Micro Lec 6 (1) PDF

Lect.2 ,3 Gm +ve P.D.Amal abo kamer Streptococci  STREPTOCOCCI MORPHOLOGY Gram-positive cocci – ovoid or spherical Arranged in chains Smaller than Staphylococci Some are capsulated CULTURAL CHARACTERISTICS Grow on enriched media – Blood agar Aerobic and facultati...

Lect.2 ,3 Gm +ve P.D.Amal abo kamer Streptococci  STREPTOCOCCI MORPHOLOGY Gram-positive cocci – ovoid or spherical Arranged in chains Smaller than Staphylococci Some are capsulated CULTURAL CHARACTERISTICS Grow on enriched media – Blood agar Aerobic and facultative anaerobic 2-5% CO2 enhance their growth Catalase – negative (Stpahylococci – positive)  CLASSIFICATION OF STREPTOCOCCI I. On the basis of haemolysis on Blood Agar A. Alpha haemolysis (partial haemolysis of RBCs) Greenish zone around colonies due to degradation of haemoglobin to biliverdin (green) e.g. S. pneumoniae, Viridans streptococci B. Beta haemolysis (complete haemolysis of RBCs) Clear zone around colonies e.g. S. pyogenes, S. agalactiae C. Gama haemolysis (no haemolysis) – misnamed Some Enterococci II. Lancefield grouping (on the basis of C- carbohydrate antigen in cell wall of streptococci) Groups A-U (group A,B & D are of great pathogenic significance. Group A = S. pyogenes Group B = S. agalactiae Group D = Enterococci Basically beta-haemolytic are grouped by this method Some alpha and non-haemolytic streptococci contain Lancefield group antigens  CLASSIFICATION ????? Group A - Streptococcus pyogenes Group B - Streptococcus agalactiae Group C - Streptococcus equi, Streptococcus zooepidemicus Group D - Enterococci, Streptococcus bovis Group E - Streptococcus milleri and mutans Group F - Streptococcus anginosus Group G - Streptococcus canis Group H - Streptococcus sanguis Group L - Streptococcus dysgalactiae Group N - Lactococcus lactis Group R&S - Streptococcus suis other Streptococcus species are classified as non-Lancefield Streptococci BETA-HAEMOLYTIC STREPTOCOCCI Medically important species 1. S. pyogenes (Lancefield gp. A) 2. S. agalactiae (Lancefield gp B) LANCEFEILD  Streptococcus pyogenes (GROUP A STREP) MORPHOLOGY Spherical / ovoid cells in chains Gram positive Some strains capsulated CULTURAL CHARACTERISTICS Pin point, beta-haemolytic colonies on blood agar Haemolysis is due to Streptolysins O & S Aerobic and facultative anaerobic ANTIGENIC STRUCTURE 1. C-carbohydrate antigen (Lancefield gp A) 2. M protein (fibronectin-binding protein )- Surface protein > 80 serotypes A major virulence factor of S. pyogenes. Antiphagocytic Due to different M-proteins some strains are rheumatogenic and others nephritogenic. 3. Lipoteichoic acid – associated with pili 4. Capsular antigen - antiphagocytic M PROTEIN M PROTEIN  Streptococcal cell wall structure Capsule M protein C-carbohydrate Lipotechoic acid Phospholipid cytoplasmic PBP PBP membrane Transport protein  HAEMOLYSINS, ENZYMES & TOXINS 1. HEMOLYSINS (STREPTOLYSINS) Streptolysin O (O is from oxygen labile) Causes lysis of WBCs, platelets, tissue cells under anaerobic condition only Antigenic – lead to formation of “antistreptolysin O antibodies” – basis of “ASO titre test”. Streptolysin S (S is from oxygen stable) Causes lysis of cells under aerobic condition Non-antigenic 2. STREPTOKINASE Causes lysis of fibrin by activating plasminogen to plasmin. Used in treatment of myocardial infarction. Antigenic-leads to antistreptokinase antibodies 3. DNAse B Antigenic : anti-DNAase B are produced 4. Hyaluronidase (spreading factor) - Lysis of hyaluronic acid- spread of infection - Cause of diffuse spreading lesions of Strep cellulitis 5. Erythrogenic Toxin (pyrogenic toxin) - Antigenic antitoxin formation - Causes scarlet fever  PATHOGENESIS OF INFECTION = Attach by lipoteichoic acid to host cell surface fibronectin (in pharynx) = Streptokinase, DNAse, hyaluronidase – invasion and spread of infection = M-protein-major virulence factor – antiphagocytic = Streptolysins produce tissue injury DISEASES CAUSED BY S.pyogenes Diseases by S. pyogenes Pyogenic Poststreptococcal Due to infections complications toxins A. INFECTIONS 1. Streptococcal pharyngitis and tonsilitis Usually in the age of 5-15 years May spread to cause sinusitis, otitis media, meningitides, pneumonia 2. Impetigo Purulent infection of skin (vesicle ,crust, pustule ) 3. Erysipelas Dermal infection (skin + S.C. tissue) with spreading erythema and oedema 4. Cellulitis – spreading inflammation of S.C. tissue due to hyaluronidase --------- bacteremia 5. Puerperal infection Infection of endometrium within 14 days after labor or abortion ------life threatening Can spread to other pelvic organs and blood. B. DISEASE DUE TO ERYTHROGENIC TOXIN Scarlet Fever Streptococcal pharyngitis - erythrogenic toxin toxemia with remote effects The disease is characterized by: Redness of face with circumoral palor Red skin rash “sand paper” feel of skin Strawberry tongue Scarlet fever; strawberry or white tongue & red face C. POSTSTREPTOCOCCAL COMPLICATIONS 1. RHEUMATIC FEVER Complication of untreated streptococcal pharyngitis (after 2-3 weeks). Acute inflammation of joints, endocardium, (fever, carditis, migratory polyarthritis) Usually at 5-15 years of age; recurrence in adults. Repeated attacks lead to progressive damage to heart valves PATHOGENESIS OF RHEUMATIC FEVER endocardium ----scaring of heart valve -- -- - -------stenosis or incompetence (Rheumatic Heart Disease -RHD) - Laboratory diagnosis of rheumatic fever Increased ESR Increased antibody titre to at least one of streptococcal antigen (ASO, anti-DNAse, antistreptokinase) Significant ASO titre: 200 IU or more / ml ASO may be up to 800 IU / ml (normal = 50-160 IU / ml) Prevention of RHD Effective treatment of strep pharyngitis Chemoprophylaxis in-patients with history of rheumatic fever 2. ACUTE GLOMERULONEPHRITIS Complication of cutaneous infection by S. pyogenes (nephritogenic strains) = Latent period 10-20 days Disease of childhood Characterized by edema, hypertension, haematuria, proteinuria and occasionally renal failure. MECHANISM = Cross reaction between anti-strep antibodies and renal tissues. = Deposition of Ag-Ab complexes in glomeruli with activation of complement --- tissue injury. TREATMENT OF S. PYOGENES INFECTIONS X Amoxycillin : drug of choice X Erythromycin and Cephalosporin in case of penicillin allergy X Adequate treatment of Strep pharyngitis for 10 days will prevent rheumatic fever  Streptococcus agalactiae (GROUP B STREP) Normal flora of vagina (30%) Capsulated – antiphagocytic Beta-haemolysis on blood agar Bacitracin resistant (S. pyogenes - sensitive) CAMP test positive ( CAMP Christie, Atkins, and Munch- Peterson)  Principle detects the production of diffusible, thermostable, extracellular protein known as CAMP factor, produced by Group B Streptococcus.form a typical arrowhead or flame-shaped clearing at the junction of the two organisms when they are placed perpendicular to each other.  If inhibited by bacitracin disk, they are likely to be group A streptococci.  CAMP Factor Test S. aureus Group B Streptococcus (Spingomyelinase C) (CAMP Factor) Group A Streptococcus Enhanced Zone of Hemolysis B) CAMP test for the identification of Streptococcus agalactiae (group B). (A) Streptococcus (group B) shows a positive CAMP reaction. (B) Streptococcus pyogenes (group A) shows a negative reaction when inoculated at a right angle to (C) Staphylococcus aureus. DISEASES CAUSED BY S. AGALACTIAE Neonatal pneumonia & meningitis Contamination from mother’s birth canal Puerperal infection and septic abortion  TREATMENT Less susceptible to penicillins than group A Combined therapy = Amoxycillin + aminoglycosides  GROUP D STREPTOCOCCI Non-haemolytic or alpha-haemolytic colonies: Two types 1. Enterococci (E. faecalis) Normal flora of colon Can grow in presence of 7.5% NaCl and bile salts Diseases Opportunistic UTI Wound infections 2. Non-Enterococci (S. bovis) Can grow in presence of bile salts (not NaCl) May cause endocarditis  ALPHA-HAEMOLYTIC STREPTOCOCCI Medically important species 1. S. pneumoniae 2. Viridans streptococci  Streptococcus pneumoniae MORPHOLOGY Gram-positive cocci – lancet / bullet shape Diplococci (arranged in pairs) – the adjacent ends of pair are rounded while other ends are pointed Virulent strains are capsulated CULTURAL CHARACTERISTIC Facultative anaerobic; 5-10% CO2 enhance growth Grow on enriched media (blood & chocolate agar) Alpha-haemolytic colonies on blood agar PATHOGENICITY FACTORS Capsule Antiphagocytic S. pneumoniae > 80 serotypes on basis of antigenic differences in polysaccharide capsule.  Toxins and extracellular enzymes IgA protease : help in colonization in respiratory tract Pneumolysin - properties like Streptolysin Neuroaminidase – spreading factor  PATHOGENESIS IN LUNGS Colonization in resp. tract due to IgA protease Antiphagocytic effect of capsule Organisms multiply rapidly & cause an acute inflammatory response  Fluid & WBCs, RBCs pour into alveoli (get filled with inflammatory fluid) ----- poor gaseous exchange Difficulty in breathing No real structural damage to lungs – complete resolution on recovery. DISEASES CAUSED BY S. pneumoniae (10-30% of normal people carry one or more serotypes in throat) Pneumococcal pneumonia High grade fever, cough, rusty sputum (reddish) Difficult breathing, chest pain Meningitis (all age groups) Sinusitis Otitis media TREATMENT Penicillins – drugs of choice Penicillin-resistant strains have been reported due to alteration in PBP Penicillin-resistant strains are treated with erythromycin  PREVENTION Vaccination with polyvalent vaccine prepared from polysaccharide capsules of 23 serotypes. Given to susceptible groups – elderly patients, diabetics, chronic pulmonary disease, immunocompromised.  VIRIDANS STREPTOCOCCI It is α-hemolytic streptococci It is a group of streptococci all found in the oral cavity as part of the normal flora of the upper respiratory tract They are opportunistic pathogens which most commonly cause sub-acute bacterial endocarditis when there is an invasive operation as in tooth extraction S. mutans, S. sanguis, and S. mitis (e.g of the viridans gp ) cause dental carries Morphology Gram positive cocci arranged in chains Culture Facultative anaerobe α-hemolytic on blood agar  DIFFERENCES BETWEEN VIRIDANS STREP AND PNEUMOCOCCI Property S. pneumoniae Viridans strep Capsule + - Optochin sensitivity + - Bile solubility + - Quellung reaction + - Inulin fermentation + -  A PROBLEM A women gave birth to a child at 32 weeks of pregnancy After 24 hours the newborn baby developed meningitis CSF culture on blood agar showed beta- haemolytic colonies Gram-stained smear showed Gram- positive cocci in chains Questions What is the likely identity of the organism ? From where the newborn baby acquired this organism ? What antibiotic will be the most appropriate for treatment ? Name one more organism which causes the same disease in newborn ?  LAB IDENTIFICATION OF S. pyogenes 1. Direct Gram Smear 2. Culture on Blood Agar , Aerobic or anaerobic incubation , beta-haemolytic colonies-identified as S. pyogenes by: i) Gram staining ii) Susceptible to bacitracin (Strep gp B - resistant) iii) Serologic detection of gp A antigen 3. Direct Group A antigen  4. Serodiagnosis (in post strep. complications) ASO titre Anti-DNAse-B antibodies Anti-Streptokinase 5. M-typing , For epidemiological purpose  LAB IDENTIFICATION OF S.agalactiae CAMP TEST (Christie, Atkins and Munch Peterson) S. agalactiae produces an extracellular diffusible protein – CAMP factor which interacts with Staphylococcal beta- haemolysin on sheep or ox RBCs.  Method: Streak a known Staph. culture across 10% sheep blood agar plate. Streak the test organism (S. agalactiae) at right angles to it Also an enterocuccus (negative control) is inoculated Incubate at 37oC overnight An arrowhead haemolytic area indicates interaction with Staph.haemolysin – Positive test.  CAMP TEST FOR STREPTOCOCCUS AGALACTIAE Arrow head of haemolysis S. agalactiae culture Enterococcus S. aureus negative control culture Blood agar plate  LAB IDENTIFICATION OF S. pneumoniae SPECIMENS : Sputum, CSF, Blood DIRECT GRAM SMEAR Gram +ve cocci in pairs in sputum and CSF. STREPTOCOCCUS PNEUMONIAE  CULTURE ON BLOOD AGAR Aerobic incubation with 5-10%, CO2 Alpha-haemolytic colonies – identified by: - Gram staining - Bile solubility test - Optochin sensitive (Viridans Strep. – resistant) Gram stain diplococcic Quelling reaction ofdiplococci  Type specific + antisera Capsulated Capsule swells & pneumococcus refractile under microscope CAPSULAR SWELLING (QUELLUNG) REACTION Capsulated pneumococci are mixed with type specific antisera The capsule absorbs water and swells Refractile under light microscope Polyvalent antiserum containing antibodies against 80 capsular types is available QUELLUNG REACTION  DETECTION OF CAPSULAR ANTIGEN IN SPECIMENS - CSF & Serum – using latex agglutination - Important when culture is –ve due to antibiotic