«(Über die Bedeutung der bakteriellen Genomplastizität für die Adaptation und Evolution asymptomatischer Bakteriurie (ABU) Escherichia coli ...»
Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular cloning. A Laboratory Manual.
Samuelsson, P., Hang, L., Wullt, B., Irjala, H., and Svanborg, C. (2004) Toll-like receptor 4 expression and cytokine responses in the human urinary tract mucosa. Infect Immun 72: 3179-3186.
Sansom, M.S. (1999) Membrane proteins: A tale of barrels and corks. Curr Biol 9: R254-257.
Sauer, F.G., Mulvey, M.A., Schilling, J.D., Martinez, J.J., and Hultgren, S.J. (2000) Bacterial pili: molecular mechanisms of pathogenesis. Curr Opin Microbiol 3: 65-72.
Savage, D.C., Howie, G., Adler, K., and Wilson, M.I. (1975) Controlled trial of therapy in covert bacteriuria of childhood. Lancet 1: 358-361.
Schilling, J.D., Mulvey, M.A., and Hultgren, S.J. (2001) Structure and function of Escherichia coli type 1 pili: new insight into the pathogenesis of urinary tract infections. J Infect Dis 183 Suppl 1: S36-40.
Schlag, S., Nerz, C., Birkenstock, T.A., Altenberend, F., and Gotz, F. (2007) Inhibition of staphylococcal biofilm formation by nitrite. J Bacteriol 189: 7911-7919.
Shaikh, N., Morone, N.E., Lopez, J., Chianese, J., Sangvai, S., D'Amico, F., Hoberman, A., and Wald, E.R. (2007) Does this child have a urinary tract infection? Jama 298: 2895Shapiro, R., and Pohl, S.H. (1968) The reaction of ribonucleosides with nitrous acid. Side products and kinetics. Biochemistry 7: 448-455.
Smyth, C.J., Marron, M.B., Twohig, J.M., and Smith, S.G. (1996) Fimbrial adhesins:
similarities and variations in structure and biogenesis. FEMS Immunol Med Microbiol 16: 127-139.
Snyder, J.A., Haugen, B.J., Buckles, E.L., Lockatell, C.V., Johnson, D.E., Donnenberg, M.S., Welch, R.A., and Mobley, H.L. (2004) Transcriptome of uropathogenic Escherichia coli during urinary tract infection. Infect Immun 72: 6373-6381.
Sokurenko, E.V., Hasty, D.L., and Dykhuizen, D.E. (1999) Pathoadaptive mutations: gene loss and variation in bacterial pathogens. Trends Microbiol 7: 191-195.
Stamm, W.E., and Hooton, T.M. (1993) Management of urinary tract infections in adults. N Engl J Med 329: 1328-1334.
Stella, C., Beckwith-Hall, B., Cloarec, O., Holmes, E., Lindon, J.C., Powell, J., van der Ouderaa, F., Bingham, S., Cross, A.J., and Nicholson, J.K. (2006) Susceptibility of human metabolic phenotypes to dietary modulation. J Proteome Res 5: 2780-2788.
Struelens, M.J., Denis, O., and Rodriguez-Villalobos, H. (2004) Microbiology of nosocomial infections: progress and challenges. Microbes Infect 6: 1043-1048.
Sunden, F., Hakansson, L., Ljunggren, E., and Wullt, B. (2006) Bacterial interference--is deliberate colonization with Escherichia coli 83972 an alternative treatment for
patients with recurrent urinary tract infection? Int J Antimicrob Agents 28 Suppl 1:
Surette, M.G., Miller, M.B., and Bassler, B.L. (1999) Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production. Proc Natl Acad Sci U S A 96: 1639-1644.
Suzuki, T., Ide, H., Yamada, M., Endo, N., Kanaori, K., Tajima, K., Morii, T., and Makino,
K. (2000) Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid:
mechanism and intermediates. Nucleic Acids Res 28: 544-551.
Svanborg-Eden, C., Hagberg, L., Hull, R., Hull, S., Magnusson, K.E., and Ohman, L. (1987) Bacterial virulence versus host resistance in the urinary tracts of mice. Infect Immun 55: 1224-1232.
Svanborg, C., Frendeus, B., Godaly, G., Hang, L., Hedlund, M., and Wachtler, C. (2001) Toll-like receptor signaling and chemokine receptor expression influence the severity of urinary tract infection. J Infect Dis 183 Suppl 1: S61-65.
Svanborg, C., Bergsten, G., Fischer, H., Godaly, G., Gustafsson, M., Karpman, D., Lundstedt, A.C., Ragnarsdottir, B., Svensson, M., and Wullt, B. (2006) Uropathogenic Escherichia coli as a model of host-parasite interaction. Curr Opin Microbiol 9: 33-39.
Svensson, M., Lindstedt, R., Radin, N.S., and Svanborg, C. (1994) Epithelial glucosphingolipid expression as a determinant of bacterial adherence and cytokine production. Infect Immun 62: 4404-4410.
Torres, A.G., and Payne, S.M. (1997) Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol 23: 825-833.
Trautner, B., Hull, R., and Darouiche, R. (2003) Escherichia coli 83972 inhibits catheter adherence by a broad spectrum of uropathogens. Urology 61: 1059-1062.
Tsai, C.M., and Frasch, C.E. (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119: 115-119.
Tsikas, D., Boger, R.H., Bode-Boger, S.M., Gutzki, F.M., and Frolich, J.C. (1994) Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography-mass spectrometry using their 15N-labelled analogs. J Chromatogr B Biomed Appl 661: 185-191.
Vaisanen, V., Elo, J., Tallgren, L.G., Siitonen, A., Makela, P.H., Svanborg-Eden, C., Kallenius, G., Svenson, S.B., Hultberg, H., and Korhonen, T. (1981) Mannoseresistant haemagglutination and P antigen recognition are characteristic of Escherichia coli causing primary pyelonephritis. Lancet 2: 1366-1369.
Victorin, K. (1994) Review of the genotoxicity of nitrogen oxides. Mutat Res 317: 43-55.
Warren, J.W., Tenney, J.H., Hoopes, J.M., Muncie, H.L., and Anthony, W.C. (1982) A prospective microbiologic study of bacteriuria in patients with chronic indwelling urethral catheters. J Infect Dis 146: 719-723.
Weiss, B. (2006) Evidence for mutagenesis by nitric oxide during nitrate metabolism in Escherichia coli. J Bacteriol 188: 829-833.
Welch, R.A., Burland, V., Plunkett, G., 3rd, Redford, P., Roesch, P., Rasko, D., Buckles, E.L., Liou, S.R., Boutin, A., Hackett, J., Stroud, D., Mayhew, G.F., Rose, D.J., Zhou, S., Schwartz, D.C., Perna, N.T., Mobley, H.L., Donnenberg, M.S., and Blattner, F.R.
(2002) Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc Natl Acad Sci U S A 99: 17020-17024.
Wheeler, M.A., Smith, S.D., Garcia-Cardena, G., Nathan, C.F., Weiss, R.M., and Sessa, W.C.
(1997) Bacterial infection induces nitric oxide synthase in human neutrophils. J Clin Invest 99: 110-116.
Wildsmith, S.E., and Elcock, F.J. (2001) Microarrays under the microscope. Mol Pathol 54:
Wink, D.A., Kasprzak, K.S., Maragos, C.M., Elespuru, R.K., Misra, M., Dunams, T.M., Cebula, T.A., Koch, W.H., Andrews, A.W., Allen, J.S., and et al. (1991) DNA
deaminating ability and genotoxicity of nitric oxide and its progenitors. Science 254:
Wink, D.A., and Mitchell, J.B. (1998) Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 25: 434-456.
Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L.H., Karch, H., Reeves, P.R., Maiden, M.C., Ochman, H., and Achtman, M. (2006) Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol 60: 1136-1151.
Wullt, B., Connell, H., Rollano, P., Mansson, W., Colleen, S., and Svanborg, C. (1998) Urodynamic factors influence the duration of Escherichia coli bacteriuria in deliberately colonized cases. J Urol 159: 2057-2062.
Wullt, B., Bergsten, G., Samuelsson, M., and Svanborg, C. (2002) The role of P fimbriae for Escherichia coli establishment and mucosal inflammation in the human urinary tract.
Int J Antimicrob Agents 19: 522-538.
Wullt, B., Bergsten, G., Fischer, H., Godaly, G., Karpman, D., Leijonhufvud, I., Lundstedt, A.C., Samuelsson, P., Samuelsson, M., Svensson, M.L., and Svanborg, C. (2003) The host response to urinary tract infection. Infect Dis Clin North Am 17: 279-301.
Wyckoff, E.E., Duncan, D., Torres, A.G., Mills, M., Maase, K., and Payne, S.M. (1998) Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria. Mol Microbiol 28: 1139-1152.
Yang, R.B., Mark, M.R., Gurney, A.L., and Godowski, P.J. (1999) Signaling events induced by lipopolysaccharide-activated toll-like receptor 2. J Immunol 163: 639-643.
Yoshimoto, T., Higashi, H., Kanatani, A., Lin, X.S., Nagai, H., Oyama, H., Kurazono, K., and Tsuru, D. (1991) Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme. J Bacteriol 173: 2173-2179.
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.
Zhou, X., Giron, J.A., Torres, A.G., Crawford, J.A., Negrete, E., Vogel, S.N., and Kaper, J.B.
(2003) Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells. Infect Immun 71: 2120-2129.
Ziebuhr, W., Ohlsen, K., Karch, H., Korhonen, T., and Hacker, J. (1999) Evolution of bacterial pathogenesis. Cell Mol Life Sci 56: 719-728.
Zogaj, X., Nimtz, M., Rohde, M., Bokranz, W., and Romling, U. (2001) The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix. Mol Microbiol 39: 1452-1463.
Zorc, J.J., Kiddoo, D.A., and Shaw, K.N. (2005) Diagnosis and management of pediatric urinary tract infections. Clin Microbiol Rev 18: 417-422.
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
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.