Transcript Gram Positive Bacilli

Aerobic Gram Positive Bacilli
Bacillus
Introduction
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The genus Bacillus includes gram positive, aerobic, sporeforming, rod shaped organisms.
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The diameter of the organism is 1 X 3-5µm
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They are arranged as single or paired bacilli in clinical
specimens and as long serpentine chains or clumps in
cultures.
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It is ubiquitous in nature and B. species are well known in
the food industries as troublesome spoilage organisms.
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A large number of species belong to the genus that
infect invertebrates but Bacillus anthracis, the
agent of anthrax, is the only obligate Bacillus
pathogen in vertebrates.
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They produce a variety of enzymes and other
metabolites during growth including the antibiotics
bacitracin and polymyxin.
Bacillus anthracis
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The causative agent of anthrax.
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Pathogenesis depends on two plasmid encoded virulence factors:
The Capsule
- A prominent poly D- glutamic acid capsule which is observed in
clinical specimens but is not produced in vitro.
- It is antiphagocytic but is a poor immunogen and antibodies
produced against it are not protective.
Toxin
- It consists of three antigenically distinct heat labile
proteins; protective antigen, lethal factor and edema
factor.
- None of the components is active alone, but the
combination of protective antigen and either lethal factor
or edema factor is active (toxic).
- Protective immunity requires antibodies against
components of the toxin, primarily the protective antigen.
Clinical Syndromes
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Bacillus anthracis is an organism found in soil and on
vegetation.
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Anthrax is a disease of herbivores and humans are
accidentally infected by exposure to infected animals or
animal products.
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Humans acquire disease by one of three routes, inoculation
leading to cutaneous anthrax, inhalation leading to
pulmonary anthrax and ingestion leading to gastrointestinal
anthrax.
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Approximately 95% of anthrax infections are due to the
inoculation of spores through exposed skin surfaces either
from contaminated soil or infected animal products such as
hides, hair or wool.
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Inhalation anthrax (wool sorters disease) results from
inhalation of spores during processing of hair or wool.
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Ingestion anthrax is very rare in humans but ingestion is a
common route of infection in herbivores.
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Person to person transmission does not occur.
Cutaneous Anthrax
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It usually occurs through contamination of a cut wound or
abrasion although in some countries biting flies may also
transmit the disease.
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After 2 to 3 days of incubation a small pimple or papule appears
at the site of entry. A surrounding ring of vesicles develops, the
central papule ulcerates, dries and blackens to form the
characteristic eschar.
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The lesion is painless and is surrounded by marked edema.
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Lesions on the face or neck are dangerous and fulminating
septicemia may develop in 20% of cases.
Pulmonary Anthrax
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Inhaled spores are transported by alveolar macrophages to
the mediastinal lymph nodes where they germinate and
multiply to initiate systemic disease.
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It may initially mimic a viral respiratory illness and then
rapidly progresses to a diffuse pulmonary involvement
leading to respiratory failure.
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It is highly fatal (> 95%) because it is not suspected until
the course is irreversible.
Gastrointestinal Anthrax
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Very rare with varied clinical presentation (mesenteric
adenopathy, hemorrhage and ascites) and high mortality
rate (95%).
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The organism probably invades the mucosa through a
preexisting lesion and spreads to the lymphatic system.
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Symptoms prior to fulminant systemic anthrax may be
absent or mild.
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During this phase the organism is multiplying and
producing toxin in the regional lymph nodes and spleen.
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Released toxin causes breakdown of organs
probably of the spleen in particular.
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This causes the sudden onset of hyperacute illness
with dyspnea, cyanosis, high fever and
disorientation which progress in a few hours to
shock, coma and death.
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This phase is marked by a high grade bacteremia
but blood culture is not always positive.
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Clinical diagnosis of anthrax is confirmed by
directly visualizing or culturing the anthrax bacilli.
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Acellular vaccines for human use are available for
individuals in high risk occupations (cell free
filtrate of culture).
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Bacillus anthracis is susceptible to penicillin and
almost all other broad spectrum antibodies.
Anthrax - Diagnosis
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Specimen
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Aspirate or swab from cutaneous lesion
Blood
Sputum
Laboratory investigation
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Gram stain
Culture
Identification of isolate
Anthrax - Epidemiology
Other Diseases
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In recent years, other B. species have been increasingly
implicated in a wide range of infections.
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They include abscesses, bacteremia/septicemia, wound and
burn infections, ear infections, endocarditis, meningitis,
ophthalmitis, osteomyelitis, peritonitis and respiratory and
urinary tract infections.
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Most of these are secondary infections in immuno compromised patients and the species most commonly
associated with these diseases are B. cereus, B.
licheniformis and B. subtilis.
Bacillus cereus
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Large, motile, saprophytic bacillus
Heat resistant spores
Preformed heat and acid stable toxin (Emetic
syndrome)
Heat labile enterotoxin (Diarrhoeal disease)
Lab diagnosis – Demonstation of large number of
bacilli in food
Food Poisoning
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B. cereus causes food poisoning by virtue of toxin
production.
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Two forms of food poisoning:
Diarrheal type (heat labile toxin).
- Characterized by diarrhea and abdominal pain occurring
8-16 hours after consumption of contaminated food.
- It is associated with a variety of foods including meat and
vegetable dishes, sauces, pastas, deserts and dairy products
Emetic type (heat stable toxin)
- Nausea and vomiting begin 1 to 5 hours after
consumption of contaminated food.
- Boiled rice that is held for prolonged periods at
ambient temperature and then quickly fried before
serving is the usual offender.
- Dairy products may also be responsible.
Bacillus cereus
clinical presentation
EMETIC FORM
Incubation period < 6 hours
Severe vomiting
Lasts 8-10 hours
Gastroenteritis
DIARRHOEAL FORM
Incubation period > 6 hours
Diarrhea
Lasts 20-36 hours
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B. cereus cause panophthalmitis by an incompletely
defined mechanism (necrotic toxin, cereolysin
which is a potent hemolysin, and phosphlipase C
which is a potent lecithinase).
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It is a post traumatic disease which is rapidly
progressive that almost universally ends in
complete loss of light perception within 48 hours.
Corynebacteria
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Gram-positive, non motile, club shaped pleomorphic bacilli
that appear in short chains [(V or Y) configurations] or in
clumps resembling Chinese letters.
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Nonencapsulated, catalase positive, and oxidase negative
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C. diphtheriae is fastidious while diphtheriods are not
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They contain metachromatic granules (polymetaphosphate)
which stain bluish purple with methylene blue.
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There are both gram-positive and gram-negative species,
although the majority of isolates are gram-positive.
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Corynebacteria are aerobes or facultative anaerobes and
generally grow slowly on enriched media.
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Four distinct cultured types exist; gravis, intermedius, mitis
and belfanti.
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These variants (biotypes) have been classified on the basis
of growth characteristics, biochemical reactions and
severity of disease.
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They colonize skin, upper respiratory tract, gastrointestinal
tract and urogenital tract of humans.
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The most important species is C. diphtheriae, the causative
agent of diphtheria which causes disease by virtue of toxin
production.
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Diphtheria toxin is a very potent exotoxin that is lethal at
doses as little as 100 to 150 ng /kg of body weight.
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Nontoxigenic (avirulent) C. diphtheriae are
morphologically indistinguishable from the virulent
(toxigenic) strains
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It is now recognized that avirulent strains may be
converted to the virulent phenotype following infection and
lysogenization by one of a number of distinct
corynebacteriophages that carry structural gene for
diphtheria toxin.
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Lysogenic conversion may occur in situ as well as in vitro.
Pathogenesis
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In 1883 Klebs demonstrated that C. diphtheriae is
the agent of diphtheria.
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The pathogenesis of diphtheria is based upon two
primary determinants:
1) The ability of a given strain to colonize in the
nasopharyngeal cavity and/or on the skin
2) Its ability to produce the toxin.
Diphtheria Toxin
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The regulation of diphtheria toxin gene expression
is mediated by an iron-activated repressor which is
encoded on the C. diphtheriae genome.
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Activation of the repressor gene derepresses the tox
gene and diphtheria toxin is synthesized and
secreted into the culture medium at maximal rates.
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It is composed of a single polypeptide chain of 535
amino acids which consists of 3 structural functional
domains:
1)
An N-terminal ADP- ribosyl transferase (catalytic
domain)
2)
A region which facilitates the delivery of the catalytic
domain across the cell membrane (transmembrane
domain).
3)
The eukaryotic cell receptor binding domain.
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Following mild digestion with trypsin and reduction under
denaturing conditions, the toxin is cleaved into two
polypeptide fragments (A and B).
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Fragment A is the N-terminal containing the catalytic
center for the ADP- ribosylation of elongation factor 2
(EF-2).
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Fragment B (the C terminal) carries the transmembrane and
receptor binding domain of the toxin.
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One molecule can inactivate the whole factor.
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The intoxication of eukaryotic cells by diphtheria toxin
involves at least four distinct steps:
1)
Binding of toxin to its cell surface receptor.
Clustering of charged receptors into coated pits and
internalization of toxin by receptors mediated endocytosis.
Insertion of transmembrane domain into the membrane
and delivery of the catalytic domain into the cytosol.
ADP- ribosylation of EF-2 irreversibly inhibiting protein
synthesis.
2)
3)
4)
Clinical Manifestations
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Incubation of 2-6 days.
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There are two types of clinical diphtheria;
nasopharyngeal and cutaneous.
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Diphtheria is most commonly an infection of the
upper respiratory tract and causes fever, sore throat
and malaise.
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A thick, gray-green fibrin membrane, the
pseudomembrane, often forms over the site(s) of
infection as a result of the combined effects of
bacterial growth, toxin production, necrosis of
underlying tissue and the host immune response.
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Symptoms of pharyngeal diphtheria vary from mild
pharyngitis to hypoxia due to airway obstruction by
the pseudomembrane.
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The involvement of cervical lymph nodes may cause
profound swelling of the neck (bull neck diphtheria).
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The skin lesions in cutaneous diphtheria are usually
covered by a gray-brown pseudomembrane.
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Life-threatening systemic complications, principally loss of
motor function (as difficulty in swallowing) and congestive
heart failure may develop as a result of the action of
diphtheria toxin on peripheral motor neurons and the
myocardium.
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Fully immune individual have asymptomatic colonization
and partially immune persons develop a mild respiratory
illness.
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Disease is followed by long lasting immunity which is also
achieved by immunization using diphtheria toxoid
(protective level is 0.01 1U/ml)/
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Diagnosis: Requires demonstration of toxigenic
C. Diphtheria in lesions (throat, nasopharynx, nose) by
Elek test.
Laboratory diagnosis
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Specimen: A throat swab
Culture: The swab is inoculated on Loeffler's serum
medium and/or on blood tellurite agar aerobically at 37C for
24.
•
On Loeffler's serum medium:
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Corynebacteria grow much more readily than other
respiratory pathogens
– Used to enhance the characteristic microscopical
appearance of corynebacteria
– The colonies of C. diphtheriae are small, granular,
grey, smooth, and creamy with irregular edges
Cultural characteristics
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On blood tellurite agar (McLeod's blood agar)
 It is a selective medium for the isolation of C. diphtheriae
(Potassium tellurite)
 3 biotypes of C. diphtheriae are characterized on BTA
 i.e. Gravis, mitis and intermedius biotypes
 The most severe disease is associated with the gravis biotype
 Colony of gravis biotype is large, non-hemolytic & grey.
 Colonies of mitis biotype are small, hemolytic and black
 Colonies of intemedius biotype are intermediate in size, nonhemolytic with black center & grey margin.
Gram stain of C.
diphtheriae
C. diphtheriae on BTA
In Vitro: Elek’s Test
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Principle:
 It is a toxin/antitoxin reaction
 Toxin production by C.diphtheriae can be demonstrated
by a precipitation between exotoxin and diphtheria
antitoxin
Procedure:
A strip of filter paper impregnated with diphtheria
antitoxin is placed on the surface of serum agar
The organism is streaked at right angels to the filter paper
Incubate the plate at 37C for 48 hrs
Filter paper saturated with diphtheria antitoxin
Lines of precipitations
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Results:
After 48 hrs incubation, the
antitoxin diffusing from filter
paper strip and the toxigenic
strains produce exotoxin,
which diffuses and results in
four precipitation lines
radiating from intersection of
the strip and the growth of
Inoculated M.O.
organism
Positive Elek’s Test
Epidemiology
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Worldwide distribution.
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Incidence has dropped dramatically since the introduction
of the vaccine.
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Crowding and low immunity promote spread and it is
maintained in the population by asymptomatic carriage in
the oropharynx or on the skin of immune individuals.
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Infections is acquired by direct (person-to-person) spread
via respiratory droplets or skin contact.
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30-60% of adults have antitoxin antibodies below the
protective level and are at risk.
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Epidemics in Russia; since 1994, 80.000 cases and 2000
deaths.
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Immunity can be assessed by serology or the Shick test.
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Control is based on immunization (adults should be
reimmunized every 10 years)
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Treatment
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Antitoxin (20.000-100.000 units)
- Antibiotics (Erythromycin)
Related Organisms
Listeria monocytogenes
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Gram positive rods, but may be coccoid in direct
smears, sometimes arranged in short chains, motile
at room temperature, and beta hemolytic.
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They can multiply at low temperature and may
accumulate in refrigerated food.
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L. monocytogenes is ingested with raw
contaminated food.
Listeria monocytogenes
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No capsules
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Resistant to cold, heat, salt, pH extremes and bile
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Virulence attributed to ability to replicate in the
cytoplasm of cells after inducing phagocytosis;
avoids humoral immune system
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They produce an invasion factor that enables them
to penetrate host cells of the epithelial lining.
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They are facultative intracellular parasites.
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Normally, the immune system eliminates the
infection before it is spread.
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L. monocytogenes infection is acquired from food
(vegetable or animal products).
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Most cases occur in immunocompromised hosts;
newborns, elderly, cancer patients and transplant recipients.
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It may be transmitted congenitally (mild flue like in
mother) and the fetus develops a serious illness.
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Depending on the stage of gestation, the fetus is either
stillborn or born with signs of congenital manifestations, a
condition known as granulomatosis infantiseptica.
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Listeriosis is a serious disease of humans with a mortality
of more than 25%.
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There are two main clinical manifestations, sepsis and
meningitis.
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Meningitis is often complicated by encephalitis which is
exceptional among bacterial infections.
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Occasionally, pyogenic infections of various organs have
been found.
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Relapses may occur after apparent recovery.
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Diagnosis is made by isolation of the organism.
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Control is achieved by hygienic food processing
and storage.
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Treatment: most antibiotics except cephalosporins
can be given (e.g.. ampicillin and aminoglycosides)
Epidemiology and Pathology
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Primary reservoir is soil and water; animal intestines
Can contaminate foods and grow during refrigeration
Listeriosis - most cases associated with dairy products,
poultry, and meat
Often mild or subclinical in normal adults
Immunocompromised patients, fetuses and neonates;
affects brain and meninges
 20% death rate
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Diagnosis and Control
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Culture requires lengthy cold enrichment process.
Rapid diagnostic tests using ELISA available
Ampicillin and trimethoprimsulfamethoxazole
(Cotrimoxazole)
Prevention – pasteurization and cooking
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Listeria monocytogenes
Erysipelothrix rhusiopathiae
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Gram-positive rod widely distributed in animals
and the environment
Similar to Listeria in morphology.
Primary reservoir – tonsils of healthy pigs
Enters through skin abrasion, multiples to produce
erysipeloid, dark red lesions
Susceptible to Penicillin or erythromycin
Vaccine for pigs
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Erysiplothrix rhusiopathiae
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Causes Erysiploid, a well defined, violet or wine-colored
inflammatory lesion of the skin of the fingers or hand.
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Itching is typical. Infrequently septicemia develops,
followed by various organ manifestations such as
endocarditis or arthritis without fever.
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Incubation is 1-4 days and healing occurs in 2-3 weeks.
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It is an occupational disease as a result of exposure to
mammals, poultry and fish.
Propionibacterium acnes
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The anaerobic counterpart of Corynebacteria.
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It normally colonizes the skin crypts
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The pathogenic role is still disputed, but in compromised
patients it may cause disease such as endocarditis.
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In skin lesions, it is found with other pathogenic bacteria
such as S. aureus and is believed to support the damaging
effects of these pathogens
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It is doubtful whether P. acnes alone is able to induce acne.
Streptobacillus moniliformis
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Gram negative rods.
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The causative agent of rat bite fever.
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At the bite site, an ulcer appears that may heal
spontaneously, occasionally spread to the regional lymph
nodes and bacteremia has been observed.
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Generalized disease (malaise, fever) may be fatal.
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Rat bite is also caused by spirillum minus, a very different
organism.
Calymmatobacterium granulomatis
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Gram negative rods that cause granuloma inguinale, an
infections localized to the genital region.
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It spreads to adjacent areas and the regional lymph nodes
also may be inflamed.
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Persistent granulomatous lesions tend to ulcerate,
destroying skin and subcutaneous tissue.
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It is normally present in the gut flora but may be
transmitted to the genital area by autoinoculation or sexual
contact.
Arcanobacterium hemolyticum
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It causes:
Pharyngitis with or without scarlet-like rash.
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Cutaneous infection.
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Endocarditis.
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Meningitis.
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