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* Corynebacteria

* Gram positive rods
Spore-forming AEROBIC Genus: Bacillus B. anthracis B. cereus B. subtilis ANAEROBIC Genus: Clostridium C. tetani C. botulinum C. difficile C. perfringens
Non spore-forming AEROBIC Corynebacteria C. diphtheriae diphtheroids Listeria monocytogenes ANAEROBIC Lactobacillus spp.

Corynebacterium spp

Gram positive bacilli, with characteristic morphology (club shaped and beaded)Non motile Non spore formingNon capsulatedNon--hemolytic on sheep blood agarFacultative anaerobicC. diphtheriae is fastidious while diphtheriods are non-fastidiousCatalase positiveOxidase negative

Species of Corynebacteria

Normal flora of RT, urethra, vagina, Skin
Other Significant Corynebacterium species C. xerosis C. pseudodiphtheriticum C. pseudotuberculosis C. jeikeium, (skin) C. ulcerans
Corynebacterium diphtheriae


* Introduction – C. diphtheriae Diphtheros – leather (tough, leathery pseudomembrane)Also known as Klebs–Loeffler bacillusCauses Diphtheria


* Important features of C. diphtheriae
Slender Gram positive bacilli Pleomorphic, non motile, non sporingChinese letter or Cuneiform arrangement Stains irregularly, tends to get easily decolorised May show clubbing at one or both ends - Polar bodies/ Metachromatic granules/ volutin or Babes Ernst granulesMetachromatic Granules:made up of polymetaphosphateBluish purple color with Loeffler’s Methylene blueSpecial stains

* Virulence factor

Exotoxin – Diphtheria toxin:Protein in naturevery powerful toxinResponsible for all pathogenic effects of the bacilliProduced by all the virulent strainsTwo fragments A & B

Mechanism of Action of Diphtheria Toxin: Inhibition of Protein Synthesis

* Epidemiology
Habitat – nose, throat, nasopharynx & skin of carriers and patientsSpread by respiratory droplets, usually by convalescent or asymptomatic carriersIncubation period of diphtheria – 3 to 4 days

* Diphtheria

Site of infection Faucial (palatine tonsil) – commonest typeLaryngealNasalOtiticConjunctivalGenital Cutaneous – usually a secondary infection on pre-existing lesion, caused by non toxigenic strains

* Pathogenesis & Clinical Manifestations

Human Disease Usually begins in respiratory tract Virulent diphtheria bacilli lodge in throat of susceptible individual Multiply in superficial layers of mucous membrane Elaborate toxin which causes necrosis of neighboring tissue cells Inflammatory response eventually results in pseudomembrane (fibrinous exudate with disintegrating epithelial cells, leucocytes, erythrocytes & bacteria) Usually appears first on tonsils or posterior pharynx and spreads upward or down In laryngeal diphtheria, mechanical obstruction may cause suffocation Regional lymphnodes in neck often enlarged (bull neck)

* Complications of diphtheria

Mechanical complications are due to the pseudomembrane, while the systemic effects are due to the toxin.Asphyxia – due to obstruction of respiratory passageToxic myocarditisCongestive heart failure Postdiphtheritic paralysis – occurs in 3rd or 4th week of disease, spontaneous recoverySepsis – pneumonia & otitis media


*

* Laboratory Diagnosis

Specimen – swab from the lesionsMicroscopy Gram stain: Gram +ve bacilli, chinese letter patternImmunofluorescenceAlbert’s stain for metachromatic granules

Biochemical Reaction

All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)

* Laboratory Diagnosis

Culture – isolation of bacilli requires media enriched with blood, serum or eggBlood agar Loeffler’s serum slope – rapid growth, 6 to 8 hrsTellurite blood agar – tellurite is reduced to tellurium, gives gray or black color to the colonies

* Growth of diphtheria bacilli

Blood agar
Loeffler’s serum slope Tellurite blood agar


3 biotypes of C. diphtheriae are characterized on BTA i.e. Gravis, mitis and intermedius biotypes The most severe disease is associated with the gravis biotype Colony of gravis biotype is large, non-hemolytic & grey. Colonies of mitis biotype are small, hemolytic and black Colonies of intemedius biotype are intermediate in size, non-hemolytic with black center & grey margin.

* Laboratory Diagnosis


* Laboratory Diagnosis
Virulence tests - Test for toxigenicityInvivo tests – animal inoculation (guinea pigs)Subcutaneous testIntracutaneous testInvitro testsElek’s gel precipitation testTissue culture test

Detection of toxin: Elek’s Test Principle: It is toxin/antitoxin reaction Toxin production by C.diphtheriae can be demonstrated by a precipitation between exotoxin and diphtheria antitoxin Procedure: A strip of filter paper impregnated with diphtheria antitoxin is placed on the surface of serum agar The organism is streaked at right angels to the filter paper Incubate the plate at 37C for 24 hrs


Resuls: After 48 hrs incubation, the antitoxin diffusing from filter paper strip and the toxigenic strains produce exotoxin, which diffuses and resulted in lines four precipitation lines radiating from intersection of the strip and the growth of organism
Filter paper saturated with diphtheria antitoxin
Inoculated M.O.
Positive Elek’s Test Lines of precipitations

* Laboratory Diagnosis Virulence tests - Invitro tests

Tissue culture test - incorporation of bacteria into agar overlay of eukaryotic cell culture monolayers. Result: toxin diffuses into cells and kills them

* Treatment

specific treatment must not be delayed if clinical picture suggests of diphtheria rapid suppression of toxin-producing bacteria with antimicrobial drugs (penicillin or erythromycin) early administration of antitoxin: 20,000 to 1,00,000 units for serious cases, half the dose being given IV

* Prophylaxis

1.active immunization (vaccination) DPT - triple vaccine given to children; contains diphtheria toxoid, Tetanus toxoid and pertussis vaccine Schedule i) Primary immunization - infants and children - 3 doses, 4-6 weeks interval - 4th dose after a year - booster at school entry ii) Booster immunization - adults -Td toxoids used (travelling adults may need more)


* Prophylaxis
Passive immunization ADS (Antidiphtheritic serum, antitoxin) - made from horse serum - 500 to1000 units subcutaneously Combined immunization First dose of adsorbed toxoid + ADS, to be continued by the full course of active immunisation

* CONTROL

isolate patients treat with antibiotics actively complete vaccination schedule should be used with booster every 5 years

Listeria

* Listeria is a genus of bacteria that contains 10 species, each containing two subspecies. Listeria species are gram-positive, rod-shaped, facultatively anaerobic, and non spore-forming. The major human pathogen in the Listeria genus is L. monocytogenes. It is usually the causative agent of the relatively rare bacterial disease listeriosis, a serious infection caused by eating food contaminated with the bacteria. The disease affects pregnant women, newborns, adults with weakened immune systems, and the elderly.

Listeria monocytogens gram staining method

*

* Listeriosis is a serious disease for humans; the overt form of the disease has a case-fatality rate of about 20%. The two main clinical manifestations are sepsis and meningitis. Meningitis is often complicated by encephalitis, when it is known as meningoencephalitis, a pathology that is unusual for bacterial infections

Treatment

* In non-invasive listeriosis, the bacteria often remain within the digestive tract, causing mild symptoms lasting only a few days and requiring only supportive care. Muscle pain and fever in mild cases can be treated with pain relievers, and diarrhea and gastroenteritis can be treated with medications if needed. In invasive listeriosis, the bacteria have spread to the bloodstream and central nervous system. Treatment includes intravenous delivery of high-dose antibiotics and in-patient hospital care





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