| E. coli is a clonal species and sometimes a clone is persistent within a host offering the opportunity to study change over a defined period. Here we sequenced genomes of 14 isolates of a uropathogenic E. coli clone that persisted in a family including a family dog for 3 years, causing a UTI episode in the dog after 2 years. The mutations observed fit a single tree that allows us to estimate the mutation rate to be about 1.1 per genome per year or 0.17 for sSNPs, with no evidence for adaptive change, including in relation to the UTI episode. However the rate is lower than is often assumed in discussion of clonal adaptation, and it may be that rates are high under some circumstances with changes in the environment. The host data show that there were at least 6 host transfer events over the 3 years. In conclusion we find that the mutation rate in E. coli is sufficient for detection by sampling a persistent clone over a few years. This approach has the advantage that variation within a species will be detected if enough clones are studied. Our study used data collected for a purpose unrlated to a study of mutation rates, and more accurate estimates of population dynamics and rates would be possible in a targeted study with higher frequency of sampling and run over a longer time frame. The study shows for the first time the the patterns of mutation and transmission between hosts of a pathogenic clone E. coli.Urinary tract infections (UTIs) are one of the most common bacterial infections and are predominantly caused by uropathogenic Escherichia coli (UPEC). E. coli strains belonging to 14 serogroups, including 01,02,04,06,07,08,015,016, 018,021,022,025,075 and 083, are the most frequently detected UPEC strains in a diverse range of clinical urine specimens. In the current study, the O-antigen gene clusters of E. coli serogroups 01,02,018 and 075 were characterized. A multiplex PCR method based on O-antigen-specific genes was developed for the simultaneous detection of all 14 E. coli serogroups. The multiplex PCR method was shown to be highly specific and reproducible when tested against 186 E. coli and Shigella O-serogroup reference strains,47 E. coli clinical isolates and 10 strains of other bacterial species. The sensitivity of the multiplex PCR method was analyzed and shown to detect O-antigen-specific genes in samples containing 25 ng of genomic DNA or in mock urine specimens containing 40 colony-forming units (CFUs) per ml. Five urine specimens from hospital were examined using this multiplex PCR method, and the result for one sample was verified by the conventional serotyping methods. The multiplex PCR method developed herein can be used for the detection of relevant E. coli strains from clinical and/or environmental samples, and it is particularly useful for epidemiologic analysis of urine specimens from patients with UTIs.Escherichia coli, which is the most important bacterial model for research, is the best material for the study of microbial pathogens'molecular evolution. O-antigen is part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, and contributes the major antigenic variability to the cell surface. Screening for the Escherichia coli O-serogroup is the conventional method for identifying E. coli clones. In this study, we sequenced 3 E. coli O-antigen gene clusters, and analyze them by combination with their respective O-antigen structures.We investigated the structural characteristics of the E. coli 099 O-antigen and the organization of the genes involved in its synthesis. The 0 unit consists of four D-rhamnose (D-Rha) moieties in the backbone and two D-glucose (D-Glc) moieties in the side chain. The O-antigen gene cluster of E. coli 099, which was located between galF and gnd, was found to contain putative genes for the synthesis of D-Rha, genes encoding sugar transferases, and ATP-binding cassette (ABC) transporter genes (wzm and wzt). Our findings indicate that in E. coli 099, the synthesis and translocation of the O-antigen occurs by an ABC transporter-dependent process.The O-polysaccharide of S. enterica 017 has the same carbohydrate backbone with E. coli 085 and, in addition, contains an O-acetyl group at position 2 of～80% β-Galf residues. The O-antigen gene cluster of E. coli 085 was found to be closely related to that of S. enterica 017, which has been reported earlier. Data presented in this report describe a previously undefined pair of S. enterica and E. coli O-serogroups with closely related O-antigens. Screening type strains of all E. coli and S. enterica O-serogroups enabled the identification of two genes specific to the E. coli 085 O-antigen gene cluster, which can be used for development of PCR-based assays for the identification and detection of E. coli 085 strains. the O-antigen structure of SalmonellaThe O-antigen structure of Salmonella 066 was established, which differs from the known O-antigen structure of Escherichia coli 0166 only in one linkage, which is, most likely, the linkage between the O-units, and 0 acetylation. The O-antigen gene clusters of Salmonella 066 and E. coli 0166 were found to have similar organizations, the only exception being that in Salmonella 066 the wzy gene is replaced by a non-coding region. The function of the wzy gene in E. coli O166 was confirmed by the construction and analysis of deletion and trans-complementation mutants. It is proposed that a functional wzy gene located outside the O-antigen gene cluster is involved in Salmonella 066 O-antigen biosynthesis, as has previously been reported in Salmonella serogroups A, B and D1. The sequence identity for the corresponding genes between the O-antigen gene clusters of Salmonella 066 and E. coli 0166 ranges from 64% to 70%, indicating that they may originate from a common ancestor. It is likely that after the species divergence, Salmonella 066 got its specific O-antigen form by inactivation of the wzy gene located in the O-antigen gene cluster and acquisition of two new genes (a wzy gene and a prophage gene for O-acetyl modification) both residing outside the O-antigen gene cluster.
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