| Escherichia coli is a diverse and remarkable bacterium.This commonly recognized harmless intestinal organism can become a highly adapted pathogen that may cause a series of diseases,ranging from intestinal diseases to extraintestinal infections of the bloodstream,brain and urinary tract.The global burden of these diseases is huge,with hundreds of millions of people affected annually.In particular,diarrheal illnesses caused by diarrheagenic E.coli is a severe public health threat and a major cause of morbidity and mortality in infants and children,especially in developing countries.Moreover,foodborne disease outbreaks caused by diarrheagenic E.coli are common all around the world,and they sometimes have fatal consequences.Rapid identification of the bacterium can aid in outbreak investigation and disease management.Therefore,this thesis focuses on rapid identification of pathotype and serotype of diarrheagenic E.coli.In chapter one,we first summarized the definition and epidemiology of diarrheagenic E.coli,the classification of pathotype and serotype,and current identification methods of E.coli.Then,we described the MeltArray approach and based on which we developed a novel molecular serotyping method for diarrheagenic E.coli.Finally,we introduced the purpose,content and significance of the proposed thesis.In chapter two:To overcome the technical barriers of current serotyping methods for E.coli,we establised a comprehensive molecular serotyping platform termed MeltArray ET that can identify 5 major diarrheagenic pathotypes,182 O groups and 53 H types of E.coli.Notably,in this assay,as many as 62 targets were detected for the first time.A series of analytical evaluations were carried out and the results showed that,all the Tm values had marginal fluctuations with maximum coefficient of variation lower than 0.42%.The assay was free from interference of other pathogens except Shigella,which cannot be distinguished from Enteroinvasive E.coli(EIEC).The limit of detection was 500 copies/reaction.When testing 176 reference strains,we obtained a 96%concordance with the traditional antibody-based serotyping and a 100%concordance with whole genome sequencing.The entire turnaourd time of MeltArray ET was less than 3.0 h.We thus concluded that MeltArray ET provides a robust,accurate and rapid approach for serotyping of diarrheagenic E.coli.In chapter three:It was a common practice to isolate bacteria through culture for identification and serotyping of an outbreak suspect strain.However,culture-based methods are cumbersome amd time-consuming,and are therefore not suitable for emergent outbreak survailence.Based on the MeltArray ET system,we propose a culture-independent method for the identification of outbreak strains,which is called the "3D Label" strategy.We first demonstrated the theoretical feasibility of this approach through simulation experiments,followed by a retrospective study to demonstrate its feasibility in real cases,and finally conducted a prospective study and reported for the first time an outbreak caused by a new serotype O120:H15 of E.coli.The identification of foodborne outbreaks can be completed within 4 hours,which can provide important guidance for patients’ diagnosis and treatment as well as timely epidemic prevention and control.Chapter 4:There exists a long lasting question regarding whether there is a correspondening relation between serotypes and pathotypes of diarrheagenic E.coli.To adress this question,we conducted a multi-center investigation covering five provinces of China,including Guangdong,Henan,Jiangsu,Sichuan and Zhejiang.A total of 637 clinical isolates were collected,and both of the pathotypes and serotypes were identified via MeltArray ET system,and the composition of pathotypes and serotypes of diarrheagenic E.coli in China was obtained.Based on this,the relationship between serotypes and pathotypes was confirmed.In chapter 2,we noticed that EIEC and Shigella were difficult to distinguish due to their identical pathogenic mechanism.We collected 18 strains of EIEC and 257 strains of Shigella and analyzied their serotypes via MeltArray ET.Meanwhile,the whole genome sequences of 360 strains of EIEC and 775 strains of Shigella were downloaded from online database for serotype determination.By comparing their serotypes distribution,we found that E.coli and Shigella can be distinguished based on their corresponding serotypes.Finally,MeltArray ET system was applied to the study of bloodstream infection of E.coli.From the serotype distribution of 106 strains of bloodstream infection of E.coli from Xiamen,we found that the two serotypes O25:H4 and O6:H1 with the highest isolation rate were also common serotypes of bloodstream infection of E.coli around the world.This indicates that MeltArray ET system is also suitable for the study of pathogenic E.coli outside the intestinal tract.In order to further study the association of the serotype with the severity of bloodstream infections,we performed a correlation analysis of data from a global multicenter study.We found that the mortality of neonatal bloodstream infection caused by different serotypes of E.coli was significantly different,suggesting a specific association between serotypes of E.coli and the severity of the disease.In chapter five,we summarized and highlighted the main points in this paper while potential research directions and clinical applications were prospected. |
