APPLIED MICROBIOLOGY

Diarrhoeal Diseases

Acute diarrhoeal diseases are important cause of morbidity and mortality throughout the world,
particularly in infants and children in the developing countries.

• Diarrhoea may be defined as an increase in the frequency, fluidity or volume of bowl movements
relative to the usual habits of each individual. As a rough guide, passage of three or more motions a
day can be taken as diarrhea

• Gastroenteritis may be defined as inflammation of the mucus membrane of the stomach and
intestine resulting in frequent loose motions with or without blood and mucous, pain in abdomen
and may be associated with vomiting

• Dysentery means presence of blood and mucous in stool, often with tenesmus

• Traveller's diarrhoea is an acute diarrhoeal illness that sometimes occurs in visitors from foreign
countries within a week or two in a developing country

• Food poisoning is an acute diarrhoea with or without vomiting caused by microbial contamination
of food

Causal Agents:
Causes of infective
diarrhoea

VIRAL: PARASITIC: BACTERIA: Others

FUNGAL:
• Rotavirus • E.histolytica • Salmonella • Candida
• Norwalk agent • Giardia intestinalis • Shigella
• Adenovirus • Cryptosporidium • Escherichia coli ANTIBIOTIC
• Coxsackie virus • Paragonimus • Vibrio cholerae ASSOCIATED:
• Coronavirus • Fasciola hepatica • V. parahaemolyticus
• Calicivirus • Fasciolopsis buskii • Campylobacter • Clindamycin
• Enteroviruses • Intestinal worms • Yersinia • Lincomycin
enterocolitica • Antimetabolites
• Aeromonas • Thyroid
• Plesiomonas replacements
• Staphylococcus
aureus
• Bacillus cereus
• Clostridium difficile
Bacterial diarrhoea can be divided in two groups:

A. Non Invasive bacterial pathogens

Vibrio cholerae---classical and El Tor

Noncholera vibrio (NAG vibrio)
Enterotoxigenic Escherichia coli (ETEC)
Enteropathogenic Escherichia coli (EPEC)
Clostridium perfringens type A
Staph. aureus
Cl. difficile
Shigella dysenteriae
Gastroenteritis or food poisoning caused by these organisms is toxin mediated.

B. Invasive bacterial pathogens

Salmonella sp.
Campylobacter sp.
Shigella sp.
Enteroinvasive Escherichia coli (EIEC)
Enterohaemorrhagic Escherichia coli (EHEC)
Yersinia enterocolitica
Vibrio parahaemolyticus
Yersinia pseudotuberculosis
Gastroenteritis or food poisoning caused by these organisms is because of their invasive mechanism
and not because of the toxins.

Collection and Transportation of Samples

Stool sample should be collected from the patient before the start of any antibiotic therapy. Specimen
should be collected in any clean container free of disinfectant. Rectal swabs can also be taken if it is
not possible to obtain stool samples.
Cary Blair medium is recommended for transport of the feces or rectal swabs. Both macro- and
microscopic examination of stool sample are done.

Macroscopic Examination

The color, consistency, blood, mucus, or pus or any parasite in the stool sample, are examined with
naked eye.

Microscopic Examination

This is done by making a saline preparation of the stool and looking for pus cells, RBCs, or any
parasitic ova and cysts. Formol ether concentration technique can be employed to concentrate
parasitic ova and cysts in stool sample

Isolation of Bacterial Pathogens

The purpose is to identify the bacterial pathogen and characterise the same. For this purpose in
addition to the routine plating media, enrichment and selective media are used to inhibit the growth of
normal flora. The various media used a,re MacConkey's agar (MA), Desoxycholate agar (DCA),
Salmonella Shigella Agar (SSA) and Selenite F broth (SeF). Selenite F enrichment permits the growth
of Salmonella and Shigella while inhibiting all other non-pathogenic enteric bacteria.

Usual biochemical reactions for presumptive identification of some of the common bacterial
enteropathogens are given in Table
Newer Diagnostic Tests

The conventional tests for the diagnosis of diarrhoeal diseases are aimed at isolation and
identification of the causative agent and demonstration of its pathogenic properties in animal models.
The newer tests are directed towards detection of pathogenic attributes of the microorganism such as
toxigenicity, adhesion, invasion, etc. or the genes responsible for these characteristics. These tests are
highly sensitive, mostly repro ducible and do not need animal models. However, these tests identify
certain properties. They fail to differentiate between the organisms showing similar properties such
as V. cholerae and ETEC, and Shigella and EIEC.

Immunological Tests

These include followings:

a. Latex agglutination
b. Co agglutination
c. Biken test
d. ELISA
e. Detection of enterotoxins

Latex agglutination test is used for detection of cholera toxin and rotavirus. The test can be
performed even in peripheral laboratories. Coagglutination test is done adopted for LT and CT and is
performed in capillary tubes. Biken test is a precipitation test based on Elek's principle and
Ouchterlony's double diffusion test. The LT toxin is liberated on culture plate by a test strain of Esch.
coli, diffuses into the agar and reacts with LT specific antiserum which has been put into well at the
centre of a cluster of colonies. If the colony produces LT, a precipitation line forms between the LT
producing colony and the well containing anti-LT. Lincomycin is added into the medium to make the
test sensitive, to further stimulate production of toxin and treatment of the colony with polymyxin B
to promote release of L T from the cell.

Enzyme linked immunosorbent assay (ELISA) is used for:

a. Detection of cholera toxin/ Esch. coli LT

b. Esch. coli heat stable toxin
c. Rotavirus

Antibiotic sensitivity test are to be done on all the isolates so as to generate data about their
sensitivity patterns in a given area. Detection of enterotoxins can be done by involving various
methods like rabbit ileal loop, infant mouse, passive immune haemolysis, DNA probs etc.

DNA Probes

DNA probes developed for hybridisation are:

a. Esch. coli heat labile toxin and cholera toxin (LT /CT)
b. Esch. coli heat stable toxin (STa and STb)
c. Esch. coli cytotoxin (VT-l and VT-2)
d. Enteroadhesive factor of Esch. coli
e. Enteroinvasive plasmid of Esch. coli and Shigella
f. Campylobacter
The advantages of DNA probes over other tests are that they identify the nucleic acid content rather
than the products it encodes thus making the method more reliable, these can be developed for
fastidious pathogens, they can be used to detect pathogenic agents directly from the specimens,
results become available in one day and large number of specimens can be processed at a time.

Detection of Viral Agents Causing Diarrhoea

Rotavirus is the most common viral agent causing diarrhoea in children. The various methods used
for demonstration of rota virus are: (a) X Latex agglutination, (b) ELISA, (c) Electron microscopy and
(d) Electropherotyping. For detection of rotavirus, stool samples should be transported to the
laboratory at the earliest. If delay is anticipated the sample can be sent by suspending small amounts
of faeces in 1 ml of PBS and freezing it to -20°C.