– LECTURE NOTES

CHAPTER 22
CLINICALLY IMPORTANT ANAEROBIC
BACTERIA

Oxygen Toxicity
There are 3 enzymes that some bacteria possess to break down these
oxygen products to non-toxic forms:
Superoxide dismutase (SOD) – breaks down the superoxide
radicals
- + Superoxide dismutase O + HO
2O + 2H 2 2 2
2

Catalase – hydrogen peroxide formed in the reaction is rapidly

destroyed by catalase; present in aerobes and aerotolerant aerobes
Peroxidase – also breaks down hydrogen peroxide Clostridium difficile
One of the most frequently encountered pathogenic anaerobes
Cause of antibiotic-associated diarrhea
Diagnostic test is usually based on the detection of the toxins produced
by the bacterium rather than by culturing the organism from feces

Anaerobes are found only in specific ecological niches

Found in soil, in freshwater and saltwater sediments, and as Anaerobes outnumber
components of endogenous microbiota of humans and other animals aerobes on mucosal
Exogenous anaerobes surfaces, such as the
Anaerobes that exist outside the bodies of animals linings of the oral
Infections these anaerobes cause are termed exogenous infections cavity, gastrointestinal
Usually caused by gram-positive, spore-forming bacilli belonging to the (GI) tract, and
genus Clostridium genitourinary (GU)
Clostridia initiate infection when spores are ingested by way of tract
contaminated food or gain access to the body through open wounds These heavily
contaminated with soil colonized surfaces are
Endogenous anaerobes the usual portals of
Anaerobes that exist inside the bodies of animals (endogenous entry into the tissues
microbiota) and bloodstream for
Source of endogenous infections endogenous
Anaerobes most frequently isolated from infectious processes in anaerobes
humans are those of endogenous origin Under ordinary
Contribute to an infectious disease in any anatomic site of the body if circumstances,
suitable conditions exist for colonization and penetration of the bacteria microorganisms that
are members of the
microbiota do not cause disease and many actually can be beneficial

CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE

BATERIA
1
 – LECTURE NOTES
However, when some of these organisms gain access to usually sterile
body sites, such as the bloodstream, brain, and lungs, they can cause
serious or even fatal infections
Factors that commonly predispose the human body 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
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
General Characteristics:
Taxonomically, anaerobic bacteria encountered in human clinical
specimens may be divided into gram-negative and gram-positive
genera
Gram-positive anaerobes can be further divided between:
Spore formers
Non-spore formers
The presence or absence of spores, coupled with the Gram stain
reaction and cellular morphology – can be helpful in making a
CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC
BATERIA
2
presumptive ID of anaerobic bacteria and determining the appropriate
identification tests to be performed
All spore-forming anaerobic bacilli are classified in the genus
Clostridium and are collectively termed clostridia
Although all clostridia are capable of producing spores, some species
do so readily, whereas others require extremely harsh conditions
Spores are often not observed in Gram-stained smears of clinical
specimens containing clostridia or in smears of colonies from an agar
plate unless the culture has been incubated for many days
Sporulation is induced by:
Heat
Alcohol shock
Clostridia can be grouped according to the location of the endospore:
Terminal
Spore is located at the end of the bacterial cell
Typically cause swelling of the cell
Subterminal
Spore is found at a location other than the end of
the cell
Central
Spore located in the center of the cell
Clostridium spp.
Gram-positive motile bacillus (by means of peritrichous flagella)
Anaerobic but some species are aerotolerant (peroxidase positive)
All are motile except C. innocuum, C. perfringens and C. ramosum
All have swollen sporangium except C. perfringens and C.
bifermentans
All are non-encapsulated except C. perfringens
Group 1: Gas gangrene group
C. perfringens
C. ramosum
COMPILED BY: CRISTALEEN BANATE
 – LECTURE NOTES
C. perfringens, however, is the most common cause
Under favorable conditions, the organisms are able to grow,
multiply, and release potent exotoxins
C. septicum Exotoxins, such as α-toxin produced by C. perfringens, cause
C. sordellei necrosis of the tissue and allow deeper penetration by the
C. histolyticum organisms
C. novyi Bacteremia
C. fallax Many of the clostridia have been recovered from blood cultures,
C. sporogenes but C. perfringens is the most common.
Group 2: Clostridium tetani When C. septicum is present in the bloodstream, it is often a
Group 3: Clostridium botulinum marker organism for a malignancy in the GI tract
Group 4: Clostridium difficile C. bifermentans and C. tertium have also been isolated from
Group 5: Miscellaneous clostridia blood cultures from patients with serious underlying disease
Biochemical Reactions
Clostridium perfringens Stormy fermentation of litmus milk
Formerly called C. welchii Lactose in the medium is fermented to
Non-motile, spore-former, obligate anaerobe organic acid and gas
Microscopically, appear as large, short, fat cells – “boxcar cells” Acid changes the color of the indicator from
Produces target hemolysis – double zone of hemolysis on BAP blue to red
(narrow inner β zone and wider outer zone of partial β-hemolysis) Acid coagulates casein to form acid clot
Due to vigorous gas production, the clot gets
disrupted
Reverse CAMP
Presumptive test for the identification of
C. perfringens
Hemolysis diminishes when β-lysin from
Staphylococcus aureus is used
However, when group B streptococci (GBS)
is used, positive reaction is production of
arrowhead type of enhanced hemolysis
Nagler’s Reaction
Clostridium perfringens produces
Virulence factors alpha toxin, which is a
Alpha toxin (lecithinase, phospholipase C) – hemolysin, anti-WBC, and lecithinase
platelet If the organism is grown on a
Beta toxin (Dambrand pigbel) – produced in Type C food poisoning – medium containing egg yolk
key factor in necrotizing colitis or enterocolitis necroticans (lecithin), enzyme
Lambda toxin – weak proteolytic toxin activity can be detected as
Theta toxin – responsible for β-hemolysis on BAP opacity
Kappa toxin – collagenase, involve in breakdown of gelatin around the line of growth (right)
Mu toxin – hyaluronidase If anti-alpha toxin is applied to
Nu toxin – RNase the surface
Epsilon and iota toxins – lead to increased capillary permeability of the plate before inoculation of the organism, the action of the toxin is
Enterotoxin – similar to V. cholerae enterotoxin inhibited (left)
Neuraminidase This test can be used to confirm the
Diseases identity of a clostridial isolate
Type A food poisoning
Mild and self-limited GI disease
C. perfringens lacks the ability to produce a number of essential
amino acids, so meats and gravies are commonly implicated in
outbreaks
Foods are often heated, which kills vegetative bacteria but allows
the spore-forming clostridia to surviveImproperly stored food
allows germination of the spores and growth of vegetative
bacteria
Caused by a C. perfringens enterotoxin linked to sporulation
Type C food poisoning
Enteritis necroticans
More serious but rarely seen disease Clostridium botulinum
Associated with strains that produce β-toxin and, less commonly, Canned-good bacillus
α-toxin Morphologically similar to Clostridium tetani
Myonecrosis Spore is oval and terminal or subterminal
Gas gangrene Resistant to heat (100oC) for 3 to 5 hours but diminished at acid pH or
Usually occurs when organisms contaminate wounds, through high salt concentration
trauma or surgery Produces botulinum toxin, which acts by blocking the release of
C. perfringens, C. histolyticum, C. septicum, C. novyi, and C. acetylcholine at synapses and neuromuscular junction resulting to
bifermentans have all been associated flaccid paralysis

CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE

BATERIA
3
 – LECTURE NOTES

Three types of botulinum

Food borne – from ingested spiced, smoked, or canned food
Wound botulism
Infant botulism – ingestion of honey with C. botulinum spores
Linked to sudden infant death syndrome (SIDS)
Eight antigenic varieties of botulinum toxin (A-H)
Types A, B, and E – causes human botulism
Type C – causes limber neck in fowl
Type D – causes botulism in cattle
Toxins are destroyed by heating for 20 minutes at 100 oC
Clostridium tetani
“Tack-head” bacillus
Spore is terminally located – called round-headed pin, drumstick, or
tennis racquet
Grow abundantly in egg yolk medium and chopped meat medium
overlaid with oil
Colonies are swarming due to pseudopodia with burnt-flesh color
(meat medium turns black)
Possesses peritrichous flagella
Exhibits Wasserman-Takaki phenomenon
Two toxins produced by Clostridium tetani
Tetanolysin – hemolytic
Tetanospasmin – neurotoxic which acts like strychnine, affecting
neuron activity
Clinical Manifestations of Tetanus
Trismus/lock jaw – spasm of masseter muscles
Risus sardonicus – “Devil’s grin” – spasm of facial muscles
Opisthotonos – arching of the back

Clostridium difficile
Most common but not the sole cause of antibiotic-associated diarrhea
and pseudomembranous colitis The group can be divided into two phyla, the Actinobacteria and the
Also causes gastroenteritis Firmicutes
Normal flora of the colon Important clinical genera of the Actinobacteria include Actinomyces,
Gives a characteristic “horse manure odor” Bifidobacterium, Eggerthella, Mobiluncus, and Propionibacterium
Bacilli with large, oval, subterminal spores Firmicutes include many genera, but Lactobacillus is the only member
Cycloserine Cefoxitin Fructose Agar encountered on a routine basis in the clinical microbiology laboratory
Colonies give a yellow fluorescence in UV light All are found as part of the endogenous microbiota of humans and can
Yellow ground-glass appearance be considered opportunistic pathogens
Clostridium ramosum Microscopic morphology varies, ranging from very short rods to long
Second most common cause of gas gangrene branching filaments
Often appear gram-negative on smears made from culture Actinobacteria can be divided into two major groups:
Spores are seen using phase contrast microscopy Produce propionic acid and acetic acid
Propionibacterium – catalase positive
Biochemical Tests for Differentiation of Clostridium Species Arachnia – catalase negative
C. perfringens C. botulinum C. tetani C. difficile
No production of propionic acid
Reverse CAMP + - - -
Bifidobacterium – produces acetic acid and lactic acid
Motility - + + +
Actinomyces – produces acetic acid and succinic acid
Lecithinase + - - -
Eubacterium – produces acetic acid and butyric acid
Lipase - + - -
Lactose + - - - Propionibacterium spp.
fermentation Gram-positive, pleomorphic rods with a diphtheroid appearance
Glucose + + - + Propionibacterium acnes
fermentation Common member of the skin microbiota
CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE
BATERIA
4
 – LECTURE NOTES

Actinomyces spp.
Straight to slightly curved
bacilli of varying lengths,
Frequently isolated from blood culture bottles as a from short rods to long
contaminant filaments
Also the most common contaminant of aspirates and CSF Short rods may have
Can cause subacute bacterial endocarditis, ventricular shunt
clubbed ends and may be
infection, and bacteremia, and thus is not always a contaminant seen in diphtheroid
Gram-positive anaerobic diphtheroid that is both catalase and arrangements, short chains,
spot indole positive can be presumptively identified as
or small clusters
Propionibacterium acnes Longer rods and filaments
may be straight or wavy and
branched
Although the Actinomyces
are gram-positive, irregular staining can produce a beaded or banded
appearance, much like that seen with Nocardia spp.
The typical branching, filamentous, Gram-stained appearance of an
Actinomyces sp. is
referred to as
Actinomyces-like
Young cultures have
spider-like and wooly
colonies
Older cultures have
Propionibacterium propionicus raspberry or molar
Can cause actinomycosis tooth colonies
Varies considerably in size and shape, ranging from coccoid and short Depending on the
diphtheroid rods to long, branched filaments species, colonies may be red, pink, tan, yellow, white, or grayish
Individual cells may be of uneven diameter and have distended or Actinomyces israelii – molar tooth colonies on solid medium; fluff ball
clubbed ends colonies at the bottom in thioglycollate
………………………………………………………………………………………. Actinomyces naeslundii – fluff ball colonies with sulfur granules
As the genus name implies, propionic acid is a major metabolic end Actinomyces viscosus – pink flocculent colonies in thioglycollate
product of Propionibacterium spp. Actinomyces odontilyticus – red-colored colonies on BAP
Bifidobacterium Cause actinomycosis
Gram-positive coccobacillus or long branching rod Other etiologic agents of actinomycosis include:
Natural part of the bacterial flora in the human body Arachnia
Has a symbiotic bacteria-host relationship with humans Bacteroides
Bifidobacterium
Bifidobacterium longum
Promotes good digestion, boosts the immune system, and Eubacterium
produces lactic acid and acetic acid that controls intestinal pH Propionibacterium
Aggregatibacter actinomycetemcomitans
Also inhibit the growth of Candida albicans, E. coli, and other
bacteria that have more pathogenic qualities Other Anaerobic, Non-Spore-Forming, Gram-Positive Bacilli
May appear as star-like aggregates, V arrangement, or palisade Eubacterium
arrangement Mobiluncus
Ends are bent, pointed, club-shaped, spatulated or bifurcated (forked) Gram-positive cell wall but stains gram-negative microscopically
Colonies of Bifidobacterium spp. are convex, entire, and cream to Causes bacterial vaginosis
white, smooth, glistening, and soft Arachnia – similar morphology in Actinomyces
Differences between Propionibacterium acnes and Bifidobacterium eriksonii
Propionibacterium acnes Bifidobacterium eriksonii
Normal flora Skin and nose Mouth and intestine
Catalase + -
Gelatinase + -
Nitrate reduction + -
Indole + -
Esculin hydrolysis - +

Lactobacillus spp.
Colony morphology of the lactobacilli varies greatly, with some species
appearing as pinpoint α-hemolytic colonies on SBA
Others have been described as medium in size, with a rough
appearance and gray color
Lactobacilli are catalase-negative and, unless a Gram stain is
performed, differentiation from Streptococcus spp. viridans group is
difficult

CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE

BATERIA
5
 – LECTURE NOTES

Bacteroides fragilis Group and other Bile-

Resistant Bacteroides spp.
Gram-negative anaerobic bacilli are all non-spore-forming and are often Include members of the Bacteroides fragilis group and the less
found as members of the endogenous microbiota frequently encountered Bacteroides splanchnicus and Bacteroides
They can be found as part of the microbiota of the oral cavity and GI eggerthii
and GU tracts Members of the Bacteroides fragilis group, which contains more than
Genera most commonly encountered in clinical specimens include the 20 species, are especially pathogenic
Bacteroides fragilis group, Porphyromonas, Prevotella, and Gram-stained smears of Bacteroides spp. colonies reveal gram-
Fusobacterium negative coccobacilli or bacilli, but cells in broth cultures are frequently
Non-motile or peritrichous flagella pleomorphic
Produce butyric acid (without isobutyric and isovaleric acid) – Bacteroides fragilis
Fusobacterium Most common species of anaerobic bacteria isolated from infectious
Produce lactic acid – Leptotrichia processes of soft tissue and anaerobic bacteremia
Produce acetic acid and H2S; reduce sulfate – Desulfomonas Responsible for more than 60% of the infections caused by the
None of the above characteristics – Bacteroides Bacteroides fragilis group
Polar flagella
Bacteroides thetaiotaomicron
Fermentative
Produce butyric acid – Butyrilvibrio Next most frequently encountered member of the Bacteroides fragilis
Produce succinic acid – Succinivibrio group
 Spiral-shaped cells – Succinimonas Often exhibits the highest degree of antimicrobial resistance
 Ovoid cells – Anaerovibrio
Produce propionic and acetic acids – Anaerovibrio
Non-fermentative, produce succinic acid from fumarate –
Wolinella
Tufts of flagella on concave side of curved cells
Fermentative – Selenomonas
Non-fermentative – Mobiluncus
Polar Tufts of flagella – Anaerobiospirillum
Clinical Infections
Frequently found in mixed infections such as abscesses occurring
beneath mucosal surfaces
As predominant members of the GI biota, the organisms are often Colonies of the Bacteroides
associated with peritoneal infections following disruption of the GI lining fragilis group on BBE agar plate
Members of the B. fragilis group are the most commonly isolated are gray and a minimum of 1 mm
anaerobes from blood cultures in diameter at 24 hours
Brain abscesses – Prevotella, Porphyromonas, and Fusobacterium The originally pale yellow
Diabetic foot ulcers and decubitus ulcer pressure sores medium turns brown to black in
Lemierre’s disease the area around the colonies
Syndrome of thrombophlebitis of the jugular vein that occurs rarely Good growth is the result of bile
following group A streptococcal pharyngitis tolerance, and darkening of the
Caused by Fusobacterium necrophorum medium is caused by esculin
The streptococcal infection produces a peritonsillar abscess containing hydrolysis
a number of bacterial species A dark precipitate (stippling) in
Abscess aids F. necrophorum in penetrating the tissue to reach the the medium around the areas of
jugular veins heavy growth is suggestive of the species Bacteroides fragilis
Some strains of Bacteroides ovatus also cause stippling
Bacteroides spp. Caution must be taken when interpreting results on BBE agar
Family Bacteroidaceae Bacteroides vulgatus, a member of the Bacteroides fragilis group, often
Genus Bacteroides was divided previously into bile-resistant (or bile- does not hydrolyze esculin and therefore may not produce a brown to
tolerant) and bile-sensitive species black discoloration of the medium
However, most of the bile-sensitive species were transferred to the Depending on the commercial source, age, and storage conditions of
genera Prevotella and Porphyromonas the medium, other organisms, such as Fusobacterium mortiferum,
Bile-resistant species grow in the presence of 20% bile, so colonies Klebsiella pneumoniae, Enterococcus spp., and some yeasts, may
will be present on BBE agar, and will exhibit robust growth on KVLB also grow on BBE agar
agar Their colony size (which tends to be smaller), Gram stain morphology,
Non-spore-forming rods, straight, curved, or helical in shape and aerotolerance will aid in the recognition of these other organisms
Motile or non-motile Members are resistant to colistin, kanamycin, and vancomycin
Metabolize carbohydrates, peptones, or metabolic intermediates, and special potency disks
most species produce organic acids Indole-positive members – Bacteroides thetaiotaomicron, Bacteroides
Indigenous flora of mucosal surfaces uniformis, and Bacteroides ovatus
Resistant to penicillin 2-unit disk (except Bacteroides ureolyticus) Indole-negative members – Bacteroides fragilis, Bacteroides vulgatus,
– shared characteristic with Prevotella, but differentiates Bacteroides Bacteroides caccae, and Bacteroides distasonis
from Porphyromonas and Fusobacterium Catalase positive members – Bacteroides distasonis, Bacteroides
fragilis, Bacteroides ovatus, and Bacteroides thetaiotaomicron

CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE

BATERIA
6
 – LECTURE NOTES
Together with B. ureolyticus, it produces pitting of the agar
medium
Bile-Sensitive Non-Pigmented Bacteroides
B. buccae, B. oris, B. disiens
Part of the normal flora on the oral cavity, GI tract, and GU tract
Involved in infections of the lower respiratory and female genital tracts
Fusobacterium spp.
F. nucleatum
F. russi
F. naveforme
F. gonidiaformans
F. mortiferum
F. varium
F. necrophorum
Often described
microscopically as long,
thin, and tapered rods, a
Bacteroides fragilis morphology
characteristically
Pale staining, gram-negative bacillus with rounded ends, with some referred to as fusiform
pleomorphism and irregularity of staining Important to note that
Possesses a capsule that can be demonstrated by ruthenium red
only Fusobacterium
staining, India ink staining, and electron microscopy nucleatum has cells that
Differentiated from other bile-resistant Bacteroides by its ability to are consistently fusiform
ferment sucrose in shape, but clinically
Resistant to most antimicrobial agents encountered bacteria
Most abundant organism in the intestinal tract
that are fusiform in shape
Virulence factors – capsule, DNase, neuraminidase, hyaluronidase are not necessarily
(fibrinolysin), heparinase, gelatinase Fusobacterium
Produces colonies with 1-3 mm diameter, smooth, white to gray, non-
Other fusobacteria, such
hemolytic, and translucent on BAP as Fusobacterium
Culture mortiferum, appear
Bacteroides Bile Esculin Agar – colonies are brown due to esculin
pleomorphic and exhibit
hydrolysis globular forms, swellings,
Kanamycin-Vancomycin-Laked Blood agar and other bizarre shapes
Laboratory Identification Tests Organisms other than
Direct examination of smear fusobacteria can also have fusiform-shaped cells, such as
Fermentation of sucrose
Campylobacter gracilis, Bacteroides forsythus, and microaerophilic
Direct gas-liquid chromatography Capnocytophaga
Immunoperoxidase method Resistant to vancomycin but susceptible to colistin and kanamycin
DNA probe
with the special potency disks
Bacteroides ureolyticus Group However, all except Fusobacterium nucleatum will grow on the KVLB
agar because the level of kanamycin is reduced from that of the special
Consists of bile-sensitive and bile-tolerant non-pigmented
potency disk
organisms
Susceptible to penicillin 2-unit
Pitting anaerobes of the Bacteroides ureolyticus group:
disks – shared characteristic
Bacteroides ureolyticus
with Porphyromonas, but
Campylobacter gracilis
differentiates Fusobacterium from
Campylobacter curvus
Bacteroides and Prevotella
Campylobacter rectus
With the exception of
Sutterella wadsworthensis
Fusobacterium mortiferum, the
Recent reports have shown that these organisms are actually
fusobacteria are indole-positive,
microaerophiles rather than obligate anaerobes
and all will fluoresce chartreuse
One useful identifying characteristic of this group is the colony
under long-wave UV light
appearance – many of the organisms have colonies that appear to pit
In addition, Fusobacterium
the agar (not all strains pit)
necrophorum is positive for
Growth in broth is enhanced by the addition of formate or fumarate,
lipase when grown on EYA
a characteristic unique to this group
Indigenous flora in the URT
All members are nitrate positive
May also be found in the GI and GU tract
B. ureolyticus is urease positive, spot oxidase positive, and bile-
May be implicated in infection throughout the body, but with some
sensitive
tendency to invade the LRT, head and neck, and CNS
Sutterella spp. are bile-tolerant, and the campylobacters are variable
in bile sensitivity Fusobacterium nucleatum
(REFER TO TABLE 22-17) Appears in pairs connected end-to-end
Campylobacter gracilis Produces convex, glistening, α-hemolytic colonies with flecked internal
Morphologically similar to Bacteroides fragilis structure or “breadcrumb-like colonies”
Encapsulated
CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE
BATERIA
7
 – LECTURE NOTES
Produces L-forms after exposure to cell wall-active antimicrobial agents
Fusobacterium mortiferum
Exhibits bizarre pleomorphism with spherical swellings along
irregularly-stained filaments and free round bodies
Colonies on BAP are non-hemolytic but often show greening of the
agar around colonies after exposure to air
Colonies vary from flat to convex with opaque centers and translucent
irregular margins – fried egg colonies of F. mortiferum and F.
necrophorum
In broth, growth occurs in balls
(REFER TO TABLE 22-17)
Leptotrichia buccalis …………………………………………………………………………………………
Prevotella loescheii produces lipase
Long, plump, straight or slightly curved gram-negative bacillus that
grows end-to-end in pairs or in chains …………………………………………………………………………………………
Ends that are joined are flattened, whereas the other ends are often
pointed
Produces 2-3 mm diameter colonies with a characteristic convoluted
appearance
Part of the normal flora of the oral cavity and has been found, on
occasion, in intestines and vagina
Recovered from dental infections, osteomyelitis of the mandible, bite
wound infections, infections of the head and neck, and from blood of a
patient with lung abscess
Bilophila wadsworthia
Bile resistant anaerobe that will grow on BBE agar with a characteristic
fish-eye appearance (clear colonies with black centers)
Also grow on KVLB agar
Strongly catalase-positive and nitrate-positive
(REFER TO TABLE 22-17)
Prevotella spp.
Bile-sensitive pigmented Bacteroides spp. are now classified under
Prevotella or Porphyromonas
Normal flora of the oropharynx and upper respiratory tract, GI, and GU
tracts
Not recovered in infections as frequently as Bacteroides fragilis
Causes aspiration pneumonia and lung abscess
Grow on KVLB agar but not on BBE agar
Resistant to vancomycin and kanamycin but are variable in their
susceptibility to the colistin special potency disk
Some species produce protoporphyrin, a dark pigment that causes
their colonies to become brown to black with age
Colony pigmentation may take 2 to 3 weeks of incubation before it
becomes evident on routine Brucella blood agar (BRU/BA) plates, but it
appears sooner on KVLB agar
Non-pigmented colonies on KVLB agar or BRU/BA should be subjected
to long-wave UV light, such as a Wood’s lamp, to detect the typical
brick red fluorescence of pigment-producing Prevotella spp. that
appears before the brown pigment
Some species of pigmented Prevotella fluoresce colors other than brick
red (e.g., brilliant red, yellow, orange, pink-orange), and some do not
fluoresce at all
Only brick red fluorescence allows presumptive identification of the
pigmented Prevotella group
CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC
BATERIA
8
In Gram stain, Prevotella spp. appear as gram-negative coccobacilli or
bacilli, very similar to Bacteroides spp.
Prevotella melaninogenica (formerly called Bacteroides
melaninogenicus)
Pale-staining, gram-negative coccobacillus
Produces black pigment (melanin), hence a black-colored
colony on BAP
Laked rabbit’s BAP allows the earliest and most reliable pigment
production
Young colonies show a brick red fluorescence when viewed in
the UV light
Prevotella intermedia
Previously named Bacteroides intermedius
Lipase and spot indole positive
Prevotella bivia
Previously called Bacteroides bivius
An endotoxin producer, in contrast to B. fragilis
Produces phospholipase A, collagenase, hyaluronidase, and
fibrinolysin
Porphyromonas spp.
Produce brick red fluorescence under UV light, similar to Prevotella,
but some species do not fluoresce.
Because most Porphyromonas strains are susceptible to vancomycin,
they will not grow on KVLB agar
However, in contrast to Prevotella, they are resistant to colistin with
the special potency disks
Most Porphyromonas spp. are spot indole positive
Porphyromonas asaccharolyticus (formerly called Bacteroides
asaccharolyticus) is encapsulated
Porphyromonas gingivalis is formerly known as Bacteroides gingivalis
Most of the gram-positive anaerobic cocci were classified previously in
the genus Peptostreptococcus, with the exception of Peptococcus
niger, an infrequent isolate
However, the genus Peptostreptococcus has been reclassified recently
into at least five different genera:
Peptostreptococcus
Anaerococcus
Finegoldia
Parvimonas
Peptoniphilus
Although several genera of gram-negative anaerobic cocci are found in
the endogenous microbiota, only Veillonella spp. are implicated as
pathogens
Veillonella are very small (0.3 to 0.5 μm in diameter) and inhabit the
oral cavity
They are usually seen in mixed biota abscesses
Clinical Infections
Anaerobic cocci are weakly pathogenic but can be isolated from a wide
variety of infections, including brain abscess, meningitis, aspiration
pneumonia, lung abscess, and gingivitis and other periodontal diseases
Usually associated with polymicrobial infections but can occasionally be
recovered from blood cultures and can cause infections following
orthopedic surgery
Finegoldia magna is the most pathogenic of the anaerobic cocci and
the one most often isolated in pure culture
Approach to Identification
Presumptive identification of gram-positive anaerobic cocci can be
reported as Peptostreptococcus spp. or as anaerobic gram-positive
cocci
COMPILED BY: CRISTALEEN BANATE
 – LECTURE NOTES
Full identification by preformed enzyme systems or other methods
should be reported with the current terminology, with
Peptostreptococcus inserted between the genus and species until
these names become more familiar to physicians – for example,
Finegoldia (Peptostreptococcus) magna
Black-pigmented, anaerobic, gram-positive coccus can be identified as
Peptococcus niger
Peptostreptococcus anaerobius can be identified by a zone of inhibition
around an SPS disk
Spot indole-positive isolates are often members of the genus
Peptoniphilus, with Peptoniphilus indolicus identified by a positive
nitrate test
Only urease-positive anaerobic coccus is Anaerococcus tetradius
Many gram-positive anaerobes can also be presumptively identified
with simple procedures
None of these organisms will grow on a BBE or KVLB medium so
identification is based on the colony appearance on nonselective
anaerobic blood and EYAs
Large, irregular-shaped colonies on SBA demonstrating a double zone
of β-hemolysis can be identified as Clostridium perfringens – stain as
large, boxcar-shaped bacilli
CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC
BATERIA
9
Large, flat colonies that produce a so-called barnyard or horse stable
odor and fluoresce chartreuse under long-wave UV light can be
identified as Clostridium difficile
A rapidly growing colony exhibiting smooth swarming (as opposed to
the waves observed with Proteus) and staining as thin rods with
subterminal spores is likely to be Clostridium septicum – colonies
often show β-hemolysis at 48 hours
Small, peaked, circular colonies appearing after 24 hours that stain as
gram-positive cocci can be considered Peptostreptococcus spp.
Peptococcus niger produces colonies that are initially black to olive
green and become light gray when exposed to air, but it is only
rarely isolated from clinical specimens
Small, opaque colonies that are catalase and indole positive and
stain as coryneform rods can be identified as Propionibacterium
acnes
Using Gram stain results, growth characteristics on primary
plating media (e.g., nonselective anaerobic blood, BBE, KVLB), and a
few rapid tests, presumptive ID is often possible within 24 hours of
inoculation
Growth of large (>1 mm) gray-black colonies on BBE agar with
growth on KVLB agar after an overnight incubation is sufficient to
identify an isolate as a member of the Bacteroides fragilis group
Translucent pitting colonies observed on the anaerobic blood agar
plate, with no growth observed on BBE or KVLB agars, are
characteristic of Bacteroides ureolyticus
Translucent colonies with a black fish eye center observed on BBE
agar (usually at 48 to 72 hours) can be used to presumptively identify
Bilophila wadsworthia
Ground-glass or breadcrumb-like colonies of long, slender, gram-
negative rods with pointed ends are usually Fusobacterium nucleatum
Organisms that grow on anaerobic blood agar and KVLB but not
BBE and fluoresce brick red can be identified as Prevotella
Small, translucent or opaque colonies of tiny gram-negative cocci or
diplococci can be identified as Veillonella (gram-negative cocci)
COMPILED BY: CRISTALEEN BANATE
 – LECTURE NOTES

Anaerobic Chambers
Glove Box
Fitted with airtight
rubber gloves
Easier to use and less
susceptible to
oxygen contamination
Technologist inserts
his/her hands in the
gloves provided

Gloveless Anaerobic
Chamber
Airtight rubber sleeves
that fit snugly against
the user’s bare forearms
are used in place of
Anaerobic Jars gloves
GasPak Dissecting microscope
Torbal may be mounted in
Buchner-Torbal the rigid Plexiglas
Filde and McIntosh chamber
Pre-reduced anaerobically sterile (PRAS)
Components
Catalyst – palladium-
coated aluminum pellets
Anatox – absorbs metabolites and prolongs catalyst life
Silica gel – desiccant
Indicator
Methylene blue – white (O2 absent) to blue (O2 present)
Resazurin – colorless to pink
Anaerobic Culture Media
CDC anaerobic agar – used for growing all anaerobes
Anaerobic Bags BHI blood agar – used for growing all anaerobes
Plates can be examined for growth without removing from the bag Schaedler blood agar – used for growing all anaerobes
Alternative to an anaerobic chamber or jar BRU/BA (Brucella Blood Agar) – used for growing all anaerobes
Oxygen removal system is activated to minimize exposure to oxygen PEA (phenylethyl alcohol) – used for all obligate anaerobes; inhibits
enterics

CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE

BATERIA
10
 – LECTURE NOTES
Double zone hemolysis or target hemolysis on BAP (narrow inner zone
of β hemolysis and wider zone of α hemolysis) – Clostridium
perfringens
Bacteroides Bile Esculin (BBE) – used for growing bile-resistant Spider-like colonies – Actinomyces israelii or Propionibacterium
Bacteroides propionicus
Kanamycin-Vancomycin-Laked Blood (KVLB) – used for growing
Bacteroides and Prevotella
Cadmium sulfate fluoride acriflavin telluride (CFAT) – used for growing
Actinomyces
Cefoxitin cycloserine egg yolk (CCEY) – used for growing Clostridium
difficile
Cefoxitin cycloserine fructose agar (CCFA) – used for growing
Clostridium difficile
Egg yolk agar (EYA) – used for growing Clostridium
Neomycin vancomycin (NV) agar – used for growing Fusobacterium
Lactobacillus selective (LS) medium
Bifidobacterium selective (BS) agar
Veillonella neomycin agar
Thioglycollate and chopped meat

Approach to Identification
Colony morphology and Gram stain reaction
Aerotolerance Testing
Rapid Tests
Fluorescence
Catalase
Spot indole
Urease
Motility
Special potency antimicrobial disks
Sodium polyanethol sulfonate
(SPS) disk
Nitrate disk
Bile disk
Lecithinase, lipase, and proteolytic reactions

Colony Characteristics of Anaerobic Bacteria

Brown-black colonies – Prevotella, Porphyromonas, Peptococcus niger,
Bacteroides
Fluorescence production
Brick-red – Porphyromonas, Prevotella (e.g., Prevotella
melaninogenicus)
Red – if gram-negative cocci, Veillonella
Chartreuse
If gram-negative, Fusobacterium nucleatum
If gram-positive, Clostridium difficile
Pitting of agar – Bacteroides urealyticus group and Wolinella

CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE

BATERIA
11
 – LECTURE NOTES

SELECTED CULTURE MEDIA

Cooked Meat (Chopped Meat Glucose) Medium
Purpose:
Useful in cultivation of anaerobes,
especially pathogenic species of
Clostridium
Useful for cultivation of mixed cultures
Principle:
Contains peptones, beef heart, and
dextrose
Contains solid meat particles which are
excellent for:
Initiating growth from a very small inoculum
Sustaining culture viability over long periods

CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE

BATERIA
12
 – LECTURE NOTES

Digestion of meat particles indicates the proteolytic activity

of cultured organisms
Saccharolytic Clostridium spp. typically produce acid with
gas
Cycloserine Cefoxitin Fructose Agar (CCFA)
Purpose: Selective, differential medium useful in
the isolation and identification of Clostridium difficile
from stool specimens of
patients suspected of
having antibiotic-associated
diarrhea with
pseudomembranous colitis
Principle:
Cycloserine and
cefoxitin – inhibit
growth of intestinal
normal biota by
interfering cell wall
synthesis in gram-
positive and gram-
negative bacteria
Fructose and neutral red are included to confirm that the
isolates can ferment this sugar
Bacteroides Bile Esculin Agar
Purpose:
Selective and
differential medium for
the isolation and
presumptive ID of
Bacteroides fragilis and
its close relatives
(Bacteroides fragilis
group)
Bacteroides fragilis is
the most abundant
bacterium found in the
human colon, reaching
densities of 1011 cells
per gram of feces
It is also the most
common anaerobic
human pathogen
Principle:
Contains a base medium of tryptic soy agar, which includes
digests of casein and soybean meal
Oxgall (bile) inhibits other anaerobes in stool sample
Gentamicin inhibits facultative anaerobes in stool
Esculin, present in medium, is hydrolyzed by Bacteroides
fragilis, producing esculetin
Esculetin reacts ferric ammonium citrate in the medium to
produce a brown coloration (insoluble iron salts) around
Bacteroides fragilis growth

CHAPTER 22: CLINICALLY IMPORTANCE ANAEROBIC COMPILED BY: CRISTALEEN BANATE

BATERIA
13