Microbiology PHT 123

2010-20112nd Term 2nd Semester

Gram positive non-sporing bacilli: Corynebacterium diphtheriae, dipherioids, and Listeria monocytogenes

1. Corynebacterium diphtheriae (Klebs-Löffler bacillus)

Points to consider:Dephtheria is a very old historical disease was called 'the strangling

angel of children‘ in the 4th century BC. The then father of medicine, Hippocrates, was the first to describe it.

Respiratory diphtheria (pseudomembrane on pharynx) and cutaneous diphtheria

Prototype A-B exotoxin acts systemically Toxoid in DPT and TD vaccines

Diphtheria toxin encoded by tox gene introduced by lysogenic bacteriophage (prophage)

Selective media: cysteine-tellurite; serum tellurite; Loeffler’s

Three speceis are known: C. diphtheriae mitis, C. diphtheriae intermedius, C. diphtheriae gravis,

and Gravis, intermedius, and mitis colonial morphology

Seminar Topics1. Staphylococcal food poisoning2. Staphylococcus aureus toxic shock

syndrome (TSS)3. Staphylococcus Scaled Skin

Syndrome (SSSS)4. Community acquired methicillin

resistant S.aureus (CA-MRSA)5. Basic principles in Strain Typing

(DNA Fingerprinting)6. Polymerase Chain Reaction (PCR)

principles7. DNA sequencing principles8. Streptococcal necrotizing fasciitis

(flesh eating disease)9. Three types of anthracis10. Case-definitions in anthracis11. Antibiotic sensitivity testing12. Supporative streptococcal diseases13. Nonsupporative streptococcal

diseases14. Gonorrhea differences in men and

women15. Waterhouse-Friderichsen Syndrome

16. Epidemics of meningitis in the Meningitis Belt in Africa

17. Local and systemic effects of diphtheria

18. Clinical Diagnosis of Tetanus (effects of tetanospasmin)

19. Physical diagnosis of botulism foodpoisoning

20. Reasons for Clostridium difficile-Associated Diarrhea (CDAD)

21. How vaccines work?22. Listeriosis: causes, reasons, risk

groups23. Laboratory Safety Protocols24. Types of human viruses and

Bactriophages 25. H1N1 26. Outline Classification of fungi

(Four classes)27. Differences between Prokaryotic

and Eukaryotic cells28. Antimicrobial resistance29. Nsoscomial Multidrug resistance30. DNA

Two types of diphtheria syndromes:1. Local toxigenic effects: elicit inflammatory response and

necrosis of the faucial mucosa cells-- formation of "pseudo-membrane“ (composed of bacteria, lymphocytes, plasma cells, fibrin, and dead cells), causing respiratory obstruction.

2. Systemic toxigenic effects: necrosis in heart muscle, liver, kidneys and adrenals. Also produces neural damage.

http://www.vaccineinformation.org/photos/index.asp

CORYNEBACTERIAPrevalent in baby’s after 3-6 months (that’s why DPT is given at 2, 4,

6 months, boosters at 18 months and at school entry), very high in young children Shick test – identiies non immune people: diluted stabilized toxin I/Derm,

localized erythema (1-3cm) in 2-4 days, means no or little antibodies

Diphtheria Skin lesion on leg

MorphologyAerobic Gram +ve bacilli, nonmotile, uncapsulated, club-shaped

rodIn stained smears bacilli are palisade Chinese letter arrangementMetachromatic granules at poles) give the rod a beaded appearance.Corynebacterium diphtheriae

Normal flora of nasopharynxDiphtheria caused when infected by lysogenic

Bacteriophage (heat labile toxin A and B)Diptheroids

Normal flora of skinCan cause disease in ‘compromised’ host

C. diphtheriae has Three biotypesGravis (severe) Inter-medius (intermediate) Mitis (mild)

Portal of entry: respiratory tract or skin abrasions.

Some Lab Diagnostic propertiesDirect smear -Gram stain- Gram positive bacilli:

Straight/curved , enlarged(club-shaped) at ends Arranged Chinese letters or V shape

Direct smear- Albert's stain, special strain for metachromatic granules Loeffler’s egg-serum agar slope for metachromatic granules, (also called polyphosphates, volutine granules, or Babes-Ernst granules)

Catalase positive

Selective media Cystine-potassium-tellulrite medium, black colonies, inhibits diphtherioids and

other Gram positivesCheck the toxigenicity Animal inoculation Guinea pigs/rabbits

Death within 96 hrs

Toxin production test by Elek's plate test (at 37C for 24 hrs}

Streak Test Strain on plate

Filter paper with antitoxin antibody

To test production of exotoxin from toxigenic strains of Corynebacterium diphtheriae to differentiate from normal flora…..diphtheriods

Precipitin lines

Brief Outline on Diphtheria Management 1. Patients–isolate patients/bed rest/antibiotic treatment/antitoxins (horse serum)

Penicillin/erythromycin/teracycline/rifampicin/clindamycin2. Contacts – immunize if not (toxoid) – adults should be schick tested or given low

dose as immunization of immune adults can result in severe reaction. prophylactic antibiotic – erythromycin swab nose and throats of contacts

3.Community – immunization

Prevention: Active immunization with toxoid (DTPa-hib)Other corynebacteria are normal flora of the skin and

URTCalled diphtherioids, cause disease only in compromised

patients

Basic principles of immunizaitonWhen small amount of diluted diphtheria toxin (0.1 ml) is injected in the skin, a skin reaction occurs ( a ring of 5–10 mm diameter) if a person is not immunized…If a person is immunized antibodies will neutralize the toxin and no rxn

Listeria monocytogenesThe only strain the infects humans

Fridge-Friendly PathogenGram positive, motile tumpling, beta hemolytic, uncapsulated

nonsporing, coccobacillihttp://www.youtube.com/watch?v=fjD_ruKmSfA Tumbling

Resistant to cold, heat, salt, pH extremes and bileGrow in refrigeration (Food poisoning risk)Most common foodborne outbreaksUsually food-borne transmission, asymptomatic intestinal carrierHigh risk:

Neonates Elderly and pregnant women

Listeriosis - most cases associated with dairy products, poultry, and meat

Virulence attributed to ability to replicate in the cytoplasm of cells after inducing phagocytosis; avoids humoral immune system

Invasive and Intracellular

Clinical signs of Listeriosis Bacteremia and sepsisCNS listeriosis (meningitis, meningoencephalitis,

cerebritis, brainstem encephalitis, and brain or spinal abscess)

EndocarditisFocal infectionsRecurrent spontaneous abortiongranulomatosis infantisepticum

Diagnostic Aids:CAMP test positive (strep)Culture requires lengthy cold enrichment process.Rapid diagnostic tests using ELISA available

Treatment and Control

For severe infections:Ampicillin (200 mg/kg/d i.v. divided in six

doses) or Penicillin (300,000 mg/kg/d i.v. divided in

six doses)Combined with gentamicin (1–2 mg/kg every

8 hours, adjusted with renal function and followed by levels)

Penicillin-allergic patients:Trimethoprim-sulfamethoxazole (20 mg/kg

per day of the Trimethoprim component IV in four divided doses)

Combination of ampicillin and trimethoprim-sulfamethoxazole might be more effective