The Origin of Faeces

In an article for the Institute of Water magazine Summer 2023 edition, WRc's Senior Consultant Microbiologist Daisy Pinn discusses how can microbial source tracking be used to protect environmental and public health.

The Challenge
Our rivers are essential to the health of every ecosystem they serve. Despite this, only 14% of English rivers meet good ecological status. The insects, fish and plants that reside within our polluted rivers – the other 86% - are at risk. This pollution is widely associated with faecal contamination from humans and other animals.

It is more than just a problem for the environment because the micro-organisms found in faeces can also be the cause of disease. It is clear that identifying, tracking and monitoring faecal contamination sources in freshwater is vital to understanding and resolving the problem.

What is Microbial Source Tracking?
Microbial source tracking refers to a method where markers associated with specific hosts (i.e., pigs, cattle, humans) are used to assess the sources of faecal contamination. In recent years, molecular techniques have advanced significantly. Tools that were once ‘out-of-reach’ are now readily available. This has implications for how we can analyse and track the source of faecal contamination in freshwaters.

What techniques are showing promise? 
Quantitative Polymerase Chain Reaction (qPCR)
The most established molecular technique used to date for microbial source tracking is qPCR. Using this technology, biological markers that are associated with the faeces of specific hosts can be quantified. For example, in the USA, the qPCR technique has been used to quantify the HF183 marker and track a human norovirus outbreak from a septic leach field. After microbial source tracking was utilised and risk management measures were implemented, no further cases were identified. The United States Environmental Protection Agency (USEPA) are also currently in the process of standardising the HF183 marker for the detection of human faecal pollution in environmental waters.

Digital Polymerase Chain Reaction (dPCR)
A suite of emerging molecular techniques known as dPCR have seen limited but promising application for microbial source tracking purposes to-date. The advantage of dPCR is that it is more resilient than qPCR to substances in the environment (pharmaceuticals, organic acids etc.) that inhibit the amplification process. The dPCR technique has been successfully used to detect in-pipe sewerage infrastructure failures for a water and wastewater treatment facility in the USA4. Deficiencies within the conveyance system could be identified by analysing for markers associated with human faecal pollution (HF183, HumM2, crAssphage and human polyomavirus). The novel approach enabled the prioritisation of investment and repair.

High-Throughput Sequencing (HTS)
In recent years, High-Throughput Sequencing (HTS) has also received interest for microbial source tracking purposes. HTS enables the detection of all micro-organisms within a sample. By comparing overlap in microbial composition between the environmental sample (i.e., sink) and the faeces of hosts (i.e., source) the relative proportion of contamination can be assessed. There are machine learning programmes available to do this, such as SourceTracker. The programme uses genetic data from thousands of organisms to estimate the likelihood of a sequence coming from the sink or source community. Interestingly, HTS has recently been applied to detect the presence of industrial effluents within wastewater samples. The study found that microbial communities were unique to the effluent of each factory tested, meaning that specific sources of contamination in complex sample-types can be attributed using this technology.

Other Techniques
There also exciting developments in rapid-field based techniques for assessing faecal contamination. This is partly being driven by the UNICEF™ Target Profile Product challenge which is seeking a rapid and cost-effective detection method for faecal pollution. There is potential for methods such as Loop-Mediated Isothermal Amplification (LAMP), and Biosensors to provide a rapid detection of host-associated markers. Such techniques could have value as an initial screening method, to assess whether further in-depth investigation would be necessary.

Conclusion
Overall, there may not be a one-size-fits-all approach to identify and track sources of faecal contamination in freshwaters. Instead, a toolbox of techniques is likely to be required. At WRc, we have recently delivered a project for Defra scoping state-of-the art microbial source tracking methods and how they could be utilised for the protection of environmental and public health of UK freshwaters. These outputs are soon to be published by Defra.

Institute of Water magazine, Spring 2023