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    Dendrogram of pulsed-filed gel electrophoresis profiles for 21 Burkholderia mallei isolates generated by the Dice method and clustering by unweighted pair group method with arithmetic mean. The scale bar represents the percentage similarity range.

  • 1

    Lopez J, Copps J, Wilhelmsen C, Moore R, Kubay J, St-Jacques M, Halayko S, Kranendonk C, Toback S, DeShazer D, Fritz DL, Tom M, Woods DE, 2003. Characterization of experimental equine glanders. Microbes Infect 5 :1125–1131.

    • Search Google Scholar
    • Export Citation
  • 2

    Godoy D, Randle G, Simpson AJ, Aanensen DM, Pitt TL, Kinoshita R, Spratt BG, 2003. Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei. J Clin Microbiol 41 :2068–2079.

    • Search Google Scholar
    • Export Citation
  • 3

    Harvey SP, Minter JM, 2005. Ribotyping of Burkholderia mallei isolates. FEMS Immunol Med Microbiol 44 :91–97.

  • 4

    Koonpaew S, Ubol MN, Sirisinha S, White NJ, Chaiyaroj SC, 2000. Genome fingerprinting by pulsed-field gel electrophoresis of isolates of Burkholderia pseudomallei from patients with melioidosis in Thailand. Acta Trop 74 :187–191.

    • Search Google Scholar
    • Export Citation
  • 5

    Holden MT, Titball RW, Peacock SJ, Cerdeno-Tarraga AM, Atkins T, Crossman LC, Pitt T, Churcher C, Mungall K, Bentley SD, Sebaihia M, Thomson NR, Bason N, Beacham IR, Brooks K, Brown KA, Brown NF, Challis GL, Cherevach I, Chillingworth T, Cronin A, Crossett B, Davis P, DeShazer D, Feltwell T, Fraser A, Hance Z, Hauser H, Holroyd S, Jagels K, Keith KE, Maddison M, Moule S, Price C, Quail MA, Rabbinowitsch E, Rutherford K, Sanders M, Simmonds M, Songsivilai S, Stevens K, Tumapa S, Vesaratchavest M, Whitehead S, Yeats C, Barrell BG, Oyston PC, Parkhill J, 2004. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci U S A 101 :14240–14245.

    • Search Google Scholar
    • Export Citation
  • 6

    Nierman WC, DeShazer D, Kim HS, Tettelin H, Nelson KE, Feldblyum T, Ulrich RL, Ronning CM, Brinkac LM, Daugherty SC, Davidsen TD, Deboy RT, Dimitrov G, Dodson RJ, Durkin AS, Gwinn ML, Haft DH, Khouri H, Kolonay JF, Madupu R, Mohammoud Y, Nelson WC, Radune D, Romero CM, Sarria S, Selengut J, Shamblin C, Sullivan SA, White O, Yu Y, Zafar N, Zhou L, Fraser CM, 2004. Structural flexibility in the Burkholderia mallei genome. Proc Natl Acad Sci U S A 101 :14246–14251.

    • Search Google Scholar
    • Export Citation
 
 
 
 

 

 

 

 

 

 

 

 

 

PULSED-FIELD GEL ELECTROPHORESIS AS A DISCRIMINATORY TYPING TECHNIQUE FOR THE BIOTHREAT AGENT BURKHOLDERIA MALLEI

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  • 1 Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Biocombinat, State-Owned Biological Enterprise, Ulaanbaatar, Mongolia; Tuberculosis Laboratory, Central Veterinary Control and Research Institute, Ankara, Turkey; Center for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical; Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom

Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) was used to type 21 laboratory strains of Burkholderia mallei. We demonstrated good resolution by PFGE together with clustering of some geographically related isolates, and confirmed previous observations that B. mallei is clonal as defined by MLST.

Burkholderia mallei is the causative agent of glanders.1 Historically, this pathogen was an important cause of morbidity and mortality in horses worldwide, but natural disease is now extremely rare in any host. The ecologic niche of this bacterium is thought to be the nasal passages of horses, and its demise is probably related to the decrease in equine numbers worldwide combined with an inability to survive in the environment. Established national strain collections maintain a limited number of isolates, most of which were originally isolated between 1930 and 1960. Burkholderia mallei is also a recognized biothreat agent, and is listed as a category B disease/agent by the U.S. Centers for Disease Control and Prevention. The organism could be spread by aerosol, has the potential to cause serious disease with a high mortality rate, and is intrinsically resistant to many antibiotics. In addition, no vaccine is available. Pre-emptive evaluation of readily available typing techniques would provide valuable information towards strategic planning of forensic investigation in the event of a deliberate release.

Typing of B. mallei has received little attention to date. Five B. mallei isolates were included in a description of the development of a mutlilocus sequence typing (MLST) scheme for B. pseudomallei, in which all five were reported to belong to a single clone (sequence type ST 40).2 A recent study reported that 17 distinct ribotypes were resolved for 25 isolates of B. mallei using two separate restriction enzymes.3 Bacterial typing using pulsed-field gel electrophoresis (PFGE) is readily and widely available, and is a straightforward technique that could be used at short notice. Typing by PFGE of the closely related B. pseudomallei is well described and has high resolution.2,4 The aim of this study was to examine 21 laboratory isolates of B mallei by PFGE and to define the utility of this technique for forensic investigation. In addition, these isolates were examined by MLST to extend the investigation of sequence types of this species.

The isolates examined are listed in Table 1. For PFGE, a single bacterial colony was streaked onto Columbia agar and incubated for 48 hour at 37°C in air. Colonies were harvested and suspended to an optical density at 600 nm of 1.2 in suspension buffer (75 mM sodium chloride, 25 mM EDTA, pH 7.5). This was mixed (1:1) with molten 2% low-melting point ultrapure agarose (Gibco-BRL, Gaithersburg, MD) and pi-petted into PFGE plug molds (Bio-Rad Laboratories, Hercules, CA). Plugs were lysed overnight at 56°C in lysis buffer (0.1% sodium dodecyl sulfate, 25 mM EDTA, pH 8.0) containing 500 μg/ml of proteinase K (Invitrogen, Carlsbad, CA) and rinsed three times with TE buffer (10 mM Tris, 10 mM EDTA). Prior to PFGE, plugs were digested overnight with 10 units of Spe I (New England Biolabs, Beverly, MA) at 37°C for 18 hours before being loaded onto a 1% agarose gel (Gibco-BRL) in 0.5× TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA). Each well was overlaid with 0.8% low-melting agarose. The PFGE was conducted on a CHEF-DRIII system (Bio-Rad Laboratories) for 44 hours at 14°C and 6 V/cm using the following parameters: initial switch time, final switch time, and run time; block I: 10–60 V for 18 hours, block II: 30–40 V for 18 hours, and block III: 50–90 V for 8 hours. Lambda concatamers were run as the standard (Promega, Madison, WI). Gels were stained with ethidium bromide, destained in water, and photographed under ultraviolet light using the Gel Doc 1000 system (Bio-Rad Laboratories). Analysis of PFGE banding patterns was conducted using the BioNumerics version 2.5 software (Applied Maths, Kortrijik, Belgium). The PFGE types were defined on the basis of DNA banding pattern. Isolates were regarded as genotypically indistinguishable if they had identical patterns, related if they differed by one to three bands, and unrelated if they differed by four or more bands. A similarity matrix was produced using the Dice coefficient and a dendrogram was generated by clustering by the unweighted pair group method with arithmetic mean with 1% band and position tolerance. Isolates with > 80% similarity were considered related.

For MLST, bacterial isolates were streaked from the freezer vial onto Columbia agar. A single colony was inoculated into tryptic soy broth and incubated overnight in air at 37°C, after which genomic DNA was extracted using the Wizard Genomic DNA purification kit (Promega). Typing was performed using the primers and conditions previously described,1 with the exception that gmhD was amplified using a nested approach with the following primers: outer primer pair: gmdD-up (5′-TCGCGCAGGGCACGCAGTT-3′) and gmhD-dn (5′-GGCTGCCGACCGTGAGACC-3′); inner primer pair gmhD-up (5′-TCGCGCAGGGCACGCAGTT-3′) and gmhD-dn (5′-GTCAGGAACGGCGCGTCGTAGC-3′). Alleles at each of the seven loci were assigned and the allelic profile (string of seven integers) was used to define sequence type (ST) using the B. pseudomallei MLST website (http://bpseudomallei.mlst.net/).

All 21 isolates were a single clone (ST40) by MLST. Analysis by PFGE yielded 13 separate types based on comparison of banding patterns. Six PFGE types contained more than one isolate, but an identical banding pattern within a given type occurred on only two occasions (T2/T3 and EY2233/EY2236). Source of origin was associated with related banding pattern in some instances, such as M1 and M2 from Mongolia that differed by a single band, and T1, T2, and T3 from Turkey. Clustering according to country of origin was not seen for the NCTC isolates. For example, NCTC 10248, NCTC 10247, and NCTC 10260 were all from Turkey but were not related by PFGE. The dendrogram is shown in Figure 1.

Burkholderia mallei is believed to have originated from B. pseudomallei.2,5,6 The lack of diversity in B. mallei MLST housekeeping genes suggests that this evolutionary divergence occurred relatively recently. The diversity in PFGE patterns for B. mallei are in marked contrast with the MLST findings, and indicates a more rapid process of genomic change. This may be related to the finding that B. mallei contains numerous insertion sequence elements that have mediated extensive deletions and rearrangements of the genome relative to B. pseudomallei.6

Our findings indicate that PFGE is a highly discriminatory typing tool for B. mallei. An initiative to assemble all known B. mallei and develop a database of PFGE banding pattern types may be an invaluable forensic resource in the event of a future outbreak of animal or human disease.

Table 1

Burkholderia mallei strains used in this study

Isolate name*Place of originDateSource
* EY isolates were kindly provided by Dr. Sumalee Tangpradubkul, (Mahidol University, Bangkok, Thailand).
Mongolia 1Mongolia1960sHorse
Mongolia 2Mongolia1960sHorse
T1 (NH insan)Turkey1970sHuman
T2 (Beygir CAU)Turkey1970sHorse
T3 (Uludag)Turkey1984Human
NCTC 3708India1931Mule lung
NCTC 3709India1932Horse
NCTC 10245China1942Horse lung and nose
NCTC 10260Ankara, Turkey1949Human
NCTC 10248Ankara, Turkey1950Human
NCTC 10247Ankara, Turkey1960Unknown
NCTC 10229Pecs, Hungary1961Unknown
NCTC 10230Pecs, Hungary1961Unknown
ATCC 23344China1942Lethal human infection
EY 100Horse
EY 2233China1942Human
EY 2235China1942Horse
EY 2236ChinaHorse
EY 2237ChinaHorse
EY 2238China1944Human
EY 2239China1951
Figure 1.
Figure 1.

Dendrogram of pulsed-filed gel electrophoresis profiles for 21 Burkholderia mallei isolates generated by the Dice method and clustering by unweighted pair group method with arithmetic mean. The scale bar represents the percentage similarity range.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 3; 10.4269/ajtmh.2006.74.345

*

Address correspondence to Sharon J. Peacock, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. E-mail: sharon@tropmedres.ac

Authors’ addresses: Narisara Chantratita, Mongkol Vesaratchavest, Vanaporn Wuthiekanun, Rachaneeporn Tiyawisutsri, Nicholas P. J. Day, and Sharon J. Peacock, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand, Telephone: 66-2-354-9172, Fax: 66-2-354-9169, E-mail: sharon@tropmedres.ac. Tsedev Ulziitogtokh, Biocombinat, State-Owned Biological Enterprise, Ulaanbaatar, Mongolia, E-mail: bio_info@mbox.mn. Erhan Akcay, Tuberculosis Laboratory, Central Veterinary Control and Research Institute, 06020 Etlik Ankara, Turkey, Telephone: 90-312-326-0090, Fax: 90-312-321-1755, E-mail: ehh.o@tr.net and etlik@vet.gov.tr.

Acknowledgments: We thank the support of staff at the Wellcome Trust-Mahidol University-Oxford University Tropical Medicine Research Program for their assistance, and Professor Don Woods (University Calgary, Calgary, Alberta, Canada) for providing bacterial strains.

Financial support: This study was supported by the Wellcome Trust. Sharon J. Peacock is supported by a Wellcome Trust Career Development Award in Clinical Tropical Medicine.

REFERENCES

  • 1

    Lopez J, Copps J, Wilhelmsen C, Moore R, Kubay J, St-Jacques M, Halayko S, Kranendonk C, Toback S, DeShazer D, Fritz DL, Tom M, Woods DE, 2003. Characterization of experimental equine glanders. Microbes Infect 5 :1125–1131.

    • Search Google Scholar
    • Export Citation
  • 2

    Godoy D, Randle G, Simpson AJ, Aanensen DM, Pitt TL, Kinoshita R, Spratt BG, 2003. Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei. J Clin Microbiol 41 :2068–2079.

    • Search Google Scholar
    • Export Citation
  • 3

    Harvey SP, Minter JM, 2005. Ribotyping of Burkholderia mallei isolates. FEMS Immunol Med Microbiol 44 :91–97.

  • 4

    Koonpaew S, Ubol MN, Sirisinha S, White NJ, Chaiyaroj SC, 2000. Genome fingerprinting by pulsed-field gel electrophoresis of isolates of Burkholderia pseudomallei from patients with melioidosis in Thailand. Acta Trop 74 :187–191.

    • Search Google Scholar
    • Export Citation
  • 5

    Holden MT, Titball RW, Peacock SJ, Cerdeno-Tarraga AM, Atkins T, Crossman LC, Pitt T, Churcher C, Mungall K, Bentley SD, Sebaihia M, Thomson NR, Bason N, Beacham IR, Brooks K, Brown KA, Brown NF, Challis GL, Cherevach I, Chillingworth T, Cronin A, Crossett B, Davis P, DeShazer D, Feltwell T, Fraser A, Hance Z, Hauser H, Holroyd S, Jagels K, Keith KE, Maddison M, Moule S, Price C, Quail MA, Rabbinowitsch E, Rutherford K, Sanders M, Simmonds M, Songsivilai S, Stevens K, Tumapa S, Vesaratchavest M, Whitehead S, Yeats C, Barrell BG, Oyston PC, Parkhill J, 2004. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci U S A 101 :14240–14245.

    • Search Google Scholar
    • Export Citation
  • 6

    Nierman WC, DeShazer D, Kim HS, Tettelin H, Nelson KE, Feldblyum T, Ulrich RL, Ronning CM, Brinkac LM, Daugherty SC, Davidsen TD, Deboy RT, Dimitrov G, Dodson RJ, Durkin AS, Gwinn ML, Haft DH, Khouri H, Kolonay JF, Madupu R, Mohammoud Y, Nelson WC, Radune D, Romero CM, Sarria S, Selengut J, Shamblin C, Sullivan SA, White O, Yu Y, Zafar N, Zhou L, Fraser CM, 2004. Structural flexibility in the Burkholderia mallei genome. Proc Natl Acad Sci U S A 101 :14246–14251.

    • Search Google Scholar
    • Export Citation
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