Bacterial community profiles within the water samples of leptospirosis outbreak areas

Abstract

Background: Leptospirosis is a water-related zoonotic disease. The disease is primarily transmitted from animals to humans through pathogenic Leptospira bacteria in contaminated water and soil. Rivers have a critical role in Leptospira transmissions, while co-infection potentials with other waterborne bacteria might increase the severity and death risk of the disease.

Methods: The water samples evaluated in this study were collected from four recreational forest rivers, Sungai Congkak, Sungai Lopo, Hulu Perdik, and Gunung Nuang. The samples were subjected to next-generation sequencing (NGS) for the 16S rRNA and in-depth metagenomic analysis of the bacterial communities.

Results: The water samples recorded various bacterial diversity. The samples from the Hulu Perdik and Sungai Lopo downstream sampling sites had a more significant diversity, followed by Sungai Congkak. Conversely, the upstream samples from Gunung Nuang exhibited the lowest bacterial diversity. Proteobacteria, Firmicutes, and Acidobacteria were the dominant phyla detected in downstream areas. Potential pathogenic bacteria belonging to the genera Burkholderiales and Serratia were also identified, raising concerns about co-infection possibilities. Nevertheless, Leptospira pathogenic bacteria were absent from all sites, which is attributable to its limited persistence. The bacteria might also be washed to other locations, contributing to the reduced environmental bacterial load.

Conclusion: The present study established the presence of pathogenic bacteria in the river ecosystems assessed. The findings offer valuable insights for designing strategies for preventing pathogenic bacteria environmental contamination and managing leptospirosis co-infections with other human diseases. Furthermore, closely monitoring water sample compositions with diverse approaches, including sentinel programs, wastewater-based epidemiology, and clinical surveillance, enables disease transmission and outbreak early detections. The data also provides valuable information for suitable treatments and long-term strategies for combating infectious diseases.

Keywords: Bacterial community; Leptospira; Leptospirosis; Next generation sequencing; Pathogenic; Public health; Water recreational.

Figure 1. The water sampling locations in four recreational areas, Hulu Langat, Selangor; (A) HP, (B) SC, (C), SL, and (D) GN.

Map were generated using ArcGIS 10.5 (Esri, Redlands, CA, USA) with the base maps was digitized from OpenStreetMap (http://www.openstreetmap.org) under CC BY-SA 2.0

Figure 2. The alpha diversity (Chao1, Shannon, and Simpson diversity indices) boxplots based on OTUs abundance in the water samples calculated with analysis of variance (ANOVA) (pairwise analysis).

The vertical axis denotes richness estimations in the number of OTUs, separate boxes are overlaid on study site combinations, and asterisks indicate significant differences: *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3. The alpha diversity of the bacterial communities in the water samples; the (A) rarefaction curves illustrate OTUs species richness in each area and (B) Venn diagram demonstrating the numbers of unique and shared OTUs between sampling areas based on 16S rR.

The OTUs were obtained with QIIME V1.9.1. with a 97% sequence similarity, overlapping colours indicate shared OTUs, non-overlapping colours represent unique OTUs, and all OTUs are defined at a 97% level of sequence similarity.

Figure 4. The Krona graph demonstrating the relative abundance of taxonomic hierarchy and percentage composition in phyla, class, family, order and species levels based on OTUs abundances in the (A) HP, (B) SL, water samples.

The samples from HP and SL had a significant relative abundance of Gammaproteobacteria, Alphaproteobacteria, Acidobacteria and Verrucomicrobiae.

Figure 5. The Krona graph demonstrating the relative abundance of taxonomic hierarchy and percentage composition in phyla, class, family, order and species levels based on OTUs abundances in the (A) SC, and (B) GN water samples.

The samples from SC and GN had a significant relative abundance of Gammaproteobacteria.

Figure 6. The beta diversity results of the water samples; the (A) PCoA between the HP, SL, SC, and GN sampling sites based on OTUs abundance (weighted UniFrac distance) and (B) heatmap based on the distance matrix analysis with weighted UniFrac.

The variance explained by each principle coordinate axis is denoted by PC1 vs. PC2 and PC1 vs. PC3. Colour hues represent the distance matrix; a darker hue indicates significant abundance, while a lighter colour represents low abundance. A high abundance reflected a significant gap relationship between samples.

Figure 7. The relative abundance of the major 10 bacteria at the (A) phyla, (B) genera, and (C) species levels based on OTUs abundance obtained from sites 1, 2, and 3 in HP, SL, SC, and GN.

Figure 8. The heatmap and hierarchical cluster results based on the relative abundance of bacterial communities in the water samples.

The numbers indicate the sample number of the corresponding sampling site, the x-axis represents similarities or dissimilarities between the sampling areas, the y-axis denotes similarities or dissimilarities between the genera, dark blue represents the more abundant genus, and light blue corresponds to the genus with less abundance in each sample.