Week 5 - Anaerobic Bacteria

Obligate anaerobes vs Facultative anearobes

Obligate anaerobes lack the enzymes needed to break down toxic products (oxygen)

Where are anaerobes found?

Exogenous - Ingestion, Trauma
Endogenous - Normal flora, Opportunistic pathogens

Anaerobe

Do not require oxygen

Microaerophilic

Do not require oxygen but grow minimally under anaerobic conditions

facultative anaerobes

Can grow in oxygen or anaerobically

obligate anaerobes

Oxygen in toxic

Ambient air incubator requirements in a Micro lab

Contains 21% Oxygen and 0.03% Carbon dioxide

CO2 incubator requirements in a Micro lab

Contains 15% Oxygen and 5-10% Carbon dioxide

Microaerophilic incubator requirements in a Micro lab

Contains 5% Oxygen

Anaerobic incubator requirements in a Micro lab

Contains 0% Oxygen

Acceptable Clinical Samples for Anaerobe Detection

Aspirated pus
Tissue
Body fluids
Transtracheal aspirates
Direct aspirates
Contents of small intestine

Unacceptable Clinical Samples for Anaerobe Detection

Areas where anaerobes are part of normal flora
Oral or respiratory tract
Intestinal and Genitourinary tract
Superficial wounds

Types of infections caused by anaerobes

Head and Neck
GI tract
Genital tract
Skin and soft tissue

Microscopic Examination of Gram positive bacilli

Large, broad, blunted ends with few or rare leukocytes

Microscopic Examination of Gram negative bacilli or coccobacilli

Pale, irregular staining

Microscopic Examination of Bizarre shaped rods

Long, slender, needle-shaped

How to properly transport anaerobic samples to the lab

Must culture within one hour of collection OR specimen be transported in a oxygen-free vials or tubes

When transporting anaerobic samples in oxygen-free vials or tubes

They must be received within 2 hours of collection

During a direct examination of anaerobes

Foul odor is common

How to tell if a sample has been oxidized and should be rejected

If the Resazurin redox indicator is Pink = oxidized, reject
If the Methylene blue redox indicator is Blue = oxidized, reject

Media for primary isolation of anaerobes

Nonselective and Enriched - provides optimal anaerobic growht

Enriched media for primary isolation of anaerobes

Shaedler blood agar
Brucella blood agar

Selective media for primary isolation of anaerobes

KV blood agar (kanamycin-vancomycin)
PEA - PV (paramomycin-vancomycin)

Enriched broths for primary isolation of anaerobes

Enriched thioglycolate broth
Chopped meat glucose broth

All specimens submitted for anaerobic culture

MUST also be aerobically cultured

Minimum incubation time

is 48 hrs for primary culture

Proof of Anaerobic culture

Each colony picked and subcultured to TWO plates of the same media
Differential disks can be placed on the media
One plate is incubated in CO2 and the other anaerobically
ONLY colonies that grow better anaerobically should be worked up

Phenylethyl alcohol blood agar (selective)

Supports growth of Gram pos and neg anaerobes; inhibits facultative gram neg anaerobes

Paromycin-vancomycin blood agar (selective)

Supports Gram negative obligate anaerobes

Egg-yolk-neomycin agar (selective)

Isolation of Clostridium spp

Anaerobic Jars or pouches

Typically used in micro labs
Gas pack systems
Cold reduction
Palladium catalyst
Contains 10% Hydrogen, 5% Carbon dioxide, and 85% Nitrogen

Glove Box

used in anaerobic research
Gas-tight chamber (hood)
Glove portals to handle specimens
Entry lock to transfer materials
Hydrogen-containing atmosphere circulated through palladium catalyst to remove oxygen

Frequently Encountered Anaerobes

Clostridium spp

Bacteroides spp

Fusonbacterium spp

Prevotella and Porphyromonas

Veillonella spp

Peptococcaceae

What causes the most anaerobe infections?

Bacteroides

Spore Forming Gram Positive bacilli

Clostridium species: C. tetani, C. botulinum, C. perfringens, C. difficile

General Characteristics of Clostridium species

Gram pos rods

Spore forming

Some are easily decolorized

Large rods

Aerotolerant to strict anaerobes

Clostridium tetani

Causes disease Tetanus

Tetanus

Toxin associated disease caused by C. tetani neurotoxin (Tetanospasmin) and hemolytic toxin (Tetanolysin)

Spore allows it to survive for years in harsh environments

Spores enter the body through a wound

Subclinical infection

Incubation 1-57 days

Preventable with vaccine (every 10 yrs)

Pathogenesis of Tetanus

Toxins reach CNS through blood, lymph, tissues, or nerves
Neurotoxin blocks neurotransmitter release which causes uncontrollable muscle contraction

Signs and Symtptoms of Tetanus

Muscle cramping and twitching
Irritability
Tachycardia
Strange facial expressions
Leads to lockjaw
Spasms of jaw, neck, trunk and limbs

Clostridium botulinum

Causes Botulism

Botulinum toxin

Produced by C. botulinum and causees botulism

Most powerful toxin known

Acts at neuromuscular junctions

Blocks release of nerve impulses

Causes Flaccid paralysis

One of the most powerful toxins produced by a natural substance

Botulinum toxin

Foodborne botulism

Ingestion of preformed toxin
Foods commonly involved: Vegetables, home-canned food, condiments
Clinical signs: Nausea, vomiting, diarrhea, Neurologic symptoms appear 18-36 hrs, Descending paralysis beginning with ocular muscles, Death due to paralysis of respiratory muscles

Infant Botulism

Ingestion of spores that germinate in intestine
Toxin production in intestine
Foods commonly involved: Honey
Affects Infants < 1yr because they have no stable gut flora to inhibit spore germination
Symptoms: Subclinical to death

Wound Botulism

Contamination of traumatic wound
Chronic drug abuse
Toxin release
Symptoms similar to foodborne botulism

Clostridium perfringens

Commonly causes anaerobic cellulitis
Gas Gangrene (myonecrosis)
Causes foul odor and pus discharge

Anaerobic cellulitis

Caused by C. perfringens

Invasion of necrotic wound

Gas accumulation

Discoloration of underlying skin

Foul odor, brown, purulent discharge

Gas Gangrene (myonecrosis)

Caused by C. perfringens

Invasion of normal healthy muscles surrounding wound o Associated with very deep wounds o

Symptoms: Drowsiness, fever, tachycardia, Painful edematous wound

Sweet or foul-odored discharge

Gas present

Clostridium difficile

Associated with pseudomembranous enterocolitis Follows antimicrobial therapy - Iatrogenic
Found in hospitals or gut flora (3% of people)
Produce toxin A (causes diarrhea) and toxin B (causes cellular damage)

Clostridium species

Habitat: Intestinal flora and Environment

Transmission: Traumatic wounds, Ingestion, Endogenous

Prevention: Vaccine (DPT), Properly canning food, Avoid giving honey to children <1 year

Treatment: Antitoxin, Debridement of wound,, Supportive therapy, Antibiotic therapy

Laboratory Diagnosis of Clostridium species

Gram positive rods (may decolorize)
Sporogenous
Anaerobic atmosphere
Blood agar
Incubation for 48 hours

Spore placement is helpful for identification of Clostridium species

C. tetani - Terminal spores

C. sordelii - Subterminal spores

Presumptive identification of C. perfringens

Production of Lecithinase causing opaque zone around colonies on egg yolk agar

Presumptive identification of C. botulinum

Production of Lipase causing iridescent sheen on or around the colonies

Identifying features of C. perfringens

Lecithinase positive
Nagler reaction
Reverse CAMP test
Biochemical testing
Double-zoned hemolysis

Non-spore forming Gram-positive rods

Actinomyces
Bifidobacterium
Eubacterium
Eggerthella
Anaerobic lactobacillus
Proprionobacterium

Actinomyces

Cause of actinomycosis: more common in brain, orofacial region, pluropulminary region, and genitals

Chronic Granulomatous Infection

Development of sinus tracts and fistulae that erupt to the surface and drain pus containing sulfur granules

Most commonly isolated anaerobe

Bacteroides fragilis

Characteristics of Gram-Neg Anaerobic Bacilli

Pale staining
Gram-neg rods
Maybe be encapsulated
Anaerobic to aerotolerant

Clinical Manifestations of Gram-Negative Anaerobes

Oral and dental infections
Throat and Pharynx infections
Bite and Wound infections
Lung and Pleural space infections
Female Genital Tract infections
Intro-Abdominal infections
Brain Abscesses
Cardiovascular infections

Oral and Dental infections caused by Gram-Negative Anaerobes

Anaerobic and polymicrobic
Endogenous
Remain localized (can disseminate)
Commonly involved organisms: Fusobacterium, Prevotella (nonpigmented), Prevotella and Porphyromonas (pigmented)

Throat and Pharynx infection caused by Gram-Negative Anaerobes

Vincent's angina:

Ulcerative lesion of the pharynx

Fusobacterium necrophorum infection

Mixed with a treponeme organism

Bite and Wound infections caused by Gram-Negative Anaerobes

Can be polymicrobic (Aerobes and Anaerobes)

Common Anaerobes involved: Porphyromonas, Prevotella, Bacteroides, Fusobacterium

Lung and Pleural space infections caused by Gram-Negative Anaerobes

Aspiration pneumonia

Lung abscesses

Emphysema

Organisms involved: Prevotella melaninogenicus and Fusobacterium nucleatum

Female Genital Tract infections caused by Gram-Negative Anaerobes

Pelvic Inflammatory Disease

Chorioamniotic infections

Endometriosis

Bacterial vaginosis

Organism involved: Prevotella, Bacteroides fragilis, Porhyromonas (pigmented)

Intro-Abdominal infections caused by Gram-Negative Anaerobes

Caused by perforation of the bowel

Organism involved: Bacteroides fragilis, bacteroides spp

Brain Abscesses caused by Gram-Negative Anaerobes

Prevotella

Fusobacterium

Cardiovascular infections caused by Gram-Negative Anaerobes

Bacteremia

Endocarditis

Organisms involved: B. fragilis, Porphyromonas, Prevotella, Bacteroides, Fusobacterium

Natural habitat of Bacteroides

Normal flora of the colon
Can be normal flora of the female genital tract

Natural habitat of Prevotella and Porphyromonas

Human mouth
Urogenital tract
Intestinal tract

Natural habitat of Fusobacterium

Normal flora of the oral cavity, colon, and genital tract

This organism is highly resistant to anitbiotics (also bile-resistant)

Bacteroides fragilis

Anaerobic Gram-Positive Cocci

Peptostreptococcus
Finegoldia
Micromonas
Anaerobic Streptococcus

Peptostreptococcus

Normal flora of mouth
Can cause brain and mouth abscesses
Cause cardiovascular infections