Experimental Design Considerations for In Vitro Microbial Endocrinology Investigations

  • Richard D. Haigh


This chapter examines and critiques the different methodological approaches, which have been adopted to investigate the interactions of catecholamine neurohormones with bacteria. Adrenergic catecholamines have been shown to promote the in vitro growth of a wide range of both gram-positive and gram-negative bacterial species using growth media, which are designed to mimic the hostile bacteriostatic environment in the host. However, in recent years, it has become apparent that neurohormones are doing more than just alleviating the iron restriction caused by the mammalian transferrin in these media. In fact, it is clear that there are specific receptors in many bacteria, which can recognise epinephrine and norepinephrine resulting in increased virulence determinant expression and alterations in bacterial transcription on a global scale. Furthermore, there is now evidence that “adrenergic” signalling events may also be involved in the bacterial growth responses to catecholamines that have been observed previously. This analysis of the currently used methodologies is intended to assist in a more concerted approach to in vitro microbial endocrinology experiments, which identifies further bacterial genes and molecules the roles of which can be verified in future in vivo investigations.


Luria Broth Virulence Determinant Iron Restriction Catecholamine Concentration Mycoplasma Hyopneumoniae 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Alverdy, J., Holbrook, C., Rocha, F., Seiden, L., Wu, R. L., Musch, M., Chang, E., Ohman, D., and Suh, S. 2000. Gut-derived sepsis occurs when the right pathogen with the right virulence genes meets the right host: Evidence for in vivo virulence expression in Pseudomonas aeruginosa. Ann. Surg. 232:480-489.PubMedCrossRefGoogle Scholar
  2. Anderson, M.T., and Armstrong, S.K. 2006. The Bordetella Bfe system: Growth and transcriptional response to siderophores, catechols, and neuroendocrine catecholamines. J. Bact.188:5731-5740.PubMedCrossRefGoogle Scholar
  3. Anderson, M. T., and Armstrong, S. K. 2008. Norepinephrine mediates acquisition of transferrin-iron on Bordetella brontiseptica. J. Bact. 190:3940-3947.PubMedCrossRefGoogle Scholar
  4. Banerjee, D., Martin, N., Nandi, S., Shukla, S., Dominguez, A., Mukhopadhyay, G., and Prasad, R. 2007. A genome-wide steroid response study of the major human fungal pathogen Candida albicans. Mycopathologia. 164:1-17.PubMedCrossRefGoogle Scholar
  5. Bansal, T., Englert, D., Lee, J., Hegde, M., Wood, T. K., and Jayaraman, A. 2007. Differential effects of epinephrine, norepinephrine, and indole on Escherichia coli O157:H7 chemotaxis, colonization, and gene expression. Infect. Immun. 75:4597-4607.PubMedCrossRefGoogle Scholar
  6. Bearson, B. L., Bearson, S. M., Uthe, J. J., Dowd, S. E., Houghton, J. O., Lee, I., Toscano, M. J., and Lay Jr., D. C. 2008. Iron regulated genes of Salmonella enterica serovar Typhimurium in response to norepinephrine and the requirement of fepDGC for norepinephrine-enhanced growth. Microbes Infect. 10:807-816.PubMedCrossRefGoogle Scholar
  7. Belay, T., Aviles, H., Vance, M., Fountain, K., Sonnenfeld, G. 2003. Catecholamines and in vitro growth of pathogenic bacteria: Enhancement of growth varies greatly among bacterial species. Life Sci. 73:1527-1535.PubMedCrossRefGoogle Scholar
  8. Borisenko, G., Kagan, A., Hsia, C., and Schor, N. F. 2000. Interaction between 6-Hydroxydopamine and Transferrin: “Let My Iron Go”. Biochemistry. 39:3392-3400.PubMedCrossRefGoogle Scholar
  9. Burton, C. L., Chhabra, S. R., Swift, S., Baldwin, T. J., Withers, H., Hill, S. J., and Williams, P. 2002. The growth response of Escherichia coli to neurotransmitters and related catecholamine drugs requires a functional enterobactin biosynthesis and uptake system. Infect. Immun. 70:5913-5923.PubMedCrossRefGoogle Scholar
  10. Clarke, M. B., and Sperandio, V. 2005. Events at the host-microbial interface of the gastrointestinal tract III. Cell-to-cell signaling among microbial flora, host, and pathogens: there is a whole lot of talking going on. Am. J. Physiol. Gastrointest. Liver Physiol. 288: G1105-G1109.PubMedCrossRefGoogle Scholar
  11. Clarke, M. B., Hughes, D. T., Zhu, C., Boedeker, E. C., and Sperandio, V. 2006. The QseC sensor kinase: A bacterial adrenergic receptor. Proc. Natl. Acad. Sci. U. S. A. 103:10420-10425.PubMedCrossRefGoogle Scholar
  12. Cogan, T. A., Thomas, A. O., Rees, L. E., Taylor, A. H., Jepson, M. A., Williams, P. H., Ketley, J., and Humphrey. T. J. 2007. Norepinephrine increases the pathogenic potential of Campylobacter jejuni. Gut 56:1060-1065.PubMedCrossRefGoogle Scholar
  13. Coulanges, V., Andre, P., Ziegler, O., Buchheit, L., and Vidon, D. J. 1997. Utilization of iron-catecholamine complexes involving ferric reductase activity in Listeria monocytogenes. Infect Immun. 65:2778-2785.PubMedGoogle Scholar
  14. Coulanges, V., Andre, P., Vidon, D. J-M. (1998) Effect of siderophores, catecholamines, and catechol compounds on Listeria spp. growth in iron-complexed medium. Biochem. Biophys. Res. Comm. 249:526-530.PubMedCrossRefGoogle Scholar
  15. Dowd, S. E. 2007. Escherichia coli O157:H7 gene expression in the presence of catecholamine norepinephrine. FEMS Microbiol. Lett. 273:214-223.PubMedCrossRefGoogle Scholar
  16. Freestone, P. P., and Lyte, M. (2008) Microbial endocrinology: experimental design issues in the study of interkingdom signalling in infectious disease. Adv. Appl. Microbiol. 64:75-105.PubMedCrossRefGoogle Scholar
  17. Freestone, P. P., Haigh, R. D., Williams, P. H., and Lyte, M. 1999. Stimulation of bacterial growth by heat-stable, norepinephrine-induced autoinducers. FEMS Microbiol. Lett. 172:53-60.PubMedCrossRefGoogle Scholar
  18. Freestone, P. P., Lyte, M., Neal, C. P., Maggs, A. F., Haigh, R. D., and Williams, P. H. 2000. The mammalian neuroendocrine hormone norepinephrine supplies iron for bacterial growth in the presence of transferrin or lactoferrin. J. Bacteriol. 182:6091-6098.PubMedCrossRefGoogle Scholar
  19. Freestone, P. P., Williams, P. H., Haigh, R. D., Maggs, A. F., Neal, C. P. and Lyte, M. 2002. Growth stimulation of intestinal commensal Escherichia coli by catecholamines: A possible contributory factor in trauma-induced sepsis. Shock 18:465-470.PubMedCrossRefGoogle Scholar
  20. Freestone, P. P., Haigh, R. D., Williams, P. H., and Lyte, M. 2003. Involvement of enterobactin in norepinephrine-mediated iron supply from transferrin to enterohaemorrhagic Escherichia coli. FEMS Microbiol. Lett. 222:39-43.PubMedCrossRefGoogle Scholar
  21. Freestone, P. P., Haigh, R. D., and Lyte, M. 2007a. Blockade of catecholamine-induced growth by adrenergic and dopaminergic receptor antagonists in Escherichia coli O157:H7, Salmonella enterica and Yersinia enterocolitica. BMC Microbiol. 7:8.PubMedCrossRefGoogle Scholar
  22. Freestone, P. P., Haigh, R. D., and Lyte, M. 2007b. Specificity of catecholamine-induced growth in Escherichia coli O157:H7, Salmonella enterica and Yersinia enterocolitica. FEMS Microbiol. Lett. 269:221-228.PubMedCrossRefGoogle Scholar
  23. Freestone, P. P., Walton, N. J., Haigh, R. D., and Lyte, M. 2007c. Influence of dietary catechols on the growth of enteropathogenic bacteria. Int. J. Food Microbiol. 119:159-169.PubMedCrossRefGoogle Scholar
  24. Freestone, P. P., Sandrini, S. M., Haigh, R. D., and Lyte, M. 2008. Microbial endocrinology: how stress influences susceptibility to infection. Trends Microbiol. 16:55-64.PubMedCrossRefGoogle Scholar
  25. Friis, N. F. 1975. Some recommendations concerning primary isolation of Mycoplasma hyopneumoniae and Mycoplasma flocculare, a survey. Nord. Vetmed. 27:337-339.Google Scholar
  26. Hughes, D. T., and Sperandio, V. 2008. Inter-kingdom signalling: communication between bacteria and their hosts. Nat. Rev. Microbiol. 6:111-120.PubMedCrossRefGoogle Scholar
  27. Kaper, J. B., and Sperandio, V. 2005. Bacterial cell-to-cell signalling in the gastrointestinal tract. Infect. Immun. 73:3197-3209.PubMedCrossRefGoogle Scholar
  28. Karavolos, M. H., Spencer, H., Bulmer, D. M., Thompson, A., Winzer, K., Williams, P., Hinton, J. C., and Khan, C. M. 2008. Adrenaline modulates the global transcriptional profile of Salmonella revealing a role in the antimicrobial peptide and oxidative stress resistance responses. BMC Genomics. 9:458.PubMedCrossRefGoogle Scholar
  29. Kendall, M. M., and Sperandio, V. 2007 Quorum sensing by enteric pathogens. Gastrointestinal infections. Curr. Opin. Gastroenterol. 23:10-15.CrossRefGoogle Scholar
  30. Kendall, M. M., Rasko, D. A., and Sperandio, V. 2007. Global effects of the cell-to-cell signaling molecules autoinducer-2, autoinducer-3, and epinephrine in a luxS mutant of enterohemorrhagic Escherichia coli. Infect. Immun. 75:4875-4884.PubMedCrossRefGoogle Scholar
  31. Kinney, K.S., Austin, C.E., Morton, D.S., Sonnenfeld, G. 1999. Catecholamine enhancement of Aeromonas hydrophila growth. Micro. Pathogen. 26:85-91.CrossRefGoogle Scholar
  32. Kinney, K. S., Austin, C. E., Morton, D. S., and Sonnenfeld, G. 2000. Norepinephrine as a growth stimulating factor in bacteria - mechanistic studies. Life Sci. 67:3075-3085.PubMedCrossRefGoogle Scholar
  33. Leinhardt, D. J., Arnold, J., Shipley, K. A., Mughal, M. M., Little, R. A., and Irving, M. H. 1993. Plasma NE concentrations do not accurately reflect sympathetic nervous system activity in human sepsis. Am. J. Physiol. 265:E284-E288.PubMedGoogle Scholar
  34. Lyte, M. 1993 The role of microbial endocrinology in infectious disease. J. Endocrinol. 137:343-345.PubMedCrossRefGoogle Scholar
  35. Lyte, M. 2004. Microbial endocrinology and infectious disease in the 21st century. Trends Microbiol. 12:14-20.PubMedCrossRefGoogle Scholar
  36. Lyte, M., and Ernst, S. 1992. Catecholamine-induced growth of gram negative bacteria. Life Sci. 50:203-212.PubMedCrossRefGoogle Scholar
  37. Lyte, M., Ernst, S. 1993. Alpha and beta adrenergic receptor involvement in catecholamine-induced growth of gram-negative bacteria. Biochem. Biophys. Res. Comm. 190:447-452.PubMedCrossRefGoogle Scholar
  38. Lyte, M., Arulanandam, B. P., and Frank, C. D. 1996. Production of Shiga-like toxins by Escherichia coli O157:H7 can be influenced by the neuroendocrine hormone norepinephrine. J. Lab. Clin. Med. 128:392-398.PubMedCrossRefGoogle Scholar
  39. Lyte, M., Arulanandam, B., Nguyen, K., Frank, C., Erickson, A., and Francis, D. 1997a. Norepinephrine induced growth and expression of virulence associated factors in enterotoxigenic and enterohemorrhagic strains of Escherichia coli. Adv. Exp. Med. Biol. 412:331-339.PubMedGoogle Scholar
  40. Lyte, M., Erickson, A. K., Arulanandam, B. P., Frank, C. D., Crawford, M. A., and Francis, D. H. 1997b. Norepinephrine-induced expression of the K99 pilus adhesin of enterotoxigenic Escherichia coli. Biochem. Biophys. Res. Commun. 232:682-686.PubMedCrossRefGoogle Scholar
  41. Lyte, M., Freestone, P. P., Neal, C. P., Olson, B. A., Haigh R. D., Bayston, R., Williams, P. H. 2003. Stimulation of Staphylococcus epidermidis growth and biofilm formation by catecholamine inotropes. Lancet 361:130-135.PubMedCrossRefGoogle Scholar
  42. Madsen, M. L., Nettleton, D, Thacker, E. L., and Minion, F. C. 2006 Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays. Microbiology 152:937-944.PubMedCrossRefGoogle Scholar
  43. Methner, U., Rabsch, W., Reissbrodt, R., and Williams, P. H. 2008. Effect of norepinephrine on colonisation and systemic spread of Salmonella enterica in infected animals: Role of catecholate siderophore precursors and degradation products. Int. J. Med. Microbiol. 298:429-439.PubMedCrossRefGoogle Scholar
  44. Nakano, M., Takahashi, A., Sakai, Y., and Nakaya, Y. 2007a. Modulation of pathogenicity with norepinephrine related to the type III secretion system of Vibrio parahaemolyticus. J. Infect. Dis. 195:1353-1360.PubMedCrossRefGoogle Scholar
  45. Nakano, M., Takahashi, A., Sakai, Y., Kawano, M., Harada, N., Mawatari, K., and Nakaya, Y. 2007b. Catecholamine-induced stimulation of growth in Vibrio species. Lett. Appl. Microbiol. 44:649-653.PubMedCrossRefGoogle Scholar
  46. Neal, C. P., Freestone, P. P. E., Maggs, A. F., Haigh, R. D., Williams, P. H., Lyte, M. 2001. Catecholamine inotropes as growth factors for Staphylococcus epidermidis and other coagulase-negative staphylococci. FEMS Microbiol. Lett. 194:163-169.PubMedCrossRefGoogle Scholar
  47. O’Donnell, P. M., Aviles, H., Lyte, M., and Sonnenfeld, G. 2006. Enhancement of in vitro growth of pathogenic bacteria by norepinephrine: Importance of inoculum density and role of transferrin. Appl. Environ. Microbiol. 72:5097-5099.PubMedCrossRefGoogle Scholar
  48. Oneal, M. J., Schafer, E. R., Madsen, M. L., and Minion, F. C. 2008. Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to norepinephrine. Microbiology 154: 2581-2588.PubMedCrossRefGoogle Scholar
  49. Reading, N. C., Torres, A. G., Kendall, M. M., Hughes, D. T., Yamamoto, K., and Sperandio, V. 2007. A novel two-component signaling system that activates transcription of an enterohemorrhagic Escherichia coli effector involved in remodeling of host actin. J. Bacteriol. 189:2468-2476.PubMedCrossRefGoogle Scholar
  50. Roberts, A., Matthews, J. B., Socransky, S. S., Freestone, P. P., Williams, P. H., and Chapple, I. L. 2002. Stress and the periodontal diseases: Effects of catecholamines on the growth of periodontal bacteria in vitro. Oral Microbiol. Immunol. 17:296-303.PubMedCrossRefGoogle Scholar
  51. Schafer, E. R., Oneal, M. J., Madsen, M. L., and Minion, C. F. 2007 Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to hydrogen peroxide. Microbiology 153:3785-3790.PubMedCrossRefGoogle Scholar
  52. Scheckelhoff, M. R., Telford, S. R., Wesley, M., and Hu, L. T. 2007. Borrelia burgdorferi intercepts host hormonal signals to regulate expression of outer surface protein A. Proc. Natl. Acad. Sci. USA. 104:7247-7252.PubMedCrossRefGoogle Scholar
  53. Sperandio, V., Torres, A. G., and Kaper, J. B. 2002. Quorum sensing Escherichia coli regulators B and C (QseBC): A novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol. Microbiol. 43:809-821.PubMedCrossRefGoogle Scholar
  54. Sperandio, V., Torres, A. G., Jarvis, B., Nataro, J. P., and Kaper, J. B. 2003. Bacteria-host communication: the language of hormones. Proc. Natl. Acad. Sci. U. S. A. 100:8951-8956.PubMedCrossRefGoogle Scholar
  55. Straub, R. H., Wiest, R., Strauch, U. G., Harle, P., and Scholmerich, J. 2006. The role of the sympathetic nervous system in intestinal inflammation. Gut 55:1640-1649.PubMedCrossRefGoogle Scholar
  56. Tarr, P. I., and Neill, M. A., 2001. Escherichia coli O157:H7, Gastroenterol. Clin. North Am. 30:735-751.PubMedCrossRefGoogle Scholar
  57. Toscano, M. J., Stabel, T. J., Bearson, S. M. D., Bearson, B. L. and Lay, D. C. 2007. Cultivation of Salmonella enterica serovar Typhimurium in a norepinephrine-containing medium alters in vivo tissue prevalence in swine. J. Exp. Anim. Sci. 43: 329-338.CrossRefGoogle Scholar
  58. Vlisidou, I., Lyte, M., van Diemen, P. M., Hawes, P., Monaghan, P., Wallis, T. S., and Stevens, M. P. 2004. The neuroendocrine stress hormone norepinephrine augments Escherichia coli O157:H7-induced enteritis and adherence in a bovine ligated ileal loop model of infection. Infect. Immun. 72:5446-5451.PubMedCrossRefGoogle Scholar
  59. Walters, M., and Sperandio, V. 2006. Autoinducer 3 and epinephrine signaling in the kinetics of locus of enterocyte effacement gene expression in enterohemorrhagic Escherichia coli. Infect. Immun. 74:5445-5455.PubMedCrossRefGoogle Scholar
  60. Zaborina, O., Lepine, F., Xiao, G., Valuckaite, V., Chen, Y., Li, T., Ciancio, M., Zaborin, A., Petrof, E. O., Turner, J. R., Rahme, L. G., Chang, E., and Alverdy, J. C. 2007. Dynorphin activates quorum sensing quinolone signaling in Pseudomonas aeruginosa. PLoS Pathog. 3:e35.PubMedCrossRefGoogle Scholar
  61. Zhang, X., Essmann, M., Burt, E. T., and Larsen, B. 2000. Estrogen effects on Candida albicans: a potential virulence-regulating mechanism. J. Infect. Dis. 181:1441-1446.PubMedCrossRefGoogle Scholar

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© Springer-Verlag New York 2010

Authors and Affiliations

  1. 1.Department of GeneticsUniversity of LeicesterLeicesterUK

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