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    eBURST population snapshot of sequence types (STs) of Burkholderia pseudomallei isolates from India, Southeast Asia, China, and Africa. Red circles indicate B. pseudomallei isolates of this study with STs labeled, blue squares are SLVs to STs 858 and 859 with the letters denoting the country of origin (“T” Thailand, “C” China, and “K” Kenya). The green triangle is the only other isolate of Indian origin currently listed in the MLST database and was isolated in 1995. The black star indicates Burkholderia mallei isolates. This diagram contains 465 isolates with 409 STs.

  • 1.

    White NJ, 2003. Melioidosis. Lancet 361: 17151722.

  • 2.

    Currie BJ, Dance DA, Cheng AC, 2008. The global distribution of Burkholderia pseudomallei and melioidosis: an update. Trans R Soc Trop Med Hyg 102 (Suppl 1): S1S4.

    • Search Google Scholar
    • Export Citation
  • 3.

    Cheng AC, Currie BJ, 2005. Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev 18: 383416.

  • 4.

    Pearson T, Giffard P, Beckstrom-Sternberg S, Auerbach R, Hornstra H, Tuanyok A, Price EP, Glass MB, Leadem B, Beckstrom-Sternberg JS, Allan GJ, Foster JT, Wagner DM, Okinaka RT, Sim SH, Pearson O, Wu Z, Chang J, Kaul R, Hoffmaster AR, Brettin TS, Robison RA, Mayo M, Gee JE, Tan P, Currie BJ, Keim P, 2009. Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer. BMC Biol 7: 78.

    • Search Google Scholar
    • Export Citation
  • 5.

    Ives JC, Thomson TJ, 1953. Chronic melioidosis: the first report of a case infected in Central India. Glasg Med J 34: 6167.

  • 6.

    Kang G, Rajan DP, Ramakrishna BS, Aucken HM, Dance DA, 1996. Melioidosis in India. Lancet 347: 15651566.

  • 7.

    Bharadwaj R, Kagal A, Deshpandey SK, Joshi SA, Khare PM, Junnarkar AR, Phadke MA, 1994. Outbreak of plague-like illness caused by Pseudomonas pseudomallei in Maharashtra, India. Lancet 344: 1574.

    • Search Google Scholar
    • Export Citation
  • 8.

    Novak RT, Glass MB, Gee JE, Gal D, Mayo MJ, Currie BJ, Wilkins PP, 2006. Development and evaluation of a real-time PCR assay targeting the type III secretion system of Burkholderia pseudomallei. J Clin Microbiol 44: 8590.

    • Search Google Scholar
    • Export Citation
  • 9.

    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: 20682079.

    • Search Google Scholar
    • Export Citation
  • 10.

    Currie BJ, Haslem A, Pearson T, Hornstra H, Leadem B, Mayo M, Gal D, Ward L, Godoy D, Spratt BG, Keim P, 2009. Identification of melioidosis outbreak by multilocus variable number tandem repeat analysis. Emerg Infect Dis 15: 169174.

    • Search Google Scholar
    • Export Citation
  • 11.

    Tuanyok A, Auerbach RK, Brettin TS, Bruce DC, Munk AC, Detter JC, Pearson T, Hornstra H, Sermswan RW, Wuthiekanun V, Peacock SJ, Currie BJ, Keim P, Wagner DM, 2007. A horizontal gene transfer event defines two distinct groups within Burkholderia pseudomallei that have dissimilar geographic distributions. J Bacteriol 189: 90449049.

    • Search Google Scholar
    • Export Citation
  • 12.

    Sitthidet C, Stevens JM, Chantratita N, Currie BJ, Peacock SJ, Korbsrisate S, Stevens MP, 2008. Prevalence and sequence diversity of a factor required for actin-based motility in natural populations of Burkholderia species. J Clin Microbiol 46: 24182422.

    • Search Google Scholar
    • Export Citation
 
 
 
 

 

 
 
 

 

 

 

 

 

 

Molecular Characterization of Clinical Burkholderia pseudomallei Isolates from India

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  • Department of Microbiology, Kasturba Medical College, Manipal, Manipal University, Karnataka, India; Menzies School of Health Research, Charles Darwin University, Darwin, Australia; Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona; Department of Infectious Diseases Epidemiology, Imperial College London, United Kingdom

Multilocus sequence typing of seven isolates of Burkholderia pseudomallei from India showed considerable diversity, with six different sequence types. Possible dissemination of melioidosis by historical trading routes is supported by links to strains from Southeast Asia, China, and Africa and the presence of the Burkholderia mallei allele of the bimA gene.

Burkholderia pseudomallei is a soil saprophyte and the causative agent of melioidosis, an infectious disease that, while predominantly restricted to Southeast Asia and northern Australia, is increasingly being recognized in other tropical and subtropical regions.1,2 In areas where the disease is endemic, the organism is commonly found in soil and surface water. Infections occur mostly by inoculation of the organism through puncture wounds in the skin and people with risk factors for melioidosis such as diabetes are especially susceptible to severe disease such as pneumonia and systemic sepsis.3 Recent phylogenetic analysis of a global set of B. pseudomallei strains has suggested an Australian origin for B. pseudomallei, with subsequent spread to Southeast Asia and beyond.4 Since 1953 melioidosis has been sporadically reported from India,5,6 with one notable cluster initially attributed to plague.7 Nevertheless, the population structure of Indian and other Asian strains of B. pseudomallei and their origins and relationships to strains from Southeast Asia and Australia remain undefined. The aim of this study was to analyze the molecular diversity of clinical B. pseudomallei strains from one location in southern India.

Seven clinical isolates of B. pseudomallei were collected from seven melioidosis patients admitted to Kasturba Hospital, Manipal, Karnataka State in southwest India between 2007 and 2009. Isolate 7 was obtained from an Austrian tourist with septicemic pneumonia, who was holidaying in India. Five patients had pulmonary involvement with septicemia and two had soft tissue infections (see Table 1). Three patients were diabetic and two had no identified risk factor for melioidosis. Ceftazidime was the most common antibiotic used. Two patients died and two were lost to follow-up.

Table 1

Summary of patient information and molecular results of Burkholderia pseudomallei isolates

Isolate no.Patient risk factorsDisease presentation (outcome)STSLVs (country of origin of SLVs*)YLF/ BTFCBps/Bm allele of bimA
1Diabetes, chronic renal diseaseSepticemic pneumonia (lost to follow-up)854NoneYLFBm
2Chronic obstructive lung diseaseSepticemic pneumonia (died)855NoneYLFBps
3DiabetesSubmandibular abscess (survived)NaNaYLFBps
4DiabetesSepticemic pneumonia (survived)856NoneYLFBm
5NilSepticemic pneumonia (died)857NoneYLFBps
6NilSubmandibular abscess (survived)858ST 859 (I, T, C, K)YLFBps
7Acute myeloid leukemiaSepticemic pneumonia (lost to follow-up)859ST 858 (I)YLFBps

Indian sequence types (STs) are listed. C = China, I = India, K = Kenya, T = Thailand.

Bps = B. pseudomallei; Bm = B. mallei.

Na = not available. Isolate 2 and 3 had the same MLV-4 pattern, which suggests that they also had the same ST.10

Genomic DNA was obtained from culture isolates using phenol-chloroform extraction and further purified by QIAamp DNA minikit (Qiagen, Victoria, Australia). Real-time polymerase chain reaction was used to target TTS1 for species confirmation.8 Molecular diversity was assessed by multilocus sequence typing (MLST)9 and multilocus VNTR (variable number tandem repeat) analysis (MLVA-4).10 A genetic marker linked to geographic origin (Yersinia-like fimbriae [YLF]/B. thailandensis-like flagellum and chemotaxis [BTFC] locus) was amplified11 and the B. pseudomallei- and Burkholderia mallei-type allele of the actin-based motility gene bimA was targeted as a potential virulence factor.12

Details of molecular results are shown in Table 1 and Figure 1. The MLST analysis resulted in six different sequence types (STs) from six B. pseudomallei isolates. All six were novel STs not previously listed on the global B. pseudomallei MLST database (http://bpseudomallei.mlst.net/). New ST numbers were assigned and comparisons were made with the global dataset. Two of the Indian STs were single locus variants (SLVs) (differing at one MLST locus) to each other and to isolates from Thailand, Kenya, and China. No MLST data were obtained from isolate 3 because of technical reasons, but isolates 2 and 3 shared the same MLVA-4 pattern suggesting an identical ST for these isolates.10 Although such an identical MLV-4 type indicates a likely geographical or epidemiological link, no such link was found, with these two patients from locations around 50 km apart. All other isolates showed different MLVA-4 patterns. All seven B. pseudomallei isolates contained the YLF marker, which is predominant in Southeast Asia11 and two of the isolates amplified the B. mallei allele of bimA, with the other five having the B. pseudomallei allele.

Figure 1.
Figure 1.

eBURST population snapshot of sequence types (STs) of Burkholderia pseudomallei isolates from India, Southeast Asia, China, and Africa. Red circles indicate B. pseudomallei isolates of this study with STs labeled, blue squares are SLVs to STs 858 and 859 with the letters denoting the country of origin (“T” Thailand, “C” China, and “K” Kenya). The green triangle is the only other isolate of Indian origin currently listed in the MLST database and was isolated in 1995. The black star indicates Burkholderia mallei isolates. This diagram contains 465 isolates with 409 STs.

Citation: The American Society of Tropical Medicine and Hygiene 85, 1; 10.4269/ajtmh.2011.11-0166

To date, only one other B. pseudomallei strain of Indian origin is listed on the MLST database. Although a small sample set, our MLST results indicate considerable diversity among B. pseudomallei collected over a small spatial and temporal range in southern India, with 6 of 7 having different STs. This suggests a non-recent introduction of B. pseudomallei into this area. The eBURST analysis clustered the Indian isolates with Asian B. pseudomallei strains and this was also supported by presence of the YLF geographical marker. The fact that some of the Indian B. pseudomallei isolates were SLVs to isolates from China and Kenya, supports the possibility of spread of B. pseudomallei along historical trading routes. BimA is involved in actin polymerization, which is crucial for intracellular motility and intercellular spread and is thus a potential virulence factor.12 Both patients with the B. mallei allele of bimA presented with septicemic pneumonia. While the B. mallei allele occurs in some Australian B. pseudomallei strains, Sitthidet and others12 found none of 64 strains from across Southeast Asia harboring this allele that is present in B. mallei, the causative bacterium of glanders. To our knowledge the two Indian B. pseudomallei strains noted here to have the B. mallei-type bimA allele are the first B. pseudomallei isolates of Asian origin reported to contain this allele. Burkholderia mallei is found in India, with sporadic reports of glanders in equines and it is recognized that B. mallei evolved from an ancestral B. pseudomallei strain.9 Of interest, B. mallei is a double locus variant of Indian isolate 6 (ST 858).

In conclusion, this is the first report on the molecular characterization of B. pseudomallei isolates from southern India. These isolates were found to be diverse at MLST level and an Asian origin is likely. More work is needed to uncover the population structure of B. pseudomallei in India and potential links to historical trading routes.

  • 1.

    White NJ, 2003. Melioidosis. Lancet 361: 17151722.

  • 2.

    Currie BJ, Dance DA, Cheng AC, 2008. The global distribution of Burkholderia pseudomallei and melioidosis: an update. Trans R Soc Trop Med Hyg 102 (Suppl 1): S1S4.

    • Search Google Scholar
    • Export Citation
  • 3.

    Cheng AC, Currie BJ, 2005. Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev 18: 383416.

  • 4.

    Pearson T, Giffard P, Beckstrom-Sternberg S, Auerbach R, Hornstra H, Tuanyok A, Price EP, Glass MB, Leadem B, Beckstrom-Sternberg JS, Allan GJ, Foster JT, Wagner DM, Okinaka RT, Sim SH, Pearson O, Wu Z, Chang J, Kaul R, Hoffmaster AR, Brettin TS, Robison RA, Mayo M, Gee JE, Tan P, Currie BJ, Keim P, 2009. Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer. BMC Biol 7: 78.

    • Search Google Scholar
    • Export Citation
  • 5.

    Ives JC, Thomson TJ, 1953. Chronic melioidosis: the first report of a case infected in Central India. Glasg Med J 34: 6167.

  • 6.

    Kang G, Rajan DP, Ramakrishna BS, Aucken HM, Dance DA, 1996. Melioidosis in India. Lancet 347: 15651566.

  • 7.

    Bharadwaj R, Kagal A, Deshpandey SK, Joshi SA, Khare PM, Junnarkar AR, Phadke MA, 1994. Outbreak of plague-like illness caused by Pseudomonas pseudomallei in Maharashtra, India. Lancet 344: 1574.

    • Search Google Scholar
    • Export Citation
  • 8.

    Novak RT, Glass MB, Gee JE, Gal D, Mayo MJ, Currie BJ, Wilkins PP, 2006. Development and evaluation of a real-time PCR assay targeting the type III secretion system of Burkholderia pseudomallei. J Clin Microbiol 44: 8590.

    • Search Google Scholar
    • Export Citation
  • 9.

    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: 20682079.

    • Search Google Scholar
    • Export Citation
  • 10.

    Currie BJ, Haslem A, Pearson T, Hornstra H, Leadem B, Mayo M, Gal D, Ward L, Godoy D, Spratt BG, Keim P, 2009. Identification of melioidosis outbreak by multilocus variable number tandem repeat analysis. Emerg Infect Dis 15: 169174.

    • Search Google Scholar
    • Export Citation
  • 11.

    Tuanyok A, Auerbach RK, Brettin TS, Bruce DC, Munk AC, Detter JC, Pearson T, Hornstra H, Sermswan RW, Wuthiekanun V, Peacock SJ, Currie BJ, Keim P, Wagner DM, 2007. A horizontal gene transfer event defines two distinct groups within Burkholderia pseudomallei that have dissimilar geographic distributions. J Bacteriol 189: 90449049.

    • Search Google Scholar
    • Export Citation
  • 12.

    Sitthidet C, Stevens JM, Chantratita N, Currie BJ, Peacock SJ, Korbsrisate S, Stevens MP, 2008. Prevalence and sequence diversity of a factor required for actin-based motility in natural populations of Burkholderia species. J Clin Microbiol 46: 24182422.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to Bart J. Currie, Menzies School of Health Research, PO Box 41096, Casuarina NT 0811, Australia. E-mail: bart@menzies.edu.au

Financial support: This work was supported by a project grant (no. 605820) from the Australian National Health and Medical Research Council, a project grant (UO1AI075568) from the National Institutes of Health, and a program grant (030662) from the Wellcome Trust.

Authors' addresses: Chiranjay Mukhopadhyay, Kalwaje Eshwara Vandana, and Krishna Sushma, Kasturba Medical College, Department of Microbiology, Kasturba Medical College, Manipal, Karnataka, India, E-mails: chiranjay.m@manipal.edu, vandanake@gmail.com, and chummu.dummu@gmail.com. Mirjam Kaestli, Mark Mayo, Leisha Richardson, and Bart J. Currie, Menzies School of Health Research, Tropical and Emerging Infectious Diseases, Menzies School of Health Research, Darwin, Northern Territory, Australia, E-mails: mirjam.kaestli@menzies.edu.au, mark.mayo@menzies.edu.au, leisha.richardson@menzies.edu.au, and bart@menzies.edu.au. Apichai Tuanyok and Paul Keim, Northern Arizona University, MG2 Centre, Northern Arizona University, Flagstaff, AZ, E-mails: Apichai.Tuanyok@nau.edu and Paul.Keim@nau.edu. Daniel Godoy and Brian G. Spratt, Imperial College London, School of Public Health, Imperial College London, London, UK, E-mails: d.godoy@imperial.ac.uk and b.spratt@imperial.ac.uk.

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