| Escherichia coli and Salmonella are two of the important pathogens of foodborne zoonoses, which have caused a significant economic impact in animal production and continue to be an increasing public health concern worldwide. Multidrug resistance in E. coli and Salmonella has become a serious threat to the treatment of infections due to the abuse of antibiotics and there is an urgent need to develop novel antibacterial agents to control these two pathogens. Both bacteriophages and bacteriophage-derived lysins have been optimized by millions of years of evolution to specifically recognize and effectively kill host cells. Thus, they are considered to be potential new antibacterial agents to replace or supplement antibiotics. The aim of this study was to isolate multivalent virulent bacteriophage infecting both E. coli and Salmonella and obtain its active lysin, which will lay the foundation for the development of phage-based and lysin-based biocontrol agents against E. coli and Salmonella.A clinical strain of E. coli from swine was used for the screening of bacteriophages and a virulent bacteriophage(named ECGD1) was isolated from sewage. The phages were negatively stained with phosphotungstic acid(PTA) for electron microscopy. The optimal multiplicity of infection(MOI) and host rang of ECGD1 was determined. The phage ECGD1 genome was sequenced and the CDSs(coding sequences) were predicted and annotated by the application of different bioinformatics tools. Whole-genome comparison of ECGD1 to related phages was performed to study their sequence homology and evolutionary relationship. The lysin gene predicted by bioinformatics softwares was cloned into p ET-32 a vector and expressed in E.coli, and the enzymatic activity of its product was tested. Furthermore, synergy testing of lysin with polymyxin B was performed in this study.Preliminary results obtained are as follows:(1) ECGD1 formed clear plaques against the E.coli host cell. Transmission electron microscopy(TEM) revealed an icosahedral capsid with a contractile tail for phage ECGD1, indicating that this phage belongs to the Myoviridae family. The optimal MOI of phage ECGD1 was determined to be 10-3.Investigation of ECGD1 host specificity showed that the host rang was not limited to clinical E.coli strains but included some laboratory strains of E.coli and many clinicalSalmonella strains.(2) The ECGD1 genome is 146,647 base pairs long, with a GC content of 37.5%, 11 t RNA genes and 2 pseudo-t RNA genes. By consecutive application of three different bioinformatics tools, a total of 234 CDSs were annotated. Among these CDSs, 78 were functionally known, while other 156 sequences corresponded to unkown putative genes. Annotation and functional analysis of the predicted CDSs revealed five goups:nucleotide metabolism/DNA replication/recombination, phage genome packaging, structure proteins, host lysis, and additional functions. Whole-genome comparative analysis of ECGD1 to related phages showed ECGD1 had a significantly high homology with phi92 and belongs to the rv-5 like genus of Myoviridae family.(3) The recombinant plasmid p ET-32a-lysin was successfully constructed. Following optimization of expression conditions, a considerable amount of soluble recombinant lysin was obtained and its lytic activity against bacterial cells was confirmed by both “lysis from within” and “lysin from without” process. Furthermore, the minimum inhibitory concentration(MIC) of polymyxin B was reduced by the combination of recombinant lysin and polymyxin B.In conclusion, virulent phage ECGD1 belonging to the rv-5 like genus of Myoviridae family has a wide host rang. The recombinant lysin expressed in this study showed broad-spectrum antimicrobial activity according to preliminary tests. Thus, both phage ECGD1 and its recombinant lysin could be developed as candidate antibacterial agents for the treatment and prophylaxis of E. coli and Salmonella infections. |
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