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Genetic Analysis and Characterization of a Bacteriophage OCJ19 Active against Enterotoxigenic Escherichia coli.

ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) is the major pathogenic E. coli that causes diarrhea and edema in post-weaning piglets. In this study, we describe the morphology and characteristics of ØCJ19, a bacteriophage that infects ETEC, and performed genetic analysis. Phage ØCJ19 belongs to the family Myoviridae. One-step growth curve showed a latent phase of 5 min and burst size of approximately 20 phage particles/infected cell. Phage infectivity was stable for 2 h between 4°C and 55°C, and the phage was stable between pH 3 and 11. Genetic analysis revealed that phage ØCJ19 has a total of 49,567 bases and 79 open reading frames (ORFs). The full genomic sequence of phage ØCJ19 showed the most similarity to an Escherichia phage, vB_EcoS_ESCO41. There were no genes encoding lysogeny, toxins, virulence factors, or antibiotic resistance in this phage, suggesting that this phage can be used safely as a biological agent to control ETEC. Comparative genomic analysis in terms of the tail fiber proteins could provide genetic insight into host recognition and the relationship with other coliphages. These results showed the possibility to improve food safety by applying phage ØCJ19 to foods of animal origin contaminated with ETEC and suggests that it could be the basis for establishing a safety management system in the animal husbandry.

SUBMITTER: Kim GH

PROVIDER: S-EPMC7492175 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Genetic Analysis and Characterization of a Bacteriophage ØCJ19 Active against Enterotoxigenic Escherichia coli.

Kim Gyeong-Hwuii GH Kim Jae-Won JW Kim Jaegon J Chae Jong Pyo JP Lee Jin-Sun JS Yoon Sung-Sik SS

Food science of animal resources 20200901 5

Enterotoxigenic Escherichia coli (ETEC) is the major pathogenic E. coli that causes diarrhea and edema in post-weaning piglets. In this study, we describe the morphology and characteristics of ØCJ19, a bacteriophage that infects ETEC, and performed genetic analysis. Phage ØCJ19 belongs to the family Myoviridae. One-step growth curve showed a latent phase of 5 min and burst size of approximately 20 phage particles/infected cell. Phage infectivity was stable for 2 h between 4° ...[more]

PMID: 32968727

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Project description:Our understanding of the mechanisms underlying phage-bacterium interactions remains limited. In Escherichia coli, RapZ regulates glucosamine-6-phosphate (GlcN6P) metabolism, the formation of which initiates synthesis of the bacterial cell envelope, including lipopolysaccharides (LPS). However, the role of RapZ, if any, on phage infectivity remains to be investigated. Here, we isolated strains of enterotoxigenic E. coli (ETEC) resistant to its specific lytic bacteriophage vB_EcoM_JS09 (JS09) in a phage aerosol spray experiment. Whole-genome analysis of phage-resistant bacteria revealed the rapZ gene acquired a premature stop mutation at amino acid 227. Here, we report that the mutation in the rapZ gene confers resistance by inhibiting 93.5% phage adsorption. Furthermore, this mutation changes the morphology of phage plaques, reduces efficiency of plating and phage propagation efficiency, and impairs the infectivity of phage JS09 against ETEC. Using scanning electron microscopy assays, we attribute the inability of the phage to adsorb to the loss of receptors in strains with defective RapZ. Analysis of the LPS profile shows that strains with defective RapZ inhibit phage infection by changing the LPS profile in E. coli Preincubation of phage JS09 with LPS extracted from a wild-type (WT) strain blocked infection, suggesting LPS is the host receptor for phage JS09 adsorption. Our data uncover the mechanism by which ETEC resists infection of phage JS09 by mutating the rapZ gene and then increasing the expression of glmS and changing the phage receptor-LPS profile. These findings provide insight into the function of the rapZ gene for efficient infection of phage JS09.IMPORTANCE The development of phage-resistant bacteria is a challenging problem for phage therapy. However, our knowledge of phage resistance mechanisms is still limited. RapZ is an RNase adaptor protein encoded by the rapZ gene and plays an important function in Gram-positive and Gram-negative bacteria. Here, we report the whole-genome analysis of a phage-resistant enterotoxigenic Escherichia coli (ETEC) strain, which revealed that the rapZ gene acquired a premature stop mutation (E227Stop). We show that the premature stop mutation of rapZ impairs the infectivity of phage JS09 in ETEC. Furthermore, our findings indicate that ETEC becomes resistant against the adsorption and infection of phage JS09 by mutating the rapZ gene, increasing the expression of glmS, and changing the phage receptor-LPS profile. It is also first reported here that RapZ is essential for efficient infection of phage JS09.

Project description:BackgroundAntibiotic resistance in Escherichia coli, a member of the Enterobacteriaceae, is of particular concern because it is the most common (Gram-negative) pathogen causing nosocomial and community infections. Researchers are now considering the use of phages for the control of various antibiotic-resistant bacterial infections.ObjectivesThe purpose of this study was to isolate and characterize a novel pathogenic/lytic phage that targets multi-drug resistant (MDR) E. coli 3, and to investigate its effectiveness at lysing this bacterium.Materials and methodsA clinical strain of E. coli 3 was identified based on its 16S rRNA sequencing and its antibiotic resistance profile was determined by the disc diffusion method. A bacteriophage was isolated from wastewater and its various characteristics, such as host range, heat tolerance, pH stability, one step growth, total protein content, and genome size, were determined. The antibacterial property of the phage was determined against log-phase bacterial planktonic cells at 37°C.ResultsThe bacteriophage, designated MJ1, was isolated by testing against a clinical MDR E. coli 3 strain. The MJ1 phage showed a wide range of heat and pH stability. The phage morphology, determined by transmission electron microscopy, revealed a structure comprised of a head (108 ± 0.2 nm long by 128 ± 0.5 nm wide) and a contractile tail (123 ± 0.5 nm long by 15 - 26 nm wide). These features placed the MJ1 phage in the family Myoviridae and the order Caudovirales. Eleven structural proteins (17 to 200 kDa) for this phage were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A double stranded DNA, approximately 32 kb, in size was detected for this phage on agarose gels. The phage efficacy against E. coli 3 planktonic cells was also investigated. The MJ1 phage demonstrated a very good capability to reduce the numbers of E. coli 3 planktonic cells, as determined by a change in the bacterial growth (an optical density decrease at 600 nm from 0.40 to 0.12).ConclusionsMJ1 phage has much potential for use in phage therapy and other applications.

Dataset Information

Genetic Analysis and Characterization of a Bacteriophage OCJ19 Active against Enterotoxigenic Escherichia coli.

Publications

Genetic Analysis and Characterization of a Bacteriophage ØCJ19 Active against Enterotoxigenic <i>Escherichia coli</i>.

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