Aerobic Gram Positive Bacilli

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Aerobic Gram Positive Bacilli

• Bacillus

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Introduction

• The genus Bacillus includes gram positive,
  aerobic, spore-forming, rod shaped organisms.
• The diameter of the organism is 1 X 3-5µm
• They are arranged as single or paired bacilli in
  clinical specimens and as long serpentine chains
  or clumps in cultures.
• 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.
• They produce a variety of enzymes and other
  metabolites during growth including the
  antibiotics bacitracin and polymyxin.

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Bacillus anthracis

• The causative agent of anthrax.
• 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.

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• 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.

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Clinical Syndromes

• Bacillus anthracis is an organism found in soil
  and on vegetation.
• Anthrax is a disease of herbivores and humans are
  accidentally infected by exposure to infected
  animals or animal products.
• 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.
• Inhalation anthrax (wool sorters disease) results
  from inhalation of spores during processing of
  hair or wool.
• Ingestion anthrax is very rare in humans but
  ingestion is a common route of infection in
  herbivores.
• Person to person transmission does not occur.

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Cutaneous Anthrax

• It usually occurs through contamination of a cut
  wound or abrasion although in some countries
  biting flies may also transmit the disease.
• 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.
• The lesion is painless and is surrounded by
  marked edema.
• Lesions on the face or neck are dangerous and
  fulminating septicemia may develop in 20 of
  cases.

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Pulmonary Anthrax

• Inhaled spores are transported by alveolar
  macrophages to the mediastinal lymph nodes where
  they germinate and multiply to initiate systemic
  disease.
• It may initially mimic a viral respiratory
  illness and then rapidly progresses to a diffuse
  pulmonary involvement leading to respiratory
  failure.
• It is highly fatal (gt 95) because it is not
  suspected until the course is irreversible.

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Gastrointestinal Anthrax

• Very rare with varied clinical presentation
  (mesenteric adenopathy, hemorrhage and ascites)
  and high mortality rate (95).
• The organism probably invades the mucosa through
  a preexisting lesion and spreads to the lymphatic
  system.
• Symptoms prior to fulminant systemic anthrax may
  be absent or mild.
• 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.
• 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.
• 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.
• Acellular vaccines for human use are available
  for individuals in high risk occupations (cell
  free filtrate of culture).
• Bacillus anthracis is susceptible to penicillin
  and almost all other broad spectrum antibodies.

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Anthrax - Diagnosis

• Specimen
• Aspirate or swab from cutaneous lesion
• Blood
• Sputum
• Laboratory investigation
• Gram stain
• Culture
• Identification of isolate

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Anthrax - Epidemiology
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Other Diseases

• In recent years, other B. species have been
  increasingly implicated in a wide range of
  infections.
• They include abscesses, bacteremia/septicemia,
  wound and burn infections, ear infections,
  endocarditis, meningitis, ophthalmitis,
  osteomyelitis, peritonitis and respiratory and
  urinary tract infections.
• 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.

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Bacillus cereus

• 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

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Food Poisoning

• B. cereus causes food poisoning by virtue of
  toxin production.
• 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

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• 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.

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Bacillus cereus clinical presentation
Gastroenteritis
EMETIC FORM
DIARRHOEAL FORM
Incubation period gt 6 hours Diarrhea Lasts 20-36
hours
Incubation period lt 6 hours Severe vomiting Lasts
8-10 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).
• It is a post traumatic disease which is rapidly
  progressive that almost universally ends in
  complete loss of light perception within 48
  hours.

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Corynebacteria

• Gram-positive, non motile, club shaped
  pleomorphic bacilli that appear in short chains
  (V or Y) configurations or in clumps resembling
  Chinese letters.
• Nonencapsulated, catalase positive, and oxidase
  negative
• C. diphtheriae is fastidious while diphtheriods
  are not
• 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.
• Corynebacteria are aerobes or facultative
  anaerobes and generally grow slowly on enriched
  media.
• Four distinct cultured types exist gravis,
  intermedius, mitis and belfanti.
• 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.
• The most important species is C. diphtheriae, the
  causative agent of diphtheria which causes
  disease by virtue of toxin production.
• 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
• 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.
• Lysogenic conversion may occur in situ as well as
  in vitro.

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Pathogenesis

• In 1883 Klebs demonstrated that C. diphtheriae is
  the agent of diphtheria.
• 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.

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Diphtheria Toxin

• The regulation of diphtheria toxin gene
  expression is mediated by an iron-activated
  repressor which is encoded on the C. diphtheriae
  genome.
• 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
• An N-terminal ADP- ribosyl transferase (catalytic
  domain)
• A region which facilitates the delivery of the
  catalytic domain across the cell membrane
  (transmembrane domain).
• 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).
• Fragment A is the N-terminal containing the
  catalytic center for the ADP- ribosylation of
  elongation factor 2
• (EF-2).
• Fragment B (the C terminal) carries the
  transmembrane and receptor binding domain of the
  toxin.
• 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
• 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.

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Clinical Manifestations

• Incubation of 2-6 days.
• There are two types of clinical diphtheria
  nasopharyngeal and cutaneous.
• 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.
• 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).
• The skin lesions in cutaneous diphtheria are
  usually covered by a gray-brown pseudomembrane.
• 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.
• Disease is followed by long lasting immunity
  which is also achieved by immunization using
  diphtheria toxoid (protective level is 0.01
  1U/ml)/
• Diagnosis Requires demonstration of toxigenic
• C. Diphtheria in lesions (throat,
  nasopharynx, nose) by Elek test.

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Laboratory diagnosis

• 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
• 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

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Cultural characteristics

• 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, non-hemolytic with black center grey
  margin.

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Gram stain of C. diphtheriae
C. diphtheriae on BTA
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In Vitro Eleks Test

• 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

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Filter paper saturated with diphtheria antitoxin
Lines of precipitations

• 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
  organism

Inoculated M.O.
Positive Eleks Test
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Epidemiology

• Worldwide distribution.
• Incidence has dropped dramatically since the
  introduction of the vaccine.
• 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.
• 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.
• Epidemics in Russia since 1994, 80.000 cases and
  2000 deaths.
• Immunity can be assessed by serology or the Shick
  test.
• Control is based on immunization (adults should
  be reimmunized every 10 years)

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• Treatment
• Antitoxin (20.000-100.000 units)
• - Antibiotics (Erythromycin)

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Related OrganismsListeria monocytogenes

• Gram positive rods, but may be coccoid in direct
  smears, sometimes arranged in short chains,
  motile at room temperature, and beta hemolytic.
• They can multiply at low temperature and may
  accumulate in refrigerated food.
• L. monocytogenes is ingested with raw
  contaminated food.

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Listeria monocytogenes

• No capsules
• Resistant to cold, heat, salt, pH extremes and
  bile
• 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.
• They are facultative intracellular parasites.
• 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).
• Most cases occur in immunocompromised hosts
  newborns, elderly, cancer patients and transplant
  recipients.
• It may be transmitted congenitally (mild flue
  like in mother) and the fetus develops a serious
  illness.
• 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.
• There are two main clinical manifestations,
  sepsis and meningitis.
• Meningitis is often complicated by encephalitis
  which is exceptional among bacterial infections.
• Occasionally, pyogenic infections of various
  organs have been found.

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• Relapses may occur after apparent recovery.
• Diagnosis is made by isolation of the organism.
• Control is achieved by hygienic food processing
  and storage.
• Treatment most antibiotics except cephalosporins
  can be given (e.g.. ampicillin and
  aminoglycosides)

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Epidemiology and Pathology

• 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

• 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
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Erysipelothrix rhusiopathiae

• 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

• Causes Erysiploid, a well defined, violet or
  wine-colored inflammatory lesion of the skin of
  the fingers or hand.
• Itching is typical. Infrequently septicemia
  develops, followed by various organ
  manifestations such as endocarditis or arthritis
  without fever.
• Incubation is 1-4 days and healing occurs in 2-3
  weeks.
• It is an occupational disease as a result of
  exposure to mammals, poultry and fish.

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Propionibacterium acnes

• The anaerobic counterpart of Corynebacteria.
• It normally colonizes the skin crypts
• The pathogenic role is still disputed, but in
  compromised patients it may cause disease such as
  endocarditis.
• 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
• It is doubtful whether P. acnes alone is able to
  induce acne.

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Streptobacillus moniliformis

• Gram negative rods.
• The causative agent of rat bite fever.
• At the bite site, an ulcer appears that may heal
  spontaneously, occasionally spread to the
  regional lymph nodes and bacteremia has been
  observed.
• Generalized disease (malaise, fever) may be
  fatal.
• Rat bite is also caused by spirillum minus, a
  very different organism.

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Calymmatobacterium granulomatis

• Gram negative rods that cause granuloma
  inguinale, an infections localized to the genital
  region.
• It spreads to adjacent areas and the regional
  lymph nodes also may be inflamed.
• Persistent granulomatous lesions tend to
  ulcerate, destroying skin and subcutaneous
  tissue.
• It is normally present in the gut flora but may
  be transmitted to the genital area by
  autoinoculation or sexual contact.

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Arcanobacterium hemolyticum

• It causes
• Pharyngitis with or without scarlet-like rash.
• Cutaneous infection.
• Endocarditis.
• Meningitis.