The following points highlight the top two types of Diarrhoeal Diseases. The types are: 1. Non-Invasive Bacterial Pathogens 2. Non-Invasive Bacterial Pathogens.
Diarrhoeal Diseases: Type # 1.
Non-Invasive Bacterial Pathogens:
A. Enterotoxigenic Escherichia coli (ETEC) Pathogens.
ETEC produce two types of toxins:
(a) Heat stable enterotoxin (ST)
(b) Heat labile enterotoxin (LT); or both.
In addition to these toxins, they also have plasma mediated colonisation factor which favour E. coli to adhere to the mucosa of small intestine. LT is related to enterotoxin of V. cholera and is a protein complex consisting of one A and five B sub-units. Sub-unit A is activated to give two fragments—A1 and A2. Sub-unit B are responsible for binding of the toxin to the epithelial cells.
A1 catalyses activation of adenylate cyclase to cause an increase in the concentration of cyclic adenosine 5′ monophosphate (cAMP) which inhibits the absorption of sodium by epithelial cells and increases sodium secretion. It causes loss of chloride and water which results into a profuse watery diarrhoea.
Whereas, the other enterotoxin, ST, exerts its effect through stimulation of guanylase cyclase (cGMP) in the intestinal mucosa. Its action is more rapid than that of LT. Some important ETEC serogroups are O6, O8, O15, O25, O115, O148 and O167.
ETEC strains cause diarrhoea in children under 5 years of age (infantile diarrhoea). They also cause Traveller’s diarrhoea in persons of developed countries who visit unhygienic underdeveloped countries.
The incubation period is 24-72 hours. In severe cases, its symptoms last for 24-72 hours- like Cholera. Patients respond well to intravenous (IV) electrolytic repletion therapy, and Oral Rehydration therapy (ORT) recommended by WHO.
B. Enteropathogenic E. coli (EPEC):
Though these pathogens are non-toxic and non-invasive, they cause infantile gastroenteritis in tropical countries, by their plasmid-encoded adherence to the mucosa of small intestine which is associated with dissolution of glycocalyx and loss of brush border microvilli. However, the exact mechanism is yet to be known. They respond to IV fluid therapy. Important EPEC strains are O26, O25, O86, O111, O126, O128, O142.
Laboratory diagnosis of ETEC and EPEC diarrhoea: Faeces from diarrhoeal patients can be inoculated in MacConkey’s agar medium. The isolates were used for Biochemical test, Agglutination test. The organism is first tested by polyvalent antisera for the EPEC serogroups, and then by individual O antisera.
C. Vibrio Cholera:
On the basis of their polysaccharide O antigen, these organism are classified into two groups:
(i) Ol Vibrio cholera;
(ii) non-Ol Vibrio cholera (non-agglutinating vibrios).
Within the group V. cholera Ol, the strains are categorized into one of the two biotypes classical or el’ tor. Both biotypes are separated into Inaba and Ogawa and Hikojima serotypes.
V. cholera cause cholera (acute diarrhoeal disease) which may result into severe and rapidly progressive dehydration causing death within hours unless quickly treated with IV electrolyte and ORT.
Some non-O1V. cholera (non-agglutinating vibrio) cause diarrhoea only, it is rarely severe.
Other vibrios, V. parahaemolyticus, V.fluvialis cause human diarrhoea. V. parahaemolyticus, V. mimicus, V. vulnificus cause invasive diarrhoea.
Pathogenesis of Vibrio Cholera:
Incubation period is 8-24 hours. V. cholera adhere to the mucosa of small intestine by mean of their adhesions. Once adhered, they produce Cholera toxin (CT) similar to LT enterotoxin of ETEC, which is plasmid-coded CT and has two sub-units, a single sub-unit A and 5 sub-units
B. These 5 sub-units B mediate binding of the toxin to receptors of jejunal mucosa. Then a single sub-unit A is transported into the cells which is responsible for the increase in the level of intracellular cyclic AMP (cAMP) causing an increase in the flow of water and electrolyte into the lumen.
This fluid contains high concentration of bicarbonate and potassium. The loss of large volumes of isotonic body fluid is the cause of Cholera.
Klebsiella and Enterobacter and other enter-toxigenic Enterobacteriaceae can sometimes cause acute diarrhoea in developing countries.
Laboratory diagnosis of V. cholera is similar to that of E.coli.
V. cholera is grown on selective media (TCBS).
D. Clostridium Perfringens Type A:
This organism is distributed in soil and in faeces of man and animal. Its heat-resistant spore can survive cooking, spores germinate into vegetative forms during slow cooking and unrefrigerated storage of food. Consumption of such contaminated food with CI. perfringens type A can cause food poisoning and gastroenteritis in man.
Pathogenesis of Clostridium Perfringens Type A:
The disease (food poisoning), caused by CI. perfringens type A, is characterised by diarrhoea, abdominal pain following ingesting of food (meat, poultry product) contaminated with this organism. Incubation period is 6 to 12 hours, and its duration is 10-24 hours, and the illness is self-limited. The heat-labile enterotoxin produced by this organism acts mainly on the membrane permeability of the small intestine.
Besides, CI. perfringens types C and D strains have been reported to produce enterotoxin which is responsible for food poisoning. Antibiotic therapy has no value. Presumptive diagnosis is done by isolation of the same serotype from a large numbers of the victims of an outbreak of food poisoning.
Laboratory diagnosis of Clostridium Perfringens Type A:
Faeces from large numbers of victims and food samples should be inoculated in aminoglycoside blood agar and incubated anaerobically colonies may be β-haemolytic or non- haemolytic. Gram-staining shows Gram-positive rod with sub-terminal spore. Identification of the organism can be done by Nagler reaction and serotyping by agglutination test.
E. Staphylococcus Aureus:
Strains of staph, aureus belonging to phage group III (type 6/47) and group IV (421) produce enterotoxin. There are five serologically distinct enterotoxins (A to E). Of these, enterotoxin A is most often incriminated in outbreaks of staphylococcal food poisoning; this enterotoxin A is toxic protein and is heat-stable.
Preformed enterotoxin (toxic-dose = lµ gm. of toxin) in contaminated food can cause acute staphylococcal food poisoning after its ingestion. During acute illness the causative organisms are absent in the stool. The illness is characterised by acute onset of nausea and vomiting within few hours after ingestion of contaminated food followed by diarrhoea.
It is self-limiting illness and rarely severe, Acute staphylococcal food poisoning differs from other non-invasive bacterial diarrhoea by its prominence of vomiting. Treatment is by intravenous electrolytic repletion therapy. Antibiotic therapy is of no value.
Pathogenesis of Staphylococcus Aureus:
Preformed toxin acts locally on the intestinal mucosa. Vomiting is apparently mediated by a direct effect of the toxin on the central nervous system. The outbreaks depend on the food habits of the country. Staph, aureus can multiply in a variable temperature, 4°-46°C.
If contaminated food is left at room temperature after cooking, the organisms multiply on potato, salad, cream, sauces. A staphylococcal lesion on the fingers of the cook or food handlers may contaminate the food.
Laboratory diagnosis of Staphylococcus Aureus:
Suspected food, vomit, or faeces specimens are inoculated on selective media, Mannitol-salt agar. Selective media are preferred for stool culture because of mixed flora. The isolate is identified by Coagulase test and epidemiology is traced by phage typing.
Demonstration of Enterotoxin:
Enterotoxin of food, vomit, or culture filtrate of isolate can be detected by ELISA and Reverse Passive Latex Agglutination (RPLA) kits which are commercially available.
F. Bacillus Cereus:
The infection occurs after ingestion of food contaminated by non-pathogenic organism, B. cereus, which is capable of producing two distinct enterotoxins—one resembling LT of E. coli and other that of staphylococcal enterotoxin.
They cause two distinct clinical syndromes:
i. Short Incubation Period (1-2 hours) Types:
Symptoms are nausea, vomiting, often followed by diarrhoea, similar to these of staphylococcal food poisoning. This type is associated with bulk prepared rice. This is also called as “emetic form of B. cereus food poisoning”.
ii. Long Incubation Period (6-16 hours) Type:
This type of gastroenteritis is characterised by sudden onset of diarrhoea and abdominal pain and is similar to those caused by enterotoxin producing E. coli (ETEC). Soups and sauces contaminated with B. cereus can cause this type of illness.
Epidemiology of Bacillus Cereus:
B. cereus are distributed in soil and cereals. Their spores resist cooking. When food is stored at a warm temperature, the spores germinate into vegetative forms which produce enterotoxin.
The short incubation period of B. cereus food poisoning is associated with contaminated fried rice served in mass feast or in restaurants. Brief rewarming of the contaminated food does not destroy heat stable toxin.
Laboratory diagnosis of Bacillus Cereus:
Suspected food, vomit, faeces can be inoculated on nutrient agar, blood agar. Overnight incubation shows “curled hair” colonies typical of B. cereus. They are Gram-positive spore- bearing bacilli seen in smear.
G. Clostridium Difficile:
CI. difficile is associate with about 20-30% cases of antibiotic associated diarrhoea and pseudomembranous colitis.
The most common antibiotics (lincomycin, clindamycin, ampicillin and cephalosporin’s) are associated with pseudomembranous colitis.
Pathogenesis of Clostridium Difficile:
When the antimicrobial therapy has suppressed the growth of competing gutflora there is overgrowth of antibiotic resistant CI. difficile—which is quite common in the faeces of neonates.
CI. difficle produces two toxins: enterotoxin (toxin A); cytotoxin (toxin B). Toxins A and B act synergistically. Toxin A causes tissue damage and accumulation of fluid in the lumen of experimental animal. Thus Toxin A has both histo-toxic and enterotoxic effects. Cytotoxin causes cytopathic effect (CPE) in cell culture which is a diagnostic marker for CI. difficile.
In antibiotic associated diarrhoea, the colonic mucosa shows a mild erythema with oedema. Sometimes the colitis is more severe and is characterised by an exudate membrane (pseudo membrane). The mucosa may be haemorrhagic. Withdrawal of antimicrobial therapy may lead to early resolution of symptoms.
Laboratory diagnosis of Clostridium Difficile:
1. Demonstration of toxin may be done by the cytopathic effect of faecal filtrate on Vero or human embryo fibroblast and by neutralisation of toxic effect by specific antiserum.
2. Culture is done by inoculating faeces on selective media and subsequent toxigenicity test.
Diarrhoeal Diseases: Type # 2.
Non-Invasive Bacterial Pathogens:
A. Salmonella Gastroenteritis:
The common food poisoning serotypes of salmonella are S. enteritidis (poultry egg), S. typhimurium (rat, mice), S. dublin (cattle), S. hador (turkey), S. choleraesuis, S. helderberg, and S. thompson.
Incubation period of salmonella food poisoning is 12-36 hours. This illness is characterised by self-limited fever and diarrhoea, vomiting and abdominal pain.
Common sources of salmonellae are food derived from domestic animals and poultry (meat, egg, sausages). Sometimes, human cases and carriers (food handlers) may transmit this infection.
Pathogenesis of Salmonella Gastroenteritis:
Ingestion of contaminated food may cause salmonella food poisoning. Salmonellae multiply in the food and later in the intestine of victim—only large doses of salmonellae are required for human infection. Sometimes septicaemia may develop due to infection with S. enteritidis.
Sporadic cases of salmonella gastroenteritis may go unnoticed; but large outbreaks—often called “food poisoning“—are investigated and diagnosed.
Laboratory diagnosis of Salmonella Gastroenteritis:
Faecal sample or food can be cultured on selective media (MacConkey’s agar, deoxycholate Citrate agar, Wilson and Blair medium) Pale non-lactose fermenting colonies appear on MacConkey’s agar and DCA; black shiny colony on Wilson and Blair medium.
Identification can be done by biochemical tests, later by agglutination test with polyvalent group sera (O and H). Epidemiology is confirmed by phage typing. Treatment consists of fluid repletion therapy. Antibiotics are not advised as they may contribute to reemergence of antibiotic-resistant strain.
Sh. dysenteriae, Sh. flexneri, Sh. boydii, Sh. sonnei cause bacillary dysentery as they are invasive pathogens; but Sh. dysenteriae type I (Sh. shigae) also produce a powerful enterotoxin (neurotoxin) and cytotoxin.
Pathogenesis of Shigella:
Infection is spread via food, flies, fomites. Ingestion of contaminated food may cause bacillary dysentery. The organisms invade the colonic mucosa. Then cell-to-cell spread of infection occurs. Infected cells are killed, DNA inflammation develops in the sub-mucosa and lamina propria resulting into necrosis and ulceration of epithelium of intestine.
They do not invade deeper layer and blood (no bacteriaemia). Stool contain blood mucus.
Laboratory diagnosis of Shigella:
Stool and rectal swabs can be inoculated on MacConkey’s agar and DCA; after 26 hours incubation non-lactose fermentating pale colonies are formed; except Sh. sonnei which is late lactose fermenter.
Identification of Shigella:
(1) Biochemical test;
(2) Agglutination test by group specific polyvalent antisera to shigella.
C. Enteroinvasive E. coli (EIEC):
They cause bacillary dysentery quite similar to that of shigellosis, but without vomiting. They invade the mucosa of large intestine and multiply intracellularly. The incidence of diarrhoea caused by EIEC is about 5% of all diarrhoea.
The illness is short lived and antibiotic therapy is of no use. Diagnosis can be done in the laboratory by culturing the stool on MacConkey’s agar and by serotyping of isolate. Serogroup 0124 is most common, others are 0128 and 0112.
Verocytotoxigenic E. coli (VTEC):
VTEC was previously called enter-haemorrhagic E. coli (EHEC). It is associated with two clinical conditions-haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). In haemorrhagic colitis there is blood stained diarrhoea preceded by abdominal pain and watery diarrhoea, sometime pyrexia.
All age group are affected. E. coli sero group 0157 is vero cytotoxigenic strain. VTEC is non-invasive and produces verocytotoxin similar to Shiga toxin and, hence, it is called “Shiga Like Toxin” (SLT)—food is the source of VTEC infection.
Laboratory diagnosis of EIEC:
Faeces can be cultured on MacConkey agar, lactose fermenting pink colonies appear on this medium after overnight incubation.
Identification of EIEC:
i. About 95% E. coli are sorbitol fermenters, whereas 0157 VTEC is sorbitol non-fermenter.
ii. Production of Vero cytotoxin (VT) is determined by its cytotoxic effects of isolated strain on vero cells.
iii. Agglutination test is done by serotyping of the strain. Rising titres of VT neutralizing antibodies may be detected in patient’s serum.
D. Campylobacter jejuni:
C. jejuni is major cause of diarrhoea. It occurs in the intestine of wild and domestic animals (poultry). Milk and water are common sources of outbreak of diarrhoea due to C. jejuni. Its incubation period is 3-10 days. The illness is characterised by diarrhoea with blood mucus in the stool with abdominal pain. It is self-limiting; septicaemia is common in severe cases.
Duration of Campylobacter jejuni:
Usually 3-4 days rarely it may persist for 3-4 weeks
Laboratory diagnosis of Campylobacter jejuni:
Culture selective media containing vancomycin, polymyxin are used. Optimum temperature for growth is 43°C. Faeces is inoculated in Thayer Martin medium and incubated at 5-10% CO2 at 42-43°C. C. jejuni and E. coli selectively grow against other faecal bacteria.
Identification of Campylobacter jejuni:
These organisms are Gram-negative, curved, slender bacilli; showing darting motility. They are oxidase positive.
Treatment of Campylobacter jejuni:
Campylobacter enteritis is self-limiting and does not require treatment. Severe cases respond to erythromycin therapy which also reduces the duration of excretion of the organism.
E. Vibrio Parahaemolyticus:
It is a Gram-negative, curved, non-motile aerobic bacillus present in coastal waters throughout the temperate zones. It is a common cause of diarrhoea (food poisoning) in Japan and Singapore. Man gets infected after ingestion of raw sea food (fish and shell fish). Other symptoms are abdominal pain and vomiting; stool may contain blood.
Kanagawa positive strains of V. parahaemolytics produce a cytotoxin which causes diarrhoea in volunteers. These strains also adhere to human intestinal cells. They produce a heat labile enterotoxin like that of E. coli.
Laboratory diagnosis of Vibrio:
Culture MacConkey’ agar, TCBS and alkaline peptone water containing 1% NaCl are used as they are halophilic vibrios. After overnight incubation, pale colonies develop.
Identification of Vibrio:
Isolates are identified by biochemical tests and agglutination test.
Other Halophilic Vibrios:
Vibrio mimicus and V. vulnificus cause sporadic cases of diarrhoeal diseases in men who ingest raw or under-cooked sea fish occurring along the Gulf Coast. V. mimicus diarrhoea follows after taking oysters while V. vulnificus occurs after ingestion of shellfish.
They do not produce cholera enterotoxin. The illness is self-limited. V. vulnificus causes septicaemia. They grow in alkaline peptone water with 1% NaCl and on thiosulphate bile salt sucrose agar.
F. Yersinia Enterocolitica:
It is common in Canada and Northern Europe (Belgium and Scandinavian countries), and is distributed worldwide. Sources of infection are birds, wild and domestic animals. The infection spreads via faecal-oral route from animals by contaminated drinking water or directly from person to person.
Symptoms vary with age. Predominant symptom in young children is acute watery diarrhoea, lasting for 3-14 days; about 5% children have blood in stool. In older children and young adults there is pain in right upper quadrant of abdomen, fever, and moderate leucocytosis which mimics appendicitis.
In debilitated patients, the infection may progress to septicaemia. This organism can be isolated from stool or from blood culture. It is non-lactose fermenting gram-negative aerobic bacilli. It is susceptible to chloramphenicol, aminoglycosides, tetracycline, and cotrimoxazole.