Anaerobic Bacteria

• Gram Positive Anaerobic Cocci

– Collinsella aerofaciens, Finegoldia magna,
Micromonas micros, Peptococcus niger,
Peptostreptococcus anaerobius, Schleiferella
asaccharolytica, Atopobium, Anaerococcus
• Gram Positive Anaerobic Bacilli
• Gram Negative Anaerobic Cocci
• Gram Negative Anaerobic Bacilli
 Anaerobic Bacteria
• Gram Positive Anaerobic Cocci
• Gram Positive Anaerobic Bacilli
– Propionibacterium, Actinomyces, Clostridium,
Bifidobacterium, Eggerthella lenta,
Eubacterium, Lactobacillus, Mobiluncus
• Gram Negative Anaerobic Cocci
• Gram Negative Anaerobic Bacilli
 Anaerobic Bacteria
• Gram Positive Anaerobic Cocci
• Gram Positive Anaerobic Bacilli
• Gram Negative Anaerobic Cocci
– Veilonella parvula
• Gram Negative Anaerobic Bacilli
– Bacteroides, Fusobacterium, Bilophila
wadsworthia, Leptotrichia, Porphyromonas,
Prevotella
 Endogenous
Anaerobic Infections
• Abscess • Endocarditis
• Actinomycosis • Meningitis (usually after brain
abscess)
• Antibiotic associated
• Necrotizing pneumonia
diarrhea and colitis
• Osteomyelitis
• Aspiration pneumonia
• Otitis media
• Complications of • Peritonitis
appendicitis/cholecystitis • Septic arthritis
• Crepitant / non crepitant • Sinusitis
cellulitis • Subdural empyema
• Clostridial myonecrosis • Tetanus
• Dental infections • Thoracic empyema
 O2 & CO2 requirements
• Obligate or strict aerobe
• Microaerophile
• Facultative anaerobe
• Aerotolerant or moderate anaerobe
• Obligate or strict anaerobe
• Capnophile
 Anaerobes
• susceptible to toxic derivatives of oxygen
• lack or low levels of protective enzymes
(superoxide dismutase and / or catalase)
• Endogenous
• Exogenous
 Sites
• Those harboring an indigenous flora (body
fluids other than urine)
• Exudates from deep abscesses
• Transtracheal aspirates
• Direct lung aspirates
• Tissue biopsy specimens
 Anaerobes part of normal flora:
• Throat / nasopharyngeal/ gingival swab
• Sputum / bronchoscopic specimens
• GI
• Surfaces of decubitus ulcers, swab samples of
encrusted walls of abscesses, mucosal linings,
eschars
• Voided urine
• Not properly decontaminated - skin
• Vaginal / cervical swab
 Macroscopic Examination
• Is it an appropriate specimen ?
• Was it submitted in an appropriate transport
container or medium ?
• Does the specimen have a foul odor ?
(Fusobacterium & Porphyromonas)
• Does the specimen fluoresce brick red when
exposed to long wave light (366 nm)?
• Is the necrotic tissue or exudate black ?
• Does the specimen have sulfur granules ?
 Selection & Use of Media –
inoculate at room temp
• Colistin & Nalidixic Acid CNA BAP
• Paromomycin-Vancomycin BA or Kanamycin-
Vancomycin BA – gr(-) non sporeforming
anaerobes
• Bacteroides bile esculin BBE
• Enriched thioglycollate medium / chopped meat
glucose broth
• Phenylethyl alcohol (PEA)
• PRAS
• CDC anaerobe blood agar
 3 ingredients – culture medium
for anaerobes
• Vitamin K
• Hemin
• Yeast extract
• Best recovery – CDC agar
 Gram’s stain
• Methanol – instead of heat fixation
• Acridine orange –blood, CSF, pleural fluid,
joint fluid, exudates
• Spores
• Branching filaments
• Spherical bodies
• Pointed ends
• Granular forms
 Gram Stain
• Methanol fixation – preserves WBC
morphology and bacteria better than heat
• Gram neg anaerobes – stain pale pink
when safranin is used - overlooked
• 0.1 % basic fuchsin as counterstain or
extending the counterstain with safranin
for 3 to 5 minutes
 Presumptive Identification of
Clinically Significant Anaerobes
Fluorescence
Catalase
Spot Indole Test
Urease Test
Motility Test
Special Potency Antimicrobial Disks
SPS, Nitrate and Bile Disks
Lecithinase, Lipase and Proteolytic Reactions
 Level / Extent
• I = preliminary report – isolate in pure
culture; colonial & microscopic morphology
• II = I + identification – B. fragilis/C.
perfringens
• III = GLC – presumptive ID
• IV = definitive
 ANAEROBES
• Exogenous Anaerobes
- spore formers
- genus Clostridium

• Endogenous Anaerobes
- polymicrobial infections
- most numerous in mucosal surfaces
- produce infections not far from mucosal surfaces
 Polymicrobial Infections
• Symbiotic relationship with:
– Obligate anaerobes
– mixtures of obligate anaerobes and
facultative organisms
 Anaerobes at Specific Anatomic Sites
• Skin
– P. acnes – contaminant / opportunistic endocarditis / surgical
wound infection
• Resp
– Prevotella, Porphyromonas, Fusobacterium
• GI (anaerobes : facultative anaerobes 1000:1)
– B. fragilis, Bifidobacterium, Clostridium, Eubacterium
– Peirtoneal cavity
• GU – pathogen or endogenous flora ?
– Anaerobic cocci, Fusobacterium, Prevotella, Bacteroides,
Lactobacillus
 Potential Virulence Factors of
Anaerobic Bacteria
• Polysaccharide Capsules
• Adherent Factors
• Clostirdial toxins / exoenzymes
– Collagenases, cytotoxins, Dnases,
enterotoxins, hemolysins, hyaluronidase,
lipases, neurotoxins (botulinum toxin,
tetanospasmin), phospholipases, proteases
 Factors that Predispose
Patients to Anaerobic Infections
• Human or animal bite wounds
• Aspiration of oral contents into the lungs
after vomiting
• Dental works; traumatic oral puncture
• GI/ Genital tract surgery; traumatic bowel
genital tract puncture
• Introduction of soil into wound
• Trauma to mucous membranes / skin
 Factors that Predispose
Patients to Anaerobic Infections
• Vascular stasis
• Decreased oxygenation of tissue 
necrosis and decreased redox potential of
tissue
 Indications of Anaerobe
Involvement in Human Disease
• +/- Foul odor
• Purulent with many PMN
• Necrotic tissue
• Gas – may also be due to E. coli
• Sulfur granules
• Absence of WBC – maybe due to enzymatic
virulence factors from C. perfringens that
destroy neutrophils, macrophages & other cells
 Clostridium
• Spores – heat or alcohol shock may be
needed to induce sporulation
• Most are catalase negative
• C. tetani – round, terminal spores (drumstick)
• C. botulinum – oval, subterminal spores
(tennis rackets)
• True exotoxins
• C. difficile – endogenous origin
 Group I Gas Gangrene
• C. perfingens, septicum, novyi
• Myonecrosis, food poisoning, postabortion
sepsis, intraabdominal and
pleuropulmonary infections, enterocolitis,
antibiotic associated diarrhea
• C perfringens – secretes both enzymes
and exotoxins  more tissue destruction
• Diabetics, endogenous infection
 C. perfringens
• Double zone hemolysis (inner theta, outer
alpha and lecithinase)
• Ferments glucose, lactose, maltose,
fructose
• Nagler reaction – zone of pptation around
the colonies on the side without antitoxin
• Reverse CAMP – arrowhead at
intersection of two streaks
 C. perfringens
• Exotoxins
• Hemolysins, lecithinase, protease,
collagenase, enterotoxin
 Clostridium perfringens
• Infections:
- food poisoning (8-12hr IP)
- myonecrosis / gas gangrene
Lab ID: non motile
• Double zone hemolysis
• Lecithinase(+)
• Reverse CAMP(+)
• Nagler(+) on EYA
• Stormy fermentation of milk
 C. perfringens Food Poisoning
• Type A – mild; self limited; enterotoxin linked to
sporulation; 8 to 12 hours incubation; no
treatment usually
• Type C – enteritis necroticans (pigbel) more
serious; rarely seen; B-toxin production; less
commonly A-toxin; 5 to 6 hours
• Ingestion of enterotoxin - producing strains in
contaminated food
• Improperly stored food allows germination of
the spores and growth of vegetative bacteria.
• Clostridium perfringens is a gram-positive
anaerobic rod that is classified into 5
toxinotypes (A, B, C, D, and E) according
to the production of 4 major toxins,
namely alpha (CPA), beta (CPB), epsilon
 (ETX) and iota (ITX).
 Myonecrosis / Gas Gangrene
•C. perfringens (most common cause)
•C. histolyticum, C. septicum, C. novyi, C. bifermentans
•when organisms contaminate wounds
•A-toxin – lecithinase (phospholipase C produced by all
strains of C. perfringnens
- pain, swelling, bullae, serous discharge, discoloration
& tissue necrosis
amputation
-
 Bacteremia
• C. perfringens – most common
• C. septicum – GI malignancy
• C. bifermentans and C. tertium – serious
underlying diseases
 Group II Tetanus
• Exogenous wound infection
• Tetanospasmin - neurotoxin
• Lockjaw, opisthotonos
• (+) gelatinase & indole
• (-) lecithinase & lipase
• Unable to ferment most CHO, motile
 Clostridium
•Tetanolysin
tetani
•Tetanospasmin
– Spores germinate into vegetative cells that produce
tetanospasmin
– Incubation pd: 7 days (3-21) depending on distance of
injury to CNS
- acts on inhibitory neurons, preventing
release of neurotransmitters
- results in continuous muscular spasms
- spastic paralysis; trismus/lockjaw, risus
sardonicus (distorted grin); dyspnea
 Clostridium tetani
Lab ID
•Tackhead bacillus; swollen terminal spores
•Swarms on BAP
•Lecithinase(-), Lipase(-)
 Group III – Botulism
• Food – ingestion of preformed toxin
• Wound – entry of spores
• Infant – colonization of GI tract and production of
toxin
• Neurotoxin – acute and flaccid paralysis
• Lipase (+) & lecithinase and indole (-)
• Ferments glucose but not lactose or xylose
• motile
 Botulism : Food-borne
• Ingestion of preformed botulinum toxin
• Toxins A to G
• Toxin A, B and E
• Neurotoxin, flaccid paralysis and death
• Prevents release of Acetylcholine
• Type A – for strabismus (wandering eye)
and frown lines
 Clostridium botulinum
• Canned-goods bacillus
• Produces botulin
- most potent toxin known
 Clostridium botulinum
Botulism
a. foodborne – ingestion of preformed toxin
b. infant botulism – ingestion of spores
c. wound – contamination with spores

Lab ID:
• lipase(+)
 Group IV –
Pseudomembranous Colitis
• Toxin A - enterotoxin
• Toxin B - cytotoxin
• Bloody diarrhea with associated necrosis
of colonic mucosa
• Health care associated / nosocomial
• Post antimicrobial therapy
 Clostridium difficile
• Endogenous anaerobe
• Most common cause of Antibiotic
Associated Diarrhea and
Pseudomembranous Colitis
Lab ID:
• Yellow colonies on CCFA
Cycloserine Cefoxitin
Fructose Agar
• Detects Toxin A at levels:
> or = 0.8 ng/mL
• Detects Toxin B at levels:
> or = 2.5 ng/mL
 n = 1,152
Tissue Culture Tissue Culture
Positive Negative

C. difficile 165 0
Tox A/B TEST
positive
C. difficile 14 973
Tox A/B TEST
Negative
 Effect of sample consistency
Test Liquid Semi solid Solid

n = 435 150 133 152

C. difficile 13 (8.7%) 11 (8.3 %) 13 (8.6 %)

Tox A/B (+)
Tissue 13 (8.7%) 14 (10.5 %) 15 (9.9 %)
Culture (+)
Sensitivity 92.2 %

Specificity 100 %

Correlation 98.8 %

Positive Predictive Value 100 %

Negative Predictive 98.6 %

Value
 Other tests :
• Selective media
• Latex agglutination
• Tissue Culture Cytotoxicity
• Distinguish between C. sordellii
• Clinical history: up to 50 % of healthy
adults and infants are toxin (+) by ELISA
or tissue culture
• 22-32% carriage in those w/ cystic fibrosis
 Group V Miscellaneous
• Brain abscess, pneumonia, gynecological
infections, bacteremia
 ACTINOMYCOSIS
• Filamantous bacteria
• Slow growers
• True bacteria; may be differentiated from
fungi microscopically
 ACTINOMYCOSIS
• Chronic, granulomatous
• Development of sinus tracts and fistulae
• “sulfur granules” (dense clumps of bacteria that
may be colored)
• Endogenous bacteria of oral cavity
• Oral cavity
• Female genital tract – long standing IUD
• Actinomyces israelli more common that
Propionibacterium and Bifidobacterium
 Bacterial Vaginosis
• Gardnerella vaginalis
Gram variable bacilli
• Mobiluncus, Bacteroides,
Prevotella
• Clue cell
• Pap smear
• Gray white, malodorous
• Absence of normal flora
– Lactobacillus acidophilus or
Doderlein’s bacillus
• Shift in normal flora
• Endogenous flora
• Clinical significance
 Lactobacillus
• Produce lactic acid from glycogen which
lowers the pH
• Maybe recovered in urine and genital
culture
• Endocarditis
Anaerobic Gram Negative Bacilli
• Mixed infections
• Abscesses beneath mucosal surfaces
 Bacteroides fragilis
• Gram-negative bacilli
• Most common cause of anaerobic infections
• Predominant flora of the intestines
• Infections in adjacent organs
• Abscess formations
Lab ID:
• Non hemolytic
• BBE(+) Bacteroides Bile Esculin
• Resistant to 3 special potency disks
(kanamycin, vancomycin, colistin)
 Bacteroides fragilis

Lab ID:
• Non hemolytic
• BBE(+)
• Resistant to 3 special potency disks
(kanamycin, vancomycin, colistin)
 Porphyromonas & Prevotella
• Gram-negative bacilli
• NF of URT and intestines
• Fluoresce brick red

• Prevotella
- will not grow on KVLB
- susceptible to vancomycin
• Agar and broth dilution
 Treatment
• Ampicillin/Sulbactam
• Chloramphenicol
• Imipenem
• Ticarcillin/Clavulanate
• On each deltoid:
– TIg = 250 IU IM
– Tetanus Toxoid = 0.5 mL
 Treatment
• High morbidity and mortality
• Treatment varies
– Antibiotic
– Surgical (debridement / amputation)
– Hyperbaric oxygen
– Antitoxins