9. E. coli in the Food Chain.

(a) E. coli in the slaughter house.

Direct ingestion of E. coli in food is most likely the source of human colonisation. Animal carcasses are generally contaminated with their intestinal E. coli, even under very good conditions in the slaughter-house. Studies in the United Kingdom in the 1970's showed that there was considerable contamination of the carcasses by the animals' own intestinal E. coli as well as there being extensive interchange of E. coli between carcasses. In another later study on a sheep slaughtering line in an abattoir in New Zealand, it was noted that the animal's own rectal E. coli tended to be washed away during the very heavy hosing down which was done after the removal of the intestines. However, the carcasses were recontaminated by other E. coli from the environment, which presumably were derived from the intestines of animals slaughtered previously. The spread of both E. coli and antibiotic resistance markers was quite extensive despite the hygienic standard of this abattoir being very high.

A United Kingdom poultry packing station was also investigated as part of the studies discussed earlier. E. coli could be isolated from the chickens and their giblets throughout the processing stages. They were also isolated from the various waters used in the feather softening processes and cooling tanks.

Similarly in an extensive study of the contamination of pig carcasses in two abattoirs, some E. coli types were found throughout the stages, while others made only transient appearances. It some ways this is reminiscent to the situation in an intestinal tract. A generally high level of antibiotic resistance was found among the strains, and as the pigs came from a variety of sources, it is suggested that this reflected the state of pig rearing at that time. As these strains were shown to be able to survive the chilling process and they thus could be considered as potential sources of human colonization. Although most of these studies were carried out around 20 years ago, and there have been no studies in modern slaughter houses, the continuing high infection rate with Gram-negative enteric pathogens, including now emergent E. coli O157, suggests that this contamination rate is still continuing.

(b) E. coli in food.

A study in 1970 in which the contamination rate of hospital food by E. coli was examined clearly demonstrated a strong correlation between the faecal types found and those contaminating the food ingested by the patients. There appeared to be a continuous flux of E. coli between the patients' food entering the ward environment and their faeces.

In a survey of over 4000 samples of retail processed foods in the United Kingdom a carriage rate of 12% overall of E. coli was noted, with over a quarter of the cakes and confectionery being contaminated and only 9% of meat and meat-based products being contaminated. However, these isolates from meat products were more likely to be antibiotic resistant. Over 103 E. coli/g. food were isolated from 27% of foods.

In the developing world food has often been associated with the transmission of pathogenic E. coli and Enterotoxigenic E. coli have been found on raw meat, as well as the hands of operatives and their equipment. Similarly dairy products yielded both pathogenic and antibiotic-resistant E. coli

A series of feeding experiments were carried out on adult human volunteers. They ingested specific E. coli types in numbers ranging from 105 to 108. These numbers were chosen, because it was considered that they would mimic the numbers, which had been found in hospital food which varied between 25 and 104 E. coli per gm. In all 25 such feeding experiments were undertaken and in 24 of them the ingested type could be recovered from the person's faeces subsequently. In four of the experiments the ingested strain was found to persist for more than a month. Thus ingested E. coli will colonize.

10. Biochemical Characteristics of Escherichia coli.

(a) Early Biochemical Studies

Most studies on bacterial metabolism and biochemical processes have been carried on E. coli. Initially E.coli were considered commensal organisms and most of the investigations on these organisms at the turn of the century were concerned with the problems of being able to distinguish the "agents of typhoid fever" and other types of Salmonella from them. It was predominantly these investigations which led during the first few decades of this century to the development of the biochemical tests which are the basis of modern bacterial taxonomy. The demonstration that there were a number of types and subtypes of these organisms, which could easily be distinguished from the typhoid bacillus by these tests, became the basis of the taxonomy of the Enterobacteriaceae in general and E. coli in particular.

(b) Carbon and Energy Sources.

All livings organisms on this planet use carbon as the basic element of their sturucture and energy in some form is required to convert carbon compounds obtained from the environment into their own structure and to maintain the continuous turnover of chemical building blocks of the cell, known as metabolism, which is the basis of life.

E. coli are typical members of the Enterobacteriaceae, which are characterised by being able to grow aerobically in the presence of air or oxygen as well as anaerobically in the absence of oxygen. They are able to utilise simple carbon and nitrogen sources for all their metabolic and energy needs. Thus, E. coli can multiply effectively in a medium containing only glucose, ammonium and mineral salts. While a variety of other carbon-containing substrates can be similarly utilized, glucose is by far the preferred substrate and specific mechanisms are available to import glucose into the cell.

Glucose is transported across the cytoplasmic membrane via a very specific transferase system and competitively inhibits the uptake of other sugars using this transport mechanism. It is also able to inhibit the uptake of sugars not using this system by a variety of mechanisms. Glucose controls the level of initiation of transcription of the enzymes required to metabolise other substrates.

In E. coli a substance cyclic AMP (cAMP) acts in many reactions as a moderating element in the same way as hormones act in humans. Most substances are broken down by a series of enzymes acting in sequence, each causing a small change, which ultimately leads to the complete breakdown of the substance. The genes controlling such a series of enzymes usually are arranged in series along the chromosome known as an operator. A promoter region at the beginning of the operator must be activated for the RNA polymerase enzyme to make the messenger-RNA (m-RNA), which causes the specific enzymes to be made.

In forming a complex with a specific protein, known as the catabolite-gene activator protein (CAP), cAMP must bind to the promoter region of an operator on the genome, to enable the binding of the RNA polymerase and the subsequent initiation of transcription to occur. The metabolism of glucose decreases the intracellular concentration of cAMP, causing an inhibition of the enzymes, which are coded for by the various operons that require activation by the cAMP-CAP complex. This phenomenon, which was termed catabolite repression as long ago as 1961 also appears to have an important role for a phosphorylated enzyme of the glucose transferase system. These two linked mechanisms, the exclusion of the inducer and prevention of induction of the catabolic enzymes by lowered cAMP concentration, cause the preferential utilization of glucose.

E. coli thus exhibits the phenomenon of diauxic growth in the presence of mixtures of glucose and another carbohydrate substrate. It will preferentially grow on glucose and not utilise the other substrate until the glucose is fully used up. Then after a lag phase, during which it adapts itself to the new substrate, it will start growing on that one instead.

E. coli can also utilize fatty acids and acetate as sole carbon and energy sources. Apart from acetate, fatty acids must be at least 12 carbon atoms long in order to be able to act as sole carbon and energy source, and then only after a distinct lag phase. This requires the coordinated induction of the synthesis of at least five fatty acid-oxidative enzymes. In addition, the glyoxylate shunt a series of enzymes designed to maintain the presence of complex carbon compounds, is required to be in operation when E. coli grows on acetate or fatty acids and the two unique enzymes of the shunt, isocitrate lyase and malate synthetase A are induced when E. coli grows on these substrates.

(c) Nitrogen Sources.

Apart from carbon, the other most important constituents of all living organisms are hydrogen, oxygen and nitrogen. Of these hydrogen and oxygen are ususally obtained from water, which acts not only as a diluent and solvent but plays an active role in all metabolic activities. There is a continuous interchange between these elements within the water environment and the cellular constituents. With nitrogen the situation is different. Although elemental nitrogen is the most common constituent of the atmosphere of this planet, it is chemically very inert in this form and generally unavailable. There are a number of bacterial groups that can utilise atmospheric nitrogen and bring it out of the atmosphere into the biosphere or the place where life maintains itself. This phenomenon is known as nitrogen fixation and the bacterial groups capable of performing this are known as nitrogen fixing organisms. E. coli cannot fix nitrogen and is therefore dependent on non-atmospheric nitrogen compounds for its nitrogen requirements.

Ammonium sulphate is the preferred nitrogen source for E. coli. There are a number of enzymes involved in the synthesis of the essential amino acid glutamine and the products of genes closely linked to the structural gene for glutamine synthetase are associated with the regulation of the various genes involved in the control of nitrogen metabolism. While a number of members of the family Enterobacteriaceae to which E. coli belongs, can grow aerobically with nitrate or nitrite compounds as the sole source of nitrogen this has not been demonstrated in E. coli. It appears to lack the ability to form an enzyme nitrate reductase, which is required to assimilate nitrate or nitrite.

However, the situation is more complex as E. coli does produce a nitrite reductase. Its induction is not controlled by the availability of ammonia, but by the absence of oxygen. The nitrite is derived from nitrate by reduction but at high concentrations nitrate suppresses nitrite reductase synthesis and E. coli grown anaerobiaclly in high concentrations of nitrate may not be able to utilise the resultant nitrite effectively. The ammonia formed by the anaerobic reduction of nitrite can be utilised by E. coli as a source of nitrogen in the usual way. Thus E. coli can grow anaerobically on nitrate as sole nitrogen source but not aerobically.

Approximately a quarter of the total nitrite reductase activity in E. coli is linked to the oxidation of formate derived from the breakdown of glucose. This pathway may be involved in energy conservation. Nitrate respiring cells resemble aerobic cells by using a respiratory chain. In addition, they contain low levels of alcohol dehydrogenase and formate-hydrogen lyase. Unlike anaerobic cells they have an incomplete tricarboxylic acid cycle and excrete acetate. These enzyme systems involved in resipiration will be discussed below.

(d) Metabolism of Glucose by E. coli.

Glucose is the main preferred metabolite as carbon and energy source for E. coli, as has been discussed ablove. Where it is unavailable, then other carbohydrates may be able to be utilised. However, in most cases these are first converted either to glucose or to a breakdown product of glucose. Thus in order to gain an understanding of how an organism like E. coli obtains its carbon and energy requirements, it is important to understand the processes involved in the breakdown of glucose.

The process of degrading glucose anaerobically will be described first. This is known as fermentation and is similar though not identical to the way yeast converts sugars to alcohol when bread or alcoholic drinks are made. E. coli uses two pathways to degrade glucose. These are the hexose monophosphate pathway or the glycolytic pathway. The former is also an important source of building blocks for biosynthesis, while the latter is mainly involved in the breakdown of glucose to provide energy. While E. coli will grow normally on glucose even if the hexose monophosphate pathway is blocked, it can only limp along if it has to rely on the complete hexose monophosphate pathway.

The basis of the glycolytic pathway is the phosphorylation of the sugar to the phosphate and by a series of steps it is oxydised to pyruvic acid. Glucose, which is a six carbon molecule becomes two molecules of pyruvic acid, which are three cabon molecules. In this process the number of complex phosphates of the cell, which are its main energy currency are increased. This conversion of glucose to pyruvic acid does not require oxygen. E. coli then completes the anaerobic fermentation by converting pyruvate to acetic acid by a process known as mixed-acid fermentation. Acetic acid has only two carbon atoms and the third is lost as carbon dioxide (CO2). An additional high energy phophate molecule is formed. As organisms such as E. coli tend to live in areas where there is not a great wealth of free carbohydrates, they can afford to have acetic acid as an end-product, because the level of acidity achieved will not be too high to inhibit the bacteria. Nevertheless, it should be noted, that E. coli growing anaerobically on glucose or another fermentable carbohydrate will significantly increase the acidity of the medium and produce gases including CO2 and hydrogen.

In the presence of oxygen, the excess acetate, which is not used for biosynthetic processes will be completely broken down to CO2 and water, with significant release of available energy. This breakdown proceeds through the tricarboxylic acid cycle. Basically in this process the acetate, linked to a large molecule is combined with a four carbon acid (oxaloacetic acid) to form a six carbon acid. This is degraded through a series of oxidative steps causing the release of CO2 at two points till oxaloacetic acid is reformed, which can accept a new molecule of acetic acid. Some of the acids of the tricarboxylic acid cycle are removed from the system because they can be used as building blocks for amino acids so a mechanism is in place in which acetate can be used to rebuild some of the acids thus mainting the operation. Acetate can also be used directly for the synthesis of lipids.

The alternative pathway used by E. coli to degrade glucose is purely oxidative and following phosphorylation, proceeds through intermediate stages of pentose phosphates, which are phosphates of five-carbon sugars. These pentoses are important for the formation of nucleic acids because both the ribose of RNA and the deoxyribose of DNA are such pentoses and thus the major backbone of the genetic basis of the E. coli is directly derived from the glucose in a process, which also provides the organism with energy. The spare carbon atom is released as CO2.

(e) How E. coli can Respond to the Environment.

By controlling the activities of existing enzymes, E. coli is well able to respond to changes in its environment. Three different types of such rapid action control mechanisms have been identified. These are modulation, covalent modification and selective inactivation.

Many enzymes have more then one area, which responds to chemical stimulation. There is the obvious area which affects the enzymic change by converting a substrate into a product. Other areas may respond specifically to certain small molecules, which can alter the three-dimensional structure of the enzyme, such that its enzymic activity is inhibited. Thus in a biosynthetic pathway, in which a series of enzymes are involved in converting a substrate 'A' to an endproduct 'G' through a series of intermediates 'B', 'C', 'D', 'E' and 'F'. the endproduct molecule 'G' may bind specifically with the enzyme which converts 'A' to 'B'. Thus if there is too much 'G' being made, or becomes available from outside, it will be present in excess bind to the 'A'-'B' converting enzyme and prevent the E. coli from continuing to make something it does not need. If, however, 'G' suddenly runs out, it will not be available to inhibit the 'A'-'B' converting enzyme and the synthetic pathway to 'G' will be open again. This process, which is also known as feedback inbition is one example of mudulation.

An example of enzyme regulation by the covalent convertion of an enzyme can be taken from the biochemical synthesis of the amino acid glutamine from ammonia and glutamate by the enzyme glutamine synthetase. This enzyme consists of 12 identical subunits. Each subunit can reversibly have a specific tyrosyl residue combined with the purine adenine, by a process known as adenylation. When it is completely unadenylated, its biosynthetic activity is at maximum level, while the fully adenylated enzyme is biosynthetically inactive. The activity varies over a wide range depending on the degree of adenylation. In addition, this enzyme is also subject to feedback inhibition and its activity is controlled by the concentrations of divalent metal cations.

When E. coli has to adapt itself to a major change in the availability of nutrients, then some enzymes are selectively inactivated. Particularly affected are those, which may no longer be required or ones which might even be harmful in the new conditions. This is known as selective inactivation. Either the enzyme protein is modified or even degraded.

There are certain categories of nutrients, which are preferred by E. coli. Thus, it will grow more rapidly with glucose as carbon source than with acetate. If the glucose is supplemented with vitamins, amino acids, nucleotide precursers, etc. it will grow even more rapidly. Every cellular building block, which it does not have to make, will be an energy saving and this saving will go straight into increased growth.

(f) Growth Rate.

With increase in nutrients, the rate of growth of the E. coli rapidly changes. Concommitant with these, there are also internal changes within each cell. As the cells become larger in the fast growing cultures, there are formed more replication forks on the chromosome. This means that the chromosomal DNA is replicating faster, with the two strands separating at the forks and new partner strands forming on each single strand.

Simultaneously there is an increase in the RNA/mass ratio with growth rate as more ribosomes are formed in the fast growing cells. These are the structures in which the messenger RNA is read and the appropriate protein synthesised from the separate amino acids. However, the DNA/mass and protein/mass ratios remain fairly constant at different growth rates. If the E. coli are subjected to a sudden change in the nutritional level such as changing of the carbon source from glucose to acetate or the removing of amino acids from the medium causing a nutritional downshift, then there occurs the so-called stringent response, which is linked with a cessation of stable RNA accumulation.

Glucose has been shown to be able to support a far higher growth rate than acetate. The explanation for this is believed to be that the energy requirement per unit molecule of pumping one molecule of acetate into the cell is the same as for one molecule of glucose. While acetate contains only two carbon atoms, glucose contains six, thus around three times more energy is obtained by the metabolism of glucose compared to the energy, which must be required for their actual pumping into the cell. Extensive observations on different substrates have revealed that an equal amount of energy per unit time is consumed by E. coli over a wide range of doubling times with a wide variety of substrates.

It is not surprising that the growth rate of E. coli increases the more the surrounding medium is supplemented with the various utilizable biosynthetic building blocks such as amino acids, nucleosides, bases and vitamins. Although E. coli is self-sufficient, only requiring glucose, an avaialble nitrogen source and some minerals for growth, being able to make all the macromolecules it needs, like all living organisms it can be considered lazy. If it does not have to make a complex series of enzymes to synthesise a substance like a vitamin, why bother. The mechanisms of feed-back inhibition and other controls will prevent enzyme systems being in operation, which are not required. Therefore, if the available carbon sources and energy are not required for the synthesis of these building blocks, then they can directly be shunted to form the more activated precursers for the biosynthesis of the macromolecules.

The genes, which code for the synthesis of the products contributing to the macromolecular apparatus of protein and RNA synthesis have very high maximal rates of expression. In a rich medium E. coli can grow very fast, because now the rate is not depenedent on the slower expression of the other enzyme system but only on the rate of these fast systems. This suddenly makes the E. coli cell dependent on a large pool of precursers. This is not available when the organisms grow in minimal conditions, and then the rate of growth decreases according to the availability of these precursers.

Some researchers have recently described the E. coli cell as an unsaturated system, which is specifically designed for rapid growth, but which is limited by the "feeder reactions" and the suspending medium. Several growth-related phenomena may be explained from medium-induced changes in the cell-elongation kinetics during protein and RNA synthesis. If cells are growing too fast in rich medium, they may not divide as often as they grow and thus form long cells.

(g) The Importance of Iron.

Of the mineral elements, which have been mentioned in passing as required by E. coli in addition to a carbon, energy and nitrogen source, available iron is probably the most important. While elements like potassium, chlorine, magnesium, zinc etc. are also essential, the requirements are in such low quantities, that great effort has to made to exclude them, they are so ubiquitous and the requirements are so comparatively low. This is not so for iron. The availability of iron is a very important factor for E. coli to be able to grow in an animate environment. Within a human or animal body all the nutrients, just discussed are available in large amounts and the E. coli certainly has feast conditions. However, an important protective strategy of humans and animals against bacterial pathogens and bacteria just living off the avaiable nutrients is to make iron unavailable. However, E. coli has the ability to absorb iron from the environment.

Within a human or animal body there is usually plenty of iron, but the amount that a microorganism like E. coli can use is generally extremely small, because most is held within cells as haem or ferritin. Even, when it is outside the cells it is attached to iron binding glycoproteins, known as transferrin and lactoferrin. These glycoproteins have an extraordinaryly strong binding power for iron, thus the concentration of free iron molecules is rarely more than 1:1018. As such concentrations cannot support the growth requirements of E. coli it has developed very specific mechanisms to obtain free iron from the environment.

One of these mechanisms is based around the substance Enterobactin (or Enterochelin). E. coli produces low-molecular weight, high-affinity iron-binding compounds known as siderophores, like many other bacteria. Under conditions of iron limitation, E. coli produces enterobactin, which is a complex cyclic triester. It removes iron from iron-binding proteins and transports it into the bacterial cell. The formation constant of ferric enterobactin is around 1052, which means it can drag iron out of a complex against a concentration gradient of 1:1052. The production of this substance is very expensive for E. coli, because the products of seven chromosomal genes are required to synthesise enterobactin. Also it can only be used once, because the molecule is cleaved within the E. coli cell and the remnants discarded for the iron to become available. It thus requires the product of seven enzymes to be synthesized for each iron atom to be brought into an E. coli cell. Also enterobactin is bound by serum albumin, which prevents its action.

Aerobactin is another type of siderophore, whose biosynthetic genes can also be plasmid mediated. The carriage of such plasmids has been associated with virulence particularly among E. coli causing extraintestinal infections. Aerobactin forms an octahedral complex with ferric iron. This siderophore can be recycled and unlike enterobactin, it is not bound by serum albumin.

E. coli can avoid making it own siderophores, being quite capable of using fungal siderophores if they are available. E. coli can also use citrate to obtain iron although this siderophore is rather inefficient.

Like all Gram-negative bacteria E. coli has Outer Membrane Proteins (OMP). An iron-loaded siderophore appears to have to be initially adsorbed on the surface of the E. coli so that the iron can be concentrated to appropriate levels for the growth requirements. These OMP receptors often also function as receptors for bacteriphage and/or colicins. Futher proteins are required by most of these receptors to complete the movement of the iron into the cells and it has been suggested that these receptors all act as 'gated porins', i.e. specific proteins, which can act as gates into the cell for specific substances.

In general E. coli will preferably take up iron from haemoglobin instead of using siderophores. The enterobactin-mediated iron-uptake systems may be involved in the transport of the haemoglobin-derived iron. E. coli makes a number of haemolysins, which break up red cells and thereby release haem and haemoglobin which are rarely present free in tissue fluids. In this way these iron-compounds become available.

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