Vergis Jess, Malik Satyaveer Singh, Pathak Richa, Kumar Manesh, Ramanjaneya Sunitha, Kurkure Nitin Vasantrao, Barbuddhe Sukhadeo Baliram, Rawool Deepak Bhiwa
Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Izatnagar, India.
Department of Veterinary Pathology, Nagpur Veterinary College, Nagpur, India.
Front Microbiol. 2020 Sep 18;11:575917. doi: 10.3389/fmicb.2020.575917. eCollection 2020.
The study evaluated the antimicrobial and antibiofilm efficacy of an antimicrobial peptide (AMP), lactoferricin (17-30) [Lfcin (17-30)], against biofilm-forming multi-drug-resistant (MDR) strains of enteroaggregative (EAEC), and subsequently, the antimicrobial efficacy was assessed in a larval model. Initially, minimum inhibitory concentration (MIC; 32 μM), minimum bactericidal concentration (MBC; 32 μM), and minimum biofilm eradication concentration (MBEC; 32 μM) of Lfcin (17-30) were determined against MDR-EAEC field isolates ( = 3). Lfcin (17-30) was tested stable against high-end temperatures (70 and 90°C), physiological concentration of cationic salts (150 mM NaCl and 2 mM MgCl), and proteases (proteinase-K and lysozyme). Further, at lower MIC, Lfcin (17-30) proved to be safe for sheep RBCs, secondary cell lines (HEp-2 and RAW 264.7), and beneficial gut lactobacilli. In the time-kill assay, Lfcin (17-30) inhibited the MDR-EAEC strains 3 h post-incubation, and the antibacterial effect was due to membrane permeation of Lfcin (17-30) in the inner and outer membranes of MDR-EAEC. Furthermore, in the experiments, larvae treated with Lfcin (17-30) exhibited an increased survival rate, lower MDR-EAEC counts ( < 0.001), mild to moderate histopathological changes, and enhanced immunomodulatory effect and were safe to larval cells when compared with infection control. Besides, Lfcin (17-30) proved to be an effective antibiofilm agent, as it inhibited and eradicated the preformed biofilm formed by MDR-EAEC strains in a significant ( < 0.05) manner both by microtiter plate assay and live/dead bacterial quantification-based confocal microscopy. We recommend further investigation of Lfcin (17-30) in an appropriate animal model before its application in target host against MDR-EAEC strains.
该研究评估了一种抗菌肽乳铁蛋白肽（17 - 30）[Lfcin（17 - 30）]对产生物膜的多重耐药（MDR）聚集性大肠杆菌（EAEC）菌株的抗菌和抗生物膜功效，随后在幼虫模型中评估了其抗菌功效。最初，针对MDR - EAEC临床分离株（ = 3）测定了Lfcin（17 - 30）的最低抑菌浓度（MIC；32 μM）、最低杀菌浓度（MBC；32 μM）和最低生物膜根除浓度（MBEC；32 μM）。测试发现Lfcin（17 - 30）在高温（70和90°C）、生理浓度的阳离子盐（150 mM NaCl和2 mM MgCl）以及蛋白酶（蛋白酶K和溶菌酶）作用下稳定。此外，在较低的MIC浓度下，Lfcin（17 - 30）对绵羊红细胞、传代细胞系（HEp - 2和RAW 264.7）以及有益肠道乳酸杆菌被证明是安全的。在时间 - 杀菌试验中，Lfcin（17 - 30）在孵育3小时后抑制了MDR - EAEC菌株，其抗菌作用归因于Lfcin（17 - 30）对MDR - EAEC内外膜的膜渗透作用。此外，在实验中，与感染对照组相比，用Lfcin（17 - 30）处理的幼虫存活率提高，MDR - EAEC数量降低（ < 0.001），组织病理学变化为轻度至中度，免疫调节作用增强，且对幼虫细胞安全。此外，Lfcin（17 - 30）被证明是一种有效的抗生物膜剂，通过微孔板测定法和基于活/死细菌定量的共聚焦显微镜检查，它能以显著（ < 0.05）的方式抑制和根除由MDR - EAEC菌株形成的预先存在的生物膜。我们建议在将Lfcin（17 - 30）应用于目标宿主对抗MDR - EAEC菌株之前，先在合适的动物模型中进行进一步研究。
