BSCI 424 — PATHOGENIC MICROBIOLOGY — Fall 2000

Streptococcus Summary

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Morphology & Physiology:

General Characteristics:

   Gram-Positive, Nonmotile Cocci (0.5-1.2um) often Arranged in Pairs or Chains (see WebLinked image; see WebLinked image)
   Exist as Commensals and Parasites for Man, Animals or Saprophytes

   Most are facultative anaerobes

   Complex nutritional requirements (fastidious): Require blood- or serum-enriched media; Inability to synthesize many basic building blocks

   Fermentative metabolism: Carbohydrates are fermented without the production of gas; Major endproducts are lactic acid, ethanol, acetate

   Catalase negative: Useful to distinguish streptococci from staphylococci

   Oxidase negative

   Rigid cell wall with typical Gram-positive peptidoglycan layer, inner plasma membrane, mesosomal vesicles, nucleoid; Divide by crosswall septation

   Some strains have hyaluronic acid capsule; Produce hyaluronidase later in growth cycle

Cellular Structure of Group A Streptococci: S. pyogenes

   Cell wall contains group- and type-specific antigens

  Group-specific carbohydrate (C-polysaccharide):

~10% dry weight of the cell

Branched polymer of L-rhamnose and N- acetyl-D-glucosamine (2:1 ratio); latter is antigenic component; linked by phosphate-containing bridges to peptidoglycan which is composed of N-acetyl-D-glucosamine, N- acetyl-D-muramic acid, D-glutamic acid, L- lysine, and D- and L-alanine

  Type-specific proteins: Two major classes, M and T antigens; Minor classes include: F, R and M-like or M-associated proteins (MAP) antigens

M Protein: Fimbriae-like, "hairy" extensions associated with virulent strains

Resistant to heat and acid; Trypsin sensitive

Carboxy (-COOH) terminal end of molecule is anchored in cell wall and sequence is highly conserved among all M serotypes

More than 80 M serotypes are due to antigenic diversity of the M protein's amino (-NH2) terminus which is surface-exposed

T protein: Useful epidemiological marker; Not identified as virulence factor

Trypsin resistant; Resistant to heat and acid

M-like proteins: Structurally similar to M protein

F protein: Binds fibronectin

Lipoteichoic acid is also antigenic

   Capsular polysaccharide: composed of hyaluronic acid (also found in host connective tissue)

Classification Schemes (Taxonomy):

Based on biochemical (Physiological) properties

Based on clinical presentation:

   Pyogenic (suppurative)
   Oral

   Enteric

Based on hemolytic patterns:

   Beta-hemolytic: beta-hemolysis often enhanced under anaerobic conditions
   Alpha-hemolytic

   Gamma-hemolytic

Based on serology: Lancefield serogrouping of beta-hemolytic streptococci; Lancefield group-specific antigens

   Lancefield (1933) developed useful serotyping system for classification of beta-hemolytic streptococci based on the group-specific antigenic composition of cell wall carbohydrates (Note: viridans streptococci and Streptococcus pneumoniae have no group-specific antigen)

   Serogroups A through H and K through V

  Major pathogens

Group A Streptococci: Streptococcus pyogenes

Group B Streptococci: S. agalactiae (Note: May also be alpha-hemolytic or nonhemolytic)

Enterococcus faecalis and E. faecium were previously classified as Group D streptococci

  Common pathogens

Group C Streptococci: S. equismilis (pharyngitis); S. anginosus (abscess formation)

Group F Streptococci: S. anginosus (abscess formation)

Group G Streptococci: S. anginosus (abscess formation)

  Uncommon pathogens

Group D Streptococci: S. bovis, S. durans, S. avium (foodborne disease)

Groups E, H, and K through

   Streptococci lacking group-specific antigens

  Viridans streptococci: Gamma or alpha-hemolysis

Streptococcus mutans group

S. sanguis group

S. salivarius group

S. mitis group

S. anginosus-milleri group

Streptococcus spp.

  Streptococcus pneumoniae

Clinical Syndromes:

Group A Streptococcus (S. pyogenes): One of the most important human pathogens; Commonly associated with a diverse group of human diseases, including both suppurative (pus-forming) and nonsuppurative diseases

   Suppurative streptococcal diseases (Acute streptococcal infection)

  Pharyngitis (& tonsilitis): "strep throat" --- Suffix "-itis" refers to inflammation of (in this case, inflammation of the pharynx or tonsils)
  Scarlet fever: complication of streptococcal pharyngitis when infecting strain is lysogenized ; Frequently develop scarletina rash on upper chest spreading to extremities

  Cutaneous & soft tissue infections:

Pyoderma (Impetigo: contagious pyoderma with superficial yellow weeping lesions)

Erysipelas: Acute superficial cellulitis of skin with lymphatic involvement; face and lower extremities, skin and subcutaneous tissues

Cellulitis: Involvement of deeper subcutaneous tissues; Deeper invasion with systemic symptoms

Necrotizing fasciitis (a.k.a., "flesh-eating bacteria"): Infection deep in subcutaneous tissues that spreads along fascial planes, destroying muscle and fat; Initially cellulitis followed by bullae (fluid filled blisters; bulla is singular), gangrene, systemic toxicity, multiorgan failure and mortality in more than 50% of patients

Streptococcal toxic shock syndrome: Multisystem toxicity following soft tissue infection progressing to shock and organ failure (not to be confused with Staphylococcal Toxic Shock Syndrome where hyperabsorbent tampons have been identified as an important risk factor); Systemic infection following soft tissue infection

  Other suppurative diseases: Puerperal sepsis (associated with childbirth); Lymphangitis (inflammation of lymphatic vessel(s)); Pneumonia

  Bacteremia: bacteria in the blood with mortality approaching 40%; Septicemia (sepsis): systemic disease associated with persistent presence of bacterial cells, bacterial toxins or other bacterial products in the blood

Acute nonsuppurative (non-pus-forming) sequelae (complications) of Group A streptococcal disease: Post-infection sequelae

Acute rheumatic fever (ARF): Inflammation of heart, joints, blood vessels, subcutaneous tissues

Nonsuppurative inflammatory reaction characterized by arthritis, carditis, chorea (disorder of nervous system with involuntary spastic movements), erythema marginatum (skin redness with defined margin), or subcutaneous nodules

Morbidity and mortality linked to subsequent valvular heart disease

Poorly understood pathogenesis with several proposed theories including cross-reactivity of heart tissues and streptococcal antigens, exotoxins, or direct invasion

  Rheumatic heart disease: Chronic, progressive heart valve damage

  Acute glomerulonephritis: Acute inflammation of renal (kidney) glomeruli

Signs include dark, smoky urine with RBC's, RBC casts, white blood cells, depressed serum complement, decreased glomerular filtration rate

Granular accumulations of immunoglobulin due to deposition of immune complexes within the kidney

Group B Streptococcus (S. agalactiae)

   Neonatal disease (Early onset and late-onset): Infection can occur in utero, at birth, or during first few months of life

  Neonatal sepsis (septicemia)
  Meningitis: Inflammation of the meninges

  Pneumonia

  Obstetric complications:

  Urinary tract infections (UTIs)
  Peurperal (postpartum) sepsis
  Amnionitis: Inflammation of amnion (membrane enveloping fetus)
  Endometritis: Inflammation of the endometrium (inner mucous membrane of the uterus)
  Wound infections
  Infections in non-pregnant adults: Primarily skin and soft tissue involvement

  Genitourinary tract infections (urosepsis)
  Wound infections
  Pneumonia
  Bacteremia

Other Beta-Hemolytic Streptococci:

  Most commonly Group C (S. equisimilis, S. anginosus), Group F (also S. anginosus), and Group G (also S. anginosus)

  Pharyngitis
  Abscess formation
  Bacteremia
  Group D: Foodborne disease similar to staphylococcal intoxication

Non-Beta-Hemolytic Streptococci: Viridans Streptococci (heterogeneous collection of alpha- and non-hemolytic)

Dental caries (cavities): S. mutans adhere to enamel via production of insoluble dextran from glucose; Allows other organisms to adhere (plaque formation) with acid production causing tooth decay; Lactobacillus spp. cause caries of dentin secondary to enamel caries caused by mutans streptococci (see also Actinomyces)

Subacute bacterial endocarditis: S.mutans, S. sanguis adhere to previously damaged heart valves via production of insoluble dextran from glucose

Abscess formation (suppurative intraabdominal infections)

Epidemiology:

General Overview:

Streptococci have a predilection for the upper respiratory tract or skin; Strains that colonize the skin are usually different "antigenic types" from those that colonize the throat
Group A streptococci commonly colonize the oropharynx of healthy children and young adults or the skin

Colonization is transient due to development of M protein-specific immune response

Rapidly killed after phagocytic ingestion, but cell walls that are not digested may lead to chronic inflammatory lesions

Elevated incidence of carriage

Non-beta-hemolytic streptococci are competitive; Produce bacteriocins which are inhibitory to Group A strains

Acute Streptococcal Infection: Disease from recently acquired strain

  Pharyngitis (peak ages 5-15 years) and scarlet fever (complication of streptococcal pharyngitis):
Transmitted person-to-person by droplets from respiratory secretions; Crowding increases risk (e.g., classrooms, day care facilities)

  Acute rheumatic fever and glomerulonephritis sequelae to pharyngitis

  Scarlet fever results from infection with a strain that is lysogenized with a temperate bacteriophage that genetically encodes for pyrogenic exotoxin (formerly known as erythrotoxin or erythrogenic toxins)

  Cutaneous & soft tissue infections:

Transmitted through breaks in skin after direct contact with infected person, fomite, or arthropod vector

T-antigen typing useful where non-M typeable

Only glomerulonephritis sequelae

Streptococcal toxic shock syndrome: caused by different M serotypes than those causing pharyngitis; pyrogenic exotoxins, particularly exotoxin A, are produced

Neonatal and puerperal disease:

Transient colonization of vagina observed in both pregnant and nonpregnant females, most commonly by limited number of serotypes

Colonize lower gastrointestinal (GI) tract and genitourinary tract

60% of colonized mothers birth colonized babies; Neonatal disease in 3/1000 live births

Risk factors:

Immune status of mother is more important risk factor than exposure to organisms for acquiring neonatal disease (maternal type-specific capsular antibodies transplacentally passed to fetus is protective during first few months of life as infant's immune system matures)

Heavy vaginal colonization during birth increases likelihood of neonatal colonization, but does not increase risk of disease

Complement deficiency

Pregnancy risks include: Premature birth; Prolonged rupture of membranes; Fever during delivery

Early onset neonatal disease: Within 7 days of birth

Acquired in utero or during delivery

Three times more frequent than late onset disease

15-30% of post-meningeal infants experience neurological sequelae that include blindness, deafness, and severe mental retardation

Late-onset neonatal disease: Occurring 1 week to 3 months after birth

Acquired from exogenous source: Mother, other infants, health care provider

Presents most commonly as bacteremia with meningitis; Neurological complications are common

Puerperal sepsis: Common cause of maternal death in pre-antibiotic era

Disease in nonpregnant adults

Risk Factors:

Diabetes mellitus

Cancer

Alcoholism

Proportionally more adult disease, but incidence is higher in neonates

Foodborne disease: Infrequent foodborne disease outbreaks

Organisms multiply when contaminated foodstuffs are improperly refrigerated and allowed to stand at room temperature for several hours between preparation and consumption

Contamination of food is result of incomplete processing or unsanitary food handling by carriers (poor hygiene, ill or asymptomatic food handler) or unpasteurized milk (frequent outbreaks before advent of pasteurization)

  High infectious dose of >10⁷ organisms

Explosive common-source Group D outbreaks (2-36h incubation period) with a clinical syndrome similar to staphylococcal intoxication

Note: Septic sore throat, scarlet fever and other pyogenic and septicemic syndromes are also infrequently acquired from ingestion (low infectious dose of <1000 organisms) of contaminated food (onset after 1-3 days)

Sequelae of Acute Streptococcal Infection: Serious suppurative and nonsuppurative sequelae in pre-antibiotic era; Nonsuppurative rheumatic fever and glomerulonephritis is still important in developing countries

Acute rheumatic fever (ARF)
Within 2-3 weeks (latent period) following respiratory infection only (e.g., pharyngitis)

For diagnosis, must demonstrate recent infection by culture or serology; Detect an increase in antibody titer to at least one of SLO, DNase B, hyaluronidase, streptokinase

Following epidemic pharyngitis: ARF in as many as 3%; Following sporadic pharyngitis: ARF in 1 per 1000

Acute post-streptococcal glomerulonephritis

Acute glomerulonephritis follows either respiratory (e.g., pharyngitis) or cutaneous streptococcal infection

Latent period: 1-2 weeks after skin infection and 2-3 weeks after pharyngitis

Associated with specific, well-defined group of serotypes

Incidence following infection varies from less than 1% to 10-15%

Most often seen in children

Viridans Streptococcal Infections: Streptococcus mutans is major dental pathogen

Acidoduric (durable in acidic environment) and acidogenic (acid generating) properties, and the ability to synthesize extracellular adherent polysaccharides enable these organisms to establish initial colonization of dental surfaces and to provide a foundation layer for the formation of a complex biofilm known as dental plaque
Enamel caries is acid demineralization of enamel caused by lactic acid production by these bacteria during fermentation of sucrose or other carbohydrates and is dependent upon a variety of physical, chemical, and mechanical factors

Dental caries can be visualized as a disease state that occurs only when three factors or conditions co-exist. These three interdependent components are:

Status of host and teeth (sites that organisms can colonize, in particular solid surfaces and interfaces)

Specific microflora

Nutritional substrate (carbon and energy source)

Gingivitis and more severe periodontal diseases are a result of plaque-derived infection at the tooth and gum interface with loss of gum tissue and underlying bone

Pyogenic oral infections can be acute or chronic in nature, caused by mixed anaerobic infection of tissue by plaque flora, e.g., by trauma, invasive dental treatments, periodontal disease, or infected dental pulps

Pathogenesis & Immunity:

  Lysogeny (Lysogenic Conversion)

  Lysogenized bacteriophages may play key role in directing synthesis of various Group A streptococcal enzymes and toxins

  Pyrogenic exotoxin (a.k.a., erythrogenic toxin): Phage-associated muralysins produced by both Group A and C streptococci

  Grp C muralysin is N-acetylmuramyl-L-alanine amidase that lyses streptococcal cell walls; Used to produce L-forms (cells lacking rigid cell walls) or to purify membranes and M protein

  Virulence Factors of Group A Streptococcus pyogenes: Structural molecules; Toxins; Enzymes

  Cellular components

  Lipoteichoic acid

  Adherence to buccal epithelial cells

  Cytotoxic for wide variety of cells

  Colonization ligand: Forms complex network with M protein and binds via lipid moiety to fibronectin on epithelial cells

  M protein

  Antiphagocytic; alpha-helical coiled dimer; fibrillar molecule

  Inhibits complement component C3b (Factor H, serum control protein) deposition on streptococcal cell surface, inhibiting alternate complement pathway and opsonization

  Cell wall is potent activator of alternate complement pathway, but M-protein prevents these reactions

  Grp A Strep with type-specific M-protein survive until type-specific antibody response develops

  Amino-terminus (distal to cell) contains antigenically variable determinants; Three large tandem repeat regions contribute to antigenic diversity with intragenic recombination events

  Abnormal host immune response against cross-reactive antigens may result in sequelae

  F protein: Binds fibronectin of host; Major adhesin to throat and skin by mediating adherence to epithelial cells

  Associated proteins: Some under control of virulence regulon and co-regulated with M-protein

  C5a peptidase destroys chemotactic signals

  Serum opacity factor

  Fc receptor protein binds to Fc region of mammalian IgG

  Antiphagocytic capsular polysaccharide

  Hyaluronic acid mimics animal tissue ground substance found in host connective tissue and is therefore nonimmunogenic; assists in avoiding phagocytosis

  Extracellular components

  Hemolysins: Two hemolytic and cytolytic toxins

  Streptolysin O (SLO)

  Prototype of oxygen labile or thiol-activated bacterial cytolytic protein toxins produced by Streptococcus, Bacillus, Clostridium, and Listeria

  Irreversibly inactivated by cholesterol

  Bind to cholesterol-containing membranes and form arc- or ring- shaped oligomers that make cell leaky (RBC's, PMN's, Platelets,etc.)

  SLO induces rapid antibody response

  SLO titer indicates recent infection (300-500 in pediatric populations)

  Streptolysin S (SLS)

  Oxygen stable, non-antigenic

  Extractable only in presence of carrier or inducer, e.g., serum, albumin, RNase-resistant fraction of RNA

  Lytic for red and white blood cells and wall-less forms (protoplast, L- forms)

  Responsible for surface hemolysis on blood agar plates; Inhibited by phospholipids

  Pyrogenic exotoxins (a.k.a., erythrogenic toxins)

  Produced by more than 90% of Grp A streptococci; 3 types Groups A, B, and C

  Heat labile; Stable to acid, alkali, pepsin

  Structural gene is carried by temperate bacteriophage, as is the case with diphtheria toxin

  Result in fever and scarlet fever rash, increase susceptibility to toxic shock, cause reticuloendothelial system blockade, act as mitogens, myocardial and hepatic necrosis, decrease in antibody synthesis

  Immunomodulators (superantigens) at low concentrations, stimulate T cells

  Type C toxin increases permeability of blood-brain barrier to endotoxin and bacteria

  Nucleases: Reduce viscosity

  Four antigenic types (A,B,C,D) in S. pyogenes assist in liquefication of pus to generate growth substrates

  Nucleases A, C have DNase activity

  Nucleases B, D also have RNase activity

  Require calcium and magnesium

  Antibody titers to DNase B useful serodiagnosis

  Other Enzymes

  Two different streptokinases that catalyze conversion of plasminogen to plasmin, leading to digestion of fibrin (dissolve blood clots); may enable rapid dissemination through infected tissues

  Hyaluronidase hydrolyzes hyaluronic acid; "spreading factor"

  DPNase, cardiohepatic toxin, proteinase, NADase, ATPase, phosphatase, etc.

  Virulence Factors of Group B Streptococcus agalactiae

  Enzymes of unknown pathogenicity: Dnases, hyaluronidase, neuraminidase, proteases, hippurase, hemolysins

  Immune Response Against Streptococcal Disease

  Antibodies against type-specific capsular antigens are protective; Infants without type-specific maternal antibodies are at increased risk of acquiring infection

  Bactericidal activity requires the presence of complement

Laboratory Identification

General Overview

  Gram stain of tissue sample can provide rapid initial diagnosis

  Bacitracin sensitivity test for presumptively distinguishing between beta-hemolytic streptococci isolated from pharyngeal swabs (95% accuracy of Grp A strep sensitivity)

  Negative catalase used to differentiate from Staphylococcus

Group A Streptococci

Specimen Collection and Processing

Throat swabs from the posterior oropharynx (back of throat, e.g., tonsils) or from skin lesion

Microscopy: Gram stain

Cocci in short chains in clinical specimens; Longer chains seen in culture

Culture

  Primary culture by pour and streak plate

  Fastidious growth requirements; Optimal growth on enriched blood agar; Growth inhibited by glucose in medium (fermented with lactic acid buildup)

  1 to 2-mm domed white (grayish to opalescent) colonies with large zone of beta-hemolysis (several times that of diameter of colony) after 18-24h incubation

  Colonies of encapsulated strains appear mucoid on moist media, wrinkled on dry media; Colonies of nonencapsulated strains appear small and glossy

Identification

  Preliminary identification

  Positive CAMP test

  Hydrolysis of hippurate

  Antigen detection

  Group Specificity

  Positive identification of group-specific carbohydrate (rhamnose, N-acetylglucosamine, galactose)

  Type Specificity

  Type-specific capsular polysaccharide (sialic acid residue)

  Type-specific proteins

Methods

  Bacitracin sensitivity

  Rapid identification tests: Based on extraction of Grp A carbohydrate directly from throat swabs

  Coagglutination

  Fluorescent Antibody

  Antigen detection

  Detection of group-specific carbohydrate (dimer of N-acetylglucosamine & rhamnose) (formerly C-Polysaccharide)

  Capillary tube precipitin tests

  Variety of agglutination tests

  Detection of M protein by Capillary Tube Precipitin Test with extracts or whole cells

  Detection of T protein by Slide Agglutination Test with trypsin treated whole cells; Adjunct to M-typing; routine surveillance

  Antibody Detection

  ASO Test: Detection of antibodies against streptolysin O confirm a recent Group A streptococcal infection

  Detection of DNase B

  Group B Streptococci

  Microscopy: Gram stain

  Cocci in short chains in clinical specimens; Longer chains in Cx

  Culture: Large, buttery-appearing colonies usually surrounded by a narrow zone of hemolysis

Treatment, Prevention & Control

Therapy: Aimed at prevention of suppurative complications and the nonsuppurative sequelae of rheumatic fever and glomerulonephritis

Group A Drug of Choice: Oral (PO) penicillin V (VK) or intramuscular (IM) benzathine penicillin; Alternatives: Erythromycin, clindamycin, cephalexin (oral cephalosporin)

Group B Drug of Choice: Intravenous (IV) penicillin G; Alternate: Vancomycin

High MIC (minimal inhibitory concentration)

Antibiotic tolerance has been observed

Tolerance: able to endure without effect

Acute Rheumatic Fever

Salicylates and corticosteroids for acute symptom reduction and control of long-term sequelae; Prevention by preventing initial strep infection or prompt treatment of pharyngitis

Acute Post-Streptococcal Glomerulonephritis

Therapy directed at secondary phenomenon of volume excess, hypertension, and seizures; Sodium restriction, diuretics, anticonvulsants

Must demonstrate recent infection by culture or serology; Detect an increase in antibody titer to SLO or DNase B

Renal biopsy, immunofluorescent exam, or electron microscopy to diagnose

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