Monitoring infectious disease threats in environmental water sources

Infectious waterborne diseases, which can be transmitted by drinking water that is contaminated by exposure to soil, sewage, and infected animals, pose a serious threat to the safety of the water supply. Global climate change increases the risk of outbreaks because higher temperatures can promote the growth of pathogens and the vectors that transmit them and increase the likelihood of exposure to contaminated water sources.

Leptospirosis is a waterborne disease caused by the Leptospira bacteria that can provoke kidney damage, meningitis, liver failure, respiratory distress, and even death. It infects humans through exposure to the urine of animals carrying these bacteria or environmental samples contaminated with urine from an infected animal. While leptospirosis has a worldwide distribution, it particularly affects countries with tropical and subtropical climates (Taniguchi and Póvoa 2019). This disease spreads easily during climate catastrophes, and extreme weather is a high-risk factor, as in the case of the leptospirosis outbreak in Puerto Rico following Hurricane Maria in 2017.

An effective method for monitoring the presence of Leptospira bacteria in the environment is crucial for preventing and controlling leptospirosis outbreaks. Since this disease can be transmitted by infected animals that contaminate water supplies, it is necessary to analyze samples from a variety of animal as well as water sources in order to identify the source of infection. In the following study, researchers harnessed the power of multiplex PCR to develop an assay that can accurately distinguish Leptospira bacteria from other species and is sufficiently robust to provide accurate results from environmental samples containing PCR-inhibitory contaminants.

Tracking the source of infection

A research group in Okinawa, Japan (Sato et al. 2019) sought to develop new tools to systematically detect Leptospira in order to prevent human infection. This group studied the bacterial ecosystem that allows the development of Leptospira during biofilm formation and investigated which animals are potential reservoirs for transmitting these pathogens to humans.

The researchers screened environmental water samples from a known endemic region in Japan for rRNA targets specific to Leptospira and animals living nearby. They performed multiplex PCR analysis with Takara Ex Taq HS DNA polymerase to detect bacteria using 16S rRNA targets, which they analyzed by NGS (on an Illumina MiSeq® platform). A similar procedure was carried out using PrimeSTAR HS DNA polymerase to detect 12S rRNA from vertebrate animals in the same environmental samples, in order to understand which vertebrate species are more likely to harbor Leptospira (Figure 1). The presence of certain animals, especially boars and eels, seemed to correlate with high levels of Leptospira, showing a potential link between pathogen and carrier. They were able to draw a correlation between the main bacteria (12 strains) that help propagate pathogenic Leptospira and the animals (10 species) that are the primary reservoirs of these bacteria. The multiplex PCR method used in the study is a powerful tool to help determine how Leptospira outbreaks can occur by showing how the environment can impact the development of this pathogen and revealing how it interacts with hosts/carriers. In addition to providing a better understanding of this phenomenon, these findings helped develop a system to better predict human infection risk.

Figure 1. Schematic view of the library preparation procedure for metabarcoding sequencing based on a two-step tailed PCR. Multiplex PCR was applied in the first step for Leptospira and bacterial detection. The procedure for vertebrate mitochondrial 12S rRNA sequencing was basically the same but slightly modified from that of Miya et al. 2015. Image and caption adapted from Sato et al. 2019 and used under a Creative Commons Attribution 4.0 International License.

Why our polymerases are the investigative tools of choice

This study highlights the use of Takara Ex Taq HS and PrimeSTAR HS DNA polymerases to detect bacteria in environmental water samples, which are typically difficult to work with due to the presence of PCR-inhibitory contaminants. It demonstrated the robustness of these two enzymes for performing DNA amplification with samples which pose such challenges. Since screening assays can help prevent health disasters provoked by weather crises, which are occurring with greater frequency and severity due to climate change, having reliable reagents to power those assays will be crucial for getting ahead of future outbreaks.

References

• High Prevalence of Deadly Bacterial Disease Found in Puerto Rico. Yale School of Medicine. Available at: https://medicine.yale.edu/news-article/20887/
• Leptospirosis | CDC. Available at: https://www.cdc.gov/leptospirosis/index.html
• Miya, M. et al. MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: Detection of more than 230 subtropical marine species. R. Soc. Open Sci. 2, (2015). Available at: https://pubmed.ncbi.nlm.nih.gov/26587265/
• Sato, Y. et al. Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan. Sci. Rep. 9, 1–11 (2019). Available at: https://www.nature.com/articles/s41598-019-42978-1
• Taniguchi, L. U. & Póvoa, P. Leptospirosis: one of the forgotten diseases. Intensive Care Med. 45, 1,816–1,818 (2019). Available at: https://link.springer.com/article/10.1007/s00134-019-05839-z