CHAPTER 13 ANAEROBIC BACTERIA 3. Pleuropulmonary infections - Esculin: B.

fragilis are esculin (+)

 RODS 4. Soft Tissue infections - K. pneumoniae, Fusobacterium mortiferum, enterococci, yeast:
1. sporeformers: Clostridium 5. Septicemia <1mm in diameter
2. nonsporeformers  Growth requirements (anaerobic culture system available) 3. KANAMYCIN VANCOMYCIN LAKED BA (KVLB)
a. Gram (+) - anaerobic jar or small O2 impermeable plastic bag - rapid isolation of Bacteroides species
b. Gram (-): most impt (2/3 of clin significant isolates) - roll tube (Hungate system) - kanamycin: inhibit most aerobic facultative & anaerobic Gram (-)
- Seleonomonas Bacteriodaceae: pigmented (bile sensitive); - anaerobic glove box (incubator) rods except Bacteroides (resistant)
non-pigmented (bile sensitive, bile resistant) - palladium: chemically reduce O2 H2O - vancomycin: inhibit most Gram (+) org
 COCCI  Specimen - laked blood: early pigmentation of pigmented Bacteroides
1. Gram (+) 1. appropriate specimen 4. PHENYLETHYL ALCOHOL sheep’s BA (PEA)
2. Gram (-) - normally sterile body fluids - inhibit enterics & other anaerobic Gram (-) bacilli
 Generalities - surgical specimen from normally sterile sites - Columbia BA w/ colistin & nalidixic acid (CAN): substitute for PEA
- 1900: bacteria would grow only in gaseous env (reduced O2 - abscess contents bloof cultures (inhibits Fusobacterium, Veillonella, B. ureolyticus, some strains
tensions) - deep wound & transtracheal aspirate of Prevotella, C. perfringens)
- Almost all are resistant to aminogkycosides - bronchoscopy aspirates 5. THIOGLYCOLATE MEDIUM (vit K, hemin, marble chips or
- 2 tetracycline derivatives are active against 65-75% - amniotic fluid at CS, amniocentesis, amnitotomy NaHCO3)
- B. fragilis strains, other anaerobes: doxycycline, minocycline - endometrium (Lochia) - freshly prepared heat
(clostridial myonecrosis) - culdocentesis fluid - boiled or steamed: drive off residual O2
 Hyperbaric O2: alternative mode of therapy - urine, catheterized or suprapubic aspirate (complicated cases) - back-up culture & enrichment med for small # of bacteria
- 7 intermittent exposure to 100% O2 in a chamber pressurized to - CSF (complicated) 6. ANARERONBIC BA (nonselective for anaerobes & fac anaerobes)
3atm absolute pressure 2. aspirated tissue specimen preferable to swabs 7. CYCLOSERINE CEFOTOXIN FRUCTOSE AGAR (C. diffcile)
- Clostridium (O-R increased) ASAP - survival, quantity, less contamination 8. nonselective cultivation of anaerobes for gas liquid chroma
 Study had lagged 3. placed in anaerobic transport device (w/in 20 mins for culture) - peptone yeast extract glucose broth (PYG
- sensitivity to O2 4. require more time (grow slowly) - CCMM,. Thio
- fastidious growth requirements (enriched media) - 48 hrs: rapid growers (anaerobes)  Examination of Primary Plates
- occurring in mixtures of anaerobic & fac anaerobic org - 18-24 hrs: aerobes & fac anaerobes - examined w. hand lens
- diffucult: anaerobic env (no pure cultures for study) 5. Grams stain - colonies described from various media & semiquantified
 Improved anaerobic technology has demonstrated 6. good communication bet microbiologis & clinician is impt - KVLB (>48 hrs)
- high incidence of anaerobic orgs in clin specimen  Direct Examination 1. Porphyromonas & Bacteroides: brick red fluorescence
- anaerobic: widespread in nature - foul odor 2. B. ureolyticus, B. braziliensis, Weilonella, Campylobacter:
- predominant part in NF (skin & mucous membrane) - sulfur granules: Actinomyces, Eubacterium, Propionibacterium pitting colonies in anaerobic but nit in CO2
- outnumber fac anaerobic bacteria in gut (1000:1) (Gram+ bacilli) 3. C. perfringengs: large colonies w/ double zone of hemolysis
- skin, mouth, URT, female lower genital tract (5:1-10:1) - blood, purulence/ other char: pigments (Porphyromonas, (target colonies) EYA & Nagler (ppt: dense oplascence)
- commensals opportunistic pathogens (ICP) Prevotella) 4. F. nucelatum: slender fusiform G (-) bacilli, breadcrumb like or
 Anaerobic bacteria as etiological agents of Infection  Gram Stain speckled colonies (greening agar)
- impt agents of infection; polymicrobic (5+ anaerobes doesn’t imply - type & #, QC measure 5. Actinomyces israelii: molar tooth colonies of G (+) branchin
contamination) - indicate need for additional media: EYA for clostridia (dense rods
- virulence factors: tissue destruction & inhibition of host defenses, opalescence, G+ sporeformer)  Subculture of Isolates
necrosis - C. perfringens: boxcar shaped 1. Aerotolerance Testing
- sporeformers: exotoxin - Fusobacterium nucleatum: scattered wheat straws 2. SPA (Sodium Polyanithol Sulfonate disc)
- nonsporeformers: no exotoxin - Actinomyces, Propionibacterium, Eubacterium: tangled masses of 3. AB ID disc
 Nonsporeformers: branching/ clubbing G+ bacilli - kanamycin, vancomycin, colistin
1. LPS  Culture - Porphyromonas, Prevotella
- Fusobacterium, Porphyromonas, Bacteroides, Prevotella,  (+) Gram, (-) culture 4. Bile disc (bile susceptibility)
Wollinella, Veillonella - poor transport method 5. NO3 discs (NO3 NO2)
- Bacteroides fragilis: abscess formation - exposure to air on processing  Susceptibility Testing
- Porphyromonas gingivalis: comparable to E. coli/ Fusobacterium - nonviable cells: AB use - not usually performed: polymicrobic nature
(more potent than LPS of Bacteroides)  PRAS broth: used w/o steaming (Prereduced Anaerobically Sterilized - delayed testing & reporting: slower growth
2. Polysaccharide Capsule broth) - performed in: life threatening infection, critical sources (blood,
- Baceteroides, Prevotella, Porphyromonas  Chopped meat CHO, Chopped meat: substitute for Thio (remove O2 CSF, surgical osteomyeltitis, no response to AB, sequelae are
- Prevents phagocytosis (PMN) overlay w/ paraffin) likely)
3. Enzymes  BHIBA: gassed w/ anaerobic gas  Differentiation & Grouping
- collagenases, heparinase, hyaluronidase, fibrinolysin, gelatinase,  Cultures must not be exposed to O2 until after 48 hrs incubation - Bacteroides & Porphyromonas divided accdg to: bile
aminopeptidase sensitive to O2 during lag phase of growth; colony morph changes susceptibility, pigment production & AB disc ID pattern
- lipases of Fusobacterium necrophorum dramatically bet 24-48 hrs - B. fragilis grp: bile resistant, nonpigmented, resistant to all 3 AB
- heparinases: Bacteroides  Exponential: not sensitive - Other Bacteroides: nonpigmented, bile sensitive
- collagenase: P. gingivalis  24 hrs: B. fragilis, C. perfringens (fastest growing) - Other bile resistant Bacteroides: B. eggerthii, B. splancnicus (not
4. short chained fatty acids (metabolic end products) - w/o growth: incubated for 1 week before discarding common)
- butyrate: dental plaque (Porphyromonas)  Thio Broith cultures: daily for 7 days; subculture when turbid at day 7 - Laked BA (rabbit): most reliable pigment production [colonies
- succinic acid: Bacteroides [ASAP] fluoresce brick red, yellow green or not at all under UV light),
5. Synergy - growth on lower half of tubes (O2 not high): anaerobes black colonies
- aspiration pneumonia & lung abscess: spirochete, fusiform - subculture: BA (anaerobic) & CA (CO2) - After pigment is produced fluorescence not detectable
bacilli, streptococci, vibrio  Culture Media - Most Porphyromonas: diffucult to grow in broth & selective agars
- Vincent’s angina – mouth: fusospirochaetal (lung abscess) - enriched & selective: fastidious containing vancomycin (KVLB) [add bicarbonate, serum &
 Clinical Infection 1. BRUCELLA 5% sheep’s BA (Vit K & hemin) hemin: enhance growth in broth]
- compromised host defense - Isolation of all bacteria Organism Gram Comments Distinguishing
- polymicrobic, synergistic mixtures - Vit K: pigmented Bacteroides reaction characteristics
- broad spectrum antimicrobial therapy generally required - Hemin: growth enhancement of B. fragilis & other Bacteroides B. fragilis - Can be Grows on
- doesn’t comply w/ KOCH’s postulate: NF of skin & membrane 2. BACTEROIDES BILE ESCULIN agar pleomorphic; BBE>1mm in
surfaces of URT, GIT gut - selection & presumptive ID of B. fragilis group safety pin feature diameter; =/-
 Clinical Manifestation - contains gentamycin: inhibit most aerobic organisms esculin
1. Intra-abdominal infections - 20% bile: inhibit most anaerobes except B. fragilis grp/ bile Pigmentrer - Can be very Foul odor; black or
2. Obstetric & Gynecologic infections resistant Bacteroides coccoid/ brown pigment;
 hemophilus like brick red  Prevotella corporis
fluorescence  Bacteroides melaninogenicus group: Prevotella loescheii &
B. - Thin, some are May pit agar or denticola
ureolyticus curved spreading - don’t develop dark pigment (exception) [light tan to buff colonies]
like transparent  B. gracilis – urease (-); pit; twitching motility
colonies  B. ureolyticus – usease (+); pit; nonmotile
B. nucleatum - Slender cells with Foul odor; 3 colony Not Included in fam but same char
pointed ends types: speckled,  Wollinella – pit; gliding
smooth &  C. concisus – darting
breadcrumb Bile sensitive; non-pigmented
Gram (-) -  B. zoogleoformans – zoogleal mass; embedded in a jelly like matrix
bacillus  B. heparinolyticus – produce viscous mass
Gram (-) - Veillonella cells FUSOBACTERIUM
coccus are tiny - originally 7
- anaerobic Gram (-) bacilli: butyric acid w/o iso acids
Gram (+) + Variable size
- nonmotile, straight/ slightly curved bacilli w/ pointed/ rounded ends
coccus
- 5 newly described species: F. alocis, F. sulci, F. periodonticum, F.
C. + Large boxcar Double zone of ß-
pseudonecrophorum, F. ulcerans
perfringens shape; no spore hemolysis
 Only indole (-): F. mortiferum, F. russii
observed, may
 Growth in bile (+): F. mortiferum, F. varium
appaear Gram (-)
 Only lipase (+): F. necrophorum
 ID
GRAM (+) BACILLI
- all are sensitive to bile
 Actinmyces
- 3 grps: AB disk ID pattern, indole reaction, colony morphology
 Bifidobacterium
BACTEROIDACAE
 Eubacterium
 B. fragilis group
 Lactobacillus (no char colonies)
- B. eggerthii (bile resistant)
 Propionibacterium
 Pigmenting Organisms/ bile sensitive
ANAEROBIC GRAM (+) COCCI
 Pigmenting Bacteroides (Prevotella)
- NF of bowel, female genital tract, oral cavity; smaller # in skin (other
- become black (melanogenicus)
sites)
- B. intermedius, B. corporis, B. bivius, B. melanogenicus, B.
- 30% of anaerobes (infection containing anaerobes)
loescheii, B. denticola, B. nigresenence
- Diabetic foot infection (IC)
 Porphyromonas (DNA homology)
- Peptococcus magnus (most virulent)
- P. asaccharolyticus, P. endodontalis, P. gingivalis
- Isolated more frequently than G (-) cocci (more pathogenic)
 Non Pigmented/ bile sensitive
 ID (biochem test)
 Prevotella / B. oralis group (pentose nonfermenters)
1. P.anaerobius
 Pentose fermenters
- susceptible to SPS
 Other Bacteroides species
- isocaproic acid: end product of metabolism
 Generalities
2. P. asaccharolyticus
- 2/3 of total anaerobic isolates from clin specimen
- (-) latter test
- Prevalent among indigenous flora of MM of humans & animals
3. Peptostreptococcus hydrogenalis
- Infection: abscess formation, tissue destruction (necrosis)
- ferments glucose; alkaline phosphatase (+)
 Morphology: pleomorphic, filamentous, vacuolated, short/ long rod,
4. Peptostreptococcus indolicius
stain irregularly, motile cells have peritrichous flagella
- rarely isolated in clin specimen
 Cultural Characteristics
- coagulase (+); reduces nitrate
- Prevotella & Porphyromonas: brown to black pigment (48
ANAEROBIC GRAM (-) COCCI
hrs)
 Veilonella (most impt): propionic & acetic acid
- B. ureolyticus: agarase, pit agar
 Acidaminococcus: butyric & acetic acid
Bacteroides fragilis group
 Megasphaera: isobutyric, butyric, isovaleric, valeric & caproic acid
 Ag structure
- thermolabile protein antigen
- thermostable LPS antigen: basis for serologic classification of
B. fragilis
 Determinants of Pathogenicity
- LPS
- Polysaccharide capsule
- heparinase
 Clinical Infection: pleuropulmonary, intra-abdominal, female genital
tract (lower)
 Dx
1. only Indole (+): B. thetaiotaomicron & B. ovatus
2. only Catalase (-): B. ovatus
3. Only Esculin (-): B. vulgatus
 B. fragilis, B. distasonis, B. uniformis
PIGMENTING GRAM (-) BACILLI
- tan to black pigmentation of colonies on BA\
 Prevotella
- normal flora
- resistant to V & K; sensitive to colistin (indole -)
 Porphyromonas
- sensitive to V & K; resistant to colistin (indole +)
 ID
 Bacteroides intermedius
- only lipase (+) ; indole (+)