Streptococci: Classification, Structure, Virulence factor, Pathogenesis and Laboratory Diagnosis

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The History of Discovery


T. Billroth described the first streptococci in patients with wound infections in 1874. L. Pasteur discovered streptococci in patients with sepsis in 1880; F. Fehleisen in 1883 and F. Rosenbach in 1884 isolated the pure culture of these bacteria.


Classification of Streptococci


  • Streptococci belong to the family Streptococcaceae and genus Streptococcus. Not long ago the family contained one more genus Enterococcus. Later it was placed into the separate family Enterococcaceae.
  • Streptococcus genus comprises more than 60 species.
  • The main microbial species that cause pathology in humans are: S. pyogenes and S. agalactiae; oral streptococci S. mutans and S. sobrinus (causative agents of caries); causative agent of pneumonia S. pneumoniae or pneumococcus.
  • Opportunistic pathogens E. fecalis and E. faecies are the main representatives of genus Enterococcus.
  • Historically all streptococci were divided according to their hemolytic activity into α-hemolytic (produce green zones of hemolysis, e.g. viridans streptococci like S. mutans), β-hemolytic that develop clear zones of complete hemolysis, e.g. S. pyogenes and S. agalactiae, and non-hemolytic streptococci without hemolysis.
  • Also streptococci are classified by their antigenic properties into serogroups (A-U), some groups are further divided into types.
  • Serological group division proposed by R. Lancefield is based on polysaccharide cell wall antigens.
  • S. pyogenes pertains to group A, S. agalactiae is the member of group B. Oral streptococcus S. sanguinis is related with group H.
  • Enterococci E. fecalis and E. faecies belong to group D.
  • Streptococci of A group are further divided into more than 80 serotypes due to the structural differences of their M protein antigen.
  • S. pneumoniae and viridans streptococci (e.g., numerous oral streptococci S. mutans, S. salivarius, S. mitis and others) are beyond of Lancefield classification. S. pneumoniae is subdivided into more than 90 serotypes on the basis of specific capsular carbohydrate antigens.

Structure and Properties of Streptococci


Morphology

  • Streptococci are gram-positive spherical microbes, 0.5-1 μm in diameter, which are usually clustered into chains or pairs. They are non-motile bacteria without flagella and spores. The cells possess pili, containing M protein and lipoteichoic acid.
  • Pneumococci are paired cocci of lancet-like shape.
  • Enterococci are the motile bacteria that carry one polar flagellum.
  • Many streptococcal species of A, B and C groups as well as pneumococci, produce the capsule. It is composed predominantly of hyaluronic acid.

Cultivation

  • Streptococci are relatively fastidious bacteria. The temperature range for their growth is rather narrow within the limits of 20-40°.
  • They are cultivated on blood, serum or sugar agar and broth, pH 7.2-7.4. On solid media streptococci develop small, gray, translucent colonies.
  • The growth in the sugar broth appears as fine precipitate near the walls and bottom of the test tubes.
  • Many strains are hemolytic (see above). Green hemolysis zone results from conversion of hemoglobin into methemoglobin (viridans streptococci and pneumococci).

Biochemical properties

  • Streptococci are facultatively anaerobic or aerotolerant microorganisms. These bacteria ferment carbohydrates (e.g. glucose, maltose, lactose, sucrose, etc.) with acid formation without gas. They lack proteolytic activity, can’t liquefy gelatin and don’t reduce nitrates into nitrites.
  • Streptococci are catalase-negative bacteria, whereas entrococci reveal minor catalase activity. Also enterococci easily grow in the presence of bile and 6.5% NaCl, hydrolyze esculin that distinguishes them from streptococci.
  • Streptococci produce great variety of invasive and toxic enzymes (see below).

Antigenic structure

  • Streptococci possess a great number of antigenic determinants within the cell wall and capsule, which are of oligosaccharide and protein nature.
  • Group-specific polysaccharide antigens of the cell wall are thermostable and contain different side residues of amine sugars and teichoic acids.
  • M protein of group A S. pyogenes is presented in more than 80 structural variations. It is heat- and acid-labile substance.
  • T protein and R protein present some other streptococcal surface antigens. They can be used for further differentiation of streptococci.
  • P substance is the nucleoprotein fraction, which is common in most hemolytic streptococci.
Antigenic Structure of Streptococcus pyogenes

Virulence factors

  • Group A streptococci are able to produce a great number of toxic substances, agressins and invasion enzymes.
  • M protein is regarded as the most significant virulence factor of streptococci. M proteins are divided now into class I and class II molecules due to the reactions with different antibodies. The patients with rheumatic fever are usually infected with class I M protein streptococci.
  • M protein, which is encoded by emm gene, inhibits phagocytosis and promotes the adhesion of streptococci to the host tissues. This is the main mechanism of streptococcal virulence, since the lack of emm gene results in efficient phagocytosis of the invaded pathogen.
  • It was elucidated also that M protein binds to H factor of alternative pathway of complement activation as well as to host fibrinogen molecules.
  • Both interactions suppress complement activation and, more important, severely reduce the opsonization of streptococci, thus inhibiting phagocytosis. Likewise, M protein is shown to activate bradykinin, stimulating tissue inflammation.
  • Adhesive capacity of M protein facilitates streptococcal entry into infected cells that results in intracellular persistence of bacteria.
  • Hyaluronic acid capsule is also required to withstand phagocytosis.
  • Streptococcal pyrogenic exotoxins A and C (or erythrogenic toxins) and streptococcal mitogens work as superantigens, resembling staphylococcal enterotoxins.
  • They trigger endotoxic shock with pyrogenic reactions that ensues from the massive release of proinflammatory cytokines (IL-1, IL-6, gamma interferon, α-TNF, etc.)
  • Streptococcal pyrogenic exotoxin B or streptococcal proteinase is an extracellular cysteine protease produced by all group A streptococci.
  • It can directly activate IL-1 via specific intramolecular proteolysis as well as host tissue metalloproteases, enhancing inflammation and bacterial invasion.
  • Streptococcal pyrogenic exotoxins A and C are encoded by genes of a lysogenic temperate bacteriophage, while exotoxin B is of chromosomal origin.
  • Streptococcal adhesins comprise great variety of virulence factors that ensure streptococcal adherence and intracellular penetration.
  • Among them are above mentioned M protein, lipoteichoic acid, fibronectin-binding protein, collagen-binding protein and many others.
  • Streptococci express different IgG- and IgA-binding proteins. These proteins prevent antibody-mediated opsonization and deregulate mucous tissue immunity.
  • The group of plasminogen-binding proteins contains several factors, including the enzyme streptokinase.
  • They convert plasminogen into plasmin on the bacterial surface. Plasmin, attached to the microbial cells, activates extracellular metalloproteases or collagenases forwarding tissiue damage and enhancing invasion.
  • Many other enzymes are produced by virulent streptococci.
  • Streptococcal hyaluronidase destroys hyaluronic acid of connective tissue facilitating microbial invasion.
  • C5a peptidase of streptococci splits C5a complement fragment, preventing efficient chemotaxis of phagocytes.
  • Streptococcal streptodornase or deoxyribonuclease hydrolyzes host DNA.
  • Different types of hemolysins are revealed in streptococci. S. pyogenes produces two main hemolysins (streptolysins): streptolysin O and streptolysin S.
  • Streptolysin O is a protein that contains free -SH groups, being sensitive to the oxygen. It induces high-titer synthesis of specific antibodies during infection.
  • Streptolysin S is of peptide nature, causing the hemolytic damage of cellular membranes. Also it may trigger apoptosis of infected cells and stimulate inflammatory response.

Resistance

Streptococci are not highly resistant bacteria, but they can withstand low temperatures, and survive for months in pus and sputum. They are killed at temperature of 70°С within one hour. Conventional disinfectants readily destroy them (e.g., phenol in concentrations of 3-5% inactivates bacteria in 15 minutes).


Pathogenesis and Clinical Findings in Streptococcal Infections


  • Group A representative Streptococcus pyogenes is the major streptococcal pathogen. It affects almost any body tissue or organ, thereby causing great variety of pyogenic local and invasive infections.
  • According to WHO data, group A streptococci account for at least 500,000 patients’ death cases annually.
  • Among local infections are streptococcal pharyngitis or sore throat, streptococcal pyoderma, erysipelas, cellulitis, wound infections and some others.
  • These disorders can be followed by serious complications, such as streptococcal pneumonia, meningitis, infectious acute and subacute endocarditis with possible fatal outcome.
  • Toxic and invasive streptococcal infections involve scarlet fever, necrotizing fasciitis, puerperal fever, streptococcal toxic shock syndrome and septicemia.
  • Non-suppurative sequelae of streptococcal infections include post-streptococcal acute glomerulonephritis and rheumatic fever.
  • The infections are transmitted by air droplet route, by direct contact, through skin lesions, etc.
  • Streptococcal sore throat is the most frequent infection caused by β-hemolytic streptococci. Bacteria attach to pharyngeal epithelium via a number of adhesins. The disease is characterized by throat pain, fever, nasopharyngitis, tonsillitis with purulent exudates, enlargement of cervical lymph nodes, etc.
  • Erysipelas is the specific streptococcal skin infection. Group A strains enter the skin through various lesions and penetrate the epidermis. The disease reproduces typical skin inflammatory damage with erythematous superficial skin layers.
  • Necrotizing fasciitis is a severe painful streptococcal disorder that affects subcutaneous tissues and fascia. It shows evident tendency to rapid spread into underlying tissues resulting in their necrosis and gangrene.
  • Scarlet fever is caused by group A streptococci that produce streptococcal pyrogenic exotoxins A, B and C. The symptoms result from systemic toxin action. They involve fever, generalized rash, bright “strawberry” tongue, skin desquamation. The disease profoundly affects cardiovascular system especially microcirculation.
  • Streptococcal toxic shock syndrome is the highest manifestation of toxigenic streptococcal infections. Hyperproduction of toxins and mitogens with superantigenic activity leads to hypotension and deep multiple organ failure that may cause patient’s death. Beta-hemolytic streptococci of M protein types 1, 3, 11, 12, 28 are predominantly associated with shock appearance.
  • Poststreptococcal acute glomerulonephritis is a typical immune complex disease that evolves 2-3 weeks after streptococcal infection. The nephritogenicity of group A streptococci is related to particular M protein serotypes of S. pyogenes, such us M 12 (predominant), 1, 2, 4, 49, 56, 57, and 60.
  • Several immune mechanisms take part in disease pathogenesis. Among them are deposition of immune complexes on glomerular basal membrane, that is followed by complement activation, the production of antibodies, cross-reactive with streptococcal and glomerular antigens, direct damage of glomeruli by streptococcal enzymes and toxins.
  • It was shown that renal glomerular membrane shares antigen epitopes with streptococcal M12 protein. This “antigen mimicry” provokes autoimmune reactions.
  • Rheumatic fever is a most serious delayed sequel of previous streptococcal infection. It arises within 1-5 weeks after group A streptococcal pharingitis (sore throat) or scarlet fever.
  • Rheumatic fever affects predominantly children or young persons. It is characterized by fever, mild polyarthritis without deformations, cardiovascular disorders that include heart inflammation (endomyocarditis and pericarditis) with systemic vasculitis, CNS involvement (chorea), skin manifestations (erythema marginatum).
  • Without adequate treatment the carditis leads to valves damage with chronic valvular heart disease progression. Finally the chronic heart failure can develop.
  • Rheumatic fever is the intermittent disease. Every secondary attack enhances valvular injury.
  • The disease is proven to be of autoimmune origin. M protein is accounted as a major streptococcal antigen that renders antigenic mimicry with host cardiac and skeletal myosin, tropomyosin, laminin, keratin and other substances.
  • Thus, rheumatic fever is provoked and supported by autoreactive antibodies and T cells that cross-react with streptococcal antigens and cardiac tissues.
  • Subsequent immune complex deposition induces complement activation. These autoimmune mechanisms lead to profound host tissue lesions.
  • In certain clinical conditions some other representatives of Streptococcaceae family can cause the diseases in humans.
  • For instance, group B Streptococcus agalactiae elicit neonatal meningitis and sepsis of newborns and infants. These bacteria colonize vaginal mucosa of 10-30% of healthy women. The newborn becomes infected during delivery and may develop severe meningitis with lethality of 30-50%.
  • S. mutans as well as S. sobrinus takes part in dental plaque formation by synthesis of long-chain polysaccharides from sucrose thereby promoting caries initiation.
  • The next progress of caries is related with fermentation of food-derived “table sugars” by S. mutans. It results in accumulation of lactic acid and tooth enamel decalcification with formation of caries lesion.
  • S. mitis and other viridans streptococci can cause individual cases of bacterial endocarditis.
  • Enterococci belong to the part of normal enteric microflora; nevertheless, they cause urinary tract infections. Being highly resistant to antimicrobial agents, they may cause severe hospital-acquired opportunistic infections in immunocompromised persons.
  • Streptococcus pneumoniae (or pneumococcus) is the major causative agent of community-acquired pneumonia in groups of all ages. According to WHO data, pneumococcal pneumonia leads to more than 1 mln death cases annually in children before the age of five.
  • The disease severity is related with multiple virulence factors of pnemococci – polysaccharide capsule that protects bacteria from phagocytosis and opsonization; membrane-affecting exotoxin pneumolysin; pneumococcal C-substance from cell wall teichoic acids that activates complement system and triggers host inflammatory response; IgA proteases suppressing mucosal immunity.
  • Besides community-acquired pneumonia, S. pneumoniae plays the substantial role in etiology of sinusitis, acute otitis media (about 40% of total cases), and bacterial meningitis in adults. It is generally ascertained that pneumococcal meningitis demonstrates extremely severe manifestations with mortality rate from 15 to 60%.
  • Post-streptococcal immunity is usually type-specific. Thus, it doesn’t prevent the reinfection with another type of bacteria. The immunity is of a moderate grade and duration.
  • Antibodies and immune T cells are directed to all major streptococcal antigens. Hypersensitivity reactions are shown to be common in most of streptococcal infections.
streptococcus pyogens

Laboratory Diagnosis of Streptococcal Infections


  • Specimens are obtained from the site of streptococcal infection. A throat swab, pus, wound discharge, blood, urine are examined.
  • Microscopy of specimens that reveals gram-positive single or short chain cocci is an auxiliary test, since the viridans streptococci may be found in clinical material as normal microflora.
  • Group A bacteria can be rapidly identified by immunofluorescence.
  • For cultivation the specimens are planted on blood agar and sugar broth. The primary growth appears in 1-2 days. Blood cultures are controlled within 5-7 days or even more.
  • The character of blood agar hemolysis is evaluated. Group A streptococci produce beta-hemolytic colonies.
  • Streptococci are catalase negative.
  • For definitive identification serologic grouping and typing of streptococci according to Lancefield classification is made by slide agglutination and precipitation tests.
  • Specific carbohydrate streptococcal antigens can be determined also by ELISA tests.
  • S. pyogenes is the single streptococcal representative rendering positive PYR-test (hydrolysis of pyrrolidonyl-β-naphthylamide substrate). In addition, S. pyogenes is sensitive to antibiotic bacitracin.
  • Serological diagnosis of group A streptococcal infections estimates the titer rise of antibodies to streptolysin O (basic test), streptokinase, hyaluronidase and DNAse (auxiliary tests). High titers of antistreptolysins (> 250 units) appear mainly in rheumatic fever patients indicating recent or relapsing infection.
  • S. agalactiae is identified by so-called CAMP test (according to R. Christie, N.E. Atkins, and E. Munch-Peterson, who proposed this method).
  • The test includes co-cultivation of Streptococcus agalactiae with S. aureus on blood agar. Usually two-streak plating of S. agalactiae is performed perpendicular to one-streak inoculation of hemolytic staphylococci. As a result, butterfly-like hemolysis enhancement of S. agalactiae appears.
  • Unlike conventional streptococci, enterococci easily grow in presence of bile and 6.5% NaCl. They can hydrolyze esculin that discriminates them from other streptococci.
  • S. pneumonia or pneumococci are gram-positive lancet-shaped diplococci. They develop alpha-hemolysis on blood agar. Their growth is inhibited by anti-microbial agent optochin. Also pneumococci are readily lysed in bile-containing media.
  • S. pneumonia is typed by capsular polysaccharide antigen into more than 90 serovars.
  • For rapid pneumococcal identification the slide microscopical test of capsule swelling is used. The specimen is treated by polyvalent antiserum that results in swelling of polysaccharide microbial capsule.
  • Streptococcal susceptibility testing is performed by disk diffusion and broth dilution methods.

Treatment and Prophylaxis of Streptococcal Infections


  • Beta-hemolytic group A streptococci are sensitive to benzylpenicillin, macrolides and azalides. Early treatment of streptococcal infections with penicillin interrupts autoimmune response against streptococcal antigens thus preventing poststreptococcal glomerulonephritis and rheumatic fever.
  • Benzylpenicillin (penicillin G) or ampicillin remain the drugs of choice for treatment of pneumococcal diseases caused by fully sensitive pneumococcal isolates; but penicillin-resistant strains gradually arise.
  • Enterococcus spp. is extremely resistant to many antibiotics. The bacteria display intrinsic resistance to most of beta-lactams, including cephalosporins.
  • Also they are resistant to sulfonamides (co-trimoxazole) and develop medium resistance to fluoroquinolones and aminoglycosides.
  • Combination of penicillinase-sensitive penicillins (benzylpenicillin, ampicillin) or vancomycin with aminoglycosides is regarded as the optimal therapy of enterococcal infections.
  • For specific prophylaxis of pneumococcal diseases in children and adults various kinds of pneumococcal vaccines are actively used now.
  • The most common are pneumococcal conjugate vaccine (PCV13) containing antigens of 13 bacterial types and pneumococcal polysaccharide vaccine (PPSV23) against 23 types of pneumococci. They successfully prevent the development of pneumococcal infections.
  • For prophylaxis of group A streptococcal infections an experimental chemical vaccine, containing various M proteins of group A streptococci is being worked out.