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 (+)