Genotyping of Burkholderia pseudomallei Isolated From Patients in South-Western Coastal Region of India.

Burkholderia pseudomallei is the causative agent of melioidosis. Various tools have been used to determine the genetic diversity in B. pseudomallei isolates. In this study, Random Amplified Polymorphic DNA (RAPD)-PCR and flagellin gene (fliC) based PCR-Restriction Fragment Length Polymorphism (RFLP) were used to genotype Indian clinical B. pseudomallei isolates. A total of 89 clinical isolates could be grouped in 6 groups (A through F) by RAPD-PCR analysis. Some of the isolates in various groups had identical banding pattern suggesting them to be epidemiologically related. The RAPD groups also correlated with MLST sequence types suggesting the utility of this easy to do typing method. The PCR- RFLP analysis suggested Type III to be the predominant type which is different from other RFLP types reported from Southeast Asia. In conclusion, the results of this study show that RAPD-PCR, a simple genotyping method, may be used for analyzing the B. pseudomallei isolates and also establish epidemiological relevant relatedness among them. The results of fliC PCR-RFLP further suggest the Indian isolates are different from other Southeast Asian isolates.

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

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.

Improvement of recombinant-truncated Burkholderia motility protein A (BimA)-based indirect ELISA for equine glanders.

Glanders is a contagious and highly fatal disease of equines with zoonotic potential. It is caused by a Gram-negative, nonmotile bacterium Burkholderia mallei. Complement fixation test (CFT) is one of the most commonly used tests for diagnosis of glanders; however, it has some limitations. A recombinant-truncated Burkholderia intracellular motility A (BimA) protein-based indirect enzyme-linked immunosorbent assay (iELISA) was previously reported by us for glanders diagnosis, which has been re-optimized in this study using a panel of glanders positive (n = 75) and glanders negative (n = 227) serum samples. The improved iELISA exhibited 96% sensitivity and 90.75% specificity. The assay had 98.56% negative predictive value. In the improved iELISA, background for negative samples was reduced and a rational assay cut-off based on ROC curves was introduced. Intra laboratory repeatability of the iELISA was tested by 3 different operators with 100% correlation. The BimA-coated ELISA plates could be used without significant decrease in diagnostic efficacy even after their storage at room temperature or 37°C for 90 days. Overall, the improved iELISA is a sensitive, specific, reproducible, and easy-to-use assay that has potential in serodiagnosis of glanders, more suitably to demonstrate freedom from B. mallei infection in a population.

Identification of a new diagnostic antigen for glanders using immunoproteome analysis.

Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n=49) and negative (n=79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.

Unravelling the Molecular Epidemiology and Genetic Diversity among Burkholderia pseudomallei Isolates from South India Using Multi-Locus Sequence Typing.

There is a slow but steady rise in the case detection rates of melioidosis from various parts of the Indian sub-continent in the past two decades. However, the epidemiology of the disease in India and the surrounding South Asian countries remains far from well elucidated. Multi-locus sequence typing (MLST) is a useful epidemiological tool to study the genetic relatedness of bacterial isolates both with-in and across the countries. With this background, we studied the molecular epidemiology of 32 Burkholderia pseudomallei isolates (31 clinical and 1 soil isolate) obtained during 2006-2015 from various parts of south India using multi-locus sequencing typing and analysis. Of the 32 isolates included in the analysis, 30 (93.7%) had novel allelic profiles that were not reported previously. Sequence type (ST) 1368 (n = 15, 46.8%) with allelic profile (1, 4, 6, 4, 1, 1, 3) was the most common genotype observed. We did not observe a genotypic association of STs with geographical location, type of infection and year of isolation in the present study. Measure of genetic differentiation (FST) between Indian and the rest of world isolates was 0.14413. Occurrence of the same ST across three adjacent states of south India suggest the dispersion of B.pseudomallei across the south western coastal part of India with limited geographical clustering. However, majority of the STs reported from the present study remained as "outliers" on the eBURST "Population snapshot", suggesting the genetic diversity of Indian isolates from the Australasian and Southeast Asian isolates.