Corynebacteria

Gram positive rods Spore-forming AEROBIC ANAEROBIC Non spore-forming 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 forming Non capsulated Non--hemolytic on sheep blood agar Facultative anaerobic C. diphtheriae is fastidious while diphtheriods are non-fastidious Catalase positive Oxidase negative

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

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

Important features of C. diphtheriae Slender Gram positive bacilli Pleomorphic, non motile, non sporing Chinese 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 granules Metachromatic Granules: made up of polymetaphosphate Bluish purple color with Loeffler’s Methylene blue Special stains

Virulence factor Exotoxin – Diphtheria toxin: Protein in nature very powerful toxin Responsible for all pathogenic effects of the bacilli Produced by all the virulent strains Two fragments A & B

Mechanism of Action of Diphtheria Toxin: Mechanism of Action of Diphtheria Toxin: Inhibition of Protein Synthesis

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

Diphtheria Site of infection Faucial (palatine tonsil) – commonest type Laryngeal Nasal Otitic Conjunctival Genital 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 passage Toxic myocarditis Congestive heart failure Postdiphtheritic paralysis – occurs in 3rd or 4th week of disease, spontaneous recovery Sepsis – pneumonia & otitis media

Laboratory Diagnosis Specimen – swab from the lesions Microscopy Gram stain: Gram +ve bacilli, chinese letter pattern Immunofluorescence Albert’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 egg Blood agar Loeffler’s serum slope – rapid growth, 6 to 8 hrs Tellurite blood agar – tellurite is reduced to tellurium, gives gray or black color to the colonies

Growth of diphtheria bacilli Blood agar Tellurite blood agar Loeffler’s serum slope

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 Biochemical reactions Ferments sugar with acid formation but not Gas ferments: glucose, galactose, maltose and dextrin, but not ferment sucrose. Resistant to light, desiccation and freezing Sterilization: sensitive to heat (destroyed in 10mins at 58°C or 1min in 100°C), chemical disinfectants

Laboratory Diagnosis Virulence tests - Test for toxigenicity Invivo tests – animal inoculation (guinea pigs) Subcutaneous test Intracutaneous test Invitro tests Elek’s gel precipitation test Tissue 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

Filter paper saturated with diphtheria antitoxin 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 Lines of precipitations Inoculated M.O. Positive Elek’s Test

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 25

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