Aerobic and Facultative GramPositive Bacilli

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Aerobic and Facultative Gram-Positive Bacilli

• Dr. John R. Warren
• Department of Pathology
• Northwestern University
• Feinberg School of Medicine
• June 2007

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An Algorithm for Aerobic Gram-Positive Bacilli

• Grams stain morphology
• Catalase reaction (positive or negative)
• ?-hemolysis with sheep blood agar (present or
  absent)
• Aerobic sporulation (present or absent)

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Grams Stain Morphology

• Irregular rods Bacilli whose longitudinal edges
  are curved and not parallel
• Coryneform rods Club-shaped bacilli
• Diphtheroid arrangement Bacilli arranged in
  palisades of parallel cells, V or L shaped pairs
  of bacilli, and clusters of these morphologies
  forming Chinese letters
• Regular rods Bacilli whose longitudinal edges
  are straight and parallel

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Irregular Gram-Positive Rods

• Corynebacterium species1
• Catalase positive
• Non-??-hemolytic
• Arcanobacterium haemolyticum2
• Catalase negative
• ?-hemolytic
• 1Coryneform rods in diphtheroid arrangement
• 2Previously Corynebacterium haemolyticum

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Regular Gram-Positive Rods

• Listeria monocytogenes (?-hemolytic, catalase
  positive)
• Bacillus species1 (aerobic sporulation, catalase
  positive2)
• Erysipelothrix rhusiopathiae (Non-? hemolytic,
  catalase negative)
• 1Bacillus anthracis non-?-hemolytic, B.
• non-anthracis species ?-hemolytic
• 2Most strains

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Taxonomy of Corynebacterium

• Belong to the class Actinobacteria and the family
  Corynebacteriaceae
• Corynebacterium species contain cell wall mycolic
  acids with 22 to 36 carbon atoms
• 16S rRNA gene sequences indicate a close
  relationship of Corynebacterium to the family
  Mycobacteriaceae (Mycobacterium, Nocardia, and
  Rhodococcus)

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Taxonomy of Corynebacterium

• Corynebacterium species not acid-fast positive
• Corynebacterium species have high DNA GC content
  in a wide range (46-74 mol) indicating genetic
  diversity
• Corynebacterium composed of 59 species of which
  36 are medically relevant

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Pathogenic Corynebacterium Species

• Corynebacterium diphtheriae1
• C. ulcerans1
• C. pseudotuberculosis1
• C. jeikeium2
• C. amycolatum2
• C. urealyticum2
• 1Toxin-producing strains cause diphtheria
• 2Variably associated with invasive infection

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Corynebacterium Natural Habitats

• Corynebacterium diphtheriae establishes a carrier
  state by infection of the human nasopharnyx or
  skin (not considered a constituent of normal
  microbial flora, no environmental or animal
  source)
• C. ulcerans associated with bovine mastitis, C.
  pseudotuberculosis associated with suppurative
  lymphadenitis, abscess formation, and pneumonia
  in cattle

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Corynebacterium Natural Habitats

• Many species normal commensals of the human skin
  (including C. jeikeium, C. amycolatum, and C.
  urealyticum)
• C. jeikeium and C. amycolatum also present in the
  inanimate hospital environment

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Corynebacterium Modes of Infection

• Corynebacterium diphtheriae spread
  person-to-person by exposure to respiratory
  droplets from convalescent or healthy carriers
• Corynebacterium ulcerans infection associated
  with cattle or raw milk
• Corynebacterium pseudotuberculosis associated
  with close animal contact or drinking raw milk,
  human infection rare

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Corynebacterium Modes of Infection

• Corynebacterium jeikeium, C. amyco- latum, and C.
  urealyticum as skin flora can be introduced
  systemically from infected catheter wounds in
  debilitated patients (prolonged hospitalization,
  multiple antibiotics, neutropenia)
• Corynebacterium urealyticum as an anterior
  urethral commensal can invade the urinary tract
  in debilitated patients

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Corynebacterium Types of Infectious Disease

• Diphtheria is an acute pharyngitis with formation
  of an inflammatory pseudomembrane. Systemic
  absorption of diphtheria toxin causes sterile
  myocarditis with a mortality of 10-30.
  Diphtheria toxin catalyzes the transfer of
  ADP-ribose from NAD to elongation factor-2 (EF-2)
  with inactivation of EF-2 in ribosomal protein
  synthesis.

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Corynebacterium Types of Infectious Disease

• Toxigenic strains of C. diphtheriae lyso- genized
  by tox ?-prophage most often cause diphtheria.
  Occasional strains of C. ulcerans and C.
  pseudotuberculosis also produce toxin, but only
  C. ulcerans is associated with diphtheria-like
  illness.
• Tox strains of C. diphtheriae cause pharyngitis
  and endocarditis.

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Corynebacterium Types of Infectious Disease

• C. jeikeium, C. amycolatum, and C. urealyticum
  produce wound infection, bacteremia, and
  endocarditis in hospital patients.
• C. urealyticum is a urease producer that causes
  alkaline-encrusted cystitis due to deposition of
  ammonium magnesium phosphate crystals in damaged
  bladder mucosa with ulceration and infection.

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Corynebacterium Resistance to ??-Lactam Drugs

• Corynebacterium amycolatum, C.
• jeikeium, and C. urealyticum are
• characteristically resistant to
• penicillin and other ??-lactam
• drugs, and uniformly vancomycin
• susceptible

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Characteristics of Corynebacterium

• Gram-positive bacilli with non-parallel sides and
  wider ends resulting in club shaped forms
  (coryneform)
• Arranged as single cells, pairs, V, L, and Y
  forms, palisades, and Chinese letters
  (diphtheroid)
• Non-?-hemolytic on sheep blood agar
• Uniformly catalase positive

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Characteristics of Corynebacterium

• Grow well on sheep blood but not enteric agar
  (MacConkey)
• Lipophilic species (C. jeikeium, C. urealyticum)
  demonstrate optimal growth in presence of the
  non-ionic detergent Tween 80 (polysorbate 80)

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Laboratory Detection of Corynebacterium
diphtheriae

• Translucent, gray, or white non-hemolytic
  colonies up to 2 mm in size after 18-24 hours in
  5 CO2 at 37oC
• Deep blue or red metachromatic granules
  (accumulated inorganic polyphosphates) by
  methylene blue stain after incubation on a
  Loefflers serum slant (heat inspissated serum
  and whole egg medium) (not specific for C.
  diphtheriae)

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Laboratory Detection of Corynebacterium
diphtheriae

• Black colonies (tellurite reductase) are
  surrounded by a brown halo (cystinase) on
  Tinsdale agar specific for C. diphtheriae.
• Tinsdale cystine-tellurite blood agar is
  selective and differential by containing
  potassium tellurite. Staphylococcus and Proteus
  can produce black colonies, but the colonies lack
  a brown halo, and the colonies demonstrate
  gram-positive cocci (Staphylococcus) or
  gram-negative rods (Proteus) by Grams stain.

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Corynebacterium diphtheriae Identification

• Forms black colonies surrounded by a brown halo
  on Tinsdale medium
• Produces acid from glucose and maltose
• Biotypes variably positive for nitrate reduction
  (biotypes gravis, intermedius, and mitis
  positive, biotype belfanti negative) and
  lipophilic growth (biotype intermedius positive)

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Corynebacterium diphtheriaeBiotypes

• Four biotypes intermedius, gravis, mitis,
  belfanti
• Biotype intermedius shows small, gray, or
  translucent, lipophilic colonies, other biotypes
  larger (up to 2 mm at 24 hours), white or opaque,
  non-lipophilic colonies
• Biotype intermedius rarely occurs in clinical
  infection and belfanti rarely contains the tox
  gene

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Elek Assay for Diphtheria Toxin

• Paper strip or disk saturated with diphtheria
  antitoxin is placed in molten agar at 55oC, and
  allowed to sink to the bottom of the plate.
• The agar is allowed to solidify by cooling to
  room temperature.
• Streaks of unknown test organisms are placed at a
  right angle to the strip, or around the periphery
  of the disk.

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Elek Assay for Diphtheria Toxin

• Development of an agar precipitin line within 1-2
  days of incubation at 35oC is a positive result
  for diphtheria toxin.
• In an Elek test using a strip, the precipitin
  line forms at a 45o angle to the strip.
• In an Elek test using a disk, the precipitin line
  forms between the disk and test organism.

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Corynebacterium amycolatum Identification

• Fermentative, growth not lipophilic
• Growth resistant to the vibriocidal compound
  O/129 (150-?g disks)
• Acid production from glucose, variable acid from
  maltose and sucrose
• Variably positive for urease

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Corynebacterium jeikeium Identification

• Oxidative, lipophilic growth
• Growth resistant to a 10 unit penicillin disk
• Acid production from glucose (broth supplemented
  with rabbit plasma or Tween 80), variable acid
  from maltose

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Corynebacterium urealyticum Identification

• Oxidative, lipophilic growth
• Positive for urease
• Asaccharolytic (glucose, maltose, sucrose,
  mannitol, xylose)

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Species Identification of Corynebacterium1

• 
  F/O LIP
  URE GLU MAL SUC
• C. jeikeium2 O
  5 /5 5
• C. amycolatum3 F /
  / /
• C. urealyticum4 O
  
• 1F/Ofermentative/oxidative LIPlipophilic
  UREurease GLU, MAL, SUCproduction of acid from
  glucose, maltose, sucrose (Andrades)
• 2Isolated from blood and vascular catheter
  wounds, growth uniformly resistant to 10-u
  penicillin disk
• 3Isolated from blood and vascular catheter
  wounds, most strains growth resistant to 150-µg
  O/129 disk
• 4Isolated from urine
• 5Andrades supplemented with 2-3 drops coagulase
  rabbit plasma

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Species Identification of Corynebacterium
Criteria1

• Isolate from normally sterile specimen
• Isolate from more than one specimen of the same
  type especially blood
• Isolate from urine as only (gt104/mL) or
  predominant (gt105/mL) organism
• Presence of coryneform bacteria by Grams stain
  with many leukocytes
• 1Including C. diphtheriae in invasive
• infection (rare)

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Arcanobacterium

• Arcanobacterium closely related but genomically
  distinct from Corynebacterium
• A. haemolyticum type species and medically
  relevant, A. pyogenes and A. bernardiae also
  clinically significant

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Arcanobacterium

• Arcanobacterium haemolyticum associated
• with streptococcal-like pharyngitis, mixed
• wound and tissue infections, A. pyogenes
• with abscess formation, wound and soft
• tissue infection, and A. bernardiae with
• polymicrobial abscess formation

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Arcanobacterium

• Irregular, sometimes branching gram-positive rods
• ?-hemolysis on sheep blood agar
• Catalase negative
• Fermentative

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Arcanobacterium

• A. haemolyticum demonstrates reverse CAMP test
• A. haemolyticum, A. pyogenes, A. bernardiae
  produce acid from glucose, only A. pyogenes
  produces acid from xylose, A. bernardiae produces
  acid more quickly from maltose than glucose

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Characteristics of Listeria

• Genetic taxonomy places Listeria closest to
  Bacillus and Staphylococcus (Listeria-Brochothrix
  family). Seven species of Listeria, of which
  only Listeria monocytogenes is pathogenic for
  humans.

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Characteristics of Listeria

• Regular short gram-positive rods, often
  coccobacillary, occur singly and in short chains
• Facultatively anaerobic, fermentative for
  glucose, Voges-Proskauer and methyl red positive,
  catalase positive
• Motile at 20o-25oC

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Listeria monocytogenes Natural Habitats

• Primary habitat is soil and decaying vegetable
  matter
• Widely present in foods including fresh and
  processed poultry and meat, raw milk, cheese, ice
  cream, raw fruits and vegetables
• Transiently colonize the gastrointestinal tract
  in 2-20 of humans without symptoms

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Pathogenesis of Listeriosis

• Two modes of infection Ingestion of food
  contaminated by L. monocytogenes (fecal
  organism) Transmission to fetus in mother
  bacteremic with L. monocytogenes
• Internalin of L. moncytogenes binds to intestinal
  epithelial cells followed by transmucosal
  invasion and lympho-hematogenous dissemination

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Pathogenesis of Listeriosis

• Infection of mononuclear phagocyte system
  (macrophages of spleen, lymph nodes, liver, and
  bone marrow)
• Listeriosis observed clinically as a bacteremia
  in pregnancy, AIDS, and renal transplantation
  with high-dose steroids
• Meningitis secondary to bacteremia in neonates
  and immunosuppresed adults

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Pathogenesis of Listeriosis

• L. monocytogenes is a facultative intracellular
  parasite that infects macrophages
• The exotoxin listeriolysin O binds to membrane
  cholesterol and disrupts phagolysosomes of
  infected macrophages

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Pathogenesis of Listeriosis

• Listeria bacilli escape phagolysosomes into the
  iron-rich cytoplasm and rapidly divide with a
  doubling time of 1 hour
• Listeria bacilli induce polymerization of actin
  in the infected cell that propels bacilli through
  the plasma membrane with penetration and
  infection of adjacent cells

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Listeria monocytogenes Types of Infectious
Disease

• Maternal bacteremia during pregnancy with
  transplacental fetal infection and abortion,
  stillbirth, or premature birth
• Bacteremia and (less frequently) meningitis in
  immune compromised individuals (AIDS, lymphoma,
  corticosteroid suppression therapy in organ
  transplantation)

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Clinical Presentation of Listerosis

• Most frequently transient bacteremia with no
  clinical signs or symptoms, or mild flu-like
  symptoms
• Bacteremia in immunocompromised host (especially
  AIDS) can manifest as sepsis with fever,
  tachycardia, and neutrophilic leukocytosis, often
  accompanied by headache, myalgia, and arthralgia

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Clinical Presentation of Listerosis

• Meningitis acute but can be subacute as a mimic
  of tuberculous meningitis
• Nuchal rigidity absent in 15-20 of adult
  patients
• Ataxia, tremors, myoclonus, and seizures occur in
  15-25
• CSF Grams stain negative in 60, glucose not
  depressed in gt60, and mononuclear cell
  pleocytosis in 1/3rd

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Listeria monocytogenes

• Gram-positive bacillus with narrow-zone
  ß-hemolysis on sheep blood agar
• Catalase positive
• Ferments glucose by butylene glycol pathway
  (Voges Proskauer positive)
• Demonstrates esculin hydrolysis
• Enhanced motility at cooler temperatures
  (20o-25oC) (end-over-end in hanging drop,
  umbrella pattern in soft agar)

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Hemolytic Patterns and CAMP Test for Listeria
monocytogenes

• L. monocytogenes produces a narrow zone of
  ?-hemolysis on sheep blood agar barely extending
  beyond the edge of colonies1
• L. monocytogenes shows a rectangular zone of
  syngergistic ?-hemolysis in the CAMP test with
  Staphylococcus aureus1
• 1Identical pattern seen for non-pathogenic
  ß-hemolytic L. seeligeri

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Fermentation Patterns of Listeria1

• Listeria monocytogenes2
• Positive for rhamnose fermentation,
  negative for xylose fermentation
• Listeria seeligeri2 and Listeria ivanovii3
  Negative for rhamnose fermentation, positive for
  xylose fermentation
• 1ß-hemolytic species of Listeria
• 2Narrow zone hemolysis
• 3Wide zone hemolysis, negative with CAMP test

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Erysipelothrix rhusiopathiae

• The genus Erysipelothrix classified as a regular
  non-spore forming gram-positive rod with Listeria
  and Lactobacillus
• Erysipelothrix rhusiopathiae carried by animals
  including mammals, birds, and fish, and most
  commonly associated with pigs

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Erysipelothrix rhusiopathiae

• E. rhusiopathiae causes erysipeloid, a localized
  cellulitis acquired by infection of skin
  abrasions, injuries, or bites in individuals who
  handle animals or animal products
• E. rhusiopathiae forms pinpoint (lt0.5 mm)
  non-hemolytic, catalase-negative colonies on
  sheep blood agar, and produces H2S in TSI agar

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Lactobacillus

• Lactobacillus is a microaerophiic or occasionally
  anaerobic lactic acid-producing organism with a
  close phylogenetic relationship to Streptococcus,
  Leuconostoc, and Weissella.
• Lactobacilli are catalase-negative and form
  pinpoint ?-hemolytic colonies on sheep blood agar
  with a strong resemblance to viridans
  streptococci.

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Lactobacillus

• Grams stain varies with long slender rods and
  parallel sides to slight curved or coryneform
  coccobacilli
• Identified by sheep blood agar colony morphology,
  Grams stain characteristics, and negative
  catalase reaction

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Lactobacillus

• Lactobacilli widely distributed in nature and
  present as mucosa-associated human saprophytes of
  the oral cavity, gastro- intestinal tract, and
  female genital tract
• Lactobacillus causes endocarditis, and is
  associated with polymicrobial abscesses of the
  mouth, abdomen, and pelvis

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Taxonomy of Bacillus

• Family Bacillaceae tentatively consists of
  Bacillus, Listeria, and Staphylococcus
• Bacillus consists of 70 species of which B.
  anthracis and B. cereus are medically important

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Bacillus Natural Habitats

• Bacillus spores ubiquitously distributed in soil
• Vegetative forms of Bacillus species other than
  B. anthracis widely present in soil and water
• Bacillus spores and vegetative forms of species
  other than B. anthracis are frequent colonizers
  of the skin and gastrointestinal tract

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Bacillus Modes of Infection

• Natural infection by Bacillus anthracis by
  introduction of spores into skin abrasions or
  cutaneous wounds by contact with infected
  herbivorous animals or animal products
  (especially wool), inhalation of spores, or
  ingestion of spores
• Spread of anthrax spores through the US postal
  system (2001) with contact and inhalational
  infection (bioterrorism)

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Bacillus Types of Infectious Disease

• Anthrax caused by Bacillus anthracis and occurs
  in three forms cutaneous, pulmonary, and
  gastrointestinal.
• Cutaneous anthrax most common natural form (95
  of human cases). Papules develop at skin
  inoculation sites, progress to hemorrhagic
  vesicles that rupture and form a black eschar
  surrounded by edema (malignant pustule).

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Bacillus Types of Infectious Disease

• Mortality of untreated cutaneous anthrax is 20.
• Pulmonary anthrax has a mortality of 100 due to
  a severe hemorrhagic mediastinitis and
  bacteremia. Pulmonary and cutaneous anthrax
  occurred during the 2001 bioterrorist attack on
  the US postal system.

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Bacillus Types of Infectious Disease

• Gastrointestinal anthrax results in a severe
  hemorrhagic ascites.
• Bacillus cereus causes a rapid, virulent, and
  destructive endophthalmitis following penetrating
  trauma of the eye or hematogenous spread.
• B. cereus bacteremia can develop secondary to
  intravenous drug abuse.

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Bacillus Types of Infectious Disease

• Most frequently (gt99) a blood culture positive
  for the growth of Bacillus is due to a skin
  contaminant.
• A food-poisoning syndrome with acute emesis is
  caused by heat-stable exotoxin of B. cereus.
  Clinically mimics staphylococcal food-poisoning.
• Secretory diarrhea is produced by heat stable
  exotoxin of B. cereus.

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Characteristics of Bacillus

• Large (0.5 x 1.2-2.5 x 10 ?m) gram-positive
  bacilli with square or concave ends containing
  endospores that by Grams stain appear as
  intracellular non-staining (clear) ovoid areas
  (spores do not stain by crystal violet, safranin,
  or carbolfuchsin)
• Bacillus becomes gram-variable or gram-negative
  with aging in culture, but unlike true
  gram-negative bacteria, Bacillus susceptible to
  growth inhibition by a 30 µg-vancomycin disk

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Characteristics of Bacillus

• Bacillus grows well on 5 sheep blood agar and
  chocolate agar but not MacConkey agar
• Catalase positive
• Aerobic growth of large spore-forming
  gram-positive rods positive for catalase strongly
  presumptive for Bacillus

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Characteristics of Bacillus

• Colonies of B. anthracis on sheep blood agar
  large (4-5 mm), flat, and white to gray with
  irregular edges and no ?-hemolysis
• Colonies of B. cereus large (3-8 mm) and raised
  with a gray to green frosted-glass appearance and
  undulate margins with distinct ?-hemolysis

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Characteristics of Bacillus

• Bacillus anthracis non-motile and sensitive to
  penicillin (growth inhibited by a 10-unit
  penicillin disk)
• Bacillus cereus motile and resistant to
  penicillin (growth inhibited by a 10-unit
  penicillin disk)

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Red Flags for Bacillus anthracis

• Flat or slightly convex 2-5 mm non-hemolytic
  tenacious colonies on sheep blood agar after
  overnight incubation at 35oC
• Catalase positive
• Gram-positive bacilli in long chains with oval
  spores that do not swell the cells
• Non-motile

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Recommended Reading

• Winn, W., Jr., Allen, S., Janda, W., Koneman,
• E., Procop, G., Schreckenberger, P. Woods,
• G.
• Konemans Color Atlas and Textbook of
• Diagnostic Microbiology, Sixth Edition,
• Lippincott Williams Wilkins, 2006
• Chapter 14. Aerobic and Facultative
  Gram-Positive
• Bacilli.

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Recommended Reading

• Murray, P., Baron, E., Jorgensen, J., Landry,
• M., Pfaller, M.
• Manual of Clinical Microbiology, 9th
• Edition, ASM Press, 2007
• Funke, G., and Bernard, K.A. Chapter 34.
  Coryneform Gram-Positive Rods.
• Bille, J. Chapter 33. Listeria and
  Erysipelothrix.
• Logan, N.A., Popovic, T., and Hoffmaster, A.
  Chapter 32. Bacillus and Other Aerobic
  Endospore-Forming Bacteria.