Screening for Sewage Contamination
By Alex Spears, EMLab P&K Senior Bacteriologist
One of the most common situations faced by environmental professionals is the possibility of a sewage contamination event. Sewage contamination can be caused by a variety of sources such as broken, backed up, or leaking sewer lines; farm runoff; flooding from storms; and septic system malfunctions. Determining whether or not a sewage contamination event has occurred is a prime concern due to public health issues associated with some organisms potentially found in sewage, such as Escherichia coli, Salmonella, and Shigella. As these organisms may be difficult to isolate and identify and would typically be found in low numbers, other organisms commonly found in sewage were sought to serve as indicator organisms to demonstrate possible sewage contamination. Historically, the indicator organisms utilized have been total coliforms and fecal coliforms. Originally developed for the examination of water, these methods can also be adapted to the examination of bulk or swab samples as well. However, these tests can often produce false positive results, potentially resulting in data misinterpretation and potentially costly and needless remediation efforts.
The total coliform test was the first of these methods to be developed. Through either a quantitative or qualitative method, the sample is examined for the presence of members of the coliform group. The coliform group (which includes E. coli, Salmonella, and Shigella) is a diverse group of organisms that share similar characteristics. These characteristics include being gram negative, aerobic or facultatively anaerobic, non-spore forming, rod shaped bacteria fermenting lactose with gas and acid production after 24 to 48 hours of incubation at 35°C. Although coliforms are found in the intestines of mammals and in sewage, they are also present in the environment as part of the natural flora. These naturally occurring coliforms, which have no sanitary significance or association with waste or sewage, can potentially cause false positive results when testing environmental samples. Total coliform test results must be interpreted with caution, and positive tests should always be confirmed by more specific testing.
The test for fecal coliforms was developed to be more definitive in indicating fecal or sewage contamination. The term "fecal coliform" is actually somewhat of a misnomer, and a more scientifically accurate term for this group of organisms is "thermotolerant coliforms." This group of organisms includes the coliforms with the ability to grow and ferment lactose with gas and acid production at 44.5°C. Included in this group are organisms such as E. coli, Klebsiella, Enterobacter, and Citrobacter. Although this test is more specific than the total coliform test in indicating potential sewage contamination, it is also capable of providing false positive results, especially when dealing with environmental samples. "Species of Enterobacteriaceae other than E. coli are associated with plants and do not indicate fecal contamination, yet they are identified as fecal coliforms by the fecal coliform assay" (Doyle & Erickson, 2006). As with the total coliform test, the results of the fecal coliform assay must be interpreted with caution, and positive results should always be confirmed.
E. coli has been demonstrated to be a very specific indicator of fecal or sewage contamination. Recent advances in detection technology has made it possible to rapidly identify and differentiate E. coli from other thermotolerant coliforms, making it one of the indicator organisms of choice in determining sewage or fecal contamination. However, the test is not 100% accurate, as E. coli has been consistently found in pristine soil samples, indicating that E. coli may not always be a reliable indicator of fecal or sewage contamination in these types of samples.
To help eliminate some of the deficiencies encountered when using members of the coliform group in determining fecal or sewage contamination, testing for enterococci was developed. The enterococci, belonging to the genus Enterococcus, are gram positive catalase negative cocci differentiated from Group D Streptococci by their ability to grow at 10° and 45°C, hydrolyze esculin, and grow in the presence of 6.5% NaCl. Enterococci testing offers several advantages, including the facts that enterococci show a high and close relationship with health hazards, are less ubiquitous than the coliforms in the environment, are always present in the feces of warm-blooded animals, and die off less rapidly than coliform organisms.
As we have seen above, the testing of the environment for a potential sewage contamination event is not as straightforward as one would like, especially in light of the public health concerns raised due to the potential contamination. At EMLab P&K, in order to minimize false positives and to make data interpretation easier, we offer sewage contamination screen assays which include analysis of the specific indicator organisms E. coli and Enterococcus, with or without total coliform results included. By offering these analyses combined, the data presented regarding a potential sewage contamination is more robust, making misinterpretation of data less likely and reducing unnecessary remediation efforts.
1. Doyle, M.P. & Erickson, M.C. (2006). Closing the door on the fecal coliform assay. Microbe, 1(4), 162-163.
2. Hurst, C.J., Crawford, R.L., Knudsen, G.R., McInerney, M.J. & Stetzenbach, L.D. (Eds.). (2002). Manual of environmental microbiology (2nd ed.). Washington, D.C.: ASM Press
This article was originally published on June 2009.