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Timo Barl(1), Xiaolei Yu(1), Ulrich Dobrindt(2), Jörg Hacker(2), Rolf D. Schmid(1),
Till T. Bachmann(1)
(1) Universität Stuttgart
Institut für Techn. Biochemie
Allmandring 31
D - 70569 Stuttgart
Fon: +49 / 711 / 685 - 64567
Fax: +49 / 711 / 685 – 63196
timo.barl@itb.uni-stuttgart.de
(2) University of Würzburg, Institute for Molecular Infection Biology, Germany
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Adhesive structures play an important role in extraintestinal infections caused by E. coli. One of the most frequently occurring adhesins expressed by extraintestinal pathogenic E. coli (ExPEC) are Type 1 fimbriae which mediate the adhesion to mannose-containing glycoprotein receptor structures of a variety of host cells. Investigations on natural variants as well as engineered random mutants of the binding-subunit FimH have shown that single mutations can result in phenotypic alterations of the binding behaviour, namely a change of the binding preference from trimannose- to monomannose-containing receptors. This interaction is essential to ExPEC for colonization, biofilm formation and invading bladder surface cells which is the first critical step of lower urinary tract infection.
In this study, we present the results of investigating fimH variants from different E. coli strains with a DNA microarray(2). We designed capture probes for SNP positions based on investigation of more than 120 fimH sequences from public databases, literature and our own isolates and spotted them on epoxy-coated glass slides. To evaluate the performance of the capture probes, genomic DNA of ten reference strains was applied as a model system and the system was optimized accordingly. The total assay can be performed in less than four hours. Finally, the DNA chip was used to genotype 50 clinical isolates. The isolates were related to a variety of severe symptoms caused by ExPEC such as chronic urinary tract infections, sepsis, meningitis and asymptomatic bacteriuria. All results of the hybridization experiments could be verified by sequencing of the fimH amplicon. In addition, we integrated the previously developed GyrA-Array(1) for the detection of mutations in Gyrase which are the primary cause of quinolone resistance.
References
(1)Yu, X. et al., Development and validation of a diagnostic DNA microarray to detect quinolone-resistant Escherichia coli among clinical isolates, JCM 42 (2004)
(2)Barl, T. et al., Genotyping pathoadaptive mutations in the type 1 fimbriae binding-subunit of extraintestinal pathogenic E. coli with a DNA microarray, in preparation
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