Molecular analysis of clinical Burkholderia pseudomallei isolates from southwestern coastal region of India, using multi-locus sequence typing

Abstract

Background: The Gram-negative soil dwelling bacterium Burkholderia pseudomallei is the etiological agent of melioidosis. The disease is endemic in most parts of Southeast Asia and northern Australia. Over last few years, there has been an increase in number of melioidosis cases from India; however the disease epidemiology is less clearly understood. Multi-locus sequence typing (MLST) is a powerful genotypic method used to characterize the genetic diversity of B. Pseudomallei both within and across the geographic regions.

Methods: In this study, MLST analysis was performed on 64 B. pseudomallei clinical isolates. These isolates were obtained between 2008-2014 from southwestern coastal region of India. Broad population patterns of Indian B. pseudomallei isolates in context with isolates of Southeast Asia or global collection was determined using in silico phylogenetic tools.

Results: A total of 32 Sequence types (STs) were reported among these isolates of which 17 STs (53%) were found to be novel. ST1368 was found as group founder and the most predominant genotype (n = 11, 17%). Most of the B. pseudomallei isolates reported in this study (or other Indian isolates available in MLST database) clustered in one major group suggesting clonality in Indian isolates; however, there were a few outliers. When analyzed by measure of genetic differentiation (FST) and other phylogenetic tools (e.g. PHYLOViZ), Indian STs were found closer to Southeast Asian isolates than Australian isolates. The phylogenetic analysis further revealed that within Asian clade, Indian isolates grouped more closely with isolates from Sri Lanka, Vietnam, Bangladesh and Thailand.

Conclusions: Overall, the results of this study suggest that the Indian B. pseudomallei isolates are closely related with lesser heterogeneity among them and cluster in one major group suggesting clonality of the isolates. However, it appears that there are a few outliers which are distantly related to the majority of Indian STs. Phylogenetic analysis suggest that Indian isolates are closely related to isolates from Southeast Asia, particularly from South Asia.

Fig 1. Genetic relationship of B. pseudomallei isolates of this study using eBURST.

Blue dot refers to group founder and yellow dot refers to sub-group founder. Each black dot represents single genotype.

Fig 2. Genetic relationship of all Indian B. pseudomallei isolates (n = 130) using eBURST.

Blue dot refers to group founder and yellow dot refers to sub-group founder. Each black dot represents single genotype. Re-sampling for bootstrapping = 10,000; minimum number of identical loci for group definition = 6; minimum number of SLV for subgroup definition = 3.

Fig 3. PHYLOViZ analysis showing the genetic relationship among global collection of sequence types (STs) of Burkholderia pseudomallei.

Each dot represents a distinct ST. Oceania and Southeast Asian dominant STs are shaded in green and pink, respectively. Indian STs (shaded in yellow) cluster in four groups- Groups A & B cluster with STs from Southeast Asia and Groups C & D cluster with STs from Australia. STs from India are colored in black. Different colored dots represent STs from Sri Lanka (red), Australia (blue), Thailand (light blue), Malaysia (orange), China (light orange), Cambodia (green), Vietnam (light mehroon), Singapore (purple), Bangladesh (light purple), Laos (mehroon) and other countries (light pink).

Fig 4. The evolutionary history inferred using the UPGMA method.

The analysis involved 128 nucleotide sequences of 65 Indian STs and 63 STs from Sri Lanka, Vietnam, Bangladesh, Cambodia, Malaysia, Thailand, China, Laos and Australia.