Oluwarinde Bukola Opeyemi, Ajose Daniel Jesuwenu, Abolarinwa Tesleem Olatunde, Montso Peter Kotsoana, Njom Henry Akum, Ateba Collins Njie
Antimicrobial Resistance and Phage Biocontrol Research Group (AREPHABREG), Department of Microbiology, School of Biological Sciences, Faculty of Natural and Agricultural Sciences, North‒West University, Private Mail Bag X2046, Mmabatho, 2735, South Africa.
Food Security and Safety Focus Area, Faculty of Natural and Agricultural Sciences, North‒West University, Private Mail Bag X2046, Mmabatho, 2735, South Africa.
BMC Microbiol. 2024 Dec 19;24(1):528. doi: 10.1186/s12866-024-03691-w.
The increase in multi drug resistance (MDR) amongst food-borne pathogens such as Escherichia coli O157:H7, coupled with the upsurge of food-borne infections caused by these pathogens is a major public health concern. Lytic phages have been employed as an alternative to antibiotics for use against food-borne pathogens. However, for effective application, phages should be selectively toxic. Therefore, the objective of this study was to characterise lytic E. coli O157:H7 phages isolated from wastewater as possible biocontrol agents and access their genomes for the absence of genes that denotes virulence, resistance, toxins, and lysogeny using whole genome sequencing. E. coli O157:H7 bacteriophages showed clear plaques ranging in size from 1.0 mm to 2.0 mm. Spot test and Efficiency of plating (EOP) analysis demonstrated that isolated phages could infect various environmental E. coli strains. Four phages; vB_EcoM_EP32a, vB_EcoP_EP32b, vB_EcoM_EP57, and vB_EcoM_EP69 demonstrated broad lytic spectra against E. coli O157:H7 strains. Transmission Electron Microscopy (TEM) showed that all phages have tails and were classified as Caudoviricetes. Growth parameters showed an average latent period of 15 ± 3.8 min, with a maximum burst size of 392 PFU/cell. The phages were stable at three distinct temperatures (4 °C, 28 °C, and 37 °C) and at pH values of 3.5, 5.0, 7.0, 9.0, and 11.0. Based on their morphological distinctiveness, three phages were included in the Whole Genome Sequencing (WGS) analysis. WGS results revealed that E. coli O157:H7 phages (vB_EcoM_EP32a, vB_EcoP_EP32b, and vB_EcoM_EP57) were composed of linear double-stranded DNA (dsDNA) with genome sizes 163,906, 156,698, and 130,723 bp and GC contents of 37.61, 37, and 39% respectively. Phages vB_EcoM_EP32a and vB_EcoP_EP32b genomes were classified under the class Caudoviricetes, Straboviridae family, and the new genus "Phapecoctavirus", while vB_EcoM_EP57 was classified under the class Caudoviricetes, Autographiviridae family. Genome analysis revealed no lysogenic (integrase), virulence, or antimicrobial resistance sequences in all three Escherichia phage genomes. The overall results provided evidence that lytic E. coli O157:H7 bacteriophages in this study, are relatively stable, can infect diverse E. coli strains, and does not contain genes responsible for virulence, resistance, toxins, and lysogeny. Thus, they can be considered as biocontrol candidates against MDR pathogenic E. coli O157:H7 strains in the food industry.
食源性病原体（如大肠杆菌O157:H7）中多重耐药性（MDR）的增加，以及由这些病原体引起的食源性感染的激增，是一个重大的公共卫生问题。裂解性噬菌体已被用作对抗食源性病原体的抗生素替代品。然而，为了有效应用，噬菌体应具有选择性毒性。因此，本研究的目的是对从废水中分离出的裂解性大肠杆菌O157:H7噬菌体进行表征，将其作为可能的生物防治剂，并通过全基因组测序分析其基因组中是否存在表示毒力、抗性、毒素和溶原性的基因。大肠杆菌O157:H7噬菌体表现出清晰的噬菌斑，大小在1.0毫米至2.0毫米之间。点滴试验和平板接种效率（EOP）分析表明，分离出的噬菌体可以感染各种环境中的大肠杆菌菌株。四种噬菌体；vB_EcoM_EP32a、vB_EcoP_EP32b、vB_EcoM_EP57和vB_EcoM_EP69对大肠杆菌O157:H7菌株表现出广泛的裂解谱。透射电子显微镜（TEM）显示，所有噬菌体都有尾部，被归类为有尾噬菌体目。生长参数显示平均潜伏期为15±3.8分钟，最大裂解量为392个噬菌斑形成单位/细胞。这些噬菌体在三个不同温度（4°C、28°C和37°C）以及pH值为3.5、5.0、7.0、9.0和11.0时都很稳定。基于它们的形态独特性，三种噬菌体被纳入全基因组测序（WGS）分析。WGS结果显示，大肠杆菌O157:H7噬菌体（vB_EcoM_EP32a、vB_EcoP_EP32b和vB_EcoM_EP57）由线性双链DNA（dsDNA）组成，基因组大小分别为163,906、156,698和130,723碱基对，GC含量分别为37.61%、37%和39%。噬菌体vB_EcoM_EP32a和vB_EcoP_EP32b的基因组被归类为有尾噬菌体目、长尾噬菌体科和新属“Phapecoctavirus”，而vB_EcoM_EP57被归类为有尾噬菌体目、肌尾噬菌体科。基因组分析显示，所有三种大肠杆菌噬菌体基因组中均不存在溶原性（整合酶）、毒力或抗菌抗性序列。总体结果表明，本研究中的裂解性大肠杆菌O157:H7噬菌体相对稳定，可以感染多种大肠杆菌菌株。并且不包含负责毒力、抗性、毒素和溶原性的基因。因此，它们可以被视为食品工业中对抗多重耐药性致病性大肠杆菌O157:H7菌株的生物防治候选物。
