Anaerobic Bacteria

Objectives
• Differentiate obligate anaerobes from facultative
organisms
• Describe how anaerobes as part of normal flora,
initiate and establish infection.
• To know the clinical infections associated with the
following organisms and how they are acquired and
manifest
 Clostridium spp.
 Bacteriodes spp
 Fusobacterium spp
 Actionmyces
 Gram-positive cocci
• To know the laboratory methods of performing
cultures and identifying anaerobes
• Acceptable and unacceptable specimens
• Antimicrobial therapy
 Anaerobic Bacteria

• Why anaerobic bacteria are important??

• Because they play a role in serious,

often fatal, infection and intoxications
 Anaerobic isolates based on Gram stain
morphology

Gram stain

Ve+ Ve -

Bacilli
Bacilli Cocci Cocci
.Peptococcus spp .Bacteriods spp
.Peptostreptococcus spp .Fusobacterium spp
Veillonella spp .Porphyromonas spp
Spore .Prevotella spp
.Actinomyces spp
No
.Bifdobacterium spp
yes .Eubacterium spp
.Lactobacillus spp
Clostridium .Propionibacterium spp
spp
 Relationship to Oxygen and carbon Dioxide

Microaerophile 5% oxygen
Obligate aerobes 15-20% oxygen

Facultative anaerobe grows equally well in the presence or absence of oxgyen

Aerotolerant anaerobe grows in reduced concentration of oxygen

Strict anaerobe 0% oxygen, most bacteriods many clostridium

 ANAEROBIC BACTERIA

A. SPORE-FORMING
Genus Clostridium
• C. perfringens
• C. tetani Are usually present as normal
flora of colon of animals
• C. botulinum
• C. difficile
B. NON-SPORING
• Bacteroides Are an important part of normal
• Fusobacteria flora of mouth, colon, skin &
lower genital tract
• Actinomyces
CLOSTRIDIUM PERFRINGENS
• Gram-positive
• Spore-forming bacilli with square
ends
• Seven (7) types (A-G) on the basis
of exotoxins
• Type A is the most important in
humans
• Spores are located in soil
• Vegetative cells are normal flora of
colon & vagina
 CLOSTRIDIUM PERFRINGENS

CULTURAL CHARACTERS
• Anaerobic
• Grow on blood agar producing two zones of
hemolysis
° Complete hemolysis due to O toxin
° Incomplete hemolysis due to alpha-toxin
• Ferments sugars with production of acids
& gas
° Much gas is also produced in vivo in necrotic
tissues hence the disease is called “Gas
Gangrene”
 CLOSTRIDIUM PERFRINGENS

ENZYMES
• Collagenase
• DNAase Responsible for invasiveness
• Protease
• Hyaluronidase

TOXINS
1. Alpha-toxin (Lecithinase)
• Hydrolyses lecithin & sphingomyelin
• Disrupts cell membranes of RBCs, WBCs & muscle cells
CLOSTRIDIUM PERFRINGENS

TOXINS
2. O Toxin
o Causes Lysis of RBCs
o Is toxic for muscles
3. Enterotoxin
• Causes food poisoning
 DISEASES By C. PERFRINGENS
1. Gas Gangrene (Myonecrosis)
Occurs in traumatic open wounds contaminated with soil

Open wounds Pathogenesis of

contaminated Gas Gangrene
with soil
Large amount
Spores of gas in
germinate subcutaneous
tissues
Reduced
Organisms
Oxygen due to
multiply
tissue necrosis

Alpha Toxin O Toxin Enzymes

cell death & Cause Break down
tissue necrosis muscle death sugars with gas
 DISEASES By C. PERFRINGENS

1. Gas Gangrene (Myonecrosis)

Prevention & Treatment
• Surgical toilet and excision of necrotic
tissues
• High doses of penicillin
• Place patient in hyperbaric oxygen
chamber to:
° Inhibit bacterial growth
° Neutralize toxicity of O toxin
• Amputation of limb if necessary to
prevent
spread of disease
 DISEASES By C. PERFRINGENS

2. Food Poisoning
• Spores from soil contaminate food
(especially meat dishes)
• Survive cooking, germinate & multiply in gut
• Produce enterotoxin - food poisoning
• Incubation Period : 8-24 hours
• Diarrhea & abdominal cramps
 LAB DIAGNOSIS

• Mainly Clinical
• Gram-stain of specimen
• Culture
 CLOSTRIDIUM TETANI

• Gram-positive bacilli
• Spore-forming with terminal spores
(drum-stick appearance)
• Spores remain viable in soil for
years
° Destroyed by autoclaving at 121oC for
15 min
Exotoxin produced
• A neurotoxin (tetanospasmin)
• Causes tetanus
 TETANUS

Mode of Transmission
• Contamination of wounds with soil
• Road-side accidents
• Nail penetration of foot
Can be contamination of :
• Umblicus & circumcision wounds - tetanus neonatorum
• Surgical wounds - Post-surgical tetanus
 Pathogenesis of Tetanus

Occurs in traumatic open wounds contaminated with soil

Wounds
contaminated Muscle spasm
with soil
• Organisms multiply
Spores locally only Continuous
germinate in • No tissue damage excitement of
dead tissues • No invasion of motor neurons
adjacent tissues

Neurotoxin Inhibits release

Reaches of inhibitory
retrograde mediators
to spinal cord
Binds to ganglioside
receptors of anterior
horn cells
 TETANUS : Clinical features

Incubation Period
• Few days to weeks, depends on:
° Site of wound
° Condition of wound
Clinical Picture
• Lock jaw (inability to open mouth)
• Intermittent spasm of muscles of
respiration & swallowing
• Generalized convulsions & opisthotonus
• Death from exhaustion & respiratory
failure
 TETANUS : Treatment

o Care of wound
o Tetanus immunoglobulins to neutralize unbound
toxin
Intramuscular & intrathecal
o Penicillin G to prevent further multiplication of
bacilli

Supportive measures:
• Care of respiration
• Sedation with benzodiazepines to prevent spasm
 TETANUS : Prevention

ACTIVE IMMUNIZATION OF:

1. Infants as part of triple vaccine (DPT)

2. Pregnant women to protect newborn

3. All road side accident wounded

patients
 CLOSTRIDIUM BOTULINUM

Morphology
• Gram-positive, motile bacilli
• Spore-forming with oval
subterminal spores
Habitat
• Spores are found in soil and
sediments of lakes world-wide
 CLOSTRIDIUM BOTULINUM

Exotoxin Production
• One of the most potent
exotoxins known
• Lethal dose may be just 1µg
• Heat-labile - destroyed at 100oC
easily
DISEASE BY C. BOTULINUM : BOTULISM

Source of Infection
• Consumption of contaminated home-
canned food (Do not grow in acidic
canned foods)
• The organisms multiply during storage
• Produce exotoxin without change of
color, odor or taste of food
 Pathogenesis of botulism
Occurs due to consumption of contaminated
home-canned food

Ingestion of
Flaccid
contaminated
paralysis
home-canned
Food

Pre-formed No excitement
exotoxin in food of motor neurons

Exotoxin & Enter blood

absorbed reach Inhibits release
from gut peripheral of acetylcholine
nerve synapses
 BOTULISM : Clinical Features

Incubation Period : 18-96 hours

• Vomiting
• Paralysis of muscles including ocular,
pharyngeal, laryngeal & respiratory
• High death rate due to paralysis of
respiratory muscles
 INFANT BOTULISM

• Organisms introduced with dietary

supplement
• Organisms multiply in colon with
absorption of small amount of toxin
• Infant suffers from constipation &
feeding problems
• One of the causes of sudden infant
death
 BOTULISM :
Treatment & Prevention

• Large doses of antitoxin

• Supportive measures
• Adequate sterilization of food during
canning
• Adequate heating before consumption
 CLOSTRIDIUM DIFFICILE

• Normal flora of adult colon (2-4%)

• Also present in environment
TOXINS PRODUCED
Toxin A (Enterotoxin)
• Causes mucosal damage with diarrhea
• Helps absorption of toxin B
Toxin B (Cytotoxin)
• Inhibits protein synthesis and leading to cell
death &
pseudomembrane formation.
The two toxins appear to act synergistically
by unknown mechanism
Clostridium difficile-associated Diarrhea
Predisposing Factors
• The risk is highest with clindamycin, the
cephalosporins, and ampicillin
• It is lowest with vancomycin, metronidazole, and
the aminoglycosides
• Increased risk with the use of proton pump
inhibitors
• Cancer chemotherapy even in the absence of
antibiotic therapy.
 CLOSTRIDIUM DIFFICILE

DISEASE: Antibiotic-Associated Pseudomembranous

Colitis

• Use of broad-spectrum antibiotics like clindamycin

alter the normal flora of colon

• Clostridium difficile will flourish

• Causes Pseudomembrane (yellow-white) formation with

poor absorption and diarrhea-like symptoms
 CLOSTRIDIUM DIFFICILE

DIAGNOSIS
• Endoscopy to see pseudomembrane
• Bacterial culture
• Detection of toxin B in stool by cell cultures
• ELISA to detect toxins A & B
• Detection of glutamate dehydrogenase, a metabolic
enzyme expressed at high levels by all strains of C.
difficile, both toxigenic and nontoxigenic.
TREATMENT
• Antibiotic withdrawal
• Oral vancomycin or metronidazole
Clostridium difficile
 GRAM-NEGATIVE ANAEROBES

BACTEROIDES
• Gram-negative small bacilli
• Grow on selective media anaerobically
• Normal flora of colon & oral cavity
• Present in enormous numbers in faeces
(1010/gm)
• Common in cervix & vaginal fornices
 GRAM-NEGATIVE ANAEROBES

BACTEROIDES : Diseases
Abscess formation
o After abdominal & gynecological surgery
o Brain abscess
o Dental caries
Treatment
o Metronidazole OR Cl
o Cefoxitin, cefotetan, and ceftizoxime have an
enhanced antianaerobic spectrum.
o Imipenem-cilastatin, meropenem, and
erzapenem are the most broadly active
 GRAM-NEGATIVE ANAEROBES
FUSOBACTERIA
• Gram-negative, spindle-shaped bacilli
• Normal flora of colon, oral cavity &
female genital tract
Diseases
• Chronic ulcerative lesions of gums
• Vincent,angina
Treatment
• Metronidazole

PREVOTELLA/PORPHYROMONAS
• Previously in genus Bacteroides
• Found in oral cavity & colon
• Cause periodontal infections
 Fusobacterium sp

Bacteroides fragilis
 LAB IDENTIFICATION OF
ANAEROBES
• Transport
• Without delay
• Use anaerobic transport media
 Anaerobic incubation
Anaerobic jars
• The most frequently used system
• Uses hydrogen and CO2 generator envelope

Anaerobic chambers
• A final gas mixture of 5% CO2, 10% hydrogen and
85% nitrogen is used
LAB IDENTIFICATION OF CLOSTRIDIA
Specimens
• Pus & infected tissue
• Blood, vomit & left-over food for detection of toxin in botulism
• Feces & food for culture in C. perfringens food poisoning
• Feces for C. difficile

Gram-staining
• Gram-positive, sporing bacilli

Culture anaerobically on:

• Anaerobic blood agar (Brucella BA)
• Cooked meat medium

Gas liquid Chromatography (GLC)

Identifying anaerobes through metabolic end product analysis
 Anaerobic, Gram-positive cocci
Peptostreptococcus sp. Propionibacterium acnes
.

Bifidobacterium bifidum Actinomyces sp

 LAB IDENTIFICATION OF CLOSTRIDIA
Biochemical Reactions by:

Culture on Egg-Yolk Agar (EYA) is used

to determine the activities of the
following enzymes

 Lecithinase
 Lipase
 Proteolyticinase activity
 Bactroides

• Specimens
o Pus (foul smelling), blood
• Gram-staining
o Gram-negative bacilli
• Culture
o Bacteriod Bile Eusculin (BBE)
o Kanamycin-vancomycin-lacked blood (KVLB)
o Anaerobic incubation for 48-96 hours
 Clinical learning assessment

A 45 years male farmer was admitted to the

hospital for complications resulting from a tractor
injury to his left leg. The patient’s leg was painful,
bluish, and edematous.. A radiograph revealed
pockets of gas in the tissue. A complete blood
count revealed a marked increase in neutrophils and
a total WBC count of 33,000/mm3 . A Gram stain
of the wound specimen revealed numerous
rectangularly-shaped Gram –positive bacilli,
moderate Gram –positive cocci in cluster, no spores,
and very few leukocytes. The attending physician
concluded that the Gram stain results were
indicative of contamination rather than infection.
Fortunately for the patient, the infectious disease
specialist thought differently
 Questions

• What disease did the infectious disease

specialist suspect (give two names for the
disease)
• What genus of bacterium do you suspect is
causing the disease?
• What species do suspect?
• What is the clinical significance of the Gram-
positive cocci?
• If this an infectious disease, why were so few
leukocytes observed on the Gram stain?
 Case
Ms. A., an 18 years-old student, was admitted to hospital with diffuse
abdominal pain, diarrhea, and nausea without vomiting. Her pain was
localized to the right side of the abdomen. Physical examination reveals
tenderness in the right lower quadrant of her abdomen. She received a
first-generation cephalosporin antibiotic (cefazolin) and was taken to
the operating room, where her ruptured appendix was removed.
Cultures of the peritoneal in the area of the appendix grew a mixture
of bacteria , typical of those found in stool. On the second after the
operation, her temperature spiked to 38.5 0C. Blood cultures obtained
preoperatively grew E. coli.
Ms. A., improved postoperatively and complained a 7-day course of
cephalosporin. Because she had no further symptoms and her blood
cultures were negative, the antibiotic was stopped. However, 36 hours
later her temperature was 38.5 0C and she felt diffuse pain over the
site of appendectomy. A CAT scan of her abdomen revealed a
retroperitoneal abscess. Cultures obtained after drainage of the
abscess grew B. fragilis. She was again treated with antibiotics. This
time metronidazole was used for 14 days, and Ms. A had recovered
 The case raises several questions

• How did the two episodes of Ms. A’s disease with

regards to pathogenesis and the kind of bacteria
involved?

• Why did B. fragilis survive the first course of

antibiotic treatment?

• Was Ms. A treated properly? What could have been

done to lessen the likelihood of abscess formation?

• How does B. fragilis facilitate intra-abdominal

abscess formation?