Causative agents of Diphtheria, Whooping

Cough and Scarlet Fever

(Prepared by Inzhevatkina S.M.,

Department of Microbiology and Virology of Russian National Research

Medical University NI Pirogov)

Causative agent of Diphtheria

Taxonomy

Kingdom: Bacteria

Phylum: Actinobacteria

Order: Actinomycetales

Family: CorynebacteriaceaeFamily: Corynebacteriaceae

Genus: Corynebacterium

Species: Corynebacterium diphtheriae

Corynebacterium diphtheriae is classified into biotypes (gravis, mitis, intermedius, and belfanti) according to their cultural and biochemical properties

Morphology of C.diphtheriae

Corynebacterium diphtheriae is a nonmotile, noncapsulated, non-spore-forming Gram-positive rods. These bacteria often have a distinctive club shape and cluster in a way resembling Chinese characters (Y, X, L, V, etc.). Corynebacterium spp.contain short chain mycolic acids in cell wall, thus, contain short-chain mycolic acids in cell wall, thus, they can’t be stained by acid-fast stains (in comparison with Mycobacterium spp). Corynebacterium spp. are non-acid fast.

Volutin granules of C.diphtheriae

The club or bar appearance of C.diphtheriae is due topockets of inorganic phosphate which form metachromaticgranules. Special stains like Loeffler's stain and Neisser'sstain are used to demonstrate the metachromatin(metachromacity is the phenomenon by which different parts of an organism can get stained in two or more different colors just by applying a single dye) granules formed by them in the polar regions (so they are also called as 'Polar Granules' along with few other names like Babes Ernst Granules, volutin, etc). Volutin is the source of phosphorus in biosynthesis of different substances, as well as energy, because the bonds between phosphates are macroergic(the same function as in ATP).

Corynebacterium diphtheriae.

Gram stain.

Corynebacterium diphtheriae.

Loeffler’s stainvolutin granules are stained in dark blue color with alkaline methylene blue

(simple stain)

Corynebacterium diphtheriae.

Neisser’s strain.volutin granules are stained in dark blue color on polar ends

Corynebacterium diphtheriae

colonies on blood agar.

Cultivation of C.diphtheriae

A selective plate known as tellurite agar allows all Corynebacteria (including C.diphtheriae) to reduce tellurite to metallic tellurium producing brown colonies and, only in the case of C.diphtheriae, a black halo around the colonies allowing for easy differentation of the organism.

Corynebacterium diphtheriae are aerobic microorganisms.

Cystine tellurite plate culture of C.diphtheriae,

displaying the gravis & mitis biotypes colonial growth pattern.Of the three biotypes of C. diphtheriae, i.e. gravis,

intermedius, and mitis, the most severe disease is associated with the gravis type, though any strain may

produce toxin.

Gravis: “daisy head” colony Mitis: “poached egg” colony

Cultivation of Corynebacterium spp. on an enrichment medium, e.g., Löffler's serum, which contains such component as equine serum,

allows them to overgrow any other organisms present in the specimen

Virulence factors of C.diphtheriae1. Adhesive and colonization factors

(nonidentified, maybe pili, microcapsule and components of cell wall);

2. Invasive factors (hyaluronidase, neuraminidase and hemolysin);

3. Diphtheria toxin3. Diphtheria toxin;

4. Cord-factor (a toxic 6,6'-diester of trehalose containing corynemycolic and corynemycolenic acids in equimolar concentrations) disrupts respiration in mitochondria of eukaryotic cells and possesses antiphagocytic activity

Diphtheria ToxinNot all strains of C. diphtheriae are toxic

because the gene for diphtheria toxin (abbreviated DT) is not found in the bacterial genome. The DT gene is actually located on the genome of a corynephage, a certain type of prophage. Not all phages carry the DT gene tox, so it is possible for a C. diphtheriae bacteria to undergo lysogenic conversion, or be infected by a prophage, without producing the diphtheria toxin. Therefore, the toxinogenicity of C.diphtheriae is dependant on phage conversion with a phage that has a functional gene for the toxin protein.

Corynebacteriophage

Diphtheria Toxin (structure)

Diphtheria toxin is produced from the diphtheria bacteria as a single protein. The toxin is usual bipartite toxin, which contains two fragments (A and B). The toxin is cleaved by bacterial proteases into two fragments, A (molecular weight 21,150) and B (molecular weight 39,000). Fragment A is catalytically active and is the sole source of toxicity of diphtheria toxin. Fragment B (binding) appears to have no enzymatic activity and is far less stable than the active A enzymatic activity and is far less stable than the active A fragment. Each fragment plays a different role in the toxin’s entry into the host cell. DT enters the host cell by binding to the extracellular EGF domain of the heparin-binding epidermal growth factor receptor (HB-EFG receptor). This binding initiates a hydrophobic domain of the B fragment to form a channel across the membrane through which the A fragment can pass into the cytoplasm. The B fragment contains the transmembrane and receptor-binding domains of the toxin, so this piece remains in the plasma membrane.

Diphtheria Toxin (mechanism)

Diphtheria Toxin (fragment A)

Diphtheria toxin is an ADP-ribosyl (ADPR) transferase. Its toxicity comes from blocking protein synthesis in the host cell by inactivating EF-2, or cellular elongation factor. EF-2 is an essential part of the translation process. Fragment A that is free in the cytoplasm catalyzes the reaction:

EF 2 + NAD > ADPR EF 2 + nicotiamide + HEF-2 + NAD+ --> ADPR-EF-2 + nicotiamide + H+

This reaction forms a covalent bond between ADPR (diphtheria toxin) and EF-2 which blocks the functional site which interacts with RNA in translation, and consequentially preventing all protein synthesis at a particular ribosome. It had been estimated, that only one exotoxin molecule can completely terminate host cell protein synthesis.

Diphtheria Toxin (mechanism)

Diphtheria Toxin (spreading)

Diphtheria is unusual as a bacterial infection

because the bacteria themselves only invade

superficial tissues. However, the toxin released

spreads throughout the entire body and attacks cells without specificity. Severe infection

with diphtheria toxin can cause death by

necrosis of the tissues of essential organs

such as the heart, kidneys, liver and lungs by

blocking all protein synthesis in infected

cells.

Diphtheria Toxin(continuation)

Source and Transmission of Diphtheria

Humans are now the only known reservoir for the disease. Human carriers are the reservoir for C. diphtheriae, andthey are usually asymptomatic.

Transmission is most often person-to-person spread from the respiratory tract. Rarely, transmission may occur from skin lesions or articles soiled with discharges from lesions of infected persons (fomites).

Pathogenesis of DiphtheriaThe pathogenesis of diphtheria is based upon two

primary determinants: (1) the ability of a given strain of C.diphtheriae to colonize in the nasopharyngeal cavity and/or on the skin, and (2) its ability to produce diphtheria toxin. Thus, there are two main types of clinical diphtheria: nasopharyngeal and cutaneous.

Toxigenic strains (approximately 90% of strains of C.diphtheriae) secrete an extraordinarily potent exotoxin. C.diphtheriae usually colonize a local lesion in the upper C.diphtheriae usually colonize a local lesion in the upper respiratory tract (although cutaneous diphtheria can occur as well) where the toxin secreted by the bacteria inducesnecrotic injury to epithelial cells. As a result, blood plasma leaks into the area and forms a fibrin network called a pseudomembrane, which is full of rapidly growing C.diphtheriae cells. At the site of the lesion the diphtheria toxin is absorbed and disseminated throughout the body via lymph channels. Most commonly affected areas include heart, muscle, peripheral nerves, adrenal glands, kidneys, liver, and spleen.

Pathogenesis of diphtheria

Symptoms of skin and pharyngealdiphtheria

Symptoms of pharyngeal diphtheria vary from mild pharyngitis to hypoxia due to airway obstruction by the pseudomembrane. The involvement of cervical lymph nodes may cause profound swelling of the neck (bull neck diphtheria), and the patient may have a fever (103 °F).and the patient may have a fever (103 °F).

The skin lesion in cutaneous diphtheria is usually chronic, non-healing ulcer, covered with a gray-brown pseudomembrane. Life-threatening systemic complications, principally loss of motor function (e.g., difficulty in swallowing) and congestive heart failure, may develop as a result of the action of diphtheria toxin on peripheral motor neurons and the myocardium.

Clinical manifestations of nasopharyngeal diphtheria

Corynebacterium diphtheria infects the nasopharynx but it can also infect the skin.

Other Corynebacterium Species

In addition to C.diphtheriae, C.ulceransand C.pseudotuberculosis, C.pseudodiphtheriticum and C.xerosis may occasionally cause infection of the may occasionally cause infection of the nasopharynx and skin.

Laboratory diagnosis of diphtheriaClinical specimen: swabs from the pharyngeal

tonsils or their beds, nasopharyngeal samples,

pseudomembrane from the surface of the lesion.

Methods:

1. Bacterioscopical

2. Bacteriological (with compulsory detection of

toxigenicity of the isolated C.diphtheriae strains by a

variety of in vitro tests)

3. Molecular-genetic method (detection the tox gene

by PCR in clinical isolates or directly in clinical

specimen).

Double diffusion in gel(Ouchterloni test).

Elek Immunodiffusion Test.Sterile filter paper impregnated with diphtheria antitoxin is

imbedded in agar culture medium. Isolates of C diphtheriae are then streaked across the plate at an angle of 90° to the antitoxin

strip. Toxigenic C.diphtheriae is detected because secreted toxin diffuses from the area of growth and reacts with antitoxin

to form lines of precipitin.

Elek Immunodiffusion Test

Schick testThe Schick test can be used to test

susceptibility. The test was invented by Hungarian-born American pediatrician Béla Schick (1877-1967) in the early twentieth century to determine whether a person is susceptible to diphtheria. For the test, a small amount of diphtheria toxin is injected amount of diphtheria toxin is injected intradermally into the arm of an individual. If the skin around the injection becomes red and swollen (a positive result), it indicates that the person does not have enough antibodies to fight off the disease. The swelling disappears after a few days. Little or no swelling and redness occur in people who are immune, indicating a negative result.

Specific Prophylaxis of Diphtheria• DPT (also DTP and DTwP) refers to a class of combination

vaccines against three infections in humans: diphtheria, pertussis (whooping cough) and tetanus. The vaccine components include diphtheria and tetanus toxoids (Toxoid can be prepared from diphtheria exotoxin after addition of 0.3-0.4% of formalin and incubation at 37-40°C during 4 weeks: such preparation lacks its toxic properties but remains its immunogenicity), and killed whole cells of the organism that causes pertussis (wP).that causes pertussis (wP).

• DTaP (also DTPa and TDaP) refers to similar combination vaccines in which the pertussis component is acellular.

• DT and DT-m are used for immunization of children elder than 2 years

• “Tetracocc” refers to a combination vaccine against whooping cough, diphtheria, tetanus, and poliomyelitis (inactivated poliomyelitis virions). Developed countries use many different combined vaccines for national immunization program (routine immunization), containing DT as a component (see lecture “Vaccines”).

Treatment of Diphtheria

1. Prompt passive immunization with diphtherial antitoxin (anti-diphtheria serum) is the most effective in reducing the fatality rate.

2. The bacterium is sensitive to the majority of antibiotics, such as the penicillins, ampicillin, cephalosporins, quinolones, chloramphenicol, tetraciclines, cefuroxime and trimethoprim.

Causative agent of whooping cough (pertussis)

Taxonomy

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gamma Proteobacteria

Order: Burkholderiales

Family: Alcaligenaceae

Genus: Bordetella

Species: B.pertussis

Bordetella pertussis is a extremely small (approximately 0.8 µm by 0.2 µm), rod-shaped, coccoid, or ovoid Gram-negative

bacterium that is encapsulated and does not produce spores.B.pertussis. Gram stain.

Growth of Bordetella pertussis on Bordet-Gengou blood agar

following 4 days media.

It is a strict aerobe.

Hemolysis phenotypes of strains of Bordetella pertussis

Virulence factors of B.pertussis(1)1. Pili(fimbriae), filamentous hemagglutinin and pertactin for adhesion and colonization on membrane of ciliated cells of trachea. Filamentous hemagglutinin and pertactin block oxidative burst after phagocytosis of B.pertussis.

2. Capsule as antiphagocytic factors

3. Toxic factors:

- heat-labile toxin is a proteinaceous dermonecrotic toxin which produces strong vasoconstrictive effects, thus, causing local ishemia, which are probably important during the initial phase of pertussis by their action on the respiratory tract

- tracheal cytotoxin is a low-molecular-weight cell wall monomer that has specific affinity for ciliated epithelial cells. At low concentration it causes ciliostasis, later at higher concentrations it causes damage of ciliated cells; tracheal toxin interferes with DNA synthesis, imparing the regeneration of damaged cells. The toxin stimulates the release of IL 1, leading to fever.

Virulence factors of B.pertussis (2)

- hemolysin/ adenylate cyclase increases intracellular cAMP massively, inhibiting leukocyte chemotaxis, phagocytosis and killing

- 2 types of endotoxin (with lipid A and lipid X) is similar in structure, chemical lipid X) is similar in structure, chemical composition, and biologic activity to other endotoxins produced by Gram-negative bacteria

- pertussis toxin (see next page)

Pertussis ToxinPertussis toxin consists of two subunits (A and B).

B subunit consists of 5 fragments and is responsible

for binding with G protein on the receptors of cell

membranes. A subunit penetrates into cytoplasm and

is responsible for toxic effects on cell, causing

inactivation of inhibitory surface G protein that inactivation of inhibitory surface G protein that

controls adenylate cyclase activity, leading to

excessive increase of cAMP levels and subsequent

increase in the secretion of viscous sputum from

respiratory epithelium. Thus, pertussis toxin is the

main inductor of paroxysmal cough due increased of

respiratory secretions and mucous. Furthermore,

pertussis toxin has adhesive properties and destroys

the phagocytic function of neutrophils.

Virulence factors of B pertussis

Virulence factors of B pertussis

Bordetella pertussis using Adhesins to Adhere to a Ciliated Epithelial cell

Synergy between pertussis toxin and the filamentous hemagglutinin in binding to ciliated respiratory

epithelial cells

Structural and functional studies of the Bordetella pertussis adenylate cyclase toxin

Anatomy of upper respiratory tract

Whooping cough:Source and Transmission

Whooping cough or pertussis, is a contagious disease that mainly affects infants and young children. The illness causes violent spells of coughing that make it hard to eat, drink, or even breathe. The cough is accompanied by a high-pitched whoop that comes from gasping for breath after a coughing episode. Whooping cough is caused by bacteria that attach to the cells in the airway. A substance is produced that inflames and narrows the airway. As a result, the lungs cannot clear the mucus. Other complications include pneumonia, ear infections, loss of appetite, and dehydration. appetite, and dehydration.

Transmission: airborne droplet (pertussis is a very contagious disease only found in humans and is spread from person to person. People with pertussis usually spread the disease by coughing or sneezing while in close contact with others, who then breathe in the pertussis bacteria).

Incubation period: 6 to 20 days and usually 7-10 days Infectivity: 1 week before the onset of the paroxysmal cough and 3

weeks after (maximal in 1ST STAGE - Catarrhal Stage)

Pathogenesis of whooping cough1. Catarrhal Stage lasts 7-10 days

prodrome of mild upper respiratory tract symptoms(The early symptoms are similar to that of a common cold) : – anorexia and listlessness

– cough - hacking and nocturnal

– coryza and rhinorrhea

– inflammed mucous membranes

– insidious and most contagious stage

Pathogenesis of whooping cough2. Paroxysmal Stage lasts a mean of 10-14 days but can last up to 6-10 weeks

paroxysmal cough – characterized by 5-15 rapid coughs followed by a

characteristic inspiratory whoop, a few normal breaths and then more of the same (chronic cough)

– paroxysms initially occur at night and then become more frequent during the day (5-50 episodes per day)

– the face may become suffused or cyanotic during an episode

– paroxysms of coughing often end in vomiting

– may be associated with copious amounts of viscid fluid leading to aspiration and vomiting

– fever absent or minimal

Pathogenesis of whooping cough3. Convalescent Stage lasts 2-4 weeks and sometimes months

paroxysmal cough – not as frequent or severe as in the

paroxysmal stage

– symptoms gradually wane but may last for 6 months

– with each acute respiratiry infection for up to a year after the acute infection, many children will develop a pertussis-like cough

Duration of illness: 6-10 weeks in uncomplicated cases

Pathogenesis of whooping cough

Photo of child suffering from whooping cough

Relationship of B pertussis to the developing antibody response during whooping cough

Collection of clinical specimen for diagnosis of whooping cough

1. The cough plate method (ancient): here a

culture plate is held about 10-15 cm in front of

patient’s mouth during a bout of spontaneous or

induced coughing so that the droplet impinge

directly on the medium.

2. The oropharyngeal swab (low concentration

of ciliated epithelium): cotton swab on a bent

wire passes though the mouth.

3. The pernasal swab (main): a swab on a

flexible nichrome wire is passed along the floor

of nasal cavity and material is collected from the

pharyngeal wall. This is the optimal method,

yelding the highest percentage of isolations.

A nasopharyngeal specimen for isolation of Bordetella pertussis

postnasal (peroral) swab

A nasopharyngeal specimen for isolation of Bordetella pertussis

(pernasal swab – the best method)

Laboratory diagnosis of Whooping Cough

Clinical specimen: nasopharyngeal swab,

“whooping plate”, etc.

Methods:

1. Bacteriological method (culture) is the main

method; method;

2. Serological method (passive agglutination test,

CFT, ELISA, );

3. Immunofluorescence (rapid diagnosis).

4. Molecular-genetic method (detection the variety of

genes by PCR in clinical isolates or directly in

clinical specimen).

Bacteriological method (culture)Whooping cough

1 stage: The swabs are plated on potato-glycerin blood medium (Bordet-Gengou medium) or casein-charcoal agar, containing methicillin (bordetellae are resistant to this antibiotic). Incubation lasts 48-72 h.

2 stage: Study of cultural properties of colonies and staining of suspected colonies, resembling “mercury drops”. Inoculation suspected colonies, resembling “mercury drops”. Inoculation of the particular isolated colonies on selective media.

3 stage: Identification: staining properties (smear, stained byGram method); cultural properties; biochemical properties;serological identification (agglutination by antibodies against factor 7 (genus-specific antigen) is typical for all bordetellae; presence of species-specific factor 1 is typical for B.pertussis; species-specific factor 14 is typical for B.parapertussis); susceptibility for antibiotics by disc method.

Comparison of B.pertussis and B.parapertussis (A similar, milder disease is

caused by B.parapertussis).

Prophylaxis of whooping cough• DTP and DTwP refers to a class of combination

vaccines against three infectious diseases in humans: diphtheria, pertussis (whooping cough) and diphtheria, pertussis. The vaccine components include diphtheria and tetanus toxoids, and killed whole cells of the organism that causes pertussis (wP).

• DTaP (also DTPa and TDaP) refers to similar combination vaccines in which the pertussis component combination vaccines in which the pertussis component is acellular (pertussis toxin, filamentous hemagglutinin, and pertactin)

• “Tetracocc” is the combined vaccine against whooping cough, diphtheria, tetanus, and poliomyelitis.

• Pediacel is the combined 'five-valent' vaccine against whooping cough, diphtheria, tetanus, poliomyelitis andHaemophilus influenza infection.

• After the first two vaccinations protection is almost 100 per cent.

• Human hyperimmune pertussis globulin is still used.

DTaP (Daptacel®, Infanrix®, Kinrix®, Pediarix®, Pentacel®, and Quadracel®)

DTaP is a combined vaccine against diphtheria, tetanus, and pertussis, in which the pertussis component is acellular. This is in contrast to whole-cell, inactivated DTP.

The acellular pertussis vaccine component uses selected antigens of the B.pertussis (detoxified pertussis toxin (PT) and pertactin; additional components for several vaccines are filamentous hemagglutinin (FHA) and fimbriae) to induce filamentous hemagglutinin (FHA) and fimbriae) to induce immunity.

DTaP uses fewer antigens than the whole cell vaccines, it is considered safer, but it is also more expensive.

Recent statistical data in Europe revealed that DTP vaccine is more effective than DTaP in conferring immunity; this is because DTaP's narrower antigen base is less effective against current pathogen strains, which increased their virulence as a result of mutations of the B.pertussis.

Treatment of whooping cough

Three macrolides (erythromycin, azithromycin and clarithromycin) are used for treatment of pertussis; trimethoprim-sulfamethoxazole is generally used when a macrolide is ineffective or is contraindicated. Usage of antibiotic shortens the infectious period but does not generally alter the outcome of the disease. If treatment is initiated during the catarrhal stage, symptoms may be less severe.

National Immunization Calendar of Russian Federation

Age / InfectionsNewborns (0–12 hours after birth) Viral Hepatitis B (1st vaccination)Newborns (3–7 days) Tuberculosis (vaccination)1 month Viral Hepatitis B (2nd vaccination)2 months Pneumococcal infection (1st vaccination)3 months Diphtheria, Pertussis, Tetanus, Polio (1st

vaccination)vaccination)

4.5 months Diphtheria, Pertussis, Tetanus, Polio (2nd vaccination)

Pneumococcal infection (2nd vaccination)

6 months Diphtheria, Pertussis, Tetanus, Polio(3rd vaccination)

Viral Hepatitis B (3rd vaccination)

12 months Measles, Rubella, Mumps (vaccination)

National Immunization Calendar of Russian Federation

(continuation)

Age / Infections15 months Pneumococcal infection (revaccination)18 months Diphtheria, Pertussis, Tetanus, Polio (1st revaccination)20 months Polio (2nd revaccination)6 years Measles, Rubella, Mumps (revaccination)7 years Diphtheria, Tetanus (2nd revaccination)7 years Diphtheria, Tetanus (2nd revaccination)

Tuberculosis (1st revaccination)13 years Rubella (vaccination for girls)

Viral Hepatitis B (vaccination)

14 years Diphtheria, Tetanus (3rd revaccination)Tuberculosis (2nd revaccination)

Adults Diphtheria, Tetanus (revaccination every 10 years after the last revaccination)

Causative agent of

scarlet fever (scarlatina)Taxonomy

Kingdom: Eubacteria

Phylum: Firmicutes

Class: Bacilli

Order: Lactobacillales

Family: Streptococcaceae

Genus: Streptococcus

Species: S.pyogenes

Streptococcus pyogenes (Group A streptococcus) is a Gram-positive, nonmotile, nonsporeforming coccus that occurs in chains or in pairs of cells. Individual cells are

round-to-ovoid cocci, 0.6-1.0 micrometer in diameter Pure culture of streptococcus, Gram stain

Streptococcus in tissue,Gram stain

Morphology of the streptococci in comparison with staphylococci.Streptococci divide in a single plane and tend not to separate, causing

chain formation.

Comparison of microscopic pictures of Staphylococcus and Streptococcus

The metabolism of S. pyogenes is fermentative; the organism is a catalase-negative aerotolerant anaerobe (facultative anaerobe), and requires enriched medium containing blood in order to grow. Group A streptococci typically have a capsule composed of hyaluronic acid and

exhibit beta (clear) hemolysis on blood agar.

S.pyogenes on blood agar

Streptococcus group A infections. Beta hemolysis

is demonstrated on blood agar media.

Antigenic composition of S.pyogenesThe cell wall of G+is composed of repeating

units of N-acetylglucosamine and N-acetylmuramic acid, the standard peptidoglycan.

Historically, the definitive identification of streptococci has rested on the serologic reactivity of "cell wall" polysaccharide antigens as originally described by Rebecca Lancefield.described by Rebecca Lancefield.

Eighteen group-specific antigens (Lancefield groups) were established.

The cell surface structure of Group A streptococci. The Group A polysaccharide is a polymer of N-acetylglucosamine and rhamnose.

Strept.structure

Virulence factors of Streptococcus

pyogenesThe cell surface of Streptococcus pyogenes accounts

for many of the bacterium's determinants of virulence, especially those concerned with colonization and evasion of phagocytosis and the host immune responses. The surface of Streptococcus pyogenes is incredibly complex and chemically-diverse. Antigenic components include capsular polysaccharide (C substance), cell wall peptidoglycanpolysaccharide (C-substance), cell wall peptidoglycanand lipoteichoic acid (LTA), and a variety of surface proteins, including M protein, fimbrial proteins, fibronectin-binding proteins, (e.g. Protein F) and cell-bound streptokinase.

The cytoplasmic membrane of S. pyogenes contains some antigens similar to those of human cardiac, skeletal, and smooth muscle, heart valve fibroblasts, and neuronal tissues, resulting in molecular mimicry and a tolerant or suppressed immune response by the host.

Summary of virulence factors of Streptococcus pyogenes

M protein.The M proteins are clearly virulence factors associated with

both colonization and resistance to phagocytosis. More than 100 types of S. pyogenes M proteins have been identified on the basis of antigenic specificity, and it is the M protein that is the major cause of antigenic shift and antigenic drift in the Group A streptococci. The M protein (found in fimbriae) also binds fibrinogen from serum and blocks the binding of complement to the underlying peptidoglycan. This allows survival of the organism by inhibiting phagocytosis.organism by inhibiting phagocytosis.

The streptococcal M protein, as well as peptidoglycan, N-acetylglucosamine, and group-specific carbohydrate, contains antigenic epitopes that mimic those of mammalian muscle and connective tissue. Cell surface of recently emerging strains of streptococci is distinctly mucoid (indicating that they are highly encapsulated). These strains are also rich in surface M protein. The M proteins of certain M-types are considered rheumatogenic since they contain antigenic epitopes related to heart muscle, and they therefore may lead to autoimmune rheumatic carditis (rheumatic fever) following an acute infection.

Streptococcus group A infections. M protein.Electron microscopy.

The Hyaluronic Acid Capsule

The capsule of S. pyogenes is non antigenic since it is composed of hyaluronic acid, which is chemically similar to that of host connective tissue. This allows the bacterium to hide its own antigens and to go unrecognized as antigenic by its host. The hyaluronic acid capsule also prevents opsonized phagocytosis by neutrophils or mancrophages.

Adhesins of S.pyogenes

Colonization of tissues by S. pyogenes is the result from a failure in the constitutive defenses (normal flora and other nonspecific defense mechanisms) which allows establishment of the bacterium at a portal of entry (often the upper respiratory tract or the skin) where the organism multiplies and causes an inflammatory purulent lesion.

S. pyogenes (like many other bacterial pathogens) produces multiple adhesins with varied specificities. Thereproduces multiple adhesins with varied specificities. Thereis evidence that Streptococcus pyogenes utilizes lipoteichoic acids (LTA), M protein, and multiple fibronectin-binding proteins in its repertoire of adhesins. LTA is anchored to proteins on the bacterial surface, including the M protein. Both the M proteins and lipoteichoic acid are supported externally to the cell wall on fimbriae and appear to mediate bacterial adherence to host epithelial cells. The fibronectin-binding protein, Protein F, mediates streptococcal adherence to the amino terminus of fibronectin on mucosal surfaces.

Invasins of S.pyogenesStreptococcal invasins lyse eukaryotic cells, including

red blood cells and phagocytes; they lyse other host macromolecules, including enzymes and informational molecules; they allow the bacteria to spread among tissues by dissolving host fibrin and intercellular ground substances.

Hyaluronidase (the original "spreading factor") can digest host connective tissue hyaluronic acid, as well as the organism's own capsule.

Streptokinases (fibrinolysin) participate in fibrin lysis.Streptokinases (fibrinolysin) participate in fibrin lysis.Streptodornases A-D (DNA-ases) possess

deoxyribonuclease activity; Streptodornases B and D possess ribonuclease activity as well.

Protease activity similar to that in Staphylococcus aureus has been shown in strains causing soft tissue necrosis or toxic shock syndrome.

Exotoxins of S.pyogenesStreptococcal protein toxins interact with

mammalian blood and tissue components in ways that kill host cells and provoke a damaging inflammatory response.

Streptolysin S is oxygen-stable exotoxin, which lyses erythrocytes, leukococytes ans which lyses erythrocytes, leukococytes ansplatelets by forming pores in their cell membranes;

Streptolysin O is an oxygen-labile exotoxin (the same mechanism of action).

NADase is leucotoxic.

Erythrogenic toxin is superantigenThree streptococcal pyrogenic exotoxins

(SPE), formerly known as Erythrogenic toxin, are recognized: types A, B, C.

These toxins act as superantigens by a mechanism similar to those described for staphylococci. As antigens, they do not requiring processing by antigen presenting cells. Rather, they stimulate T cells by binding class II MHCthey stimulate T cells by binding class II MHCmolecules directly and nonspecifically. With superantigens about 20% of T cells may be stimulated (vs 1/10,000 T cells stimulated by conventional antigens) resulting in massive detrimental cytokine release.

SPE A and SPE C are encoded by lysogenic phages; the gene for SPE B is located on the bacterial chromosome.

Pathogenesis of S.pyogenes

infections

Scarlet Fever

Scarlet fever (also called scarlatina in older literature) is an infectious disease which most commonly affects 4-8 year old children. Symptoms include sore throat, fever and a characteristic red rash.

Scarlet fever is usually spread by inhalation. Transmission usually occurs via airborne respiratory particles that can be spread from infected patients and asymptomatic carriers.

Patients are contagious during the acute illness and during the subclinical phase.

Clinical manifestations of scarlet fever• Sudden fever with sore throat.• Flushed face with circumoral pallor.• Tonsils - edematous, erythematous, and covered with an

exudates.• Petechiae on the soft palate.• Anterior cervical lymphadenopathy.

On day 1 or 2, a white strawberry tongue.• On day 1 or 2, a white strawberry tongue. • By day 4 or 5, a raspberry tongue.• The body rash appears 12-24 hours after onset of

illness.Lastly, as a person recovers from the illness,desquamation (peeling of the skin) may occur in areaswhere the rash was. Desquamation is usually mostprominent on the skin of the hands.

Skin rash, typical for scarlet fever (scarlatiniform rash) on the chest

and abdomen

Bright Red Tonsils, typical for scarlet

fever

Flushed face, typical for scarlet fever

Strawberry Tongue , typical for

scarlet fever

Complications of scarlet feverAlthough most cases are mild, some children

and adults can become very sick with scarlet fever. If left untreated for long enough, the infection can spread to the blood and cause bacteremia, pneumonia, sepsis, or meningitis.

If left untreated, even if the illness resolves, the individual can be at risk of developing the individual can be at risk of developingrheumatic fever (autoimmune disease) or rheumatic heart disease.

Streptococcal glomerulonephritis can occur after a case of strep throat (streptococcal pharyngitis), impetigo, or scarlet fever, usually about 7-14 days afterwards.

Laboratory diagnosis of streptococcal infections

Clinical specimen: pus, sputum, blood, urine, cerebrospinal fluid, mucus from nasopharynx, etc.Methods:

1. Bacterioscopical method (microscopy) is the preliminarymethod (Gram stain of clinical specimen);

2. Bacteriological method (culture) is the main method;2. Bacteriological method (culture) is the main method;3. Serological method (passive agglutination test, ELISA,

toxin neutralization tests) for detection of ASO (anti-streptolysin O, an antibody to streptolysin O (toxinproduced by S.pyogenes). The ASO level stays high formonths following an infection with Streptococcus. TheASO titer is specifically useful since it can detect arecent illness of strep/ infection, even if the infection hasalready resolved;

4. Molecular-genetic method (polymerase chain reaction);5. Methods of rapid diagnosis (latex-agglutination).

Bacteriological method (culture)Streptococcal infections

Preliminary stage is used only for special cases (sepsis) for study of blood: inoculation of blood on sugar broth.

1 stage: Inoculation of clinical specimen (pus, sputum, etc.) on serum or blood agar.

2 stage: Study of cultural properties of colonies & staining of particular colonies by Gram method. Inoculation of the particular isolated colonies on agar slant.

3 stage: Identification staining properties (smear, stained

by Gram method); cultural properties; biochemical properties on differential--diagnostic system API-20 Strept. and catalase test; virulence factors; serological identification (detection of group-specific polysaccharide antigen) ;susceptibility for antibiotics by disc method.

Principles of treatment of streptococcal infections

Treatment is given for ten days in order to prevent

future complications of rheumatic fever and rheumatic heart disease. Ten days of treatment has been proven to prevent these complications.prevent these complications.

Antibiotics:

penicillin G and V, amoxicillin;

erythromycin, clindamycin, or azithromycin.

Prevention of Scarlet Fever

No vaccines are currently available to protect against S. pyogenes infection, although there has been research into the development of one. Difficulties indevelopment of one. Difficulties indeveloping a vaccine include the wide variety of strains of S. pyogenes present in the environment.

Staphylococcal Scarlet Fever

A more rare form of scarlet fever is staphylococcal scarlet fever. The signs and symptoms are indistinguishable from scarlet fever caused by Streptococcusexcept that throat infection is usually absent. Culture of the eyes, nose, or mouth will show Staphylococcus aureusinstead of Streptococcus pyogenes. Stronger antibiotics are usually necessary in these cases.