Jeyarajan Sivakumar, Ranjith Sukumar, Veerapandian Raja, Natarajaseenivasan Kalimuthusamy, Chidambaram Prahalathan, Kumarasamy Anbarasu
Microbial Biotechnology Laboratory, Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli 620024, India.
Transgenic Animal Model Core, Biomedical Research Core Facilities, University of Michigan, Ann Arbor, MI 48109, USA.
Infect Dis Rep. 2024 Dec 12;16(6):1214-1229. doi: 10.3390/idr16060096.
Indwelling intrauterine contraceptive devices (IUDs) have surfaces that facilitate the attachment of spp., creating a suitable environment for biofilm formation. Due to this, vulvovaginal candidiasis (VVC) is frequently linked to IUD usage, necessitating the prompt removal of these devices for effective treatment. In this study, we evaluated the susceptibility of antimicrobial peptides in vitro against biofilm forming, Amphotericin B (MIC50 > 2 mg L) resistant and isolated from IUD users who had signs of vaginal candidiasis (hemorrhage, pelvic pain, inflammation, itching, and vaginal discharge). Three antimicrobial peptides, namely, epinecidin-1 (epi-1) and its two variants, namely, variant-1 (Var-1) and variant-2 (Var-2), which were reported to have enhanced antibacterial activity were tested against IUD isolates ( and ) with pathogenic form of as control. Variants of epi-1, namely, Var-1 and Var-2 were created by substituting lysine in the place of histidine and alanine. The antimicrobial activity was measured using the microbroth dilution method to determine the minimum inhibitory concentration (MIC) of peptides against , and . The MIC of each peptide was used for biofilm assay by Crystal violet staining, Scanning Electron Microscopy, and Reactive Oxygen Species (ROS) assay. To find the possible mechanism of anti-biofilm activity by the peptides, their ability to interact with spp. cell membrane proteins such as Exo-β-(1,3)-Glucanase, Secreted Aspartic Proteinase (Sap) 1, and N-terminal Domain Adhesin: Als 9-2 were determined through PatchDock. The MIC values of peptides: epi-1, var-1 and var-2 against are 128 μg mL, 64 μg mL and 32 μg mL, are 256 μg mL, 64 μg mL and 32 μg mL and are 128 µg mL, 128 µg mL and 64 µg mL, respectively. Both the variants outperformed epi-1. Specifically for tested spp., var-1 showed two- to four-fold enhancements and var-2 showed two- to eight-fold enhancements compared to epi-1. Electron microscopy confirmed that the mechanism of action involves pore formation thus inducing reactive oxygen species in spp. cell membrane. Computational analysis showed that the peptides have a high tendency to interact with spp. cell membrane proteins such as Exo-β-(1,3)-Glucanase, Secreted Aspartic Proteinase (Sap) 1, and N-terminal Domain Adhesin: Als 9-2, thereby preventing biofilm formation. The in vitro evidence supports the potential use of epi-1 and its variants to be used as an anti-biofilm agent to coat IUDs in the future for therapeutic purposes.
宫内节育器（IUD）的表面有利于某些物种的附着，为生物膜形成创造了适宜的环境。因此，外阴阴道念珠菌病（VVC）常与IUD的使用有关，需要及时取出这些装置以进行有效治疗。在本研究中，我们评估了抗菌肽对形成生物膜的、对两性霉素B耐药（MIC50>2mg/L）且从有阴道念珠菌病症状（出血、盆腔疼痛、炎症、瘙痒和阴道分泌物）的IUD使用者中分离出的白色念珠菌的体外敏感性。测试了三种抗菌肽，即表皮抗菌肽-1（epi-1）及其两个变体，即变体-1（Var-1）和变体-2（Var-2），据报道它们具有增强的抗菌活性，以致病性白色念珠菌作为对照，对IUD分离株（白色念珠菌和光滑念珠菌）进行测试。epi-1的变体，即Var-1和Var-2，是通过用赖氨酸取代组氨酸和丙氨酸而产生的。使用微量肉汤稀释法测量抗菌活性，以确定肽对白色念珠菌、光滑念珠菌和热带念珠菌的最低抑菌浓度（MIC）。每种肽的MIC用于通过结晶紫染色、扫描电子显微镜和活性氧（ROS）测定进行生物膜测定。为了找出肽的抗生物膜活性的可能机制，通过PatchDock确定它们与白色念珠菌细胞膜蛋白如外-β-（1,3）-葡聚糖酶、分泌天冬氨酸蛋白酶（Sap）1和N端结构域粘附素：Als 9-2相互作用的能力。肽epi-1、Var-1和Var-2对白色念珠菌的MIC值分别为128μg/mL、64μg/mL和32μg/mL，对光滑念珠菌的MIC值分别为256μg/mL、64μg/mL和32μg/mL，对热带念珠菌的MIC值分别为128μg/mL、128μg/mL和64μg/mL。两种变体均优于epi-1。具体针对测试的白色念珠菌物种，与epi-1相比，Var-1显示出2至4倍的增强，Var-2显示出2至8倍的增强。电子显微镜证实其作用机制涉及孔形成从而在白色念珠菌细胞膜中诱导活性氧。计算分析表明，这些肽具有与白色念珠菌细胞膜蛋白如外-β-（1,3）-葡聚糖酶（Exo-β-(1,3)-Glucanase）、分泌天冬氨酸蛋白酶（Sap）1和N端结构域粘附素：Als 9-2相互作用的高度倾向，从而防止生物膜形成。体外证据支持epi-1及其变体在未来有可能用作抗生物膜剂来包被IUD以用于治疗目的。
