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«(Über die Bedeutung der bakteriellen Genomplastizität für die Adaptation und Evolution asymptomatischer Bakteriurie (ABU) Escherichia coli ...»

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8. Appendix  

8.1. Legends to figures and tables   Table 1: Prevalence of asymptomatic bacteriuria in selected populations 

Table 2: Bacterial strains used in this study 

Table 3: Plasmids used in this study. 

Table 4: Oligonucleotides used in this study. 

Table 5: Antibiotic substances used in this study. 

Table 6: Parameters of the ArrayVision pre-set protocols (Wizard) used for reading intensity values. 60  Table 7: Genotypic characterization of asymptomatic bacteriuria E. coli strains by comparative genomic hybridization 

Table 8: Genome size of ABU E. coli isolates 

Table 9: Geno- and phenotypic characterization of selected virulence traits of asymptomatic bacteriuria E. coli strains 

Table 10: Identification of the Gln472 → Leu substitution critical for FocD function 

Table 11: Geno- and phenotypic characterization of in vivo re-isolates of ABU strain 83972. .......... 106  Table 12: Motility of in vitro 83972 re-isolates on urine soft agar plates. 

Table 13: Total number of de-regulated genes in the in vivo and in vitro re-isolates of ABU strain

83972. 

Table 14: Differently expressed cytoplasmic proteins in the in vivo re-isolate KA25 and the ancestor strain 83972. 

Table 15: Differently expressed cytoplasmic proteins in the in vivo re-isolate SR12 and the ancestor strain 83972. 

Table 16: Differently expressed cytoplasmic proteins in the in vivo re-isolate CK12 and the ancestor strain 83972. 

Table 17: Changes in the outer membrane subproteome of the in vivo re-isolates of strain 83972 following in vitro culture in pooled human urine. 

Table 18: Genes with up-regulated expression in in vivo re-isolate SR12 

Table 19/1: Genes with down-regulated expression in in vivo re-isolate SR12 

Table 20/1: Genes with up-regulated expression in in vivo re-isolate CK12 

Table 21/1: Genes with down-regulated expression in in vivo re-isolate CK12 

Table 22: Genes with up-regulated expression in in vivo re-isolate KA25 

Table 23/1: Genes with down-regulated expression in in vivo re-isolate KA25 

Table 24: Genes with up-regulated expression in in vitro re-isolate 4.9 

Table 25: Genes with down-regulated expression in re-isolate in vitro 4.9 

 

 

Fig. 1: Pathogenesis of urinary tract infection caused by uropathogenic E. coli

Fig. 2: Phenotypic comparison of E. coli strains CFT073, Nissle 1917 and 83972 

Fig. 3.: Bacterial genome plasticity 

Fig. 4: DNA markers used for electrophoresis 

Fig. 5: Triplex PCR profiles specific for the four E. coli phylogenetic groups. 

Fig. 6: Agarose gel showing PCR products from the multiplex virulence factor–PCR assay .............. 48  Fig. 7: Strategy for inactivation of chromosomal genes using PCR products 

Fig. 8: Schematic construction of the single flow culture unit 

Fig. 9: Four-chamber microfermenter setup 

Fig. 10: Analysis of the genome content of ABU E. coli isolates. 

178    Appendix     Fig. 11: Genomic fingerprints of asymptomatic bacteriuria E. coli isolates. 

Fig. 12: Assessment of the genome size of asymptomatic bacteriuria E. coli isolates by PFGE .......... 86  Fig. 13: Genetic structure of the fim determinant and adjacent KpLE2 phage-like chromosomal region in asymptomatic bacteriuria E. coli isolates 

Fig. 14: Allelic variation of the FimH type 1 fimbrial adhesins among asymptomatic bacteriuria E. coli isolates 

Fig. 15 Amino acid alignment of FocD, SfaF and FimD fimbrial ushers 

Fig. 16: Inactivation of the hly determinant in strains 27 and 83972 

Fig. 17: Analysis of the LPS phenotype among asymptomatic bacteriuria E. coli isolates .................. 93  Fig. 18: Analysis of biofilm formation of asymptomatic bacteriuria E. coli isolates in urine .............. 93  Fig. 19: Growth characteristics of E. coli isolates in pooled human urine. 

Fig. 20: Schematic representation of the experimental design of patients colonisation study .............. 96  Fig. 21: Mean of host response parameters in urine samples collected from patients during the time of colonization with strain 83972. 

Fig. 22: Levels of IL 8 and PMNs at each sampling time point. 

Fig. 23: Genetic organization of the fim loci in E. coli K-12 and E. coli 83972 

Fig. 24: Verification of the patient re-isolates of E. coli strain 83972. 

Fig. 25 Genomic fingerprints of in vivo re-isolates of strain 83972 from different human patients ... 100  Fig. 26: Motility of in vivo re-isolates of strain 83972 on urine soft agar plates incubated overnight at 37 °C. 

.

Fig. 27: Growth characteristics of in vivo re-isolates of strain 83972 and their parent strain in pooled human urine. 

Fig. 28: Competitiveness in urine of in vivo re-isolates of E. coli 83972 against their parent strain derivative 83972cat. 

Fig. 29: Analysis of biofilm formation of in vivo re-isolates of ABU strain 83972 at 37 °C .............. 105  Fig. 30: Growth dynamics of E. coli strain 83972 during continous culture experiments. ................. 108  Fig. 31: Biofilm formation of ABU strain 83972 in continous cultures 

.

Fig. 32: Comparison of the genome structure of in vitro re-isolates of E. coli strain 83972  ............. 111  Fig. 33: Growth characteristics of in vitro re-isolates of strain 83972. 

Fig. 34: Analysis of biofilm formation of in vitro re-isolates of ABU strain 83972 

Fig. 35: Functional grouping of the de-regulated genes in in vivo and in vitro re-isolates of ABU strain 83972 upon in vitro growth in pooled human urine. 

Fig. 36: Hierarchical cluster analysis of all de-regulated genes in three ABU 83972 in vivo re-isolates CK12, KA25 and SR12 and one in vitro re-isolate 4.9 relative to their parent strain 83972 .............. 117  Fig. 37: Altered expression of sugar transport and degradation pathways in the in vivo re-isolate SR12 compared to strain 83972. 

Fig. 38: Hierarchical cluster analysis of commonly de-regulated genes in at least two re-isolates of strain 83972 relative to their parent strain. 

Fig. 39: Real Time-PCR-based quantification of transcript levels of selected genes in ABU re-isolates  

Fig. 40: Comparison of 2D cytoplasmic protein profiles from ABU strain 83972 and the in vivo reisolates KA25, CK12 and SR12 upon growth in vitro at 37 °C in pooled human urine. 

Fig. 41: Comparison of the cytoplasmic proteome of ABU strain 83972 and the in vivo re-isolate KA25 

Fig. 42: Comparison of the cytoplasmic proteome of ABU strain 83972 and the in vivo re-isolate SR12  

Fig. 43: Quantification of FrmA and FrmB protein expression in in vivo re-isolates of ABU strain 83972 

Fig. 44: Comparison of the cytoplasmic proteome of ABU strain 83972 and the in vivo re-isolate CK12 

Fig. 45: Adaptation of the ribonucleoside degradation pathway in in vivo re-isolate CK12 of ABU strain 83972 

179    Appendix     Fig. 46: Adaptation of the deoxy-ribonucleoside degradation pathway in in vivo re-isolate CK12 of ABU strain 83972. 

.

Fig. 47: Comparison of the outer membrane proteome of ABU strain 83972 and the in vivo re-isolate KA25 

Fig. 48: Comparison of the outer membrane proteome of ABU strain 83972 and the in vivo re-isolate CK12 

Fig. 49: Comparison of the outer membrane proteome of ABU strain 83972 and the in vivo re-isolate SR12 

Fig. 50: Geno- and phenotypic diversity among closely related members of E. coli clonal group (ST 73)

Fig. 51: Model of D-serine catabolism and nitrogen assimilation in strain SR12 

Fig. 52: Model of nitric oxide detoxification based on hierarchical cluster analysis of genes differently de-regulated in in vivo re-isolates SR12 and KA25 relative to their parent strain 83972 

–  –  –

8.2. Expression profiling data   Data derived from expression profiling of three ABU 83972 in vivo re-isolates CK12, KA25 and SR12 and one in vitro re-isolate 4.9, sorted according to their level of expression.

–  –  –

                       

8.4. Publications Publications Zdziarski, J., Svanborg, C., Wullt, B., Hacker, J., and Dobrindt, U. (2008) Molecular basis of commensalism in the urinary tract: low virulence or virulence attenuation? Infect Immun 76: 695-703.

Bielaszewska, M., Dobrindt, U., Gartner, J., Gallitz, I., Hacker, J., Karch, H., Muller, D., Schubert, S., Alexander Schmidt, M., Sorsa, L.J., and Zdziarski, J. (2007) Aspects of genome plasticity in pathogenic Escherichia coli. Int J Med Microbiol 297: 625-639.

Presentations Zdziarski, J., Dobrindt, D., Svanborg, C., and Hacker, J. (2006) Geno- and phenotypic analysis of asymptomatic bacteriuria Escherichia coli isolates. Jahrestagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie (DGHM), Würzburg. Poster presentation Zdziarski, J., Hacker, J., Svanborg, C., and Dobrindt, U. (2007) Geno- and phenotypic analysis of asymptomatic bacteriuria Escherichia coli isolates. Vereinigung für Allgemeine und Angewandte Mikrobiologie (VAAM), Göttingen. Poster presentation Zdziarski, J., Hacker, J., Svanborg, C., and Dobrindt, U. (2007) Diversity makes a difference – genome plasticity and its consequences for the evolution of asymptomatic bacteriuria strains. Leopoldina, Staffelstein. Poster presentation Zdziarski, J. (2008) Asymptomatic Bacteriuria (ABU) - An Adaptive Challenge for Escherichia Coli within the Urinary Tract. 3rd Student’s Meeting EuroPathoGenomics, Graduate Academy, Innsbruck, Austria. Oral presentation.

–  –  –

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