Highly Discriminatory Single-Nucleotide Polymorphism Interrogation Of Source Of Escherichia Coli In Food

Food safety has been already considered as the fourth social problem followingPopulation, Resource and Environment now in our country. It was one of the mainreasons that foods were contaminated by pathogenic bacteria or conditional pathogens.And food poisoning caused by pathogenic E. coli was worth particularly concern.E. coli O157:H7,which has raged across world for30years is the main serotypeof Enterohemorrhagic Escherichia coli, which can cause diseases such as diarrhea,Hemorrhagic enteritis.in2011, there was an outbreak of the E. Coli O104:H4strain inGermany that caused dozens to die, and landed hundreds of people in the hospital.Therefore, pathogenic E. coli is concerned more and more in the world for itsdestructive force to health.Rapid trace to the source of pathogenic E. coli is the key step to control foodborne infections and interrogation of geographical source of E. coli in food. There area lot of bacterial tracing methods mainly used for host-specific research, but have itsbottlenecks. Multilocus sequence typing (MLST) is becoming more widely used as agenotyping and tracing method and has been applied successfully to MST (microbialsource tracking), provides reproducibility, comparability and transferability betweenlaboratories. However, this sequencing procedure remains expensive and timeconsuming for routine food monitoring and may not be optimal for distinguishinghighly closely related strains and not be amenable for screening a large number ofisolates.Single Nucleotide Polymorphism (SNP) based genotyping and tracing methodcould serve as a relatively inexpensive, avoids the sequencing and highly reproduciblescreening test for MST. But few studies have paid any attention to how can trace thegeographical source of E. coli in food by SNP.In this study, SNP technology was first used in geographical research of the E.coli in foods.Studies are summarized by the parts below:1. The choice of the E. coli housekeeping genes. In this study, four kinds ofhousekeeping genes in E. coli were selected, they are clpX(caseinolytic peptidase X), fadD(acyl-CoA synthetase), mdh(malate dehydrogenase), uidA(beta-glucuronidase).2. Screening of the polymorphism sites in housekeeping genes. Highlydiscriminatory polymorphism sites were identified by using software program calledMinimum SNPs. The software processes the allelic sequences and MLST profile datatogether, executing a Simpson's index of diversity (D) function. The score of SNP setswith increasing D values is displayed as the name of the gene locus with the numberof the informative nucleotide at that locus. Four SNPs, with D values of up to0.86,were determined by the program, previous study and sequence alignment for thedifferentiation of E. coli isolates. At last,12SNP loci were confirmed.3. A total of253E. coli isolates were usedin this study.The bacteria were isolatedfrom livestock meat, processed foods and food material from12country or regionsfrom4continents. Europe: Denmark(63isolates), France(17isolates),Ireland(17isolates),Germany (20isolates)Spain(25isolates). North America: USA(24isolates),Canada (20isolates). Oceania:Australia(9isolates), New Zealand(3isolates).Asia: China (6isolates),Taiwan(4isolates) India (45isolates).And all of isolates were identified and purified.4. Sequencing was used to get interrogation of SNP loci of253E. coli isolates.The following steps were taken: designing the sequencing primer for fourhousekeeping gene of E coli, PCR amplification of four housekeeping genesrespectively, sequencing, and bioinformatics analysis of polymorphism sites.According to clustering analysis,253strains of E. coli can be divided into61SNPGroups. Custer analysis results based on SNP profile showed9Groups were dividedby different continents. There were6Groups coming from Europe,1Groups comingfrom North America,1Groups coming from Asia,1Groups coming from Oceania. Inthe groups that contained2or less isolates,35specific geographical SNP profile werefound. In83%countries and regions, specific geographical SNP profile were found.In the rest of groups, there are not specific geographical SNP profile that show geneticdiversity existed.5. The blind test performed on10hidden source E. coli isolates. The accuracy ofsource tracking of E. coli isolates based on continents was up to80%;and theaccuracy of source tracking based on countries was up to60%.6. The results suggested that SNP sets in conservative housekeeping genes of E.coli can identify a specific geographical origin of E. coli. The SNP tracing methodwas rapid and robust. 7. In summary, there was good consistency between the SNP profile found in thisstudy and the eBURST–defined clonal coplexes. eBURST analysis is good at geneticdiversity investigation for E. coli population. And eBURST results demonstrated thereare same origin clonal between E. coli from different geographical sources. The sameclone complex exsited not only in same country, but also in different geographicorigin.The E. coli population is widely distributed and highly diversity, which belongto same origin clonal. It pointed out that food E. coli have genetic diversity whichbelong to same origin.