Theme: Microbiological diagnosis of anaerobic diseases. - презентация

1 Theme: Microbiological diagnosis of anaerobic diseases

2 Spore- forming (Clostridia) Nonspore-forming Gram-negativeGram-positive C.botulinumRod-shaped C.tetaniBacteroidesActinomyces C.perfringensPrevotellaBifidobacterium C.novyiFusobacteriumEubacterium C.septicumLeptotrichiaPropionibacterium C.histolyticumCocci C.dificilleVeilonellaPeptococcus (about 100 species) Peptostreptococcus Spiral forms Treponema, Borrelia

3 Effects of oxygen on aerobic, anaerobic, and facultative anaerobic bacteria

4 Chemical dynamics in anaerobic bacteria

5 Anaerobic culture system (anaerobic jar or GasPak jar)

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7 Nutrient media Milk under oilKitt-Tarozzi medium Wilson-Blair agar Sugar blood agar

8 Family Bacillaceae Genus Clostridium Causative agents: tetanus - C.tetani gas gangrene: C.perfringens, C.novyi, C.septicum, C.histolyticum botulism - C.botulinum pseudomembranous enterocolitis -C.difficile

9 C.tetani. Tetanus risus sardonicus opisthotonos Trismus or lockjaw

10 Mechanism of Action of Tetanus Toxin

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12 Clostridia of gas gangrene C.histolyticum С. septicum C.novyiC.perfringens

13 Micro & Macroscopic C. perfringens NOTE: Large rectangular gram-positive bacilli Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α toxin NOTE: Double zone of hemolysis

14 C. perfringens Virulence Factors Major Minor

15 C. perfringens Nagler Reaction NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes phospholipids in egg-yolk agar around streak on right. Antibody against α-toxin inhibits activity around left streak.

16 Gas gangrene

17 Microbiological diagnosis of anaerobic infection Specimen: wound exudate, edema liquid, necrotic tissues. Methods: 1.IF. 2.Microscopy. 3.Bacteriological. 4.Biological (neutralization test). 5.PCR.

18 Scheme of microbiological diagnosis of anaerobic infection 1 step 2 step 3 step 4 step Microscopic examination Bacteriological method Biological method Gram stain result Express test – lecitinase test Seeding in Kitt- Tarozzi medium, milk under oil result Gram stain Seeding on sugar- blood agar in Petri dish Guinea pig inoculation Morphology of colonies, Gram stain Seeding in Kitt-Tarozzi medium resultSeeding in Hiss media Neutralization test to detect exotoxin (mouse inoculation) Definitive diagnosis Specimen: wound exudate, edema liquid, necrotic tissues

19 Lecitinase test Lecitinase positive colonies of C.perfringens on egg yolk agar

20 Differentiation of Clostridia due to culture properties Sugar-blood agar: –С. perfringens –С. novyi –С. septicum –С. histolyticum

21 C. botulinum Gram stain Growth on sugar-blood agar

22 Botulism toxin

23 Mechanism of Action of Botulinum Toxin

24 Botulism toxin action

25 Botulism Ptosis Wound botulism

26 Laboratory diagnosis of botulism Specimens: food, feces, vomit, may be patients blood. Culture: Isolation of organism. Biological: Demonstration of C.botulinum toxin on the guinea pigs. Detection of toxin: PHAT.

27 Scheme of laboratory diagnosis of botulism Culture

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29 Scheme of laboratory diagnosis of botulism

30 Neutralization test for detection of toxin Control (specimen) Specimen + antibodies against toxin А Specimen + antibodies against toxin В Specimen + antibodies against toxin E

31 BoNT A (Botox) Botox injection patient 13 weeks after injection Sadick, N. and A.R. Herman (2003). Comparison of Botulinum Toxins A and B in the Aesthetic Treatment of Facial Rhytides. Dermatologic Surgery 29:

32 C. difficile

33 C. dificile Virulence Factors

34 Laboratory diagnosis of pseudomembranous enterocolitis Specimens: feces. Culture: Isolation of organism. Detection of toxin: ELISA, cell culture. PCR.