一株C3形态多重耐药大肠杆菌噬菌体生物学特性和基因组分析

Biological characteristics and genome of a C3-morphotype phage against multidrug-resistant Escherichia coli

摘要:

【目的】多重耐药菌的出现对公共卫生安全构成严重威胁，本研究分离多重耐药大肠杆菌噬菌体，研究其生物学特性和基因组特征，为耐药菌的噬菌体疗法提供理论依据。【方法】使用双层平板法从污水样本中分离纯化大肠杆菌噬菌体；磷钨酸染色后通过透射电镜观察形态；测定其宿主范围，测定温度和pH稳定性、一步生长曲线和体外抑菌效果等生物学特性；体内抑菌试验评估噬菌体对多重耐药大肠杆菌N1203-1Af感染的大蜡螟幼虫的保护作用；基于全基因组测序对其基因组特点进行分析。【结果】本研究分离共得到5株大肠杆菌噬菌体，分别命名为pEC-S163-2.1、pEC-S163-2.2、pEC-M1167-5Ar.1、pEC-m1291-2Dr.1和pEC-N1203-2Af.1；电镜结果显示噬菌体pEC-N1203-2Af.1属于短尾噬菌体中罕见的C3形态型，头部较长，长是宽的2-3倍；pEC-N1203-2Af.1可裂解受试15株大肠杆菌中的3株；感染10 min后进入指数增长期，-20-50 ℃、pH值为4.0-10.0的环境下均能够保持稳定活性；大蜡螟幼虫感染大肠杆菌N1203-2Af后噬菌体pEC-N1203-2Af.1治疗感染复数(multiplicity of infection, MOI)=100、1和0.01在48 h内存活率均达到70%以上(7/10）；噬菌体pEC-N1203-2Af.1基因组全长77 334 bp，(G+C)%含量为42.18%，不携带耐药基因和毒力因子；功能基因预测表明，噬菌体pEC-N1203-2Af.1基因组共有121个CDS，其中CDS53-CDS64是编码噬菌体结构和裂解模块的序列。【结论】多重耐药大肠杆菌噬菌体pEC-N1203-2Af.1具有良好的抑菌活性，生物学特性稳定，可能为Kuravirus属的新成员，其具有罕见的C3形态，这种头部细长的特殊形态可能与CDS63编码的主要头部蛋白有关。不同地区的C3形态噬菌体长尾纤维远端三聚体蛋白一致性小于50%，推测该形态噬菌体为适应不同的环境可能发生了不同方向的进化。

Abstract:

[Objective] The emergence of multidrug-resistant bacteria represents a serious challenge to public health security. In this study, we isolated the phages against multidrug-resistant Escherichia coli and studied their biological and genomic features, aiming to provide a theoretical foundation for the development of phage therapies against drug-resistant bacteria. [Methods] We used the double plate method to isolate E. coli phages from sewage. We employed phosphotungstic acid staining and transmission electron microscopy to observe the phage morphology, and subsequently assessed the biological properties, including host range, thermal and pH stability, one-step growth curve, and in vitro antibacterial effect of the phages. Furthermore, we conducted an in vivo bacteriostasis test to evaluate the protective effects of the phages on Galleria mellonella larvae infected with multidrug-resistant E. coli N1203-1Af. Finally, we carried out whole genome sequencing to analyze the genomic characteristics of the phages. [Results] Five E. coli phages (pEC-S163-2.1, pEC-S163-2.2, pEC-M1167-5Ar.1, pEC-m1291-2DR.1, and pEC-N1203-2Af.1) were isolated. The phage pEC-N1203-2Af.1 presented a C3 morphotype rare among short-tailed phages and was characterized by a long head with the length 2–3 times that of the width. Furthermore, pEC-N1203-2Af.1 demonstrated lytic activity against three out of the 15 tested E. coli strains. Ten minutes after infection, the phage entered an exponential growth phase and exhibited stable activity within the temperature range of –20 to 50 ℃ and pH 4.0–10.0. After treatment with pEC-N1203-2Af.1 at the multiplicity of infection (MOI) levels of 100, 1, and 0.01 for 48 h, the survival of G. mellonella larvae infected with E. coli N1203-2Af reached over 70% (7/10). The genome of phage pEC-N1203-2Af.1 had a total length of 77 334 bp and the G+C content of 42.18%. Notably, the phage did not carry any drug resistance gene or virulence factor. Functional gene prediction revealed a total of 121 CDS in the phage genome, with CDS53–CDS64 encoding the phage structure and lysis module. [Conclusion] The phage pEC-N1203-2Af.1 against multidrug-resistant E. coli exhibits potent antibacterial activity and stable biological characteristics. Our findings suggest that pEC-N1203-2Af.1 may represent a new member of the Kuravirus genus, characterized by a rare C3 morphotype that may be attributed to the main head protein encoded by CDS63. Notably, the distal trimeric protein of bacteriophages with a C3 morphotype exhibits less than 50% consistency across different regions, which indicates that the bacteriophages with this morphotype may have evolved in diverse directions to adapt to varying environmental conditions.