Analysis Of The Whole Genome Sequencing To Salmonella And The Development And Application Of DNA Isothermal Amplification Assays For Detecting Salmonella

Salmonella is one of the major pathogens that cause a global foodborne illness,which has been listed as foodborne pathogen with medium hazard and serious harm by World Health Organization(WHO).Egg,egg products,vegetables,and meat are important vehicles for Salmonella infections.Among over 2 500 Salmonella serotypes,Salmonella ser.Enteritidis 、Salmonella ser.Typhimurium and Salmonella ser.Heidelberg are the top pathogenic serotypes.Therefore,the development of new detection methods for these serotypes will play an important role on the guarantee of food safety,the surveillance of public health,and the prevention and control of poultry disease,as well.In addition,increasing antibiotic resistance in Salmonella is a global concern.Monitoring the evolution of antibiotic resistance of Salmonella will not only provide scientific basis for the prevention and control of Salmonella associated outbreaks,but also create more reference data for the establishment of relevant laws and regulations.Recently,there are breakthroughs in the whole genome sequencing(WGS)fields frequently,especially for the detection and surveillance of foodborne outbreaks.WGS obviously will play a very important role in the near future for food safety.Among a variety of other detection methods for Salmonella,molecular methods like real-time PCR have been widely used.But some of them are time-consuming,complicated to operate,and expensive,there is still room for improvement.Due to its advantages of specificity,sensitivity,speed,accuracy,simplicity and low cost,DNA isothermal amplification technology,especially loop-mediated isothermal amplification(LAMP),has been widely investigated for the detection of microbial pathogens in many fields in recent years.In this project,firstly,we studied the evolutionary diversity of 147 Salmonella isolates from egg,egg products,and chickens by using WGS technology.Three frequently occurring serotypes of Salmonella(Salmonella ser.Enteritidis,Salmonella ser.Typhimurium,and Salmonella ser.Heidelberg)has been analyzed comprehensively and systematically by phylogenetic tree,single nucleotide polymorphism(SNP),and antibiotic resistance genes,etc.The aim was to provide theoretic basis and data support for detection,surveillance and epidemiological investigation for Salmonella.Secondly,we firstly compared and analyzed the DNA sequence and protein structure prediction of prot6 E and sef A genes,which are unique to Salmonella ser.Enteritidis to providemore information for developing Salmonella ser.Enteritidis detection methods.Thirdly,we developed new LAMP assays for the detection of Salmonella ser.Enteritidis and Salmonella ser.Typhimurium using Genie III instruments,providing powerful tools for detecting and monitoring Salmonella in foods and food environments.In addition,as compared to the Food and Drug Administration’s Bacteriological Analytical Manual(BAM),3M Molecular Detection System(MDS),ANSR Pathogen Detection System(PDS)and Genie III were evaluated for their effectiveness in the detection of Salmonella,and the results also compared with real-time PCR.It would provide highly valuable support for the selection,use and evaluation of these technologies,and design new detection methods,as well.Presently,a variety of broths and media for pre-enrichment,enrichment,plating are used in China and abroad,and the lack of comparison study on the effectiveness of these broths and media causes confusion and problems.We compared the effectiveness of buffered peptone water(BPW)and lactose broth(LB)for pre-enrichment of Salmonella.Here,BPW was the recommend pre-enrichment broth by 3M MDS and ANSR PDS instruments,and LB was the recommend pre-enrichment broth by FDA BAM.And both pre-enrichment and enrichment samples were used in our study and their effect on the efficacy of 3M MDS and ANSR PDS assays were compared with FDA BAM.The objective of this part of our study was to select the better broth.Finally,we explored the influence of storage time and temperature on the survival of Salmonella in the enriched samples(Rappaport-Vassiliadis,RV;Tetrathionate,TT).The aim was to provide theoretical and technical supports for improving Salmonella detection methods,developing new detection kits,and reducing costs for industry,thus improving the abilities of detection and surveillance for foodborne disease outbreaks,in order to ensure the food safety.Mainly results and discoveries are listed below:1.By analyzing of the whole genome sequences of 147 isolates of Salmonella,it was found that an evolution diversity of Salmonella ser.Enteritidis,Salmonella ser.Typhimurium,and Salmonella ser.Heidelberg varied over the years,geographic regions,and origins.And the results also showed the horizontal gene transfer(HGT)among the isolates.2.Results by analysis of the antibiotic resistance genes(ARG)of Salmonella ser.Enteritidis,Salmonella ser.Typhimurium,and Salmonella ser.Heidelberg isolates(total 147 isolates)using WGS technology indicated different ARG in different serotypes and source.The numbers of ARG phenotypes in Salmonella ser.Enteritidis and Salmonella ser.Typhimurium isolates were higher than that in Salmonella ser.Heidelberg isolates,and the numbers of ARG phenotypes in chicken-origin isolates are higher than egg-origin(including egg products)isolates.Salmonella ser.Enteritidis isolates showed a high resistance level to aminoglycosides.And Salmonella ser.Typhimurium isolates were highly resistant to aminoglycosides,tetracycline,and sulfadiazine.Salmonella ser.Heidelberg was highly resistant to aminoglycosides and fosfomycin.Meanwhile,the results also indicated that there were more multi-drug resistant isolates in Salmonella ser.Typhimurium than in Salmonella ser.Enteritidis and Salmonella ser.Heidelberg.In addition,thereis an increasing tendency of antibiotic resistance rate and spectrum of Salmonella in recent years.3.Analyzing the DNA sequence and protein structure of prot6 E and sef A genes among 68 Salmonella ser.Enteritidis isolates,results indicated that these two genes had high conservatism during evolution.There were two variations in prot6 E gene,and both variations were nonsynonymous mutations.However,no visible protein structure changes were predicted by the model used in this study.For sef A gene,only one variation were found among the Salmonella ser.Enteritidis isolates used in this study,the mutation caused an obvious change of the protein structure.4.We successfully developed a novel LAMP method based on prot6 E gene for the detection of Salmonella ser.Enteritidis.The method was evaluated using Genie III system.It was time-saving and easy to handle.It also had high specificity and sensitivity.As for the detection of Salmonella ser.Enteritidis in egg products,the results showed that this LAMP assay could be used as a rapid detection tool for Salmonella ser.Enteritidis in egg products.5.We successfully developed a new LAMP assay to detect Salmonella ser.Typhimurium,using Genie III.Results indicated that this method had high specificity,which can differentiate Salmonella ser.Typhimurium from Salmonella ser.Enteritidis and Salmonella ser.Heidelberg;and the sensitivity of our newly designed LAMP method was 100 times higher than the real-time PCR.As for the detection of Salmonella ser.Typhimurium in egg products,the results indicated that this LAMP assay could be used as a rapid detection tool for Salmonella ser.Typhimurium in egg products.6.By comparing culture method,isothermal amplification detection system(3M MDS,ANSR PDS,and Genie III),real-time PCR for detecting Salmonella in pure cultures and Salmonella in egg,egg products and leafy greens,we concluded that 3M MDS,ANSR PDS,and Genie III methods were all suitable for rapidly detecting Salmonella in food samples.And 3M MDS was more accurate.7.By comparing the effect of BPW and LB pre-enrichment broths on the effectiveness of LAMP assays,using BPW as preenrichment broth,3M MDS and ANSR PDS produced the exact same results as the culture method.With LB as preenrichment broth,both methods have less positive results than culture method,but there is no significant difference statistically between the two molecular methods and culture method.Therefore,BPW was a better preenrichment broth for3 M MDS and ANSR PDS assays.When using RV and TT enriched samples as DNA templates for3 M MDS and ANSR PDS analysis,results showed that RV enriched samples were better than TT enriched samples.8.RV and TT enriched samples(136 egg products)were stored at 4 ℃ for up to 60 days.They were streaked onto XLD,He and BS media at different time intervals.The results showed that there is no change for RV enriched samples;however,12 positive TT enriched samples at time 0changed to negative at day 60.In addition,the results by XLD and HE plates were better than BS plates.