噬菌体A149的生物学特性及其在小鼠肠道耐万古霉素粪肠球菌感染模型中的治疗评价

Biological characterization of phage A149 and evaluation of its therapeutic effect in a mouse model intestinal infection with vancomycin-resistant Enterococcus faecalis

摘要:

【背景】粪肠球菌(Enterococcus faecalis)是一种革兰氏阳性兼性厌氧细菌，可以引起菌血症、心内膜炎、牙髓感染和尿路感染等疾病。由于抗生素耐药性日趋严重，噬菌体治疗已成为一种有效针对粪肠球菌感染的替代策略。【目的】分离针对粪肠球菌的烈性噬菌体，为开发粪肠球菌噬菌体疗法奠定基础。【方法】分离出针对耐万古霉素粪肠球菌V583的噬菌体A149，通过透射电镜观察噬菌体的形态，测定其最佳感染复数、一步生长曲线及温度和pH稳定性，并进行全基因组测序分析，以阐释该噬菌体的基本生物学特性；测定其裂解谱和体外抑菌曲线，并进一步通过治疗耐万古霉素粪肠球菌肠道感染小鼠模型评价其抗菌性能。【结果】噬菌体A149的最佳感染复数为0.001，具有短尾噬菌体科(Podoviridae)的典型形态特征。噬菌体A149在较宽的温度范围(20-50 ℃)稳定存活，60 ℃滴度略有下降，70 ℃及以上完全失活；在pH 4.0-10.0范围内稳定存活，在pH 11.0时滴度略有下降，pH低于3.0或高于12.0时完全失活。噬菌体A149潜伏期短(≤5 min)，暴发量高(约912 PFU/cell)，基因组不含抗生素抗性基因和毒力基因，在体外可有效抑制粪肠球菌生长达11 h，对所测试的粪肠球菌具有86.4%的裂解能力(51/59)，在小鼠肠道感染模型中可降低粪肠球菌约1.3个数量级。【结论】本研究表明噬菌体A149对于治疗粪肠球菌感染具有一定的应用潜力，为开发粪肠球菌噬菌体防控技术奠定了基础。

Abstract:

[Background] Enterococcus faecalis is a Gram-positive facultative anaerobic bacterium that can cause bacteremia, endocarditis, pulp infection, and urinary tract infection. Due to the aggravated antibiotic resistance, phage therapy has become an alternative strategy for treating E. faecalis infections. [Objective] To isolate virulent phages against E. faecalis and lay a foundation for the development of phage therapy targeting E. faecalis. [Methods] In this study, a phage A149 targeting vancomycin-resistant E. faecalis V583 was isolated, and it was observed by transmission electron microscopy. The phage was then characterized in terms of the optimal multiplicity of infection (MOI), one-step growth curve, thermal and pH stability, and whole genome sequence. The lytic spectrum and anti-bacterial curve in vitro were measured for the phage. Furthermore, the antibacterial properties of the phage were evaluated by treatment of the mouse model of intestinal infection with vancomycin-resistant E. faecalis. [Results] A phage A149 targeting vancomycin-resistant E. faecalis V583 was isolated, with the optimal MOI of 0.001. It had the typical morphological characteristics of the Podoviridae family. Phage A149 remained stable within a wide temperature range (20-50 ℃), with a slight decrease in titer at 60 ℃ and complete inactivation at 70 ℃ and above. Phage A149 survived stably within the range of pH 4.0-10.0, with a slightly decreased titer at pH 11.0, and it was completely inactivated below pH 3.0 or above pH 12.0. Phage A149 had a short incubation period (≤5 min) and a high burst size (about 912 PFU/cell). Its genome did not contain antibiotic resistance genes or virulence genes. Phage A149 can effectively inhibit the growth of E. faecalis for 11 h in vitro. It demonstrated the cleavage capacity of 86.4% for tested E. faecalis (51/59) and reduced the count of E. faecalis by about 1.3 orders of magnitude in a mouse model of intestinal infection. [Conclusion] Phage A149 demonstrates the potential for treating E. faecalis infection, which lays a foundation for the development of phage therapy for the prevention and control of E. faecalis.