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Abstract：
Streptococcus agalactiae (S.agalactiae), also known as group B Streptococcus (GBS), is a highly infectious pathogen. Prolonged antibiotic usage leads to significant issues of antibiotic residue and resistance. Chelerythrine (CHE) is a naturally occurring benzophenidine alkaloid and chelerythrine chloride (CHEC) is its hydrochloride form with diverse biological and pharmacological activities. However, the antibacterial mechanism of CHEC against GBS remains unclear. Thus, this study aims to investigate the in vitro antibacterial activity of CHEC on GBS and elucidate its underlying mechanism. The antibacterial effect of CHEC on GBS was assessed using inhibitory zone, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays, as well as by constructing a time-kill curve. The antibacterial mechanism of CHEC was investigated through techniques such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), measurement of alkaline phosphatase (AKP) activity, determination of Na+ K+, Ca2+ Mg2+-adenosine triphosphate (ATP) activity, observation of membrane permeability, and analysis of intracellular reactive oxygen species (ROS) and mRNA expression levels of key virulence genes. The results demonstrated that the inhibition zone diameters of CHEC against GBS were 14.32 mm, 12.67 mm, and 10.76 mm at concentrations of 2 mg/mL, 1 mg/mL, and 0.5 mg/mL, respectively. The MIC and MBC values were determined as 256 μg/mL and 512 μg/mL correspondingly. In the time-kill curve, 8 × MIC, 4 × MIC and 2 × MIC CHEC could completely kill GBS within 24 h. SEM and TEM analyses revealed significant morphological alterations in GBS cells treated with CHEC including shrinkage, collapse, and leakage of cellular fluids. Furthermore, the antibacterial mechanism underlying CHEC\'s efficacy against GBS was attributed to its disruption of cell wall integrity as well as membrane permeability resulting in extracellular release of intracellular ATP, AKP, Na+ K+, Ca2+ Mg2+. Additionally CHEC could increase the ROS production leading to oxidative damage and downregulating mRNA expression levels of key virulence genes in GBS cells. In conclusion, CHEC holds potential as an antimicrobial agent against GBS and further investigations are necessary to elucidate additional molecular mechanisms.
摘要：
无乳链球菌（S.无乳)，也称为B组链球菌（GBS），是一种高度传染性的病原体。长期使用抗生素会导致抗生素残留和耐药性的重大问题。白屈菜红碱（CHE）是一种天然存在的二苯甲定生物碱，而白屈菜红碱氯化物（CHEC）是其盐酸盐形式，具有多种生物和药理活性。然而,CHEC对GBS的抗菌机制尚不清楚.因此，本研究旨在研究CHEC对GBS的体外抗菌活性，并阐明其作用机制。使用抑制性区评估CHEC对GBS的抗菌作用，最小抑制浓度（MIC），和最小杀菌浓度（MBC）测定，以及通过构建时间杀伤曲线。通过扫描电子显微镜(SEM)和透射电子显微镜(TEM)等技术研究了CHEC的抗菌机理,碱性磷酸酶（AKP）活性的测量，Na+K+的测定,Ca2+Mg2+-三磷酸腺苷(ATP)活性,膜通透性的观察，并分析细胞内活性氧（ROS）和关键毒力基因的mRNA表达水平。结果表明，CHEC对GBS的抑制区直径为14.32mm，12.67mm,和10.76毫米，浓度为2毫克/毫升，1mg/mL,和0.5mg/mL，分别。MIC和MBC值分别确定为256μg/mL和512μg/mL。在时间杀伤曲线中，8×MIC,4×MIC和2×MICCHEC可以在24小时内完全杀死GBS。SEM和TEM分析显示，用CHEC处理的GBS细胞的形态学改变包括收缩，崩溃,和细胞液体的泄漏。此外，CHEC对GBS功效的抗菌机制归因于其细胞壁完整性的破坏以及膜通透性导致细胞内ATP的细胞外释放。AKP,Na+K+,Ca2+Mg2+。此外，CHEC可以增加ROS的产生，从而导致氧化损伤并下调GBS细胞中关键毒力基因的mRNA表达水平。总之,CHEC具有作为抗GBS的抗微生物剂的潜力，需要进一步的研究来阐明其他分子机制。
