医学微生物学
Medical Microbiology
病原生物学教研室
Department of pathogenic Biology of
Gannan Medical University
张文平
Chapter 11
Enteric Bacilli
(Enterobacteriaceae)
Classification
•Enterobacteriaceae (肠杆菌科细菌）
•Vibrio cholerae(霍乱弧菌)
•Vibrio parahaemolyticus (副溶血性弧菌)
•Helicobacter pylori (幽门螺杆菌)
•Campylobacter(弯曲菌属)
Characteristics
large Gram- negative rods
associated with intestinal infections
(bacillary dysentery, typhoid, and food
poisoning )
oxidase negative
glucose fermenters & nitrate reducers
the pathogenicity of a particular enteric
bacterium can be determined by its
ability to metabolize lactose
Escherichia coli
Coliform —
enterobacteria
which ferment
lactose
Characteristics
Morphology
usually motile, produce
peritrichous flagella
some produce polysaccharide
capsules
grow well on non-selective media,
forming smooth, colourless
colonies 2-3 mm in diameter in 18h
on nutrient agar
temperature (15-45℃)
Characteristics
Colonies on selective media
blue and violet
colonies on
EMB agar
red colonies on
MacConkey
agar
Characteristics
Biochemical reaction
aerobic or anaerobic --can grow in the
presence or absence of O2.
typically oxidase-negative
most strains ferment lactose & glucose
with the production of acid and gas
IMViC reaction : + + - -
distinguish!
Reactions on TSI agar slantsi
Characteristics
Antigenic structure
Over 700 antigenic types (serotypes) are
recognized based on O, H, and K antigens.
Lipopolysaccharide LPS (heat-stable somatic
antigens )
-- O antigens
flagellar antigens
-- H antigens
"capsular" antigens
-- K antigens
Diseases caused by E. coli
E. coli is responsible for three types of
infections in humans:
urinary tract infections (UTI),
neonatal meningitis,
intestinal diseases
(gastroenteritis).
Pathogenic E. coli
Five classes (virotypes) of E. coli that
cause diarrheal diseases are now
recognized:
enterotoxigenic E. coli (ETEC),
enteroinvasive E. coli (EIEC),
enterohemorrhagic E. coli (EHEC),
enteropathogenic E. coli (EPEC),
enteroaggregative E. coli (EAggEC).
Pathogenic E. coli
Pathogenic E. coli
Enterotoxigenic E. coli (ETEC)
•cause diarrhea in
infants and travelers
Pathogenic E. coli ---- ETEC
pathogenesis of ETEC
involves two steps:
intestinal colonization,
fimbrial adhesins e.g. CFA I, CFAII, K88. K99
non invasive
diarrheagenic enterotoxin(s) -- LT and/or ST
toxin
both traits are plasmid-encoded
Pathogenic E. coli ---- ETEC
Enterotoxins
LT
(heatlabile)
toxin
ST
(heatstable)
toxin
Pathogenic E. coli ---- ETEC
heat-labile toxin(1)
similar to Vibrio cholerae enterotoxin.
two types of subunits.
the B subunit binds the toxin to the
target cells via a specific receptor that
has been identified as Gm1 ganglioside.
the A subunit is then activated by
cleavage of a peptide bond and
internalized
Pathogenic E. coli ---- ETEC
heat-labile toxin(2)
The actived subunit A then catalyzes the
ADP-ribosylation (transfer of ADP-ribose from
nicotinamide adenine dinucleotide [NAD]) of
a regulatory subunit of membrane-bound
adenylate cyclase, the enzyme that converts
ATP to cAMP.
This activates the adenylate cyclase, which
produces excess intracellular cAMP, which
leads to hypersecretion of water and
electrolytes into the bowel lumen.
Pathogenic E. coli ---- ETEC
heat-stable toxin
ST enterotoxin causes an increase in cyclic
GMP in host cell cytoplasm.
Two major classes -- Sta & STb
Those termed STa stimulate intestinal
guanylate cyclase, the enzyme that converts
guanosine 5'-triphosphate (GTP) to cyclic
guanosine 5'-monophosphate (cGMP).
Increased intracellular cGMP inhibits intestinal
fluid uptake.
Those termed STb do not seem to cause
diarrhea by the same mechanism.
Pathogenic E. coli ---- ETEC
Laboratory methods for isolation and identification of ETEC
Pathogenic E. coli ---- ETEC
Control
controlled by preventing transmission
and by stressing the importance of
breast-feeding of infants
The best treatment is oral fluid and
electrolyte replacement (intravenous in
severe cases).
Antibiotics are not recommended ----antibiotic-resistant pathogenic E coli
Pathogenic E. coli
Enteroinvasive E. coli (EIEC)
Shigella-like E coli strains
dysentery-like diarrhea (mucous, blood),
severe inflammation, fever
nonfimbrial adhesins, possibly outer
membrane protein
invasive (penetrate and multiply within
epithelial cells)
does not produce shiga toxin
Pathogenic E. coli ---- EIEC
Cellular pathogenesis of invasive E coli
Pathogenic E. coli ---- EIEC
Sereny test -- Invasive E coli, like
Shigella,
causes
a
rapid
keratoconjunctivitis when placed on the
conjunctiva of the guinea pig eye.
Virulent Sereny test-positive isolates
carry a large (usually 140-megadalton)
plasmid responsible for this property.
Pathogenic E. coli
Enteropathogenic E. coli (EPEC)
non fimbrial adhesin (intimin)
moderately invasive (not as invasive as
Shigella or EIEC) "attaching and
effacing"
does not produce LT or ST; some
reports of shiga-like toxin
usually infantile diarrhea; watery
diarrhea similar to ETEC, some
inflammation, no fever; symptoms
probably result mainly from invasion
rather than toxigenesis
Pathogenic E. coli
Enterohemorrhagic E. coli (EHEC)
represented by a single strain (serotype O157:H7)
adhesins not characterized, probably fimbriae
moderately invasive
produces shiga toxin but not LT or ST
copious bloody discharge (hemorrhagic colitis),
intense inflammatory response, may be complicated
by hemolytic uremia
pediatric diarrhea caused by this strain can be
fatal due to acute kidney failure (hemolytic
uremic syndrome [HUS]).
Pathogenic E. coli
Enteroaggregative E. coli (EAggEC)
adhesins not characterized
non invasive
produce ST-like heat-labile plasmidencoded toxin (EAST) and a hemolysin
persistent diarrhea in young children
without inflammation, no fever
Pathogenic E. coli
Treatment
treatment is based
on symptomatology.
fluid replacement is
the primary
treatmen
Antibiotics are
generally not used
except in severe
disease or disease
that has progressed
to a systemic stage
(e.g.hemolyticuremia syndrome).
Shigella
agents of bacillary dysentery
Shigella
Structure
Gram-negative,
nonmotile,
facultatively anaerobic,
non-spore-forming rods
failure to ferment lactose or
decarboxylate lysine
closely related with Escherichia coli
Shigella
Classification
four serogroups with multiple serotypes
A (S dysenteriae, 12 serotypes);
B (S flexneri, 6 serotypes);
C (S boydii, 18 serotypes);
D (S sonnei, 1 serotype).
Shigella
bacillary dysentery--shigellosis
Shigella
Virulence
1. invasin
encoded by large extra-chromosomal
elements
(plasmids)--Invasion
Plasmid
Antigens (Ipa) B and C.
induces the endocytic uptake of shigellae by
M cells, epithelial cells, and macrophages.
deform the plasma membrane of contiguous
cells.
IcsB plasmid-encoded protein lyses the
plasma membranes, resulting in intercellular
bacterial spread.
Shigella
Histopathology of acute colitis following peroral infectionwith shigellae
Shigella
Virulence
2. Endotoxin
cause fever, shock, bloody, mucoid
stools, and abdominal pain (cramps and
tenesmus) .
Shigella
Virulence
3. exotoxin-- Shiga toxin(vero toxin)
chromosomally-encoded
neurotoxic, enterotoxic and cytotoxic
The toxin inhibits protein synthesis
(acting on the 70S ribosome and lysing
28S rRNA). Its enterotoxicity can make
the disease clinically appear as a
diarrhea.
Shigella
Diagnosis -- Sampling
Positive cultures are most often
obtained from blood-tinged plugs of
mucus in freshly passed stool
specimens obtained during the acute
phase of disease
Rectal swabs may also be used if the
specimen is deposited in a buffered
glycerol saline holding solution
Shigella
Diagnosis -- Isolation
primary
differential/se
lective media:
SalmonellaShigella (SS)
Agar(contain
bile salts& pH
indicators)
MacConkey,
Hektoen
Enteric Agar,
Shigella
Diagnosis -- Isolation
colorless, non-lactose-fermenting
colonies
Secondly tubed slants of Kligler's Iron
Agar or Triple Sugar Iron Agar.
Shigella species produce an alkaline
slant and an acid butt with no bubbles
of gas in the agar.
Shigella
Diagnosis -- Identification
slide agglutination tests with antisera
for serogroup and serotype confirm the
identification
polymerase chain reaction (PCR).
Enzyme-linked immunosorbent assay
(ELISA)
Shigella
Epidemiology
occurring by fecal-oral contact
it can be transmitted by infected adult
food handlers who contaminate food.
Man is the only "reservoir"
Shigella
Treatment and Control
Managing of dehydration is of primary
concern.
Patients with severe dysentery are
usually treated with antibiotics (e.g.
ampicillin).
Patients respond to antibiotic therapy
and disease duration is diminished
Salmonella
salmonellosis
Salmonella
Structure & Antigenic Types
Gram-negative, flagellated, facultatively
anaerobic bacilli
three major antigens:
H or flagellar antigen;
O or somatic antigen;
Vi antigen (only a few serovars)
Salmonella
Virulence Factors
(1) the ability to invade cells
---- invasin: Vi (capsular) antigen
(2) a complete lipopolysaccharide coat
---- LPS (endotoxin)
(3) the ability to replicate intracellularly,
(4) possibly the elaboration of toxin(s)
Salmonella
Salmonella
Invasion of intestinal mucosa by Salmonella.
Salmonella -- Clinical Manifestations
1) Gastroenteritis--food poisoning
 Symptoms usually begin 6 to 48 hours after



ingestion of contaminated food or water
the cardinal manifestation is diarrhea.
nausea, vomiting, abdominal cramps,
myalgia, headache, fever (38oC to 39oC)
and chills are common
The duration of fever and diarrhea is usually
2 to 7 days
Salmonella -- Clinical Manifestations
2) Septicemia
an intermediate stage of infection –
no intestinal symptoms and the bacteria
cannot be isolated from fecal specimens.
it may remain localized in the intestine
or disseminate to the bloodstream
Salmonella -- Clinical Manifestations
3) Enteric fevers ---- typhoid
severe systemic form, may be fatal
The best studied enteric fever is typhoid
fever, the form mainly caused by S typhi
may be preceded by gastroenteritis
an incubation period of 10 to 14 days
symptoms of enteric fevers are
nonspecific:fever, anorexia, headache,
myalgias, and constipation
Salmonella -- Clinical Manifestations
3) Enteric fevers ---- typhoid
primary bacteraemic phase:
(7- 10 days of the incubation period )
invade the epithelium
spread to mesenteric lymph nodes &
throughout the body
be taken up by the reticuloendothelial
cells
infect the liver, spleen, gallbladder,
bones, meninges
invade bloodstream via thoracic duct
Salmonella -- Clinical Manifestations
3) Enteric fevers ---- typhoid
second and heavier bacteraemic phase
(2-3 weeks)
pass into the blood( the onset of the fever
and other signs of clinical illness)
From the gallbladder a further invasion of
the intestine results.
Peyer’s patches & other gut lymphoid tissues
become involved in an inflammatory reaction
infiltration with mononuclear cells
followed by necrosis, sloughing and the
formation of characteristic typhoid ulcers
Salmonella -- Clinical Manifestations
3) Enteric fevers ---- typhoid
Onset: 2 weeks/ insidious/ vague early symptoms
Progression :
the temperature shows a stepladder rise over the 1st week
of the illness, remains high for 7-10 days and then falls
by lysis during the 3rd or 4th week.
physical signs include a relative bradycardia for the
height of the fever, hepatomegaly, splenomegaly and
often a rash of rose spots.
Relapse: shorter and of milder.
Complications:
severe intestinal haemorrhage and intestinal perforation
Salmonella -- Clinical Manifestations
4) The prolonged carrier state
continue to excrete the
salmonellae for a year or more
The bacilli are most commonly
present in the gallbladder
Salmonella -- laboratory diagnosis
Isolation & identification 1
Blood culture
the definitive diagnosis of enteric fever
most commonly found during the first 7-10
days and during relapses
Stool and urine culture
In typhoid fever, stool cultures are usually
positive form the 2nd week and urine cultures
from the 3rd week of the infection.
Salmonella-laboratory diagnosis
Identification & identification
2
Specimens should
be plated on several
nonselective and
selective agar(EMB)
media.
Laboratory
identification of the
genus Salmonella is
done by biochemical
tests.
Salmonella-laboratory diagnosis
Identification & identification
3
Biochemical reactions of suspicious
colonies are then determined on triple
sugar iron agar and lysine-iron agar.
serologic type is confirmed by serologic
testing.
It can be confirmed by antigenic analysis
of O and H antigens using polyvalent and
specific antisera.
Salmonella-laboratory diagnosis
Widal test
a tube test for determining the
quantity of agglutinating
antibodies, or agglutinins, in the
serum of a patient with typhoid
fever
Salmonella-laboratory diagnosis-widal test
The procedure involves
adding a suspension of
dead typhoid bacterial
cells to a series of tubes
containing the patient’s
serum, which has been
diluted out to various
concentrations.
After the tubes have
been incubated for 30
minutes at 37℃, they
are centrifuged and
examined to note the
amount of agglutination
that has occurred.
The reciprocal of the
highest dilution at which
agglutination is seen
designated as the
antibody titer of patient’s
serum.
Naturally, the higher the
titer, the greater is the
antibody response of the
individual to the disease
Salmonella-laboratory diagnosis-widal test
Generally, in typhoid cases, it is
valuable that the titre of specific
O antibodies is ≥1:80 or the titre of
specific H antibodies is ≥1:160.
In paratyphoid cases, if the titre of
specific H antibodies is ≥1:80, the
result is positive.
Salmonella-laboratory diagnosis-widal test
Interpretation
The level of ‘enteric’ antibodies in the healthy
population must be known and may be variable.
Previous inoculation with TAB(typhoid,
paratyphoid A, paratyphoid B)vaccine can give
relatively high titres of specific antibodies, as
can previous infection, although only the H
antibodies tend to persist at detectable levels.
Cross-reating antibodies from previous
exposure to other salmonellae sometimes
confuse the picture.
Salmonella-laboratory diagnosis-widal test
Interpretation
As with other serological tests for acute
infections, the usefulness of the Widal test is
greatest when a four-fold or greater rise in
antibody levels is detected on testing a second
specimen of the patient’s serum some days after
the first’ by this time the diagnosis has usually
been made by isolation of the offending
organism from blood culture.
The detection of persisting Vi antibody in
significant titre may be of limited value in the
search for long-term carriers after an outbreak
of infection.
Salmonella
Control and Treatment
Vaccines are available for typhoid fever
and are partially effective.
Typhoid fever and enteric fevers should
be treated with antibiotics.
VIBRIO
Characteristics
Gram-negative, highly motile
curved rods, polar flagella
oxidase-positive
most species require 2-3% NaCl or
a sea water base for optimal
growth
tolerant to alkali but not to acid
Grow rapidly in the pH ranger 7.4-
9.6
Classification
two groups
1.non-halophilic vibrios, including V.
cholerae and other species that are able
to grow in media without added salt.
2.halophilic vibros: species which do not
grow in these media.
V. cholerae and V. parahaemolyticus are
pathogens of humans.
Vibrio cholera
History and spread of epidemic
cholera
classic biotypes/ from 1817 to the
early 20th/six global waves
"El Tor" biotype / In 1961 /
Philippines /seventh global/hemolysins
O139 "Bengal" /In 1992/Bangladesh
/at least 11 countries in southern Asia
Antigenic Variation
three distinct O1 biotypes
Bengal strain – O139 biotype -- "non-
Pathogenesis
Cholera Toxin
Cholera toxin
activates the
adenylate cyclase
enzyme in cells of
the intestinal
mucosa leading to
increased levels of
intracellular cAMP,
and the secretion of
H20, Na+, K+, Cl-, and
HCO3- into the lumen
of the small intestine
The toxin has been
characterized and
contains 5 binding (B)
subunits of 11,500
daltons, an active
(A1) subunit of
23,500 daltons, and
a bridging piece (A2)
of 5,500 daltons that
links A1 to the 5B
subunits.
Pathogenesis
-- Cholera
Toxin
Colonization of the Small
Pathogenesis
Intestine
determinants of the
colonization :
adhesins: fimbriae
Tcp pili,
hemagglutinin ,
acf (accessory
colonization factor)
genes
motility: polar
flagella
LPS
Clinical
Manifestations
"rice-water stool"
Immunity to Cholera
Secretory IgA, as well as IgG and
IgM in serum exudate, can be
detected in the intestinal mucosa
of immune individuals.
Vibriocidal antibodies reach a
peak 8-10 days after the onset of
clinical illness, and then decrease,
returning to the baseline 2 - 7
months later.
Diagnosis
rapid diagnosis -wet mount of
liquid stool is
examined
microscopically.
culture of stool or
rectal swab
samples on TCBS
agar (yellow
colonies) and
other selective
and nonselective
slide agglutination
test of colonies
with specific
antiserum
fermentation tests
(oxidase positive)
fluorescent
antibody tests
PCR
Treatment & Control
rapid intravenous replacement
Most antibiotics and chemotherapeutic
agents have no value in cholera therapy.
A parenteral vaccine of whole killed
bacteria has been used widely
Vibrio parahaemolyticus
a halophilic (salt-loving) vibrio
associated with enteritis
acquired by ingestion of raw
or improperly cooked seafoods..
Morphology
won’t grow in the
absence of sodium
chlorid.
Strains from clinical
specimens generally
form green, nonsucrose-fermenting
colonies on TCBS
agar.
Strains associated
with gastroenteritis
usually cause
haemolysis of human
red cells in
Wagatsuma’s agar, a
special medium
containing mannitol.
This haemolysis is
known as the
Kanagawa
phenomenon
Pathogensis
Kanagawa-positive strains produce
a heat-stable cytotoxin and
adhere to human intestinal cells .
A heat-lable haemolysin and
causes mophologyical changes in
tissue culture cells resembling
those caused by cholera toxin and
the heat-labile enterotoxin of Esch.
coli.
Diseases and Treatment
V. parahaemolyticus
can cause explosive
diarrhoea, but
symptoms usually
abate after 3 days
Patients with
diarrhoea generally
require only fluid
replacement therapy.