Burkholderia thailandensis: Growth and Laboratory Maintenance

Erin C. Garcia1, Peggy A. Cotter1

1 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 4C.1
DOI:  10.1002/cpmc.15
Online Posting Date:  August, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Burkholderia thailandensis is a nonpathogenic Gram‐negative bacterium found in tropical soils. Closely related to several human pathogens, its ease of genetic manipulation, rapid growth in the laboratory, and low virulence make B. thailandensis a commonly used model organism. This unit describes the fundamental protocols for in vitro growth and maintenance of B. thailandensis in the laboratory. © 2016 by John Wiley & Sons, Inc.

Keywords: Burkholderia thailandensis; Burkholderia pseudomallei; growth; model organism

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of B. thailandensis from a Frozen Stock
  • Basic Protocol 2: Growth of B. thailandensis in Liquid Medium
  • Basic Protocol 3: Preparation of B. thailandensis Frozen Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Growth of B. thailandensis from a Frozen Stock

  Materials
  • B. thailandensis frozen stock (see protocol 3)
  • Low salt LB agar plate (see recipe), with antibiotics if necessary (see Table 4.1.1)
  • Wooden or plastic applicator stick or inoculating loop, sterile
  • 37°C incubator
Table 4.0.1   MaterialsAntibiotic Concentrations for Use with B. thailandensis

Antibiotic Solvent Stock concentration a Final concentration b
Chloramphenicol Ethanol 20 mg/ml 20 μg/ml
Glyphosate c N/A 50% (w/v) 0.04% (w/v)
Kanamycin Water 125 mg/ml 250 μg/ml
Tetracycline d Ethanol 10 mg/ml 50 μg/ml
Trimethoprim d DMSO 50 mg/ml 50 μg/ml

 aAll stock solutions should be filter‐sterilized (0.22‐μm filter) and stored at −20°C.
 bFinal working concentration in liquid medium or agar plates. Media should be cooled to 55°C before addition of antibiotics.
 cGlyphosate is a nonantibiotic herbicide optimized for use with B. pseudomallei, B. mallei, and B. thailandensis. It is only compatible for use with minimal medium and can be purchased as a 50% (w/v) solution from hardware stores (sold as Roundup; Norris et al., ).
 dLight‐sensitive. Antibiotic stock solution and prepared media should be protected from light.

Basic Protocol 2: Growth of B. thailandensis in Liquid Medium

  Materials
  • Low salt LB medium (see recipe) or M63 minimal medium (see recipe)
  • Selective antibiotics (see Table 4.1.1)
  • B. thailandensis grown on agar plates (see protocol 1)
  • Capped test tubes, sterile (glass or plastic)
  • Sterile wooden or plastic applicator stick or heat‐sterilized inoculating loop
  • 37°C incubator with shaker or drum roller

Basic Protocol 3: Preparation of B. thailandensis Frozen Stocks

  Materials
  • 20% (v/v) glycerol LB medium (see recipe)
  • B. thailandensis grown on agar plates (see protocol 1)
  • Screw‐cap cryovials
  • Sterile wooden or plastic applicator stick
  • Vortex
  • −80°C freezer
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

  Anderson, M.S., Garcia, E.C., and Cotter, P.A. 2012. The Burkholderia bcpAIOB genes define unique classes of Two‐Partner secretion and contact dependent growth inhibition systems. PLoS Genet. 8:e1002877. doi: 10.1371/journal.pgen.1002877.
  Anderson, M.S., Garcia, E.C., and Cotter, P.A. 2014. Kind discrimination and competitive exclusion mediated by contact‐dependent growth inhibition systems shape biofilm community structure. PLoS Path. 10:e1004076. doi: 10.1371/journal.ppat.1004076.
  Biot, F.V., Valade, E., Garnotel, E., Chevalier, J., Villard, C., Thibault, F.M., Vidal, D.R., and Pagès, J.‐M. 2011. Involvement of the efflux pumps in chloramphenicol selected strains of Burkholderia thailandensis: Proteomic and mechanistic evidence. PLoS ONE 6:e16892. doi: 10.1371/journal.pone.0016892.
  Brett, P.J., Deshazer, D., and Woods, D.E. 1997. Characterization of Burkholderia pseudomallei and Burkholderia pseudomallei‐like strains. Epidemiol. Infect. 118:137‐148. doi: 10.1017/S095026889600739X.
  Brett, P.J., Deshazer, D., and Woods, D.E. 1998. Burkholderia thailandensis sp. nov., a Burkholderia pseudomallei‐like species. Int. J. Sys. Bacteriol. 48:317‐320. doi: 10.1099/00207713‐48‐1‐317.
  Chandler, J.R., Duerkop, B.A., Hinz, A., West, T.E., Herman, J.P., Churchill, M.E.A., Skerrett, S.J., and Greenberg, E.P. 2009. Mutational analysis of Burkholderia thailandensis quorum sensing and self‐aggregation. J. Bacteriol. 191:5901‐5909. doi: 10.1128/JB.00591‐09.
  Fisher, N.A., Ribot, W.J., Applefeld, W., and DeShazer, D. 2012. The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis. BMC Microbiol. 12:117. doi: 10.1186/1471‐2180‐12‐117.
  French, C.T., Toesca, I., Wu, T.‐H., Teslaa, T., Beaty, S. M., Wong, W., Liu, M., Chiou, P.‐Y., Teitell, M.A., and Miller, J.F. 2011. Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade. Proc. Natl. Acad. Sci. U.S.A. 108:10295‐12100. doi: 10.1073/pnas.1107183108.
  Gee, J.E., Glass, M.B., Novak, R.T., Gal, D., Mayo, M.J., Steigerwalt, A.G., Wilkins, P.P., and Currie, B.J. 2008. Recovery of a Burkholderia thailandensis‐like isolate from an Australian water source. BMC Microbiol. 8:1. doi: 10.1186/1471‐2180‐8‐54.
  Ginther, J.L., Mayo, M., Warrington, S. D., Kaestli, M., Mullins, T., Wagner, D.M., Currie, B.J., Tuanyok, A., and Keim, P. 2015. Identification of Burkholderia pseudomallei near‐neighbor species in the northern territory of Australia. PLoS Negl. Trop. Dis. 9:e0003892. doi: 10.1371/journal.pntd.0003892.
  Glass, M.B., Gee, J.E., Steigerwalt, A.G., Cavuoti, D., Barton, T., Hardy, R.D., Godoy, D., Spratt, B.G., Clark, T.A., and Wilkins, P.P. 2006. Pneumonia and septicemia caused by Burkholderia thailandensis in the United States. J. Clin. Microbiol. 44:4601‐4604. doi: 10.1128/JCM.01585‐06.
  Haraga, A., West, T.E., Brittnacher, M.J., Skerrett, S.J., and Miller, S.I. 2008. Burkholderia thailandensis as a model system for the study of the virulence‐associated type III secretion system of Burkholderia pseudomallei. Infect. Immun. 76:5402‐5411. doi: 10.1128/IAI.00626‐08.
  Johnson, S.L., Bishop‐Lilly, K.A., Ladner, J.T., Daligault, H.E., Davenport, K.W., Jaissle, J., Frey, K.G., Koroleva, G.I., Bruce, D.C., Coyne, S.R., Broomall, S.M., Li, P.E., Teshima, H., Gibbons, H.S., Palacios, G.F., Rosenzweig, C.N., Redden, C.L., Xu, Y., Minogue, T.D., and Chain, P.S. 2015. Complete genome sequences for 59 burkholderia isolates, both pathogenic and near neighbor. Genome Annouc. 3:e00159‐15. doi: 10.1128/genomeA.00159‐15.
  Kespichayawattana, W., Rattanachetkul, S., Wanun, T., Utaisincharoen, P., and Sirisinha, S. 2000. Burkholderia pseudomallei induces cell fusion and actin‐associated membrane protrusion: A possible mechanism for cell‐to‐cell spreading. Infect. Immun. 68:5377‐5384. doi: 10.1128/IAI.68.9.5377‐5384.2000.
  Nikolakakis, K., Amber, S., Wilbur, J.S., Diner, E.J., Aoki, S.K., Poole, S.J., Tuanyok, A., Keim, P.S., Peacock, S., Hayes, C.S., and Low, D.A. 2012. The toxin/immunity network of Burkholderia pseudomallei contact‐dependent growth inhibition (CDI) systems. Mol. Microbiol. 84:516‐529. doi: 10.1111/j.1365‐2958.2012.08039.x.
  Norris, M.H., Kang, Y., Lu, D., Wilcox, B.A., and Hoang, T.T. 2009. Glyphosate resistance as a novel select‐agent‐compliant, non‐antibiotic‐selectable marker in chromosomal mutagenesis of the essential genes asd and dapB of Burkholderia pseudomallei. Appl. Environ. Microbiol. 75:6062‐6075. doi: 10.1128/AEM.00820‐09.
  Russell, A.B., Singh, P., Brittnacher, M., Bui, N.K., Hood, R.D., Carl, M.A., Agnello, D.M., Schwarz, S., Goodlett, D.R., Vollmer, W., and Mougous, J.D. 2012. A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 11:538‐549. doi: 10.1016/j.chom.2012.04.007.
  Schwarz, S., West, T.E., Boyer, F., Chiang, W.‐C., Carl, M.A., Hood, R.D., Rohmer, L., Tolker‐Nielsen, T., Skerrett, S.J., and Mougous, J. D. 2010. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Path 6:e1001068. doi: 10.1371/journal.ppat.1001068.
  Schweizer, H.P. 2012. Mechanisms of antibiotic resistance in Burkholderia pseudomallei: Implications for treatment of melioidosis. Future Microbiol. 7:1389‐1399. doi: 10.2217/fmb.12.116.
  Sim, B.M.Q., Chantratita, N., Ooi, W.F., Nandi, T., Tewhey, R., Wuthiekanun, V., Thaipadungpanit, J., Tumapa, S., Ariyaratne, P., Sung, W.‐K., Sem, X.H., Chua, H.H., Ramnarayanan, K., Lin, C.H., Liu, Y., Feil, E.J., Glass, M.B., Tan, G., Peacock, S.J., and Tan, P. 2010. Genomic acquisition of a capsular polysaccharide virulence cluster by non‐pathogenic Burkholderia isolates. Genome. Biol. 11:R89. doi: 10.1186/gb‐2010‐11‐8‐r89.
  Smith, M.D., Angus, B.J., Wuthiekanun, V., and White, N.J. 1997. Arabinose assimilation defines a nonvirulent biotype of Burkholderia pseudomallei. Infect. Immun. 65:4319‐4321.
  Stevens, J.M., Ulrich, R.L., Taylor, L. A., Wood, M.W., Deshazer, D., Stevens, M.P., and Galyov, E.E. 2005. Actin‐binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin‐based motility defect of a Burkholderia pseudomallei bimA mutant. J. Bacteriol. 187:7857‐7862. doi: 10.1128/JB.187.22.7857‐7862.2005.
  Thongdee, M., Gallagher, L.A., Schell, M., Dharakul, T., Songsivilai, S., and Manoil, C. 2008. Targeted mutagenesis of Burkholderia thailandensis and Burkholderia pseudomallei through natural transformation of PCR fragments. Appl. Environ. Microbiol. 74:2985‐2989. doi: 10.1128/AEM.00030‐08.
  Wiersinga, W.J., de Vos, A.F., de Beer, R., Wieland, C.W., Roelofs, J.J.T.H., Woods, D.E., and van der Poll, T. 2008. Inflammation patterns induced by different Burkholderia species in mice. Cell. Microbiol. 10:81‐87. doi: 10.1111/j.1462‐5822.2007.01016.x.
  Yu, Y., Kim, H.S., Chua, H.H., Lin, C.H., Sim, S.H., Lin, D., Derr, A., Engels, R., DeShazer, D., Birren, B., Nierman, W.C., and Tan, P. 2006. Genomic patterns of pathogen evolution revealed by comparison of Burkholderia pseudomallei, the causative agent of melioidosis, to avirulent Burkholderia thailandensis. BMC Microbiol. 6:46. doi: 10.1186/1471‐2180‐6‐46.
Internet Resources
  http://www.burkholderia.com
  The Burkholderia Genome Database website contains a comprehensive collection of sequences and annotations for completed genomes within the Burkholderia genus (including B. thailandensis), as well as comparative tools.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library