Have you ever gone to the beach, ready for a day of sun and sand, only to find a warning sign? One of the most common reasons beaches close is due to the presence of coliform bacteria. These indicator bacteria, such as Escherichia coli, are used as surrogates for fecal waste, since their presence can suggest the presence of other, more pathogenic species, such as Shigella or Salmonella. E. coli is used as an indicator species because it occurs at a higher concentration in fecal waste compared to other pathogens of public concern, making it easier to identify potentially unsafe conditions. Currently, most surveillance is done through culture-based methods, but this method of measuring bacterial contamination has some limitations.
These limitations are mainly due to the lack of E. coli animal source specificity. Human waste contamination can increase fecal coliform numbers, but so can waste from deer, birds, or other animals surrounding recreational waterways. In fact, waste from almost any warm-blooded animal will elevate E. coli levels; however, some animal sources, such as human fecal waste, pose a higher public health risk compared to others. “Human waste represents one of the greatest public health risks in the U.S. and worldwide,” says Environmental Protection Agency (EPA) Research Geneticist Orin Shanks. He and his EPA research team are working to generate standard genetic biomarkers that indicate the presence of human-associated fecal bacteria.
Shanks and Mathematical Statistician, Mano Sivaganesan, work with a team that is developing molecular tests to quickly differentiate human-associated from non-human-associated contamination with fecal bacteria. Their research focuses on quantitative PCR (qPCR), which measures the relative amount of a given gene in a sample. In the past few years, their research team has published studies demonstrating that qPCR of particular bacterial genes can identify bacterial contamination from human sources in water samples.
Two of their assays (the HF183/BacR287 and HumM2 assays) measure the relative abundance of human-associated Bacteriodes species as indicators for human waste contamination. “These methods have been tested against a large collection of materials from wastewater facilities and animal species across the U.S. and suggest a strong association with human fecal waste,” said Shanks. “In rare cases, these bacteria can be present in non-human animal waste, but typically at much lower concentrations compared to human sources.” Shanks notes that because of these rare cases, it’s important to test each assay using local waste samples prior to testing water quality.
Protocols vary in efficacy from research lab to research lab, which is why Shanks and Sivagenesan are developing a repeatable, step-by-step protocol. “Standardized procedures with data acceptance criteria are necessary to ensure proper method implementation and verify technical quality of findings,” says Sivaganesan. “In addition, standardized procedures facilitate the adoption of modified or new technologies by providing an established benchmark to compare performance.” A molecular detection method like qPCR is more expensive and more technically challenging compared to a culture-based method, hurdles which a standardized protocol may help overcome. The challenge is now to standardize the qPCR protocol, so surveillance labs around the U.S. can confidently run the same assay and trust the results.
One challenging aspect of standardization is in the different conditions of waterways sampled - salt concentrations, pH conditions, and even sediments in local water sources can vary between sampling locations, and these in turn may affect molecular detection methods. “Substances present in some environmental waters may hinder DNA recovery,” says Shanks. “For example, kaolinite clay readily binds to DNA, and, when bound, prevents qPCR amplification. This suggests that when this type of clay is present in environmental samples, it could introduce bias in methods that rely on isolation of DNA such as PCR, qPCR, digital PCR, and microarray technologies.”
To address this concern, the research team used a sample processing control to identify possible matrix interference - interference from the sample itself. “In this study, a control DNA spike was able to identify matrix interference due to the presence of clay particles. Other strategies are available using bacterial or plant cells, as well as plasmid constructs,” says Shanks. “This is a research area that clearly warrants additional work to identify other compounds that induce matrix interference, and establish which control strategies are most suitable for environmental applications.” Improved controls means the molecular assay is more accurate, and trusted results are important when public health is at stake.
In addition to better reliability, these assays offer the benefit of speedier results. Using traditional culture methods, samples are commonly collected in morning, cultured in the afternoon, and the resultant colony-forming units analyzed the subsequent day. Not only does this mean that beachgoers may have a full day of enjoying potentially contaminated swimming water, but the conditions can also change during the sample processing time. The immediacy of qPCR greatly speeds sample identification, with the potential to produce same-day results. Surveillance with this method could help officials act more quickly to both raise the red flag when bacterial contamination occurs and give the all-clear signal.
The effort to standardize qPCR, a common technique used in research labs, illustrates the rigorous verification necessary for water quality surveillance applications. But with hard work and a little luck, molecular detection shows promise to change the way bacterial contamination of waterways is assessed. Shanks and Sivaganesan concur: “A key challenge is the implementation of these methods across laboratories. The path forward begins with the standardization of methods and the development of data acceptance criteria.”