In Situ Monitoring of Bacterial Presence and Activity

Claus Sternberg; Micheal Givskov; Søren Molin; Leo Eberl; Karen A. Krogfelt

doi:10.1128/9781555817619.ch4

Chapter 4 : In Situ Monitoring of Bacterial Presence and Activity

Authors: Claus Sternberg¹, Micheal Givskov¹, Søren Molin¹, Leo Eberl², Karen A. Krogfelt³

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Affiliations: 1: Centre for Biomedical Microbiology, BioCentrum-DTU, Technical University of Denmark, Building 301, 2800 Lyngby, Denmark; 2: Department of Microbiology, Institute of Plant Biology, University of Zürich, CH-8008 Zürich, Switzerland; 3: Department of Gastrointestinal Infections, Statens Serumsinstitut, 2300 Copenhagen S, Denmark

Content Type: Monograph

Publication Year: 2005

Category: Clinical Microbiology; Bacterial Pathogenesis

Book DOI: 10.1128/9781555817619

Chapter DOI: 10.1128/9781555817619.ch4

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Abstract:

The transfer of molecular biology methods and concepts to environmental microbiology and microbial ecology has provided new insights into microbial complexity and activity in many different types of natural settings, and the rapidly expanding genomic databases have further accelerated the development. Comparative genomics, meta-genomes, techniques for in situ metabolic activity monitoring, and DNA chips for rapid identification of hundreds of species as well as for transcriptomic investigations are tools which have recently been added to those of fluorescent in situ hybridization (FISH) and reporter gene techniques. This chapter reviews the application of some of these in situ methods and tools in the context of mucosal colonization, after which one specific example—monitoring of quorum sensing-based cell-cell communication in colonized lungs of cystic fibrosis (CF) animal models. The first fluorescent protein to be useful as a tool for gene expression was the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The last 10 years of development of new molecular tools for microscopic investigations, along with the rapid development and dispersal of advanced fluorescence microscopy methods, have resulted in greatly improved techniques for studies of microbial performance in very complex settings, including those found in connection with both commensal and pathogenic bacteria in animals and even human patients.

Citation: Sternberg C, Givskov M, Molin S, Eberl L, Krogfelt K. 2005. In Situ Monitoring of Bacterial Presence and Activity, p 49-58. In Nataro J, Cohen P, Mobley H, Weiser J (ed), Colonization of Mucosal Surfaces. ASM Press, Washington, DC. doi: 10.1128/9781555817619.ch4

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References

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1. Albus, A. M.,, E. C. Pesci,, L. J. Runyen-Janecky,, S. E. West,, and B. H. Iglewski. 1997. Vfr controls quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179: 3928– 3935.

2. Amann, R. I.,, L. Krumholz,, and D. A. Stahl. 1990. Fluorescent- oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172: 762– 770.

3. Andersen, J. B.,, C. Sternberg,, L. K. Poulsen,, S. P. Bjørn,, M. Givskov,, and S. Molin. 1998. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64: 2240– 2246.

4. Andersen, J. B.,, A. Heydorn,, M. Hentzer,, L. Eberl,, O. Geisenberger,, B. B. Christensen,, S. Molin,, and M. Givskov. 2001. gfp based N-acyl-homoserine-lactone monitors for detection of bacterial communication Appl. Environ. Microbiol. 67: 575– 585.

5. Ando, R.,, H. Hama,, M. Yamamoto-Hino,, H. Mizuno,, and A. Miyawaki. 2002. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99: 12651– 12656.

6. Apostolou, E.,, L. Pelto,, P. V. Kirjavainen,, E. Isolauri,, S. J. Salminen,, and G. R. Gibson. 2001. Differences in the gut bacterial flora of healthy and milk-hypersensitive adults, as measured by fluorescence in situ hybridization. FEMS Immunol. Med. Microbiol. 30: 217– 221.

7. Bevis, B. J.,, and B. S. Glick. 2002. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20: 1159– 1159.

8. Boye, M.,, T. K. Jensen,, K. Moller,, T. D. Leser,, and S. E. Jorsal. 1998. Specific detection of the genus Serpulina, S. hyodysenteriae and S. pilosicoliin porcine intestines by fluorescent rRNA in situ hybridization. Mol. Cell. Probes 12: 323– 330.

9. Boye, M.,, T. K. Jensen,, P. Ahrens,, T. Hagedorn-Olsen,, and N. F. Friis. 2001. In situ hybridization for identification and differentiation of Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae and Mycoplasma hyorhinis in formalin-fixed porcine tissue sections. APMIS 109: 656– 664.

10. Camara, M.,, P. Williams,, and A. Hardman. 2002. Controlling infection by tuning in and turning down the volume of bacterial small-talk Lancet Infect. Dis. 2: 667– 676.

11. Chalfie, M.,, Y. Tu,, G. Euskirchen,, W. W. Ward,, and D. C. Prasher. 1994. Green fluorescent protein as a marker for gene expression. Science 263: 802– 805.

12. Chudakov, D. M.,, V. V. Belousov,, A. G. Zaraisky,, V. N. Novoselov,, D. B. Staroverov,, D. B. Zorov,, S. Lukyanov,, and K. A. Lukyanov. 2003. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 91: 191– 194.

13. Connell, H.,, L. K. Poulsen,, and P. Klemm. 2000. Expression of type 1 and P fimbriae in situ and localisation of a uropathogenic Escherichia coli strain in the murine bladder and kidney. Int. J. Med. Microbiol. 290: 587– 597.

14. Cormack, B.,, R. H. Valdivia,, and S. Falkow. 1996. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33– 38.

15. Costerton, J. W.,, P. S. Stewart,, and E. P. Greenberg. 1999. Bacterial biofilms: a common cause of persistent infections. Science 284: 318– 1322.

16. Davies, D. G.,, M. R. Parsek,, J. P. Pearson,, B. H. Iglewski,, J. W. Costerton,, and E. P. Greenberg. 1998. The involvement of cell-to- cell signals in the development of a bacterial biofilm. Science 280: 295– 298.

17. Davies, J. C.,, D. M. Geddes,, and E. W. Alton. 1998. Prospects for gene therapy for cystic fibrosis Mol. Med. Today 4: 292– 299.

18. De Kievit, T. R.,, and B. H. Iglewski. 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 68: 4839– 4849.

19. Dennis, P. P.,, and H. Bremer. 1973. Regulation of ribonucleic acid synthesis in Escherichia coli B-r: an analysis of a shift-up. J. Mol. Biol. 75: 145– 159.

20. Drouault, S.,, G. Corthier,, D. Ehrlich,, and P. Renault. 1999. Survival, phyisology, and lysis of Lactococcus lactis in the digestive tract. Appl. Environ. Microbiol. 65: 4881– 4886.

21. Eberl, L.,, S. Molin,, and M. Givskov. 1999. Surface motility of Serratia liquefaciens MG1. J. Bacteriol. 181: 1703– 1712.

22. Favre-Bonté, S.,, T. R. Licht,, C. Forestier,, and K. A. Krogfelt. 1999. Klebsiella pneumoniae capsule expression is necessary for colonization of large intestines of streptomycin-treated mice. Infect. Immun. 67: 6152– 6156.

23. Franks, A. H.,, H. J. Harmsen,, G. C. Raangs,, G. J. Jansen,, F. Schut,, and G. W. Welling. 1998. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 64: 3336– 3345.

24. Gotschlich, A.,, B. Huber,, O. Geisenberger,, A. Togl,, A. Steidle,, K. Riedel,, P. Hill,, B. Tummler,, P. Vandamme,, B. Middleton,, M. Camara,, P. Williams,, A. Hardman,, and L. Eberl. 2001. Synthesis of multiple N-acylhomoserine lactones is wide-spread among the members of the Burkholderia cepacia complex. Syst. Appl. Microbiol. 24: 1– 14.

25. Gurskaya, N. G.,, A. F. Fradkov,, A. Terskikh,, M. V. Matz,, Y. A. Labas,, V. I. Martynov,, Y. G. Yanushevich,, K. A. Lukyanov,, and S. A. Lukyanov. 2001. GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507: 16– 20.

26. Harmsen, H. J.,, A. C. Wildeboer-Veloo,, G. C. Raangs,, A. A. Wagendorp,, N. Klijn,, J. G. Bindels,, and G. W. Welling. 2000. Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J. Pediatr. Gastroenterol. Nutr. 30: 61– 67.

27. Harmsen, H. J.,, G. R. Gibson,, P. Elfferich,, G. C. Raangs,, A. C. Wildeboer-Veloo,, A. Argaiz,, M. B. Roberfroid,, and G. W. Welling. 2000. Comparison of viable cell counts and fluorescence in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria. FEMS Microbiol. Lett. 183: 125– 129.

28. Heim, R.,, A. B. Cubitt,, and R. Y. Tsien. 1995. Improved green fluorescence. Nature 373: 663– 664.

29. Heim, R.,, and R. Y. Tsien. 1996. Engeneering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6: 178– 182.

30. Heim, R.,, D. C. Prasher,, and R. Y. Tsien. 1994. Wavelength mutations and posttranslational autooxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91: 12501– 12504.

31. Hentzer, M.,, H. Wu,, J. B. Andersen,, K. Riedel,, T. B. Rasmussen,, N. Bagge,, N. Kumar,, M. Schembri,, Z. Song,, P. Kristoffersen,, M. Manefield,, J. W. Costerton,, S. Molin,, L. Eberl,, P. Steinberg,, S. Kjelleberg,, N. Høiby,, and M. Givskov. 2003. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J. 22: 1– 13.

32. Hentzer, M.,, K. Riedel,, T. B. Rasmussen,, A. Heydorn,, J. B. Andersen,, M. R. Parsek,, S. A. Rice,, L. Eberl,, S. Molin,, and M. Givskov. 2002. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148: 87– 102.

33. Hentzer, M.,, L. Eberl,, J. Nielsen,, and M. Givskov. 2003. Quorum sensing: a novel target for the treatment of biofilm infections. BioDrugs 17: 241– 250.

34. Hentzer, M.,, M. Givskov,, and L. Eberl,. 2004. Quorum sensing in biofilms: gossip in slime city, p. 118– 140. In M. A. Ghannoum, and G. O’Toole (ed.), Microbial Biofilms. ASM Press, Washington, D.C..

35. Høiby, N.,, and B. Frederiksen,. 2000. Microbiology of cystic fibrosis, p. 83– 107. In M. E. Hodson, and D. M. Geddes (ed.), Cystic Fibrosis. Arnold, London, United Kingdom.

36. Høiby, N.,, B. Giwercman,, T. Jensen,, H. K. Johansen,, G. Kronborg,, T. Pressler,, and A. Kharazmi,. 1993. Immune response in cystic fibrosis—helpful or harmful? p. 133– 144. In H. Scobar (ed.), Clinical Ecology of Cystic Fibrosis. Exerpta Medica, Amsterdam, The Netherlands.

37. Hold, G. L.,, A. Schwiertz,, R. I. Aminov,, M. Blaut,, and H. J. Flint. 2003. Oligonucleotide probes that detect quantitatively significant groups of butyrate-producing bacteria in human feces. Appl. Environ. Microbiol. 69: 4320– 4324.

38. Huber, B.,, K. Riedel,, M. Hentzer,, A. Heydorn,, A. Gotschlich,, M. Givskov,, S. Molin,, and L. Eberl. 2001 The cep quorumsensing system of Burkholderia cepacia H111 controls biofilm formation and swarming motility. Microbiology 147: 2517– 2528.

39. Isles, A.,, I. Maclusky,, M. Corey,, R. Gold,, C. Prober,, P. Fleming, and H Levison. 1984. Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. J. Pediatr. 104: 206– 210.

40. Jacobi, C. A.,, S. Gregor,, A. Rakin,, and J. Heesemann. 2001. Expression analysis of the yersiniabactin receptor gene fyuA and the heme receptor hemA of Yersinia entericolitica in vitro and in vivo using the reporter genes for green fluorescent protein and luciferase. Infect. Immun. 69: 7782– 7772.

41. Jensen, T. K.,, K. Moller,, M. Boye,, T. D. Leser,, and S. E. Jorsal. 2000. Scanning electron microscopy and fluorescent in situ hybridization of experimental Brachyspira (Serpulina) pilosicoli infection in growing pigs. Vet. Pathol. 37: 22– 32.

42. Jensen, T. K.,, M. Boye,, P. Ahrens,, B. Korsager,, P. S. Teglbjaerg,, C. F. Lindboe,, and K. Moller. 2001. Diagnostic examination of human intestinal spirochetosis by fluorescent in situ hybridization for Brachyspira aalborgi, Brachyspira pilosicoli, and other species of the genus Brachyspira (Serpulina). J. Clin. Microbiol. 39: 4111– 4118.

43. Lam, J.,, R. Chan,, K. Lam,, and J. W. Costerton. 1980. Production of mucoid microcolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis. Infect. Immun. 28: 546– 556.

44. Latifi, A.,, M. Foglino,, K. Tanaka,, P. Williams,, and A. Lazdunski. 1996. A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol. Microbiol. 21: 1137– 1146.

45. Lewenza, S.,, B. Conway,, E. P. Greenberg,, and P. A. Sokol. 1999 Quorum sensing in Burkholderia cepacia: identification of the LuxRI homologs CepRI. J. Bacteriol. 181: 748– 756.

46. Li, X.,, X. N. Xiao,, Y. Fang,, X. Jiang,, T. Doung,, C. Fan,, C. C. Huang,, and S. R. Kain. 1998. Generation of destabilized green fluorescent protein transcription reporter. J. Biol. Chem. 273: 34970– 34975.

47. Licht, T. R.,, K. A. Krogfelt,, P. S. Cohen,, L. K. Poulsen,, J. Urbace,, and S. Molin. 1996. Role of lipopolysaccharide in colonization of the mouse intestine by Salmonella typhimurium studied by in situ hybridization. Infect. Immun. 64: 3811– 3817.

48. Licht, T. R.,, T. Tolker-Nielsen,, K. Holmstrøm,, K. A. Krogfelt,, and S. Molin. 1999. Inhibition of Escherichia coli precursor- 16S rRNA processing by mouse intestinal contents. Environ. Microbiol. 1: 23– 32.

49. Lukayanov, K. A.,, A. F. Fradkov,, N. G. Gurskaya,, M. V. Matz,, Y. A. Labas,, A. P. Savitsky,, M. L. Markelov,, A. G. Zaraisky,, X. Zhao,, Y. Fang,, W. Tan,, and S. A. Lukyanov. 2000. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275: 25879– 25882.

50. Lyczak, J. B.,, C. L. Cannon,, and G. B. Pier. 2002. Lung infections associated with cystic fibrosis. Clin. Microbiol. Rev. 15: 194– 222.

51. Matz, M. V.,, A. F. Fradkov,, Y. A. Labas,, A. P. Savitsky,, A. G. Zaraisky,, M. L. Markelov,, and S. A. Lukyanov. 1999. Fluorescent proteins from nonbioluminescent Anthozoa species Nat. Biotechnol. 17: 969– 973.

52. McCartney, A. L. 2002. Application of molecular biological methods for studying probiotics and the gut flora. Br. J. Nutr. 88( Suppl. 1): S29– S37.

53. Meyvis, T. K. L.,, S. C. De Smedt,, P. van Oostveldt,, and J. Demeester. 1999. Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research. Pharmacol. Res. 16: 1153– 1162.

54. Molin, S.,, and M. Givskov. 1999. Application of molecular tools for in situ monitoring of bacterial growth activity. Environ. Microbiol. 1: 383– 391.

55. Møller, A. K.,, M. P. Leatham,, T. Conway,, P. J. M. Nuijten,, L. A. M. de Haan,, K. A. Krogfelt,, and P. S. Cohen. 2003. An Escherichia coli MG1655 lipopolysaccharide deep-rough core mutant grows and survives in mouse cecal mucus but fails to colonize the mouse large intestine. Infect. Immun. 71: 2142– 2152.

56. Morin, J. G.,, and J. W. Hastings. 1971. Energy transfer in a bioluminescent system. J. Cell. Physiol. 77: 313– 318.

57. Morise, H.,, P. Shimomura,, F. H. Johnson,, and J. Winant. 1974. Intramolecular energy-transfer in bioluminescent system of Aequorea. Biochemistry 13: 2656– 2662.

58. Moser, C.,, H. K. Johansen,, Z. J. Song,, H. P. Hougen,, J. Rygaard,, and N. Hoiby. 1997. Chronic Pseudomonas aeruginosa lung infection is more severe in Th2 responding BALB/c mice compared to Th1 responding C3H/HeN mice. APMIS 105: 838– 842.

59. Ormö, M.,, A. B. Cubitt,, K. Kallio,, L. A. Gross,, R. Y. Tsien,, and S. J. Remington. 1996. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273: 1392– 1395.

60. Passador, L.,, J. M. Cook,, M. J. Gambello,, L. Rust,, and B. H. Iglewski. 1993. Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260: 1127– 1130.

61. Patterson, G. H.,, and J. Lippincott-Schwartz. 2002. A photoactviatable GFP for selective photolabeling of proteins and cells. Science 297: 1873– 1877.

62. Pearson, J. P.,, C. Van Delden,, and B. H. Iglewski. 1999. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J. Bacteriol. 181: 1203– 1210.

63. Pedersen, S. S.,, G. H. Shand,, B. L. Hansen,, and G. N. Hansen. 1990. Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres. APMIS 98: 203– 211.

64. Pesci, E. C.,, J. P. Pearson,, P. C. Seed,, and B. H. Iglewski. 1997. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179: 3127– 3132.

65. Poulsen, L. K.,, F. Lan,, C. S. Kristensen,, S. Molin,, and K. A. Krogfelt. 1994. Spatial distribution of Escherichia coli in the mouse large intestine inferred by rRNA in situ hybridization. Infect. Immun. 62: 5191– 5194.

66. Poulsen, L. K.,, T. R. Licht,, C. Rang,, K. A. Krogfelt,, and S. Molin. 1995. The physiological state of Escherichia coli BJ4 growing in the large intestine of streptomycin treated mice. J. Bacteriol. 177: 5840– 5845.

67. Purevdorj, B.,, J. W. Costerton,, and P. Stoodley. 2002. Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 68: 4457– 4464.

68. Ramos, C.,, C. Sternberg,, T. R. Licht,, K. A. Krogfelt,, and S. Molin. 2001. Monitoring bacterial growth activity in biofilms from laboratory flow-chambers, plant rhizosphere and animal intestine. Methods Enzymol. 337B: 21– 42.

69. Riedel, K.,, M. Hentzer,, O. Geisenberger,, B. Huber,, A. Steidle,, H. Wu,, N. Hoiby,, M. Givskov,, and L. Eberl. 2001. N-Acylhomoserine- lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms. Microbiology 147: 3249– 3262.

70. Rumbaugh, K. P.,, J. A. Griswold,, B. H. Iglewski,, and A. N. Hamood. 1999. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections. Infect. Immun. 67: 5854– 5862.

71. Russmann, H.,, A. Feydt-Schmidt,, K. Adler,, D. Aust,, A. Fischer,, and S. Koletzko. 2003. Detection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluorescent in situ hybridization. J. Clin. Microbiol. 41: 813– 815.

72. Russmann, H.,, V. A. Kempf,, S. Koletzko,, J. Heesemann,, and I. B. Autenrieth. 2001. Comparison of fluorescent in situ hybridization and conventional culturing for detection of Helicobacter pylori in gastric biopsy specimens. J. Clin. Microbiol. 39: 304– 308.

73. Shimomura, O.,, F. H. Johnson,, and Y. Saiga. 1962 Extraction, purification and properties of Aequorin, a bioluminescent protein from the luminous hydromedusan Aequorea. J. Cell. Comp. Physiol. 59: 223– 239.

74. Siemering, K. R.,, R. Golbik,, R. Sever,, and J. Haselhoff. 1996. Mutants that suppress the thermosensitivity of green fluorescent protein. Curr. Biol. 6: 1653– 1663.

75. Singh, P. K.,, A. L. Schaefer,, M. R. Parsek,, T. O. Moninger,, M. J. Welsh,, and E. P. Greenberg. 2000. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407: 762– 764.

76. Steidle, A.,, K. Sigl,, R. Schuhegger,, A. Ihring,, M. Schmid,, S. Gantner,, M. Stoffels,, K. Riedel,, M. Givskov,, A. Hartmann,, C. Langebartels,, and L. Eberl. 2001. Visualization of N-acylhomoserine lactone-mediated cell-cell communication between bacteria colonizing the tomato rhizosphere. Appl. Environ. Microbiol. 67: 5761– 5770.

77. Struve, C.,, and K. A. Krogfelt. 2003. Role of capsule in Klebsiella pneumoniae virulence: lack of correlation between in vitro and in vivo studies. FEMS Microbiol. Lett. 218: 149– 154.

78. Struve, C.,, and K. A. Krogfelt. 1999. In vivo detection of Escherichia coli type 1 fimbrial expression and phase variation during experimental urinary tract infection. Microbiology 145: 2683– 2690.

79. Tan, M. W.,, L. G. Rahme,, J. A. Sternberg,, R. G. Tompkins,, and F. M- Ausubel. 1999. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc. Natl. Acad. Sci. USA 96: 2408– 2413.

80. Tan, M. W.,, S. Mahajan-Miklos,, and F. M. Ausubel. 1999. Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc. Natl. Acad. Sci. USA 96: 715– 720.

81. Tang, H. B.,, E. DiMango,, R. Bryan,, M. Gambello,, B. H. Iglewski,, J. B. Goldberg,, and A. Prince. 1996. Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection. Infect. Immun. 64: 37– 43.

82. Terskikh, A.,, A. Fradkov,, G. Ermakova,, A. Zaraisky,, P. Tan,, A. V. Kajava,, X. Zhao,, S. Lukyanov,, M. Matz,, S. Kim,, I. Weissman,, and P. Siebert. 2000. “Fluorecent timer”: protein that changes color with time. Science 290: 1585– 1588.

83. Trebesius, K.,, K. Adler,, M. Vieth,, M. Stolte,, and R. Haas. 2001. Specific detection and prevalence of Helicobacter heilmannii- like organisms in the human gastric mucosa by fluorescent in situ hybridization and partial 16S ribosomal DNA sequencing. J. Clin. Microbiol. 39: 1510– 1516.

84. Trebesius, K.,, K. Panthel,, S. Strobel,, K. Vogt,, G. Faller,, T. Kirchner,, M. Kist,, J. Heesemann,, and R. Haas. 2000. Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridization. Gut 46: 608– 614.

85. Urdaci, M. C.,, B. Regnault,, and P. A. Grimont. 2001. Identification by in situ hybridization of segmented filamentous bacteria in the intestine of diarrheic rainbow trout ( Oncorhynchus mykiss) . Res. Microbiol. 152: 67– 73.

86. Van Bost, S.,, S. Roels,, and J. Mainil. 2001. Necrotoxigenic Escherichia coli type 2 invade and cause diarrhoea during experimental infection in colostrum-restricted newborn calves. Vet. Microbiol. 81: 315– 329.

87. Van Delden, C.,, and B. H. Iglewski. 1998. Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerg. Infect. Dis. 4: 551– 560.

88. Williams, P.,, M. Camara,, A. Hardman,, S. Swift,, D. Milton,, V. J. Hope,, K. Winzer,, B. Middleton,, D. I. Pritchard,, and B. W. Bycroft. 2000. Quorum sensing and the population-dependent control of virulence. Philos. Trans. R. Soc. Lond. Ser. B 355: 667– 680.

89. Winson, M. K.,, M. Camara,, A. Latifi,, M. Foglino,, S. R. Chhabra,, M. Daykin,, M. Bally,, V. Chapon,, G. P. Salmond,, B. W. Bycroft,, A. Lazdunski,, G. S. A. B. Stewart,, and P. Williams. 1995. Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 92: 9427– 9431.

90. Wouters, F. S.,, P. J. Verveer,, and P. I. H. Bastiaens. 2001. Imaging biochemistry inside cells. Trends Cell. Biol. 11: 203– 211.

91. Wu, H.,, Z. Song,, M. Hentzer,, J. B. Andersen,, A. Heydorn,, K. Mathee,, C. Moser,, L. Eberl,, S. Molin,, N. Høiby,, and M. Givskov. 2000. Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa. Microbiology 146: 2481– 2493.

92. Yoon, S. S.,, R. F. Hennigan,, G. M. Hilliard,, U. A. Ochsner,, K. Parvatiyar,, M. C. Kamani,, H. L. Allen,, T. R. DeKievit,, P. R. Gardner,, U. Schwab,, J. J. Rowe,, B. H. Iglewski,, T. R. McDermott,, R. P. Mason,, D. J. Wozniak,, R. E. Hancock,, M. R. Parsek,, T. L. Noah,, R. C. Boucher,, and D. J. Hassett. 2002. Pseudomonas aeruginosa anaerobic respiration in biofilms. Relationships to cystic fibrosis pathogenesis. Dev. Cell 3: 593– 603.

93. Zhao, M.,, M. Yang,, E. Baranov,, X. Wang,, S. Penman,, A. R. Moosa,, and R. M. Hoffman. 2001. Spatial-temporal imaging of bacterial infection and antibiotic response in intact animals. Proc. Natl. Acad. Sci. USA 98: 9814– 9818.

94. Zhu, J.,, J. W. Beaber,, M. I. More,, C. Fuqua,, A. Eberhard,, and S. C. Winans. 1998. Analogs of the autoinducer 3-oxooctanoyl- homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J. Bacteriol. 180: 5398– 5405.

95. Zhu, X. Y.,, and R. D. Joerger. 2003. Composition of microbiota in content and mucus from cecae of broiler chickens as measured by fluorescent in situ hybridization with group-specific, 16S rRNA-targeted oligonucleotide probes. Poult. Sci. 82: 1242– 1249.

96. Zoetendal, E. G.,, K. Ben-Amor,, H. J. Harmsen,, F. Schut,, A. D. Akkermans,, and W. M. de Vos. 2002. Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNA-targeted probes. Appl. Environ. Microbiol. 68: 4225– 4232.