A Report on Diarrhea and Its Impact on Health of Public Life
1. Introduction
1.1: Diarrhea- a potential killer:
Diarrhea is defined as the phenomenon with unusually frequent bowel movements and excessive watery evacuations of fecal material. Diarrheal diseases are major causes of morbidity, with attack rates ranging from 2 to 12 or more episodes per person per year, especially in developing countries. It is the second leading cause of death in children under five years old, and is responsible for killing 1.5 million children every year. Nearly one in five children under the age of five dies as a result of dehydration, weakened immunity or malnutrition associated with diarrhea (UNICEF/WHO, 2009). Dehydration is a common phenomenon among patients with acute diarrhea and most people who die from diarrhea actually die from severe to moderate dehydration and fluid loss.
Diarrhea is usually a symptom of gastrointestinal infection, which can be caused by a variety of bacterial, viral and parasitic organisms. Infection is spread through contaminated food or drinking-water or from person to person as a result of poor personal hygiene especially in the developing world. Typically, diarrhea can be classified into two categories based on the duration and severity of the disease:
· Acute diarrhea: Diarrhea that lasts less than a week and is usually related to a bacterial, viral or parasitic infection.
· Chronic diarrhea: Chronic diarrhea lasts more than four weeks and is usually related to functional disorders like irritable bowel syndrome or inflammatory bowel diseases like Crohn’s disease.
Diarrhea has both short-term and long-lasting effects ranging from severe dehydration to malnutrition. Since dehydration is the most lethal consequence of diarrhea; particularly in children and it must be treated promptly to avoid serious health problems.
1.2: Pathophysiologic classification of Diarrhea:
Whatever the cause of diarrhea it is, this disorder belongs to one of the following clinical manifestations which are given below.
Secretory diarrhea
Secretory diarrhea means that there is an increase in the active secretion, or an inhibition of absorption with little to no structural damage. This is most commonly caused by cholera toxin – a protein secreted by the bacterium Vibrio cholerae that stimulates the secretion of anions, especially chloride ions (Kaper, 1996b).
Osmotic diarrhea
This condition arises when excess amount of water is drawn into the bowels. This may be the result of celiac disease, pancreatic disease, or laxatives. Too much magnesium, vitamin C, undigested lactose, or undigested fructose can also trigger osmotic diarrhea. If excessive amounts of solutes are retained in the intestinal lumen, water will not be absorbed and diarrhea will result.
Exudative diarrhea
Exudative diarrhea occurs with the presence of blood and pus in the stool. This occurs with inflammatory bowel diseases such as Crohn’s disease or ulcerative colitis.
Inflammatory diarrhea
It occurs due to damage to the brush border or mucosal lining. This condition causes a passive loss of protein rich fluids. The absorption of the lost fluids is also decreased. Its causes may be viral, bacterial or parasitic infection. In some individuals, disturbed autoimmunity such as inflammatory bowel diseases can cause inflammatory diarrhea
Motility-related diarrhea
Motility-related diarrhea occurs owing to a rapid movement of food through the intestinal area (hypermotility). If the food moves too quickly there is not enough time to absorb sufficient nutrients and water. Sometimes, it causes severe pain in the lower abdomen right after eating any edible.
Dysentery
The presence of blood in the stools is usually a sign of dysentery, rather than diarrhea. Any diarrheal episode in which the loose or watery stools contain visible red blood will be termed as Dysentery. It is most often caused by Shigella species (bacillary dysentery) or Entamoeba histolytica (amoebic dysentery).
1.3: Causes of diarrhea
Acute diarrhea is usually caused by bacterial, viral, or parasitic infection while chronic diarrhea is usually related to a functional disorder. The most common causes of diarrhea include the following (NIH, 2011):
· Bacterial infections: Several types of bacteria consumed through contaminated food or water can cause diarrhea. Common culprits include diarrheagenic Escherichia coli (E. coli), Campylobacter, Salmonella spp. and Shigella spp.
· Viral infections: Many viruses cause diarrhea, including rotavirus, norovirus, cytomegalovirus, herpes simplex virus, and viral hepatitis. Infection with the rotavirus is the most common cause of acute diarrhea in children.
· Parasitic infections: Parasites can enter the body through food or water and settle in the digestive system. Parasites that cause diarrhea include Giardia lamblia, Entamoeba histolytica, and Cryptosporidium.
· Functional bowel disorders: Diarrhea can be a symptom of irritable bowel syndrome.
· Intestinal diseases: Inflammatory bowel disease, ulcerative colitis, Crohn’s disease, and celiac disease often lead to diarrhea.
· Food intolerances and sensitivities: Some people have difficulty digesting certain ingredients, such as lactose, the sugar found in milk and milk products. Some people may have diarrhea if they eat certain types of sugar substitutes in excessive quantities.
· Reaction to medicines: Antibiotics, cancer drugs, and antacids containing magnesium can cause diarrhea as well.
1.4: Acute infectious diarrhea
Acute diarrhea, defined as an increased frequency of defecation (three or more times per day or at least 200 g of stool per day) lasting less than 14 days, may be accompanied by nausea, vomiting, abdominal cramping or malnutrition (Thielman and Guerrant, 2004). Bacteria, virus and parasites are the major enteric pathogens or causative agents of infectious diarrhea in people of all ages but responsible for a high level of mortality, particularly in children below 5 years of age (Cheng et al., 2005).
1.4.1: Etiological agents
Our knowledge of infectious diarrheal disease has expanded enormously over the past decade, particularly with regard to understanding the pathogenesis of infectious diarrhea. A broad spectrum of pathogens is responsible for causing diarrhea which appears to occur worldwide, although some seem to be more frequent in developing countries (Black, 1984). An overview of the agents in diarrhea is given in Figure: 1.1.
Figure 1.1: Overview of causative agents in diarrhea.
1.4.2: Transmission of diarrheal disease
Most of the pathogenic organisms that cause diarrhea and all the pathogens that are known to be major causes of diarrhea are transmitted primarily or exclusively by the fecal–oral route (Feache, 1984). Diarrheal disease may spread through contaminated food and drinking water or from person to person as a result of poor hygiene and sanitation. Younger children fed weaning foods prepared under unhygienic conditions are exposed to food-borne pathogens and are at a higher risk of being affected with diarrheal disease (Barrel, 1979). According to a report published by WHO and UNICEF in 2010, an estimated 2.6 billion people in the developing world lack improved sanitation facilities, and nearly one billion people do not have access to safe drinking water. These unsanitary environments allow diarrhea-causing pathogens to spread more easily.
1.5:Pathogenic Escherichia coli:
Escherichia coli named after its discoverer Theodore Escherich, alsotermed as E. coli are gram negative, rod shaped bacilli under the family Enterobacteriaceae (Figure 1.2) that are commonly found in the gut of warm blooded organisms. Most of them are commensal which benefit the host by providing vitamins (Bentley and Meganathan, 1982) and preventing other pathogenic bacteria within the intestine (Hudault et al., 2001). However, there are several highly adapted E. coli strains that have acquired specific virulence attributes, which confers an increased ability to adapt to new niches and allows them to cause a broad spectrum of disease (Kaper et al., 2004). Horizontal gene transfer events have allowed the transition of some E. coli strains from commensals to pathogens (Baumler, 1997) usually through the acquisition of a pathogenicity island (Lee, 1996). E. coli has been implicated as an agent of diarrheal disease since the 1920s (Nataro et al., 1998) and particular gene clusters such as the attaching and effacing genes (now commonly found in EPEC) are transferred from pathogenic bacteria to commensal E. coli enabling E. coli to become fully pathogenic (McDaniel and Kaper, 1997).
Figure 1.2: Pathogenic Escherichia coli.
(Source: http://edweb.tusd.k12.az.us)
Three general clinical syndromes result from infection with inherently pathogenic E. coli strains: (i) urinary tract infection, (ii) sepsis/meningitis, and (iii) enteric/diarrheal disease (Nataro and Kaper, 1998). This study will particularly focus on the diarrheagenic Escherichia coli (here abbreviated as DEC) which include several emerging pathogens of worldwide public health importance.
1.6:Diarrheagenic Escherichia coli (DEC):
Diarrheagenic Escherichia coli (DEC) strains are major pathogens associated with enteric disease worldwide. Diarrheagenic Escherichia coli pathotypes represent a leading bacterial cause of pediatric diarrhea in developing regions (Nataro and Kaper, 1998), with some responsible for traveler’s diarrhea (Ericsson, 2003; Qadri F, 2005), and are also an emerging cause of diarrhea in industrialized countries (Cohen et al., 2005; Robins-Browne, 2004). However, some strains of DECcan cause severe and life-threatening diarrhea in children and adults.
DEC strains can be divided into six main categories on the basis of distinct molecular, clinical and pathological features (Levine, 1987; Nataro and Kaper, 1998). The main pathotypes of DEC are:
Ø Enterotoxigenic E. coli (ETEC)
Ø Enteropathogenic E. coli (EPEC)
Ø Enteroaggregative E. coli (EAEC)
Ø Enterohemorrhagic E. coli (EHEC, also known as Shiga toxin–producing E. coli [STEC])
Ø Enteroinvasive E. coli (EIEC)
Ø Diffusely adherent E. coli (DAEC)
Figure 1.3: Pathogenic schema of diarrhoeagenic E. coli.
The six recognized categories of diarrhoeagenic E. coli each have unique features in their interaction with eukaryotic cells. Here, the interaction of each category with a typical target cell is schematically represented. AAF, aggregative adherence fimbriae; BFP, bundle-forming pilus; CFA, colonization factor antigen; DAF, decay-accelerating factor; EAST1, enteroaggregative E. coli ST1; LT, heat-labile enterotoxin; ShET1, Shigella enterotoxin 1; ST, heat-stable enterotoxin.
Table 1.1. Diarrheagenic E. coli: virulence determinants and characteristics of disease
ETEC
fimbrial adhesins e.g. CFA/I, CFA/II, K88. K99
non invasive
produce LT and/or ST toxin
watery diarrhoea in infants, adults and travelers to ETEC endemic countries; no inflammation, no fever
EPEC
non fimbrial adhesin (intimin)
EPEC adherence factor (EAF) enables localized adherence of bacteria to intestinal cells
moderately invasive (not as invasive as Shigella or EIEC)
does not produce LT or ST; some reports of Shiga-like toxin
usually infantile diarrhea; watery diarrhea with blood, some inflammation, no fever; symptoms probably result mainly from invasion rather than toxigenesis
EAEC
adhesins not fully characterized
non invasive
produce ST-like toxin (EAST) and a hemolysin
persistent diarrhea in young children without inflammation or fever
EHEC
adhesins not characterized, probably fimbriae
moderately invasive
does not produce LT or ST but does produce Shiga toxin
pediatric diarrhea, copious bloody discharge (hemorrhagic colitis), intense inflammatory response, may be complicated by hemolytic uremia
EIEC
nonfimbrial adhesins, possibly outer membrane protein
invasive (penetrate and multiply within epithelial cells)
does not produce Shiga toxin
dysentery-like diarrhea (mucous, blood), severe inflammation, fever
DAEC:
produce a fimbrial adhesin or a related adhesin.
patients infected with DAEC hadwatery diarrhea without blood or fecal leukocytes
induce a cytopathic signal transduction effect.
E. coli strains whose study has advanced mostly over the last decade, i.e., enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC) and enteropathogenic E. coli (EPEC). Since the categories of diarrheagenic E. coli are differentiated on the basis of pathogenic features, emphasis will be placed on the mechanisms of disease and the major virulence factors of the agents associated with their pathogenicity.
1.7: Enterotoxigenic Escherichia coli (ETEC):
ETEC is defined as E. coli strains that elaborate at least one member of two defined groups of enterotoxins: heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST) (Levine, 1987). Although ETEC strains were first recognized as causes of diarrheal disease in piglets, where the disease continues to cause lethal infection in newborn animals (Alexander, 1994), its ability to cause diarrhea in humans was first demonstrated in human volunteers (DuPont, 1971). Infection with ETEC can cause watery diarrhea that can range from a mild, self-limiting illness to a severe purging disease similar to cholera. It is associated with childhood diarrhea in the developing world and travelers’ diarrhea (TD) among those visiting developing countries or regions of poor sanitation (Nataro and Kaper, 1998; Qadri F, 2005).
ETEC possess a wide variety of O (somatic), K (capsular) and H (flagellar) antigens on its surface. After the discovery of several different molecular structures, including fimbriae, the K phenotype led experts to suggest restructuring the K antigen designation to include only acidic polysaccharides (Lior, 1996). Since then proteinaceous fimbrial antigens have been removed from the K series and have been given F designations (Orskov I, 1982). Specific virulence factors differentiate ETEC from other categories of diarrheagenic E. coli such as enterotoxins and colonization factors (CFs) (MK., 1997; Sjöling A, 2007). Characteristically, ETEC colonize the small intestine by adhering to the epithelium and induce secretion by elaborating toxins without invasion or damage to cells. CFs allow the organisms to readily colonize the small intestines and stimulate the lining of the intestines causing them to secrete excessive fluid thus causing diarrhea.
Figure 1.4: Enterotoxigenic E. coli adhereing to cultured human epithelial cells.
(Source: Laboratoire de Bactériologie, Faculté de Pharmacie, Clermont Ferrand, France).
1.8: Virulence factors of ETEC
The distinguishing virulence factors of ETEC that make the organism pathogenic are enterotoxins and colonization factor antigens. At the beginning of pathogenesis, ETEC must adhere and colonize to small bowel enterocytes and elaborate enterotoxins that provoke intestinal secretion and diarrhea. Colonization is mediated by one or more proteinaceous fimbrial or fibrillar colonization factors (CFs), which are designated by CFA (colonization factor antigen), CS (coli surface antigen) or PCF (putative colonization factor) followed by a number (Kaper, 2004). Hence, the major virulence factors of ETEC are:
· Enterotoxins (LT and/or ST)
· Colonization factors (CFs)
Figure 1.5: Virulence factors of entertoxigenic E. coli.
(Source: Open Courseware of Johns Hopkins Bloomberg School of Public Health.)
1.8.1: Toxins
Enterotoxins are major virulent factors that are released by ETEC after successful fimbrial adhesion to mammalian intestinal cells. Following colonization, ETEC strains produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, and strains may express either or both (Nataro and Kaper, 1998).
1.8.1.1: Heat-labile toxins (LT):
Heat-labile toxins (LTs) are a class of enterotoxins that are closely related in structure and function to cholera toxin (CT), which is expressed by Vibrio cholerae (Spangler, 1992). LT and CT share many characteristics including holotoxin structure, protein sequence (80% identity), primary receptor identity, enzymatic activity, and activity in animal and cell culture assays (Dickinson, 1995). LT enterotoxins are further classified into two major groups: LT-I and LT-II based on their pathogenicity in human and other animals and do not cross-react immunologically.
- LT-I:
LT-I, expressed by ETECstrains that are pathogenic for both humans and animals is an oligomeric toxin of 86 kDa composed of one 28-kDa A subunit and five identical 11.5-kDa B subunits (Streatfield, 1992). The B subunits are arranged in a ring or “doughnut” shape andbind strongly to the ganglioside GM1 and weakly to GD1b and some intestinal glycoproteins (Teneberg, 1994). The A subunit is responsiblefor theenzymatic activity of the toxin. Since LT-I is expressed by ETEC strains that are pathogenic for both humans and animals,these two variants are calledLTh (LTh-I) and LTp (LTp-I) after their initial discovery in strainsisolated from humans and pigs, respectively.
- LT-II:
LT-II is found primarily in animal ETECisolates and rarely in human isolates, but it has not been associated with disease in either organism. The LT-II serogroup of the LT family shows 55 to 57% identity to LT-I and CT in the A subunit but essentially no homologyto LT-I or CT in the B subunits (Fukuta, 1988; Spangler, 1992). Two antigenic variants of LT-II, LT-IIa and LT-IIb, are detected so far.
Figure 1.6: Architecture of the heat labile enterotoxin (LT) of ETEC.
(Source: http://www.bmsc.washington.edu)
1.8.1.2: Heat-stable (ST) toxins:
Heat stable enterotoxins (STs) of ETEC are small, monomeric single peptide toxins that contain multiple cysteine residues, whose disulfide bonds account for the heat stability of these toxins. It include two unrelated classes, STa and STb which differ in both structure and mechanism of action (Kaper, 2004).
- STa:
STa is an 18- or 19-amino-acid peptide with a molecular mass of 2 kDa in which six cysteine residues form three intramolecular disulphide bridges. There are two variants of STa, designated STp (ST porcine or STIa) and STh (ST human or STIb) after their initialdiscovery in strains isolated from pigs or humans, respectively.Both variants can be found in human ETEC strains (Nataro and Kaper, 1998).
Figure 1.7: The crystal structure of heat stable toxin (STa) of E. coli.
(Source: OPM database)
- STb:
STb is associated primarily with ETEC strains isolated from pigs, although some human ETEC isolates expressing STb have been reported. STb is initially synthesized as a 71-amino-acid precursorprotein, which is processed to a mature 48-amino-acid protein(Dreyfus, 1992). The STb proteinsequence has no homology to that of STa, although it does containfour cysteine residues which form disulfide bonds (Arriaga, 1995).
1.9: Mechanism of ETEC Pathogenesis
ETEC infection causes a toxin-mediated diarrhea that is similar to, but less severe than cholera. ETEC initially adhere to the surface of small intestinal enterocytes through ligand–receptor interactions via fimbriae, known as adherence antigens or colonization factor antigens (CFs) (Evans, 1975 ).
After colonization, ETEC secrete plasmid-encoded heat labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). LT toxin, closely related to cholera toxin gets endocytosed and translocated through the cell in a process involvingtrans-Golgi vesicular transport (Lencer, 1995).
The cellular target of LT is adenylate cyclase located on the basolateral membrane ofepithelial cells. The B subunit of LT toxin binds to the target cells via a specific receptor that has been identified as GM1 ganglioside and the A subunit acts by transferring an ADP-ribosyl moiety from NADto the alpha subunit of the GTP-binding protein, GS, which stimulatesadenylate cyclase activity. ADP-ribosylation of the GS subunitresults in adenylate cyclase being permanently activated, leadingto increased levels of intracellular cyclic AMP (cAMP). Excess intracellular cAMP leads to hypersecretion of water and electrolytes into the bowel lumen, when these actions exceed, resulting in secretion of anions (predominantly Cl by a direct effect, and HCO3 indirectly) by crypt cells and a decrease in absorption of Na+ and Cl by absorptive cells (Kaper, 1996a).
The main receptors for the secreted STh toxin is a transmembrane enzyme guanyl cyclase (GC) located in the apical membrane of the intestinal cells. When ST binds to GC it promotes an increase in intracellular levels of cyclic guanosine monophosphate (cGMP) (Mezoff AG, 1992 ; Mooi FR, 1994). The increase in cGMP allows activation of CFTR through phosphorylation-dependent cGMP protein kinase II generating an increase in salt and water secretion and inhibition of sodium absorption via the apical Na/H channel (Mezoff AG, 1992 ).
Figure 1.8: Mechanism of virulence of enterotoxigenic Escherichia coli (ETEC).
1.10: Colonization Factors (CFs):
To cause diarrhea, ETEC strains must first adhere to enterocytes of the small intestine by means of bacterial appendages known as colonization factors (CFs). These adhesions are host specific and exclusively found in human porcine or bovine strains (Mooi FR, 1994). More than 25 colonization factors (CFs) have been recognizedamong human ETEC and many more are about to be characterized(Gaastra, 1996).
Majority of the colonization factors are fimbriae, i.e. rigid hair like pertinacious appendages. Fibrillae, in contrast are thinner and more flexible filaments and some CFs are composed of two fibrillae arranged in a helix, called helicoidal. Furthermore, some other CFs has been reported as non-fimbrial (Hultgren SJ, 1993). On the basis of their morphological characteristics, the CFs can be subdivided on the following: rigid rods, bundle forming flexible rods, and thin flexible wiry structures. Human ETEC strains possess their own array of colonization fimbriae, the CFAs (Mooi FR, 1994).
Figure 1.9: CF expressing ETEC.
Source: (Dolores G. Evans, 1977)
A nomenclature for the CFs designating them as coli surface antigen (CS) was introduced in the mid-1990s (Gaastra, 1996); list showing the old and new CFs is shown in Table 1.2.
Table 1.2: Past and present designations for colonization factors of ETEC
| Nomenclature OF CFs | |
| Old name New name | |
| CFA/I | CFA/I |
| CS1 | CS1 |
| CS2 | CS2 |
| CS3 | CS3 |
| CS4 | CS4 |
| CS5 | CS5 |
| CS6 | CS6 |
| CS7 | CS7 |
| CFA/III | CS8 |
| PCFO159 | CS12 |
| PCFO9 | CS13 |
| PCFO166 | CS14 |
| 8786 | CS15 |
| CS17 | CS17 |
| PCFO20 | CS18 |
| CS19 | CS19 |
| CS20 | CS20 |
| Longus | CS21 |
| CS22 | CS22 |
CFA/I is composed of a single protein assembled in a tight helical configuration. CFA/II is composed of three separate antigens named coli surface antigen 1 (CS1), CS2, and CS3 of which CS3 is expressed either alone or concomitantly with CS1 or CS2. Similarly, CFA/IV is composed of the three antigens CS4, CS5, and CS6. CS6 is usually expressed alone or in conjunction with either CS4 or CS5 or with CS8. Putative colonization factors that are found with varying frequencies include CS7, CS8 (CFA/III), CS12 (PCFO159), CS14 (PCFO166) and CS17. Within each of these families there are cross-reactive epitopes that have been considered as candidates for vaccine development (Rudin A, 1994).
1.11: Enteroaggregative Escherichia coli (EAEC):
Enteroaggregative Escherichia coli (EAEC) is a pathotype of diarrheagenic E. coli defined by its characteristic aggregative, or stacked brick, pattern of adherence to HEp-2 epithelial cells in culture (Nataro and Kaper, 1998). The first association of EAEC with diarrheal disease was reported in 1987 (Nataro JP, 1987). Since then, EAEC has emerged as an important pathogen in several clinical scenarios, including traveler’s diarrhea (Adachi JA, 2001), endemic pediatric diarrhea among children in developing (Okeke IN, 2000) and developed (Pabst WL, 2003) countries.
Figure 1.10: HEp-2 cell assay for enteroaggregative E. coli (EAEC). EAEC has a characteristic aggregative pattern of adherence on the surface of the human epithelial tissue culture (HEp-2) cells (Huang and Dupont, 2004).
EAEC characteristically produce watery, often prolonged diarrhea, which can be associated with abdominal pain and low-grade fever (Huppertz HI, 1997). The largest outbreak of EAEC diarrhea was described in Japan, where a total of 2697 of 6636 school children from 16 different schools became ill after consuming contaminated school lunches (Itoh Y, 1997).
1.12: Virulence factors of EAEC
EAEC initially adhere to intestinal mucosa and form a mucoid biofilm then induce toxic effects on the intestinal mucosa that result in diarrhea (Nataro, 2001). EAEC colonization can occur in the mucosa of both the small and large bowels, which can lead to mild inflammation in the colon (Cheng, 2008). Adhesins, toxins, and several other factors, which could contribute to disease are considered as EAEC virulence factors.
1.12.1: Adhesins
The EAEC-defining criterion-aggregative adherence-suggests that adhesins have an important role in pathogenesis. Microscopy, genetic, and phenotypic studies show that EAEC adhesins are multiple and diverse. The first EAEC adhesin described at the molecular level was the aggregative adherence fimbriae I (AAFI), expressed by EAEC strain (EAEC-17-2). The cloned AAFI fimbriae conferred the aggregative phenotype and agglutination of human erythrocytes on non-pathogenic E. coli (Nataro JP, 1992). Expression of AAF/I needs two regions of the EAEC virulence plasmid (pAA), structural subunit gene aggA (Savarino SJ, 1994) and gene that encodes an AraC-type transcriptional activator called AggR (Nataro JP, 1994). Some of the EAEC strains also contain AAFII which is 25% identical and 47% similar to AAFI (Czeczulin JR, 1997). AAF fimbriae have been shown to mediate aggregative adherence to epithelial cells, haemagglutination, and to be necessary for biofilm formation (Nataro JP, 1992; Sheikh J, 2001).
1.12.2: Cytotoxins
The first EAEC virulence factor that was implicated as a potential cause of diarrhea in laboratory studies was the enteroaggregative heat-stable toxin EAST-1 (Vial PA, 1988). Plasmid encoded EAST-1 is a 38-amino acid peptide with homology to the heat-stable enterotoxin of enterotoxigenic E coli (ETEC) and the endogenous signaling peptide guanylin (Nataro, 2001). Another plasmid encoded toxin (Pet), a serine protease autotransporter is capable of reducing resistance and increasing short-circuit current across rat jejunal tissue (Navarro-Garcia F, 1998). In addition to this enterotoxin activity, Pet has cytotoxic activity against cultured intestinal epithelial cells and erythrocytes (Navarro-Garcia F, 1998).
1.13: Mechanism of EAEC Pathogenesis
EAEC is associated with a complex pathogen-host immune interaction which has some common mode of action in the infected individual. The common features of EAEC pathogenesis can be described in following stages:
Stage I involves initial adherence to the intestinal mucosa and/or the mucus layer which is denoted as aggregative adherence (AA) (Okeke IN, 2000). AAF/I and AAF/II are the structural subunits of aggregative adherence fimbriae and are leading candidates for factors that may facilitate initial colonization.
Stage II involves enhanced mucus production, apparently leading to deposition of a thick mucus containing biofilm encrusted with EAEC. The blanket may promote persistent colonization and perhaps nutrient malabsorption.
In the final stage of EAEC pathogenesis, an inflammatory response with cytokine release, mucosal toxicity and fluid secretion is observed. Plasmid mediated toxin encoded by pet gene on pAA plasmid induces enterotoxic and cytotoxic effects leading to host innate immune system to respond by releasing high level of IL-8 (Steiner TS, 1998).
Figure 1.11: Pathogenesis of EAEC infection. Enteroagreggative E. coli (EAEC) attaches to enterocytes in both the small and large bowels through aggregative adherence fimbriae (AAF) that stimulate a strong interleukin-8 (IL-8) response, allowing biofilms to form on the surface of cells. Plasmid-encoded toxin (Pet) is a serine protease autotransporter that targets ?-fodrin which disrupts the actin cytoskeleton and induces exfoliation (Source: Macmillan Publishers Limited).
1.14: Clinical manifestation of EAEC infection
EAEC-infected patients develop watery diarrhea, without fecal blood or leukocytes. Few patients experience prolonged illness, and most cases resolve without antibiotic therapy. Most infants and children infected with EAEC experience a self-limiting illness resembling that seen in adults. However, a minority of infants may develop persistent diarrhea (longer than 14 days), which may require antibiotics and nutritional support (Cobeljic M, 1996).
1.15: Enteropathogenic Escherichia coli (EPEC):
Enteropathogenic Escherichia coli (EPEC) is an important category of diarrheagenic E. coli which has been linked to infant diarrhea in the developing world. In particular, EPEC was the first strain of E. coli incriminated as the cause of outbreaks of infantile diarrhea in the 1940s and 1950s (Bray, 1945). EPEC are characterized by their ability to induce attaching-effacing (A/E) lesions in the intestine (Moon et al., 1983). EPEC colonizes the small intestine and causes typical attaching-and-effacing lesions characterized by the degeneration of microvilli and intimate adherence of bacteria to epithelial membranes. The genes required for the production of these lesions are located on a pathogenicity island known as the locus for enterocyte effacement (LEE), which encodes (i) intimin, an outer membrane protein product of the eae gene that acts as an adhesin, (ii) a type III protein secretory system, and (iii) several effector proteins secreted by the type III system, including a translocated intimin receptor, Tir, which, once bound to intimin, serves as an anchor for host cytoskeletal proteins (Celli J, 2000).
Figure 1.12: Attaching and effacing histopathology caused by EPEC. The attaching and effacing histopathology results in pedestal-like structures (Source: Nature © Macmillan Magazines Ltd, 1992).
EPEC are divided into two subtypes:
· Typical EPEC and
· Atypical EPEC (ATEC)
· Typical EPEC
In addition to having LEE, typical EPEC strains carry a 90-kb EPEC adherence factor (EAF) plasmid that encodes type IV-like bundle-forming pili (BFP) (Tobe T, 1999). BFP facilitate the adherence of bacteria to the intestinal mucosa and to each other, allowing them to form micro-colonies on epithelial cells in vitro and in vivo (Cleary J, 2004; Tobe T, 2001). Studies with adult volunteers have demonstrated that intimin, EAF plasmid and BFP are essential virulence determinants of EPEC (Donnenberg MS, 1993).
Atypical EPEC (ATEC)
Atypical EPEC (ATEC) (Daniel Mu¨ller, 2006) strains harbor the LEE pathogenicity island but, due to the lack of the EAF plasmid, they mostly adhere in a diffuse pattern to epithelial cells (Ga¨rtner, 2004). There is evidence that atypical EPEC (ATEC), which have eae gene but lack EAF plasmid and bfp gene, are also pathogenic (Trabulsi LR, 2002).
1.16: Virulence factors of EPEC
Different virulent factors of EPEC have been identified based on their ability to show localized adherence to intestinal epithelial cell or attaching and effacing (A/E) lesion on epithelial cells. The main virulence factors that are responsible for the pathogenicity of EPEC include locus of enterocyte effacement (LEE) and EPEC adherence factor (EAF plasmid).
- Locus of enterocyte effacement (LEE)
Virulent EPEC strains contain a 35.5 kb chromosomal region in which cluster of genes (eae, espB, and esc) responsible for pathogenicity occur in close proximity (McDaniel, 1995). This region which encodes a type III secretion system, multiple secreted proteins, and a bacterial adhesin called intimin is called locus of enterocyte effacement (LEE). The LEE contains the eae (stands for E. coli attaching and effacing) gene, encoding the outer membrane protein intimin. This protein mediates intimate adherence to target eukaryotic cells upon interaction with its translocated receptor Tir (stands for translocated intimin receptor), a protein encoded upstream of the eae gene on the LEE.
- EAF plasmids
The BFP (bundle forming pilli) is encoded on plasmids which range in size from 50 to 70 MDa, called the EAF plasmids. Downstream of the bfp gene there is a cluster of three genes encoding a transcriptional activator (Per), which positively regulates several chromosomal and plasmid genes necessary for the pathogenesis of EPEC. Beyond the per genes is a 1-kb restriction fragment that has been extensively used as a diagnostic DNA probe, called the EAF probe.
1.17: Mechanism of EPEC Pathogenesis
A three-stage model of EPEC pathogenesis, comprising of localized adherence, signal transduction and intimate attachment, was first proposed in 1992 (Donnenberg MS, 1993).
The first stage in EPEC pathogenesis involves the initial adherence of bacteria to epithelial cells. Previous studies have implicated the BFP as the initial EPEC attachment factor (Giron, 1991).
The second stage of EPEC infection is characterized by signal transduction. One key finding which has facilitated the study of EPEC pathogenesis was that EPEC secreted a number of proteins via a type III secretion pathway (Jarvis, 1995). The type III secretion system appears dedicated to the secretion of specific proteins, including Tir, EspA, EspB and EspD which are essential for the subversion of host cell signal transduction pathways and the formation of A/E lesions (Kenny, 1995).
The third stage of EPEC infection is characterized by enterocyte effacement, pedestal formation at the apical enterocyte–cell membrane and intimate bacterial attachment to the host cell. This is mediated by a 94 kDa outer membrane protein, intimin. Intimin, encoded by eae gene(E. coli attaching-and-effacing) can modulate a strong antibody response and responsible for full virulence of EPEC (Donnenberg MS, 1993). Then intimin binds to Tir (translocated intimin receptor) of host cell which clusters Tir beneath adherent EPEC. Thus, directly linking extracellular EPEC to the epithelial membrane and anchoring it to the host cell actin and cytokeratin cytoskeleton networks (Batchelor, 2004 ). The type III secretion system is then activated and various effector proteins- including Tir, EspF, EspG, EspH and Map- are translocated into the host cell. EPEC binds through the interaction of intimin with Tir inserted in the membrane and numerous cytoskeletal proteins accumulate underneath the attached bacteria. Protein kinase C (PKC), phospholipase C?, myosin light-chain kinase and mitogen-activated protein (MAP) kinases are activated, which leads to several downstream effects, including increased permeability of cell membrane. Diarrhea results from multiple mechanisms, including active ion secretion, increased intestinal permeability, intestinal inflammation and loss of absorptive surface area resulting from microvillus effacement (Figure: 1.13).
Figure 1.13: Pathogenesis of enteropathogenic Escherichia coli (EPEC).
(Source: Nature © Macmillan Magazines Ltd).
1.18: Clinical manifestation of EPEC infection
The incubation period is variable for EPEC infection. Clinical features of EPEC infection in children include:
– severe acute diarrhea
– low grade fever
– vomiting
– may be persistent diarrhea resulting weight loss and malnutrition even death.
1.19: Treatment and management of Diarrhea
The treatment for diarrhea caused by the three categories of diarrheagenic E. coli (DEC) includesrehydration strategies andantimicrobial therapy. Treatment is primarily supportive and directed toward maintaining hydration and electrolyte balance. Antibiotic therapy is also indicated in case of severe diarrhea. The adjustment and maintenance of hydration is always most important.
Adequate dietary management (including breastfeeding), micronutrient supplementation (zinc, vitamin A, folic acid, copper, and selenium), rehydration (especially with low osmolar ORS), and antimicrobials (to cover bacteria and parasites) are key parts of therapy to manage children with persistent diarrhea (Ochoa TJ, 2004).
- Rehydration
Supplement of fluid and minerals in the form of oral saline (ORS) is the simplest way of rehydration until the diarrhea ceases. Intravenous fluids (such as Ringer’s lactate) are required initially for all patients with severe dehydration.
- Antimicrobials
Diarrhea in children is caused not only by ETEC, EAEC and EPEC but also by other bacterial and viral agents. Therefore it has been difficult to study the effect of antimicrobials in children with different pathotypes of diarrheagenic E. coli. In adults following severe cases, ciprofloxacin, azithromycin, tetracycline, erythromycin, cotrimoxazole are the usual antibiotic of choice.
1.20: Aims and objectives of the study:
1.20.1: General objective:
The objective of the study was to determine the prevalence of three categories of diarrheagenic Escherichia coli (DEC) – enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAEC) and enteropathogenic Escherichia coli (EPEC) infection among the patients of Kumudini hospital, in Mirzapur from January 2010to August 2011.
1.20.2: The specific objectives of the present study were:
1) Determination and confirmation of ETEC, EAEC and EPEC from diarrheal stool specimens by microbiological culture methods.
2) Rapid detection of ETEC organisms in stool samples by genotypic method using polymerase chain reaction (PCR) and characterization of ETEC by phenotypic methods (ELISA, dot blot immunoassay using specific monoclonal antibodies).
3) Detection of EAEC and EPEC from diarrheal stool specimens genotypically using specific primers in multiplex PCR.
4) Association of ETEC, EAEC and EPEC with demographic data of the patients in a particular area in Bangladesh.
1.20.3: Future prospective of the study:
Future prospective of this study is to assist development of vaccine against diarrhea caused by ETEC, EAEC and EPEC. In case of ETEC, various purified CFs have been considered as oral immunogens are less suitable since they are expensive to prepare and sensitive to proteolytic degradation (Levine, 2001). For this reason ETEC toxins (LT, ST) are the best target for the development of vaccine together with formalin inactivated whole cell bacteria where the CFs are still immunogenic but not as labile as purified antigen. Since there are currently no vaccines for prevention of EAEC and EPEC infection, it needs to have extensive studies and knowledge on the pathogenesis and virulence pattern in human for successful introduction of interventions against these pathogens.
| 2. Methods and Materials |
2.1: Study campus:
This study was carried out at the International Centre for Diarrheal Disease Research, Bangladesh (icddr,b) in Dhaka. Stool specimens were collected from January, 2010 to August, 2011 from diarrheal patients at Kumudini Hospital, Mirzapur (Tangail district, Bangladesh) to screen diarrheagenic Escherichia coli (ETEC, EAEC and EPEC) and Vibrio cholerae. This study was approved by the Research Review Committee (RRC) and Ethical Review Committee (ERC) of the centre.
2.2: Clinical specimens and screening:
Stool samples collected from patients and controls were placed in Cary-Blair transport medium and in sterile plastic containers, transported to the laboratory, and inoculated on MacConkey agar media within 24 h. In addition, information regarding age, sex, and clinical features (fever, vomiting and dehydration status) as well as data on the duration of diarrhea, etc. was also collected from the patients. These specimens were then screened for Diarrheagenic Escherichia coli (DEC) and Vibrio cholerae. PCR (Polymerase Chain Reaction) and ganglioside GM1-ELISA (Enzyme-linked immunosorbent assay) techniques were performed to confirm genotypic and phenotypic detection of ETEC toxin. Later, ETEC toxin positive colonies were tetsed by dot-blot method to detect the colonization factors. Detection of diarrheagenic Escherichia coli was performed by multiplex PCR. Detection of V. cholerae O1/O139was done using specific monoclonal antibodies.
| Diarrheal stool
Diarrheal stool |
| Immunological method: ELISA, Dot blot |
| Molecular method: PCR |
| Microbiological analysis
aanalysis |
Figure 2.1 Screening of clinical samples using different methods.
2.3: Study design:
The underlying flow-chart is the overview of the study plan to detect diarrheagenic Escherichia coli (ETEC, EAEC and EPEC) and Vibrio cholerae from the samples of patients:
| Stool and Carry Blair from diarrheal patient of Kumudini Hospital |
| For V. cholerae detection |
| E. coli specific colony was tested for Diarrheagenic E. coli |
| Multiplex PCR to detect ETEC |
| If PCR positive |
| GM1 ELISA to detect ETEC toxin phenotype |
| Dot blot to detect ETEC CFs |
| Streaked on TTGA plate |
| Serological conformation with serogroups of V. cholerae O1/O139 using monoclonal antibodies |
| Multiplex PCR to detect EAEC and EPEC |
| For E. coli detection |
| Streaked on MacConkey agar plate |
Figure 2.2: Flowchart for detection of DEC and Vibrio cholerae.
2.4: Identification of Escherichia coli:
E. coli was recovered easily from clinical specimens on MacConkey agar, on the basis of theirmorphology. To confirm detection of E. coli freshly collected stool specimens were plated on to MacConkey agar plates and incubated at 37°C overnight. Six individual lactose-fermenting colonies with deep pink colour from each clinical sample were tested. The different categories of diarrheagenic E. coli were detected by PCR method and the ETEC toxins were detected by ganglioside GM1-ELISA method.
| Deep pink colonies of lactose fermenting E. coli |
Figure 2.3: E. coli colonies growing on MacConkey agar plate.
2.4.1: Multiplex PCR for the detection of ETEC genes (LT and/or ST)
Ø Reference strains :
Table 2.1: Reference ETEC strains used for the detection of LT and/or ST genes
| Strains Toxin types |
| E. coli ST 64111 STh+
E. coli 286C2 LT+ E. coli 195 STp E. coli VM 75688 LT+, STh+ E. coli E34420C ST-, LT- |
Multiplex Polymerase chain reaction (MPCR) was performed to amplify desired genes of ETEC (LT and/or ST) where LT and ST gene specific primers were used together in one master mixture preparation. After completion of master mixture preparation genomic DNA added to appropriate volume of master mixture and then PCR was done using thermal cycler. Genomic DNA was prepared using following steps:
Ø Template Preparation:
· 100 mL of PBS was poured to each Eppendorf tube.
· One loop of bacteria was taken from MacConkey agar plates (from a pool of six colonies) and suspended in an eppendorf tube containing 100mL of phosphate buffered saline (PBS) for the detection of LT or ST by PCR.
· The suspension was heated at 100°C on water bath for 10 minutes.
· Transferred the tubes on ice and kept for a minute.
· Centrifuge the suspension at 12,000 rpm for 10 minutes.
· The supernatant was the template, ready to use; 3.5mL of supernatant was used for each PCR reaction.
Ø Specific primer sequence used:
| Toxins | Primers | Primer Sequence |
| LT | Forward | 5’-ACG GCG TTA CTA TCC TCT C-3’ |
| Reverse | 5’- TGG TCT CGG TCA GAT ATG TG-3’ | |
| STp | Forward | 5’-TCT TTC CCC TCT TTT AGT CAG-3’ |
| Reverse | 5’-ACA GGC AGG ATT ACA ACA AAG-3’ | |
| STh | Forward | 5’-TAC AAG CAG GAT TAC AAC AC-3’ |
| Reverse | 5’-AGT GGT CCT GAA AGC ATG-3’ |
Ø Master mixture preparation :
Table 2.2: Preparation of master mixture for ETEC detection
| Component | Volume |
| PCR buffer, with MgCl2 (10X) | 2.5 ?L |
| MgCl2 (25 mM) | 0.5 mL |
| dNTPs (2.5 mM each) | 4.0 ?L |
| Primer LT mixture (4 pm/ml) | 2.0 ?L |
| Primer STp mixture (4 pm/ml) | 2.0 ?L |
| Primer STh mixture (4 pm/ml) | 2.0 ?L |
| Taq. Polymerase (5U/ml) | 0.15 mL |
| Deionized water | 10.85 mL |
| Total | 23.65 ?L |
· 1.5 ?L DNA templates added in each tube.
Ø The thermal cycle
Table 2.3: The thermal cycle of ETEC Multiplex PCR reaction
| First step | 95°C for 5 minutes (initial denaturation) | |
| Second step |
94°C for denaturation-30 seconds |
|
| 54°C for primer annealing-30 seconds | ||
| 72°C for elongation-30 seconds | ||
| Third step | 72°C for 5 minutes (final extension step)
4°C until use |
Ø Size of toxin products:
| LT | 250bp |
| STp | 163bp |
| STh | 100bp |
Ø Agarose Gel Preparation (2%):
Amplified PCR products were analyzed by agarose gel electrophoresis on 2% agarose gel. Agarose gel was prepared by adding 2g Ultra pure agarose in 100 ml 1X TBE buffer (Invitogen, ultra pure) and melted at med high temperature in micro oven for 3-4 minutes.
• 4mL of ethidium bromide was added to the gel, mixed well and poured on a gel casting.
• 30-40 minutes allowed for gel formation.
Ø Agarose Gel Electrophoresis:
• 4mL of loading dye was mixed with 26 mL of PCR product and loaded into the gel.
• Amplified PCR products were separated at 150 volt for 30 minutes.
• The band was observed on a Gel documentation system (BIORAD) under UV light.
2.4.2: Detection of LT and ST toxin producing isolates by ELISA:
The procedure for detection of LT is based on the binding of the B-subunit of the toxin to GM1 ganglioside and the detection of ST is based on its ability to inhibit the binding of ST to the GM1 and ST-CTB conjugates by ELISA technique.
Ø Procedure
• For coating purpose 100 mL of ganglioside GM1 solution, 0.3mg/mL, was added to each well of an ELISA plate and incubated at room temperature overnight (these plates could be stored at +4°C for about 2 weeks until used).
• GM1-coated plates were washed twice with PBS and then concentrated solution of non-interacting protein, bovine serum albumin (BSA) (0.1% BSA-PBS, 200 mL /well) is added to plates and incubated at 37°C for 30 minutes. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
• Plates were washed once with PBS and 100 mL of Luria-Bertani (LB) broth containing 45 µg/mL of lincomycin was added to each well of the plate.
• Six individual colonies (for one sample) from MacConkey plates (using which PCR was done) were inoculated, single bacterial colony/well, with wooden sticks. The outside rows and columns of the ELISA plate were excluded to avoid background.
• The plates were covered with a plastic film (to prevent evaporation) and incubated with shaking at 250 rpm overnight at 37oC. These plates were used for the detection of LT toxin.
• On next day, another GM1 coated ELISA plate (for each to be tested for LT) was washed twice with PBS and blocked with 200 mL 0.1% BSA-PBS at 37oC for 30 minutes. These plates were used for the detection of ST.
• The plates were washed once with PBS and then 100 mL of recombinant ST-CTB conjugate solution was added. Plates were incubated at room temperature for 60 min.
• The plates were washed three times with PBS and then 50 mL volumes of the overnight cultures from the plates for detection of LT were transferred to the corresponding wells in the plates used for the detection of ST. Then 50 mL of anti-ST mAb (1:3 dilution) was immediately added and incubated at room temperature for 90 min.
• Following this LT plates were washed three times with PBS contained 0.05% Tween and 100 mL of anti-LT mAb (LT 39:13:1) was added immediately and incubated at room temperature for 90 min.
• Both types of plates (LT and ST) were washed three times with PBS-Tween.
• After that 100 mL of conjugate (anti-mouse IgG-HRP, 1:1000 dilutions in BSA-PBS-Tween) was added into each well and incubated at room temperature for 90 minutes.
• All plates were washed three times with PBS-Tween.
• Developed with OPD (orthophenyl diamine), prepared by dissolving in 10 mg of 0.1 M sodium citrate buffer, (pH 4.5), to which was added 4 mL of 30% H2O2 immediately before use. The substrate was added 100 mL/well and i