Organisms causing diarrhea & dysentery, shigella spp

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: Sterptococciin oral cavity &
Lactobcillusin 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.flexnerithan 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.flexneriserotype 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 solu
n
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.dysenterytype 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.dysenterytype 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 propertyof 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 intestineMultiply in them
Spread laterally to infect adjacent cellPenetrate 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)
Intussusceptionin 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
brothless 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 2adeve 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.flexnerior S.sonneiproteosome,
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