食源性致病性大肠杆菌O157:H7和O55:H7特异性噬菌体的分离与鉴定

Isolation and identification of phages of food-borne pathogenic Escherichia coli O157:H7 and O55:H7

摘要:

【背景】肠出血性大肠杆菌(enterohemorrhagicEscherichia coli，EHEC) O157:H7和肠致病性大肠杆菌(enteropathogenicE. coli，EPEC) O55:H7是2株常见食源性致病菌，能导致腹泻及肠道外疾病，其特异性噬菌体具有制备新型抗菌制剂的应用前景。【目的】分离能特异裂解O157:H7和O55:H7的噬菌体，并分析其生物学特性和基因组特征，探索致病性大肠杆菌防控的抗生素替代方法。【方法】利用双层平板法从环境水样中分离噬菌体，对其形态、感染复数、宿主范围、一步曲线等生物学特性进行鉴定，使用Illumina MiSeq平台对其全基因组进行测序，利用RAST、Prokka、BLASTp等软件进行生物信息学分析。【结果】分别以E. coli O157:H7和O55:H7为宿主分离出2株特异性烈性噬菌体：vB_EcoM_P251和vB_EcoM_P255，均属于肌尾病毒科(Myoviridae)。最佳感染复数均为1，在培养15 min内能以91.9%和90.8%的速率吸附到宿主细胞上，而且在37-60 ℃、pH 4.0-11.0条件下保持高且稳定的活性；P251仅对大肠杆菌O157:H7和O78:H11菌株具有感染性，P255对O55:H7、O157:H7等11株不同血清型的EHEC和EPEC均具有感染性。P251基因组全长为136 254 bp，P255基因组全长为111 068 bp，GC含量分别为37%和35%；分别含有227、173个开放阅读框(open reading frame，ORF)，其中，80、73个ORF与已知功能基因具有显著相似性；P251还含有2个tRNA。比较基因组显示，P251和P255基因组在其48%的长度上共享72.24%的核苷酸同一性。【结论】分离鉴定了2株新的O157:H7和O55:H7噬菌体P251和P255，宿主范围广泛，具有防控食源性致病性大肠杆菌的潜力。

Abstract:

[Background] Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O55:H7 are common food-borne pathogens, causing intestinal infection and other diseases. The specific bacteriophages are of great potential for the development of new antibacterial agents. [Objective] To isolate the phages of O157:H7 and O55:H7 and to characterize their biological and genomic characteristics for future development of phage therapy. [Methods] Phages were separated from environmental water samples by the double agar overlay plaque assay. Their biological characteristics, including morphology, multiplicity of infection (MOI), host range, as well as one-step growth curve, were studied. Their genomes were sequenced by the Illumina MiSeq and then analyzed with RAST, Prokka, BLASTp. [Results] We isolated two potent phages of Myoviridae family, vB_EcoM_P251 and vB_EcoM_P255, using E. coli O157:H7 and O55:H7 as host, respectively. Their optimal MOI were both 1, and 91.9% and 90.8% of them adsorbed to the respective host within 15 min. Their lysis activity was high and stable at 37–60 ℃ and pH 4.0–11.0. P251 was only infectious to E. coli O157:H7 and O78:H11, while P255 was infectious to 11 EHEC and EPEC strains, including O55:H7 and O157:H7. The P251 genome and P255 genome are 136 254 bp and 111 068 bp in length separately, with GC content of 37% and 35%, respectively. Their genomes contain 227 and 173 open reading frames (ORFs), separately, 80 and 73 of which share significant similarities to functional genes. Besides, P251 contains 2 tRNAs. In addition, genomes of P251 and P255 share 72.24% nucleotide identity over 48% of their length. [Conclusion] Two new O157:H7 and O55:H7 phages, P251 and P255, with strong lysis activity, wide host range, as well as a great application potential in food-borne pathogenic E. coli control, were isolated and identified.