Classification and Importance of Enteropathogenic E.coli.

Isolation Method from Pediatric Stool Samples

Presenter:Ms. Sana Muslim

Research Associate

Escherichia coli typically colonizes the gastrointestinal tract of human infants within a few hours after birth.

Escherichia coli (E. coli) bacteria normally live in the intestines of people and animals.

Most E. coli are harmless and actually are an important part of a healthy human intestinal tract.

Some E. coli are pathogenic, meaning they can cause illness, either diarrhea or illness outside of the intestinal tract.

These commensal E. coli strains rarely cause disease except in immunocompromised hosts.

Introduction

E. coli consists of a diverse group of bacteria. Pathogenic E. coli strains are categorized into pathotypes.

Six pathotypes are associated with diarrhea and collectively are referred to as Diarrheagenic E. coli.

Enterohemorrhagic E. coli (EHEC) Enterotoxigenic E. coli (ETEC) Enteropathogenic E. coli (EPEC) Enteroaggregative E. coli (EAEC) Enteroinvasive E. coli (EIEC) Diffusely adherent E. coli (DAEC)

Classification of E.coli

The types of E. coli that can cause diarrhea can be transmitted through contaminated water or food, or through contact with animals or persons.

Transmission

First recognized as a cause of human disease in 1982

EHEC causes bloody diarrhea (haemorrhagic colitis), non-bloody diarrhea and haemolytic uremic syndrome (HUS).

The principal reservoir of EHEC is the bovine intestinal tract.

Associated with foodborne outbreaks. The most commonly identified in North America is

E. coli O157:H7.

Enterohemorrhagic E. coli (EHEC)

The key virulence factor for EHEC is Stx, which is also known as verocytotoxin (VT).

EHEC also induce the attaching and effacing lesion, but in the colon. The distinguishing feature of EHEC is the elaboration of Shiga toxin (Stx), systemic absorption of which leads to potentially life-threatening complications.

The Shiga toxin (Stx) of EHEC cleaves ribosomal RNA, thereby disrupting protein synthesis and killing the intoxicated epithelial or endothelial cells

EHEC Pathogenesis

EHEC Pathogenesis

Diagnosis Diagnosis of E. coli 0157:H7 must be reported to the Nationally

Notifiable Diseases Surveillance System (NNDSS) at CDC. In all cases of acute community-acquired diarrhea stool samples should be cultured for E. coli 0157:H7 on cefixime tellurite-sorbitol MacConkey agar (CT-SMAC), or CHROMagar O157. Sorbitol non-fermenting colonies should be assayed for Shiga toxin using EIA or PCR. The PCR technique has been extensively used to detect stx genes

Treatment: The disease usually is self-limiting and most patients recover

without specific treatment within five to 10 days. Antibiotics are counter-indicated for treatment of E. coli 0157:H7 infections

Diagnosis and Treatment

ETEC are bacteria that colonize the small intestine and cause severe diarrhea, dysentery, abdominal cramps, and fever

ETEC can be life threatening due to significant fluid loss and severe dehydration

ETEC is transmitted when a person eats food, or drinks water or ice contaminated with ETEC bacteria. Human or animal wastes (e.g., feces) are the main source of ETEC contamination.

Enterotoxigenic E. coli (ETEC)

ETEC colonizes the surface of the small bowel mucosa and elaborates enterotoxins, which give rise to intestinal secretion

ETEC enterotoxins belong to one of two groups: the heat-labile enterotoxins (LTs) and the heat-stable enterotoxins (STs)

heat-labile (LT) and heat-stable (ST) toxins cause intestinal epithelial cells to secrete excess fluid. Some strains produce only one of the toxins while others produce both.

heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST), are secreted and cause diarrhea through cyclic AMP (cAMP)- and cyclic GMP (cGMP)-mediated activation of cystic fibrosis transmembrane conductance regulator (CFTR).

ETEC Pathogenesis

LT acts by stimulating adenylate cyclase and increasing intracellular cyclic AMP, which results in secretion of chloride from intestinal crypt cells and inhibition of absorption of sodium chloride at the villus tips

ST activates enterocyte cyclic GMP and also leads to the stimulation of chloride secretion and inhibition of sodium chloride absorption. The end result is secretion of free water into the intestinal lumen, which manifests clinically as watery diarrhea

ETEC Pathogenesis

Diagnosis The diagnosis of ETEC is currently performed in the

research or reference laboratory using DNA probes that identify the genes for LT or ST.

Treatment: Most infected persons will recover within a few days,

without requiring any specific treatment. Clear liquids are recommended for persons with diarrhea to prevent dehydration and loss of electrolytes. For adults, packaged oral rehydration salts or premixed oral rehydration solutions (both available over-the-counter) may be used

Diagnosis and treatment

EPEC have been associated with sporadic diarrheal illness and diarrhea outbreaks, most commonly among children less than six months of age in developing countries

Enteropathogenic E. coli (EPEC) strains are defined by the characteristic "attaching and effacing" effect they elicit upon interaction with epithelial cells and by the fact that they do not produce Shiga toxin

Enteropathogenic E. coli (EPEC)

EPEC proteins do not behave like classic toxins; the following observations have furthered our understanding of how bacterial organisms can affect cell physiology through adherence, even in the absence of toxin production

EPEC adhere to small bowel enterocytes, but destroy the normal microvillar architecture, inducing the characteristic attaching and effacing lesion.

Cytoskeletal derangements are accompanied by an inflammatory response and diarrhea.

EPEC Pathogenesis

1. Initial adhesion,

2. Protein translocation by type III secretion,

3. Pedestal formation.

EPEC Pathogenesis

Diagnosis The gold standard for identification of EPEC is the

detection by DNA probe or polymerase chain reaction (PCR) of the EPEC adherence factor (EAF). EPEC strains would possess the eae gene for A/E and the EAF probe or bfp sequences. These diagnostic assays are available only in research or reference laboratories.

Treatment: Treatments for EPEC (Enteropathogenic E. Coli

infection) include Rehydration and antibiotic therapy in susceptible patients

Diagnosis and treatment

EAEC are increasingly recognized as a cause of often persistent diarrhea in children and adults in both developing and developed countries

identified as the cause of several outbreaks worldwide

EAEC known as auto aggregative in which bacteria adhere to each other in a ‘stacked-brick’ configuration

Enteroaggregative E. coli (EAEC)

The basic strategy of EAEC infection seems to comprise colonization of the intestinal mucosa, probably predominantly that of the colon, followed by secretion of enterotoxins and cytotoxins

EAEC prototype strains adhere to HEp-2 cells and intestinal mucosa by virtue of fimbrial structures known as aggregative adherence fimbriae (AAFs)

(EAEC) Pathogenesis

EAEC adheres to small and large bowel epithelia in a thick biofilm and elaborates secretory enterotoxins and cytotoxins.

the formation of a heavy biofilm may be related to the diarrheagenicity of the organism and, perhaps, to its ability to cause persistent colonization and diarrhea

(EAEC) Pathogenesis

Diagnosis: EAEC infection is diagnosed definitively by the

isolation of E. coli from the stools of patients and the demonstration of the AA pattern in the HEp-2 assay. A PCR with oligonucleotide primers derived from the probe sequence has also been developed.

Treatment: Broad-spectrum antibiotics are recommended in

chronic and/or life-threatening cases.

Diagnosis and treatment

EIEC are biochemically, genetically and pathogenically closely related to Shigella Spp

EIEC might cause an invasive inflammatory colitis, and occasionally dysentery.

EIEC infection is thought to represent an inflammatory colitis, although many patients seem to manifest secretory, small bowel syndrome

Enteroinvasive E. coli (EIEC)

The current model of Shigella and EIEC pathogenesis comprises

(i) epithelial cell penetration, (ii) lysis of the endocytic vacuole, (iii) intracellular multiplication, (iv) directional movement through the cytoplasm, and (v) extension into adjacent epithelial cells

(EIEC) Pathogenesis

Diagnosis: The ial PCR is also effective in a multiplex

PCR system to identify EIEC strains simultaneously with other E. coli categories.

Treatment: Broad-spectrum antibiotics are

recommended

Diagnosis and Treatment

DAEC are defined by the presence of a characteristic, diffuse pattern of adherence to HEp-2 cell monolayers.

DAEC have been implicated as a cause of diarrhea in several studies, particularly in children >12 months of age

DAEC infection could be proinflammatory;this effect could potentially be important in the induction of inflammatory bowel diseases

Diffusely adherent E. coli (DAEC)

(DAEC) Pathogenesis

DAEC elicits a characteristic signal transduction effect in small bowel enterocytes that manifests as the growth of long finger-like cellular projections, which wrap around the bacteria.

DAEC strains produce a fimbrial adhesin called F1845 or a related adhesin which use DAF, a cell-surface glycosylphosphatidylinositol anchored protein, which normally protects cells from damage by the complement system.

Diagnosis: daaC gene has been used as a DAEC DNA

probe; daaC encodes a molecular usher necessary for expression of the F1845 fimbriae.  No PCR assay has yet been described to identify DAEC.

Treatment:Broad-spectrum antibiotics are recommended

Diagnosis and Treatment

Day 1 Fresh stool samples

were plated onto MacConkey agar the plates were incubated for 18–24 h at 37 °C.

Isolation Methods

Day 2Observation of coloniesRemove your plates from the incubator. Visually examine your plates for Lactose fermenting colonies (3-5 colonies)

Restreaking for isolation We have to obtain a pure culture from plates

contain a mixture of organisms. To obtain a pure culture, we will restreak a single Lactose fermenting colony on Tryptone soy agar plate. Touch the needle end of a sterile loop to a well isolated colony. Streak the needle across the plate. Using a new, sterile needle, streak for isolation for 3-5 colonies. Incubate the plate at 37oC.

Isolation Methods

Day 2Set Biochemical Identification Test From the same colonies inoculate Sufide indole motility medium and Tripple Sugar Iron Medium.

Day 3After 18-24 hours, remove the plate from the incubator and examine it. All of the colonies on this plate should look the same. Record the Biochemical Test Result.Isolates identified as E.coli then saved in Glycerol Peptone Broth and Growth plates are used for PCR.

This medium is both selective and differential. MacConkey agar is used for the isolation of

gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria

The selective ingredients are the bile salts and the dye, crystal violet which inhibit the growth of Gram-positive bacteria.

The differential ingredient is lactose. Fermentation of this sugar results in an acidic pH and causes the pH indicator, neutral red, to turn a bright pinky-red color. Thus organisms capable of lactose fermentation such as Escherichia coli, form bright pinky-red colonies

MacConkey agar

Biochemical Test