| Urinary tract infection (UTI) is a common and frequently occurring disease. Escherichia coli is its pathogen. Most E.coli isolated from urine of patients with upper urinary tract infections posses P pili, and are called Uropathogenic Escherichia coli (UPEC). P pili formation requires transcription of the pap cluster consisting of 11 genes (papA-K). P pili are composed of many different polypeptides, of which the PapA form the bulk of the pilus structure and of which the tip protein PapG mediate specific binding. Polymorphisms of PapA are of considerable epidemiological, phylogenetic and immunotherapeutic importance.In the present study, we developed and validated a novel PCR-based assay for papC and used the assay to identify UPEC strains from 147 E.coli strains isolated from urine of patients with UTIs. The detection rates of papC PCR positive strains in the tested isolates were 31.98%. As compared with hemagglutination test, there was no statistical significance between the results of the two assays (P>0.05). The papC PCR assay was specific and sensitive and may have a good application prospect in the identification ofUPEC strains.Secondly, we used multiplex PCR-based assays to detect the variant alleles of papA and papG, papA and papG genotypes. Among the 45 strains, all the strains carried class II genotypes of papG, 44 (97.8%) were found by the multiplex papA PCR assay (F-PCR) to contain one or more of 11 known papA alleles (F types). Only one strain (UPEC4030) was negative according F-PCR assay and by PCR contained papC and papG, hence, it was suspected of harboring novel variant of papA. Of the F-PCR positive strains, 24 (54.55%) had a single F type, 13 (29.55%) had two types, 3 (6.82%) had three F types and 3 (6.82%) had four types. F7-2 (25.00%), Fll (6.82%) and F16 (9.09%) tended to occur alone. Among the 11 known F types, nine F types occurred at least once in the population, in decreasing order of prevalence, as follows: F10 and F13 27.27% (12/44), F7-2 and F11 25.00% (11/44), F14 22.27% (10/44), F16 20.45% (9/44), F8, F9 and F14 6.82% (3/44). The strains containing four F types (F8, F9, F13, and F14) were firstly found.Lastly, the papA and papG genes of UPEC 4030 strain were amplified and then were cloned into plasmid pUC18 and pMD-18T vectors. The recombinant plasmids pCA31 and pMG20 were identified and subjected to DNA sequencing. The sequence analysis of papA revealed a 720bp gene encoding 112 amino acid polypeptide. The overall homology between UPEC 4030 and the standard strains of ten F types were 36.11-77.95% at nucleotide level and 21.11-78.34% at deduced amino acid. The homology between the sequence of the reverse primers and the corresponding pap sequence of UPEC4030 was 10-66.67%. The result confirmed that UPEC4030 strain contained a novel papA variant. These findings indicated that F PCR assay were extremely sensitive and specific, and a new molecular tool available to epidemiological and phylogenetic investigations that should make rapid, specific detection of papA alleles. The sequence analysis of papGrevealed a 1100bp gene encoding 337 amino acid polypeptide. The homology of papG between UPEC 4030 and UPEC 132 was 98.89% at nucleotide level and 99.01% at deduced amino acid. Both strains carried class II genotypes of papG.Above all, the papG sequences of UPEC strains in China were highly conserved and the papA sequences possessed polymorphisms. Furthermore, in this report, we found a novel papA variants by revealing the absence of any of the 11 known papA alleles in UPEC 4030 strain. These findings provide novel insight into the papA alleles of UPEC for epidemiological phylogenic and immunotheraputic investigations.
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