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Fecal contamination observed near wastewater plant along Florida’s coast was from non-human sources

Fecal contamination observed near wastewater plant along Florida’s coast was from non-human sources

Fecal contamination observed near wastewater plant along Florida’s coast was from non-human sources

A municipal wastewater treatment plant (City WWTP) fielded complaints regarding fecal matter on local beaches. In review of the potential sources of fecal contamination with Microbe Detectives (MD), and MD technical advisor Alison Ling, PhD, three had been contemplated including animal feces, human feces from the City’s homeless population, and the City WWTP.  The objectives of this sampling and analysis program were to:

  • Evaluate the presence/absence of overall fecal contamination and human fecal contamination in samples collected from stormwater outfalls.
  • Evaluate the relative contribution of human fecal contamination in samples collected.
  • Evaluate how presence/absence and relative contribution of human fecal contamination change with time.

Fecal Coliform Measurements

Fecal coliform and similar culture-based microbiology testing methods count how many bacteria grow on a specific type of food under specific laboratory conditions. As such, the term “fecal coliform” is misleading in that it describes bacteria that grow in a certain way rather than bacteria of a specific origin. Fecal coliform tests, including Standard Method 9222D, account for bacteria that can be present in animal feces as well as naturally occurring environmental bacteria. These methods were developed to indicate whether fecal contamination of any original exists in drinking water sources (typically very low in bacterial counts).  They routinely detect bacteria that are not fecal in origin, including bacteria common in plant materials and industrial effluents not associated with sewage (Reference 1).

Bacteria that proliferate during a controlled fecal coliform test can be found naturally in numerous environmental settings, including soils, sediments, algae, and lake water columns.  These “indigenous” fecal coliforms can be present in natural waters, even in cold climates.  When those waters are sampled, the fecal coliform laboratory method can sometimes indicate that fecal coliforms are present, in effect presenting a false positive (Reference 2).

The EPA states that “fecal coliform” test results do not meet the World Health Organization criteria for effective fecal indicators.  The EPA recommended in 1986 that states use either E. coli or enterococci tests instead of fecal coliforms to set water quality criteria, as fecal coliforms can present false positives with no association to fecal pollution (References 2, 3).

Molecular methods using qPCR Bacteroides spp. Markers

Due to these well documented method deficiencies, there has been an emphasis in recent years on the development of molecular biology methods to measure specific types of bacteria associated with fecal contamination rather than relying on culture-dependent methods with high false positive rates.  In addition to being more precise (only about 1% of bacteria can be grown in the lab), these molecular biology methods are less subject to false positives.  Specific methods used for evaluating fecal contamination are designed to measure specific types of bacteria that are prevalent in animal guts, but they are not good at growing in the environment, including Prevotella and Bacteroides genera.  Bacteriodes, specifically, include several types that evolve with the host animal and thus present host-specific targets for analysis.  These targets have been used to track the source of fecal contamination to different sources, including bovine, waterfowl, and human wastes (Reference 5).  Bacteriodes also have the benefit of comprising a significant portion of the bacteria present in human guts, and are 1,000 times more abundant in feces than fecal coliforms (References 2, 6).  Numerous studies have shown that Bacteriodes concentrations were better predictors of human pathogens than fecal coliforms (References 2, 7).

Bacteriodes spp. bacteria can be detected and quantified using quantitative polymerase chain reaction (qPCR) of the 16S ribosomal RNA gene (rRNA).  The 16S rRNA gene is the industry standard for identifying what types of microorganisms are present in a given sample, because it falls in the sweet spot of being conserved enough to be present in all organisms but variant enough to detect differences between specific species.  The qPCR method uses specific “primers” segments of DNA that bookend a targeted portion of the gene.  The primers are designed to be specific to the targeted organism (in this case Bacteroides spp.).  When combined with extracted DNA from the environmental sample and DNA replication enzymes and supplies, the qPCR process replicates DNA from only that target and back-calculates the initial concentration of the targeted DNA fragment.

Specific qPCR primers for general Bacteriodes (indicating fecal contamination) and host-specific Bacteriodes species and strains (Bacteriodes spp.) have been developed to assist with fecal source tracking efforts in environmental settings (References 5, 7). The HF183 marker targets a subset of Bacteriodes bacteria that evolved specifically with humans and is thus only present in human guts. HF183 was developed in 2005 (Reference 8) and an updated version of the assay is generally considered the most accurate method for detecting human fecal contamination (References 5, 9).

Samples collected during the first and fifth sampling events occurred after a rain event. All other samples were collected in the absence of rain before or during sampling. Fecal coliforms were analyzed by the City WWTP according to their methods. Host specific analysis for fecal bacteria were analyzed using molecular biology methods that target DNA of specific host-associated gut bacteria, specifically Bacteroides spp. (general fecal origin) and HF183 (human fecal origin).  The method involves collection of water in DNA-free containers, overnight shipment to the lab, filtration, DNA extraction, and quantitative polymerase chain reaction (qPCR) using primers developed for the specific targets.

Sampling Steps

Microbiology methods are especially sensitive to contamination from other materials. Samples were collected by authorized personnel at the City of Client WWTP, avoiding disturbance to the ground surface near or under the water close to the sampling location. The following steps were followed during sample collection.

  • Each sample container was submerged in the water at the outfall to fill the container. A one liter of water per sample was collected using sterile bottles provided by Microbe Detectives.  The inside of the bottle cap or rim of the sample bottle was not touched with anything.  If touched, the sample bottle was discarded and a new one was used.
  • The required volume of salt water was collected for each sample for fecal coliform analysis.
  • Gloves were used while sampling and were replaced often.  If gloves touched dirt or skin or clothing, they were replaced with new, clean gloves straight from the box.
  • Each sample bottle was clearly labelled with the sample name, Sample ID, date and time collected.
  • The labels on the bottles were covered with clear tape to protect them from getting wet and rubbing off from the ice and shipping.  “Bacteriodes spp.” and “HF183” were recorded on the COC for the host-specific fecal markers.

Findings

The below table summarizes test results from the first five rounds of sampling.

CT value reflects the threshold cycle number for a qPCR assay, which is the cycle number where the measured fluorescence exceeds a set threshold.  Smaller CT values correspond to larger concentrations of the target gene (Reference 11).

The below table compares results of the fecal coliform test versus the qPCR Bacteriodes spp test on the basis of whether fecal associated microbes were detected or not detected. A comparison of results is summarized by sampling event, and by sample location. The results confirm the stated testing inaccuracies of the fecal coliform test that is summarized above, as compared to the more accurate qPCR Bacteriodes spp test.

Takeaways

Results from the first five rounds of molecular testing suggested the following main takeaways:

  • Human-specific fecal contamination (Human Specific HF183) was not detected at any of the four sites during the five sampling events.
  • Samples from all four sites tested positive for fecal contamination using the molecular method (for Bacteroides spp.)
  • Positive fecal coliform results did not always correspond to positive fecal marker detection, suggesting that some fecal coliform tests may be subject to false positives.  Specifically, false positives were observed in the first sampling event (1 of 4) and the fifth sampling event (4 of 4). 
  • Positive fecal coliform and negative molecular results suggested a potential false positive fecal coliform test.  False positives on fecal coliform tests are a common concern and can be caused by naturally occurring bacteria from soil or other sources that can also grow on fecal coliform media.
  • In the third sampling event, molecular fecal markers were detected despite no detection of fecal coliforms in 3 of 4 sample locations.  This likely reflects the higher sensitivity of the molecular method over the culture-based fecal coliform method, and therefore is a false negative result.

References

  1. Doyle, MP and Erickson, MC. Closing the door on the fecal coliform assay. Microbe. 2006. Vol. 1, 4.
  2. US Environmental Protection Agency. Assessment of Fecal Indicators in Ambient Waters. 2015.
  3. World Health Organization. Guidelines for drinking-water quality. Recommendations – First addendum to the third edition. Geneva, Switzerland: World Health Organization.
  4. Zhang, Y. & Liu, WT (2019). The application of molecular tools to study the drinking water microbiome – Current understanding and future needs, Critical Reviews in Environmental Science and Technology, 49:13,  1188-1235,  DOI: 10.1080 /10643389.2019.1571351
  5. Ahmed, W, Hughes, B and Harwood, V. Current status of marker genes of Bacteroides and related taka for identifying sewage pollution in environmental waters. Water. s.l. : 8, 2016. Vol. 6.
  6. King, CH, et al. Baseline human gut microbiota profile in healthy people and standard reporting template. PLoS ONE. 2019.
  7. Savichtcheva, O, Okayama, N and Okabe, S. Relationship between Bacteroides 16S rRNA genetic markers and presence of bacterial enteric pathogens and fecal indicators. Water Research. 2007. Vol. 41.
  8. Seurinck, S, et al. Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human fecal pollution in freshwater. Environmental Microbiology. 2005. Vol. 7, 2.
  9. Green, HC, et al. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples. Applied and Environmental Microbiology. 2014. Vol. 80, 10.
  10. Gronewold, AD and Wolpert , RL. Modeling the relationship between most probable number (MPN) and colony-forming unit (CFU) estimates of fecal coliform concentration. Water Research. 2008. Vol. 42, 13.