Differentation of Anaerobic Bacteria According to the Wadsworth Method

Differentation of anaerobic bacteria according to the Wadsworth method

Peptostreptococcus Veillonella Anaerococcus Peptoniphilus Parvimonas Finegoldia

Clostridium-Eubacterium B. fragilis Actinomyces © by author C. ureolyticus Propionibacterium Prevotella ESCMIDBifidobacterium Online Lecture PorphyromonasLibrary Lactobacillus-Atopobium Fusobacterium Bilophila (non-) selective media

BBA isolation of all anaerobic bacteria Brucella Blood Agar

PEA isolation of almost all anaerobic bacteria PhenylEthyl Alcohol blood agar

BBE isolation of Bacteroides fragilis sp. and Bacteroides Bile Esculin agar Bilophila wadsworthia, inhibition of gal- sensitive bacteria

BBKV © by isolation author of Bacteroides sp. and Brucella Blood agar with Prevotella sp., inhibition of Gram-positive Kanamycin and Vancomycin bacteria, promotes pigmentation (lakedESCMID blood) Online Lecture Library

Additional media

CCFA isolation of Clostridium difficile Cycloserine Cefoxitin inhibition of almost all anaerobic bacteria Fructose Agar

MMBA isolation of Actinomyces sp. Metronidazole Mupiro- inhibition of most anaerobic bacteria cine Blood Agar

EYA isolation of lipase- and/or lecithinase positive Egg Yolk Agar anaerobic© by authorbacteria

ESCMID Online Lecture Library Level II identification

Ka Va Ct bile cat ind

B. fragilis group R R R R V V Porphyromonas sp. R S R S V +- Prevotella sp. RS R V S - V B. ureolyticus S R S S - - Bilophila wadsworthia S R S R + - Fusobacterium sp. S R S V - V Veillonella sp. © by S author R S S V -

Ka = kanamycin ESCMID Online Lecture LibraryVa = vancomycin Ct = colistin kat = catalase Wadsworth manual, Table 4-2, p. 58 ind = indole Prevotella sp./ Porphyromonas sp.

Identification is based pigment formation

ESCMID Onlinepigmented colonies Lecture Library Fusobacterium sp.

CellESCMID morphology is Onlineimportant for identificationLecture Library F. nucleatum, thin rods with pointed ends

Ka 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 by S author R V V -+ positive cocci Ka = kanamycin ESCMID Online Lecture LibraryVa = vancomycin Ct = colistin kat = catalase ind = indole Wadsworth manual, Table 4-3, p. 60 nitr = nitrate

Clostridium sp.

Anaerobic Gram-positive rods which can be Gram-variable

Spore demonstration: spore-stain alcohol-shock

C. botulinum with spores Lecithinase-positive clostridia

ESCMID Online LectureC. perfringens Library: Nagler-test Swarming clostridia

Lipase reaction C. septicum

For phenotypic identification clostridia are divided into 3 groups:

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

Identification is based on sugar fermentation and other supplemental tests © by author ESCMID Online Lecture Library Clostridia who do not follow the rules

C. tertium, grown anaerobically. Spores are present

C. tertium, grown aerobically. SporesESCMID are not present, Online morphology Lecture Library resembles Lactobacillus © by author

ESCMIDC. clostridioforme Online, stains Gram-negative,Lecture cellsLibrary are fusiform Characteristics C. difficile

Selective medium: CCFA

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

Specific odour: horse stable (production IC)

Chartreuse fluorescence

Proteolytic+ saccharolytic© by author ESCMID Online Lecture Library C. difficile a blood agar and Gram-stain Clostridia who do not follow the rules

C. tertium, grown anaerobically. Spores are present

C. tertium, grown aerobically. SporesESCMID are not present, Online morphology Lecture Library resembles Lactobacillus 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. © by -author - -+ ESCMID Online Lecture Library

Most reliable method: gas-chromatography B. breve in pus from a liver abscess

- indole + - indole +

Eubacterium sp. P. acnes

- ADH +

E. lenta

P GLC S

main product GLC

SuccinateESCMID Propionate Online Butyrate Lecture Acetate Library Lactate Actinomyces Propionibacterium Eubacterium Bifidobacterium Lactobacillus Collinsella Atopobium Eggerthella Eubacterium Gram-positive anaerobic cocci (GPAC)

Level II: Ka Va Ct SPS

F. magna Cell morphology GPAC

Small cocci: P. micra

Large cocci: F. magna A. vaginalis Pn. ivorii

Other GPAC: “normal” size ESCMID Online Lecture Library most GPAC can be identified using enzym reactions

Most reactions are present in the Rapid ID 32A

The code obtained with© bythis API-stripauthor is not suitable for identification. ESCMID Online Lecture Library Use table from Wadsworth manual.

Features

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

In practice, this discrimination is difficult.

Bacterium Color of fluorescence

P. gingivalis, P. catoniae, P. levii none Other Porphyromonas sp. red, orange 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 © by author chartreus C. innocuum chartreus C.ESCMID ramosum Online Lecture red Library © by author ESCMID Online Lecture Library Red fluorescence of a pigmented gram-negative rod Anaerobes are:

 likeable  lethargic  silly  cool  on holiday