Cultivation and Laboratory Maintenance of Chlamydia pneumoniae

Lee Ann Campbell1, Cho‐Chou Kuo1

1 Department of Epidemiology, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 11B.1
DOI:  10.1002/9780471729259.mc11b01s12
Online Posting Date:  February, 2009
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Abstract

Chlamydiae are Gram‐negative obligate intracellular parasites. Chlamydia pneumoniae is a human respiratory pathogen that causes pneumonia, bronchitis, sinusitis, and pharyngitis. C. pneumoniae has also been associated with cardiovascular disease. C. pneumoniae can only be grown in cell culture and is more difficult to isolate from specimens than Chlamydia trachomatis. Commonly used cell lines for isolation of C. trachomatis are not sensitive for C. pneumoniae. The most sensitive cell lines for isolation of C. pneumoniae are HL and HEp‐2. Centrifugation of the inoculum onto the monolayer and inclusion of cycloheximide in the medium enhance isolation. Inclusions are smaller than those of other chlamydiae and are visualized by staining with FITC‐conjugated genus‐ or C. pneumoniae–specific monoclonal antibodies. Slow expansion and use of a small inoculum are key to successful culture. Infectious organisms can be purified by use of Hypaque‐76 gradients to titers >1 × 108/ml. Curr. Protoc. Microbiol. 12:11B.1.1‐11B.1.23. © 2009 by John Wiley & Sons, Inc.

Keywords: Chlamydiae; Chlamydia pneumoniae; respiratory pathogen; pneumonia; cell culture; elementary bodies; inclusion staining

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of C. pneumoniae from Clinical Specimens
  • Basic Protocol 2: Continuous Passage in Cell Culture to Raise the Titers
  • Basic Protocol 3: Infection of Monolayer Tissue Culture Cells by Centrifugation
  • Basic Protocol 4: Purification of Elementary Bodies by Linear Gradient Density Centrifugation
  • Support Protocol 1: Determination of Bacterial Contamination in EB Preparations or Cell Culture
  • Support Protocol 2: Staining of Inclusions and Determination of Inclusion‐Forming Units (IFU)
  • Support Protocol 3: Preparation of Cell Monolayers for Inoculation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of C. pneumoniae from Clinical Specimens

  Materials
  • HL cells (can be obtained through ), freshly trypsinized ( protocol 7)
  • Eagle's Minimal Essential Medium (EMEM)/10% FBS (see recipe) supplemented with 10 µg/ml gentamicin or 100 µg/ml vancomycin plus 100 µg/ml streptomycin
  • Clinical specimens stored in Chlamydia transport medium (see for collection of specimens and see recipe for Chlamydia transport medium)
  • Sucrose/phosphate/glutamate buffer (SPG, see recipe) or 2 sucrose/phosphate buffer (2SP, see recipe)
  • Hanks' balanced salt solution (HBSS, appendix 2A)
  • Chlamydial culture medium [e.g., Eagle's Minimal Essential Medium (EMEM)/10% FBS, see recipe] supplemented with 0.6 to 1.0 µg/ml cycloheximide (see recipe and ) and 10 µg/ml gentamicin or 100 µg/ml vancomycin plus 100 µg/ml streptomycin plus 3.75 µg/ml amphotericin B or 25 U/ml mycostatin
  • Chlamydia‐specific FITC‐conjugated antibody (either C. pneumoniae species‐specific antibody or Chlamydia genus‐specific antibody; can be obtained through ; unconjugated antibodies are available commercially)
  • 1–dram flat‐bottom glass shell vials (outside diameter, 15 mm; height, 45 mm containing a round cover slip, diameter, 12 mm) and plugged with a No. 0 nontoxic silicone rubber stopper, or 24‐well tissue‐culture plates
  • 35°C, 5% CO 2 humidified incubator
  • Sonic dismembrator (e.g., Fisher Model 50)
  • Low‐speed centrifuge with vial holders or tissue culture plate adaptors and temperature control
  • Forceps and scissors (for tissue samples only)
  • Tissue homogenizer or mortar and pestle
  • Additional reagents and equipment for preparing cell monolayers for chlamydial inoculation ( protocol 7) and fixing and staining chlamydial inclusions ( protocol 6)

Basic Protocol 2: Continuous Passage in Cell Culture to Raise the Titers

  Materials
  • Chlamydia in culture ( protocol 1)
  • HL cells (can be obtained through ), freshly trypsinized ( protocol 7)
  • Chlamydial culture medium [e.g., Eagle's Minimal Essential Medium (EMEM)/10% FBS, see recipe] supplemented with 0.6 µg/ml cycloheximide (see recipe and ) and 10 µg/ml gentamicin or 100 µg/ml vancomycin plus 100 µg/ml streptomycin plus 3.75 µg/ml amphotericin B or 25 U/ml mycostatin
  • Hanks' balanced salt solution (HBSS, appendix 2A)
  • 4‐mm sterile glass beads (Kimble Glass)
  • Sucrose/phosphate/glutamate buffer (SPG, see recipe)
  • 75‐cm2 and 150‐cm2 culture flasks
  • 35°C, humidified 5% CO 2 incubator
  • 50‐ml conical centrifuge tubes
  • Low speed centrifuge with appropriate adaptors for vials or tissue culture plates
  • Sonic dismembrator (e.g., Fisher Model 50)
  • Additional reagents and equipment for isolation and passaging of C. pneumoniae ( protocol 1)

Basic Protocol 3: Infection of Monolayer Tissue Culture Cells by Centrifugation

  Materials
  • Cell line, freshly trypsinized (see protocol 7)
  • Eagle's Minimal Essential Medium (EMEM)/10% FBS (see recipe) supplemented with 10 µg/ml gentamicin or 100 µg/ml vancomycin plus 100 µg/ml streptomycin
  • Stock of titered C. pneumoniae EBs ( protocol 4), frozen at −75°C
  • Chlamydial culture medium [e.g., Eagle's Minimal Essential Medium (EMEM)/10% FBS, see recipe] supplemented with 0.6 µg/ml cycloheximide (see recipe and ) and 10 µg/ml gentamicin or 100 µg/ml vancomycin plus 100 µg/ml streptomycin plus 3.75 µg/ml amphotericin B or 25 U/ml mycostatin
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • SPG (see recipe)
  • 1–dram flat‐bottom glass shell vials (outside diameter, 15 mm; height, 45 mm containing a round cover slip, diameter, 12 mm) and plugged with a No. 0 nontoxic silicone rubber stopper, or 24‐well tissue‐culture plates
  • 35°C, humidified 5% CO 2 incubator
  • Sonic dismembrator (optional; e.g., Fisher Model 50)
  • Low‐speed centrifuge with appropriate adaptors for vials and tissue culture plates
  • Additional reagents and equipment for preparation of cell monolayers for chlamydial inoculation ( protocol 7)

Basic Protocol 4: Purification of Elementary Bodies by Linear Gradient Density Centrifugation

  Materials
  • 30% and 65% Hypaque‐76 (Diatrizoate Meglumine and Diatrizoate Sodium, Nycomed) or RenoCal‐76 (Bracco)
  • Cell harvest frozen at −75°C (harvested from eight infected monolayers in 150‐cm2 flasks, see protocol 6)
  • Sucrose/phosphate/glutamate buffer (SPG, see recipe)
  • Hanks' balanced salt solution (HBSS, appendix 2A)
  • Gradient maker (e.g., Gradient Mixer GM‐1, GE Healthcare)
  • Sonicator (e.g., Microson ultrasonic cell disruptor with 4 mm in diameter probe; Misonix)
  • High‐speed centrifuge and rotor (e.g., a Sorvall RC5C and a SS‐34 rotor)
  • 25 × 89–mm Ultraclear centrifuge tubes (Beckman) and tubes for high‐speed centrifuge
  • Ultracentrifuge and a swinging bucket rotor (e.g., Beckman L7‐55 centrifuge and SW 28 rotor)
  • Syringe
  • 18‐G and 21‐G needles
  • 50‐ml sterile plastic centrifuge tubes

Support Protocol 1: Determination of Bacterial Contamination in EB Preparations or Cell Culture

  Materials
  • Trypticase soy broth or agar (Difco)
  • Thioglycolate broth or agar (Difco)
  • Cell culture and or purified EB preparations to be tested for bacterial contamination

Support Protocol 2: Staining of Inclusions and Determination of Inclusion‐Forming Units (IFU)

  Materials
  • HL cells (Gordon and Quan, ; Kuo et al., ; Ripa and Mardh, ), freshly trypsinized ( protocol 7)
  • Eagle's Minimal Essential Medium (EMEM)/10% FBS (see recipe) supplemented with 10 µg/ml gentamicin or 100 µg/ml vancomycin plus 100 µg/ml streptomycin
  • Hanks' balanced salt solution (HBSS; see recipe)
  • Sucrose/phosphate/glutamate buffer (SPG, see recipe)
  • Inoculum: purified chlamydial organisms
  • Chlamyidial culture medium [e.g., Eagle's Minimal Essential Medium (EMEM)/10% FBS, see recipe] supplemented with 0.6 µg/ml cycloheximide (see recipe and ) and 10 µg/ml gentamicin or 100 µg/ml vancomycin plus 100 µg/ml streptomycin plus 3.75 µg/ml amphotericin B or 25 U/ml mycostatin
  • Absolute methanol
  • C. pneumoniae species‐specific or Chlamydia genus‐specific monoclonal antibody conjugated to FITC, for staining chlamydial inclusions
  • 0.2% (w/v) Evans blue counterstain in PBS (see appendix 2A for PBS)
  • Mounting medium (e.g., Permount; Fisher Scientific)
  • 1–dram flat‐bottom glass shell vials (outside diameter, 15 mm; height, 45 mm containing a round cover slip, diameter, 12 mm) and plugged with a No. 0 nontoxic silicone rubber stopper or 24‐well tissue‐culture plates
  • 35°C, 5% CO 2 humidified incubator
  • Low‐speed centrifuge with vial holders or tissue culture plate adaptors and temperature control
  • Moist chamber e.g., large petri dish with moistened paper towel on the bottom
  • Forceps
  • Three beakers containing PBS (see appendix 2A for PBS)
  • Three beakers containing distilled H 2O
  • Microscope slides
  • Epi‐fluorescence microscope containing a counting reticle in the eyepiece
  • Hand‐held counter
  • Additional reagents and equipment for seeding cell monolayers for inoculation ( protocol 7)

Support Protocol 3: Preparation of Cell Monolayers for Inoculation

  Materials
  • Cell line
  • Appropriate culture medium for cell type: for example, for HEp2 (ATCC no, CCL‐23) and HL (can be obtained from investigators in the field) cell lines, use Eagle's Minimal Essential Medium /10% FBS (see recipe) supplemented with 10 µg/ml gentamicin or 100 µg/ml vancomycin plus 100 µg/ml streptomycin
  • GKNP: HBSS ( appendix 2A) without calcium and magnesium
  • Trypsin solution: dilute 10 ml of 0.5% trypsin/EDTA (Invitrogen) per 90 ml GKNP
  • 70% (v/v) ethanol
  • 0.4% (w/v) trypan blue in PBS (see appendix 2A for PBS)
  • 75‐ or 150‐cm2 culture flasks
  • 35°C, 5% CO 2 humidified incubator
  • Hemacytometer ( appendix 4A)
  • Hand‐held counter
  • Additional reagents and equipment for counting cells ( appendix 4A)
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Figures

Videos

Literature Cited

   Campbell, L.A. and Kuo, C.‐C. 2004. Chlamydia pneumoniae, an infectious risk factor for atherosclerosis? Nature Med. Rev. Microbiol. 2:23‐32.
   Campbell, L.A., Kuo, C.‐C., and Gaydos, C.A. Chlamydial infections. 2006. In Manual of Molecular and Clinical Laboratory Immunology, 7th Edition (B. Detrick, R.G. Hamilton, and J.D. Folds, eds.) pp. 518‐525. ASM Press, Washington, D.C.
   Cavallaro, J.J. and Monto, A.S. 1972. HL cells, a sensitive line for the isolation and propagation of respiratory syncytial virus. Proc. Soc. Exp. Biol. Med. 140:507‐510.
   Cles, L.D. and Stamm, W.E. 1990. Use of HL cells for improved isolation and passage of Chlamydia pneumoniae. J. Clin. Microbiol. 28:938‐940.
   Dowell, S.F., Peeling, R.W., Boman, J., Carlone, G.M., Fields, B.S., Guarner, J., Hammerschlag, M.R., Jackson, L., Kuo, C.‐C., Maass, M., Messmer, T.O., Talkington, D., Tondella, M.L., Zaki, R., Apfalter, P., Bandea, C., Black, C., Campbell, L.A., Cohen, C., Deal, C., Fong, I., Gaydos, C., Leionen, M., Mahony, J., O'Connor, S., Ossewaarde, J.M., Papp, J., Saikku, P., Schindler, L., Schuchat, A., Stevenes, V., Talkington, D., Taylor, C., Tondella, M.L., Van Benenden, C.A., Wang, S.‐P., and Zell, E. 2001. Standardizing Chlamydia pneumoniae assays: Recommendations from the Centers for Disease Control and Prevention (USA), and the Laboratory Centre for Disease Control (Canada). Clin. Infect. Dis. 33:492‐502.
   Furness, G., Graham, D.M., and Reeve, P. 1960. The titration of trachoma and inclusions blennorrhoea viruses in cell cultures. J. Gen. Microbiol. 23:613‐619.
   Godzik, K.L., O'Brien, E.R., Wang, S.‐P., and Kuo, C.C. 1995. In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae. J. Clin. Microbiol. 33:2411‐2444.
   Gordon, F.B. and Quan, A.L. 1965. Isolation of the trachoma agent in cell culture. Proc. Soc. Exp. Biol. Med. 118:354‐359.
   Grayston, J.T. 2005. Chlamydia pneumoniae and atherosclerosis. Clin. Infect. Dis. 40:1131‐1132.
   Grayston, J.T., Kuo, C.‐C., Campbell, L.A., and Wang, S.‐P. 1989. Proposal to create the Chlamydia sp. nov. for Chlamydia strain TWAR. Int. J. Syst. Bacteriol. 39:88‐90.
   Grayston, J.T., Kronmal, R.A., Jackson, L.A., Parisis, A.F., Muhlestein, J.G., Cohen, J.D., Rogers, W.J., Crouse, J.R., Borrowdalek, S.L., Schron, M.S., and Knirsch, C. for the ACES Investigators. 2005. Azithromycin for the secondary prevention of coronary events. N. Engl. J. Med. 352:1706‐1709.
   Kuo, C.‐C. 1999. Chlamydia pneumoniae: Culture methods. In Chlamydia pneumoniae: The Lung and the Heart (L. Allegra and F. Blasi, eds.) pp. 9‐15. Springer‐Verlag, Milan, Italy.
   Kuo, C.‐C. and Grayston, J.T. 1988. Factors affecting viability and growth in HeLa 229 cells of Chlamydia sp. strain TWAR. J. Clin. Microbiol. 26:812‐815.
   Kuo, C.‐C. and Grayston, J.T. 1990. A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR. J. Infect. Dis. 162:755‐758.
   Kuo, C.‐C., Wang, S.‐P., Wentworth, B., and Grayston, J.T. 1972. Primary isolation of TRIC organisms in HeLa 229 cells treated with DEAE‐dextran. J. Infect. Dis. 125:665‐668.
   Kuo, C.‐C., Wang, S.‐P., and Grayston, J.T. 1977. Growth of trachoma organisms in HeLa 229 cell culture. In Nongonococcal Urethritis and Related Infections (D. Hobson and K.K. Holmes, ed.) pp. 328‐336. American Society for Microbiology, Washington, D.C.
   Kuo, C.‐.C, Chen, H.‐H, Wang, S.‐P., and Grayston, J.T. 1986. Identification of a new group of Chlamydia psittaci strains called TWAR. J. Clin. Microbiol. 24:1034‐1037.
   Kuo, C.C., Jackson, L.A., Campbell, L.A., and Grayston, J.T. 1995. Chlamydia pneumoniae (TWAR) Clin. Microbiol. Rev. 8:451‐461.
   Maass, M. and Dalhoff, K. 1995. Transport and storage conditions for cultural recovery of Chlamydia pneumoniae. J. Clin. Microbiol. 33:1793‐1796.
   McComb, D.E. and Puzniak, C.I. 1974. Micro cell culture method for isolation of Chlamydia trachomatis. Appl. Microbiol. 28:727‐729.
   O'Connor, C.M., Dunne, M.W., Pfeffer, M.A., Muhlestein, J.B., Yao, L., Gupta, S., Benner, R.J., Fisher, M.R., Cook, T.D. and Investigators in the WIZARD Study. 2003. Azithromycin for the secondary prevention of coronary heart disease events: The WIZARD study: A randomized controlled trial. JAMA 290:1459‐1466.
   Ripa, K.T. and Mardh, P.‐A. 1977. Cultivation of Chlamydia trachomatis in cycloheximide‐treated McCoy cells. J. Clin. Microbiol. 6:328‐331.
   Roblin, P.M., Dumornay, W., and Hammerschlag, M.R. 1992. Use of HEp‐2 cells for improved isolation and passage of Chlamydia pneumoniae. J. Clin. Microbiol. 30:1968‐1971.
   T'ang, F., Chang, H., Huang, Y., and Wang, K. 1957. Studies on the etiology of trachoma with special reference to isolation of the virus in chick embryo. Chin. Med. J. 75:429‐447.
   Taylor‐Robinson, D. and Boman, J. 2005. The failure of antibiotics to prevent heart attacks: It's not necessarily the end of the road. BMJ 331:461‐362.
   Wong, K.H., Skelton, S.K., and Chan, Y.K. 1992. Efficient culture of Chlamydia pneumoniae with cell lines derived from the human respiratory tract. J. Clin. Microbiol. 30:1625‐1630.
   Yoder, B.L., Stamm, W.E., Koester, C.M., and Alexander, E.R. 1981. Microtest procedure for isolation of Chlamydia trachomatis. J. Clin. Microbiol. 13:1036‐1039.
Key References
   Kuo and Grayston, 1988. See above.
  These two early publications provide details for handling and growing C. pneumonia.
   Kuo and Grayston, 1990. See above.
  This resource contains several helpful chapters on cell culture methods for different cell types, and basic techniques. In addition, the chapter by C. Uphoff and H.G. Drexler entitled “Detection of Mycoplasma contamination” (pp.13‐23) provides PCR methods for detection of Mycoplasma contamination in cell culture.
   Helgason, C.D. and Miller, C.L. (eds.). 2005. Basic cell culture protocols. In Methods in Molecular Biology, Vol. 290. Humana Press, Totowa, N.J.
  This resource is essential for following the appropriate biosafety procedures required for handling a pathogenic organism.
   Chosewood, L.C. and Wilson, D.E. (eds.). 2007. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition. U. S. Government Printing Office, Washington, D.C.
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