Download Causetive agents of bacterial intestinal diseases

Tvorko M. S.
Enterobacteriaceae
Escherichia
Shigella
Edwardsiella
Salmonella
Citrobacter
Enterobacter
Serratia
Providencia
Yersinia
Klebsiella
Hafnia
Proteus
Morganella
Erwinia
Escherichia coli Characteristics
• Discovered in 19th century by Bavarian
pediatrician Theodor Escherich
• Gram negative eubacteria
• Facultative anaerobe
• Usually motile
• Universal inhabitant of human (mammalian)
colon
Escherichia coli
Found in the intestines of humans and animals, this bacterium
is usually harmless, but some strains can cause food
poisoning and more serious illnesses. Most outbreaks involve
contaminated beef that was not cooked thoroughly. The strain
known as O157:H7 is considered a potential biological weapon.
E. coli Lives in Colon of Healthy People
(member of commensal flora)
Includes E. coli
Scanning electron micrograph
Cultivation.
Colonies of E. coli on meat-peptone agar
Colonies of E. coli on Endo's medium
Colonies of E. coli on Ploskirev's medium
Colonies of E. coli on blood agar
Escherichia coli
is highly motile
and will show turbidity
throughout the tube.
Fermentative properties.
“+” -ve test
“—” -ve test
Positive (left)
reactions of
isolates E. coli
in glucose
fermentation
broth. Note the
formation of
acid (yellow
color) and gas.
Observe the
bubble in the
Durham tube.
Indole reaction
A. Salmonella
B. E. coli
is the positive microbe.
A
B
E. coli can reduce nitrate
to nitrite.
Note the bubble formation.
Catalase positive
Antigenic structure.
The antigenic structure of E. coli is characterized by
variability and marked individuality. Along with the H- and
O-antigens, the presence of other antigens has been
shown in some strains, i.e. the surface somatic
(membranous, capsular) K-antigens which contain the
thermolabile L- and B-antigens and the thermostable Aand M-antigens.
On the basis of antigenic structure an antigenic
formula is derived which fully reflects the antigenic
properties of the strain For example, one of the most
widely spread serotypes is designated 0111 : K58 : H2.
Enteropathogenic E. coli (EPEC)
• First E. coli pathotype described
• UK pediatrician John Bray, 1945
• Causes potentially fatal infant diarrhea in developing areas
• Contract organism by ingestion of contaminated water,
food or fomites
EPEC Colonization and Lesion Development
• patchy colonization of the small intestine
• Generates unique histopathology termed “attaching and
effacing” lesion
• destroys microvilli
• Expresses numerous toxins/effectors that manipulate host
cell systems to serve the pathogen
• EPEC, like other pathogenic E. coli strains, is a master cell
biologist
Toxin production.
a gluco-lipo-protein complex with which their
toxic, antigenic, and immunogenic properties
endotoxins
thermolabile neurotropic exotoxins
haemotoxins
pyrogenic substances,
proteinases,
deoxyribonucleases, urease,
phosphatase
hyaluronidase
aminoacid decarboxylases
Pathogenic E. coli Virulence Mechanisms
Generalities
• Virulence systems frequently encoded on mobile genetic
elements
• successful combinations reflected in pathotypes
• Like other mucosal pathogens, pathogenic E. coli use
multi-step strategy to infect host
• attachment
• evasion of host defenses
• multiplication
• damage host
• Pathogenic E. coli often colonize host niches not normally
inhabited by E. coli
EPEC A/E Lesion and Pedestal Formation
Fig. 1. Scanning electron micrograph
showing microcolonies
of EPEC displaying the characteristic
localized adherence
pattern of adherence to HEp-2 cells.
Fig. 2. High power scanning
electron micrograph of EPEC
displaying localized adherence
to
HEp-2 cells. Note the
elongated microvilli to which
the bacteria appear to attach.
Enterohaemorrhagic E. coli (EHEC)
• First described in 1982
• Causes bloody diarrhea (haemorrhagic colitis), non-bloody
diarrhea and haemolytic uremic syndrome (HUS)
• ~5% of EHEC infections result in HUS; predominantly in
children <5 years old and elderly
• ~5% of HUS cases are fatal
• Contract organism by ingesting contaminated food
• common inhabitant of bovine gut
• low infectious dose for humans (<100 organisms)
• organism may be resistant to stomach acid
EHEC Virulence Factors
• O157:H7 serotype dominant in North America, UK, Japan
• O26 and O111 serogroups more prominent in other countries
• Evolved from LEE containing EPEC serogroup O55
• Like EPEC, generates A/E lesion
• Colonizes the large intestine
• Expanded repertoire of adhesion factors
• Encodes toxins/effectors in EPEC LEE
Additional virulence factors (plasmid and chromosomal)
• RTX toxin similar to haemolysin
• StcE activates host Complement cascade
• Stx (Shiga) toxin
Stx (Shiga) Toxin
• Toxin encoded on bacteriophage
• >200 E. coli serotypes encode Stx
• Shiga toxin E. coli (STEC)
• most do not encode LEE (not virulent)
• A/B toxin
• pentameric B subunits bind holotoxin to host cell surface
• A subunit cleaves host ribosomal RNA arresting protein
synthesis and cell death (apoptosis)
• Stx produced in colon travels through bloodstream to kidney
• Damages renal endothelium and induces inflammation that
may lead to acute renal failure and death
Shiga toxin
• Distinguishing virulence factor
• Subunit toxin:
A: acts at ribosomal level,
inhibits protein synthesis
B: binds glycolipid receptor in
mammalian cells (renal
endothelium)
• Stx1, Stx2
– Stx2 variants: 2c,2d,2e,2f
HUS: Stx (Shiga) Toxin Activity
Direct and indirect contact
transmission
EHEC Ecology and Transmission
Has HACCP led to a reduction
in human incidence?
Excretion
Re-colonization
Death
Environment
Direct contact transmission
Indirect contact (environmental)
transmission
B.Enterotoxigenic E coli ( ETEC)
– Common cause for travelers diarrhoea, and watery diarrhoea in
children.
• Colonisation factor facilitates the attachment to the
intestinal epithelium.
– Some ETEC produces heat labile exotoxin LT and heat stable or
either of the toxins
•
•
LT has two sub units A B
Action -Activate Adenylate cyclase
Increase local CAMP
Intense, prolonged hypersecretion of water ,
Lumen of gut fill with water
Hypermobility and diarrhoea results.
LT is antigenic and cross reacts with the enterotoxin of
Vibrio cholerae.
Some ETEC produces heat stable enterotoxin
STa/b
STa activates guanylyl cyclase.
STb activates cyclic nucleotides.
Releases water
Enteroinvasive E coli (EIEC)
• Produces disease similar to shigellosis.
•In adults this has been isolated with Shigella
•Commonly affect children in developing countries,
and travelers.
•Disease is due to invasion into mucosal cells of the intestine
multiply inside the cells and destruction /inflammation/ulceration
diarrhoea with blood
EIEC are nonlactose fermenter, or late lactose fermenter
and non motile.
E. coli
Enteroinvasive E. coli
• member normal gut flora
• Agent of bacillary dysentery like disease
Normal Colon
Dysenteric Colon
• healthy GI tract
• diseased GI tract
• Fever
• Severe abdominal pain
• Bloody discharge with mucous
E. Enteroaggregative E coli (EAEC)
•
Produce acute/chronic diarrhoea in persons in
developing countries.
•
Sepsis When normal host defense is poor
sepsis can happen.
• Common in new born babies whose IgM level is
low.
Treatment of E.coli related diarrhoea
1st Line
• Nitrofurantoin
• Nalidixic acid
• Norfloxacin
ABST’ SHOULD BE DONE
• Ampicillin
• Cotrimoxazole
2nd line
• Ciprofloxacin/Ceftriaxone/Cefuroxime
Gentamicin
Salmonella
•Salmonella enterica
–one species, ~2000 serovars
–Non standard nomenclature
•S. enterica serovar Typhimurium
•or S. typhimurium
•rod-shaped, non-spore-forming Gramnegative bacterium
•belongs to the family Enterobacteriaceae
–close relative of E. coli
•Motile by peritrichous flagella (H antigen).
–nonmotile exceptions: S. gallinarum and S.
pullorum
Salmonella infections
in humans
• Enteric fever
– typhoid and
paratyphoid fevers
– typhi, paratyphi A, B, C
– systemic infection
– infects only humans
– GI symptoms may not
be evident
• Salmonella
gastroenteritis
– non-typhi serovars
– zoonosis: predominantly
food-borne
– can be complicated by
septicaemia
• more common with some
serovars, e.g. S. dublin
(15% mortality rate when
septicemic in the elderly)
• Metastatic disease, e.g.
osteomyelitis
10 most frequently isolated Salmonella
strains causing human disease
–
–
–
–
–
–
–
–
–
–
S typhimurium (22.1%)
S enteritidis (26.1%)
S enteritidis heidelberg (4.8%)
Salmonella enteritidis newport (4.3%)
Salmonella hadar (2.7%)
Salmonella enteritidis agona (2.0%)
Salmonella enteritidis montevideo (1.7%)
Salmonella oranienburg (1.6%)
Salmonella muenchen (1.5%)
Salmonella enteritidis thompson (1.5%)
Salmonella typhi
Scanning electron micrograph
Gram’s staining
Cultivation.
Colonies of S. paratyphi on Ploskirev's medium
Colonies of S. typhus on Ploskirev's medium
Colonies of Salmonella on Mac-Conkey medium
Colonies of Salmonella on CLED medium
Colonies of S. typhus on on bismuth-sulphite agar
Fermentative properties.
Antigenic Structure
• Kauffmann-White antigenic scheme
– agglutination reactions with specific antisera against
Salmonella antigens
• O antigens
– characteristic sequence of repeating polysaccharide
units in LPS.
• H antigens
– flagellar antigens (protein) and may occur in one of
two phase variations.
• Vi antigen
– a capsular polysaccharide homopolymer of N-acetyl
galactosamineuronic acid
Toxin production.
S. typhi contains gluco-lipo-protein complexes. The endotoxin is
obtained by extracting the bacterial emulsion with trichloracetic
acid. This endotoxin is thermostable, surviving a temperature of
120° C for 30 minutes, and is characterized by a highly specific
precipitin reaction and pronounced toxic and antigenic properties.
Investigations have shown the presence of exotoxic substances in
S. typhi which are inactivated by light, air, and heat (80° C), as well
as enterotropic toxin phosphatase, and pyrogenic substances.
Infectious dose
• typically about 1,000,000 bacteria
• much lower if the stomach pH is raised
• much lower if the vehicle for infection is chocolate
– protects the bacteria in their passage through
the stomach
– an infectious dose of about 100 bacteria
Clinical Features
Enteric Fever
• incubation period 10 to 14 days
• septicaemic illness
– myalgia and headache
– fever
– splenomegaly
– leukopenia
– abdominal pain
– Rose spots (macular rash on abdomen)
• 10% fatal
• positive blood, urine, and stool cultures
• Sequelae: intestinal haemorrhage and perforation
Pathogenesis and diseases in man.
• The causative agent is primarily located in the intestinal tract.
Infection takes place through the mouth (digestive stage).
• Salmonellae penetrate into the intestine, during which inflammatory
processes develop in the isolated follicles and Peyer's patches
(invasive stage).
Salmonellae enter the blood (bacteriemia stage). Here they are
partially destroyed by the bactericidal substances contained in the
blood, with endotoxin formation.
• During bacteraemia typhoid salmonellae invade the patient's body,
penetrating into the lymph nodes, spleen, bone marrow, liver, and
other organs (parenchymal diffusion stage).
During the second week of the disease endotoxins
accumulate in Peyer's patches, are absorbed by the blood, and
cause intoxication. The general clinical picture of the disease is
characterized
by
status
typhosus,
disturbances
of
thermoregulation, activity of the central and vegetative nervous
systems, cardiovascular activity, etc.
•On the third week of the disease a large number of typhoid bacteria
enter the intestine from the bile duct. This results in the development of
hyperergia and ulcerative processes. (excretory and allergic stage).
Clinical recovery (recovery stage) does not coincide with the elimination
of the pathogenic bacteria from the body. The majority of convalescents
become carriers during the first weeks following recovery, and 3-5 per
cent of the cases continue to excrete the organisms for many months
and years after the attack and, sometimes, for life.
Epidemiology
carrier states
• carrier state may last from many weeks to years
with faecal shedding
– convalescent carrier
• chronic carrier
– ~3% of persons infected with S. typhi
– ~0.1% of those infected with non-typhoidal
salmonellae
• potential for cross-contamination of foods by the
infected handler
– “Typhoid Mary” Mallone
– but more common in textbooks than in real life
Typhoid Mary's real name was Mary Mallon.
Irish immigrant who made her living as a cook.
Mallon was the first person found to be a "healthy
carrier" of typhoid fever in the United States.
She herself was not sick – but over 30% of the
bacteria in her feces were S. typhi
Mallon is attributed with infecting 47 people with
typhoid fever, three of whom died.
Laboratory Diagnosis
• Isolated from stool, blood and urine in
enteric fever (blood cultures need to be
taken!) Isolation of haemoculture.
• Isolated from stool in gastroenteritis
• Appears as a non-lactose fermenter
– on MacConkey agar or similar selective agar
Blood (5 - 10 ml)
BHI
Incubate 37 0C
Subculture
(18-24hrs/every 2nd day for 7days)
MacConkey
Stools and urine first should be
cultured in enrichment media
Laboratory Diagnosis
• Biochemical tests and serological tests must be done in
parallel
– Some other bacteria, e.g. Citrobacter, may have similar
serological profiles
The isolated culture is identified by inoculation into a series of
differential media and by the agglutination reaction. The latter is
performed by the glass-slide method using monoreceptor sera or by the
test-tube method using purified specific sera
LABORATORY DIAGNOSIS
1. Isolation of the bacteria
2. Demonstration of antibodies
3. Demonstration of circulating
antigens
4. Blood picture
Isolation of bacteria
Specimens - stools/urine/blood
Blood culture - before giving chloramphenicol
- repeat the culture
- 80-90% +ve 1st week up to10days
- +ve during relapses
- can culture bone marrow +ve
1-2 days after drug therapy ?
good
-
• When blood culturing is done there is
diluting of the drug
Stool and urine culture
Stools - during the second and third week
Urine - during the third and the fourth week,
less frequently +ve than stools
Repeated culture is necessary when blood
culture is -ve
Serological test
Detection of antibodies to H and O antigens
Widal test
• Antibodies appear at the end of the first
week
• Rises during the 3rd week of the enteric
fever
• Two specimens are taken at interval of 7 to
10 days
Interpretation of Widal test
1. Antibody appears at the end of 1st
week/rises during 2nd and 3rd/steady at the
4th week then decline
2. Four fold rise of both H and O antibody in an
interval of 7 - 10 days (between 1st and the
3rd week is highly significant)
Commercial kits commonly used, e.g.
API20
Phage typing done for epidemiological
purposes
E.g. to find source of outbreak
Certain phage types predominate nationally
S. typhimurium PT4
S. enteritidis DT109
Salmonella vaccines
• Vaccination of travellers against typhoid recommended,
but does not remove need for good hygiene
• Three licensed vaccines
– Traditional heat-killed
• very reactogenic
– Vi subunit vaccine
– live oral vaccine, S. typhi Ty21A
• Salmonellas can act as live attenuated carriers for other
antigens
– So far only experimental
• No vaccines for gastroenteritis
Enterocolitis
•
•
•
•
•
•
Common manifestation of Salmonella
Common pathogen is S typhimurium
8-48 hrs after ingestion
nausea/ headache/ vomiting and profuse diarrhoea,
low grade fever Resolution 2-3 days.
Inflammatory lesions in bowel are present
S. typhimurium in the mouse
• S. typhimurium
– causes gastroenteritis in humans
– causes typhoid-like disease in mice
• infection can be established orally or
systemically
• used as model of typhoid
• primary mechanisms of pathogenesis
– invasion of the intestine
– survival and growth in macrophages
Salmonella in eggs
• various Salmonella serovars isolated from the
outside of egg shells
• S. enteritidis PT4 present inside the egg, in
the yolk
• vertical transmission
– deposition of the organism in the yolk by an
infected layer hen prior to shell deposition.
Sources of in infection
•
•
•
•
•
contaminated water
contaminated dairy products
shellfish
dried or frozen eggs
meat and meat products of infected
animals
• Household pets
Clinical features
Gastroenteritis
• incubation period depends on dose
• symptoms usually begin within 6 to 48 hours
–
–
–
–
–
Nausea and Vomiting
Diarrhoea
Abdominal pain
Myalgia and headache
Fever
• duration varies, usually 2 to 7 days
• seldom fatal, except in elderly or
immunocompromised
Pathogenesis
Gastroenteritis
• Pathogenic salmonellae ingested in food survive
passage through the gastric acid barrier
• invade intestinal mucosa
• invasion of epithelial cells stimulates the release
of proinflammatory cytokines
• induces an inflammatory reaction
• causes diarrhoea and may lead to ulceration
and destruction of the mucosa
• Bacterimia rare
• Blood culture -ve
• Stools remain +ve for sometime after
recovery
Diagnosis
1.Faeces, vomitus, food stuffs
(mainly a single source)
2. Convalescent serum often
agglutinates the suspension of
casual serotype
Prevention and control
•Sanitation
Thorough cooking
•Carriers should not handle food
•Vaccination not successful long-term.
Vibrio cholerae: 1-pure culture; 2- flagellate vibrios
sacharose
mannose
arabinose
Sheep erythrocyte
hemolysis
Lysis by specific O1 subgroup phages
Agglutination by O1 cholera serum
Sensitivity to
polymixin B
Vibrio cholerae
biovar cholerae
A
A
–
–
+
+
+
Vibrio cholerae
biovar El Tor
A
A
–
+
+
+
–
Vibrio cholerae
biovar Proteus
A
A
–
+
–
–
–
Vibrio cholerae
biovar albensis
А
–
–
–
–
–
–
Fermentation
within 24 hrs
Vibrio
The golden rules for prevention of
food poisoning
1- Choose foods processed for safety.
2- Cook food thoroughly.
3- Eat cooked food immediately.
4- Store cooked food carefully.
5- Reheat cooked food thoroughly.
6- Avoid contact between cooked and raw food.
7- Wash hands repeatedly. using lots of friction
W—warm
A—and
S—soapy
8- Keep all kitchen surfaces meticulously clean.
Wash dishes and utensils after contact with raw
meat or eggs.
9- protect foods from
insects,
rodents and other
animals.
10- use safe water.
Keep hot food hot and
cold food cold.