ANAEROBIC BACTERIA

Prepared By: nahaeminrmt

Gram Positive Anaerobic Bacilli

Actinomyces
 Normal flora of mucosal surfaces
 granulomatous disease with formation of sinus tracts containing sulfur granules
 Oral and dental infections,aspiration events, and adominal and pelvic inflammations
 Affected sites: liver, breasts, soft-tissues,breast, liver, and brain

 Actinomycoses israelii
 Gram stain: short rods; branching or beading, associated with sulfur granules
 Colony: white, opaque, with molar tooth
 Biochem tests: catalase (-); esculin (+)
- to differentiate with A. naeslundii (urease pos) and A. odontolyticus (urease neg), A. turicensis= esculin (-)
 A. naeslundii, and A. odontolyticus, A. turicensis = oral flora; associated with IUD infections
Cutibacterium acnes
 Formerly known as: Propionibacterium acnes
 Commonly isolated anaerobic bacillus
 Normal flora of the skin
 Causative agent of acnes vulgaris
 Production of biofilm = neurosurgical shunt infections, prosthetic joint infections, and endolphthamitis after cataract
surgery
 Biochem tests: catalase positive; indole positive
Bifidobacterium spp.
 Diphtheroid-like; bifurcated or forked ends
 Normal flora of gastrointestinal tract and genitourinary tract
 Polymicrobial bacteremia (abdomen, pelvis) and pulmonary infections
 Acidophilic
Lactobacillus spp.
 Gram pos, long and slender rods; singly or in pairs and chains
 Normal flora of the vagina and decreased amount may lead to BV; dental caries, rarely bacteremia and endocarditis
 Biochem test: catalase negative and oftenly vacomycin resistant
Mobiluncus spp.
 Curved with tapered ends; gram variable
 Normal vaginal flora but increased amount may lead to BV

Gram Positive Anaerobic Bacilli

Peptococcus anaerobius
 Large cocci often in chains
 Colony morph: non-hemolytic; gray with raised center; sweet odor
 Infections in abdominal cavity and female urogenital tract
Anaerococcus prevotii
 Gram Stain: clumps and tetrads
 Non-glistening ; gray; convex
 Infections in abdominal cavity and female urogenital tract
Finegoldia magna
 Predominant gram positive anaerobic cocci of the skin and oral flora
 Gram Stain: pairs, tetrads, and clusters
 Colony: small, white, convex
 Skin, soft tissue, bone, and joint infections
Parviomonas micra
 Predominant gram positive anaerobic cocci of the skin and oral flora
 Gram Stain: clusters or short chains
 Colony: small, dull color; halo around the colonies
 Endodontic disease and peritonsillar abscess
Anaerococcus tetradius
 Formerly known as Peptostreptococcus tetradius
 Gram Stain: clumps and tetrads

Species Indole PYR

Peptococcus anaerobius - -
Anaerococcus prevotii - +
Finegoldia magna - +
Parviomonas micra - +
Anaerococcus tetradius - +

Medium Comment
Anaerobic Blood Agar (CDC) Components: sheep blood, vitamin K (Porphyromonas spp.), and yeast extract
Bacteroides Bile Esculin (BBE) agar Components:
-selective medium  Gentamicin - inhibits the most aerobic organisms
 20% bile - inhibits most anaerobes
 Esculin
(+) light-yellow will turn into black (esculin hydrolysis)
Bile tolerant species: Bacteroides spp., Fusobacterium mortiferum, Klebsiella
pneumoniae, enterococci, and yeast
Brucella Blood Agar Enrichment media: sheep RBC, casein peptones, dextrose, yeast extract, vitamin K,
and hemin
Kanamycin-vancomycin-laked blood = Components:
selective medium  Kanamycin - inhibits most facultative gram-negative bacilli
 Vancomycin - inhibits most gram positive organisms and vancomycin-
sensitive strains Porphorymonas spp.
 Laked blood - accelerates production of brown-black pigmented colonies by
certain Prevotella spp.
Phenylethyl Alcohol (PEA) agar Components:
 Sheep RBC and PEA - suppress the growth of any facultative, gram negative
bacilli (swarming Proteus spp.)
Colistin Nalidixic Acid (CNA) blood agar  Colistin and Nalidixic acid - suppress the growth of any facultative , gram-
plate negative bacilli
Anaerobic broth (thioglycollate and  Supports the growth of obligate aerobes (at the top), microaerophilic (near
chopped or cooked meat) the top), obligate anaerobes (at the bottom) and facultative anaerobes
(dispersed throughout the broth)

**organisms - reviewers

PRESUMPTIVE TESTS FOR ANAEROBES

Fluorescence Test Organism Color of Fluorescence
Prevotella (pigmented) Brick-red
P.bivia and P.disiens Light orange to pink (coral)
Porphyromonas Brick-red (some no fluorescence)
Fusobacterium, C.difficile, C. innocuum Chartreuse
Veillonella Red but fades rapidly upon exposure
to air (5-10 mins)
Catalase Test  Rgt: 15% H2O2
 (+) = bubbling
 To differentiate Clostridium (catalase-negative) from Bacillus (catalase-
positive)
Spot Indole Test  Rgt: p-dimethylaminocinnamaldehyde disck
 (+) = blue or green [production of indole from amino acid tryptophan]
 Propionibacterium acnes, C. sordelii, C. tetani
 (-) = pink or orange color
  Propionibacterium spp.
Urease test  Rgt: Wee-Tab Urease Test Tablet to 1mL of distilled water
 Incubation temperature: 37°C
 (+) after 15 mins = Clostridium sordelii
Motility test  Wet mount of young colonies
 4-6 hrs old = broth cultures
 24-48 hrs old colonies on agar
 (+) distinct motility NOT BROWINIAN MOVEMENT
 Campylobacter (C. concisus, C. curvus, and C. rectus) and Mobiluncus
spp.
Special Potency Antimicrobial Disk  Kanamycin(1000μg), Vancomycin (5μg), and Colistin (10μg)
Kanamycin Vancomycin Colistin Species
R R R Bacteroides (or prevotella)
R R S Prevotella spp.
S S R Clostridium and other gram positive
anaerobes
S R S FUsobacterium, B. urealyticus,
Veilonella, Bilophilia wadsworthia
R S R Porphyromonas spp.

DEFINITIVE ID OF ANAEROBES
Biochemical-Based Multitest systems  API; bioMerieux, Durham, ND
 Alternative to conventional tubes media
 Almost the same with enzyme based system
 Requires anaerobic incubation
 In form of plastic strip or tray
 24-48 hrs of incubation
 Specific code for each isolate
Pre-formed Enzyme-Based Systems  Vitek ANI card, AN-IDENT, MicroScan Rapid Anaerobe Identification Panel
(Siemens Biomedical Diagnostics, Deerfield, IL), BBL Crystal Anaerobe ID
System (BD Diagnostic System) and RapID-ANA II
 Rgt: nitrophenyl and naphthylamide compounds
 It detects the preexisiting bacterial enzymes
 Incubation - 4 hrs
 Do not require anaerobic incubation
 Disadvantage: The codebook is based and divided by the gram stain therefore accurate gram stain should be observed
Conventional Tubed Biochemical  PRAS or non-PRAS biochemical test media
Indentification Systems  It measures the pH
 Probe
 Color change - Bromthymol blue (indicator)
 Disadvantage: time consuming and expensive
Gas Liquid Chromatography  Gold standard but dismissed with rapid ID identification
 Peptone-yeast extract glucose (PYG medium) = culture media
 Principles is GLC (extraction → injected to chromatograph → volatilized →
carried as a gas
 Organic compounds for extraction process
 Ether extracted - volatile
 Chloroform extracted - non-volatile
 Short chain volatile acids by anaerobes: formic, acetic, propionic, isobutyric,
isovaleric, valeric, isocaproic, butyric. Caproic, and hepatonic acids
 Nonvolatile and low molecular weight aliphatic and aromatic acids - pyruvic,
lactic, oxaloacetic, malonic, oxalic, fumaric, siccinic, benzoic, phenylactic, and
hydrocinamic acids
 Disadvantage: cost, safety, and time-consuming
Cellular fatty acid by high resolution  Cellular fatty acid - fatty acids and compounds (aldehydes, hydrocarbons,
GLC dimethylacetals)
 PYG broth as media
  Centrifugation → saponification → release of fatty acids from bacteria
 Code for bacterial chormosomes and not affected by mutations or
plasmid loss
 MIDI Sherlock Microbial Identification System
16 s rRNA Gene sequencing

Identification of Clostridium
Gram stain  Gram pos with some gram variable spp (C. ramosum and C. clostridioforme)
 Clostridium difficile - box-car shaped, gram positive anaerobic bacillus with
double zone of hemolysis in SBA
Spores location  Terminal Spore - Clostridium tetani
 Subterminal Spore - C. septicum
ELISA  Screening test C. Difficile
 Identification of glutamate dehydrogenase
 Toxins A and B
NAAT  Determine the presence of toxins A and B in feces
 Gold standard for detection of toxin producing C. difficile
 Positive EIA for glutamate dehydrogenase should be confirmed with NAAT for
toxin genes