Han Yijia, Zhang Yi, Zhang Xiaodong, Huang Zeyu, Kong Jingchun, Wang Xiuxiu, Chen Lijiang, Wang Yue, Cao Jianming, Zhou Tieli, Shen Mo
Department of Clinical Laboratory, Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, China.
Front Microbiol. 2024 Apr 30;15:1291876. doi: 10.3389/fmicb.2024.1291876. eCollection 2024.
Antibiotic misuse and overuse have led to the emergence of carbapenem-resistant bacteria. The global spread of resistance to the novel antibiotic combination ceftazidime-avibactam (CZA) is becoming a severe problem. Antimicrobial peptide PAM-1 offers a novel approach for treating infections caused by antibiotic-resistant bacteria. This study explores its antibacterial and anti-biofilm activities and mechanisms against CZA-resistant , evaluating its stability and biosafety as well.
The broth microdilution method, growth curve analysis, crystal violet staining, scanning electron microscopy, and propidium iodide staining/N-phenyl-1-naphthylamine uptake experiments were performed to explore the antibacterial action and potential mechanism of PAM-1 against CZA-resistant E. coli. The biosafety in diverse environments of PAM-1 was evaluated by red blood cell hemolysis, and cytotoxicity tests. Its stability was further assessed under different temperatures, serum concentrations, and ionic conditions using the broth microdilution method to determine its minimum inhibitory concentration (MIC). Galleria mellonella infection model and RT-qPCR were used to investigate the in vivo antibacterial and anti-inflammatory effects.
antibacterial experiments demonstrated that the MICs of PAM-1 ranged from 2 to 8 μg/mL, with its effectiveness sustained for a duration of 24 h. PAM-1 exhibited significant antibiofilm activities against CZA-resistant ( < 0.05). Furthermore, Membrane permeability test revealed that PAM-1 may exert its antibacterial effect by disrupting membrane integrity by forming transmembrane pores ( < 0.05). Red blood cell hemolysis and cytotoxicity tests revealed that PAM-1 exerts no adverse effects at experimental concentrations ( < 0.05). Moreover, stability tests revealed its effectiveness in serum and at room temperature. The Galleria mellonella infection model revealed that PAM-1 can significantly improve the survival rate of (>50%)for treatment. Lastly, RT-qPCR revealed that PAM-1 downregulates the expression of inflammatory cytokines ( < 0.05). Overall, our study findings highlight the potential of PAM-1 as a therapeutic agent for CZA-resistant infections, offering new avenues for research and alternative antimicrobial therapy strategies.
抗生素的滥用和过度使用导致了碳青霉烯耐药菌的出现。对新型抗生素组合头孢他啶-阿维巴坦（CZA）耐药性的全球传播正成为一个严重问题。抗菌肽PAM-1为治疗由耐药菌引起的感染提供了一种新方法。本研究探讨了其对CZA耐药菌的抗菌和抗生物膜活性及机制，并评估了其稳定性和生物安全性。
采用肉汤微量稀释法、生长曲线分析、结晶紫染色、扫描电子显微镜以及碘化丙啶染色/N-苯基-1-萘胺摄取实验，以探究PAM-1对CZA耐药大肠杆菌的抗菌作用及潜在机制。通过红细胞溶血和细胞毒性试验评估PAM-1在不同环境中的生物安全性。使用肉汤微量稀释法在不同温度、血清浓度和离子条件下进一步评估其稳定性，以确定其最低抑菌浓度（MIC）。利用黄粉虫感染模型和RT-qPCR研究其体内抗菌和抗炎作用。
抗菌实验表明，PAM-1的MIC范围为2至8μg/mL，其有效性持续24小时。PAM-1对CZA耐药菌表现出显著的抗生物膜活性（P<0.05）。此外，膜通透性试验表明，PAM-1可能通过形成跨膜孔破坏膜完整性来发挥其抗菌作用（P<0.05）。红细胞溶血和细胞毒性试验表明，PAM-1在实验浓度下无不良影响（P<0.05）。此外，稳定性试验表明其在血清和室温下均有效。黄粉虫感染模型表明，PAM-1可显著提高感染CZA耐药菌的黄粉虫的存活率（治疗后>50%）。最后，RT-qPCR表明，PAM-1可下调炎性细胞因子的表达（P<0.05）。总体而言，我们的研究结果突出了PAM-1作为治疗CZA耐药菌感染治疗剂的潜力，为研究和替代抗菌治疗策略提供了新途径。
