Antimicrobial peptide isolated from Bacillus amyloliquefaciens K14 revitalizes its use in combinatorial drug therapy

Abstract

The present study was performed to evaluate the antibacterial activities of an antimicrobial peptide (CSpK14) and the synergies thereof with β-lactams against vancomycin-resistant Staphylococcus aureus (VRSA) and Enterococci (VRE). Our strain was isolated from fermented food (kimchi), which is 99.79 % homologous with Bacillus amyloliquefaciens subsp. plantarum FZB42(T). CSpK14 was purified to homogeneity by diammonium sulfate precipitation, concentration, dialysis, and followed by two-stage chromatographic separation, i.e., Sepharose Cl-6B and Sephadex G-25 chromatography, and had a molar mass of ~4.6 kDa via Tricine SDS-PAGE and in situ examination. It was stable at pH 6.0-11.5 and temperature up to 80 °C. In addition, it was also stable with various metal ions, solvents, and proteases. The N-terminal amino acid sequence was H-Y-D-P-G-D-D-S-G-N-T-G and did not show any significant homology with reported peptides. However, it shows some degrees of identity with alpha-2-macroglobulin and ligand-gated channel protein from different microorganisms. CSpK14 significantly reduced the minimum inhibitory concentrations (MICs) of β-lactams and had no effect on non-β-lactams against VRSA and VRE. MICs of CSpK14/oxacillin and CSpK14/ampicillin were reduced by 8- to 64-fold and 2- to 16-fold, respectively. The time killing assay between CSpK14/oxacillin (2.29-2.37 ΔlogCFU/mL at 24 h) and CSpK14/ampicillin (2.30-2.38 ΔlogCFU/mL at 24 h) being >2-fold and fractional inhibitory concentration index ˂0.5 revealed synergy. Furthermore, the biofilms formed by VRSA and VRE were reduced completely. CSpK14 was simple to purify, had low molecular mass, was stable over a wide pH range or tested chemicals, had broad inhibitory spectrum, and possessed potent synergistic antimicrobial-antibiofilm properties. CSpK14 synergistically enhanced the efficacy of β-lactams and is therefore suitable for combination therapy.

Evaluation of the Antibacterial Material Production in the Fermentation of Bacillus amyloliquefaciens-9 from Whitespotted Bamboo Shark (Chiloscyllium plagiosum)

“…Seven nitrogen sources were assessed based on the antibacterial activity. As shown in Figure 1c, NH 4 Cl, yeast extract, and peptone had similar antibacterial effects against TG1 and BS168. The addition of urea and beef cream led to the loss of antibacterial activity.…”

“…As shown in Figure 2, increasing each component increased the antibacterial activity of GBacillus-9. The optimum concentrations of sucrose, Na 2 HPO 4 , KH 2 PO 4 , NH 4 Cl, MgSO 4 , and NaCl were at 30, 8.5, 3.0, 1.0, 0.5, and 0.5 g L −1 , respectively. The results of various cultures with antibacterial activity are shown in Table 6.…”

“…As shown in Tables 3 and 4, the p-values obtained using ANOVA and the F-test were less than 0.05, demonstrating that the model term was significant (p < 0.05). Tables 3 and 4 showed that the coefficients for A, B, and C were extremely significant (p < 0.05), indicating that sucrose, NH 4 Cl, and MgSO 4 had a remarkable influence on the antibacterial production of GBacillus-9. The model was significant with a very low p-value < 0.01 (p-value Probability > F).…”

“…The addition of urea and beef cream led to the loss of antibacterial activity. NH 4 Cl was selected as the nitrogen source because of its advantages, such as low cost and source availability. , nutrient yeast beef glucose (NYBD), M9, beef peptone yeast (BPY), yeast sucrose peptone (YSP), and yeast peptone glucose (YPG)) on the antibacterial activity of metabolites.…”

“…Marine microorganisms are regarded as major sources of antimicrobial agents [2]. Bacillus amyloliquefaciens, a widely distributed and environmentally friendly aerobic Gram-positive Bacillus bacterium, is rich in metabolites and can secrete bacteriostatic substances, such as lipopeptides [3,4] and glycosidases [5]. B. amyloliquefaciens has been used in animal feeding [6] or the therapy of plant diseases due to its ability to produce broad-spectrum antibacterial activity [7,8].…”