Objectives: The mannose phosphotransferase system (Man-PTS) plays crucial roles in the adaptive metabolic activity of Enterococcus faecalis (E. faecalis) in adverse environments. The aim of this study was to evaluate the role of Man-PTS in the alkaline resistance of E. faecalis against calcium hydroxide (CH) and the effect of metformin (Met) on the alkaline resistance of E. faecalis to CH.

Materials and methods: The regulatory role of Man-PTS EII in the alkaline resistance of E. faecalis was firstly investigated using a wild-type highly alkaline-resistant E. faecalis XS 003, standard ATCC 29212 and Man-PTS EIID gene deficient (△mptD) and overexpressing (+mptD) strains of E. faecalis. RNA sequencing of Met-treated E. faecalis was performed to further validate the effect of Met on Man-PTS. The effect of Met on CH resistance of E. faecalis was verified by evaluating the survival, membrane potential and permeability, intracellular pH and ATP, and the expression of Man-PTS EII and membrane transporter-related genes of E. faecalis. The effect of Met on the ability of CH to remove E. faecalis biofilm on the dentin surface was also tested. The in vivo therapeutic effect of Met plus CH (CHM) was further investigated in a rat apical periodontitis model induced by E. faecalis XS 003.

Results: Man-PTS EII significantly promoted the survival ability of E. faecalis in CH and enhanced its resistance to CH. The inhibition of Man-PTS EII by Met resulted in reduced alkaline resistance of E. faecalis in the presence of CH, while also enhancing the antimicrobial properties of CH against E. faecalis biofilm on dentin. Additionally, Met plus CH showed the synergistically promoted intra-canal E. faecalis infection control and healing of periapical lesion in rats.

Conclusions: Met could significantly reduce the alkaline resistance of E. faecalis against CH through the modulation of Man-PTS EII, and improved the antibacterial effect of CH against E. faecalis infection both in vitro and in vivo.

Clinical relevance: Met could significantly enhance the ability of CH to control E. faecalis infection through reducing the alkaline resistance of E. faecalis.

Keywords: Enterococcus faecalis; Alkaline resistance; Calcium hydroxide; Mannose phosphotransferase system; Metformin.

© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

目标: 甘露糖磷酸转移酶系统（Man-PTS）在不利环境下的适应性代谢活动中对肠球菌属粪肠球菌（E. faecalis）起着关键作用。本研究旨在评估Man-PTS在E. faecalis对抗氢氧化钙（CH）碱性抗性中的作用，以及二甲双胍（Met）对E. faecalis对抗CH的碱性抗性的效果。

材料和方法: 首先使用野生型高度耐碱的E. faecalis XS 003、标准ATCC 29212以及缺乏Man-PTS EIID基因（△mptD）和过表达（+mptD）的E. faecalis菌株，调查了Man-PTS EII在E. faecalis碱性抗性中的调节作用。通过进行Met处理后的RNA测序来进一步验证Met对Man-PTS的影响。通过评估E. faecalis在CH中的存活率、膜电位和通透性、细胞内pH值以及ATP水平，以及E. faecalis的Man-PTS EII和膜转运相关基因的表达情况，验证了Met对E. faecalis对抗CH耐药性的影响。此外还测试了Met对E. faecalis在牙本质表面生物膜去除能力的影响。进一步通过使用E. faecalis XS 003诱导的大鼠根尖周炎模型研究了Met加CH（CHM）的体内治疗效果。

结果: Man-PTS EII显著提高了E. faecalis在CH中的存活能力和对抗CH的耐药性。通过使用Met抑制Man-PTS EII，在存在CH的情况下，导致了E. faecalis碱性抗性的降低，并且增强了CH对牙本质表面E. faecalis生物膜的抗菌性能。此外，Met加CH在大鼠根管内E. faecalis感染控制和根尖周病灶愈合方面也表现出协同促进的效果。

结论: 通过调节Man-PTS EII，Met能够显著降低E. faecalis对抗CH的碱性抗性，并且提高了CH在体外和体内对E. faecalis感染的抗菌效果。

临床相关性: 通过降低E. faecalis的碱性抗性，Met能够显著增强CH控制E. faecalis感染的能力。

关键词: 粪肠球菌；碱性耐受性；氢氧化钙；甘露糖磷酸转移酶系统；二甲双胍。