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. 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.
Criteria for indicator organisms The US Environmental Protection Agency (EPA) lists the following criteria for an organism to be an ideal indicator of fecal contamination:
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.
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.
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.
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.
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. 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.
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