ANAEROBIC BACTERIA

CHARMAINE C MIRANDILLA
 AEROBES
FACULTATIVE ANAEROBES
CAPNOPHILIC
• preferentially use oxygen as an
• grow best when the concentration of
electron acceptor if it is available but
carbon dioxide is increased to 5% to
can grow well in the absence of
10% in a CO2
oxygen
• Example: Capnocytophaga spp
• Example: Enterobacteriaceae
MICROAEROPHILIC
OBLIGATE AEROBE
• require the oxygen concentration to
• require oxygen for metabolism, and
be reduced to 5% or less.
they can grow well in an ambient air
• Example: Campylobacter spp incubator
 ANAEROBES

• OBLIGATE ANAEROBES
• Killed in the presence of Oxygen
• AEROTOLERANT OR MODERATE ANAEROBES
• can survive some oxygen exposure but will not be able to
perform metabolic processes unless placed into an anaerobic
environment.
• Example: Bacteroides fragilis
 WHY SOME ORGANISM ARE ANAEROBES?

•Toxicity of the Oxygen

•Superoxide anion + Hydrogen Peroxide + Fe3+/Fe2+ 🡪
Hydroxyl Radical (OH-)
•Hydroxyl Radical
• the most potent biologic oxidant known.
• Damaging to the cells particularly protein and nucleic
acid
 WHY ORGANISM IS ANAEROBES?

•They do not contain the two enzyme that protects the

bacteria to superoxide anions and toxic derivatives
• Superoxide dismuthase
• Catalase
 WHERE ANAEROBES
FOUND?
• Endogenous microbiota
• Human and other animals

• Exogenous Anaerobes
• Soil, freshwater, saltwater
• Gram positive spore forming bacilli
like Clostridium
• Contamination
ANATOMIC SITE
VIRULENCE FACTORS
FREQUENTLY ISOLATED ANAEROBES
SPORE FORMING
 CLOSTRIDIUM SPP.
• Obligate anaerobes, catalase negative spore forming Gram positive bacilli
• Exogenous anaerobic infection
• Identify by where the spores is located
• Terminal: C. tetani
• Subterminal: C. sordellii

• Motile with peritrichous flagella except: C. perfringens

• Have swollen sporangia except: C. perfringens and C. bifermentans
• Single hemolytic reaction except: C. perfringens
• Carbohydrates fermentation except: C. tetani and C. bistolyticum
SPORES
 CLOSTRIDIUM PERFRINGENS
(GANGRENE BACILLUS)
• associated with two types of food poisoning
• type A, a relatively mild and self-limited GI
illness
• type C, a more serious but rarely seen
disease and can cause enteritis necroticans
• Virulence factor: alpha toxin and enterotoxin
• Microscopy: Boxcar shaped bacilli (subterminal
spores)
• Infections
• Gas gangrene (Myonecrosis)
• Enteritis necroticans
 GAS GANGRENE

• LECITHINASE (Phospholipase C) the

alpha toxin of C. perfrigens
• Clinical Manifestation: Bullae
(fluid-filled blisters), serous discharge,
discoloration, and tissue necrosis are
observed
• Other organism: C. histolyticum, C.
septicum, C. novyi, and C. bifermentans
MICROSCOPY

• “Box car shaped bscilli

with oval, subterminal
spores
 DIAGNOSTICS

• Culture
• Blood Agar plate: Dome
shaped and grayish
white with double zone
of hemolysis
• Litmus milk
• Stormy fermentation of
milk
DOUBLE ZONE
HEMOLYSIS
 BIOCHEMICAL TEST

• Lecithinase (+) using Egg

Yolk Agar (EYA)
• Naegler Test (+):
Lecithovitellin on EYA
• Reverse CAMP test (+):
Arrow head shaped towards
the test organism
NAEGLER TEST
REVERSE CAMP TEST
 CLOSTRIDIUM BOTULINUM
“CANNED FOODS BACILLUS”
• Found in soil and acquatic sediments
• Presence of subterminal spores
• Virulence factor: botulism toxin
• extremely potent neurotoxin; it takes
only a small amount to produce
paralysis and death
 BOTULISM

• FOOD BORNE
• cause by botulism A

• INFANT BOTULISM
• Ingesting organism thru honey or
breast feeding
 CLOSTRIDIUM TETANI

• Tack head bacillus

• Found in soil
• Virulence factor: Tetanospasmin (neurotoxin)
• acts on inhibitory neurons, preventing the release of
neurotransmitters
• trismus (lockjaw), risus sardonicus (distorted grin),
and difficulty breathing.

• Microscopically: Drumstick or tennis racket

appearance
 LAB DIAGNOSIS

•BLOOD AGAR PLATE: matte surface with

narrow zone of Beta hemolysis
•Gelatinase and indole (+)
•Lecithinase and Lipase (-)
 CLOSTRIDIUM DIFFICILE

•Most common cause of

antibiotic associated and
pseudomembranous colitis.
•Produce two toxin:
•Toxin A: Enterotoxin; Toxin
B: cytotoxin
LABORATORY DIAGNOSIS

• Ferment fructose
• Cycloserine cefoxitin fructose agar (CCFA)
• Exhibit yellow color and ground glass appearance

• Blood Agar plate

• “Horse stable odor”
• Non hemolytic
• Fluorescent chartreuse in UV light
GRAM-POSITIVE, NON–SPORE-FORMING
ANAEROBIC BACILLI
 ACTINOMYCES ISRAELII

• Microbiota of oral cavity

• Common cause of Actinomycosis
• a chronic, granulomatous, infectious
disease characterized by the
development of sinus tracts and fistulae,
which erupt to the surface and drain pus
that may contain so-called sulfur granules
• Seen in Maxillary region, neck, thorax, female
genital tract because of Intrauterine dvices
 LACTOBACILLUS SPP
• Comprises the largest portion of the •Lactobacillus spp
vagina
• Produce Lactic acid from glycogen • L. fermentum, L. vaginalis, L.
• Bacterial vaginosis salivarius, L. Plantarum
• a shift in the vaginal biota occurs, • Lactobacillus acidophilus
resulting in the overgrowth of other
endogenous anaerobes of the vagina
• Mobiluncus spp., Bacteroides spp.,
Prevotella spp., anaerobic
gram-positive cocci, and the aerobic
bacterium Gardnerella vaginalis
• Treatment: Penicillin + Aminoglycoside
 LABORATORY DIAGNOSIS

•Tomato juice agar

•Catalase, H2S, Esculin
Hydrolysis (-)
ANAEROBIC GRAM-NEGATIVE BACILLI
 BACTEROIDES FRAGILIS

• isolated anaerobes from blood cultures

• Beta lactamase producer
• Non Motile and saccharolytic
• Cause Intra abdominal abscesses
• Culture:
• Bacteroides Bile Esculin Agar: Gray color
and growth in 20% bile, blackening of the
agar
• Biochemical test
• Esculin hydrolysis positive
 PREVOTELLA, PORPHYROMONAS,
FUSOBACTERIUM
• Found in the oral cavity
• associated with mixed biota in diabetic foot ulcers and decubitus pressure sores.
• Fusobacterium necrophorum
• Caused Lemierre disease
 ANAEROBIC COCCI

• Gram Positive Anaerobic Cocci • Gram Negative Anaerobic Cocci

• Peptostreptococcus • Veilonella
• Anaerococcus • very small (0.3 to 0.5 µm in diameter) and
• Pepostreptococcus inhabit the oral cavity
• Finegoldia • Mixed biota absceses
• F. magna
• Parvimonas
• Peptoniphilus
SPECIMEN SELECTION AND COLLECTION
 SPECIMEN

• Aspirates
• injected into an oxygen-free transport tube
or vial, preferably one containing a
prereduced, anaerobically sterilized
(PRAS) transport medium
• PRAS - prepared by boiling (to remove
dissolved oxygen), autoclaving (to sterilize
the mixture), and replacing any air with an
oxygen-free gas mixture
 SPECIMEN

• Swabs
• not appropriate for anaerobic culture
• If biopsy is unavailable, transported
under anaerobic conditions
• swab should be placed into a tube
containing about 0.5 mL of sterile
thioglycollate broth
• Amies liquid that can maintain aerobic
and anaerobic organisms for up to 48
hours at room temperature can be used.
• Tissue
• placed in anaerobic transport tubes or
vials containing PRAS medium to keep
the tissue moist such a transport
medium.
• Larger tissues of more than 1 cm2 can
maintain a reduced atmosphere.
• following procedures should be performed on clinical specimens for the recovery of
anaerobic bacteria:
• Macroscopic examination of the specimen
• Preparation of Gram-stained smears for microscopic examination
• Inoculation of appropriately plated and tubed media, including media specifically
designed for culturing anaerobes
• Anaerobic incubation of inoculated media
 DIRECT EXAMINATION OF SPECIMEN
• Morphotypes might provide a presumptive identification of organisms and serve as a
guide to media selection
• The Gram stain often reveals the presence of leukocytes, indicating an inflammatory
response at the site of the infection.
• The Gram stain may also reveal the presence of squamous epithelial cells that would
suggest mucosal surface contamination during specimen collection.
• Gram stain can serve as a valuable quality control tool.
• Note: Chemically fixed (Methanol)
• Pale in Safranin: To enhance the red color of gram-negative anaerobes, the use of 0.1%
basic fuchsin as the counterstain or extending the counterstaining with safranin for 3 to 5
minutes is recommended
 APPROPRIATE CULTURE MEDIA

• Anaerobes have special nutritional requirements for vitamin K, hemin, and yeast
extract,
• CDC blood agar provides the best recovery
 ANAEROBIC CULTIVATION

• Use special culture media with thioglycolate and cysteine (reducing agents)
• Boiling is done to remove oxygen
• Use of Anaerobic chamber with vacuum pump and nitrogen to remove the oxygen
• Gaspak must contain Palladium Catalyst
• Pouches and plastic bags contain Calcium carbonate and Catalyst
 ANAEROBIC CHAMBER
• Nitrogen gas
• Filler for anaerobic atmosphere

• Palladium Pellets
• Used to remove residual Oxygen
• Combine Oxygen with Hydrogen to form Water

• Dessicant or silica gel

• Absorbed water

• Methylene blue or reazurin

• Oxygen reduction indicator
• Colorless in absence of O2
• Blue with Presence of O2
 GAS PAK JAR

• Needs 30 – 45 minutes to
obtain an anaerobic
environment