Enterobacteriaceae.
Lesson 1.
Theme: «Escherichiae.
Klebsiella».
1. Escherichiae
coli.
Genus Escherichiae subdivide into:
common Escherichia coli and diarrheal Escherichia coli.
Common E.coli – normal resident
inhabitant of colon.
Morphology of common E.coli.
E.coli is a Gram negative
rods. It is motile by peritrichate flagella, though some strains may be nonmotile.
Capsules are found in some strains. Spores are not formed.
Cultural characteristics of
common E.coli.
It is a facultative anaerobe. Good
growth occurs on ordinary media (MPA,MPB). On MacConkey s (Endo) medium,
colonies are bright pink (lactose positive) due to lactose fermentation.
Biochemical reactions of
common E.coli.
Glucose, lactose, maltose and
many other sugars are fermented with the production of acid and gas.
The main positive functions of
common E.coli for macroorganism:
· Antagonistic
function with respect to some pathogenic bacteria (Shigella, Salmonella,
Vibrio).
· Immunomodulator and
immunostimulant function.
· Participate in
synthesis of vitamins (Vitamin B, K, nicotinic acid).
· Participate in
lipid and water-salt metabolism.
Common E.coli can cause
different endogenous infections, if human organism have lowering of resistance.
Clinical forms of diseases
which can cause common E.coli: cystitis, pyelonephritis, cholecystitis,
meningitis, sepsis, pneumoniae, tonsillitis, appendicitis, etc.
Diarrheal E.coli have morphological,
cultural and biochemical characteristics such as common E.coli. Diarrheal
E.coli differ from common E.coli on based their antigenic structure.
Antigenic structure of
diarrheal E.coli.
Serotyping of E.coli is based
on three antigens – the somatic antigen O, the capsular antigen K and the
flagellar antigen H. The antigenic pattern of a strain (serovars) is recorded
as the number of the particular antigen it carries, as for example E.coli O111:
K58: H2. In practical work diarrheal
E.coli identify by only O antigen (named as serogroup).
Diarrheal E.coli cause coli-infections
(exogenous infections).
Source of infection: patient or
bacteriocarrier.
The mode of infection: infection acquired
by ingestion of contaminated water and food.
Four different types of
diarrheagenic E.coli are now recognised enteropathogenic, enterotoxigenic,
enteroinvasive and enterohemorrhagic (shigatoxigenic) E.coli.
Table
1.
|
Category
|
Serogroups
|
Clinical forms
|
Mechanism
|
|
1. EPEC (enteropathogenic E.coli)
|
O55, O86, O111
|
Diarrhea. Newborn and babies (on artificial feeding) are ill more
often than other children.
|
1. Adhesion to cell of small intestine.
2. Multiply on the surface of cells.
3. Cytotoxic action
|
|
2. ETEC (enterotoxigenic E.coli)
|
O6, O8, O15
|
Its severity varies from mild watery diarrhea to fatal disease
indistinguishable from cholera.
|
1. Adhesion and multiply on cell of small intestine.
2. production exoenterotoxin (cholera-like toxin).
|
|
3. EIEC (enteroinvasive E.coli)
|
O28, O143, O124
|
Clinically EIEC infection resembles shigellosis ranging from mild
diarrhea to typical dysentery, and occurs, in children as well as adults.
|
1. Adhesion to cell of colon.
2. Invasion and multiply.
3. Ulcerative lesion.
|
|
4. EHEC (enterohemorrhagic E.coli)
|
O157, O26, O145
|
E.coli strains producing shiga-like toxin can give rise to diarrheal
disease ranging in severity from mild diarrhea to fatal hemorrhagic colitis
and hemorrhagic uremic syndrom particularly in young children and the
elderly.
|
1. Adhesion to cell of colon.
2. Invasion.
3. Production shiga-like toxin.
|
Laboratory diagnosis.
Material for dignostics: feces.
1. Bacteriological method.
·
The feces is inoculated on Endo medium and incubated
at 370C.
·
Colonies appear after 18 – 24 hours and may be
identified by biochemical reactions
sugar media ( for determination of genus – Escherichia) and antigenic structure
(for determination: common or diarrheal E.coli) – agglutination test.
2. Klebsiella.
3 species. Klebsiella
pneumoniae, Kl.ozaenae, Kl.rhinoscleromatis.
Morphology.
Gram negative rods. They are nonmotile and
nonsporing. They are capsulated (in vivo and in vitro).
Cultural characteristics.
Grow well on ordinary media
(MPB, MPA). MPA – mucous colonies, MPB – turbidity.
Pathogenicity.
1. Adhesion.
2. Capsule – protects from
phagocytosis.
3. R – plasmids (K.pneumoniae).
4. Endotoxins.
The main disease
which can cause Klebsiella.
|
Species of
Klebsiella
|
Diseases
|
Material for
diagnostics
|
|
K.pneumoniae
|
1. Pneumonia, bronchopneumonia.
2. Meningitis, sepsis, pyelonephritis, cystitis.
3. Enterocolitis, enteritidis.
|
1. Sputum.
2. Liquor, blood, urine.
3. Feces.
|
|
K.ozaenae
|
Ozaenae – disease characterised by damage (atrophy) of mucous membrane
of nose and foul smelling nasal discharge.
|
Nasal discharge.
|
|
K.rhinoscleromatis
|
Rhinoscleroma – chronic granulomatous hypertrophy of the nose.
|
|
Laboratory diagnosis.
1. Bacteriological method.
2. Microscopic method
(Burri-Gins stain).
3. Serological method –
complement fixation test, reaction passive hemagglutination, agglutination
test.
Lesson 2.
Theme: «Salmonella.
Shigella.»
1. Salmonella.
Salmonellae currently comprise
above 2000 serotypes or species, all of them potentially pathogenic. For
practical purposes, they may be divided into two groups:
1) The enteric fever group
consisting of the typhoid and paratyphoid bacilli that are exclusively or
primarily human parasites;
2) The food poisoning group,
which are essentially animal parasites but which can also infect, human beings,
produsing gastroenteritis, septicemia or localised infections.
Salmonella typhi,, Salmonella
paratyphi A, Salmonella paratyphi B (S.schottmuelleri), Salmonella paratyphi C
(S.hirschfeldii) – agents of enteric fever.
Morphology.
Gram negative rods. They are
motile with peritrichate flagella, but noncapsulated and nonsporing.
Cultural characteristics.
Salmonellae facultatively
anaerobic, growing readily on simple media (MPA, MPB). On Endo medium colonies
are colourless (lactose negative) due to the absence of lactose fermentation.
On bismuth sulphite medium, jet black colonies with a metallic sheen are formed
due to production of H2S.
Biochemical reactions. S.typhi ferment
some sugars, forming acid. S.paratyphi A and B ferment some sugars forming acid
and gas. Indole is not produced.
Antigenic structure.
1) O-antigen (somatic) –
phospholipid-protein-polysaccharide complex.
2) H-antigen. This antigen
present on the flagella is a heat labile protein.
3) K-antigen.
4) Vi-antigen (surface
antigen) – found in some species. The Vi-antigen affords a method of
epidemiological typing of the S.typhi strains based on specific
Vi-bacteriophage.
Classification and
nomenclature.
Classification within the
genus is on antigenic characterisation based on the Kauffmann – White scheme.
This scheme depends on the identification, by agglutination, of the structural
formulae of the O and H antigens of the strains (Table 1).
Table 1.
Kaufmann – White
scheme.
|
Serogroups
|
Antigen O
|
Antigen H (Phase I)
|
Antigen H (Phase II)
|
|
Group A (O2)
|
|
|
|
|
S.paratyphi A
|
1, 2, 12
|
a
|
(1, 5)
|
|
Group B (O4)
|
|
|
|
|
S.schottmuelleri
|
1, 4, (5), 12
|
b
|
1, 2
|
|
S.abony
|
1, 4, (5), 12, 27
|
b
|
e, n, x
|
|
S.typhimurium
|
1, 4, (5), 12
|
i
|
1, 2
|
|
S.heidelberg
|
1, 4, (5), 12
|
r
|
1, 2
|
|
Group C1,
C2, C3 (O7, 8)
|
|
|
|
|
S.hirschfeldi
|
6, 7 (Vi)
|
c
|
1, 5
|
|
S.choleraesuis
|
6, 7
|
(c)
|
1, 5
|
|
S.tompson
|
6, 7
|
c
|
1, 5
|
|
S.newport
|
6, 8
|
e, h
|
1, 2
|
|
Group D1 (O9, 12)
|
|
|
|
|
S.typhi
|
9, 12 (Vi)
|
d
|
-
|
|
S.enteritidis
|
1, 9, 12
|
g, m
|
(1, 7)
|
|
Group E1 (O3,
10)
|
|
|
|
|
S.oxford
|
3, 10
|
a
|
1, 7
|
|
S.anatum
|
3, 10
|
e, h
|
1, 6
|
|
S.london
|
3, 10
|
l, v
|
1, 6
|
Pathogenicity.
1. Pili – facilitates adhesion
of Salmonella to the host cell surface.
2. Invasion – they enter the
lymphoid follicles of the ileum.
3. Endotoxin.
4. R – plasmids.
Enteric fever.
The term enteric fever
includes typhoid fever caused by S.typhi and paratyphoid fever caused by
S.paratyphi A, B, and C. The clinical course may vary from a mild undifferentiated
pyrexia (ambulant typhoid) to a rapidly fatal disease. The onset is usually
gradual, with headache malaise, anorexia a coated tongue and abdominal
discomfort with either constipation or diarrhea, rose-rash (roseola) on skin.
Source of infection: patient or
bacteriocarrier – for enteric fever, cattle and hen – for S.schottmuelleri.
The mode of infection: acquired by
ingestion of contaminated food (milk, egg, meat).
Stages of pathogenesis of
enteric fever.
|
Date of disease
|
Stages
|
Material for
diagnostics
|
|
1. Incubation
period (6-14 days)
|
Digestive stage – transit of microbe from oral cavity to small
intestine with food.
Invasion stage – bacteria penetrate to the lamina propria of submucosa,
then mesenteric lymph nodes.
|
|
|
2. 1 week
|
Bacteremia – bacteria multiply in the blood.
Intoxicating:
·
Destruction of salmonella
·
Endotoxin go out from bacteria.
|
1. Hemoculture (blood from ulnar vena – 10 ml)
2. Urineculture (urine).
3. Coproculture (feces).
|
|
3. 2 week
|
Parenchymal diffussion – bacteria multiply in the organ (liver,
spleen, lung, skin, bone marrow, kidney).
Secretory stage – secretion of salmonella with bile, feces, urine,
breast milk.
|
1. Serum for agglutination test (Widal reaction).
2. Coproculture.
3. Urineculture.
4. Hemoculture (20 ml)
5. Roseoloculture (lymph from roseola).
6. Lactoculture (breast milk from mother).
7. myeloculture (bone marrow) – after death of the patient.
|
|
4. 3 week
|
Secretory stage.
Allergological stage – necrosis of small intestine →appear ulcer.
Ulseration of the bowel leads to the two major complications of the disease –
intestinal perforation and hemorrhage.
|
1. Coproculture.
2. Urineculture.
3. Serum (Widal reaction).
|
|
5. 4 week
|
Convalescence.
|
1. Coproculture.
2. Urineculture.
|
Laboratory diagnosis.
1. Bacteriological method.
Identification bacteria by biochemical and antigenic properties (agglutination
test). Blood inoculate on liquid media (Rappaport media). Feces, urine and
other materials for diagnostics inoculate on Endo medium and bismuth sulphite
agar.
2. Serological method (Widal
reaction).
Prophylaxis.
1. Heat killed typhoid
bacillus vaccine.
2. Salmonellosis.
The main agent. Salmonella
typhimurium.
Human infection results from
the ingestion of contaminated food. The most frequent sources of salmonella
food poisoning are poultry, meat, milk, and milk products. Of great concern are
eggs and egg products.
Food contamination may also
result from the droppings of rats or other small animals. Gastroenteritis may
occur without food poisouing as in cross infection in hospitals (direct contact
– by hands of medical personnel medical apparatus etc.
Clinically the disease
develops after a short incubation period of 24 hours or less with diarrhea,
vomiting, abdominal pain and fever.
3. Shigella.
The causative agents of
bacillary dysentery belong to the genus shigella. Shigella are classified into
four species:
·
Shigella dysenteriae
·
Shigella flexneri
·
Shigella boydii
·
Shigella sonnei
Morphology.
Shigellae are short, Gram
negative rods. They are nonmotile, nonsporing and noncapsulated.
Cultural characteristics.
They are aerobes and
facultative anaerobes. They grow on ordinary media but less readily than other
enterobacteria. Colonies on MacConkey (Endo) agar are colourless due to the
absence of lactose fermentation.
Biochemical reactions.
Glucose is fermented with the
production of acid, witho ut gas.
Antigenic structure.
·
O-antigen (somatic)
·
K-antigen
Pathogenicity.
1. Adhesion (pili).
2. Invasive property
(penetrate and multiply in colonic mucosa) – plasmids coding.
3. Exotoxin:
·
Enterotoxic action
·
Cytotoxic action
·
Neurotoxic action
4. Endotoxin.
5. R-plasmids.
Shigellae cause bacillary
dysentery. Infection occurs by ingestion. The minimum infective dose is
low, as few as 10-100 bacilli being capable of initiating the disease, probably
because they survive gastric acidity better than other enterobacteria.
The main clinical features
are frequent passage of loose, scanty feces containing blood and mucus, along
with abdominal cramps and tenesmus. Fever and vomiting may be present.
The source of infection: patient or
bacteriocarrier.
The mode of infection: acquired by
ingestion of contaminated water and food.
Laboratory diagnosis.
Material for diagnostics:
feces.
1. Bacteriological method.
Identification bacteria by biochemical and antigenic properties.
2. Reaction passive hemagglutination,
immunofluorescence test – for determination of shigella antigen in the feces.
Lesson 3.
Theme: «Vibrio. Campylobacter. Helicobacter».
1. Vibrio cholerae.
3 agents: Vibrio cholerae
biovar classical, Vibrio cholerae biovar El-Tor, Vibrio cholerae biovar O-139 «Bengal».
Morphology.
The cholera vibrio is Gram
negative, curved rods. Organization in cells – comma shaped. They are
nonsporing and noncapsulated. Monotrichous.
Cultural characteristics.
The cholera vibrio is strongly
aerobic. Growth is better in alkaline medium (optimum pH 8,5 - 9,0). On alkaline agar, after overnight
growth colonies are moist, translucent, round discs with a bluish tinge in
transmitted light.
In 1% peptone water, growth
occurs in about six hours as a fine surface pellicle.
Biochemical reactions.
Heiberg classified vibrios
into six groups based on the fermentation of mannose, sucrose and arabinose.
Two more groups were added later. Cholera vibrios belong to Group I (Table 3).
Table
3.
Heiberg grouping of
vibrios.
|
Group
|
Fermentation of
mannose
|
Fermentation of Sucrose
|
Fermentation of Arabinose
|
|
I
|
A (+)
|
A (+)
|
-
|
|
II
|
-
|
+
|
-
|
|
III
|
+
|
+
|
+
|
|
IV
|
-
|
+
|
+
|
|
V
|
+
|
-
|
-
|
|
VI
|
-
|
-
|
-
|
|
VII
|
+
|
-
|
+
|
|
VIII
|
-
|
-
|
+
|
Antigenic structure.
O-antigen (somatic)
H-antigen (flagella)
Based on the major (specific)
somatic (O) antigen, Genus Vibrio was classified into serogroups, 150 of which
are currently known. Vibrio cholerae classical and V.cholerae El-Tor belong to
serogroups O-1, which include three serovars: Ogawa, Inaba and Hikojima.
Serovar «Bengal» belong to serogroup O-139.
Pathogenicity.
1. Adhesion (pili).
2. Cholera exoenterotoxin
(choleragen). The toxin molecule consists of one A and B subunits. The B
(binding) unit attach to the ganglioside receptors on the surface of jejunal
epithelial cells. The A (active) subunit causes prolonged activation of
cellular adenylate cyclate and accumulation of cAMP, leading to outpouring into
the small intestinal lumen, of large quanfities of water and electrolytes and
the consequent watery diarrhea.
Cholera – is an acute
diarrheal disease.
The source of infection: patient or
vibriocarrier.
The mode of infection: infection is
acquired through fecally contaminated water or food.
The main clinical forms.
1. Choleraic enteritis and
gastroenteritis
·
Profuse painless watery diarrhea.
·
Copious effortless vomiting.
Stools may be from 3 to 10
times (mild cases of disease) and from 200 to 300 times (serious illness). As a
result massive loss of fluid and electrolytes from patient.
2. Choleraic algid (without
treatment)
·
Fall of temperature (below 36,60).
·
Common complications: muscular cramps, renal and
cadiac failure, pulmonary edema, etc.
Laboratory diagnosis.
1. Bacteriological method
(main).
2. Genetic method – PCR.
3. Express method: immunofluorescence
test (direct), reaction passive hemagglutination.
Treatment.
The treatment of cholera
consists essentially of the prompt and adequate replacement of lost fluid and
electrolytes (by special solutions which injection per os and intravenous).
Specific prophylaxis.
1. Vaccine V.cholerae
corpuscular inactivated.
2. Chemical vaccine (mono- and
bivalent).
2. Campylobacter.
The Genus Campylobacter
contains spirally curved Gram negative rods. They are typically comma shaped
but may occur as «S». They are nonsporing and motile with a single polar
flagellum at one or both poles.
Cultural characteristics.
Growth occurs under
microaerophilic conditions, 5% oxygen concentration being optimal. They grow on
nutritional media with blood.
Campylobacters of medical
importance are the following:
·
Causing diarrheal disease (gastroenteritis,
enterocolitis): C.jejuni, C.coli.
·
Causing extraintestinal infection: C.fetus.
Campylobacter jejuni.
Medically, this is the most
important campylobacter species as it causes attacts of diarrhea worldwide. The
infection is zoonotic, the source being food or animal origin, especially raw
milk. It is part of the normal intestinal flora of domestic animals and birds,
and is shed in their feces.
Campylobacter coli.
C.coli causes an infection
clinically indistinguishable from that due to C.jejuni. C.coli is commonly
found in intestinal tract of healthy pigs.
Campylobacter fetus.
The source of infection: sheep
and cattle.
It is a very important
veterinary pathogen.
Human infection occurs by
ingestion of contaminated food: milk and meat.
C.fetus may lead to
bacteremia, sepsis and meningitis.
Helicobacter pylori.
They were originally named
Campylobacter pylori. They morphological, cultural characteristics such as
C.coli and C.jejuni.
Today, helicobacters colonise
the stomachs of half the human population of the world.
The sole source of H.pylori is
the human gastric mucus. The axact mechanism of transmission is not clear, but
it is likely to be oral-oral or fecal-oral mechanisms.
H.pylori causes:
·
Gastritis
·
Ulcer of the stomach
·
Cancer of the stomach
Laboratory diagnosis:
1. Microscopic method – use
Gram stain.
2. Bacteriological method.
3. Serological method: CFT,
immunofluorescence test, EIA).
4. For H.pylori – urease test.
H.pylori is strongly urease positive.
Исследование выделений на степень чистоты (на
флору)
