Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
Arch Microbiol. 2024 Feb 8;206(3):93. doi: 10.1007/s00203-023-03823-2.
The present work aimed to examine the intracellular antibacterial efficacy of Recombinant Lactobacillus acidophilus/antimicrobial peptides (AMPs) Melittin and Alyteserin-1a, specifically targeting Gram-negative bacteria. The first assessment was to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Recombinant L. acidophilus/AMPs versus Gram-negative and Gram-positive bacteria. In addition, the researchers examined the in vitro viability and safety of AMPs generated by L. acidophilus. The experiments included exposing the AMPs to elevated temperatures, proteases, cationic salts at physiological levels, and specific pH settings. The safety aspect was evaluated using hemolytic analysis utilizing sheep erythrocytes; cytotoxicity assays employing cell lines, and experiments on beneficial gut lactobacilli. An experiment was done using a time-kill method to assess the intracellular antibacterial efficacy of Recombinant L. acidophilus/AMPs compared to pathogenic varieties in HEp-2 cells. Previous investigations have shown that the MBC levels of recombinant L. acidophilus/AMPs were consistently two to four times higher than the equivalent MIC values when evaluated versus Gram-negative bacteria. Furthermore, the stability of the Recombinant L. acidophilus/AMPs showed variability when exposed to elevated temperatures (70 and 90 ℃), treated with protease enzymes (proteinase K, lysozyme), exposed to higher concentrations of physiological salts (150 mM NaCl and 2 mM MgCl), and varying pH levels (ranging from 4.0 to 9.0). The recombinant L. acidophilus/AMPs are non-hemolytic towards sheep erythrocytes, exhibit little cytotoxicity in RAW 264.7 and HEp-2 cells, and are considered safe when compared to beneficial gut lactobacilli. The research examined the intracellular bacteriostatic effects of recombinant L. acidophilus/AMPs on Gram-negative bacteria inside HEp-2 cells. Nevertheless, no notable bactericidal impact was seen on Gram-positive bacteria (P > 0.05). The research shows that recombinant L. acidophilus/AMPs, namely (L. acidophilus/melittin/Alyteserin-1a) as the focus of the investigation, effectively eliminate Gram-negative bacteria. Therefore, more investigation is necessary to elaborate on these discoveries.
本研究旨在探讨重组嗜酸乳杆菌/抗菌肽（AMPs）蜂毒素和 Alyteserin-1a 对革兰氏阴性菌的细胞内抗菌功效。首先，确定重组嗜酸乳杆菌/AMPs 对革兰氏阴性和革兰氏阳性菌的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）。此外，研究人员还研究了嗜酸乳杆菌产生的 AMPs 的体外活力和安全性。实验包括将 AMPs 暴露于高温、蛋白酶、生理浓度的阳离子盐和特定 pH 值下。通过使用绵羊红细胞进行溶血分析、采用细胞系进行细胞毒性测定以及对有益的肠道乳杆菌进行实验来评估安全性。采用时间杀伤法评估重组嗜酸乳杆菌/AMPs 与 HEp-2 细胞中致病性菌相比的细胞内抗菌功效。先前的研究表明，在评估重组嗜酸乳杆菌/AMPs 对革兰氏阴性菌时，MBC 水平始终比 MIC 值高 2 到 4 倍。此外，重组嗜酸乳杆菌/AMPs 的稳定性在暴露于高温（70 和 90℃）、用蛋白酶（蛋白酶 K、溶菌酶）处理、暴露于更高浓度的生理盐（150mM NaCl 和 2mM MgCl）和不同 pH 值（4.0 至 9.0）时会发生变化。重组嗜酸乳杆菌/AMPs 对绵羊红细胞无溶血作用，在 RAW 264.7 和 HEp-2 细胞中细胞毒性较小，与有益的肠道乳杆菌相比被认为是安全的。研究检测了重组嗜酸乳杆菌/AMPs 对 HEp-2 细胞内革兰氏阴性菌的细胞内抑菌作用。然而，对革兰氏阳性菌没有明显的杀菌作用（P>0.05）。研究表明，重组嗜酸乳杆菌/AMPs（以嗜酸乳杆菌/蜂毒素/Alyteserin-1a 为研究重点）能有效消除革兰氏阴性菌。因此，需要进一步研究来阐述这些发现。
