Detection of Viral DNA in Water Reveals Fecal Contamination

Fecal contamination of clean water can be detected via microbial source tracking, a method that identifies specific biomarkers in the water. In a new study, scientists from Chungnam National University and Gyeongsang National University developed a new method for detecting viruses found exclusively in human gut and feces. They also identified and created specific viral DNA markers that can be used for improved microbial source tracking of fecal contamination in sewage, wastewater, and water bodies.

The presence of human waste in sewage overflow or stormwater runoffs that ultimately find their way into rivers and lakes is a major public health hazard. As a result, it is imperative that water bodies and samples are continuously tested for the presence of human fecal contamination. Conventionally, testing for fecal contamination involves detecting fecal indicator bacteria (FIB), such as E. coli, by adding a drop of the sample water into a microbial culture medium. While FIB detection is cheap and simple, the method cannot be used to detect low-level contamination. This method is also incapable of determining the source of the contamination.

Microbial source tracking (MST) has the potential to overcome these challenges by detecting biomarkers—usually a protein, DNA, or RNA segment—that are specific to a host species. This allows MST to detect low-level contamination in wastewater, drinking water, lakes, etc., and identify the hosts, even when feces from multiple species are involved.

CrAss-like phages (CLPs), a class of viruses that infect bacteria (bacteriophages), are being hailed as a promising group of MST markers. CLPs are the most abundant bacteriophages in the human gut, and many CLPs are only found in human intestines (and feces). While there are several known groups of CLPs, only a single group, known as genus I, has been used as an MST marker. This prompts questions about whether the other group CLPs also function as MST markers and their viability compared to genus I.

Now, in a new study, Dr. Ok Kyung Koo and Dong Woo Kim from Chungnam National University, South Korea, in collaboration with Yang Jae Kang and Dong U Woo from Gyeongsang National University, developed a novel MST detection method using CLPs that was capable of specifically detecting human feces-contaminated water. The method uses polymerase chain reaction (PCR) to detect CLP DNA, making the method cost-effective, selective, and easy-to-perform. The study was published in Volume 266 of Water Research on 15 Nov 2024 and made available online on 24 Aug 2024.

“The primary aim of this study was to improve the efficacy and accuracy of detection methods used to assess specific fecal contamination,” said Dong Woo Kim. Adding further, he says, “A robust MST marker would greatly help in our ability to mitigate health risks from fecal-contaminated water.”

To test the efficacy of this method, the researchers collected fecal samples from selected human volunteers. Fecal samples from wild animals, such as dogs, deer, cats, birds, and raccoons, were also included in the experiment to test the specificity of the detection method. DNA extraction and sequencing was then performed to detect and classify CLP viral genes. 13 distinct CLP groups were identified in human gut viruses. Using these CLPs as a template, the scientists then developed specific markers for their detection via PCR and tested them for their capacity to detect human fecal contamination.

The results of this study were encouraging. CLPs were found in 91.52% of human feces samples and were absent in all animal samples, except racoons. Of the 13 CLP groups classified identified, genus VI was present in 64.4% of samples, nearly double that of genus I (37.28%). Furthermore, all 13 CLP groups showed some similarity in their gene sequences, called the major head protein gene. This implied that a single marker could be used to detect multiple groups of contaminants.

“Our method shows that genus VI is a potent MST marker in the Korean population. Using PCR to detect MHP genes of genus VI CLPs, or even genus I, can be a practical approach towards monitoring human fecal contamination of water. I expect that our method can, over time, improve hygiene regulations and lower public health costs through the strategic screening of sewage, wastewater, and various environmental samples,” Dr. Ok Kyung Koo concludes.

In summary, the scientists concluded that CLPs, especially genus VI, could be used as viable MST markers that are able to specifically detect human fecal contamination. This was notable as human-specific and selectively targeted MST markers can significantly impact hygiene regulations, lowering public health costs through their application in screening liver, sewage, wastewater, and various environmental samples.  

Reference: Kim DW, Woo DU, Kim UI, Kang YJ, Koo OK. Development of a novel crAss-like phage detection method with a broad spectrum for microbial source tracking. Water Res. 2024;266:122330. doi: 10.1016/j.watres.2024.122330

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