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DNA points to likely septic contamination of potable water system

DNA points to likely septic contamination of potable water system

DNA points to likely septic contamination of potable water system in residential apartment building

A resident of an apartment building in New York City contacted Microbe Detectives to see if the potable water supplied to his apartment could be analyzed for microbiological contamination. He said people are getting sick in the building and he thinks the problem could be with their water.

One water sample was analyzed using Microbe Detectives’ 16S and 18S rRNA sequencing methodology. This analysis identified nearly all Bacteria, Archaea, and Eukarya (e.g., fungi, algae, amoeba, etc.) known in environmental science that were present in the water sample and the % relative abundance of each. The data was then checked for the presence of genus identities that contain pathogens and microbes known to be associated with fecal contamination.

Potential pathogens observed

Fifteen (15) genus identities that contain pathogens were observed to be present as shown below. Since the pathogens are identified at the species level, and this DNA analysis identifies at the genus level, this analysis could not confirm the presence of pathogens. However, a healthy potable water system should be free from all of these organisms. Therefore, the presence of these organisms was rated as a warning that pathogens species may be present.

Yersinia – The 4th most abundant microbe observed @7.2% relative abundance

Yersinia are bacteria that can cause illnesses in humans. Y. enterolitica are the most common species causing human enteric (intestinal) yersiniosis.(1) The organism is transmitted to humans as a foodborne or waterborne pathogen, and infection results in an acute gastrointestinal condition known as yersiniosis.(2)

DNA points to likely septic contamination

The gut is the most colonized human organ with up to 100 trillion microbes, about 10 times the number of the human cells. The predominance of these microbe groups consists of only 4 major phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria (3).

A phylum analysis of the clients potable water sample observed the presence of all four human gut associated phyla, as shown below. This is the first major clue that the potable water system may be contaminated with human feces.

Enterobacterales are a large order of different types of bacteria that commonly cause infections in healthcare settings, and are generally associated with microbes found in the human gut.(4) Nine bacteria grouped under the Enterbacterales Order were observed to be present, as shown below.
A few descriptions of Enterobacterales genera observed present include the following.

Citrobacter – @1.3% relative abundance

Citrobacter spp. primarily are inhabitants of the intestinal tract of mammals and other vertebrates. Their isolation from environmental sources such as water and soil likely is the result of fecal excretion.(5)

Clostridium – @0.7% relative abundance

Clostridium bacteria are found in soil, water, and the intestinal tracts of humans and other animals. Most species grow only in the complete absence of oxygen.(6)

Klebsiella – @0.2% relative abundance

Klebsiella is a type of gram-negative bacteria that can cause different types of healthcare-associated infections. Increasingly, Klebsiella bacteria have developed antimicrobial resistance, most recently to the class of antibiotics known as carbapenems. Klebsiella bacteria are normally found in the human intestines and in the human stool (feces).(7)

Other clues of fecal

Bacteroidia – @0.5% relative abundance

Bacteroidia is the Class that contains Bacteriodes which is a standard fecal contamination indicator. While Bacteriodes was not observed to be present, detection of the Class Bacteroidia strengthens the case suggesting that the potable water system may have been contaminated with fecal matter.

Unveil the Truth: Analyzing Water Contamination

Take advantage on modern DNA tools to uncover hidden fecal and pathogenic contamination in your water supply. If fecal or pathogenic contamination exists, WaterTrust’s advanced DNA methods provide very detailed evidence, giving you the knowledge you need to protect yourself and your loved ones. Contact us today to learn more.


  1. Centers for Disease Control and Prevention, website (
  2. Rogers, Kara. “Yersinia”. Encyclopedia Britannica, 28 Apr. 2016, (
  3. Rizzatti G, Lopetuso LR, Gibiino G, Binda C, Gasbarrini A. Proteobacteria: A Common Factor in Human Diseases. Biomed Res Int. 2017;2017:9351507. doi: 10.1155/2017/9351507. Epub 2017 Nov 2. PMID: 29230419; PMCID: PMC5688358.
  4. Centers for Disease Control and Prevention website. (
  5. Stella Antonara, Monica I. Ardura, 141 – Citrobacter Species,
    Editor(s): Sarah S. Long, Charles G. Prober, Marc Fischer, Principles and Practice of Pediatric Infectious Diseases (Fifth Edition), Elsevier, 2018, ISBN 9780323401814, (
  6. Britannica, The Editors of Encyclopaedia. “clostridium”. Encyclopedia Britannica, 5 Jan. 2022, Accessed 12 May 2024.
  7. Centers for Disease Control and Prevention, website (
The journey to a healthier lake

The journey to a healthier lake

Microbial source tracking of fecal contamination in Baker Lake from recycled wastewater used for Lakeview Golf Course irrigation.

By: Hunter Bailey, Grand Prize Winner of the Montana 2024 State Science Fair competition, AP Biology Student, Baker High School, Baker, MT, Class of 2024

Purpose of Experiment

The purpose of this experiment was to find the main cause of contamination of fecal bacteria in Baker Lake, using fecal indicator bacteria and gene marker HF183 tests.


  • Null: There is no connection between the golf course and Baker Lake in terms of the source of fecal contamination in the lake.
  • Alternative: There is a connection between the golf course and Baker Lake in terms of the source of fecal contamination in the lake.

Illness in Baker Lake

  • Since the summer of 2022, there have been reports of swimmers getting sick after the recreational use of Baker Lake in Baker, MT.
  • Illnesses reported to the Fallon County Public Health department included swimmer’s itch and gastrointestinal illnesses.
  • DEQ identified a toxic algae bloom in summer of 2022, even though the county commission tests were negative.
  • In 2022, a student group partnered with the county and DEQ and found the lake had way over the recommended level of coliforms and high levels of E. coli, which could be causing the human illness.
  • The source(s) of contamination were unknown.
  • One possibility could be runoff from irrigation of the nearby golf course.

Lakeview Golf Course

  • Located 635 m from the lake and 720 m from the upper lake.
  • The upper and lower lakes are 8 m lower in altitude.
  • The golf course is irrigated with recycled wastewater from city lagoons.
  • The city of Baker chlorinates the lagoons with 0.5 lbs of chlorinated gas per
    100,000 gal (0.6 mg/L).
  • Surface waters should receive a dosage of 5.0 to 10.0 mg/L (Migliaccio et al., 1992).
  • The lagoon pumps 200 gal/min to the golf course holding pond during the summer months.
  • The golf course must follow irrigation rules for recycled wastewater but Montana DEQ denied renewal of the plan in 2023.

Recycled Wastewater Compliance

  • An operation plan for the disposal of wastewater effluent to be used for Lakeview golf course irrigation was received by the Montana DEQ and denied.
  • The report indicated that the city treats lagoon water to Class C, even though golf course irrigation requires Class A.
  • Coliform tests show a total coliform count in the effluent at over >2400 CFU/ml.
  • The effluent does not comply with the maximum coliform concentration for even Class C.
  • Class C can only be used for landscape irrigation on sites that have restricted access.
  • The golf course is open to the public, so Fallon County must show that the golf course is adequately restricted from people during irrigation hours.

Runoff from Recycled Wastewater

  • Based on these reports of high coliform counts in the irrigation water and the location of the golf course compared to the lake, it is possible that the recycled irrigation wastewater from the Lakeview golf course is running off into the lake.
  • On September 4, 2023, in less than six hours the storm had dumped off more than two inches of rain, creating severe flooding across Fallon County, especially the golf course area.
  • This event gives evidence that stormwater runoff from the golf course does runoff into the lake and could point to a possible contamination and explanation of the high coliform and E. coli levels in the lake after heavy rain (Abu-Ashour & Lee, 2000).

Determining the Source of Contamination

  • “Microbial source tracking is a tool that can be used to obtain information on whether the fecal contamination in water came from human or animal wastewater or both” (Ahmed et al., 2014, p. 1305).
  • Fecal indicator bacteria test (FIB) – used for stormwater and freshwater samples for coliform, E. coli, and fecal indicators.
  • Gene markers in stormwater and freshwater samples. It can indicate whether the gene is associated with human or animal feces. The human fecal gene is HF183.
  • Biotracer is the use of a specific E. coli strain that is put on the surface of the soil and tested in other locations to detect movement (Sandrin et al., 2004).
  • In this study, FIB tests and the gene marker MST methods were used to determine whether irrigation of the recycled golf course water was causing the Baker Lake contamination.

Part 3

North Sample

  • Metagenomic testing was done on two one-liter samples from the north and south end of Baker Lake by Microbe Detectives, along with the HF183 Microbial source tracking.
  • In the north sample, there were 91 bacterial strains identified with two genes for antimicrobial resistance, along with 12 genes for virulence factors.
  • The highest relative abundance was in Alphaproteobacterium and Betaproteobacterium along with the Actinobacterium.
  • The two genes for antibiotic resistance are Tetracycline and Fosfomycin. The relative abundance of tetracycline is 54.96% and Fosfomycin 45.04%.
  • Some virulence factors included Bacteroides fragilis GENE IS942, Bacteroides fragilis GI 46242810, and Bacteroides fragilis GI 46242805.
  • The HF183 test was negative for both samples.

South Sample

  • In the South sample there was 45 bacterial strains identified with two genes for antimicrobial resistance, along with 3 genes for virulence factors.
  • The highest relative abundance was in Alphaproteobacterium and Betaproteobacterium.
  • The two genes for antibiotic resistance are Aminoglycoside and Macrolide. The Relative abundance of Aminoglycoside is 55.45% and Macrolide is 44.56%.
  • Some virulence factors Bacteroides fragilis GI 46242807, Bacteroides fragilis GI 46242810, Bacteroides fragilis GI 7899274.


  • One source of fecal coliforms in Baker Lake is the golf course irrigation of wastewater. There could also be other sources of contamination.
  • I recommend the city treating the lagoons with more chlorinated gas, at the recommended level of 5.0 to 10.0 mg/L, while continuing to test the effluent weekly for coliforms and adjusting chlorinated gas to counterbalance the levels of coliform.
  • Then, the county could ensure that the surrounding area, including the Baker Lake, is tested for coliforms and E.coli due to runoff and other sources.
  • The Fallon County commission should keep supporting and funding the future of this project for this ongoing issue, to try to eventually resolve this issue.

Take advantage of modern DNA tools to properly diagnose fecal contamination and identify the source(s)

It is no longer necessary to be unsure of whether fecal contamination is present in fresh water lakes, rivers, and saltwater estuaries, nor of the specific contributing sources. These questions can be answered in detail with modern DNA tools applied at WaterTrust. In addition, we can inform on other pathogenic microbes, antibiotic resistent genes, and even viruses. Reach out to us today for more information.


  1. Abu-Ashour, J. & Abu-Zreig, M. (2005). Effect of interstitial velocity on the adsorption of bacteria onto soil. Adsorption Science & Technology, 23(7), 535-542.
  2. Abu-Ashour, J. & Lee, H. (2000). Transport of bacteria on sloping soil surface by runoff. Environmental Toxicology, 15(2), 149-153.
  3. Ahmed, W., Hamilton, K., Toze, S., Cook., S., & Page, D. (2019). A review on microbial contaminants in stormwater runoff and outfalls: Potential health risks and mitigation strategies. Science of the Total Environment, 692, 1304-1321.
  4. Brosz Engineering. (nd). Baker Lake Clean-up, Dredging and Restoration. Accessed 10 Jan 2024.
  5. Hudson, M. (2017). A Year after Tornado Devastated Town, Baker Looks Ahead of Its Drained, Debris-Filled Lake. Billings Gazette.
  6. s-drained-debris-filled-lake/article_da9e4578-33ca-5569-adc7-703b77d2fa7a.html. Accessed 10 Jan 2024.
  7. Layton, A., McKay, L., Williams, D., Garrett, V., Gentry, R., & Sayler, G. (2006). Development of Bacteroides 16s rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water. Applied Environmental Microbiology, 72(6), 4214-4224.
  8. Marsh, J. (2022). More Golf Courses Need to Use Recycled Water. Environmental Protection. 2013%20percent%20of%20greenskeepers,supply%20from%20used%20water%20sources. Accessed 10 Jan 2024.
  9. Menger, R. (2023). Operation and maintenance plan: Spray irrigation of Lakeview Golf Course using recycled city of Baker wastewater. Plan submitted to the Montana DEQ by Fallon County for approval.
  10. Miggliaccio, K. W., Boman, B., & Clark, G. A. (1992). Treating irrigation systems with chlorine. Institute of Food and Agricultural Sciences Extension University of Florida.
  11. Montana Department of Environmental Quality. (2018) Design Standards For Public Sewage Systems. DEQ Montana Department of Environmental Accessed 10 Jan 2024.
  12. Rost, L. (2022). Baker High School Baker Lake E. coli and coliform monitoring sampling and analysis plan. Montana DEQ.