In this article we will discuss about:- 1. Introduction to Escherichia Coli 2. The Organism of Escherichia Coli and its Characteristics 3. Pathogenesis and Clinical Features 4. Isolation and Identification 5. Association with Foods.
- Introduction to Escherichia Coli
- The Organism of Escherichia Coli and its Characteristics
- Pathogenesis and Clinical Features of Escherichia Coli
- Isolation and Identification of Escherichia Coli
- Escherichia Coli’s Association with Foods
1. Introduction to Escherichia Coli:
Since 1885, when it was first isolated from children’s faeces and described by the German bacteriologist Theodor Escherich, scientific attention has been lavished on Escherichia coli to such an extent that it is today probably the best understood free-living organism.
Escherichia Coli is an almost universal inhabitant of the gut of humans and other warm-blooded animals where it is the predominant facultative anaerobe though only a minor component of the total microflora.
Generally a harmless commensal, it can be an opportunistic pathogen causing a number of infections such as Gram-negative sepsis, urinary tract infections, pneumonia in immunosuppressed patients, and meningitis in neonates.
Its common occurrence in faeces, ready culturability, generally non-pathogenic character, and survival characteristics in water led to the adoption of Escherichia Coli as an indicator of faecal contamination and the possible presence of enteric pathogens such as S. typhi in water. This usage has been transferred to foods where greater circumspection is required in interpreting the significance of positive results.
Strains of Escherichia Coli were first recognized as a cause of gastroenteritis by workers in England investigating summer diarrhoea in infants in the early 1940s. Until 1982, strains producing diarrhoea were classified into three types based on their virulence properties: enteropathogenic Escherichia Coli (EPEC), entero-invasive Escherichia Coli (EIEC), and entero-toxigenic Escherichia Coli (ETEC).
They are not very common causes of foodborne illness in developed countries, but an important cause of childhood diarrhoea in less developed countries. ETEC is also frequently associated with so-called traveller’s diarrhoea.
However since 1982, Escherichia Coli serotype 0157:H7 has been recognized as the cause of a number of outbreaks of haemorrhagic colitis and haemolytic uraemic syndrome, particularly in North America, where foods such as undercooked ground meat and raw milk have been implicated.
An exponential rise in isolations of 0157:H7 was reported in Canada between 1982 and 1986 and a study in the UK between 1985 and 1988 suggested that the increased reporting of isolations there (118 in England and Wales and 86 in Scotland) represented a real increase.
A fifth type, entero-adherent Escherichia Coli, identified by their characteristic pattern of adherence to Hep-2 cells, has been described more recently. Their significance as a cause of diarrhoea is presently uncertain. They have been implicated largely on epidemiological grounds, although a challenge trial with volunteers showed at least one strain caused a mild diarrhoea.
2. The Organism of Escherichia Coli and its Characteristics:
Escherichia is the type genus of the Enterobacteriaceae family and Escherichia Coli is the type species of the genus. It is a catalase-positive, oxidase-negative, fermentative, short, Gram-negative, non-sporing rod.
Genetically, Escherichia Coli is closely related to the genus Shigella, although characteristically it ferments the sugar lactose and is otherwise far more active biochemically than Shigella spp. Late lactose fermenting, non-motile, biochemically inert strains of Escherichia Coli can however be difficult to distinguish from Shigella.
Escherichia Coli can be differentiated from other members of the Enterobacteriaceae on the basis of a number of sugar-fermentation and other biochemical tests. Classically an important group of tests used for this purpose are known by the acronym IMViC.
These tested for the ability to produce:
1. indole from tryptophan (I);
2. sufficient acid to reduce the medium pH below 4.4, the break point of the indicator methyl red (M);
3. acetoin (acetyl-methyl carbinol)(V); and
4. the ability to utilize citrate (C).
Since production of mixed acids and acetoin are alternative pathways for the metabolism of pyruvate, most species of the Enterobacteriaceae are either VP positive or methyl red positive.
In the IMViC tests, most strains of Escherichia Coli are indole and methyl red positive and VP and citrate negative (Table 7.6). The tests are still used for identification purposes but nowadays usually as part of the larger range of tests available in modern miniaturized test systems.
Escherichia Coli is a typical mesophile growing from 7-10 °C up to 50 °C with an optimum around 37 °C, although there have been reports of some ETEC strains growing at temperatures as low as 4 °C.
It shows no marked heat resistance, with a D value at 60 °C of the order of 0.1 min, and can survive refrigerated or frozen storage for extended periods. A near-neutral pH is optimal for growth but growth is possible down to pH 4.4 under otherwise optimal conditions. The minimum aw for growth is 0.95.
A serotyping scheme for Escherichia Coli based on lipopolysaccharide somatic O, flagellar H, and polysaccharide, capsular K antigens was proposed by Kauffman in the 1940s. As currently applied in the O:H system, principal serogroups are defined by Q antigens and then subdivided into serovars on the basis of H antigens.
Strains of each category of pathogenic Escherichia Coli tend to fall within certain O:H serotypes, so the scheme plays an important role in detecting pathogens as well as in epidemiological investigations.
3. Pathogenesis and Clinical Features of Escherichia Coli:
There are four major categories of diarrhoea-genic Escherichia Coli based on distinct, plasmid- encoded virulence properties.
Enterotoxigenic Escherichia Coli (ETEC):
Illness caused by ETEC usually occurs between 12 and 36 h after ingestion of the organism. Symptoms can range from a mild afebrile diarrhoea to a severe cholera-like syndrome of watery stools without blood or mucus, stomach pains and vomiting. The illness is usually self-limiting, persisting for 2-3 days, although in developing countries it is a common cause of infantile diarrhoea where it can cause serious dehydration.
The ingested organism resists expulsion from the small intestine with the rapidly flowing chyme by adhering to the epithelium through attachment or colonization factors in the form of fimbriae on the bacterial cell surface. These can have different morphology and be either rigid (6-7 nm diameter) or flexible (2-3 nm diameter) structures composed of 14-22 kDa protein subunits.
They are mannose resistant, i.e. they mediate haemagglutination in the presence of mannose, and particular colonization fimbriae are restricted to certain O:H serotypes. They are encoded on plasmids which frequently also encode for the diarrhoeagenic toxins.
Two toxin types are produced: the heat-stable toxins (ST), which can withstand heating at 100 °C for 15 min and are acid resistant, and the heat-labile toxins (LT) which are inactivated at 60 °C after 30 min and at low pH.
LTI bears a strong similarity to cholera toxin; it consists of five B subunits (Mr 11.5 kDa) which are responsible for binding of the toxin to the epithelial cells and an A subunit (Mr 25 kDa) which is translocated into the epithelial cell where it activates adenylate cyclase.
The subsequent increase in cAMP levels then inhibits Na+, Cl– and water absorption by the villus cells and stimulates their loss from intestinal crypt cells thus leading to profuse watery diarrhoea. LTII toxin produced by certain ETEC strains has similar biological activity to LTI but does not cross react with antiserum to LTI or cholera toxin.
Two types of ST have been recognized; the most common, STA, is a low molecular weight, poorly antigenic polypeptide produced from a 72 amino acid precursor. The reported size of the extracellular molecule has ranged from 18-42 amino acids and its resistance to heat, low pH and proteolytic digestion probably derive from its compact three-dimensional structure.
It acts by stimulating the production of cGMP by guanylate cyclase in epithelial cells. The mechanism of action of STB, which can be distinguished from STA by its inability to produce fluid secretion in the intestines of suckling mice, is not known but does not appear to operate through the stimulation of cyclic nucleotide production.
Enteroinvasive Escherichia Coli (EIEC):
Infection by EIEC results in the classical symptoms of an invasive bacillary dysentery normally associated with Shigella. Like Shigella, EIEC invades and multiplies within the epithelial cells of the colon causing ulceration and inflammation. Clinical features are fever, severe abdominal pains, malaise and often a watery diarrhoea which precedes the passage of stools containing blood, mucus, and faecal leukocytes.
Invasiveness is determined by a number of outer membrane proteins which are encoded for on a large plasmid (a 140 MDa). The infective dose of EIEC appears to be substantially higher than for Shigella and this is thought to be a reflection of the organism’s greater sensitivity to gastric acidity.
Enteropathogenic Escherichia Coli (EPEC):
When the properties of ETEC and EIEC were established it was noted that these strains were rarely of the same serotypes first associated with Escherichia Coli diarrhoea in the 1950s. Subsequent investigation of some of these earlier strains in most cases failed to demonstrate the property of entero-invasiveness or the ability to produce ST or LT and yet they retained the ability to cause diarrhoea in volunteers.
Symptoms of EPEC infection, malaise, vomiting and diarrhoea with stools containing mucus but rarely blood, appear 12-36 h after ingestion of the organism. In infants, the illness is more severe than many other diarrhoeal infections and can persist for longer than two weeks in some cases.
Pathogenesis appears to be related to the ability of EPEC strains to adhere closely to the enterocyte membrane causing cell loss from the villus tips. It is hypothesized that this causes diarrhoea by disrupting the balance between absorption and secretion in the small intestine.
Enterohaemorrhagic Escherichia Coli (EHEC):
EHEC, sometimes also known as Verotoxin-producing Escherichia Coli (VTEC), was first described in Canada where in some areas it rivals Campylobacter and Salmonella as the most frequent cause of diarrhoea. A number of serotypes have this capacity but 0157:H7 is the one most frequently isolated from humans.
It has attracted attention not only because foodborne transmission is more common than with other diarrhoeagenic Escherichia Coli, but because it can also cause the life-threatening conditions, haemorrhagic colitis, haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura.
Haemorrhagic colitis is typically a self-limiting, acute, bloody diarrhoea that begins with stomach cramps and watery diarrhoea after an incubation period of 3-8 days. It can be distinguished from inflammatory colitis by the usual lack of fever and absence of leukocytes in the stools. It affects mainly adults, with a peak incidence in the summer months, and can be life-threatening in the elderly.
Haemolytic uraemic syndrome is characterized by three features, acute renal failure, haemolytic anaemia (reduction in the number of red blood cells) and thrombocytopenia (a drop in the number of blood platelets), sometimes preceded by a bloody diarrhoea. It is most common in children among whom it is the leading cause of acute renal failure in western Europe and North America.
In 70 cases seen in London between 1980 and 1986 the fatality rate was 6%, with 13% of cases showing some long term kidney-damage. In one outbreak in a North American nursing home, the fatality rate among the 55 affected residents was 31%.
Thrombotic, thrombocytopenic purpura is related to the haemolytic uraemic syndrome but includes fever and neurological symptoms. Adhesion is an important factor in virulence and O157:H7 strains have been shown to possess a 60 MDa plasmid encoding for fimbrial colonization factors which produce a similar, but not identical, lesion to EPEC strains on the villus tips.
EHEC strains produce the cytotoxin Verotoxin (so-called because of its ability to kill Vero (African Green Monkey Kidney) cells). Studies have revealed the presence of at least two toxins VTI and VTII which because of their similarity to Shiga toxin have also been called Shiga-like toxins, SLTI and SLTII.
In fact, VTI bears the closest resemblance to Shiga toxin; it cross reacts with antisera to Shiga toxin, is also composed of A (Mr 32 kDa) and B (Mr 7.7 kDa) subunits and the B units are structurally identical.
VTII also comprises an A and B subunit but these are larger than in VTI (Mr 35 kDa and 10.7 kDa respectively) and do not cross- react immunologically, though they do share a 60% amino acid sequence homology, with Shiga toxin. Both toxins have been shown to be phage encoded in a number of strains.
4. Isolation and Identification of Escherichia Coli:
Selective techniques for Escherichia Coli mostly exploit the organism’s tolerance of bile and other surfactive compounds, a consequence of its natural habitat, the gut. Aniline dyes and the ability of many strains to grow at temperatures around 44°C are also used as relative agents.
The first selective and differential medium was that originally devised by MacConkey in 1905. It has been variously modified since but its essential characteristics have remained unchanged. Bile salts (and sometimes the aniline dye, crystal violet) act as inhibitors of Gram-positive and some fastidious Gram- negative bacteria.
Lactose is included as a fermentable carbohydrate with a pH indicator, usually neutral red. Strong acid producers like Escherichia, Klebsiella, and Enterobacter produce red colonies, non-lactose fermenters such as Salmonella, Proteus, and Edwardsiella, with rare exceptions produce colourless colonies.
MacConkey agar is not however strongly selective and will support the growth of a number of non-Enterobacteriaceae including Gram-positives such as enterococci and staphylococci.
Eosin/methylene blue agar is a popular selective and differential medium in North America. The aniline dyes eosin and methylene blue are the selective agents but also serve as indicator for lactose fermentation by forming a precipitate at low pH. Strong lactose fermenters produce green-black colonies with a metallic sheen.
A biochemical feature of Escherichia Coli increasingly being used in diagnostic media is β- glucuronidase activity, which is possessed by around 95% of Escherichia Coli strains but by only a limited number of other bacteria.
A fluorogenic or chromogenic glucuronide is incorporated into a conventional medium and enzyme activity detected by the production of colour or fluorescence. Most widely used is the fluorogen 4- methylumbelliferyl-β-D-glucuronide (MUG) which is hydrolysed to produce fluorescent 4-methylumbelliferone.
Suspect colonies from selective and differential media can be confirmed by further biochemical testing.
Detection of Escherichia Coli O157:H7 is based on phenotypic differences from most other serotypes: its inability to ferment sorbitol on MacConkey-sorbitol agar and absence of β-glucuronidase activity in most strains. Presumptive Escherichia Coli O157:H7 from these tests must then be confirmed serologically for which a latex agglutination kit is commercially available.
Identification of diarrhoeagenic Escherichia Coli can be based on detection of their associated virulence factors. For example, procedures are available to detect the ST and LT of ETEC serologically, and LTI and VT genes in ETEC and EHEC using gene probes and the polymerase chain reaction (PCR).
5. Escherichia Coli’s Association with Foods:
Faecal contamination of water supplies and contaminated food handlers have been most frequently implicated in outbreaks caused by EPEC, EIEC and ETEC. A number of foods have been involved, including a coffee substitute in Romania in 1961, vegetables, potato salad, and sushi.
In the United States, mould-ripened soft cheeses have been responsible for outbreaks in 1971, associated with EIEC in which more than 387 people were affected, and in 1983, caused by ETEC (ST). Escherichia Coli would not be expected to survive well in a fermented dairy product with a pH below 5 but, where contamination is associated with mould-ripening, the local increase in pH as a result of lactate utilization and amine production by the mould would allow the organism to grow.
Outbreaks caused by EHEC serotype O157:H7 have mostly involved undercooked ground meat and occasionally raw milk. Six hundred people became ill and four children died in a major US outbreak in 1993 caused by undercooked hamburgers.
This outbreak caused a major public outcry over meat hygiene and resulted in, amongst other things, the introduction of new meat-labelling regulations. Surveys have isolated the serotype from 3.7% (6/164) samples of retailed fresh beef, and a significant percentage (1-2%) of other fresh meat products such as pork, poultry and lamb. In one UK outbreak, a cold turkey roll was implicated on epidemiological grounds.