II MBBS CBME Pattern

1. Gastrointestinal system
MI 3.1
Organisms causing
Diarrhea & Dysentery
Dr V S Vatkar
Associate Professor
Department of Microbiology
D Y Patil Medical College, Kolhapur

2. Microbial flora
 Upper GIT: Sterptococci in oral cavity &
Lactobcillus in stomach
 Lower GIT: microbial load gradually
increases towards lower part of GIT, highest
in the distal ileum

3. Gastrointestinal infective
syndrome
 Diarrheal diseases:
 Diarrhea
 Dysentery
 Traveler's diarrhea
 Persistent diarrhea
 Gastroenteritis
 Food poisoning

4. Other gastrointestinal infective
syndrome
 Acute vomiting
 Necrotizing enterocolitis
 Necrotizing enteritis
 Pseudomembranous enterocolitis
 Peptic ulcer disease
 Infections of other GI structures like
appendicitis, diverticulitis, typhlitis

5. Diarrheal diseases
 Diarrhea: (by WHO) defined as passage of three or
more liquid stools per day, in excess than the usual
habit for that person
 Acute diarrhea: lasts for <14 days , mostly caused
by Viral agents followed by bacterial or parasitic
agents
 Dysentery: defined as stool with blood & mucus
often associated with fever, abdominal pain &
tenesmus (feeling of constant need to pass stools
despite of an empty colon)

6. Traveler’s diarrhea
 Travel related infection. Occurs in about 20-50% of
travelling people from temperate to tropical regions
of Africa, Asia & Central and South America.
 Case presentation: first 3-5 days : sudden onset of
abdominal cramps, anorexia & watery diarrhea . Ds
is self limiting lasts for 1-5 days
 Organisms: enterotoxogenic E.coli , Compylobcter
jejuni . Norovirus diarrhea : associated with
travelling on Cruise ships.

7. Persistent And Chronic Diarrhea
 Lasts for more than ˃ 14 days (usually 2-4 weeks)
 Infections due to various organisms like
 Parasites: Cryptosporidium, Cyclospora, Entamoeba
histolytica, Giardia
 Bacteria: areomonas, Compylobacter, Clostridium
difficile
 Other various infections: pancreatic disorders,
intestinal disorders (Crohn’s ds, Irritable bowel
syndrome) or tumors

8.  Gastroenteritis: defined as inflammation of mucus
membrane of stomach & intestine resulting diarrhea,
vomiting & abdominal pain, with/without mucus or
blood in stool
 Food poisoning: consumption of food or drink
contaminated either with microorganisms or their
toxins

9. Pathogenic Mechanism
 Inoculum size: infective dose
 Shigella, enterotoxogenic E.coli, Giardia ,
Entamoeba histolytica: 10-100 bacteria or cysts
 Vibrio cholerae: 10^5- 10^8
 Salmonella: 10^3-10^5 bacilli
 Adherence: many microorganisms adhere to
intestinal mucosa e.g. enterohaemorrhagic E.coli,
entropathogenic E.coli, enterotoxogenic E.coli &
V.cholerae

10. Pathogenic mechanism---- cont
 Toxin production: Entero toxins: causes watery
diarrhea, Cytotoxins: causes destruction of mucosal
cells leading to inflammatory diarrhea, Neurotoxin:
act directly on CNS producing vomiting.
 Invasion: bacteria invades inside the mucosa
resultingto dysentery
 Predisposing factors: alteration of host’s defense
mechanism: suppression of normal flora,
neutralization of gastric acidity(V.cholerae)

11. Pathogenic mechanism------- cont
 Inhibition of intestinal motility: interfere with the clearance of
the bacteria from the small intestine
 Age: children below 5 yrs age : at risk (mostly during weaning
period)
 Location: Closed or semi-closed areas like day care centers,
schools, residential area, ship cruise (responsible for outbreaks
of diarrheal diseases)
 Antibiotic associated : prolong antibiotic treatment
 Impaired host immunity: Immunocompromised conditions:
AIDS, hypogammaglobulinemia especially Cl deficile & giardia
 Genetic factors: O bl gr people : more susceptible to
V.cholerae, E.coli O 157, Norovirus

12. Organisms Causing Non-
Inflammatory Diarrhea
 Usually proximal
small intestine
 Watery diarrhea
 No leukocytosis
(pus cells : not
increased)
 Bacteria : (mostly
enterotoxine mediated)
 V.cholerae
 E coli: Entropathogenic ,
Enterotoxogenic,
Enteroaggregative
 Clostridium perfringens
 Bacillus cereus
 Staphylococcus aureus
 Areomonas hydrophila
 Pleciomonas shigelloides

13. Organisms Causing Non-
Inflammatory Diarrhea----- cont.
 Viruses:
 Rotavirus
 Noro virus
 Enteric Adenovirus 40, 41
 Calcivirus
 Astrovirus
 Fungus:
 Microsporidia
 Parasites:
 Protozoa : Giardia lamblia,
Cryptosporidium parvum,
Cryptosporidium
cayetanensis, Isospora belli
 Helminths : Ascaris
lumbricoides, Hookworm,
Enterobious vermicularis,
Taenia solium, Taenia
saginata, H.nana,
Diphyllobothrium latum,
Fasciolopsis buski

14. Organisms Causing Inflammatory
Diarrhea
 Usually colon & distal
small intestine
 Dysentery or
inflammatory diarrhea
 Increased pus cells in
feces
 Predominantly
dysentery
 Bacteria:
 Dysentery:
 Shigella spp
 Compylobacter jejuni
 Enterohaemorrhagic E.coli
 Vibrio parahemolyticus
 Diarrhea:
 Non-typhoidal Salmonella
 Yersinia enterocolitica
 Listeria monocytogen
 Clostridium deficile
 Klebsiella oxytoca
 Pleciomonas spp

15. Organisms Causing Inflammatory
Diarrhea------ cont
 Parasites: predominantly dysentery
 Entamoeba histolytica
 Balantidium coli
 Trichuris trichura
 Schistosoma hematobium
 Schistosoma japonicum

16. Organisms Causing Traveler's
Diarrhea
 Bacteria:
 Enterotoxogenic E.coli:
10-45% cases
 Enteroaggregative E.coli :
5-35% cases
 Compylobacter jejuni :
5-25% cases
 Enteroinvasive E.coli
 Shigella
 Non typhoidal salmonella
 Viruses: common in
children (< 20%)
 Norovirus
 Rotavirus
 Parasites: 0-10% cases
 Giardia lamblia
 Cryptosporidium entamoeba
histolytica
 cyclospora

17. Toxin Producing Organisms
 Toxin production:
 Enterotoxins :
 cholera toxin
 Vibrio parahemolyticus
 E.coli
 Heat labile toxin (LT)
 Heat stable toxin (ST)
 vero toxin
 Toxin A: Cl deficile
 NSP4: Rotavirus
 Cytotoxin:
 Shigella dysentery type I
 Enterohaemorrhagic E.coli
 Clostridium deficle: toxin B
 Neurotoxin:
 Staphylococcus aureus-
enterotoxin
 Bacillus cereus toxin
 Clostridium botulinum toxin
E.coli

18. GENUS SHIGELLA
 Causes Shigellosis (dysentery): blood &
mucus in stool. Named after Shiga : isolated
organism in 1896.

19. Scientific Classification
• Kingdom : Bacteria
• Phylum : Proteobacteria
• Class : Gamma Proteobacteria
• Order : Enterobacteriales
• Family : Enterobacteriaceae
• Genus : Shigella
• Species : S.dysentery, S.sonnei, S.flexneri, S.boydii

20. MORPHOLOGY
 Short, GNB, size: 0.5×
1-3µm
 Non motile
 Non capsulated
 Some strains : type 1
fimbria (S.flexneri)

21. CULTURAL CHARACTERISTICS
 Aerobes & facultative anaerobes.
 Optimum temp : 37ºC. S.sonnei : grow at
10ºC & 45ºC.
 Grow on ordinary media less readily.
Colonies on B.A. & N.A.= smooth, grayish or
colourless, translucent, 2-3mm dia. S.sonnei:
slightly larger, more opaque, 2 antigenic
forms called PHASES.

22.  M.A: pale yellowish (NLF), S.sonnei: LLF on prolong
incubation.
 DCA medium: selective medium for faeces. Small,
pale colonies. S.sonnei: pink papillae on prolong
incubation.
 XLD medium: red colonies. Less inhibitory to
S.dysentery, S.flexneri than DCA medium
 H E agar: green colonies
 S-S agar: colourless colonies

23.  Peptone water & Nutrient broth : uniform
turbidity on overnight incubation. Fimbriated
strains form surface pellicle on prolong
incubation.
 Enrichment broth : Selenite F broth:
S.sonnei, S.flexneri serotype 6 grow well,
other Shigella strains inhibited. Weakly
inhibitory GN broth : less inhibitory

25. Colonies on Congo Red Agar

27. BIOCHEMICAL REACTIONS
 Ferment Glucose : acid
no gas (S.flexneri type 6
produces acid & gas).
 Do not ferment salicin,
adonitol, inositol &
lactose (S.sonnei: LF).
 Reduces Nitrates to
Nitrites
 Inhibited by KCN
 Urease: - ve
 Indole +ve: some
memb of Gr A,B & C,
not Gr D.
 M-R : +ve
 Catalase +ve: except
S.dysentery type 1
 H2S : - ve
 Citrate : - ve

28. Antigenic structure
 1 or more Major Ag. Large no of Minor
Somatic Ag
 Some strains : K Ag
 Fimbrial Ag : S.flexneri
 Identification of Shigella: made by
combination of Agenic & biochemical
properties.

29. VIABILITY
 Viable in faeces for few hrs, but survive for few days
in faeces kept in Buffered glycerol solun or preserved
at 4ºC
 Survive for several days (5-20) on :
 Faeces dried on cloth
 Soiled lavatory seats, fomites
 In cool, damp & dark conditions
 Cult. remain viable for many yrs on Dorset egg agar

30. RESISTANCE
 Killed at 56ºC in 1 hr & 1% phenol : 30 min
 In ice: last for 1-6 mths
 Die rapidly on drying
 S.sonnei : most resistant spp than other
shigella

31. VIRULENCE FACTORS
 Shiga toxin: S.dysentery type 1: known as
Verotoxin or shiga like toxin. MOA : act like
toxin produced by EHEC. Responsible for
complications like Hemorrhagic colitis &
Hemolytic Uremic Syndrome.
 Shigella enterotoxin 1: S.flexneri ,
S.dysentery type 1. Chromosomally
encoded, responsible for early diarrheal
phase.

32.  Shigella enterotoxin 2 : S.flexneri ,
S.dysentery : encoded on plasmid,
responsible for early diarrheal phase.
 Type II secretion system(T2SS):
S.dysentery : encoded by gene, show
similarity to genes of ETEC & cholera toxin.
Function: unknown.

33.  Invasion plasmid Ag: responsible for
attachment & penetration of bacilli to the
mucosal epith cells of colon, also resist
phagocytosis.
 Intracellular spread proteins: increases cell
to cell spread of bacteria.

34.  Invasive property of Shigella demonstrated
by : ability to penetrate HeLa cells & Hep-2
cells and by Congo Red binding test.
Plasmid mediated:proteins present on OMP
responsible for cell penetration. These
proteins are known as Virulence Marker Ag
(VMA). Detected by ELISA.

35. CLASSIFICATION
 Antigenically divided into 4 grs. On the basis
of serologically somatic O Ag & on
biochemical characteristics.
 S.sonnei: classified by Colicin typing.
 Subgroup A :S.dysentery type 1, described
by Shiga. Consist of 12 serotypes. Type 1
bacilli: Shigella shiga (synonym) : mannitol
non fermenter, Indole –ve, Catalase –ve.

36.  3 types of toxins produce by it :
1) Neurotoxin: damages endothelial cells of
small bl vs of CNS, causes polyneuritis,
meningitis, coma. 2) Enterotoxin 1 & 2: fluid
accumulation in lumen 3) Cytotoxin :
causes cytopathic changes in Vero cells
 Type 2 bacilli : S.schmitzi: Indole +ve,
ferment Sorbitol & Rhamnose.

37.  Subgroup B: S.flexneri
 Flexner (1900): described mannitol
fermenting Shigella from Philippines.
 6 serotypes (1-6) & 2 Agenic variants X & Y.
 Several subtypes: 1a, 1b, 2a,2b, 3a, 3b,
4a,4b,5a, 5b. Biochemically heterogeneous
& Agenically more complex
 Serotype 6: Indole –ve.

38.  Serogroup C: S.boydii
 Boyd (1931): discovered this strain from
India.
 Resembles with S.flexneri biochemically but
not Agenically.
 18 serotypes: yet identified

39. Serogroup D: S.sonnei
 Described by Sonne (1915) in Denmark.
 Indole –ve, Catalase +ve
 Colonies: S to R variation, called Phase I &
Phase II (may present in patients & carriers,
loss of variability in subcult.)
 On subcult. produce phase II type of
colonies.
 Causes mildest type of infection.

40. PATHOGENESIS
 Causes bacillary dysentery, Shigellosis
 Mode of infection: i) thr’ contaminated food
& water. 10-100 bacilli capable of causing inf
(survival in gastric acidity). ii) thr’ fomites.
 Incubation period: short upto 48hrs
(1-7days)
 Source of inf: human beings (cases), less
often carriers.

43. Organism reaches to terminal ileum & colon
Attaches to epith.cells of villi of large intestine
Multiply in them
Spread laterally to infect adjacent cell Penetrate into lamina propria
Inflammatory reaction with capillary
thrombosis
Necrosis of epith cells.
Cells: soft & friable Serpinginous ulcer
Bleeding from mucosa

44.  Endotoxins irritates bowel causes diarrhoea.
 Ulcer surface: covered with pseudomemb
made up of fibrin, leucocytes, cell debris,
necrosed epith & bacteria.
 Clinical features: loose, scanty, faeces
frequently with blood & mucus
 Abdominal cramps, tenesmus.
 Fever, vomiting may present.

45. complications
 Hemorrhagic colitis
 Hemorrhagic Uremic Syndrome
(S.dysentery type 1) convulsions in children.
 Reiter’s Syndrome: conjunctivitis, toxic
neuritis, arthritis(3% pt suffered with
S.flexneri inf)
 Intussusception in children

46. Dysentery carriers
 Acquired after Ac.attack of dysentery. Person
excretes bacilli for few days in faeces.
 Diagnosis done : culture on DCA or S-S
agar.
 Healthy carriers may be seen.

47. Laboratory Diagnosis
 Specimen: fresh stool, rectal swab
 Inoculate immediately or transport media:
Sach’s buffered glycerol saline / Selenite F
broth.
 Microscopy: to exclude amoebic dysentery.
 Culture : loopful of sample is inoculated in
Enrichment broth: Weakly inhibiting GN
broth less inhibitory than Selenite F broth.
Incubated at 37ºC

48.  Subcultured on M.A./ DCA/ XLD/ S-S agar
after 6hrs & 18-24hrs.
 Biochemical reactions : for primary
identification of organism.
 Slide agglutination test: serological test.
Polyvalent sera : available for spp
identification.

49.  Tube agglutination test: done if
biochemical reactions & slide agglutination
tests are atypical.
 Ag is prepared. Dilutions : like Widal test
 Incubate for 4 hrs at 50ºC
 Agglutination titer of serum is observed.

50. Colicin typing of S.sonnei
 Helpful in epidemiological study : done by
phage typing & colicin typing.
 Bacteriosins produced by these bacilli have a
wide range of activity against enteric bacilli &
on this basis S.sonnei strains : subdivided
into 17 colicin types against 15 indicator
strains characterized by production of
specific colicin.

51. Bacteria confused with shigella
 Salmonella: NLF, non fermenter,
agglutinated by polyvalent O & H sera.
 Alkalescens Dispar gr: NLF, non gas
producers, non-motile
 Hafnia: NLF, motile, citrate +ve.
 Providencia: NLF, motile, deamination of
phenyl alanine
 Plesiomonas shigelloides: NLF,
anaerogenic

52. Treatment & prophylaxis
 Uncomplicated shigellosis: self limiting
 Ac cases ORT: no vomiting. I/V fluids to
correct dehydration.
 Ampicillin, Trimethoprim-Sulfamethoxazole,
Ciproflox, Norflox, Nitrofurantoin are useful.
 Improvement in personal hygiene
 Environmental sanitation
 No effective vaccine.

53. VACCINES under trial
 Live attenuated strains:
 i) two prototype attenuated vaccine strains of
S.flexneri 2a & S.dysentery type 1 . Phase 1
trials recently begun in USA (Baltimore).
Stimulates secretory IgA Ab (sIgA Ab).
 ii) Attenuated mutant S.flexneri 2a deve in
France. Double attenuation: capacity to
survive intra & intercellularly.

54.  iii) Lypopolysaccharide in live attenuated vector :
under deve. in Mexico.
 iv) strains used as a carrier for expression of O-Ag.
Ist generation cholera vaccine(CH3), expresses both
encoded Inaba & heterologous (S.sonnei) O-
serotype.
 Subunit vaccine:
 i) Conjugate vaccine: S.sonnei-rEPA (recombinant
exoprotein A) & S.flexneri 2a-rEPA. Parenteral
under trial in Israel.

55.  ii) Proteosome vaccine: proteosome :
multimolecular vesicle form from Neisseria
OMP, used as mucosal vaccine to induce
systemic & mucosal immune response. e.g.
after oral / nasal immunization in mice/guinea
pig with S.flexneri or S.sonnei proteosome,
high levels of Ab against LPS.
 Under phase I trial.

56. Shigella nucleoprotein (ribosomal)
vaccine:
 Lyophilized Shigella vaccine: stable, low
cost, under development.
 Non-covalent complex of O-polysaccharide &
ribosomal particles from Shigella.
 Single does :S/C
 IgA Ab secretion.

58. Mode Of Action of LT
Toxin binds to Gm1 receptors of intestinal epithelium with subunit
B subunit A activated
activates Adenyl
cAMP cyclase.
increase outflow of
water & electrolytes leads to DIARRHOEA
in intestinal lumen
* MOA of Cholera toxin is same as LT