The Study Of Examination, Typing Methods And Analysis Of 16S RDNA Gene Sequence Of Enterobacter Sakazakii

It has attracted great attention to E.sakazakii infection due to consumption ofcontaminated powdered formula for infant and young children in recent years.However,the methods of examination and typing of this pathogen were quiteinsufficiency and incompleted because of the little research in this field.The systemicstudy of E.sakazakii,including the establishment of examination,investigation ofcontamination,typing methods and tracing technologies,was started in 2004 in ourlaboratory.All the research included following four parts.PartⅠStudy on the examination and investigation of contamination orE.sakazakiiTwo detection methods were established according to the US FDA method andISO method of examination for E.sakazakii respectively.To compare and analysetwo different methods of detection for E.sakazakii in infant formula,the pathogenfrom 194 powdered formula sampled from the market was isolated and identifiedusing the both method.After selective enrichment and isolation,there are only eightpresumptive isolates of E.sakazakii in the second method,which is significantlylower than the first method,and the characteristic colony on the DFI agar is mucheasier to identify.Four and five positive samples were detected by the first methodand the second method respectively.One sample was identified as positive by thesecond method but negative by the first method.The selectivity of the mlST-Vm andE.sakazakii chromogenic media used in the second method is much better than thosein the first method,and the second method is more simple and less laborious andtime-consuming.Based on much comparison and research,the qualitative andquantitative examination methods of E.sakazakii were established according tomethods of ISO and US FDA,and the national standard Microbiological examinationof food hygiene-Examination of E.sakazakii is under the approval of Standardization Administration of the People’s Republic of China after beening approved by relativeexperts.Two national trainings on examination of relative foodborne pathogens were heldin 2006 and 2007 to improve the implementation of Microbiological examination offood hygiene that will be issuing.To study the presence of E.sakazakii in powderedformula in retail markets and find out the ability of examination of E.sakazakii,20provinces or municipalities were selected to investigate the contamination of E.sakazakii in infant powdered formula by the method established in this research.Sixhundred and thirty samples of infant food were sampled,and ten positive sampleswere detected from 610 infant formula and one from rice flour(1.64%).All thispositive samples were sampled from Fujian,Gansu,Hunan,Jilin,Neimenggu,Ningxia and Shanxi provinces.Through there have been good management and greatimprovement in the market of powdered formula for the infant and young children inthe past few years,the contamination ofE.sakazakii is still existing.It is necessary toevery manufacturers to improve the product hygiene by strengthening inspection andcontrol ofE.sakazakii during the production and finished product.To study the contamination of E.sakazakii and other Enterobacteriaceae inpowdered formula in retail markets of China.E.sakazakii and otherEnterobacteriaceae from 212 powdered formulas were isolated and identified usingthe conventional method.The procedure included incubation in sterile water,selectiveincubation in EE broth,growth on Violet Red Bile Glucose Agar,Isolation onTrypticase Soy Agar,then chemical identification.E.sakazakii was isolated from 11powdered infant formulas.Members of the family Enterobacteriaceae were culturedfrom 48.58% of 212 powdered formulas.The species which were isolated mostfrequently were Pantoea spp,Klebsiella pneumoniae,Enterobacter cloacae,Acn.Calo-baumannii complex,E.sakazakii,Leckercia adecarboxylata,Escherichiavulneris,and Escherichia coli 1.The formula with members of theEnterobacteriaceae might result in serious infection of infant and young children, especially infant.The survey in this study is useful to the hygienic precaution andcontrol of the formula.PartⅡStudy on the typing ofE.sakazakiiCommercial identification systems were used to determine differences in biotypesaccording to the manufacturer’s instructions.Two type strains and twenty-nineisolates of E.sakazakii could be divided into five and fifteen biotypes by API 20E andVITEK GNI+respectively.Compared with API 20E,the discriminatory power ofVITEK GNI+was better because of including 30 different biochemical tests.ThoughE.sakazakii could be divided into different biotypes by more biochemical test,biotype couldn’t be an accurate typing method because expressed characteristics ofbacteria are easy to change because of outside influence.Two type strains and twenty-nine isolates of E.sakazakii were evaluated forantibiotic susceptibility with 16 kinds of antibiotics by broth microdilutionrecommended by the Clinical and Laboratory Standards Institute(CLSI).There wasno resistant to any antibiotics and only four isolates(ie.ES 002,ES 010,ES 011 andES 026)showed to be intermediate susceptible to streptomycin.It was proved thatantibiotic susceptibility test couldn’t be an effective typing method because of thehigh antibiotic susceptibility of isolates ofE.sakazakii from food.To analyze the fingerprint ofE.sakazakii,two type strains and twenty-nineisolates were analyzed with the pulsed-field gel electrophoreses(PFGE).All of strainswere digested with restriction endonucleases XbaⅠand SpeⅠrespectively,and thePFGE patterns were analyzed with BioNumerics software.PFGE with restrictionendonucleases XbaⅠand SpeⅠgave 30 kinds of PFGE patterns respectively.Twenty-nine strains ofE.sakazakii gave different patterns except ES 004 and ES 005.ES 004 and ES 005,which were isolated from the infant formula with the same brandand same lot but different package,showed the same PFGE pattern withendonucleases XbaⅠand SpeⅠ.According to the analysis of BioNumerics software, there is no significant correlation between PFGE patterns and source information ofcorresponding samples except ES 004 and ES 005.Though the average number ofPFGE bands with XbaⅠis less than that with SpeⅠ,it proved that PFGE with XbaⅠstill has a high discriminatory power.The above two PFGE methods both can beused to the molecular typing and tracing ofE.sakazakii.To analyze the fingerprint ofE.sakazakii,two type strains and twenty-nineisolates were analyzed with the DuPont RiboprinterTM microbial characterizationsystem.Ribotyping pattems were analyzed with BioNumerics software and comparedwith the result of PFGE.This system divided 31 strains into 27 ribotypes and itsdiscriminatory power was poorer than that of PFGE.According to the sampleinformation and analysis of BioNumerics software,there is no significant correlationbetween ribotyping pattems and source information of corresponding samples exceptES 004 and ES 005 from the same brand and same lot but different package.In conclusion,PFGE were found to be the most discriminatory typing method ofE.sakazakii,followed by ribotyping,biotyping and antibiograms.PartⅢAnalysis of 16S rRNA gene sequence of E.sakazakiiIn this study 16S rRNA gene of twenty-nine E.sakazakii strains from food aswell as the type strain ATCC 51329 and ATCC 29544 were sequenced.By Mutipulecluster analysis and phylogenetic analysis of the 16S rRNA gene sequence dataobtained we could find all the strains but ATCC 51329 and ES 014 cluster togetherwith the E.sakazakii type strain ATCC 29544,whereas the E.sakazakii strain ATCC51329 and ES 014 form a different branch respectively.From the similarity maxtirxcalculated by BioNumerics software,the most similar sequences with ATCC 29544are ES 001 and ES 016,and the similarity arrives to 99.97%.The similarity is 100%between ES 004,ES 005 and ES 019,so as ES 021 and ES 029.However,there is nosignificant correlation between 16S rRNA gene sequence and source information ofcorresponding samples except ES 004 and ES 005.From the analysis of 16S rRNA gene sequence,all these isolates but ES 014 can be identified accurately to be E.sakazakii by gene analysis.PartⅣEstablition of tracing database ofE.sakazakiiBy the BioNumerics database software,the database of E.sakazakii(includingphenotying,molecular typing and 16S rRNA gene sequence)was established.Themethods and increasing data in this database will provide effective technical warrantyfor the tracing of outbreaks,sporadic cases and routine surveillance E.sakazakii.According to the database,all these 2 type strains and 29 isolates were analysed,itwas proved that there was no significant relationship except ES 004 and ES 005without other epidemiological information such as source of raw material,PFGE is apractical and exact method to the typing of E.sakazakii.It is necessary to use otherepidemiological information and not less than two typing methods during the tracingof E.sakazakii.