Chapter 022

Chapter 22
Anaerobes of Clinical Importance
Copyright © 2015 by Saunders, an imprint of Elsevier Inc.

1
Introduction
 Anaerobes of clinical importance and their
role in disease
 Serious and fatal intoxications and infections
 Endogenous bacteria
 Proper techniques for recovery and
identification of anaerobes
 Procedures for identifying and testing their
susceptibility
 Treatment
Copyright © 2015 by Saunders, an imprint of Elsevier Inc. 2

Anaerobes Defined
 Anaerobes
 Able to replicate in the absence of oxygen
 Obligate (strict) anaerobes
 Killed in the presence of oxygen
• Almost immediately
 Capnophilic
 Increased carbon dioxide (CO2) (5% to 10%),
reduced oxygen (O2) to 15%
 Microaerophilic
 Oxygen reduced to 5% or less

Anaerobes Defined (Cont.)
 Facultative anaerobes
 Preferentially use oxygen if available but can grow well
without oxygen
 Obligate (strict) anaerobes
 Killed in the presence of oxygen
• Oxygen free radicals lead to cell destruction
 Bactericidal
• Require low oxidation-reduction (redox) potential
 Aerotolerant (moderate anaerobes)
 Oxygen steals electrons used in metabolism.
• Bacteriostatic

Classification of Bacteria on the Basis of
Their Relationship to Oxygen and CO2

 Oxygen has a direct toxic effect
 Lack enzymes to break down toxic products
• O2- : superoxide anion
• H2O2: hydrogen peroxide
• OH- : hydroxyl radical
 O2 + e-O2- (superoxide anion)
 O2 + e- + 2H+H2O2 (hydrogen peroxide)

 Strict aerobic and facultative anaerobic
bacteria can protect themselves.
 Possess enzymes to break down toxic products
• Superoxide dismutase
• Catalase
 4O2- + 4H+  2H2O2 + O2 via superoxide
dismutase
 2H2O2  2H2O + O2 via catalase

Where Anaerobes Are Found
 Exogenous  Endogenous
 Organisms exist inside
 Organisms exist outside
of the bodies of animals. the bodies of animals
and become sources
 Infections develop when
of infection.
the agent is ingested or
 Example: Bacteroides,
has gained entrance
through trauma. Porphyromonas,
Prevotella,
 Example: Clostridium,
Fusobacterium,
Sarcina, Fusobacterium,
Clostridium,
and others
Propionibacterium, and
anaerobic cocci

Endogenous Anaerobes
Commonly Involved in
Human Infections

Incidence of Anaerobes at a
University Medical Center

Endogenous Anaerobes of
Various Anatomic Sites

Anaerobes at Specific
Anatomic Sites
 Skin
 Propionibacterium acnes, Peptostreptococcus
 Respiratory tract
 Prevotella, Porphyromonas, Fusobacterium, and
anaerobic cocci
 Gastrointestinal (GI) tract
 Bacteroides, Bifidobacterium, Clostridium, Eubacterium
 Genitourinary (GU) tract
 Fusobacterium, Prevotella, Bacteroides, Lactobacillus,
and anaerobic cocci

Factors That Predispose Patients
to Anaerobic Infections
 Trauma to protective barriers
 Vascular stasis
 Prevention of oxygen flow to site
 Gains entry and has a reduced environment
that allows for a perfect growth site
 Bite wounds
 Aspiration of oral flora into deep lung
 Oral surgery
 Wounds or burns with soil contamination

Potential Virulence Factors of
Anaerobic Bacteria

Indications of Involvement of
Anaerobes in Infectious
Processes

Examples of Predisposing
Conditions

Frequently Encountered
Anaerobes
 Clostridium spp.
 Bacteroides spp.
 Fusobacterium spp.
 Prevotella and Porphyromonas
 Veillonella spp.
 Peptococcaceae

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

Classification of Clostridia by
Endospore Location

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

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

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

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

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

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

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

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

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

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

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”

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.

Gram Stain of
Actinomyces israelii

Anaerobic Gram-Negative Bacilli
 Bacteroides fragilis group
 Most commonly isolated anaerobe from blood cultures
 Porphyromonas
 Prevotella
 Fusobacterium
 Clinical infections
 Frequently found in mixed infections, such as abscesses
occurring beneath mucosal surfaces, peritoneal
infections, diabetic foot ulcers, decubitus pressure sores
 Lemierre disease: Fusobacterium necrophorum

Anaerobic Cocci
 Formerly Peptostreptococcus
 Now Peptostreptococcus, Anaerococcus,
Finegoldia, Parvimonas, Peptoniphilus
• Still not settled
 Veillonella spp.
 Clinical infections
 Brain abscess, meningitis, aspiration pneumonia,
lung abscess, and gingivitis and other periodontal
diseases
• Most often associated with polymicrobial infections

Acceptable Specimens for
Anaerobic Bacteriology

Unacceptable Specimens for
Anaerobic Bacteriology

Specimen Quality, Transport, and
Processing
 Need proper education about which sites are
appropriate for anaerobes
 Includes collection of actual site of infection, not
just the surface
 Prevent misleading and nonuseful clinical results
 Requires rapid processing
 Why?
• Toxicity of oxygen
• Drying of specimen

Processing (Cont.)
 Aspirates
 Needle and syringe
• Less contamination and can expel any air
 Best to inject specimen into prereduced anaerobic
ally sterilized (PRAS) media
 Vortex to mix sample and plate to appropriate
isolation media
 Use a few drops on PRAS media and streak for
isolated colonies
 Vital since most anaerobic infections are polymicrobial
• Need isolated colonies to verify relevant organisms

PRAS Transport Medium

Processing (Cont.)
 Swabs
 Not recommended and only under prereduced
conditions
 Tissue
 Place small piece into PRAS media or reduced
transport medium
 Blood
 Aseptic inoculation of both anaerobic and aerobic
blood culture bottles

Processing (Cont.)
 Anaerobic chambers
 Glove boxes
 Anaerobe jars
 Biobags
• Allow working with anaerobes in an oxygen-free
environment
• All tests can be done inside chamber
• Reduces the likelihood of oxygen toxicity killing relevant
organisms

Processing (Cont.)
 Procedures
 Macroscopic and microscopic examination of
specimen
 Incubation of appropriately plated and tubed
media

Characteristics to Note During
the Macroscopic Examination of
a Specimen

Direct Microscopic Examination
of Specimen
 Direct examination
 Helps rule in and rule out pathogens
 Establishes a polymicrobial or monomicrobial
infection
 Provided presumptive identification
 Aids in selection of culture media
 Determines the quality of specimen collected

Direct Microscopic Examination
of Specimen (Cont.)
 Gram stain
 Use methanol as fixative instead of heat
 Recognize morphology with presumptive identification
 Gram-positive bacilli
• Clostridium
 Thin gram-negative bacilli
• Fusobacteria
 Tiny gram-negative cocci that are gram variable
• Veillonella
 Gram-negative coccobacilli
• Bacteroides, Porphyromonas, Prevotella

Primary Setup Media Recommended
for Recovery of Anaerobes

Media and Media Inoculation
 Media for aerobic isolation
 Inoculation procedures
 Anaerobic incubation of inoculated media

PRAS Media

Anaerobic Chambers
 Basic components of anaerobic chambers
 Gases
• Hydrogen gas (5%-10%)
• CO2 gas (5%-10%)
• Nitrogen gas (80%-90%)
 Catalysts
• Palladium-coated alumina pellets
 Remove any residual oxygen

Anaerobic Chambers (Cont.)
 Desiccant
• Absorb excess water produced when catalyst removes
oxygen to form water
 Indicator
• Colorless when no oxygen present
• Blue or pink when oxygen present, depending on dye

Glove Boxes and Other
Chambers

Glove Boxes and Other
Chambers (Cont.)

Options Available for Identifying
Anaerobic Bacteria

Indications of the Presence of
Anaerobes in Cultures
 Foul odor when opening an anaerobic jar or bag
 Characteristic colonial morphology on anaerobic
agar plates but not on aerobic culture plates
 Unique morphology on Gram stain of clinical
specimens or isolates
 Good growth on Bacteroides bile-esculin agar
(BBE)
 Double zone of hemolysis on blood agar
 Brick-red fluorescence on kanamycin
vancomycin laked blood (KVLB) agar

Processing Colonies Suspected
of Being Anaerobes
 Describe colony morphology and growth on
selective media
 Describe the Gram-stain reaction and cell
morphology
 Set up an aerotolerance test
 Inoculate pure culture/subculture plate and
add appropriate disks

Interpretation of Aerotolerance
Test Results
 Aerotolerance test
 Determines whether a microorganism isolated
under anaerobic conditions is a strict anaerobe or
a facultative anaerobe
 Inoculated with a short streak onto a chocolate
agar plate for incubation in a CO2 incubator and
an anaerobic blood agar incubated anaerobically

Schematic Diagram for Initial
Identification of Anaerobic Isolates
Based on Gram-Stain Morphology

Fluorescence
 Ultraviolet (UV) light
 Porphyromonas and Prevotella spp.
• Brick-red fluorescence
 Fusobacterium nucleatum and C. difficile
• Chartreuse fluorescence
 Veillonella spp.
• Red fluorescence
 Dependent on culture medium

Fluorescent Bacteria

Fluorescence Under Long-Wave
UV Light

Other Tests
 Catalase test
 15% hydrogen peroxide added to organism on slide
• Bubbling is positive.
 Separates Clostridium from Bacillus
 Spot indole test

 Dilated cardiomyopathy with ataxia (DCMA) reagent
• Filter paper saturated with para-dimethylaminocinnamaldehyde
• Turns a blue or green color (positive)
• Pink or orange color (negative)
 Rapid urease test
 Rapid motility test

Special Potency Disks
 Vancomycin disk
 Susceptibility indicates gram-positive bacillus or
Porphyromonas
 Kanamycin disk
 Susceptibility indicates Bacteroides other than
fragilis group
 Also Bilophila, Fusobacterium, Veillonella
 Colistin disk
 Susceptibility indicates same as kanamycin disk
plus Prevotella

Interpretation of Special-Potency
Antimicrobial Disk Results

Antimicrobial Disk Results (Cont.)

Antimicrobial Disk Results (Cont.)

Special Potency Disks (Cont.)
 Nitrate disk
 Ability to reduce nitrate
 Sodium polyanethol sulfonate (SPS) disk
 Peptostreptococcus anaerobius is susceptible.
 Peptoniphilus asaccharolyticus is resistant.
 Bile disk
 Growth in 20% bile
 B. fragilis group

Lecithinase and Lipase
Reactions
 Egg yolk agar (EYA)
 Lecithinase cleaves lecithin found in egg yolk
• Releases insoluble fat
 Forms opaque zone around colony
 C. perfringens
 Lipase
 Hydrolyzes triglycerides and diglycerides to fatty
acids and glycerol
• Iridescent, multicolored sheen
 F. necrophorum

Positive Lecithinase and Lipase
Reactions

Presumptive Identification of
Gram-Positive Anaerobes

Colonies on Sheep Blood Agar
(SBA)

Presumptive Identification of
Gram-Negative Anaerobes

Colonies on BBE Agar

F. nucleatum

Definitive Identifications
 Biochemical and enzyme-based minisystems
 PRAS and non-PRAS tubed biochemical test
media
 Gas-liquid chromatographic analysis of
metabolic end products
 Cellular fatty acid analysis by gas-liquid
chromatography (GLC)
 16S ribosomal sequencing

Identification of Clostridium
Species
 Double zone hemolysis
 Two zones of hemolysis around colonies
 Cycloserine-cefoxitin-fructose agar (CCFA)
 Selective and differential for C. difficile
• Yellow “ground glass” colonies
• Horse stable odor
 Lecithinase and lipase reactions
 Spot indole, rapid urease, gelatin hydrolysis
 GLC

Identification Algorithm for Some
Clinically Encountered
Clostridium Species

Characteristics of Some Clinically
Encountered Clostridium Species

Double Zone of Hemolysis

Identification of Non–Spore-
Forming Gram-Positive Bacilli
 Actinomyces
 Bifidobacterium
 Lactobacillus
 Propionibacterium

Characteristics of Gram-Positive Non–
Spore-Forming Anaerobic Bacilli

A. israelii

P. acnes

Identification of Anaerobic
Gram-Negative Bacilli
 Bacteroides
 Bile-tolerant species
• B. fragilis group
 Bile-sensitive pigmented species
• Porphyromonas
• Prevotella
 Bile-sensitive nonpigmented species
 Bacteroides ureolyticus
 Campylobacter spp.
 Bilophila wadsworthia
 Fusobacterium

Phenotypic Characteristics of
Anaerobic Gram-Negative Bacilli

B. fragilis Group
 KVLB agar plate
 BBE agar plate
 Usually biplate

B. wadsworthia

Other Pigmented Species
 Prevotella spp.
 Produce protoporphyrin, causing a brown-black
color shift of macroscopic colonies over 2 to 3
weeks
 Gram-negative coccobacilli or bacilli
 Brick-red fluorescence under long-wave UV light
 Porphyromonas spp.
 Brick-red fluorescence under long-wave UV light
 No growth on media containing vancomycin

Bile-Sensitive Nonpigmented
Species
 B. ureolyticus
 Isolated from blood following tooth extraction
 Urease positive
 Prevotella spp.
 Vagina and oral cavities
 P. denticola esculin positive
 P. intermedia indole positive, lipase positive
 Some bile-sensitive pigmented species can be
categorized as pitting or nonpitting colonies.

Fusobacterium spp.
 Generally considered gram-negative long,
thin, tapered rods (fusiform)
 May appear pleomorphic
 F. nucleatum
 Classic fusiform shape
 Indole positive
 Butyric acid from peptone yeast glucose (PYG)
media

Fusobacterium spp. (Cont.)
 F. mortiferum
 Indole negative, bile resistant, esculin positive
 F. russi
 Indole negative, bile sensitive

Example of Fusobacterium

Anaerobic Gram-Positive Cocci
 Many clinical specimens may contain these
organisms.
 Vagina, abscesses are most likely sites
• Cocci in a variety of arrangements
 Tetrads, chains, clusters
 P. anaerobius
 SPS sensitive
 Iso: acid production in PYG
 P. asaccharolyticus
 SPS resistant
 Indole positive

Gram Stain of Anaerobic
Gram-Positive Cocci

Identification of Anaerobic
Gram-Positive Cocci

Characteristics of Some Clinically
Encountered Anaerobic Cocci

Antimicrobial Susceptibility
Testing
 Clinical and Laboratory Standards Institute
(CLSI)
 Methods for Antimicrobial Susceptibility Testing of
Anaerobic Bacteria (Approved Standard, M11-A8)
 Performed in
 Brain abscess
 Endocarditis
 Infection of a prosthetic device or vascular graft
 Joint infections
 Osteomyelitis
 Bacteremia

Antimicrobial Susceptibility
Testing (Cont.)
 Highly virulent and/or commonly resistant
 B. fragilis group
 Prevotella
 Clostridium
 Fusobacterium
 B. wadsworthia
 Sutterella wadsworthensis

CLSI Recommendations for the
Antimicrobial Agents

Problems in Susceptibility
Testing of Anaerobic Isolates
 Issues
 Clinical significance of isolates
 Predictability of susceptibilities and reproducibility
 Cost-effectiveness
 Difficulty in performing susceptibility testing

Treatment of Anaerobe-
Associated Diseases
 Destroy reduced environment
 Débridement, draining wounds, improving
circulation
 Provide a hyperbaric oxygen environment
 Administer antimicrobials to prevent invasion
to surrounding healthy tissue
 Use antitoxins to reduce virulence factors

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Chapter 22

Anaerobes of Clinical Importance

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 Spot indole test

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