7.1. The Rationale of the Use of Fecal Indicator Bacteria
The most important bacterial gastrointestinal diseases transmitted through water are cholera, salmonellosis and shigellosis. These diseases are mainly transmitted through water (and food) contaminated with feces of patients. Drinking water can be contaminated with these pathogenic bacteria, and this is an issue of great concern. However, the presence of pathogenic bacteria in water is sporadic and erratic, levels are low, and the isolation and culture of these bacteria is not straightforward. For these reasons, routine water microbiological analysis does not include the detection of pathogenic bacteria. However, safe water demands that water is free from pathogenic bacteria .
The conciliation of the two needs was met by the discovery and testing of indicator bacteria. Water contaminated with pathogenic species also has the normal inhabitants of the human intestine. A good bacterial indicator of fecal pollution should fulfill the following criteria: (1) exist in high numbers in the human intestine and feces; (2) not be pathogenic to humans; (3) easily, reliably and cheaply detectable in environmental waters. Additionally, the following requisites should be met if possible: (4) does not multiply outside the enteric environment; (5) in environmental waters, the indicator should exist in greater numbers than eventual pathogenic bacteria; (6) the indicators should have a similar die-off behavior as the pathogens; (7) if human fecal pollution is to be separated from animal pollution, the indicator should not be very common in the intestine of farm and domestic animals [1,4,6,57,58]. The usefulness of indicator bacteria in predicting the presence of pathogens was well illustrated in many studies, namely by Wilkes et al. .
7.2. The Composition of Human and Animal Feces
Microbiological analysis of the human feces was important in order to structure and validate the use of fecal indicator bacteria in environmental waters. Bacteria present in feces are naturally derived from the microbiota of the human gastrointestinal tract.
Although bacteria are distributed throughout the human gastrointestinal tract, the major concentration of microbes and metabolic activity can be found in the large intestine. The upper bowel (stomach, duodenum, and jejunum) has a sparse microbiota with up to 105 CFU/ml of contents. From the ileum on, bacterial concentrations gradually increase reaching in the colon 1010 to 1011 CFU/g .
It has been estimated that at least 500–1,000 different microbial species exist in the human gastrointestinal microbiota, although on a quantitative basis 10–20 genera usually predominate (Table 6). The total number of microbial genes in the human gastrointestinal tract has been estimated as 2–4 million. This represents an enormous metabolic potential which is far greater than that possessed by the human host [60,64].
Total viable count in feces of healthy humans (children, adults and elderly)a.
The composition of feces from an individual is stable at genus level, but the species composition can vary markedly from day to day. The relative proportion of intestinal bacterial groups can vary between individuals [60,64].
The microflora of the human gastrointestinal tract is dominated by obligate anaerobes, which are ca. 103 more abundant than facultative anaerobes. The main anaerobic genera are Bacteroides, Eubacterium and Bifidobacteria. These organisms account for ca. 90% of the cultivable human fecal bacteria. Bacteroides (mainly B. thetaiotaomicron and B. vulgatus) are the most abundant organism in the human feces and account for 20–30% of cultivable bacteria. The most abundant facultative anaerobes are Enterococci and Enterobacteriaceae. The main Enterobacteriaceae genera are Escherichia, Citrobacter, Klebsiella, Proteus and Enterobacter. Citrobacter and Klebsiella are present in most individuals although in low numbers. Proteus and Enterobacter are only present in a minority of humans .
A variety of molecular techniques have been used to study the microbial composition of the human gastrointestinal tract. Results yielded by these studies have shown that many microbes detected by molecular techniques are not isolable by conventional culture-based methods. The presence of high proportions of bifidobacteria detected by culture-based methods is not supported by the results of molecular-based studies. However, the results of molecular-based approaches support many of the findings derived from culture-based methods: the dominance of the obligate anaerobes over facultative anaerobes; the presence of high counts of Bacteroides, Clostridium and Eubacterium .
Anaerobic bacteria such as Bacteroides and Eubacterium are not easily cultured by conventional techniques since require incubation chambers with nitrogen atmosphere. Bifidobacterium and Lactobacillus tolerate some oxygen but are fastidious bacteria growing very slowly in culture media. Therefore, these four genera are not adequate to be used as indicators of fecal pollution (the introduction of molecular techniques may improve the situation). Citrobacter, Klebsiella and Enterobacter are present in low numbers in the human intestine and are widespread in environmental waters, and therefore are also not suitable as indicators of fecal pollution. Clostridium, Streptococcus and Escherichia do not suffer from these drawbacks. Therefore, their suitability as fecal indicators has been tested since several decades.
7.3. Fecal Bacteria in Their Hosts and in the Environment
The traditional genus Bacteroides included Gram-negative, non-sporeforming, anaerobic pleiomorphic rods. Many species have been transferred to other genera—Mitsuokella, Porphyromonas, Prevotella, Ruminobacter. Bacteroides are the most abundant bacteria in human feces. In animal feces, on the contrary, Bacteroides are present at low numbers. Although anaerobic, Bacteroides are among the most tolerant to oxygen of all anaerobic human gastrointestinal species. B. thetaiotaomicron is one of the most abundant species in the lower regions of the human gastrointestinal tract. Bacteroides have a high pathogenic potential and account for approximately two-thirds of all anaerobes isolated from clinical specimens. The most frequently isolated species has been B. fragilis. The survival of Bacteroides in environmental waters is usually much lower than the survival of coliforms [64,65].
The traditional genus Eubacterium included anaerobic non-sporeforming Gram-positive rods. Some species have been transferred to other genera—Actinobaculum, Atopobium, Collinsella, Dorea, Eggerthella, Mogibacterium, Pseudoramibacter and Slackia. Cells are not very aerotolerant. Species isolated from the human gastrointestinal tract include: E. barkeri, E. biforme, E. contortum, E. cylindrioides, E. hadrum, E. limosum, E. moniliforme, E. rectal and E. ventricosum .
Bifidobacteria are Gram-positive, non-sporeforming, pleiomorphic rods. Bifidobacteria are anaerobic (some species tolerate oxygen in the presence of carbon dioxide) or facultative anaerobic. The optimum growth temperature is 35–39 °C. The genus Bifidobacterium contains ca. 25 species, most of which have been detected in the human gastrointestinal tract [64–66].
Bifidobacteria are present in high numbers in the feces of humans and some animals. Several Bifidobacterium species are specific either for humans or for animals. B. cuniculi and B. magnum have only been found in rabbit fecal samples, B. gallinarum and B. pullorum only in the intestine of chickens and B. suis only in piglet feces. In human feces, the species composition changes with the age of the individual. In the intestine of infants B. breve and B. longum generally predominate. In the adult, B. adolescentis, B. catenulatum, B. pseudocatenulatum and B. longum are the dominant species. In both human and animal feces, bifibobacteria are always much more abundant than coliforms [64–66].
Bifidobacteria have been found in sewage and polluted environmental waters, but appears to be absent from unpolluted or pristine environments such as springs and unpolluted soil. This results from the fact that upon introduction into the environment, bifidobacteria decrease appreciably in numbers, probably due to their stringent growth requirements. Bifidobacteria grow poorly below 30 °C and have rigorous nutrient requirements. Reports on the survival of bifidobacteria in environmental waters indicate that their survival is lower than that of coliforms [64–66].
The presence of bifidobacteria in the environment is therefore considered an indicator of fecal contamination. Since some species are specific for humans and animals, the identification of Bifidobacterium species present in the polluted water could, in principle, provide information on the origin of fecal pollution [64–66].
A study carried out in a highly contaminated stream near Bologna, Italy, revealed that B. adolescentis, B. catenulatum, B. longum, B. pseudocatenulatum and B. thermophilum were the most representative species, whereas B. angulatum, B. animalis subsp. animalis (B. animalis), B. breve, B. choerinum, B. minimum, B. pseudolongum subsp. globosum (B. globosum) and B. subtile occurred only in low numbers .
Bifidobacteria are the less studied of all fecal bacteria, due to the technical difficulties in their isolation and cultivation. Other Gram-positive bacteria, such as Streptococcus and Lactobacillus, which may occur in higher numbers than bifidobacteria, can inhibit their growth. Although selective media has been designed for the isolation of bifidobacteria from environmental waters, the outcome is still unsatisfactory, with appreciable numbers of false positives and low recovery percentages [64–66].
The genus Clostridium is one of the largest genera of the prokaryotes containing 168 validly published species. From these, 77 (including C. perfringens) are considered to belong to a united group—Clostridium sensu stricto [64,67,68].
Clostridia are Gram-positive rods, forming endospores. Most of the clostridial species are motile with peritrichous flagellation. Cells are catalase-negative and do not carry out a dissimilatory sulphate reduction. Clostridia usually produce mixtures of organic acids and alcohols from carbohydrates and proteins. Many species are saccharolytic and proteolytic. Some species fix atmospheric dinitrogen [64,67,68].
The genus Clostridium includes psychrophilic, mesophilic, and thermophilic species. The major role of these organisms in nature is in the degradation of organic material to acids, alcohols, CO2, H2, and minerals. Frequently, a butyric acid smell is associated with the proliferation of clostridia. The ability to form spores that resist dryness, heat, and aerobic conditions makes the clostridia ubiquitous [64,67,68].
Most species are obligate anaerobic, although tolerance to oxygen occurs. Oxygen sensitivity restricts the habitat of the clostridia to anaerobic areas or areas with low oxygen tensions. Growing and dividing clostridia will, therefore, not be found in air saturated surface layers of lakes and rivers or on the surface of organic material and soil. Clostridial spores, however, are present with high probability in these environments, and will germinate when oxygen is exhausted and when appropriate nutrients are present [64,67,68].
C. perfringens ferment lactose, sucrose and inositol with the production of gas, produce a stormy clot fermentation with milk, reduce nitrate, hydrolyze gelatin and produce lecithinase and acid phosphatase. The species is divided into five types, A to E, on the basis of production of major lethal toxins [68,69].
C. perfringens appears to be a universal component of the human and animal intestine, since has been isolated from the intestinal contents of every animal that has been studied. Humans carry C. perfringens as part of the normal endogenous flora. The main site of carriage is the distal gastrointestinal tract. The principal habitats of type A are the soil and the intestines of humans, animals, and birds. Types B, C, D, and E appears to be obligate parasites of animals and occasionally are found in humans [68,69].
Clostridium perfringens is the most frequently isolated Clostridium in clinical microbiology laboratories, although it seldom causes serious infections. C. perfringens is isolated from infections in humans and the organism most commonly found in gas gangrene in humans. C. perfringens is most commonly isolated from infections derived from the colonic flora, namely peritonitis or abdominal abscess [68,69].
This organism is a common cause of food poisoning due to the formation of the enterotoxin in the intestine. C. perfringens food poisoning is seldom fatal, being marked by diarrhea and nausea, with no vomiting and no fever [68,69].
Sources yielding C. perfringens include soil and marine sediment samples worldwide, clothing, raw milk, cheese, semi-preserved meat products, and venison. Like E. coli, C. perfringens does not multiply in most water environments and is a highly specific indicator of fecal pollution. Berzirtzoglou et al.  reported a comparative study on the occurrence of vegetative cells and spores of Clostridium perfringens in a polluted station of the lake Pamvotis, in rural North-West Greece. The numbers of C. perfringens varied according to the water depth. Sporulated forms were found in all sampling sites with the exception of the surface sampling.
Lactobacilli are non-sporeforming Gram-positive long rods. There are more than thirty species in the genus. Most are microaerophillic, although some are obligate anaerobes. Cells are catalase-negative and obtain their energy by the fermentation of sugars, producing a variety of acids, alcohol and carbon dioxide. Lactobacilli have complex nutritional requirements and in agarized media may need the supplementation with aminoacids, peptides, fatty-acid esters, salts, nucleic acid derivatives and vitamins. Lactobacilli very rarely cause infections in humans .
Enterococci are Gram-positive, non-sporeforming, catalase-negative ovoid cells. Cells occur singly, in pairs or short chains. Optimal growth for most species is 35–37 °C. Some will grow at 42–45 °C and at 10 °C. Growth requires complex nutrients but is usually abundant on commonly used bacteriological media. Cells are resistant to 40% bile, 0.4% azide, 6.5% sodium chloride, have β-glucosidase and hydrolyze esculin. The enterococci are facultative anaerobic but prefer anaerobic conditions [64,71].
The genus was separated from Streptococcus in the 1980s. Enterococci form relatively distinct groups. Members of such groups exhibit similar phenotypic characteristics and species delimitation can be difficult. The E. faecalis group contains, among others, E. faecalis. The E. avium group contains, among others, E. avium. The E. faecium group contains, among others, E. faecium, E. durans and E. hirae. The E. gallinarum group contains, among others, E. gallinarum [64
Water microbiology is concerned with the microorganisms that live in water, or can be transported from one habitat to another by water.
Water can support the growth of many types of microorganisms. This can be advantageous. For example, the chemical activities of certain strains of yeasts provide us with beer and bread. As well, the growth of some bacteria in contaminated water can help digest the poisons from the water.
However, the presence of other disease causing microbes in water is unhealthy and even life threatening. For example, bacteria that live in the intestinal tracts of humans and other warm blooded animals, such as Escherichia coli, Salmonella, Shigella, and Vibrio, can contaminate water if feces enters the water. Contamination of drinking water with a type of Escherichia coli known as O157:H7 can be fatal. The contamination of the municipal water supply of Walkerton, Ontario, Canada in the summer of 2000 by strain O157:H7 sickened 2,000 people and killed seven people.
The intestinal tract of warm-blooded animals also contains viruses that can contaminate water and cause disease. Examples include rotavirus, enteroviruses, and coxsackievirus.
Another group of microbes of concern in water microbiology are protozoa. The two protozoa of the most concern are Giardia and Cryptosporidium. They live normally in the intestinal tract of animals such as beaver and deer. Giardia and Cryptosporidium form dormant and hardy forms called cysts during their life cycles. The cyst forms are resistant to chlorine, which is the most popular form of drinking water disinfection, and can pass through the filters used in many water treatment plants. If ingested in drinking water they can cause debilitating and prolonged diarrhea in humans, and can be life threatening to those people with impaired immune systems. Cryptosporidium contamination of the drinking water of Milwaukee, Wisconsin with in 1993 sickened more than 400,000 people and killed 47 people.
Many microorganisms are found naturally in fresh and saltwater. These include bacteria, cyanobacteria, protozoa, algae, and tiny animals such as rotifers. These can be important in the food chain that forms the basis of life in the water. For example, the microbes called cyanobacteria can convert the energy of the sun into the energy it needs to live. The plentiful numbers of these organisms in turn are used as food for other life. The algae that thrive in water is also an important food source for other forms of life.
A variety of microorganisms live in fresh water. The region of a water body near the shoreline (the littoral zone) is well lighted, shallow, and warmer than other regions of the water. Photosynthetic algae and bacteria that use light as energy thrive in this zone. Further away from the shore is the limnitic zone. Photosynthetic microbes also live here. As the water deepens, temperatures become colder and the oxygen concentration and light in the water decrease. Now, microbes that require oxygen do not thrive. Instead, purple and green sulfur bacteria, which can grow without oxygen, dominate. Finally, at the bottom of fresh waters (the benthic zone), few microbes survive. Bacteria that can survive in the absence of oxygen and sunlight, such as methane producing bacteria, thrive.
Saltwater presents a different environment to microorganisms. The higher salt concentration, higher pH, and lower nutrients, relative to freshwater, are lethal to many microorganisms. But, salt loving (halophilic) bacteria abound near the surface, and some bacteria that also live in freshwater are plentiful (i.e., Pseudomonas and Vibrio). Also, in 2001, researchers demonstrated that the ancient form of microbial life known as archaebacteria is one of the dominant forms of life in the ocean. The role of archaebacteria in the ocean food chain is not yet known, but must be of vital importance.
Another microorganism found in saltwater are a type of algae known as dinoflagellelates. The rapid growth and multiplication of dinoflagellates can turn the water red. This "red tide" depletes the water of nutrients and oxygen, which can cause many fish to die. As well, humans can become ill by eating contaminated fish.
Water can also be an ideal means of transporting microorganisms from one place to another. For example, the water that is carried in the hulls of ships to stabilize the vessels during their ocean voyages is now known to be a means of transporting microorganisms around the globe. One of these organisms, a bacterium called Vibrio cholerae, causes life threatening diarrhea in humans.
Drinking water is usually treated to minimize the risk of microbial contamination. The importance of drinking water treatment has been known for centuries. For example, in pre-Christian times the storage of drinking water in jugs made of metal was practiced. Now, the anti-bacterial effect of some metals is known. Similarly, the boiling of drinking water, as a means of protection of water has long been known.
Chemicals such as chlorine or chlorine derivatives has been a popular means of killing bacteria such as Escherichia coli in water since the early decades of the twentieth century. Other bacteria-killing treatments that are increasingly becoming popular include the use of a gas called ozone and the disabling of the microbe's genetic material by the use of ultraviolet light. Microbes can also be physically excluded form the water by passing the water through a filter. Modern filters have holes in them that are so tiny that even particles as miniscule as viruses can be trapped.
An important aspect of water microbiology, particularly for drinking water, is the testing of the water to ensure that it is safe to drink. Water quality testing can de done in several ways. One popular test measures the turbidity of the water. Turbidity gives an indication of the amount of suspended material in the water. Typically, if material such as soil is present in the water then microorganisms will also be present. The presence of particles even as small as bacteria and viruses can decrease the clarity of the water. Turbidity is a quick way of indicating if water quality is deteriorating, and so if action should be taken to correct the water problem.
In many countries, water microbiology is also the subject of legislation. Regulations specify how often water sources are sampled, how the sampling is done, how the analysis will be performed, what microbes are detected, and the acceptable limits for the target microorganisms in the water sample. Testing for microbes that cause disease (i.e., Salmonella typhymurium and Vibrio cholerae) can be expensive and, if the bacteria are present in low numbers, they may escape detection. Instead, other more numerous bacteria provide an indication of fecal pollution of the water. Escherichia coli has been used as an indicator of fecal pollution for decades. The bacterium is present in the intestinal tract in huge numbers, and is more numerous than the disease-causing bacteria and viruses. The chances of detecting Escherichia coli is better than detecting the actual disease causing microorganisms. Escherichia coli also had the advantage of not being capable of growing and reproducing in the water (except in the warm and food-laden waters of tropical countries). Thus, the presence of the bacterium in water is indicative of recent fecal pollution. Finally, Escherichia coli can be detected easily and inexpensively.
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