Indicator bacteria
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Indicator bacteria are types of bacteria used to detect and estimate the level of fecal contamination of water. They are not dangerous to human health but are used to indicate the presence of a health risk.
Each gram of human feces contains approximately ~100 billion (1×1011) bacteria.[1] These bacteria may include species of pathogenic bacteria, such as Salmonella or Campylobacter, associated with gastroenteritis. In addition, feces may contain pathogenic viruses, protozoa and parasites. Fecal material can enter the environment from many sources including waste water treatment plants, livestock or poultry manure, sanitary landfills, septic systems, sewage sludge, pets and wildlife. If sufficient quantities are ingested, fecal pathogens can cause disease. The variety and often low concentrations of pathogens in environmental waters makes them difficult to test for individually. Public agencies therefore use the presence of other more abundant and more easily detected fecal bacteria as indicators of the presence of fecal contamination. Aside from bacteria being found in fecal matter, it can also be found in oral and gut contents.[2]
The US Environmental Protection Agency (EPA) lists the following criteria for an organism to be an ideal indicator of fecal contamination:[citation needed]
- The organism should be present whenever enteric pathogens are present
- The organism should be useful for all types of water
- The organism should have a longer survival time than the hardiest enteric pathogen
- The organism should not grow in water
- The organism should be found in warm-blooded animals' intestines.
None of the types of indicator organisms that are currently in use fit all of these criteria perfectly, however, when cost is considered, use of indicators becomes necessary.
Types of indicator organisms
Commonly used indicator bacteria include total coliforms, or a subset of this group, fecal coliforms, which are found in the intestinal tracts of warm blooded animals. Total coliforms were used as fecal indicators by public agencies in the US as early as the 1920s. These organisms can be identified based on the fact that they all metabolize the sugar lactose, producing both acid and gas as byproducts. Fecal coliforms are more useful as indicators in recreational waters than total coliforms which include species that are naturally found in plants and soil; however, there are even some species of fecal coliforms that do not have a fecal origin, such as Klebsiella pneumoniae. Perhaps the biggest drawback to using coliforms as indicators is that they can grow in water under certain conditions.
Escherichia coli (E. coli) and enterococci are also used as indicators.
Current methods of detection
Membrane filtration and culture on selective media
Indicator bacteria can be cultured on media which are specifically formulated to allow the growth of the species of interest and inhibit growth of other organisms. Typically, environmental water samples are filtered through membranes with small pore sizes and then the membrane is placed onto a selective agar. It is often necessary to vary the volume of water sample filtered in order to prevent too few or too many colonies from forming on a plate. Bacterial colonies can be counted after 24 to 48 hours depending on the type of bacteria. Counts are reported as colony forming units per 100 mL (cfu/100 mL).
Fast detections using chromogenic substances
One technique for detecting indicator organisms is the use of chromogenic compounds, which are added to conventional or newly devised media used for isolation of the indicator bacteria. These chromogenic compounds are modified to change color or fluorescence by the addition of either enzymes or specific bacterial metabolites. This enables for easy detection and avoids the need for isolation of pure cultures and confirmatory tests.[3]
Application of antibodies
Immunological methods using monoclonal antibodies can be used to detect indicator bacteria in water samples. Precultivation in select medium must preface detection to avoid detection of dead cells. ELISA antibody technology has been developed to allow for readable detection by the naked eye for rapid identification of coliform microcolonies. Other uses of antibodies in detection use magnetic beads coated with antibodies for the concentration and separation of the oocysts and cysts as described below for immunomagnetic separation (IMS) methods.[3]
IMS/culture and other rapid culture-based methods
Immunomagnetic separation involves purified antigens biotinylated and bound to streptoavidin-coated paramagnetic particles. The raw sample is mixed with the beads, then a specific magnet is used to hold the target organisms against the vial wall and the non-bound material is poured off. This method can be used to recover specific indicator bacteria.[3]
Gene sequence-based methods
Gene sequence-based methods depend on the recognition of exclusive gene sequences particular to specific strains of organisms. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are gene sequence-based methods currently being used to detect specific strains of indicator bacteria.[3]
Water quality standards for bacteria
Drinking water standards
World Health Organization Guidelines for Drinking Water Quality state that as an indicator organism Escherichia coli provides conclusive evidence of recent fecal pollution and should not be present in water meant for human consumption.[4] In the U.S., the EPA Total Coliform Rule states that a public water system is out of compliance if more than 5 percent of its monthly water samples contain coliforms.[5]
Recreational standards
Early studies showed that individuals who swam in waters with geometric mean coliform densities above 2300/100 mL for three days had higher illness rates.[6] In the 1960s, these numbers were converted to fecal coliform concentrations assuming 18 percent of total coliforms were fecal. Consequently, the National Technical Advisory Committee in the US recommended the following standard for recreational waters in 1968: 10 percent of total samples during any 30-day period should not exceed 400 fecal coliforms/100 mL or a log mean of 200/100 mL (based on a minimum of 5 samples taken over not more than a 30-day period).[7]
Despite criticism, EPA recommended this criterion again in 1976, however, the Agency initiated numerous studies in the 1970s and 1980s to overcome the weaknesses of the earlier studies. In 1986, EPA revised its bacteriological ambient water quality criteria recommendations to include E. coli and enterococci.
| Single Sample Maximum Allowable Density per 100 mL | |||||||
|---|---|---|---|---|---|---|---|
| Water Type | Indicator | Acceptable Swimming-Associated Gastroenteritis Rate per 1000 Swimmers | Steady State Geometric Mean Indicator Density per 100 mL | Designated Beach Area (upper 75% one-sided C.L.) | Moderate Full Body Contact Recreation (upper 82% C.L.) | Lightly Used Full Body Contact Recreation (upper 90% C.L.) | Infrequently Used Full Body Contact Recreation (upper 95% C.L.) |
| Freshwater | E. coli | 8 | 126 | 235 | 298 | 409 | 575 |
| enterococci | 8 | 33 | 61 | 78 | 107 | 151 | |
| Marine Water | E. coli | 19 | 35 | 104 | 158 | 276 | 501 |
Canada's National Agri-Environmental Standards Initiative's approach to characterizing risks associated with fecal water pollution bacterial water quality at agricultural sites is to compare these sites with those at reference sites away from human or livestock sources. This approach generally results in lower levels if E. coli being used as a standard or “benchmark” based on a study that indicated pathogens were detected in 80% of water samples with less than 100 cfu E. coli per 100 mL.[8]
