Valencian Institute of Microbiology
(I V A M I)

Escherichia coli - Main enteropathogenic virotypes (ETEC, EPEC, EaggEC-EAEC, EIEC, EHEC and DAEC; Escherichia coli K1: Molecular diagnosis (PCR).           

Escherichia coli is a kind of facultative aerobic-anaerobic gram-negative bacillus included in the Enterobacteriaceae family. This species of bacteria is an inhabitant of the normal intestinal flora of man and animals. Occasionally, it can produce diarrhea and even many types of extraintestinal infections (urinary tract infections, sepsis, pneumonia, meningitis, etc.). The knowledge of their participation in the production of an intestinal process is hindered by the need to differentiate between the isolates of E. coli that are part of the normal flora of the intestine and those capable of producing a condition that affects the functionalism of the digestive tract. Initially, those corresponding to some specific serotypes that were found in cases or outbreaks of gastroenteritis (infection with inflammation of the digestive tract mainly manifested by diarrhea and vomiting) in young children were differentiated. Next, those varieties that were producing toxins were differentiated, which altered the functioning of the enterocytes (cells of the intestinal epithelium) producing episodes of diarrhea. These varieties were those that produced one of two types of enterotoxins: thermolabile enterotoxin (so called because it is destroyed by heating) or thermostable enterotoxin (so called because it resists heating). Other varieties endowed with other determinants of pathogenicity were subsequently differentiated by the acquisition of certain virulence genes.

Nowadays, in addition to the intestinal processes of some E. coli strains, it is known that E. coli strains can cause infections of different locations that can occur at the urinary, respiratory, systemic, etc. levels. In certain circumstances, neonatal infections caused by K1 strains are being of interest, so we explain after the intestinal conditions, the situation with the problem of these strains.

In order to better understand the relationships between the structure of the bacterium and its participation in certain processes, it is necessary to know what the surface antigenic structure of this bacterium is with its somatic antigens O, its flagellar antigens (H) and the capsular antigens (K).

Lipopolysaccharide (LPS) (≈ O antigen)

The outer membrane of the E. coli wall contains many polysaccharides:

• O antigen, an immunogenic polymer of repeated units of oligosaccharides (1–40 units).
• The central region of non-repeated oligosaccharides.
• Lipid A (endotoxin)

The O antigen is used to serotype E. coli and differentiates the O groups, differentiating between O1 and O181, with the exception of some groups that have been eliminated O31, O47, O67, O72, O93 (now K84), O94, and O122; Groups 174 to 181 are provisional (O174 = OX3 and O175 = OX7) or are under investigation (176 to 181 are STEC/VTEC). In addition, there are some subtypes of many O groups (e.g. O128ab and O128ac). It should be noted that if antibodies are used to perform typing, some of them may cross-react with other O antigens and also with some K antigens, not only of E. coli, but also of other Escherichia species and species of Enterobacteriaceae

The O antigen is encoded by the rfb gene group. The rol gene (cld) encodes the chain length regulator of the lipopolysaccharide O.

Flagella (H antigen)

The H antigen is the main component of flagella and is encoded by the fliC gene. There are 53 identified H antigens, numbered from H1 to H56 (H13 and H22 were not from E. coli but from Citrobacter freundii, and H50 was found to be the same as H10).

Capsule (K antigen)

Capsular acidic polysaccharide is a layer of thick and mucous polysaccharide that surrounds the bacteria. There are two separate groups of K antigens, called group I and group II, with a small subgroup between them (K3, K10, and K54 / K96) that has been classified as group III. Group I is a capsular polysaccharide of about 100 kDa (large), while group II is associated with extraintestinal diseases and is less than 50 kDa in size.

In total there are 60 different K antigens (K1, K2a/ac, K3, K4, K5, K6, K7 (= K56), K8, K9 (= O104), K10, K11, K12 (K82), K13 (= K20 and = K23), K14, K15, K16, K18a, K18ab (= K22), K19, K24, K26, K27, K28, K29, K30, K31, K34, K37, K39, K40, K41, K42, K43, K44, K45, K46, K47, K49 (O46), K50, K51, K52, K53, K54 (= K96), K55, K74, K84, K85ab/ac (= O141), K87 (= O32), K92, K93, K95 , K97, K98, K100, K101, K102, K103, KX104, KX105, and KX106).

Escherichia coli as intestinal pathogen

From the point of view of intestinal pathogenesis, the currently well-defined groups of Escherichia coli producing diarrheal symptoms are:

• ETEC (Enterotoxigenic Escherichia coli).
• EPEC (Enteropathogenic Escherichia coli).
• EaggEC-EAEC (Enteroaggregative Escherichia coli).
• EIEC (Enteroinvasive Escherichia coli).
• VTEC = EHEC = STEC (Vero Toxin Escherichia coli = Enterohemorrhagic Escherichia coli = Shiga Toxin producing Escherichia coli). 
• DAEC (Diffusely-Adherent Escherichia coli).

Microbiological characteristics

Since most E. coli are non-pathogenic and in view of the need to differentiate the strains that cause pathological processes, before using molecular methods and identifying virulence factors, microbiologists identified surface antigens. This resulted in a serological classification based on the reaction of the bacterial surface molecules with different antibodies (serotyping). Subsequently, a classification scheme based on virulence factors (virotyping) has been developed. A virotype can therefore include more than one serogroup or serotype. This scheme is more related to the pathological process caused than the serotyping was. Virotyping is mainly performed by PCR methods (polymerase chain reaction) that detect the genes that produce virulence factors.

For the serological classification (serotyping) of Escherichia coli, the identification of the antigens O (lipopolysaccharides) and the antigens H (flagella; H of "hauch, German word of flagella) is used. The O antigens identify the serogroup of a strain, and the H antigens the serotype. More than 160 serogroups of E. coli are known.

There are several virulent types (virotypes) of Escherichia coli, each of them producing pathology through a different mechanism.

• Enterotoxigenic Escherichia coli (ETEC), causing pathology due to alteration of the hydroelectrolytic balance of intestinal mucosa cells due to the production of thermolabile toxin (LT), and/or thermostable toxin (ST), encoded, respectively by locus genetic lt and st. These toxins increase the content of mucosal cells in AMP-cyclic, or in GMP-cyclic, by induction of the enzymes adenyl cyclase or guanylate cyclase, respectively. In effect, they do not alter the morphology of the mucosal cells, nor cause inflammation in the mucosa, causing diarrhea and vomiting, with or without fever. 

• Enteropathogenic Escherichia coli (EPEC): This virotype causes damage to the cells of the mucosa, with an elongation of the microvilli, inducing in the cells a reorganization of the actin fibrils. The effect is accompanied by an inflammatory response. Those affected have prolonged, non-bloody diarrhea and fever. These strains possess the genetic locus bfpA. 

• Enteroagregative Escherichia coli (EAggEC): This virotype adheres in groups to the cells of the mucosa, without invading them, and without causing them a morphological change, but alters them functionally, just as the ST (thermostable) toxin performs. These strains possess the genetic locus bfpA. 

• Enteroinvasive Escherichia coli (EIEC): This virotype invades the cells of the mucosa of the colon, extending from one cell to another, similar to how Shigella spp. causes damage, without toxin production. It manifests as an aqueous diarrhea or as a bloody diarrhea. These strains possess the ial genetic locus.

• Enterohemorrhagic Escherichia coli, producer of verotoxin, or Shiga toxin (EHEC, VTEC, STEC): This virotype is fixed to the cells of the mucosa without invading them, but produces a Shiga toxin (from Shigella dysenteriae). The process manifests itself as moderate diarrhea or hemorrhagic diarrhea, and in the most severe cases as a hemolytic-uremic syndrome with hemolytic anemia, thrombocytopenia and renal failure. Serotypes O157: H7, O157: NM have been implicated, but other serotypes that are not O157 are known today. It is acquired primarily from meat products, dairy products, contaminated vegetables, and there is even the possibility of interhuman transmission. The causative strains are producing the toxins sxt1 and/or sxt2, or other sxt types, encoded by their corresponding genetic locus.

• Diffusely-Adherent Escherichia coli (DAEC): ADHE gene.

Strains of E. coli VTEC (EHEC, STEC) have been shown to be related to a severe form of intestinal infection with the possibility of causing extraintestinal complications, such as hemolytic-uremic syndrome.

Interest of E. coli O157

In 1982, Escherichia coli O157:H7 caused two outbreaks of an enteric disease called "hemorrhagic colitis," characterized by severe abdominal pain with watery diarrhea followed by hemorrhagic diarrhea, and evidence of colonic inflammation with or without moderate fever. Since then, it is considered an important pathogen, which exceeds Salmonella or Shigella in some areas. Some infected people may remain asymptomatic. The individuals most susceptible to infection are children and the elderly. The infection can cause a serious complication known as hemolytic-uremic syndrome (HUSD: Hemolytic uremic syndrome). Hemolytic-uremic syndrome is a process in which red blood cells are destroyed and renal failure occurs.

The clinical manifestations of the infection usually occur after three days of infection (1 to 9 days). The infection can cause a wide variety of clinical conditions that can occur as moderate non-bloody diarrhea, severe bloody diarrhea (hemorrhagic colitis), and the complication of hemolytic-uremic syndrome (in approximately 2 to 7% of cases). About 30% of people who have suffered a hemolytic-uremic syndrome continue with impaired renal function for many years. In the US, this syndrome is the leading cause of acute renal failure in children.

E. coli O157:H7 or E. coli O157:non-mobile (O157 STEC) or other E. coli NM (non-mobile) can produce one or more Shiga toxins called verotoxins. The producing strains of these toxins are more frequent in the US and Europe.

There are at least 100 E. coli STEC serotypes that have been related to these processes other than E. coli O157. Of these, E. coli O111: NM (non-mobile), and E. coli O26: H11 are the most frequent, although there is little knowledge of the possible participation of others because they are not usually sought.

Strains of enterohemorrhagic E. coli have adhesins similar to those of other E. coli (eg EPEC - enteropathogens), with a gene called eaeA (intimin coding), which is similar to the eaeA gene of EPEC strains and that performs the same function, mediate the intimate fixation of the bacteria to the intestinal wall cell. They also usually produce an enterohemolysin encoded by a plasmid gene - hlyA-.

The main difference of strains of enterohenorrhagic E. coli (EHEC) is that they produce a toxin that is almost identical to Shiga toxin (Stx), a toxin that is responsible for the dysentery picture caused by Shigella spp. There are two types of toxins: Stx - Stx1, which is very similar to the toxin of Shigella spp., and Stx2 that is similar to the toxin Stx1 but that differs from it in a sufficient number of amino acids with what generates different antibodies that allow differentiate them. Stx2 toxin producing strains are more likely to produce hemolytic-uremic syndrome. There are receptors for these toxins in both intestinal cells and kidney cells, and hence when diffused from the kidney can cause kidney damage. The genes of these toxins are found in a tempered bacteriophage and through them they could pass to other strains of E. coli that do not possess these genes.

In the usual MacConkey plate cultures (with incorporated lactose), it can be confused with other E. coli, since it ferments lactose rapidly and is indistinguishable from other colonies of E. coli. One way to differentiate most E. coli O157:H7 isolates is because of their inability to ferment sorbitol, unlike what other E. coli do, so this characteristic is used to differentiate them in medium plates. MacConkey containing sorbitol.

Habitat and transmission of E. coli O157

E. coli O157 colonizes the digestive tract of cattle. Human infection is caused by contaminated food or drinks. From cattle, it can contaminate meat products, mainly minced meat, and dairy products, because the bacteria are often found in the udders of cows and contaminate milk and through it to dairy products. Similarly through animal droppings it can contaminate water.

Minced meat has been the food most related to epidemic outbreaks of this infection, mainly when ingested undercooked, as with hamburgers. Outbreaks have also been produced by fresh milk, sausages, roast beef, unchlorinated water or bath in contaminated water, consumption of raw vegetables (salads), mayonnaise, etc. Cases of occasional transmission through unusual products have been described, for example, from apple juices, possibly due to having been prepared with apples fallen from trees that could have been contaminated with cattle droppings used as fertilizer. Cases of infection through mayonnaise have been attributed to the fact that this bacterium could colonize small chickens and persist in them for a long time, subsequently contaminating the egg envelope. Similarly, keep in mind that this bacteria, unlike others, can survive in acidic foods (yogurt, some vegetable juices, etc.) when they are not pasteurized

It can pass from person to person through contact with dirty hands, mainly in schools, families, nurseries or nursing homes. People who have had an infection with this bacterium can continue to eliminate it for a week or three, or even longer.

Diagnosis of E. coli O157

To detect human infection, a stool culture must be performed. To detect its presence in a contaminated food, a suspected food culture should be performed. Subsequently, some of the recommended procedures for detection in culture are described in more detail. Some methods that can be followed to detect the ability to produce enterohemorrhagic toxins (Shiga toxin = verotoxin) (toxicity in cell culture or immunological tests), or the genes that produce them (PCR: polymerase chain reaction) are also indicated.

E. coli O157 treatment

Most people get better without antibiotics in about 5 to 10 days, and there is no evidence that antibiotics improve the course of the infection. On the other hand, antibiotic administration is not recommended because an increase in renal complications has been observed when patients are treated with them. Also, some antidiarrheals such as loperamide should not be used. Only sufficient fluid intake is recommended to avoid dehydration.

When the complication of hemolytic-uremic syndrome occurs it is recommended to administer blood transfusions or coagulation factors, as well as renal dialysis when necessary. Although most people affected by this syndrome recover, in some cases it can be fatal in 3 to 5% of patients.

E. coli O157 prevention

 

The most recommended is to avoid the intake of hamburgers, or other products made with minced meat, undercooked. A simple way to know is that they are not pink inside, but brown or gray and that the juice they release is clear, and the inside is hot. In this sense, experiments have been carried out and it has been observed that the change in color that appeared during cooking may not be significant for internal heating, so the use of special thermometers to measure meat heating is recommended in the US.

Likewise, only milk or dairy products that are pasteurized should be ingested. You also have to make sure that infected people, especially children, wash their hands with soap after using the toilet to avoid spreading the infection.

In addition, it is recommended: wash any fruit or vegetable eaten without cooking, carefully wash your hands before and after preparing food, keep meat products in the refrigerator, avoid swallowing water during baths in lake or pool waters, and that people With diarrhea do not use swimming pools or prepare food.

On the other hand, because the bacteria are eliminated by feces, children, mainly those who attend daycare centers, health workers or food handlers, should not go to their respective nurseries or workplace while diarrhea persists. When the diarrhea disappears, great care should be taken in handwashing after using the toilet as they can continue to eliminate the bacteria by feces for one to three weeks or more.

 

Escherichia coli K1 and its relationship with neonatal infections

Some Escherichia coli isolates possess the K1 capsular antigen, a linear homopolymer of α-2,8-linked N-acetyl-neuraminic acid that is subject to various types of O-acetylation. These strains of E. coli K1 are characterized by causing neonatal sepsis and meningitis with mortality rates of 5 to 30% of those infected and those who survive usually have permanent neurological dysfunctions such as hearing loss, mental retardation or cortical blindness. This problem is increasing due to the multiresistance that can present to antibiotics.

Neonates have a special predisposition to suffer infection from these strains by being transmitted vertically from the mother during childbirth or shortly thereafter, since in adults they can be part of the intestinal microbiota. In the neonates, gastrointestinal colonization would occur, and in some cases there could be a translocation from the intestinal lumen to the systemic circulation, from the reach to the central nervous system and meninges. However, in adults, E. coli K1 meningitis can occur when there is an alteration of defenses, head injuries, neurosurgical interventions or gram-negative sepsis.

E. coli K1 strains interact with the endothelial cells of the microvascular network of the brain (BMEC: Brain Microvascular Endothelial Cells). This interaction is mediated by outer membrane protein A (OmpA) and a glycoprotein receptor (Ecgp96) of endothelial cells. Initially, OmpA binds to GlcNAc1-4GlcNAc epitopes of the Ecpg96 glycoprotein receptor. In addition, the same OmpA protein prevents the binding of the C4bp protein of the serum complement (C4b binding protein) that participates in the natural immune defense, so it cannot act to eliminate these strains of E. coli K1. Infants because they have lower concentrations of C4bp have a higher risk of suffering from E. coli K1 meningitis. Some other structures such as Ibe proteins, or cytotoxic necrotizing factor 1 would be necessary for the bacterium to cross the blood-brain barrier or the blood-cerebrospinal fluid barrier.

 

Tests performed in IVAMI

• Escherichia coli, species confirmation to differentiate from other Escherichia species, by amplification of the dnaA gene.

• Escherichia coli K1, molecular diagnosis.

• Enterotoxigenic Escherichia coli (ETEC)

Detection of thermolabile enterotoxin (LT) gene (locus lt) and/or

Detection of thermostable enterotoxin (ST) gene (locus st).

• Enteropathogenic Escherichia coli (EPEC)

• Detection of locus bfpA gene.

• Enteroagregative Escherichia coli (EAggEC-EAEC)

• Detection of locus aggR and aggA genes.

• Enteroinvasive Escherichia coli (EIEC)

• Detection of locus ial gene.

• Enterohemorrhagic Escherichia coli, with verotoxin or with Shiga toxin (EHEC = VTEC = STEC)

Detection of genes of toxins sxt1 and/or sxt2, or other types (rfbE, wbdl, wzx, ihp1, wzx, fli).

Serogroup detection (O26, O45, O103, O111, O121, O145 and O 157).

• Diffusely-Adherent Escherichia coli (DAEC): ADHE gene

Sample recommended:

• Diarrheal stools.

• Food samples (meats or others). These samples require a previous enrichment culture for 24 hours.

• Samples for molecular detection of Escherichia coli K1: depending on clinical suspicion, CSF sample in case of meningitis, blood in case of bacteraemia, urine in the case of urinary infections and pyelonephritis, respiratory samples in suspected pneumonia and biopsies in case of localized infections, such as cellulite.

• Isolated strains, if required for typing.