Molecular Typing And Virulence Related Islands Analysis Of Citrobacter Freundii

In this study, we explored the potential of Citrobacter freundii as a cause of infection diarrhea. Using the sequence information of CF72 complete genome sequence and following the principles on designing of the MLST, we developed a MLST scheme for Citrobacter freundii by choosing 7 house keeping genes. A total of 12 different STs were assigned in the 36 isolates. Phylogenetic tree based on the concatenated nucleotide sequences of the seven genes showed that the 36 isolates could be divided into 2 Clusters, namely CluterA and ClusterB. The ClusterA contained ST9, only one isolated from human in this Cluster. Cluster B contained the rest of the 11 STs, which could be subdivided into B1 and B2. B1 contained two STs , 2 isolates in B1, namely ST8 and ST12. B2 contained 9 STs of 33 isolates. ST1, ST3 and ST4 as well as ST2 and ST5 were closely related. The 4 STs contained 26 isolates and belonged to the common STs. From the original source of the strains, seven STs only contained the isolates from humans, one ST only contained the isolate from animals, four STs (ST1, ST2, ST3, ST5) contained the isolates from both humans and animals.Based on the complete genome sequence, we found 49 genomic islands(GI). It could be divided into 10 categories: 17 GIs related to metabolism, 7 GIs encoding transport proteins, 7 GIs related to phage related genes, 5 GIs related to DNA/RNA processing gene, 3 GIs encoding cell structure proteins, 1 GI encoding DNA recombination, 1 GI related to function unknown, 1 GI encoding antibiotic resistance, 6 GIs ecoding virulence related proteins . Screening the 49 GIs in the 36 isolates, only 6 ST2 isolates contained all the GIs. Phages took important function in the evolution of the bacteria. The distribution of the phage related GIs in ST1, ST2, ST3, ST4 isolates were distinctly different from other STs. Phage related GI22 encoded the protein which was related to the biofilm. The STs that having GI22 (19 isolates) included ST1(7/13), ST2(7/7), ST3(3/4), ST4(l/3), ST8(1/1). Only 6 ST2 isolates had the phage related GIB, GI29, GI30. Phage related GI36 could be detected in 7 isolates, in which six belonged to ST2 and one belonged to ST1. The observation about distribution of phage related GIs suggested that the isolates of ST1, ST2, ST3, ST4 were relatively more active in horizontal gene transfer by phage. ST1, ST3, ST4 were more related to G122, while ST2 more related to others GIs. The distribution of phage related GIs was closely related to STs, in which existed in the possible virulence related islands. Three virulence related GIs could be detected in all isolates, and they were GI5 encoding OmpX-SfaABC ABC-transporter, GI7 encoding Tol-PaL inner member protein, GI9 encoding Curli pilus. One virulence related GI encoding invasion related Fim proteins could be detected in isolates except strain CF5(ST9). One virulence related GI encoding O-antigen could be detected in 29 isolates. The isolates belonged to the common STs. One virulence related GI encoding the tellurite resistance could be detected in 8 strains, in which six existing in ST2, one in ST6 and one in ST7.The observation about the adhesive properties of Citrobacter freundii showed that 34 isolates could adhere to Hela cell. Adhesion index indicated that 3 isolates had the strong adhesive ability, which belonged to ST1, ST3, ST12. Eighteen isolates had middle adhesive ability, in which 16 isolates belonged to ST1, ST3, ST2, ST5. The representative ST9 from ClusterA was relatively less pathogenic strain for the reason that the Virulence related GIs (GI6,GI24,GI44) were not detected in ST9 and no adhesive ability was observed. The results of the cytotoxicy of Hela cell showed that the CF74 (ST12) induced significantly different level after 10 hours incubation than others, and it also had strong adhesive ability to Hela cell.In summery, we developed a MLST method for Citrobacter freundii. 12 different STs were assigned to 36 isolates. 5 STs (ST1, ST2, ST3, ST4, ST5) were the common STs which were distinctly different from others on the aspects of distribution of GIs especially virulence related islands, phage related islands.Other project during PHD:We report that EPEC O125:H6, which represent a large category of strains, lack the ability to utilize either Nck or TccP and hence trigger actin polymerization in vitro only inefficiently. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck and TccP independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence. Typical enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli colonize the gut epithelium via formation of a distinct histopathological lesion name attaching and effacing (A/E).A/E lesions are characterized by loss of microvilli, an intimate adherence of bacteria adjacent to the host cell membrance and the generation of organized cytoskeletal structures containing filamentous actin beneath sites of bacterial attachment,termed actin polymerization.The gene necessary for A/E lesion formation in vitro are carried on the locus of enterocyte effacement, which encodes a type III secretion systerm. Although EPEC and EHEC prodece highly similar lesions by LEE, the mechanisms are different. The actin polymerization induced by EPEC is in a Nck-dependent pathway while EHEC in a TccP-dependent pathway to activate N-WASP and to trigger actin polymerization in cultured cells. This phenotype is used as a marker for the pathogenic potential of EPEC and EHEC strains.In this paper we report that EPEC O125:H6, which represent a large category of strains, lack the ability to utilize either Nck or TccP and hence trigger actin polymerization in vitro only inefficiently. However, we show that infection of human intestinal biopsies with EPEC O125:H6 results in formation of typical attaching and effacing lesions. Expressing TccP in EPEC O125:H6, which harbor an EHEC O157-like Tir, resulted in efficient actin polymerization in vitro and enhanced colonization of human intestinal in vitro organ cultures with detectable N-WASP and electron dense material at the site of bacterial adhesion. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck and TccP independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence.