericidal).
Inhibition assays indicated that the biotechnologically produced ethanolic extract has efficacy comparable to commercial GrS against Staphylococcus aureus ATCC6538, achieving 100% inhibition at concentrations of 5 µg/mL and above. Conversely, a differential inhibitory profile was observed against Staphylococcus epidermidis ATCC1457. The biotechnological GrS extract exhibited higher inhibitory power against this commensal microorganism than the commercial standard. Kinetic analyses revealed that at inhibitory concentrations (5 µg/mL) against S. aureus, growth is suppressed during the initial 24–48 hours; nevertheless, after 48 hours the pathogen initiates an exponential growth phase. This was confirmed by plate streaking from inhibited wells, which demonstrated microbial recovery for both commercial and biotechnologically produced GrS.
The biotechnological extract of GrS analogs demonstrates highly satisfactory antimicrobial activity against S. aureus, paralleling the commercial standard containing TFA. Moreover, the biotechnologically produced GrS (free of TFA) showed higher inhibition of S. epidermidis, suggesting that easily extractable GrS analogs could inhibit a broader spectrum of Gram-positive microorganisms. Therefore, GrS analogs produced biotechnologically by A. aneurinilyticus can compete with commercial GrS containing TFA against Gram-positive bacteria. However, more assays are needed to determine the bactericidal dose.
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