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
Micro-organisms Micro-organisms (or microbes) are literally microscopic organisms, which can only be seen properly with the aid of a microscope. These include bacteria, microscopic fungi (moulds) and protoctists. Although viruses, which are even smaller than bacteria, are generally considered to be non- living entities, they might also be included here as they are important disease-causing agents. Micro-organisms are the most numerous organisms in any ecosystem. There are about 159,000 known species, although this is thought to be less than 5% of the total in existence. There is vast genetic diversity among micro-organisms, which is not surprising as they began evolving over a billion years before land plants. This, coupled with their small size and reproduction, helps explain why micro-organisms, particularly bacteria, are the most widely distributed forms of life on the planet. While many are cosmopolitan species, others exist in habitats totally inhospitable to larger organisms. There are species of bacteria able to grow in hot springs up to 90° C, others live below freezing point in Antarctica, in soda lakes, anaerobic situations, and sites with high concentrations of metals, sulphur and other normally toxic compounds.
and people Micro-organisms are of immense importance to the environment, to human health and to our economy. Some have profound beneficial effects without which we could not exist. Others are seriously harmful, and our battle to overcome their effects tests our understanding and ingenuity to the limit. However, certain micro-organisms can be beneficial or harmful depending on what we want from them: saprophytic decomposers play an important role in breaking down dead organic matter in ecosystems, but these same micro- organisms can be responsible for food spoilage (rotting, going bad, going off) and subsequent illness.
micro-organisms Disease and decay are not inherent properties of organic objects, nor do they result from physical damage or being eaten by insects, it is micro- organisms that bring about these changes. We are surrounded by bacteria, viruses, protoctists and fungi. Many cause disease in farm animals and commercial crops, many others are capable of invading our bodies and causing human disease.
Examples of familiar human diseases include:
Bacteria: salmonella, tetanus, typhoid, cholera, gangrene, bacterial dysentery, diphtheria, tuberculosis, bubonic plague, meningococcal meningitis, pneumococcal pneumonia
Viruses: rabies, influenza (flu), measles, mumps, polio, rubella (german measles), chicken pox, colds, warts, cold sores
Protoctists: malaria, amoebic dysentery
Fungi: athlete's foot, ringworm
These disease-causing organisms are called pathogens and we often refer to them in everyday, non-scientific terms as germs or bugs. Each disease has a specific pathogen, i.e. different diseases are caused by different kinds of germ. If the disease organism can be transmitted from one person to another it is said to be infectious. Non-infectious diseases, such as allergies, cancer, vitamin deficiency, and mental illness may develop when the body is not functioning properly.
Common infectious diseases can be spread (or be caught) by consuming food or water containing pathogens or their toxic products (e.g. salmonella, typhoid, cholera ); by droplet infection, which is inhaling or ingesting droplets of moisture which have been breathed, coughed or sneezed out by an infected person (e.g. colds, flu); by entry through a wound or sore (e.g. tetanus); by direct contact with an infected person (e.g. athletes foot, ringworm). Some pathogens are carried by vectors from one organism to another. For example: mosquitoes carry the malaria protoctistan; rat fleas carried the bacterium that caused the Black Death; houseflies can spread micro-organisms from faeces to our food. The vectors should not be confused with the pathogenic organisms that they are carrying.
We are usually able to develop immunity to infections by virtue of our immune system. Our blood produces specific antibodies in response to the presence of specific foreign bodies called antigens. These antibodies gradually proceed to destroy the invading organisms. However, over 40% of all deaths in developing countries, including the annual deaths of 14 million children, are caused by infectious diseases. In developed countries, where there are good medical services, people seldom die from infectious diseases. Diseases can be prevented or cured. Prevention is principally through improved standards of hygiene, personal health and the development of vaccinations. Vaccines contain killed or non-virulent (less pathogenic) strains of bacteria and viruses, and when these are injected into the blood, or swallowed, the body has a mild form of the disease, and is able to manufacture sufficient antibodies to acquire immunity. This is the process of immunisation, and vaccinations are an effective way of stimulating the body's defence against such diseases as diphtheria, polio, measles, mumps, german measles, tetanus, tuberculosis and hepatitis B. Vaccinations do exist for flu, but these have to be continually developed, because flu virus antigens are frequently changing, producing new strains of virus to which people are not immune. New strains can result in a flu epidemic.
Most bacterial infections can be treated with antibiotics which are chemicals extracted from fungi or other bacteria. Penicillin was the first antibiotic drug. It was discovered by Alexander Fleming (1881 - 1955), isolated from the Penicillium mould, and commercially produced using biotechnology. They can be swallowed or injected to kill internal bacteria or prevent them from multiplying, although this is not an instantaneous process. However, as we use more and more antibiotics, some bacteria are becoming resistant to them. One strain of Staphylococcus aureus is resistant to all known antibiotics except one, but this drug can have dangerous side-effects. Contributing factors to this resistance include the over-prescribing of antibiotics for people and for farm animals, and patients not finishing their course of the drugs. Antibiotics cannot treat viral infections, and yet many people expect their doctors to prescribe antibiotics for colds and flu, which of course are viral.
Disinfectants, such as bleach, are powerful chemicals used to kill micro-organisms in the environment. Antiseptics are weaker chemicals applied to wounds and sores to prevent micro-organisms from multiplying. Specific fungicidal chemicals are effective against the few fungal micro-organisms that live on our skin such as ringworm and athletes foot.
Decomposers Fungi and most bacteria are saprotrophic and have an important role in an ecosystem as decomposers, breaking down dead or waste organic matter and releasing inorganic molecules. These nutrients are taken up by green plants which are in turn consumed by animals, and the products of these plants and animals are eventually again broken down by decomposers.
Sewage treatment employs bacteria which break down harmful substances in sewage into less harmful ones. Aerobic bacteria decompose organic matter in sewage in the presence of oxygen. Once the oxygen is used up the aerobic bacteria can no longer function, and anaerobic bacteria continue the decomposition of organic matter into methane gas and carbon dioxide, along with water and other minerals. The digested sludge is rich in nitrates and phosphates and can be spread on the land as fertiliser. Some sewage treatment plants have used the methane as a cheap form of fuel (biogas). Anaerobic micro-organisms are also being used to convert carbohydrate-rich crops, such as cane sugar and maize, into ethanol which is used as a substitute for petrol in cars. This biofuel (or gasohol) is used widely in Brazil, which has meagre oil resources.
The carbon cycle Fats, carbohydrates and proteins all contain carbon atoms, so dead and waste organic matter contains a lot of carbon. In breaking this down, saprophytic bacteria and fungi take up some carbon to build their own bodies, and release some as carbon dioxide during respiration. However, the carbon cycle need not involve decomposers because autotrophs can access carbon from the abundant carbon dioxide in the air.
The nitrogen cycle All living things need nitrogen, it is an essential component of all proteins. It makes up 79% of the air we breathe, but the N2 molecules are very stable and unreactive, and are not readily accessible to plants and animals in this form. Nitrogen-fixing bacteria are able to convert (or fix) nitrogen gas from the air into nitrogen compounds. Plants take up these nitrogen compounds through their roots, combine them with products of photosynthesis, and make proteins. Animals obtain the protein they need by eating the plants or other animals. Some nitrogen-fixing bacteria are free- living in the soil, others live in small swellings, or nodules, on the roots of some plants, particularly members of the legume family (such as clover, peas and beans). This is a symbiotic arrangement, the plant gets nitrogen compounds and the bacteria receive carbohydrates from the plant. Dead and waste organic matter contains ammonium compounds which are converted by nitrifying bacteria into nitrates, and these are assimilated by plants. Denitrifying bacteria remove nitrates and ammonium compounds from the soil by converting them into nitrogen gas.
Digestion Despite the vast quantities of cellulose eaten by herbivores, mammals themselves cannot digest cellulose and rely entirely on the action of carbohydrate-digesting bacteria in their guts. These secrete the enzyme cellulase which splits the cellulose into monosaccharides which can be absorbed by the gut. Ruminants (cud-chewing) mammals such as cows have a large chamber in the stomach called a rumen which contains huge numbers of these bacteria. Non-ruminant herbivores such as rabbits and horses have cellulose-digesting micro-organisms in their appendix and caecum which act as fermentation-chambers. Huge numbers of bacteria, particularly Escherichia coli, also inhabit the human colon. There are an estimated four hundred species and it has been suggested that the action of some of these on carbohydrate can contribute up to 10% of our energy requirements. Other bacteria synthesise vitamins and amino acids, and others may contribute to our resistance to disease by competing for space in the gut with harmful bacteria. It is important therefore to maintain a healthy gut flora.
Biotechnology The manipulation of cells, particularly micro-organisms, to produce useful substances is referred to as biotechnology. Micro-organisms are exploited extensively in the fields of medicine, agriculture, food production, waste disposal and many other industries. We make use of some saprophytic bacteria which do not produce waste products harmful to humans. The bacterium Lactobacillus feeds on milk, turning it into yoghurt. Other bacteria and fungi help in cheese-making and are responsible for distinctive flavours. Most industrial enzymes (protein catalysts) come from micro-organisms. Special strains of fungi and bacteria are developed by genetic engineering. They are grown in large fermenters where they secrete enzymes into their nutrient solution. The enzyme is isolated and concentrated for use. Examples of such enzymes include amylases for producing chocolates, fruit juices and syrups; cellulases for softening vegetables; proteases for tenderising meat and for removing biological stains when put in biological washing powders.
Yeast is a single-celled fungus that lives naturally on the surface of fruit. It is economically important in brewing and bread-making. Yeast respires anaerobically (i.e. without the use of oxygen) and breaks down glucose with the production of carbon dioxide, ethanol (alcohol) and energy. In wine-making the yeast feeds on fruit sugars in the grapes, and in beer-making it feeds on the maltose sugar in germinating barley. The term fermentation, is usually applied to this process of anaerobic respiration in which alcohol is produced. Controlled oxidation of alcohol can be carried out to produce vinegar (ethanoic acid). Bread-making uses the carbon dioxide produced by anaerobic respiration, not the ethanol. Starch in the dough breaks down to sugar, which feeds the yeast. The carbon dioxide bubbles make the dough rise before it is baked into bread.
Yeast, including that left over from brewing, and other micro-organisms are also cultivated as an important food source for farm animals, and for humans. When fed on simple sugars and inorganic salts in controlled conditions, these micro-organisms can double their mass within hours (plants and animals may take weeks). They are rich in protein and contain most of the essential vitamins and amino acids required by animals. The mould Fusarium is grown in this way to form a mycoprotein which is as nutritious as meat, but lower in cholesterol and higher in fibre. It is marketed as the meat substitute Quorn. This kind of high protein food produced from micro-organisms is called single-cell protein, and it is increasingly grown on the nutrients present in industrial waste (e.g. from food, paper-making and agricultural industries).
micro-organisms Micro-organisms can be grown in a sterile Petri dish on agar jelly which contains appropriate nutrients. After introducing a small sample of water, soil, leaf, etc., the lid should be permanently sealed. After several days the micro-organisms will have grown and multiplied. The colonies become visible due to the multiplication of the cells, not due to cells getting larger! Fungi usually appear as furry clumps and bacterial colonies are often smooth and shiny-looking. After inspection, the sealed dishes should be sterilised in an autoclave or strong disinfectant, in case any pathogens have been incubated.
ContentsDiversity of organisms
Ecosystems and habitats
Self assessment (1)
Self assessment (2)