L2 - Strep&Diptheria

Streptococcus

Corynebacterium diphtheriae

Borrelia burgdorferiG-ve

AnaerobicSpirochetesIxodes ticks (nymph stage)IP : ? Early stages:- amoxicillin- doxycycline- cefuroxime

Late stages (IV):- ceftriaxone- penicilin

1. Microscopy, Stain

Giemsa or Wright stainDarkfield microscopy

2. Molecular

PCRSerology (main) – ELISA Western blot

Streptococcus - description

• Gram positive oval or spherical cocci

• pairs or chains

• 0.5 to 1.0m• blood agar - haemolysis

• colonies small (0.5-2.0mm), grey to greyish white

• catalase negative

streptococcus

classification– based on presence or absence of haemolysis

around colonies growing on blood agar

haemolysis beta (complete)

alpha (incomplete)

gamma (non-haemolytic)

appearance distinct clear zone

greenish discoloration

no change

Lancefield classification (1933)

• beta-haemolytic streptococci

• group-specific carbohydrate of cell wall

• 20 Lancefield groups (A-H and K-V)

• latex agglutination

Streptococci of importance in human infection

Lancefield group Type of haemolysis

S. pyogenes A

S. agalactiae B

S. pneumoniae none

S. sanguis none

S. mitior none

Streptococcus pyogenes

Description– Gram-positive cocci in chains– facultative anaerobes– blood agar

• small grey/greyish white colonies• complete/beta haemolysis

Habitat– nasopharynx– children 15-20% carriage rate (adults lower rate)

S. pyogenes - virulence

i. M protein• major surface protein and virulence factor

– two polypeptide chains complexed in alpha-helical coiled coil configuration anchored in cell membrane

• more than 80 serotypes• antiphagocytic

– binding to factor H and fibrinogen impede binding of C3b to bacterial surface

• adhesin (skin keratinocyte)• type-specific opsonic antibodies against M protein

provide protective immunity


ii. Lipoteichoic acid (LTA) Protein F1(PrtF1) aka streptococcal fibronectin binding proteins (sfbI)

• cell surface proteins• adheres to fibronectin on respiratory

epithelial cells

• other proteins– Protein F2, SbfII, FBP54, vitronectin-binding protein,

collagen-binding protein,


iii. Capsule• composed of hyaluronic acid

– chemically similar to human connective tissue

• antiphagocytic• degree of encapsulation variable

– mucoid colonies and more virulent if exuberant encapsulation

• adherence factor in pharynx as binds to CD44 on epithelial cells


iv. Streptolysin O (SLO) Streptolysin S (SLS)

• pore-forming cytolysins• haemolytic and cytotoxic• both responsible for haemolysis on blood agar

• SLO is antigenic– antibodies to SLO (ASOT) used in serodiagnosis


v. Streptococcal pyrogenic exotoxins (SPE)• family of superantigens• SpeA, SpeC, SpeG, SpeH, SpeJ, SpeK, SpeL, SpeM,• Streptococcal superantigen (SSA)• Streptococcal mitogenic exotoxin Z (SMEZ, SMEZ-2)

• streptococcal toxic shock syndrome• scarlet fever


vi. “Spreading factors”• Hyaluronidase - degrades hyaluronic acid

• Deoxyribonucleases - hydrolyse nucleic acid and nucleoproteins

• Streptokinase - converts plasminogen to plasmin - breaks down fibrin

vii. C5a peptidase• degrades complement component C5a• destroy C5a ability to act as chemo-attractant of polymorphonuclear leucocytes

S. pyogenes - infections

• Acute pharyngitis / tonsillitis

• Scarlet fever

• Impetigo, erysipelas, cellulitis, sepsis in burns, necrotizing fasciitis

• Toxic Shock Syndrome

• Puerpural sepsis, endocarditis, pneumonia-post viral

• Non-suppurative post-streptococcal sequelae: rheumatic fever, acute glomerulonephritis

S. pyogenes - laboratory diagnosis

• Specimens– throat swab– pus swab– blood culture

• Direct examination by gram stain• Culture on blood agar at 37C

• Bacitracin sensitivity test (rapid presumptive diagnosis)

• Lancefield grouping by latex agglutination

S. pyogenes - laboratory diagnosis

• Serological diagnosis– antibodies to streptolysin O, DNase B, hyaluronidase,

NADase and streptokinase

– diagnosis of ARF and APSGN to confirm antecedent streptococcal infection

• ASOT – peak 2-4 weeks after acute infection– tonsillar-associated diseases

• Anti-DNase B in pyoderma-associated diseases

Streptococcus pneumoniae

• Pneumococcus

Description– Gram-positive ovoid or lanceolate cocci– pairs (diplococci)– grow well on blood agar– colonies

• small (1mm), circular, raised, smooth• haemolysis• Optochin sensitive• older cultures : “draughtsman” appearance• mucoid due to excessive capsular production

S. pneumoniae - virulence

Capsule– most significant virulence factor– polysaccharide– >90 capsular / serotypes

• antigenic differences in polysaccharides• 90% due to 23 serotypes

– antiphagocytic– anticapsular antibodies protective


Choline-binding proteins– pneumococcal surface adhesin A and choline-binding

protein C binds to epithelial cell receptors

Pneumococcal surface proteins A (PspA)– binds to complement factor B and prevents deposition of C3b

Pneumococcal surface proteins C (PspC)– binds to complement factor H and prevents deposition of C3b


Pneumolysin– transmembrane pore-forming toxin– cytotoxic for phagocytic and respiratory epithelial cells– activate classical complement pathway

Autolysin– causes bacterial disintegration and release of cell wall

components (peptidoglycan and teichoic acid)

Neuraminidase– cleave sialic acid on mucous membrane surfaces– contribute to adherence

S. pneumoniae - pathogenic mechanisms

• adherence• colonization• infection if carried into cavities and not readily cleared by

clearance mechanisms (ciliary action)– coexisting viral infection causing oedema– damage to ciliated bronchial cells– increased mucus production

• invasion across mucosal barriers into bloodstream• escape from phagocytosis• activation of complement and inflammatory cytokines

S. pneumoniae - epidemiology

• human only known reservoir• nasopharynx

– 20 - 40% children; 5 -10% adults

• transmission by droplets or contact with respiratory secretions

• infections throughout life but most common in <2 year olds and elderly

• predisposed by defects in antibody, complement and splenic function

• also at risk are patients with chronic diseases– cirrhosis, renal failure, diabetes, congestive heart failure

S. pneumoniae - infections

• Infection of middle ear, sinuses, trachea, bronchi and lungs by direct spread from nasopharynx (Non-invasive diseases)

• Infection of CNS, heart valves, bones, joints and peritoneal cavity by haematogenous spread (Invasive diseases)

• Infection of CNS, pleura or peritoneal cavity may occur by direct extension or haematogenous spread

S. pneumoniae - infections

• lobar pneumonia (commonest bacterial cause)

• acute exacerbation of COPD• acute otitis media• sinusitis• meningitis• septicaemia (*splenectomised patients)

• empyema• septic arthritis• osteomyelitis• endocarditis

S. pneumoniae - treatment

• Was susceptible to penicillin but increasing resistance to penicillin and other antibiotics– mutations in penicillin-binding proteins (PBPs)

• Definitive therapy guided by antibiotic susceptibility testing results

S. pneumoniae - prevention

• Vaccination– Pneumococcal capsular polysaccharide vaccines

• incorporates 23 of most common capsular types• recommended for older than 2 year olds at

increased risk of developing pneumococcal infection or a serious complication

– Conjugate pneumococcal vaccine• heptavalent polysaccharide vaccine• serotypes responsible for 70% invasive diseases• 2, 4, 6 months and booster at 12-15 months

Corynebacteria

Corynebacteria

Greek: korynee = clubbacterion = little rod

• Corynebacterium diphtheriae – major human pathogen

• non-diphtheria corynebacteria(coryneforms / diphtheroids)– diverse– increasing pathogenic role

C. diphtheriae - description

• pleomorphic Gram positive slender bacilli

– pleomorphism • often club-shaped

– long and slender with rounded or swollen ends

• some coccoid• some globular

Description - characteristic features

1. Metachromatic (volutin) granules– intracellular long chain polymerised phosphate– 2-3 granules per cell– polar– Neisser or Albert stain beaded appearance

Description - characteristic features

2. “Chinese lettering”– on Gram stain – snapping fission

• incomplete separation at moment of division and daughter cells remain attached at one point

• adjacent cells lie at various angles to one another and appear as angled pairs or parallel rows (palisades)

V, L, W forms

C. diphtheriae - culture

1. Loeffler’s serum slopes– rapid growth at 12 to 18 hours

2. Culture media containing potassium tellurite e.g. chocolate tellurite agar– selective medium – characteristic grey-black colonies

C. diphtheriae - epidemiology

• humans only known natural host and reservoir• transmission primarily by

i. airborne respiratory droplets ii. direct contact with respiratory secretions

• primarily affects < 15 year olds– maximal 2nd to 5th year

• incidence decreased steadily and now rare in developed countries– major impact due to immunisation

Diphtheria - definition

“a localised inflammation of the throat with greyish white adherent exudate (pseudomembrane) and a generalised toxaemia due to secretion and dissemination of a highly potent toxin”

C. diphtheriae - virulence

Diphtheria toxin– potent exotoxin– tox+ gene– only strains lysogenic for bacteriophage– fragment B

• attachment to host cell membrane and transport of fragment A into cell

– fragment A• inhibits polypeptide chain elongation

C. diphtheriae - Diphtheria toxin

• primary effects within first few days, toxin elaborated locally

induces a dense necrotic coagulum “pseudomembrane”

» fibrin, WBC, RBC, dead epithelial cells and bacteria

cardiac muscle structural and functional damage demyelination in both peripheral and cranial

nerves

Agar gel precipitation (Elek) test

• a wide filter paper strip impregnated with antitoxin is implanted in a serum agar plate while medium is still fluid

• when agar has set, the organism is streaked at right angles to strip

• precipitate / precipitin reaction in the form of double arrow-headed lines after incubation

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L2 - Strep&Diptheria