Anaerobes of Clinical Importance • contribute to an infectious disease in any anatomic site of the body if

suitable conditions exist for colonization and penetration of the bacteria

Anaerobes at Specific Anatomic sites

Exogenous anaerobes
Portal of entry of anaerobes into the tissues and bloodstreams
• exist outside the body
• linings of the oral cavity
• causes exogenous infection
• gastrointestinal (GI) tract
• caused by gram (+), spore-forming bacilli belonging to genus Clostridium
• genitourinary (GU) tract
Endogenous anaerobes
➢ finding site-specific organisms at a distant and/or unusual site can serve as a
• exist inside the body (endogenous microbiota) clue to the underlying origin of an infectious process
• source of endogenous infection
• anaerobes most frequently isolated from infectious processes
 ✓ Vascular stasis – prevents oxygen from entering a particular site,
which results in an environment conducive to growth and
multiplication of any anaerobe that might be present at that site
✓ Decreased oxygenation of tissue – leading to tissue necrosis and a
decrease in the redox potential of tissue
• Precise mechanisms whereby anaerobic bacteria cause disease are not
always known

Factors that predispose patients to anaerobic Infections

Factors Predisposing Patient to Anaerobic Infections

✓ Trauma to mucous membranes or the skin – allows anaerobes of the
endogenous biota (or in some cases, soil anaerobes) to gain access to
deeper tissues
 Gram (+), spore-forming anaerobic bacilli
All spore-forming anaerobic bacilli are classified under the genus Clostridium
(collectively termed clostridia)
➢ spores are not often observed in Gram stained smears unless the
culture has been incubated for many days
➢ sporulation is induced by:
✓ heat
✓ alcohol shock
➢ Clostridia are grouped according to the
location of the endospores:
Terminal spores – produced at the end of the
bacterial cells typically cause swelling of the cell
ex. Clostridium tetani
Subterminal spores – produced within the cell and whether or not it causes a Group 1: Gas gangrene group
swelling of the cell serves as clues to the identity of the organism
✓ C. perfringens Group 2: Clostridium tetani
ex. Clostridium sporogenes
✓ C. ramosum Group 3: Clostridium botulinum
Central – ex. Clostridium botulinum
✓ C. septicum Group 4: Clostridium difficile
✓ C. sordellei Group 5: Miscellaneous
✓ C. histolyticum clostridia
✓ C. novyi
✓ C. fallax
✓ C. sporogenes.

Clinical Infection
Clostridia gain access to the body through:
✓ ingestion
✓ open wounds that have become contaminated with soil
Genus Clostridium ➢ Spores enter open wounds and germinate in vivo in:

Clostridium spp. ✓ tetanus

General Characteristics ✓ gas gangrene (myonecrosis)

• Gram-positive motile bacillus (by means of peritrichous flagella) ✓ botulism

• Anaerobic but some species are aerotolerant (peroxidase positive) ➢ Clostridium perfringens - contaminated food that has been improperly
stored:
• All are motile except C. innocuum, C. perfringens and C. ramosum
✓ food poisoning (foodborne intoxication)
• All have swollen sporangium except C. perfringens and C.
bifermentans ➢ Clostridium difficile

• All are non-encapsulated except C. perfringens ✓ antibiotic-associated pseudomembranous colitis

Clostridium perfringens ➢ seven antigenically different botulinum toxins (A through H)
• Formerly called C. welchii ➢ Toxin A, B and E are associated with human disease
• Non-motile, spore-former, ➢ Botulinum toxin
obligate anaerobe
• extremely potent neurotoxin
• Microscopically, appear as
large, short, fat cells –
“boxcar cells”
• Produces target hemolysis –
double zone of hemolysis on Infant botulism
BAP (narrow inner β zone and
wider outer zone of partial β- ➢ follows ingestion of spores
hemolysis) Wound botulism
Food poisoning ➢ contamination of wounds with spores
Two types of food poisoning: Potential agents of bioterrorism:
✓ botulism toxin
✓ Clostridium botulinum

Clostridium tetani
tetanus

Clostridium botulinum
food borne botulism
➢ ingestion of preformed botulinum toxin
 ✓ Clostridium bifermentans
➢ occurs when organisms contaminate wounds through trauma or surgery
➢ α-Toxin – a lecithinase (phospholipase C) produced by all strains of Clostridium
perfringens
✓ Allows deeper penetration by the organism
✓ Cause necrosis of tissue

Bacteremia
Clostridium perfringens
✓ most common caused
Clostridium septicum
2 toxins produced by C. tetani
✓ if present in bloodstream, marker organism for a malignancy in the GI tract
➢ tetanospasmin
✓ neurotoxin produced by Clostridium tetani
Clostridium difficile – associated diseases
➢ tetanolysin - hemolytic
Antibiotic – associated diarrhea and pseudomembranous colitis
tetanus
✓ occurs when spores enter the skin through skin puncture wounds

Myonecrosis or Gas gangrene

➢ Clostridia associated with Gas gangrene:
✓ Clostridium perfringens – most common
✓ Clostridium histolyticum
✓ Clostridium septicum
✓ Clostridium noyvi
Gram (+), non-spore-forming anaerobic Bacilli

Actinomycosis in the female genital tract

➢ associated with the use of long-standing intrauterine devices (IUDs)
Genera associated with Actinomycosis

Bacterial vaginosis (BV)

➢ arise because of a shift in the ecology of the endogenous microbiota of the
vagina
➢ shift in the vaginal biota occurs, resulting in the overgrowth of other
endogenous anaerobes of the vagina such as:
✓ Mobiluncus spp.
✓ Bacteroides spp.
✓ Prevotella spp.
✓ Anaerobic gram (+) cocci
Clinical Infections ✓ Gardnerella vaginalis
Actinomycosis
➢ a chronic, granulomatous, infectious disease characterized by development
Lactobacillus
of sinus tracts and fistulae
➢ comprise the largest portion of vaginal biota
➢ gram (+), highly pleomorphic bacilli
 ➢ appears in gram stain as a coccoid or spiral-shaped organism
➢ aerotolerant anaerobes
➢ normal microbiota of human:
✓ mouth
✓ GI tract
✓ female genital tract – mostly Lactobacillus acidophilus
Lactobacillus spp.
✓ help protect the host from urogenital infections
✓ produce lactic acid from glycogen which lowers
vaginal flora and suppresses the overgrowth of:
• Mobiluncus
• Prevotella Clinical infection
• Gardnerella vaginalis ➢ frequently found in mixed infections such as abscesses occurring beneath
mucosal surfaces
➢ treatment – penicillin plus an aminoglycoside
➢ associated with peritoneal infections following disruption of the GI lining
➢ Lemierre’s disease
Anaerobic Gram-Negative Bacilli
✓ a syndrome of thrombophlebitis of the jugular vein that occurs
➢ gram (-) anaerobic bacilli are: rarely following group A streptococcal pharyngitis
✓ non-sporeforming ✓ causative agent – Fusobacterium necrophorum
✓ members of the endogenous microbiota
✓ part of the microbiota of the oral cavity, GI and GU tracts Anaerobic cocci
➢ genera encountered in clinical specimen: ➢ only Veillonella spp. are implicated as pathogens in anaerobic gram negative
cocci
➢ most are weakly pathogenic
 ➢ Finegoldia magna – most pathogenic of the anaerobic gram positive cocci
Specimen Quality
➢ specimen collected must be from the actual site of infection and not just a
swab of a mucosal surface
➢ avoid prolong exposure to oxygen

Specimen Transport and Processing

➢ must be transported as quickly as possible to the laboratory under
anaerobic conditions
➢ avoid shocking the anaerobes by exposing them to oxygen or permitting
them to dry out
➢ should not be refrigerated
➢ amount of time they remain at room temperature should be minimized
ASPIRATES Blood
• collected by needle and syringe Prepare venipuncture site with a bactericidal agent:
• better than those collected by swab ➢ Tincture of iodine
• injected into an oxygen-free transport tube or vial containing a ➢ Iodophor
prereduced, anaerobically sterilized (PRAS)
➢ Chlorhexidine gluconate in combination with 70% isopropyl alcohol
• PRAS media are prepared by:
✓ boiling – to remove dissolved oxygen
Inoculation of appropriate plated and tubed media
✓ autoclaving – to sterilize the mixture
Anaerobes have special nutritional requirements for:
✓ replacing any air with an oxygen-free gas mixture
➢ vitamin K
Swabs
➢ hemin
➢ in general , are not appropriate for
➢ yeast extract
anaerobic culture
CDC blood agar – provides the best recovery for anaerobic organisms
➢ should be use only when:
✓ aspiration of material is not possible
✓ biopsy specimen is not available
➢ should always be transported under
anaerobic condition

Tissue
➢ Tissue specimen collected by biopsy or at autopsy from usually sterile sites
are acceptable specimens for anaerobic culture
➢ Do not tip the container when inserting swabs or small pieces of tissue into
an anaerobic transport container
• this would cause the oxygen-free gas mixture to be displaced by room air
 GLOVE BOX
➢ Fitted with airtight rubber
gloves
➢ Easier to use and less
susceptible to oxygen
contamination
Disadvantage:
✓ accommodate different
hand sizes into one pair
of gloves

GLOVELESS ANAEROBIC CHAMBER

➢ Airtight rubber sleeves that fit snugly
against the user’s bare forearms are
used in place of gloves

Anaerobic chambers contain:

Anaerobic chamber
➢ provides an oxygen-free environment for inoculating media and incubating
cultures
➢ Available as:
✓ sealed glove boxes
✓ gloveless chambers
Anaerobic jars Indication of the presence of Anaerobes in culture
GasPak jar

Gas Pak Jar

Steps must be performed and recorded to initiate presumptive

identification of the plates

Anaerobic bags or pouches

➢ alternative to an anaerobic chamber or jars
➢ oxygen removal system is activated to minimize exposure to oxygen
 Treatment of Anaerobe-Associated Diseases
Aerotolerance testing Four approaches to the management of anaerobic infections
➢ determines whether a microorganism isolated under anaerobic conditions is
a strict or facultative anaerobe
➢ suspected isolate is incubated in aerobic and anaerobic environments
➢ CHOC plate for CO2 incubator
➢ anaerobic blood agar incubated anaerobically