Prevalence and genetic diversity of Burkholderia pseudomallei isolates in the environment near a patient's residence in Northeast Thailand

Abstract

Background: Burkholderia pseudomallei is the causative agent of melioidosis, a severe infectious disease in tropical regions. It is necessary to understand the risk of acquiring this infection from the environment.

Methodology /principal findings: The prevalence, concentration and genetic diversity of B. pseudomallei isolates collected from two sites in Buriram, Northeast Thailand were investigated. Forty-four environmental samples (18 from soil, 14 from rice rhizosphere, and 12 from water) were collected; of those 44 samples, 19 were collected from near a patient's residence and 25 from suspected exposure sites and compared with 10 clinical isolates of the patient. Quantitative culture was performed, and B. pseudomallei was identified using the latex agglutination test and matrix-laser absorption ionisation mass spectrometry. Genotyping was performed in 162 colonies from clinical (N = 10) and environmental samples (N = 152) using pulse-field gel electrophoresis (PFGE) followed by multi-locus sequence typing (MLST) of the clinical strain. B. pseudomallei was detected in 11 of the 44 environmental samples (1 from soil, 4 from rice rhizosphere, and 6 from water). The bacterial count in the positive soil sample was 115 CFU/g. The mean concentrations ± SDs of B. pseudomallei in the positive water and rhizosphere samples were 5.1 ± 5.5 CFU/ml and 80 ± 49 CFU/g, respectively. Six water samples with positive results were collected from a pond and water sources for drinking and daily use. All colonies isolated from the patient shared the same PFGE type (PT) indicating monoclonal infection of ST99. Although the 152 colonies from environmental isolates exhibited 25 PTs, none were identical to the patient's isolates. PT5 and PT7 were most common genotype among the environmental samples.

Conclusions/significance: Diverse genotypes of B. pseudomallei were prevalent in the environment. However, the patient may have been infected with a low-density genotype. Intervention strategies for preventing B. pseudomallei infection are required.

Fig 1. Location of the patient’s residence in Buriram, Northeast Thailand.

Maps of the study region were created using ArcGIS software version 10.3.1.

Fig 2. Geographic location of the sampling sites and points.

(A) The locations at which the patient was suspected to be exposed (suspected to be exposed), the patient’s house (patient’s house) and the community-dug well used by the patient and community for drinking and cooking (dug well). (B) The pins denote the sampling points for the locations near the patient’s house. (C) The pins denote the sampling points around the pond and the rice paddy field to which the patient was suspected to be exposed. (D) The pins denote the water samples and the enclosed area surrounding the dug well. The sampling points were tracked using GPSMAP 60CSx, and the map was created using LandsatLook Viewer (http://landsatlook.usgs.gov/). Pins with black spots represent B. pseudomallei culture-positive samples.

Fig 3. Prevalence of B. pseudomallei positive samples at two sampling sites among different sample types.

(A) Prevalence of B. pseudomallei positive samples between two sampling sites. (B) Prevalence of B. pseudomallei positive samples in the soil, rice rhizosphere and water.

Fig 4. Concentrations of B. pseudomallei from two sampling sites and three different sample types.

(A) Concentration of B. pseudomallei in all environmental samples from two sampling sites: near the patient’s house and to which the patient was suspected to be exposed. (B) Concentration of B. pseudomallei in the soil, rhizosphere, and water samples.

Fig 5. Dendrogram of the 26 representative PFGE types of B. pseudomallei isolated from the patient (PT1), soil, rice rhizosphere and water (PT2 to PT26) collected from the environment near patient’s residence (PT7, PT8 and PT9) and to which the patient was suspected to be exposed (PT2, PT3, PT4, PT5, PT6, PT7, PT8 and PT10 to PT26) in Buriram, Northeast Thailand.

A dendrogram was created using the BioNumerics software (version 7.6).

Fig 6. Venn diagram of 25 representative PFGE types from environmental B. pseudomallei isolates in the soil, rice rhizosphere and water samples.

PT7, PT8 and PT9 were collected from the environment near the patient’s residence, whereas PT2, PT3, PT4, PT5, PT6, PT7, PT8 and PT10 to PT26 were collected from the sites to which the patient was suspected to be exposed in Buriram, Northeast Thailand.