Indicator bacteria
Encyclopedia
Indicator bacteria
are types of bacteria
used to detect and estimate the level of fecal contamination of water.
Each gram of human feces
contains approximately ~100 billion bacteria . These bacteria may include species of pathogenic bacteria, such as Salmonella
or Campylobacter
, associated with gastroenteritis
. In addition, feces may contain pathogenic virus
es, 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.
Indicator bacteria are not themselves dangerous to the health but are used to indicate the presence of a health risk.
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.
, 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.
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 24-48 hours depending on the type of bacteria. Counts are reported as colony forming units per 100 mL (cfu/100 mL).
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.
(PCR) and fluorescence in situ hybridization (FISH) are gene sequence-based methods currently being used to detect specific strains of indicator bacteria.
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 water system is out of compliance if more than 5% of its monthly water samples contain coliforms.
Despite criticism, the US Environmental Protection Agency (EPA) recommended this criterion again in 1976, however, numerous studies were initiated by the EPA in the 1970’s and 80’s to overcome the weaknesses of the earlier studies. In 1986, the EPA revised its bacteriological ambient water quality criteria recommendations to include E. coli and enterococci.
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.
and Campylobacter
; however, it is also important to understand the risk of exposure to pathogens via recreational waters. This is especially the case in watersheds where human or animal wastes are discharged to streams and downstream waters are used for swimming or other recreational activities. Other important pathogens other than bacteria include viruses such as rotavirus
, hepatitis A
and hepatitis E
and protozoa like giardia
, cryptosporidium
and Naegleria fowleri
. Due to the difficulties associated with monitoring pathogens in the environment, risk assessments often rely on the use of indicator bacteria.
Data on water exposure are generally collected using questionnaires, but may also be determined from actual measurements of water ingested, or estimated from previously published data. Respondents are asked to report the frequency and timing and location of exposures, detailed information about the amount of water swallowed and head submersion, and basic demographic characteristics such as age, gender, socioeconomic status and family composition. Once sufficient data are collected and determined to be representative of the general population, they are usually fit with distributions, and these distribution parameters are then used in the risk assessment equations. Monitoring data representing occurrence of pathogens, direct measurement of pathogen concentrations, or estimations deriving pathogen concentrations from indicator bacteria concentrations, are also fit with distributions. Dose is calculated by multiplying the concentration of pathogens per volume by volume. Dose-responses can also be fit with a distribution.
of 1977. Once water quality standards are approved, states are tasked with monitoring their surface waters to determine where impairments occur, and watershed plans called Total Maximum Daily Loads
(TMDLs) are developed to direct water quality improvement efforts including changes to allowable bacteria loading by point sources and recommendations for changes to practices that reduce nonpoint-source
contributions to bacteria loads. Also, many states have beach monitoring programs to warn swimmers when high levels of indicator bacteria are detected.
Indicator bacteria
Indicator bacteria are types of bacteria used to detect and estimate the level of fecal contamination of water.Each gram of human feces contains approximately ~100 billion bacteria . These bacteria may include species of pathogenic bacteria, such as Salmonella or Campylobacter, associated with...
are types of bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
used to detect and estimate the level of fecal contamination of water.
Each gram of human feces
Feces
Feces, faeces, or fæces is a waste product from an animal's digestive tract expelled through the anus or cloaca during defecation.-Etymology:...
contains approximately ~100 billion bacteria . These bacteria may include species of pathogenic bacteria, such as Salmonella
Salmonella
Salmonella is a genus of rod-shaped, Gram-negative, non-spore-forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella which grade in all directions . They are chemoorganotrophs, obtaining their energy from oxidation and reduction...
or Campylobacter
Campylobacter
Campylobacter is a genus of bacteria that are Gram-negative, spiral, and microaerophilic. Motile, with either unipolar or bipolar flagella, the organisms have a characteristic spiral/corkscrew appearance and are oxidase-positive. Campylobacter jejuni is now recognized as one of the main causes...
, associated with gastroenteritis
Gastroenteritis
Gastroenteritis is marked by severe inflammation of the gastrointestinal tract involving both the stomach and small intestine resulting in acute diarrhea and vomiting. It can be transferred by contact with contaminated food and water...
. In addition, feces may contain pathogenic virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es, protozoa
Protozoa
Protozoa are a diverse group of single-cells eukaryotic organisms, many of which are motile. Throughout history, protozoa have been defined as single-cell protists with animal-like behavior, e.g., movement...
and parasites
Parasitism
Parasitism is a type of symbiotic relationship between organisms of different species where one organism, the parasite, benefits at the expense of the other, the host. Traditionally parasite referred to organisms with lifestages that needed more than one host . These are now called macroparasites...
. 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.
Indicator bacteria are not themselves dangerous to the health but are used to indicate the presence of a health risk.
Criteria for Indicator Organisms
The US 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 coliformsFecal coliforms
A fecal coliform is a facultatively-anaerobic, rod-shaped, gram-negative, non-sporulating bacterium. Fecal coliforms are capable of growth in the presence of bile salts or similar surface agents, are oxidase negative, and produce acid and gas from lactose within 48 hours at 44 ± 0.5°C.Coliform...
, 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
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
(E. coli) and enterococci are also used as indicators.
Membrane Filtration and Culture on Selective Media
Indicator bacteria can be cultured on mediaGrowth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....
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 24-48 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 antibodiesMonoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
can be used to detect indicator bacteria in water samples. Precultivation in select medium must preface detection to avoid detection of dead cells. ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
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 Mehtods
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 reactionPolymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) and fluorescence in situ hybridization (FISH) are gene sequence-based methods currently being used to detect specific strains of indicator bacteria.
Drinking Water Standards
World Health OrganizationWorld Health Organization
The World Health Organization is a specialized agency of the United Nations that acts as a coordinating authority on international public health. Established on 7 April 1948, with headquarters in Geneva, Switzerland, the agency inherited the mandate and resources of its predecessor, the Health...
Guidelines for Drinking Water Quality state that as an indicator organism Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
provides conclusive evidence of recent fecal pollution
Water pollution
Water pollution is the contamination of water bodies . Water pollution occurs when pollutants are discharged directly or indirectly into water bodies without adequate treatment to remove harmful compounds....
and should not be present in water meant for human consumption. In the U.S., the EPA total coliform rule states that a water system is out of compliance if more than 5% of its monthly water samples contain coliforms.
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. In the 1960’s, these numbers were converted to fecal coliform concentrations assuming 18% of total coliforms were fecal. Consequently, the National Technical Advisory Committee (NTAC) in the US recommended the following standard for recreational waters in 1968: 10 % 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).Despite criticism, the US Environmental Protection Agency (EPA) recommended this criterion again in 1976, however, numerous studies were initiated by the EPA in the 1970’s and 80’s to overcome the weaknesses of the earlier studies. In 1986, the 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% 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.
Risk Assessment for Exposure to Pathogens in Recreational Waters
Most cases of bacterial gastroenteritis are caused by food-borne enteric microorganisms, such as SalmonellaSalmonella
Salmonella is a genus of rod-shaped, Gram-negative, non-spore-forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella which grade in all directions . They are chemoorganotrophs, obtaining their energy from oxidation and reduction...
and Campylobacter
Campylobacter
Campylobacter is a genus of bacteria that are Gram-negative, spiral, and microaerophilic. Motile, with either unipolar or bipolar flagella, the organisms have a characteristic spiral/corkscrew appearance and are oxidase-positive. Campylobacter jejuni is now recognized as one of the main causes...
; however, it is also important to understand the risk of exposure to pathogens via recreational waters. This is especially the case in watersheds where human or animal wastes are discharged to streams and downstream waters are used for swimming or other recreational activities. Other important pathogens other than bacteria include viruses such as rotavirus
Rotavirus
Rotavirus is the most common cause of severe diarrhoea among infants and young children, and is one of several viruses that cause infections often called stomach flu, despite having no relation to influenza. It is a genus of double-stranded RNA virus in the family Reoviridae. By the age of five,...
, hepatitis A
Hepatitis A
Hepatitis A is an acute infectious disease of the liver caused by the hepatitis A virus , an RNA virus, usually spread the fecal-oral route; transmitted person-to-person by ingestion of contaminated food or water or through direct contact with an infectious person...
and hepatitis E
Hepatitis E
Hepatitis E is a viral hepatitis caused by infection with a virus called hepatitis E virus . HEV is a positive-sense single-stranded RNA icosahedral virus with a 7.5 kilobase genome. HEV has a fecal-oral transmission route. It is one of five known hepatitis viruses: A, B, C, D, and E...
and protozoa like giardia
Giardia
Giardia is a genus of anaerobic flagellated protozoan parasites of the phylum Metamonada in the supergroup "Excavata" that colonise and reproduce in the small intestines of several vertebrates, causing giardiasis, commonly known as Beaver fever...
, cryptosporidium
Cryptosporidium
Cryptosporidium is a protozoan that can cause gastro-intestinal illness with diarrhea in humans.Cryptosporidium is the organism most commonly isolated in HIV positive patients presenting with diarrhea...
and Naegleria fowleri
Naegleria fowleri
Naegleria fowleri is a free-living excavate form of protist typically found in warm bodies of fresh water, such as ponds, lakes, rivers, and hot springs. It is also found in soil, near warm-water discharges of industrial plants, and unchlorinated swimming pools in an amoeboid or temporary...
. Due to the difficulties associated with monitoring pathogens in the environment, risk assessments often rely on the use of indicator bacteria.
Epidemiological Studies
In the 1950s, a series of epidemiological studies were done in the US to determine the relationship between water quality of natural waters and the health of bathers. The results indicated that swimmers were more likely to have gastrointestinal symptoms, eye infections, skin complaints, ear, nose, and throat infections and respiratory illness than non-swimmers and in some cases, higher coliform levels correlated to higher incidence of gastrointestinal illness, although the sample sizes in these studies were small. Since then, studies have been done to confirm causative relations between swimming and certain health outcomes. A review of 22 studies in 1998 confirmed that the health risks for swimmers increased as the number of indicator bacteria increased in recreational waters and that E. coli and enterococci concentrations correlated best with health outcomes among all the indicators studied. The relative risk (RR) of illness for swimmers in polluted freshwater versus swimmers in unpolluted water was between 1-2 for the majority of the data sets reviewed. The same study concluded that bacterial indicators were not well correlated to virus concentrations.Fate and Transport of Pathogens
Survival of pathogens in waste materials, soil, or water, depends on many environmental factors including temperature, pH, organic matter content, moisture, exposure to light, and the presence of other organisms. Fecal material can be directly deposited, washed into waters by overland runoff, transported through the ground, or discharged to surface waters via sewer lines, pipes, or drainage tiles. Risk of exposure to humans requires pathogens to (1)survive and be present (2) recreate in surface waters and (3) Individuals to come in contact with water for sufficient time, or ingest sufficient volumes of water to receive an infectious dose. Die-off rates of bacteria in the environment are often exponential, therefore, direct deposition of fecal material into waters generally contribute higher concentrations of pathogens than material that must be transported overland or through the subsurface.Human Exposure
In general, children, the elderly, and immunocompromised individuals require a lower dose of a pathogenic organism in order to contract an infection. Unfortunately, there are very few studies which are able to quantify the amount of time people are likely to spend in recreational waters and how much water they are likely to ingest. In general, children swim more often, stay in the water longer, submerge their heads more often, and swallow more water.Quantitative Microbiological Risk Assessment (QMRA)
Quantitative Microbial Risk Assessments (QMRA) combine pathogen concentrations in water with dose-response relationships and data reflecting potential exposure to estimate the risk of infection.Data on water exposure are generally collected using questionnaires, but may also be determined from actual measurements of water ingested, or estimated from previously published data. Respondents are asked to report the frequency and timing and location of exposures, detailed information about the amount of water swallowed and head submersion, and basic demographic characteristics such as age, gender, socioeconomic status and family composition. Once sufficient data are collected and determined to be representative of the general population, they are usually fit with distributions, and these distribution parameters are then used in the risk assessment equations. Monitoring data representing occurrence of pathogens, direct measurement of pathogen concentrations, or estimations deriving pathogen concentrations from indicator bacteria concentrations, are also fit with distributions. Dose is calculated by multiplying the concentration of pathogens per volume by volume. Dose-responses can also be fit with a distribution.
Risk management and policy implications
The more assumptions that are made, the more uncertain estimates of risk related to pathogens will be. However, even with considerable uncertainty, QMRAs are a good way to compare different risk scenarios. In a study comparing estimated health risks from exposures to recreational waters impacted by human and non-human sources of fecal contamination, QMRA determined that the risk of gastrointestinal illness from exposure to waters impacted by cattle were similar to those impacted by human waste, and these were higher than for waters impacted by gull, chicken, or pig faeces. Such studies could be useful to risk managers for determining how best to focus their limited resources, however, risk managers must be aware of the limitations of data used in these calculations. For example, this study used data describing concentrations of Salmonella in chicken feces published in 1969. Methods for quantifying bacteria, changes in animal housing practices and sanitation, and many other factors may have changed the prevalence of Salmonella since that time. Also, such an approach often ignores the complicated fate and transport processes that determine bacteria concentrations from the source to the point of exposure.Addressing Bacterial Water Quality Problems
In the US, individual states are allowed to develop their own water quality standards based on EPA’s recommendations under the Clean Water ActClean Water Act
The Clean Water Act is the primary federal law in the United States governing water pollution. Commonly abbreviated as the CWA, the act established the goals of eliminating releases of high amounts of toxic substances into water, eliminating additional water pollution by 1985, and ensuring that...
of 1977. Once water quality standards are approved, states are tasked with monitoring their surface waters to determine where impairments occur, and watershed plans called Total Maximum Daily Loads
Total Maximum Daily Load
A Total Maximum Daily Load is a regulatory term in the U.S. Clean Water Act, describing a value of the maximum amount of a pollutant that a body of water can receive while still meeting water quality standards...
(TMDLs) are developed to direct water quality improvement efforts including changes to allowable bacteria loading by point sources and recommendations for changes to practices that reduce nonpoint-source
Nonpoint source
Nonpoint source, or non-point source, or NPS, is a source that does not come from a single point.* Point source, contrasts with nonpoint source* Nonpoint source pollution, water pollution...
contributions to bacteria loads. Also, many states have beach monitoring programs to warn swimmers when high levels of indicator bacteria are detected.