El-Telbany Mohamed, Wang Chen, Clokie Martha, Lwin Su Zar Chi, Abdelaziz Marwa Nabil Sayed, Maung Aye Thida, Linn Khin Zar, Hofilena Damaso Catherine, Lin Yunzhi, Ye Haomin, Masuda Yoshimitsu, Honjoh Ken-Ichi, Miyamoto Takahisa
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 744 Motooka, Nishi-Ku, Fukuoka, 819-0395, Japan.
Department of Microbiology and Botany, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt.
Probiotics Antimicrob Proteins. 2025 Jun 7. doi: 10.1007/s12602-025-10616-0.
A gene encoding endolysin LysEf-9 from the phage PEF9, a lytic enterococcal phage, was cloned and overexpressed in Escherichia coli. The expressed LysEf-9 protein with a concentration of 62.5 μg/mL reduced the turbidity and viability of E. faecalis JCM 7783 after 3 h of incubation. LysEf-9 showed stability at different pH (2-12) and withstood different temperature treatments up to 60 °C. In addition, it showed a wide spectrum against Enterococci. Phage vB_EfKS5 with antibacterial and antibiofilm properties against E. faecalis was isolated previously. The phage-resistant mutant was isolated and used as an indicator strain in the present study. A bacteriocin-producing E. faecalis strain was also isolated, and the cell-free supernatant (crude bacteriocin) was partially purified by ammonium sulfate precipitation. The crude bacteriocin showed a wide spectrum against Enterococcus spp. and lysed the phage-resistant mutant. Using LysEf-9 with the crude bacteriocin reduced the viable count of E. faecalis JCM 3378 by 2.45 log after 24 h. In addition, LysEf-9 with phage vB_EfKS5 retarded the growth of the phage-resistant mutant. The antibiofilm efficacy of LysEf-9 and crude bacteriocin was also determined. LysEf-9 lowered the viable count of E. faecalis JCM 7783, E. faecium No. 26, and the phage-resistant mutant in the 48-h biofilm by 3 log, 2 log, and 1.5 log, respectively. The combination of LysEf-9 with crude bacteriocin reduced the total viable count of the indicator strains by at least 3 logs. This study shows the strong antibacterial and antibiofilm activity of LysEf-9, and the activity was enhanced when used with crude bacteriocin or phage.
来自裂解性肠球菌噬菌体PEF9的编码内溶素LysEf-9的基因被克隆并在大肠杆菌中过表达。浓度为62.5μg/mL的表达的LysEf-9蛋白在孵育3小时后降低了粪肠球菌JCM 7783的浊度和活力。LysEf-9在不同pH值（2-12）下表现出稳定性，并且在高达60°C的不同温度处理下仍能耐受。此外，它对肠球菌具有广谱活性。先前已分离出对粪肠球菌具有抗菌和抗生物膜特性的噬菌体vB_EfKS5。在本研究中分离出噬菌体抗性突变体并将其用作指示菌株。还分离出了一株产细菌素的粪肠球菌菌株，其无细胞上清液（粗细菌素）通过硫酸铵沉淀进行了部分纯化。粗细菌素对肠球菌属具有广谱活性，并裂解了噬菌体抗性突变体。将LysEf-9与粗细菌素一起使用在24小时后使粪肠球菌JCM 3378的活菌数减少了2.45个对数。此外，LysEf-9与噬菌体vB_EfKS5一起抑制了噬菌体抗性突变体的生长。还测定了LysEf-9和粗细菌素的抗生物膜功效。LysEf-9使48小时生物膜中粪肠球菌JCM 7783、屎肠球菌No. 26和噬菌体抗性突变体的活菌数分别降低了3个对数、2个对数和1.5个对数。LysEf-9与粗细菌素的组合使指示菌株的总活菌数减少了至少3个对数。本研究表明LysEf-9具有强大的抗菌和抗生物膜活性，并且与粗细菌素或噬菌体一起使用时活性增强。
