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Arrangement Differentation of anaerobic Classification bacteria according to the Selective media Wadsworth method Level II identification

Level III identification of Gram-negative anaerobes Porphyromonas/Prevotella Fusobacterium sp.

Level III identification of Gram-positive anaerobes Clostridium sp. Linda Wildeboer-Veloo non-sporeforming rods anaerobic cocci Library

Classification (non-) selective media

BBA isolation of all anaerobic bacteria Peptostreptococcus Veillonella Brucella Blood Agar Anaerococcus PEA isolation of almost all anaerobic bacteria Peptoniphilus LecturePhenylEthyl Alcohol blood agar Parvimonas Finegoldia BBE isolation of Bacteroides fragilis sp. and Bacteroides Bile Esculin agar Bilophila wadsworthia, inhibition of gal- sensitive bacteria Clostridium-Eubacterium B. fragilis Actinomyces B. ureolyticus BBKV isolation of Bacteroides sp. and Brucella Blood agar with Prevotella sp., inhibition of Gram-positive Propionibacterium Prevotella authorKanamycin and Vancomycin bacteria, promotes pigmentation Bifidobacterium Porphyromonas (laked blood) Lactobacillus-Atopobium Fusobacterium OnlineBilophila by ©

Additional media Level II indentification

CCFA isolation of Clostridium difficile Ka Va Ct bile cat ind Cycloserine Cefoxitin inhibition of almost all anaerobic bacteria Fructose Agar B. fragilis groep RRRRVV Porphyromonas sp. R S R S V +- MMBA isolation of Actinomyces sp. Prevotella sp. RS RVS-V Metronidazole Mupiro- inhibition of most anaerobic bacteria B. ureolyticus SRSS-- ESCMIDcine Blood Agar Bilophila wadsworthia S R S R + - Fusobacterium sp. SRSV-V EYA isolation of lipase- and/or lecithinase positive Veillonella sp. SRSSV- Egg Yolk Agar anaerobic bacteria

Ka = kanamycin Va = vancomycin Ct = colistin kat = catalase Wadsworth manual, Table 4-2, p. 58 ind = indole

1 Bacteroides fragilis B. ureolyticus, coloniesLibrary with “pitting”

B. wadsworthia on BBA, small translucent colonies. Lecture

Gram-stain of B. wadsworthia, straight rods of uniform size and internal vacuoles author Veillonella sp. Onlineby ©

Prevotella sp./ Porphyromonas sp.

Identification is based pigment formation ESCMID

2 Fusobacterium sp.

Cell morphology is important for identification pigmented colonies Library

Level II identification

F. nucleatum, thin rods with pointed ends LectureKa Va Ct cat ind nitr Clostridium sp. S S R - V V Propionibacterium sp. S S R +- -+ -+ P. acnes S S R + +- + Eggerthella lenta S S R + - + Actinomyces sp. S S R -+ - +- Anaerobic Gram- V S R V V -+ positive cocci F. necrophorum, pleomorph (looks like F. mortiferum) author Ka = kanamycin Va = vancomycin Ct = colistin kat = catalase ind = indole Onlineby Wadsworth manual, Table 4-3, p. 60 nitr = nitrate ©

Lecithinase-positive clostridia Clostridium sp.

Anaerobic Gram-positive rods which can be Gram-variable

Spore demonstration: spore-stain ESCMIDalcohol-shock

C. perfringens: Nagler-test

C. botulinum with spores

3 C. perfringens on a blood agar showing double β- hemolysis

S. agalactiae

Reverse CAMP-test C. perfringens Gram-stain C. perfringens, cellsLibrary are “box-car shaped”

Swarming clostridia For phenotypic identification clostridia are divided into 3 groups:

- Proteolytic clostridia gelatin: + glucose: - - Saccharolyticclostridia gelatin: - glucose: + Lecture- Proteolytic + saccharoly- gelatin: + glucose: + tic clostridia

Identification is based on sugar fermentation and other supplemental tests author Lipase reaction OnlineC. septicum by ©

Clostridia who do not follow the rules Characteristics C. difficile

C. tertium, grown anaerobically. Selective medium: CCFA Spores are present

Colonies: 4 mm, yellow, shine of ground-glass, pleomorphic

Specific odour: horse stable (production IC) ESCMIDChartreuse fluorescence Proteolytic+ saccharolytic

C. tertium, grown aerobically. Spores are not present, morphology resembles Lactobacillus C. difficile a blood agar and Gram-stain

4 Gram-positive non-sporeforming rods

Difficult to distinguish from each other

cat ind nitr

Propionibacterium sp. +- -+ -+ Actinomyces sp. -+ - +- Bifidobacterium sp. -+ -- Eubacterium sp. --+ V Lactobacillus sp. ---+

C. clostridioforme, stains Gram-negative, cells are fusiform Most reliable method:Library gas-chromatography

- catalase +

- indole + - indole +

Eubacterium sp. P. acnes A. viscosus, branching - ADH + E. lenta Lecture P GLC S

Propionibacterium sp. A. viscosus A. neuii A. radicidentis A. israelii, molar shaped main product GLC authorcolonies Succinate Propionate Butyrate Acetate Lactate Actinomyces Propionibacterium Eubacterium Bifidobacterium Lactobacillus Collinsella Atopobium Eggerthella OnlineEubacterium by ©

B. breve in pus from a liver abscess

ESCMID Lactobacillus sp., straight Gram-positive rods

Best growth was obtained anaerobically on a chocolate agar, however

also reasonable growth at 5 % CO2

5 Gram-positive anaerobic cocci (GPAC)

Level II: Ka Va Ct SPS

Most GPAC S S R R P. anaerobius RS S R S P. stomatis S Pa. micra SSRV Pn. ivorii SSRV

When SPS: S cell morphology P. anaerobiusLibrary

Cell morphology GPAC most GPAC can be identified using enzym reactions

Most reactions are present in the Rapid ID 32A Small cocci: P. micra

Large cocci: F. magna A. vaginalis ESCMIDPn. ivorii In chains: P. anaerobius P. stomatis The code obtained with this API-strip is not suitable for Other GPAC: “normal” size identification.

Use table from Wadsworth manual.

6 Features

Phenotypically Pn. asaccharolyticus cannot be distinguished from Pn. harei.

Pn. harei has an irregular cell- and colony morphology, Pn. asaccharolyticus is more regular.

In practice, this discrimination is difficult.

Library

Fluorescence

Bacterium Color of fluorescence

P. gingivalis, P. catoniae, P. levii none Other Porphyromonas sp. red, orange Lecture Pigmented Prevotella sp. red Non-pigmented gr- bacilli none, pink, orange, yellow Fusobacterium sp. chartreus Veillonella sp. red, none E. lenta red, none C. difficile chartreus C. innocuum chartreus C. ramosum red author Red fluorescence of a pigmented gram-negative rod Onlineby ©

Growth stimulation Some anaerobes require growth supplements for growth in a broth

The growth of several anaerobes in broth is stimulated by the addition of growth supplements Bacterium Supplement

Bacterium Supplement E. lenta 0.5 % arginine B. ureolyticus formate/fumarate (both 0.3%) Bilophila/Veillonella 1 % pyruvate B. fragilis group hemin Pigmented gr. – rods hemin ESCMIDGr.- rods 1 % sodiumbicarbonate Gr. + bacteria 0.5 % Tween-80 All anaerobes 1 % serum

7 Antibiotic sensitivity

E-tests: Anaerobes are: Inoculate a BBA agar with the bacterial suspension (0.5 McFarland) likeable using a cotton swab lethargic silly The MIC of fast growers can be determined after 18 hours, however cool definitively after 48 hours on holiday

If the isolate forms small colonies the agar plate can be inundated with the bacterial suspension Library

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