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Detection and differentiation of Burkholderia species with pathogenic potential in environmental soil samples

Sujintana Janesomboon1, Veerachat Muangsombut1, Varintip Srinon2

  • 1Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.

Plos One
|January 7, 2021
PubMed

Insights

A new multiplex PCR assay accurately detects pathogenic Burkholderia species, including B. pseudomallei and its variants, in soil. This sensitive method aids in the epidemiological study of melioidosis and related diseases.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Epidemiology

Background:

  • The Burkholderia pseudomallei phylogenetic cluster contains pathogenic species (B. pseudomallei, B. mallei) and non-pathogenic relatives.
  • Pathogenic strains and specific variants (BPBM, BTCV) pose significant public health concerns, necessitating accurate detection methods.
  • Current detection methods may lack the sensitivity or specificity required for comprehensive epidemiological studies in environmental samples.

Purpose of the Study:

  • To develop and validate a novel multiplex PCR assay for the rapid and accurate detection of pathogenic Burkholderia species and their variants.
  • To differentiate between pathogenic and non-pathogenic Burkholderia strains, including specific variants like BPBM and BTCV.
  • To assess the assay's sensitivity, specificity, and utility in environmental soil samples for epidemiological surveillance.

Main Methods:

  • Design of five PCR primers targeting species-specific sequences within the bimA gene.
  • Development of a multiplex PCR assay to amplify and differentiate target Burkholderia species and variants.
  • Specificity testing against a panel of related and unrelated bacterial species.
  • Determination of the assay's limit of detection (LOD) in spiked soil samples.
  • Comparative analysis of the multiplex PCR assay against standard bacterial culture for B. pseudomallei detection in environmental samples.

Main Results:

  • The multiplex PCR assay successfully distinguished pathogenic B. pseudomallei and BPBM from non-pathogenic B. thailandensis and BTCV strains.
  • A singleplex PCR variant effectively differentiated BTCV from B. thailandensis.
  • The assay demonstrated high specificity, with negative results against a broad range of other bacteria, excluding B. humptydooensis.
  • The detection limits in spiked soil were as low as 5 CFU/g for B. pseudomallei and 6 CFU/g for B. thailandensis.
  • The multiplex PCR showed high sensitivity and specificity when compared to standard culture methods for detecting B. pseudomallei in soil samples from a melioidosis-endemic area.

Conclusions:

  • A robust, sensitive, and specific multiplex PCR assay has been developed for the detection of pathogenic Burkholderia species and their variants.
  • This assay is a valuable tool for epidemiological studies, enabling accurate identification of B. pseudomallei and related pathogenic species in environmental matrices like soil.
  • The developed PCR method can significantly contribute to understanding the distribution and transmission dynamics of melioidosis and glanders-causing agents.

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