Laboratory Maintenance of Bartonella quintana

James M. Battisti1, Michael F. Minnick1

1 Division of Biological Sciences, The University of Montana, Missoula, Montana
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3C.1
DOI:  10.1002/9780471729259.mc03c01s10
Online Posting Date:  August, 2008
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Abstract

Trench fever is the common name for the acute febrile syndrome associated with a Bartonella quintana bacterial infection. The focus of this unit is to describe reliable methods for cultivation and cryopreservation of B. quintana and can be applied to cultivation and preservation of all Bartonella. Detailed recipes for preparation of three types of semisolid media are also included. Curr. Protoc. Microbiol. 10:3C.1.1‐3C.1.13. © 2008 by John Wiley & Sons, Inc.

Keywords: Bartonella; trench fever; culture; HIB‐B; BB‐H; chocolate agar; cryopreservation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Inoculation of B. quintana from a Frozen Stock onto Semisolid Media
  • Basic Protocol 2: Inoculation of B. quintana from a Liquid Suspension onto Semisolid Media
  • Basic Protocol 3: Inoculation of B. quintana from a Solid Plate Culture onto Semisolid Media
  • Basic Protocol 4: Harvesting B. quintana from Semisolid Media
  • Basic Protocol 5: Cryopreservation of B. quintana
  • Support Protocol 1: Preparation of Workspace and Tools
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Inoculation of B. quintana from a Frozen Stock onto Semisolid Media

  Materials
  • B. quintana glycerol stock ( protocol 5)
  • Petri plates with solidified medium of choice (see and )
  • 250 ml glass beaker containing ∼100 ml 95% ethanol
  • Ice bucket
  • Kimwipes, moistened with 70% (v/v) ethanol
  • Aerosol‐barrier pipet tip
  • Glass culture spreader, sterilized with ethanol
  • 5% CO 2 incubator with a pan of H 2O to maintain humidity
  • Additional reagents and equipment for preparing a biological safety cabinet ( protocol 6) and using the spread technique ( appendix 4A)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after preparation of workspace and tools (see protocol 6 and appendix 4D).

Basic Protocol 2: Inoculation of B. quintana from a Liquid Suspension onto Semisolid Media

  Materials
  • Liquid suspension of B. quintana ( protocol 4)
  • Petri plates with solidified medium of choice (see and )
  • Aerosol‐barrier pipet tip
  • Glass culture spreader, sterilized with ethanol
  • 5% CO 2 incubator with a pan of H 2O to maintain humidity
  • Additional reagents and equipment for using the spread technique ( appendix 4A) and preparing a biological safety cabinet ( protocol 6)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after preparation of workspace and tools (see protocol 6 and appendix 4D).

Basic Protocol 3: Inoculation of B. quintana from a Solid Plate Culture onto Semisolid Media

  Materials
  • B. quintana colony or confluent lawn (from protocol 1 or 2)
  • Standard inoculation loop or bent inoculation wire (see Fig. )
  • Kimwipe, moistened with 70% (v/v) ethanol
  • Petri plates with solidified medium of choice (see and )
  • 5% CO 2 incubator with a pan of H 2O to maintain humidity
  • Additional reagents and equipment for sterilizing the inoculation loop or bent wire using a flame ( appendix 4A)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after preparation of workspace and tools (see protocol 6 and appendix 4D).

Basic Protocol 4: Harvesting B. quintana from Semisolid Media

  Materials
  • Plate(s) containing B. quintana clonal populations (Basic Protocols protocol 11, protocol 22, or protocol 33)
  • Heart infusion broth (HIB; see recipe)
  • Single‐edged razor blades with grooved handle (Smith Brand, cat. no. 67‐0238)
  • Test tube holder (see Fig. )
  • 250‐ml glass beaker containing ∼100 ml 95% ethanol
  • 1000‐µl micropipettor
  • Sterile collection tube
  • Additional reagents and equipment for sterilizing harvesting tool using a flame ( appendix 4A)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after preparation of workspace and tools (see protocol 6 and appendix 4D).

Basic Protocol 5: Cryopreservation of B. quintana

  Materials
  • Cryopreservation solution (see recipe)
  • 1‐ml screw‐capped cryovials (Nunc, cat. no. 375353)
  • Fine‐tipped permanent marker (to label the cryovials)
  • −80°C freezer
  • Additional reagents and equipment for growing and harvesting a clonal population of B. quintana ( protocol 4)
NOTE: Cultivation of B. quintana should be performed in a biosafety cabinet after preparation of workspace and tools (see protocol 6 and appendix 4D).

Support Protocol 1: Preparation of Workspace and Tools

  Materials
  • Laminar‐flow type biosafety cabinet equipped with germicidal UV bulb
  • Spray bottle with 70% ethanol (v/v)
  • Kimwipes
  • Latex, vinyl, or nitrile protective gloves
  • Laboratory coat
  • Touch‐O‐Matic (Hanau) bunsen burner
  • 10‐µl, 100‐µl, and 1000‐µl micropipettors
  • Aerosol‐barrier micropipet tips, sterile
  • Biohazard bag
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Figures

Videos

Literature Cited

   Alsmark, C.M., Frank, A.C., Karlberg, E.O., Legault, B.A., Ardell, D.H., Canbäck, B., Eriksson, A.S., Näslund, A.K., Handley, S.A., Huvet, M., La Scola, B., Holmberg, M., and Andersson, S.G. 2004. The louse‐borne human pathogen Bartonella quintana is a genomic derivative of the zoonotic agent Bartonella henselae. Proc. Natl. Acad. Sci. U.S.A. 101:9716‐9721.
   Battisti, J.M., Sappington, K.N., Smitherman, L.S., Parrow, N.L., and Minnick, M.F. 2006. Environmental signals generate a differential and coordinated expression of the heme receptor gene family of Bartonella quintana. Infect. Immun. 74:3251‐3261.
   Battisti, J.M., Smitherman, L.S., Sappington, K.N., Parrow, N.L., Raghavan, R., and Minnick, M.F. 2007. Transcriptional regulation of the heme binding protein gene family of Bartonella quintana is accomplished by a novel promoter element and iron response regulator. Infect. Immun. 75:4373‐4385.
   Birtles, R.J., Harrison, T.G., Saunders, N.A., and Molyneux, D.H. 1995. Proposals to unify the genera Grahamella and Bartonella, with descriptions of Bartonella talpae comb. nov., Bartonella peromysci comb. nov., and three new species, Bartonella grahamii sp. nov., Bartonella taylorii sp. nov., and Bartonella doshiae sp. nov. Int. J. Syst. Bacteriol. 45:1‐8.
   Boonjakuakul, J.K., Gerns, H.L., Chen, Y.T., Hicks, L.D., Minnick, M.F., and Koehler, J.E. 2007. Proteomic and immunoblot analysis of Bartonella quintana total membrane proteins identifies antigens recognized by serum from infected patients. Infect. Immun. 75:2548‐2561.
   Breitschwerdt, E.B. and Kordick, D.L. 2000. Bartonella infection in animals: Carriership, reservoir potential, pathogenicity, and zoonotic potential for human infection. Clin. Microbiol. Rev. 13:428‐438.
   Eremeeva, M.E., Gerns, H.L., Lydy, S.L., Goo, J.S., Ryan, E.T., Mathew, S.S., Ferraro, M.J., Holden, J.M., Nicholson, W.L., Dasch, G.A., and Koehler, J.E. 2007. Bacteremia, fever, and splenomegaly caused by a newly recognized Bartonella species. N. Engl. J. Med. 356:2381‐2387.
   Fournier, P.E., Minnick, M.F., Lepidi, H., Salvo, E., and Raoult, D. 2001. Experimental model of human body louse infection using green fluorescent protein‐expressing Bartonella quintana. Infect. Immun. 69:1876‐1879.
   Huang, K.Y. 1967. Metabolic Activity of the Trench Fever Rickettsia, Rickettsia quintana. J. Bacteriol. 93:853‐859.
   Koehler, J.E., Quinn, F.D., Berger, T.G., LeBoit, P.E., and Tappero, J.W. 1992. Isolation of Rochalimaea species from cutaneous and osseous lesions of bacillary angiomatosis. N. Engl. J. Med. 327:1625‐1631.
   La Scola, B. and Raoult, D. 1999. Culture of Bartonella quintana and Bartonella henselae from human samples: A 5‐year experience (1993 to 1998). J. Clin. Microbiol. 37:1899‐1905.
   La Scola, B., Fournier, P.E., Brouqui, P., and Raoult, D. 2001. Detection and culture of Bartonella quintana, Serratia marcescens, and Acinetobacter spp. from decontaminated human body lice. J. Clin. Microbiol. 39:1707‐1709.
   Maggi, R.G., Duncan, A.W., and Breitschwerdt, E.B. 2005. Novel chemically modified liquid medium that will support the growth of seven Bartonella species. J. Clin. Microbiol. 43:2651‐2655.
   Marsh, F. and Buxton, P.A. 1937. Measurements of the temperature and humidity between clothes and body. J. Hyg. 37:254‐260.
   Mason, R.A. 1970. Propagation and growth cycle of Rickettsia quintana in a new liquid medium. J. Bacteriol. 103:184‐190.
   Maurin, M. and Raoult, D. 1996. Bartonella (Rochalimaea) quintana infections. Clin. Microbiol. Rev. 9:273‐292.
   Mellannby, K. 1932. The conditions of temperature and humidity of the air between the skin and shirt of man. J. Hyg. 37:268‐274.
   Minnick, M.F. and Barbian, K.D. 1997. Identification of Bartonella using PCR; genus‐ and species‐specific primer sets. J. Microbiol. Meth. 31:51‐57.
   Minnick, M.F., Sappington, K.N., Smitherman, L.S., Andersson, S.G., Karlberg, O., and Carroll, J.A. 2003. Five‐member gene family of Bartonella quintana. Infect. Immun. 71:814‐821.
   Myers, W.F., Cutler, L.D., and Wisseman, C.L. Jr. 1969. Role of erythrocytes and serum in the nutrition of Rickettsia quintana. J. Bacteriol. 97:663‐666.
   Myers, W.F., Osterman, J.V., and Wisseman, C.L. Jr. 1972. Nutritional Studies of Rickettsia quintana: Nature of the Hematin Requirement. J. Bacteriol. 109:89‐95.
   Norman, A.F., Regnery, R., Jameson, P., Greene, C., and Krause, D.C. 1995. Differentiation of Bartonella‐like isolates at the species level by PCR‐restriction fragment length polymorphism in the citrate synthase gene. J. Clin. Microbiol. 33:1797‐1803.
   Ohl, M.E. and Spach, D.H. 2000. Bartonella quintana and urban trench fever. Clin. Infect. Dis. 31:131‐135.
   Rolain, J.M., Brouqui, P., Koehler, J.E., Maguina, C., Dolan, M.J., and Raoult, D. 2004. Recommendations for treatment of human infections caused by Bartonella species. Antimicrob. Agents Chemother. 48:1921‐1933.
   Vinson, J. 1966. In vitro cultivation of the rickettsia agent of trench fever. Bull. World Health Organ. 35:155‐164.
Key References
   Anderson, B.E. and Neuman, M.A. 1997. Bartonella spp. as emerging human pathogens. Clin. Microbiol. Rev. 10:203‐219.
  Two reviews regarding molecular pathogenesis of several Bartonella species.
   Dehio, C. 2004. Molecular and cellular basis of bartonella pathogenesis. Annu. Rev. Microbiol. 58:365‐390.
  An extensive and comprehensive review of the epidemiology, clinical manifestations, diagnosis and treatment of B. quintana infections.
   Maurin, M. and Raoult, D. 1996. Bartonella (Rochalimaea) quintana infections. Clin. Microbiol. Rev. 9:273‐292.
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