M BACTE: STREPTOCOCCUS

• Streptococcus, Enterococcus, and Other Catalase-Negative, Gram-Positive Cocci


  A large and biologically diverse group of gram-positive cocci that grow in pairs or chains
• Streptococcus, Enterococcus, and Other Catalase-Negative, Gram-Positive Cocci

  • Normal flora of humans and animals
  • Inhabit various sites, notably the upper respiratory tract
  • Live harmlessly as commensals
• First described by Billroth in exudates from erysipelas (St Anthony's fire) and wound infections, who called them streptococci


  1874
• Streptococci


  Streptos, meaning twisted or coiled; coccus, a grain or berry
• Enterococci


  Formerly known as group D streptococci, have been classified in their own genus, enterococcus
• Lactococci


  Previously classified as group N streptococci, now belong in the genus lactococcus
• Weak false-positive catalase reactions can be seen when growth is taken from media containing blood, owing to the peroxidase activity of hemoglobin
• Pasteur found similar organisms in the blood of a patient with puerperal sepsis


  1879
• Ogston isolated them in acute abscesses, distinguished them from staphylococci and by animal inoculation established their pathogenicity


  1881
• Genera of catalase-negative, gram-positive cocci

  • Streptococcus
  • Enterococcus spp.
• 17 genera of catalase-negative, gram-positive cocci, including Streptococcus, and Enterococcus spp. belong to the family Streptococcaceae
• Streptococcus and Enterococcus


  Behave like facultative anaerobes - they grow in the presence of oxygen but are unable to use oxygen for respiration, considered aerotolerant anaerobes
• Carbohydrates


  Metabolized fermentatively with lactic acid as the major end product; gas is not produced
• Some species


  • Capnophilic, requiring increased concentration of carbon dioxide (CO2), whereas the growth of other species is stimulated by increased concentration of CO2, but CO2 is not required
• Growth


  • Poor on nutrient media such as trypticase soy agar (TSA), but more pronounced on media enriched with blood or serum
  • Colonies are usually small and transparent
• Most streptococci, except for many of the viridans group, have a group or common C carbohydrate (polysaccharide), which can be used to serologically classify an isolate
• Streptococci are first divided into

  • Obligate anaerobe
  • Facultative anaerobes
• Obligate anaerobe


  Designated as Peptostreptococci
• Streptococcus


  Facultative anaerobe
• Alpha-hemolytic (α) Streptococci


  • Produce a zone of partial hemolysis with a greenish discoloration around the colonies on blood agar
  • The zone of lysis is small (1–2 mm wide) with indefinite margin, within this zone unlyzed erythrocytes can be made out microscopically
  • Also known as Viridans streptococci
• Beta (ß) Hemolytic Streptococci


  • Produce a sharply defined, clear, colorless zone of hemolysis (2–4 mm wide) around the colony, caused by complete lysis of red blood cells in the agar medium induced by bacterial hemolysins
  • No red blood cell is visible on microscopic examination in clear zone of complete hemolysis
  • ß-hemolysis constitutes the principal marker for potentially pathogenic streptococci in cultures of throat swabs or other clinical samples
• Gamma (γ) or Non-hemolytic Streptococci


  • Produce no hemolysis on blood agar
  • Enterococcus faecalis is an important organism of this group
• Lancefield Grouping


  • Basis of group specific carbohydrate (c) antigens in the cell wall, divided into 21 serological groups from A to W (without I and J)
  • Groups A, B, C, D, and G are most commonly found associated with human infections
• Griffith Typing - S. pyogenes

  Hemolytic streptococci of Group A are known as S. pyogenes, further divided into types based on the protein (M, T and R) antigens present on the cell surface
• About 80 types of S. pyogenes have been recognized so far (types 1, 2, 3 and so on)
• Biochemical and other criteria are also used in defining various species within a single serogroup, and some species contain strains of more than one serogroup
• Streptococcus pyogenes


  • Gram-positive, spherical to ovoid organisms 0.5–1.0 mm in diameter
  • Grows in short or moderately long chains, the chain length being dependent on the strain and culture medium
  • Nonmotile and non spore former
  • Some strains produce a capsule of hyaluronic acid, while polysaccharide capsules are encountered in members of Group B and D
• Cultural Characteristics of Streptococcus pyogenes

  • Aerobe and facultative anaerobes
  • Best at a temperature of 37°C (range 22–42°C)
  • The optimal pH for growth is 7.4–7.6
  • Demanding in nutritive requirements, growth occurring only in media containing fermentable carbohydrates or enriched with blood or serum
• Cultural Characteristics - BAP


  • S. pyogenes colonies are small (0.5–1 mm in diameter), circular, semitransparent, low convex disks surrounded by a wide zone of β-hemolysis, several times greater than the diameter of the colony after incubation for 24 hours
  • Enhancement of growth and hemolysis are promoted by 10% CO2
  • Matt (finely granular) colonies contain M antigen, which are virulent strains, while avirulent strains form glossy colonies
  • Mucoid colonies may occur when a strain is heavily capsulated
  • Very rarely, non hemoloytic group A streptococci are encountered
• Cultural Characteristics


  • Crystal violet blood agar and PNF medium (blood agar containing polymyxin-B, neomycin and fusidic acid) are selective for beta hemolytic streptococci
  • Pike's medium is a transport medium for clinical specimens containing Group A streptococci, prepared by adding crystal violet (1 in 1,000,000) and sodium azide (1 in 16,000) to blood agar
  • In liquid media, such as glucose or serum broth, growth occurs as a granular turbidity with a powdery deposit
• Biochemical Reactions


  • S. pyogenes is catalase negative
  • Insoluble in 10% bile unlike S. pneumoniae (soluble)
  • It ferments several sugars producing acid and no gas
  • Hydrolysis of pyrrolidonyl naphthylamine (PYR test) is positive
  • Failure to ferment ribose distinguishes it from non-group A hemolytic streptococci
• Resistance


  • Can be killed by heating at 54°C for 30 minutes
  • Can survive in dust for several weeks, if protected from sunlight
  • More resistant to crystal violet than many other bacteria, it is used for preparation of selective media
  • Sensitive to benzylpenicillin and a wide range of antimicrobial drugs
  • Susceptible to sulfonamides, but does not develop resistance to drugs
  • Sensitive to bacitracin, and this property is employed as a convenient method for differentiating S.pyogenes from other hemolytic streptococci
• Antigenic Structure - Inner Layer of Peptidoglycan

  • Peptidoglycan: mucoprotein is responsible for cell wall rigidity, pyrogenic and thrombolytic activity
• Antigenic Structure - Group-specific Polysaccharide Antigen

  • Serologic classification of β-hemolytic streptococci is based on their cell wall polysaccharide antigen
  • As this antigen is integral part of the cell wall, it has to be extracted for grouping by a precipitation test with group antisera
  • Techniques for the extraction of the group antigens: Lancefield's acid extraction method, Formamide (Fuller's method), by enzyme produced by Streptococcus albus (Maxted's method), By autoclaving (Rantz and Randall's method)
• Antigenic Structure - Outer Layer - M protein

  • Acts as virulence factor by inhibiting phagocytosis
  • Antigenic and specific anti-M antibody develops after infection
  • Resistant to heat and acid but susceptible to trypsin
  • Encoded by the gene emm
  • Can be extracted by the Lancefield acid extraction method and M typing is performed by capillary tube precipitation test using type-specific antisera and acid extract
  • Causes the streptococcal cell to resist phagocytosis and bacterial cell plays a role in adherence of the bacteria to mucosal cells
  • More than 200 different serotypes and subtypes of M protein exist, identified as M1 (emm1), M2 (emm2), and so on
  • Resistance to infection with S. pyogenes appears to be related to the presence of type-specific antibodies to the M protein
  • M1 serotype is the most common serotype seen in pharyngitis
• Antigenic Structure - Outer Layer - R protein

  • Some strains of S. pyogenes (types 2, 3, 28 and 48) and some strains of groups B, C and G contain third antigen R protein
  • R protein has no relation to virulence
• Antigenic Structure - Outer Layer - M-like proteins, lipoteichoic acid, and F protein

  • Facilitate binding of host cells by complexing with fibronectin, which is present on the host cell surface
• Antigenic Structure - Outer Layer - Capsule

  • Composed of hyaluronic acid and is not immunogenic
  • It has an antiphagocytic effect like other bacterial capsule
• Toxins and Enzymes - Hemolysins - Streptolysin O


  • So called because it is oxygen labile hemolysin
  • Inactivated in the oxidized form but may be reactivated by treatment with mild reducing agents
  • Heat-labile protein, cytolytic and capable of lysing erythrocytes, leukocytes, platelets and cultured cells
  • Lethal on intravenous injection into animals and has a specific cardiotoxic activity
• ASO Test


  • Strongly antigenic and antistreptolysin O (ASO) antibodies appears in sera following streptococcal infection
  • An ASO titer in excess of 200 Todd units/mL is considered significant and suggests either recent or recurrent infection with streptococci