Isolation, molecular characterization and phylogeny of Naegleria species in water bodies of North-Western Province, Sri Lanka.

Nuwan Gunarathna,Devika Iddawela,Sunil Wijesundara,Anjalie Amarasinghe,Susiji Wickramasinghe,Supram Hosuru Subramanya

doi:10.1371/journal.pone.0248510

Nuwan Gunarathna, Devika Iddawela + Show 4 more

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https://doi.org/10.1371/journal.pone.0248510

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Abstract

BackgroundThe inland freshwater bodies in the North-Western Province of Sri Lanka have ideal environmental conditions for the Naegleria species. Therefore, the presence and prevalence of Naegleria species in the water bodies of North-Western Province were determined by molecular characterization and phylogenetic analysis in this study.MethodsA total of 104 water bodies were selected from Kurunegala and Puttalam districts in the North-Western Province of Sri Lanka. Mean turbidity, pH, and temperature were recorded in each water body from three selected site. Centrifuged samples were cultured on non-nutrient agar plates with Escherichia coli. Enflagellation test positive isolates were subjected to DNA extraction and polymerase chain reaction using genus and species-specific primers targeting the internal transcribed spacer region (ITS) and Mp2CL5 gene. Phylogenetic analysis was performed using Bayesian and maximum likelihood (ML) methods.ResultsThe prevalence of Naegleria species and N. fowleri in the study area were 23.07% and 1.92%. The prevalence of Naegleria species and the physicochemical parameters of the water bodies showed no significant correlation. Bayesian analysis of the ITS region revealed the Naegleria Sri Lankan (SL) isolates 1, 3, and 4 in a single clade separated from the 2 and 5. Furthermore, Bayesian analysis identified isolates 2 and 5 in the same clade with Naegleria sp. samples and N. Philippinensis forming a sister clade. However, in the ML tree, all isolates were in the same clade with Naegleria sp. samples and N. Philippinensis.ConclusionsThe present study reports the first isolation of pathogenic N. fowleri from Sri Lanka. Based on Bayesian analysis, SL isolates 2 and 5 form a separate clade from 1, 3, and 4. However, in ML analysis, all isolates are grouped in one clade with Naegleria sp. samples and N. philippinensis. Further investigations are required to confirm these findings.

Highlights

The genus Naegleria is a group of free-living amoebae (FLA) with more than 47 species [1]
Enflagellation test positive isolates were subjected to DNA extraction and polymerase chain reaction using genus and species-specific primers targeting the internal transcribed spacer region (ITS) and Mp2CL5 gene
In the maximum likelihood (ML) tree, all isolates were in the same clade with Naegleria sp. samples and N

Summary

Introduction

The genus Naegleria is a group of free-living amoebae (FLA) with more than 47 species [1]. Naegleria fowleri is the only species in this genus that is known to cause human disease. Trophozoites of N. fowleri can penetrate the cribriform plate and cause fulminant and primary amoebic meningoencephalitis (PAM) in humans. This disease is acquired while swimming, diving, or total submersion in water bodies contaminated with the infective stage of N. fowleri [2]. The inland freshwater bodies in the North-Western Province of Sri Lanka have ideal environmental conditions for the Naegleria species. The presence and prevalence of Naegleria species in the water bodies of North-Western Province were determined by molecular characterization and phylogenetic analysis in this study

Methods

Results

Conclusion