microbiology corynebacterium-11 fcm (1).pptx

microbiology corynebacterium

1. C ORYNEBACTERIUM Presented By : Rahul Shreya Rimsha Akshita

2. C ONTENTS

3. I NTRODUCTION Gram – positive, non – acid fast, non – motile rods Frequently show club – shaped swellings { hence, named “corynebacterium” ( coryne means club )} Most important member of the genus is n “ CORYNEBACTERIUM DIPHTHERIAE ” , the causative agent of the diphtheria.

4. CORYNEBACTERIUM DIPHTHERIAE

5. MORPHOLOGY Are pleomorphic, non-capsulated, thin, slender and gram – positive bacilli Measure about 3-6 µm x 0-.6-0.8 µm club shaped ( presence of metachromatic granules) bacilli are usually seen in angular fashion, resembling letters V or L ( called as Chinese letter or cuneiform arrangement) Bacilli look green and metachromatic granules appear bluish black on Albert staining

6. CULTURAL CHARACTERISTICS Grown best on media enriched with “ BLOOD, SERUM OR EGG ” Aerobic and facultatively anaerobes Optimum temperature for growth is 37ºC & optimum pH is 7.2 Media employed for cultivation of diphtheria bacillus are: HISS’S SERUM WATER LOEFFLER’S SERUM SLOPE TELLURITE BLOOD AGAR MEDIUM

7. HISS’S SERUM WATER Liquid medium containing serum Growth is seen as a turbidity and pellicle formation

8. LOEFFLER’S SERUM SLOPE  Diphtheria bacilli grows rapidly on this medium Colonies can be seen in 6-8 hours of incubation colonies are small, circular, white or creamy and glistening

9. TELLURITE BLOOD AGAR MEDIUM Potassium tellurite selective agent Organisms grow slowly on this medium and form grey or black colonies Colonies may take two days to appear on this medium Based on colony morphology on tellurite medium and other properties, three main biotypes can be seen- gravis, intermedius and mitis can be distinguished

10. BIOCHEMICAL REACTIONS Ferment glucose and maltose with the production of acid but without gas GRAVIS : ferments starch and glycogen INTERMIDIUS AND MITIS : have no such action Reduces nitrates to nitrites Do not hydrolyse urea or form phosphatase Gelatin is not liquefied

11. TOXIN

12. R ESISTANCE Readily dies when exposed to a temperature of 58ºC for 10 minutes or 100ºC for 1 minute Resistant to drying Susceptible to penicillin, erythromycin and broad spectrum antibiotics

13. P ATHOGENESIS Diphtheria is mostly seen in 2 to 10 years of children Infection is confined to humans only Incubation period is 3 to 4 days Infection occurs by way of droplet spread Diphtheria is of the following clinical types : Faucial Laryngeal Nasal Conjunctival Otitic Vulvovaginal Cutaneous (mainly around mouth and nose)

14. LABORATORY DIAGNOSIS Laboratory confirmation is necessary for control measures and epidemiological studies but not for the treatment of cases Laboratory diagnosis consists of isolation of organism and demonstration of it’s toxicity

15. ISOLATION OF ORGANISMS COLLECTION OF SPECIMEN two swabs from the lesions are collected 1. smear examination 2. culture swabs are collected prior to start of antibiotics and application of antiseptics Swabs are rubbed over the affected area and pseudomembrane , if formed If there’s no definite localized lesion, swabs should be rubbed over tonsils and posterior pharyngeal wall

16. ISOLATION OF ORGANISMS Direct Microscopy smears are stained with both Gram and Albert stain Albert staining: diphtheria bacilli shows beaded slender green rods in typical Chinese letter pattern Gram staining: done to identify Vincent’s spirochaetes and fusiform bacilli

17. ISOLATION OF ORGANISMS CULTURE Swabs are inoculated in the following culture media: Loeffler’s serum slope Growth appears within 6-8 hrs Subculture from Loeffler’s is made on tellurite blood agar Plate is incubated at 37ºC for 48 hours Tellurite blood agar Incubation at 37ºC for at least 48 hours before declaring these as negative, as growth can sometimes be delayed Blood agar useful for differentiating streptococcal or staphylococcal pharyngitis, which can stimulate diphtheria

18. ISOLATION OF ORGANISMS COLONY MORPHOLOGY AND STAINING Loeffler’s serum slope: Colonies are small, circular, white or creamy Tellurite blood agar: Diphtheria bacilli grow as black or grey coloured colonies Albert staining: green bacilli with bluish black metachromatic granules is seen Gram staining: reveals gram positive bacilli

19. ISOLATION OF ORGANISMS Biochemical reactions Hiss’ s serum water is used for testing fermentation of carbohydrates *only gravis biotype is positive GLUCOSE A LACTOSE - MANNITOL - SUCROSE - MALTOSE + NO 3 REDUCTION + INDOLE - UREASE - PHOSPHATASE + CATALASE + OXIDASE - GLYCOGEN* + STARCH* +

20. VIRULENCE TESTS Demonstrate the production of exotoxin by bacteria isolated on culture Can be done by two methods : 1) IN VIVO TESTS 2) IN VITRO TESTS 20

21. VIRULENCE TESTS In vivo tests Subcutaneous and intracutaneous test Guinea pigs and rabbits In vitro tests Elek’s gel precipitation test and tissue culture tests

22. S CHICK TEST An intradermal test Done to demonstrate circulating diphtheria antitoxin Antitoxin may be present either due to previous infection or immunisation Results are read after 1, 4 and 7 days There could be four types of reaction Positive reaction Negative reaction Pseudoreaction Combined reaction

23. Positive reaction signifies that the person is susceptible to diphtheria due to either absence or lack of adequate amount of circulating antitoxin Negative reaction Indicates that toxin has been neutralised by sufficient amount of antitoxin present in the blood and that the person is immune to diphtheria Pseudoreaction Indicates that the individual is immune to diphtheria and also hypersensitive to the components of diphtheria bacilli Combined reaction Indicates that the individual is susceptible to diphtheria & hypersensitive to bacillus Necessary to immunise such persons but vaccine may likely to induce reaction *SCHICK TEST IS NOT USED NOWADAYS

24. P ROPHYLAXIS Active Immunisation Started at 6 weeks of by toxoid in combination with tetanus toxoid and pertussis vaccine (DPT, Triple vaccine) 3 doses are given by intramuscular route at an interval of 4-6 weeks Booster doses of DPT are given at 18 months and at 5 years Passive Immunisation 500-1000 units of antitoxin [ anti diphtheric serum, ADS ] is administered subcutaneously Combined immunisation All persons receiving ADS as prophylactic measure should receive this type of immunisation

25. T REATMENT Erythromycin (orally or by injection) for 14 days (40 mg/kg per day with a maximum of 2 g/d) or, Procaine penicillin G given IM for 14 days ( 300,000 U/d for patients weighing < 10 kg and 600,000 U/d for those weighing > 10 kg) Patients with allergies to penicillin G or erythromycin can use rifampin or clindamycin t