Gram-Positive Bacilli

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Schematic Diagram for Presumptive
Identification

2
Corynebacterium Species: General
Characteristics

• Found as free-living saprophytes

• Water, soil, air
• Resistant to drying
• 80 species, of which 50 are clinically significant
• Members of the usual flora of humans and animals
• Closely related to Mycobacteria and Nocardia
• Clinically significant isolates
• Catalase positive and nonmotile

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 Corynebacterium Species:
General Characteristics (Cont.)
• Facultatively anaerobic
• Gram-positive, non–spore-
forming rods
• Arranged in palisades
• Club-shaped
• Babes-Ernst granules
• Polymerized phosphates

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Corynebacterium Species: General
Characteristics (Cont.)

• Corynebacterium diphtheriae is the most

significant pathogen.
• Causes diphtheria
• Difficult to speciate other species
• Other species may cause infections in
immunocompromised hosts.
• Generally thought of as contaminants

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C. diphtheriae

• Toxigenic Corynebacterium diphtheriae

• Worldwide distribution
• Rare in places where vaccination programs exist
• Exotoxin is a major virulence factor.
• Diphtheria toxin
• Toxin is produced by certain strains.
• Lysogenized by bacteriophage with toxin gene (tox+)
• Only toxin-producing bacteria can cause diphtheria.
• Toxin production is inversely proportional to iron availability.
• Toxin is antigenic.
• Thus can use immunoglobulin G (IgG) to block toxin effects

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Toxigenic Corynebacterium diphtheriae

• Two fragments
• A: active fragment
• Inhibits protein synthesis
• NAD+ + EF-2 → ADPR-EF-2 + Nicotinamide + H+
• Catalyzes transfer of ADPR to link with EF2 ADPR.EF (inactive)
• Leads to cell/tissue death
• B: binds to specific cell membrane receptors
• Mediates entry of fragment A

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Clinical Infections of Diphtheria

• Respiratory
• Acquired by droplet spray
• Aerosol or hand-to-mouth contact
• Unimmunized individuals are susceptible.

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Clinical Infections of Diphtheria (Cont.)

• Symptoms
• Incubation period lasts 2 to 4 days
• Low-grade fever, malaise, mild sore throat
• Local toxin production causes tissue necrosis.
• Forms a tough white to gray pseudomembrane
• Starts at tonsils and spreads downward into the larynx and trachea
• May obstruct breathing

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Clinical Infections of Diphtheria (Cont.)

• Systemic effects
• Toxin is absorbed in the bloodstream and carried
systemically.
• Affects the kidneys, heart, and nervous system
• Sometimes causes demyelinating peripheral neuritis,
resulting in paralysis
• Death occurs due to cardiac failure

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Treatment of Diphtheria

• Administer antitoxin
• Relieves toxin symptoms, but must use antibiotics to
clear the infection
• Produced in horses
• Occasionally a hypersensitivity reaction

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Nonrespiratory Disease of
C. diphtheria

• Cutaneous form
• More prevalent in the tropics
• Infections occur at the site of minor
abrasions.
• Nonhealing ulcers with a dirty gray
membrane

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 Culture Characteristics

• Loeffler's serum agar

• Used to demonstrate
pleomorphism and
metachromatic granules
• Babes-Ernst granules
• Cystine-tellurite blood
agar (CTBA)
• Modified Tinsdale medium
• Selective and differential
• Black colonies with brown
halos around the colonies

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Identification of Corynebacteria

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Identification of Corynebacteria (Cont.)

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 Laboratory Diagnosis

• Identification
• Confirm identification by fermentation reactions
• Urease
toxigenicity testing
• Must have toxin for disease
• Elek’s test
• Immunodiffusion test
• Polymerase chain reaction (PCR) for Tox gene

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 Other Corynebacteria
• C. amycolatum
• Common on skin and most frequently recovered species
• Opportunistic infections
• Prosthetic joint infections, sepsis, and endocarditis
• In nosocomial or immunocompromised patients
• C. jeikeium
• Infections from catheters or prosthetic devices
• Prosthetic valve endocarditis
• C. pseudodiphtheriticum
• Opportunistic infection
• Often endocarditis

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Other Corynebacteria (Cont.)

• C. pseudotuberculosis
• Contact with sheep
• Dermonecrotic toxin
• C. striatum
• Rare infections
• Normal flora
• C. ulcerans
• Mastitis in cattle
• Contact with animals or unpasteurized milk
• C. urealyticum
• Urinary pathogen
• Highly urease positive (within minutes) 18
Rothia

• Rothia dentocariosa
• Normal oral flora
• Endocarditis
• Undesignated Centers for Disease Control and Prevention
(CDC) coryneform groups
• Bacteremia
• Endocarditis
• Wound infections
• Eye infections

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Listeria monocytogenes

• Gram-positive, nonsporulating forming rods

• Coccobacillus that often appears like cocci
• Widespread in nature
• Primarily infect animals
• Human exposure is limited.
• Generally dairy products because it grows at low temperature
• Processed meats also can contain Listeria.

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Listeria monocytogenes Virulence Factors

• Hemolysin
• Listeriolysin O
• Damages phagocytic membrane and prevents killing of the organism
• Catalase
• Superoxide dismutase
• Phospholipase C
• Surface protein p60
• Induces phagocytosis

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Listeriosis Clinical Infections

• Adults
• Likely develop disease from penetration of the intestine
and systemic spread
• Septicemia/meningitis in the compromised/elderly
• Also endocarditis
• Mild flulike syndrome in pregnant women could be fatal to
fetus
• Premature labor, spontaneous abortion

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Listeriosis Clinical Infections (Cont.)

• Disease in pregnant women

• Neonatal
• Early onset from intrauterine transmission results in sepsis; high
mortality rate
• Late onset manifests as meningitis; lower mortality rate
• Immunocompromised
• Central nervous system (CNS) infection
• Endocarditis

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Laboratory Diagnosis:
L. monocytogenes

• Identification
• Microscopic morphology
• Gram-positive non–spore-forming coccobacillary

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Laboratory Diagnosis:
L. monocytogenes (Cont.)

• Cultural characteristics
• Grows well on sheep blood agar (SBA) and chocolate
agar
• Prefers slightly increased carbon dioxide (CO2)
• Colonies produce a narrow zone of hemolysis.
• Similar to group B Streptococcus

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Identifying L. monocytogenes

• Identification
• Cold enrichment
• Grows at 4°C
• Catalase positive
• Rules out Streptococcus
• Negative for hippurate hydrolysis
• Motility
• Motile at 25°C
• “Umbrella" type motility
• Tumbling motility in hanging drop method
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Differentiation of Listeria monocytogenes

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Umbrella Motility

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Identifying L. monocytogenes

• Identification
• Christie Atkins Munch-Petersen (CAMP) test
• Produces a “block” type of hemolysis in contrast to “arrow”
shape produced by group B Streptococcus
• Can use hippurate hydrolysis test as well

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Erysipelothrix rhusiopathiae
• Gram positive, non–spore-forming, pleomorphic
rods
• Distributed in nature
• Can cause disease in animals (swine, turkey, sheep)
• Infection through occupational exposure
• Erysipeloid
• Self-limiting localized infection at the site of inoculation
• Produces painful swelling, usually on the hands or fingers
• Heals within 3 to 4 weeks
• Endocarditis
• May occur in those who have had valve replacements

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Erysipelothrix rhusiopathiae (Cont.)

• Culture
• CO2 is required.
• Grows on blood or chocolate agar
• Colonies may appear gray or translucent with alpha hemolysis.
• Pleomorphic, gram-positive thin rods that may form long filaments
• Singly, in short chains, or in a V shape

• Identification
• Catalase negative
• Production of hydrogen sulfide (H2S) on triple sugar iron (TSI)

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Gram Stain and Colony Growth
of E. rhusiopathiae

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Characteristics of Organisms

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Arcanobacterium

• Catalase negative
• Narrow zone β-hemolysis
• Black dot on agar surface under the colony
• Significant species
• A. haemolyticum
• Pharyngitis and pruritic scarlatiniform rash with desquamation of the skin of the hands
and feet
• A. pyogenes
• Sepsis and wound infections in those with animal contact
• A. bernardiae
• Bacteriemia, wound infections, urinary tract infections (UTIs), and septic arthritis
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A. haemolyticum

- Lipase and lecithinase positive

- Exhibits a CAMP inhibition reaction
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Gardnerella vaginalis

• Short, pleomorphic gram-positive rod or coccobacillus

• Often stains gram-variable or gram-negative
• Infections
• Bacterial vaginosis (BV)
• Malodorous discharge
• Vaginal pH of greater than 4.5
• Wet mounts of vaginal fluid
• Observe clue cells
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Culture Characteristics

• SBA or chocolate agar

• 5% to 7% CO2 at 35°to 37°C
• Human blood bilayer Tween (HBT) agar
• Medium of choice
• Treatment
• Metronidazole
• Clindamycin

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Aerobic Actinomycetes:
Nocardia Species

• General characteristics
• Aerobic
• Gram-positive filamentous rods
• Weakly acid-fast and may stain gram-variable
• Sometimes resembling branched hyphae
• Morphologically resemble fungi
• In culture and in types of infections
• Generally found in the environment and mostly affect
immunocompromised individuals

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Stains of Nocardia

 Sulfur granules

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Significant Nocardia Species
• Slow growers, may take 3 to 6 days
• Primarily affect immunocompromised
• Rare but high mortality rate (40%)
• N. cyriacigeorgica and N. farcinica
• Confluent bronchopneumonia
• Necrosis with abscesses
• No “sulfur granules”
• N. brasiliensis
• Cutaneous infection termed actinomycotic mycetomas
• Trauma to skin
• Major destruction of the tissue and bone
• “Sulfur granules” present
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Identification of Nocardia and Similar Species

• Wet mounts
• Crush granules to see cellular morphology
• Microscopy
• Gram-positive branching filaments are seen in direct smears from
sputum or aspirated material.
• May show beading appearance
• May take 3 to 6 days to grow on media
• Nocardia can be identified using paraffin bait test.
• Only Nocardia can break it down as a substrate.

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Identification of Nocardia and Similar Species
(Cont.)

• Methods of identification
• Substrate hydrolysis (casein, tyrosine, xanthine, and
hypoxanthine)
• Other substrate and carbohydrate utilization (arylsulfatase,
gelatin liquefaction, and carbohydrate utilization)
• Antimicrobial susceptibility profile
• Fatty acid analysis by high-performance liquid chromatography

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Other Actinomycetes

• Actinomadura
• Streptomyces
• Gordonia
• Tsukamurella
• Rhodococcus
• Tropheryma whipplei
• Whipple’s disease

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Colony Appearance and Identification

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Bacillus Species

• Found in nature
• Most are saprophytic and are isolated as contaminants
• Gram-positive large rods with “empty” spaces
• Endospores
• Highly resistant to heat, drying, and chemicals
• Catalase positive

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Bacillus anthracis

• Agent of anthrax, a disease in livestock

• Humans acquire infection by contamination of wound or
ingestion or inhalation of spores.
• Morphology
• Large, spore-forming, gram-positive bacilli
• Spores viable for over 50 years (even restored from fossils)
• Nonhemolytic on SBA

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Bacillus anthracis (Cont.)

• Virulence factors
• Polypeptide capsule
• Potent exotoxin
• Edema factor (EF)
• Protective antigen (PA)
• Lethal factor (LF)
• Vaccination is available to those with high risk of
exposure.

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Infections of Bacillus anthracis

• Incidence of infection is very low.

• Fewer than five cases per year
• Woolsorter’s disease; Ragpicker’s disease
• Handling fibers, hides, or other animal products
• Most infections are cutaneous anthrax.
• Enter through cuts causing a localized infection
• Malignant pustule or black eschar
• Painless and non–pus-producing
• Produces a permanent scar

48
Infections of Bacillus anthracis (Cont.)

• Pulmonary anthrax or "woolsorter's disease”

• Acquired through inhalation of spores
• Mild fever, fatigue, and malaise 2 to 5 days after exposure
• Similar to flu or common cold, lasting 2 to 3 days
• Sudden respiratory distress, disorientation, coma, and
death
• May last less than 24 hours from onset to death

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Infections of Bacillus anthracis (Cont.)

• Gastrointestinal anthrax
• Acquired by ingestion of contaminated raw meat
• Inoculates into a lesion on the intestinal mucosa
• Abdominal pain, nausea, anorexia, vomiting, and
sometimes bloody diarrhea
• More likely to be fatal but less likely to occur than
cutaneous form

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Infections of Bacillus anthracis (Cont.)

• Injectional anthrax
• Acquired by direct injection of the endospores into tissue
• Injection drug use
• Does not form black eschar
• Causes necrotizing fasciitis, organ failure, shock, coma, and
meningitis

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Identification of Bacillus anthracis

• Microscopic • Colonial morphology

morphology • Nonhemolytic on 5%
• Large, square-ended blood agar; raised,
gram-positive rods large, grayish-white,
• Bamboo appearance irregular, fingerlike
edges
• “Medusa head” or
“beaten egg whites”

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Characteristics to Differentiate
B. anthracis from B. cereus

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Suspecting Anthrax

• Work in biologic safety cabinet

• Nonhemolytic on blood agar plate (BAP)
• Is nonmotile
• Produces lecithinase
• These are in Table 17-1.
• Exposure to penicillin causes large, spherical bacilli in chains.
• String of pearls morphology

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Treatment of Anthrax

• Most isolates are susceptible to penicillin.

• Resistance can occur due to β-lactamase production.
• Ciprofloxacin was approved by the U.S. Food and Drug
Administration (FDA) for treatment.
• Until susceptibility results are known

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B. cereus

• Food poisoning
• Caused by distinct enterotoxins
• Diarrheal syndrome
• Associated with meat, poultry, and soups
• Incubation period of 8 to 16 hours
• Fever uncommon
• Resolves within 24 hours
• Emetic form
• Associated with fried rice
• Abdominal cramps and vomiting
• Incubation period of 1 to 5 hours
• Resolves in 9 hours

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B. cereus (Cont.)

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Comparison of Enterotoxins Produced by B.
cereus

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B. cereus (Cont.)

• Infections in the immunosuppressed hosts

• Opportunistic infections of the eye
• Endophthalmitis, panophthalmitis, and keratitis
• Forms abscesses
• Meningitis, septicemia, and osteomyelitis

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Other Bacillus Species

• Usually contaminants
• B. subtilis
• B. licheniformis
• B. circulans
• B. pumilus
• B. sphaericus

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Gram-Positive, Spore-Forming Anaerobic
Bacilli

• All in the genus Clostridium

• C. perfringens
• Boxcar-shaped, gram-negative bacillus
• Produces a double zone of hemolysis on Brucella blood agar or blood agar
• C. tetani
• Heavily swarming with terminal spores
• C. septicum
• Heavily swarming with subterminal spores

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Classification of Clostridia by Endospore
Location

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Clinical Infections

• Most frequently encountered in exogenous infections

• Wounds, ingesting toxins
• Endogenous infections
• Clostridium difficile
• Antibiotic-associated diarrhea and pseudomembranous colitis
• Diseases
• Tetanus
• Gas gangrene
• Botulism
• Other food poisoning

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Clostridium perfringens
Food Poisoning
• Type A
• Ingestion of enterotoxin causing diarrhea and cramps
• Usually self-resolving
• Type C
• Enteritis necroticans
• Acute onset of severe abdominal pain and diarrhea, which is
often bloody, and may be accompanied by vomiting
• Followed by necrotic inflammation of the small intestines, at times
leading to bowel perforation
• Without treatment it can be fatal

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Botulism

• Botulinum toxins
• A through G, but only types A, B, and E are associated with human
disease
• Toxin A is now used in medical treatments, including Botox®.
• Clostridium botulinum
• Causes flaccid paralysis
• In contrast to tetanus
• Ingestion of preformed botulin toxin
• Extremely potent
• Causing paralysis and sometimes death
• Prevents the release of acetylcholine

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Botulism (Cont.)

• Sources of infection
• Home canning
• Home-cured meats
• Symptoms
• 2 hours to 8 days later
• Infant botulism
• Ingestion of spores in contaminated honey
• Colonize the colon and produce toxins

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Tetanus

• Causative agent
• Clostridium tetani
• Develops from tetanospasmin neurotoxin
• Inhibits neurons by inhibiting neurotransmitters
• Symptoms
• Begin about 7 days after inoculation
• Muscular rigidity of the jaw, neck, and lumbar region
• Difficulty in swallowing
• Sometimes involves limbs as well
• Diphtheria, pertussis, tetanus (DPT) vaccine
• Very uncommon disease in the United States
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Myonecrosis

• Also known as gas gangrene

• Contaminated wounds from trauma or surgery
• Caused by several species
• C. perfringens: most common cause
• C. histolyticum
• C. septicum
• C. novyi
• C. bifermentans

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Myonecrosis (Cont.)

• Grow, multiply, and produce toxin

• Alpha toxin causes tissue necrosis and deeper invasion
• Causes death of tissue that can require amputation
• Symptoms
• Pain and swelling with obvious tissue necrosis
• Treatment
• Antibiotic and surgical débridement of necrotic tissue
• Hyperbaric oxygen can help destroy anaerobes

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Bacteremia

• C. perfringens
• Most common isolate from blood cultures
• C. septicum
• Marker organism for malignancy of GI tract
• C. bifermentans
• C. tertium
• Both isolated from patients with underlying disease

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Antibiotic-Associated Diarrhea

• C. difficile
• Most common isolate in antibiotic-associated diarrhea
• Can cause pseudomembranous colitis
• Necrosis of colon tissue and bloody diarrhea
• Develops when normal flora are destroyed by antimicrobials that
do not affect clostridium
• Increased growth causing toxin production
• Toxin A: enterotoxin
• Toxin B: cytotoxin

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Antibiotic-Associated Diarrhea (Cont.)

• Commonly transmitted as a nosocomial infection

• Transferred among patients
• Hands of hospital personnel
• Generally do not culture for this pathogen
• Test for toxin production
• Virulent strain B1/NAP1/027
• Produces larger amounts of toxins A and B, plus a third toxin,
binary toxin, and has high level resistance to fluoroquinolones

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Gram-Positive Non–Spore-Forming Anaerobic
Bacilli

• Actinomyces spp.
• Bifidobacterium
• Propionibacterium spp.
• All three can cause
• Actinomycosis
• Chronic, granulomatous disease characterized by sinus tracts and fistulae
that erupt to the surface and drain pus that contains “sulfur granules”

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Gram-Positive Non–Spore-Forming Anaerobic
Bacilli (Cont.)
• Mobiluncus spp.
• Bacterial vaginosis
• Lactobacillus decreases, causing overgrowth of Mobiluncus,
Bacteroides, Prevotella, anaerobic gram-positive cocci, and
Gardnerella vaginalis
• Lactobacillus spp.
• Gram-positive, highly pleomorphic bacilli
• Usually a contaminant
• Serious infections
• Bacteremia and endocarditis
• Immunocompromised
• Endocarditis has a high mortality rate.
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