Escherichia coli

1) Introduction

There is probably more known about these bacteria then about any other living creature on this planet including us human beings. We know most of the full structure of their nucleic acid, we know about a larger number of their proteins and carbohydrates, about their cell walls and nevertheless we are still learning more about them every day. More scientific papers are still being published about this species of bacteria then about any other living creature, averaging about 100-200 papers per week. So, what is it that makes these bacteria so well known and so much studied.

Where they live is not the most exciting place! They live in our intestines and are excreted daily with our faeces. There are present 108 to 109 individuals of the species of Escherichia coli (E. coli) in one gram of faeces of an average individual. As the daily faecal mass averages per individual about 200 g, this will contain at least 2 x 1010 to 2 x 1011E. coli. Therefore the human global population of about five billion (5 x 109) will excrete every day 1020 to 1021 E. coli into our environment. As in most parts of the world human sewage is not adequately treated it can be estimated that about 90% of these will initially survive. If they go straight out to sea, they will not survive very long in the salt water, but many will survive long enough to be ingested by seabirds foraging near a sewage outflow. These seabirds are likely to drop their E. coli onto pastures or other places where humans or their domestic animals may reacquire them.

2) What are E. coli?

E. coli are cylindrically shaped bacteria, which are between 0.3-1.0 µm in diameter and between 1.0-6.0 µm long. Such cylindrical bacteria are usually termed rods or baccilli, as opposed to spherical bacteria, which are known as cocci. They do not form spores. They do not retain dyes in the presence of organic solvents or acids, which are means of distinguishing the Gram-positive bacteria, which retain dyes in the presence of organic solvents and acid-fast bacteria which retain them in the presence of acids. Thus E. coli are described by bacteriologists as Gram-negative, non-acid-fast non-sporing rods or bacilli.

Most E. coli are motile by means of flagellae, which are strings of protein made in the shape of a corkscrew. They are continuously replenished from inside as they may be broken off. They are attached to a hook like structure embedded in the cell wall which rotates around 360° causing the screw like flagellae to push the bacteria through the water. To change direction the direction of rotation is reversed. The flagellae are arranged all over the body of the E. coli unlike other bacteria where they may be attached at one pole. This type of flagellation found in E. coli is known as peritrichous as opposed to polar.

E. coli can also have long straight or protein structures extruding from their cell wall. These are known as fimbriae or pili. There are a number of different types of fibriae, dependent on the differences in specificity of the material to which they attach. They are mainly used to attach to surfaces and to interact with other members of E. coli.

They have a firm cell wall comprising of complex lipopolysaccharide molecule, i.e. made up of fats and carbohydrates. It maintains the physical inegrity of the cell. The cell wall has on its surface, differing structures of sugar complexes, which give each type of E. coli a different chemical face it shows to the world. There are nearly 200 different types of such carbohydrate "outer decorations" known among the E. coli. They confer on the carrying bacterium a unique immunological specificity. Thus if a person has been infected with an E. coli of type "1", and have made antibody to that type, the antibody will only react and bind to type "1" and not any of the other nearly 200 types.

Many E. coli also have on their outer surface a capsular material. This may be only a layer of slime or it may ba a defined chemical structure. This can make the E. coli slippery and aid it in eluding the capture by a protozoon in water or a macrophage during an infection process.

Exterior to the cell wall there is also an outer membrane, which plays an important role jointly with the membrane internal to the cell wall, in controlling the entry and exit of substances. E. coli can release into the environment substance such as enzymes, which will break down molecules or substances too large to be internalised directly. Haemolysins, which are enzymes capable breaking down red blood cells to release the haemolglobin and iron, are an important group of enzymes that some E. coli can release. Others include toxins involved in disease process which will discussed separately and protein and carbohydrate splitting enzymes.

An important factor for an organism like E. coli to grow, particularly in an animate environment, is the availability of iron. E. coli has the ability to absorb iron from the environment. These iron-uptake abilities of E. coli were dependent on the production of specific proteins specific for the uptake of iron, such aerobactin and enterochelin. Iron is also strongly bound in a human body or animal and generally unavailable to bacteria. Thus these E. coli iron-uptake proteins strongly compete with the host for iron. If an E. coli can make a haemolysin then iron may be available in excess not just for it but other strains of E. coli as well as other bacteria.

Within the bacterial cell are all the structures involved with growth and multiplication. These include the ribosomes on which all the specific proteins are made according to the instructions on the messenger RNA copied from the genomal DNA. The DNA is present as a supercoiled circular strand, which if streched out would be up to 1.4 mm long. In addition there may be present in some E. coli small separate coils of DNA derived from bacterial viruses (bacteriophages) or obtained by transfer from other E. coli. These separate DNA pieces may confer on the E. coli resistance to a number of antibiotics, the ability to grow on certain unusual substrates, to produce certain virulence factors, and many other characteristics many not known or any combination of these.

3) E. coli in Natural Waters

E. coli are widely distributed in waters including natural waters, particularly in tropical regions of the globe. High densities of E. coli have been detected in pristine streams and in groundwater samples in many parts of the world. They have even been found in epiphytic vegetation 10 m above ground in rain forests. Detailed analyses have shown many of these isolates to be indistinguishable from clinical isolates. A number of studies from many parts of the world have shown the presence of viable E. coli, which cannot be cultured on the media currently available. Even the non-chromosomal DNA is stably maintained in these viable but non-culturable forms of E. coli in a variety of waters. These non-culturable forms, which develop as a result of starvation conditions, may revert to culturable forms following ingestion into human or animal intestinal tracts.

In lakes or rivers, they can survive for quite long times, but are usually in a near or actual starvation state. They can live with very little nutrient (food). All they need to survive is some form or carbohydrate like a sugar and a source of nitrogen such as an ammonium compound. E. coli have been found in pristine waters in tropical rain forests and in arctic lakes. They are found in water reservoires providing potable water for cities. Numbers in such reservoires which have been reported vary from 1000 bacteria per ml of water upwards to a few thousand per ml. It is only subsequent treatment and chlorination, which largely eliminates them from the tap water which we in the developed world drink. However, in many parts of the developing world the only water available for drinking will be highly contaminated with E. coli.

In lakes and other still waters including reservoirs E. coli have been shown to be able to grow and multiply at a temperature range of 15-40°C and numbers as high as 3000 per ml. have been observed. It should be noted that before there would be seen any cloudyness in water to the naked eye, there have to be at least 1,000,000 per ml. While in some cases such numbers can develop as a result of faecal pollution, it should be apparent that under the right conditions of nutrients and temperature the E. coli can bloom and multiply to such numbers by themselves.

In a study in Finnland at 69°N in small brooks, rivers and small lakes, in an area devoid of human settlements, agriculture, industry or any type of human activity or technology found around 1-100 E. coli per ml. The numbers were high in winter and spring and decreased in summer rising again in autumn. The summer decrease is probably due increased bacterial die-off at higher temperatures, while the reduced light intensity in winter and the runoff due to melting snow increase E. coli survival.

The E. coli can protect themselves from the adverse effects of sunlight by sheltering in shade provided by floating mats of algae, in sand, amongs sea grasses and on tropical coral reefs. In addition, attachment and association with such natural agents provides E. coli with protection from the lethal effects of such antibacterial agents like chlorine. As an example, it has been shown that E. coli is 2400 times more resistant to chlorine when attached to such surfaces, then when free in water.

4) E. coli in Starvation Conditions.

When E. coli are deprived of nutrient, as can happen in such waters, they enter into a stationary phase, in which no multiplication occurs. This development is under strict genetic control. A chemical signal molecule (ppGpp) is induced, which switches the transcription from the situation during normal growth to a specific starvation level. This induces the production of a series of stress proteins which give the E. coli ability to resist a multiplicity of stresses. When these proteins have been made, protein synthesis drops off markedly drooping by 11 days to around 0.05% of that found in a normally growing culture. The proportion of individual E. coli staying alive varies with the conditions underwhich starvation occurs.

Initially during starvation, there are also a number of cell divisions, without increase in cell mass. This causes the cells to become smaller and more round than normal. The internal cytoplasm is more condensed and the cell envelope becomes covered with the type of water-resistant molecules, which, which promote bacterial adhesion leading to aggregation. Some E. coli even produce curli fibres, which cause them to clump together and aids adherence to human or other host cells.

While cell wall synthesis ceases, the wall nevertheless thickens and thus the cells become less likely to autolyse. Also membrane synthesis ceases and the membranes become less fluid and less permeable. These and other factors make the E. coli more resistant to a number of stresses, which would normally significantly affect them. They become more resistance to further starvation, temperature shifts, acid, high osmotic pressure, including salinity, UV radiation, oxidative stress and antibiotic uptake.

It should not, however, be considered that they are not metabolically active in this starvation state. They are able to respond immediately, if nutrients become available As they grow they rapidly lose the various resistances to the stresses, which they acquired during the starvation phase. It is often believed by the lay public that when bacteria lack nutrient, they die. Of course, ultimately they will die, but as this section has shown they are not only very active under starvation conditions, but are also more resistant to other stresses and thus can survive situations, which normally growing E. coli would not be able to survive. This should be particularly taken into consideration, when untreated waters even apparently pristine waters are used in such activities as the preparation and cleaning of foods.

These E. coli, after extensive starvation can enter into a dormat state, in which they remain alive, in that they respire and under certain conditions will revive and multiply. In general these dormant E. coli cannot easily be cultured under normal circumstances on the sort of media, on which E. coli will normally be grown. For these E. coli the term viable but not culturable has been coined.

5) E. coli Survival under Stress.

E. coli are very good at surviving stresses and have developed different mechanisms to respond to different sub-lethal stresses. While the survival mechanisms are tailored to specific stresses, they often involve similar networks of factors and it is for that reason that E. coli which have responded to one stress are often also more resistant to other stresses.

Exposure to increased sub-lethal temperature has been shown to cause the rapid production of a series of special proteins. These have been called the heat-shock proteins and their function is to alleviate the natural damage, which heat is likely to induce in other proteins. In a way they act like chaperones to the normal proteins, which may have become unfolded, folded into unnatural ways or aggregated and refold them and deaggregate them. If proteins have been altered such that they cannot now go through membranes, they assist in this process. Inactivated proteins are reactivated and they even prevent inactivation of proteins. If, however, proteins have been inactivated into some form, which cannot be repaired, then the heat-shock proteins will degrade them so that they can do no further damage.

If E. coli in the viable but not culturable form at room temperature as would be found in inadequately treated drinking water are given a heat shock of only 35°C for twenty minutes such as would occur if that water were ingested there will be a sudden thousand fold increase in numbers. This clearly shows that ingestion of water containing a thousand per ml. apparently non-culturable E. coli and thus appearing apparently clean, will contain twenty minutes after ingestion a million fully viable E. coli per ml.

It has to be noted that bacteria like E. coli have to maintain their shape and size. For this purpose they use osmoregulation, i.e. they regulate their internal osmotic pressure with respect to the external osmotic pressures. Not only can they detect unfavourable osmotic pressures such as high salt concentrations, they will swim away from them if able to, as most E. coli are, which have flagellae.

Apart from controlling the increased flagellar synthesis and enhancing the flight response, increased osmotic pressure causes two further responses. The protein composition in the membrane is altered so that the uptake of molecules into the cell is more strictly controlled. In addition the uptake of small molecules and ions, which are non-toxic and would increase the internal osmotic pressure, if present outside the cell is enhanced. The type of molecule or ion taken up under such conditions particularly includes potassium ions and some amino acids. The carbohydrate trehalose is synthesised and it acts as both a protecting substance for increased osmotic pressure and temperature.

As has been written earlier, E. coli is not particularly well adapted to sea water. In a marine environment, the growth of E. coli is reduced and the viable but not culturable state is induced. If the type of substances, which have been discussed above as protecting E. coli from osmotic pressure are present, then the E. coli will be protected. It is noteworthy, that it is just these substances, which are excreted by the various algae and photosynthetic marine microorganisms and thus the presence of these in the marine ecosystem will also protect E. coli.

Similarly, protective substances are present in the urine and these are able to protect the uropathogenic E. coli from the higher salt concentration present in urine and the kidneys. Mechanisms thus designed to protect E. coli from adverse environmental pressures in the inanimate environment, have been harnessed to protect pathogenic E. coli from host defense mechanisms.

As has been aluded before, E. coli has to protect itself from sunlight and especially the ultraviolet and particularly the UVB (280-320 nm), component aspect of sunlight. This is why they survive better in natural waters under algal masses. The seasonal factors of sunlight are an important consideration and thus winter sunlight in the latitudes far from the poles has a far less lethal effect on E. coli than in the tropical latitudes. Exposure to sunlight will also induce the viable but not culturable state as starvation will and this can confound determination of survival after sunlight exposure.

Apart from UVB, UVC (200-280nm) which is absorbed by the nucleic acids and chemically damages them also UVA (320-400 nm) and visible light (400-700nm) effects E. coli. While the detrimental effects on the E. coli are greatest by the lower wavelength radiation, their penetration into water is also reduced and thus emeliorated. The lethal and detrimental effects of sunlight on E. coli should therefore be seen as a balance between the very lethal but low penetration UVC and the more penetrating but less detrimental visible light.

In darkness, the E. coli are able to repair the DNA and other nucleic acids damaged by sublethal exposure to visible and ultraviolet light. The most important mechanisms occurring in darkness are the excision and removal of the damaged nucleic acids. Also recombining the nucleic acids and repairing them are used by E. coli to reduce the damage of the radiation. Visible light is also required to affect some of the repair mechanisms, which E. coli can harness to revive itself from sublethal damage caused particularly by exposure to UVC and UVB. Both UVA and visible light induce the formation of active oxygen molecules. These in turn induce the response to oxidative stress and its concomitant repair mechanisms.

Active oxygen molecules, which include the superoxide radical [O2-], hydrogen peroxide [H2O2], the hydroxyl radical [OH], singlet oxygen [1SO2], hydroperoxyl radicle [HOO] and ozone [O3], can damage nucleic acids (RNA and DNA) as well as proteins and lipids particularly membranes. Light, particularly sunlight, and heat will cause the production of these active oxygen molecules.

E. coli has mechanisms both to remove these active oxygen species as well as repairing the damage they caused before their removal. Two types of enzyme are produced by E. coli to remove these active oxygen molecules. Enzymes like the superoxidedismutases convert substances like the superoxide radical [O2-] to hydrogen peroxide [H2O2] and then catalases convert hydrogen peroxide [H2O2] to water [H2O] and ordinary oxygen [O2].

In addition, if the oxygen stress is such that the above mechanisms cannot cope, E. coli can bring two further mechanisms into play. Under the action of one genetically controlled mechanism responding to the stress, the DNA is more actively repaired and increased resistance to hydrogen peroxide is developed. A second different process similarly under genetic control also induces increased DNA repair capacity when the stressor is the superoxide radical.

Also within each cell there are a number of antioxidants, which help to maintain the reduced environment essential for cellular metabolism. These are able to deal with the active oxygen molecules produced under normal metabolic activity. The special response mechanisms are only brought into play under more extreme conditions.

In general E. coli can survive very well in the inanimate environment and even multiply whenever sufficiant nutrients are available. This environment thus provides an ideal reservoire for these organisms for them to recolonise the intestinal tract of humans and animals, which is their main habitat. One should not only consider rivers, lakes, seas, oceans, water reservoirs and other large bodies of water in this connection, cattle troughs, puddles, spilled water and damp areas in a part of a kitchen, where food is prepared are also areas where E. coli can and do survive and not all these E. coli may necessarily be benign.

© Copyright Microbionet.

All literary matter in Microbionet is covered by copyright, and must not be reproduced, stored in a retrieval system, or transmitted in any form by electronic or mechanical means, photocopying, or recording, without written permission. This page is to be read in conjunction with the Disclaimer.

Use of this site signifes your agreement to the Legal Notices. Legal Notices © Copyright Microbionet. All rights reserved.