Construction And Application Of EGFP-Labeled Cell Wall Binding Domain Derived From VB＿SarS＿BM31 In Detection Of Staphylococcus

Coagulase negative staphylococci(CoNS)are often responsible for nosocomial infections in clinical,and they are also the major pathogen which could cause mastitis in dairy cows.Ingestion of related animal products which were contaminated by CoNS may also cause foodborne infections in humans and promote the spread of drug resistance among staphylococci.The biofilms forming ability and multi-drug resistance of CoNS had posed intractable problems for treatment and prevention.Therefore,rapid,accurate,and efficient detection of CoNS is of great significance to public health.Phages and their derived proteins have been favored for detection applications of bacteria based on their advantages such as easy availability and high host specificity.In this study,a highly specific phage was isolated using Staphylococcus arlettae,an emergency CoNS pathogen as an indicator.After analyzing of its biological characteristics and genome sequences,the fusion protein of enhanced green fluorescent protein(EGFP)and cell wall binding domain(CBD)of phage was constructed by prokaryotic expression for detection of staphylococci,providing technical reserves for establishing rapid and effective detection method for staphylococci.1.Isolation,identification,and genome analyzing of S.arlettae phageIn this study,a long-tailed phage vB_Sar S_BM31 which had a high host specificity was isolated from the milk sample of cows suffering from clinical mastitis in a dairy farm in Sichuan Province,using S.arlettae SAR31 as an indicator.The results of biological characterization tests showed that phage BM31 was tolerant to higher temperature,intolerant to acid or alkaline environment,with an optimal multiplicity of infection(MOI)of 0.001.The result of one-step growth curve assay showed that phage BM31 has a latency period of 20 min,with a burst size of 49 phage particles per infected cell.Sequencing and annotation of the whole genome showed that phage BM31 was a doble-strand(ds)virus consists of 42,271 base-pairs(bp),with a GC content of 34.59%,and 26 of 65 open reading frames(ORFs)had predictable functions.Symbolized gene of lysogeny phage such as integrase was contained,pem K and maz F-like toxin were also coded in BM31.Analysis of both phylogenetic trees and average nucleotide identity(ANI)had shown that BM31 had the closest genetic relationship with phiRS7(accession number: KF589919),and then was classified within the staphylococcal phage B14 subcluster.2.Prokaryotic expression,activity assays,and preliminary application of fusion proteins in detection of artificial contaminationRecombinant fusion proteins of EGFP with CBDs contain different amino acid(aa)lengths,EGFP-CBD,EGFP-CBDpro,and EGFP-Lys BM31 were successfully constructed by Over-lap PCR and prokaryotic expression according to the predicted CBD aa sequence from gene sequence of lysin.At the same time,recombinant protein EGFP,and Lys BM31 were also constructed to serve as control groups.After then,EGFP-CBDpro was screened out to be the preferred fusion protein with optimal activity from binding and lysis assay.Results of working conditions assay showed that EGFPCBDpro was tolerant to higher temperatures,strong acids,and strong alkaline environment.Results of activity assay showed that SAR31 can be detected by EGFPCBDpro within 10 min with a minimum detection limit of 1 CFU / m L in PBS buffer.SAR31 could be recognized at different growth periods,and EGFP-CBDpro could strongly recognize SAR31 at logarithmic phase.EGFP-CBDpro could recognize a wide range of Staphylococcus,including S.aureus(SA848).Preliminary application of EGFP-CBDpro had been achieved in artificially contaminated ultra-high temperature sterilized milk,pasteurized milk,and fresh beef.It could detect both SAR31 and SA848 at an initial contamination concentration of 0.4 CFU/g in no more than 4 hours.This result suggests that EGFP-CBDpro has a good application prospect in the detection of Staphylococcus.In a word,the findings not only expand the data of CoNS phage in the staphylococcal phage library,but also provides theoretical basis and technical reserves for the establishment of efficient detection methods for staphylococci.