STREPTOCOCCI

General characteristics

Gram positive cocci in chains

hemolytic classes and serologic groups used for classification

facultative anaerobes

lactic acid bacteria

fermentation of sugars --> lactic acid in the media

lowers the pH so that growth is limited unless the pH is corrected.

Hemolysis

1. beta hemolysis

major human pathogens

some beta-hemolytic strep are not pathogenic

wide clear zones of complete hemolysis

2. alpha hemolysis

incomplete hemolysis

green discoloration - formation of an unidentified reductant of red cell hemoglobin

narrow zone of hemolysis

viridans group of streptococci

3. Gamma hemolysis = no hemolysis

heterogeneous group; few are considered primary pathogens

commensals in humans and animals.

Enterococcus (formerly Streptococcus) faecalis is a common and important nonhemolytic species.

The classification of streptococci based on hemolysis is not so reliable since there is considerable variation within some of the groups. For example, although E. faecalis is commonly nonhemolytic, it could also produce alpha or beta hemolysis.

Strep may also be classified by their antigenic properties: surface polysaccharides:

In 1933 Rebecca Lancefield and her co-workers developed a technique for differentiation of the streptococci. She placed them into 18 serological groups A through R on the basis of the antigenic characteristics of a cell wall carbohydrate called the C substance. The main pathogenic groups for humans are A, B, C, D, and G, and each group is given a species name.

GROUP A STREPTOCOCCI

 

More than 90% of streptococcal disease in humans is caused by group A beta-hemolytic strep: S. pyogenes.

Transmission - person to person - usually by contact with respiratory secretions or fomites from an asymptomatic carrier who harbors the organism in the upper respiratory tract, skin, or rectum (reservoir). Between 5 and 20% of asymptomatic individuals carry group A strep in their throats: "carrier state."

Clinical

Classification of infections caused by S. pyogenes (Group A strep):

a. noninvasive infections: cutaneous, mucous membranes

streptococcal pharyngitis (strep throat) may have complications including otitis media, peritonsillar abscesses, meningitis, peritonitis, scarlet fever (rash from exotoxin), and pneumonia.

Impetigo - superficial; highly contagious skin disease primarily in children. The infection begins as small blisters that can spread to adjacent areas.

Cellulitis - deeper; an inflammatory condition associated with strep invasion of connective tissue.

Erysipelas - infection of the skin or mucous membrane; characterized by spreading inflammation. Cellulitis w/ fever and systemic toxicity.

Puerperal fever (or sepsis) - a post-partum infection of the uterus. May accompany childbirth if aseptic technique is not followed. Rare now.

b. invasive infections

streptococcal toxic shock syndrome. Onset is abrupt and severe. Fever is a common early sign. Patients generally have no predisposing underlying diseases (not compromised).

The most common presentation is acute localized pain in an extremity with a paucity of physical findings. Risk factors: trauma, varicella, use of nonsteroidal anti-inflammatory agents. Often associated with SPE-A and SPE-B production and M protein types M1 and M3. Epidemiologic data indicate that a highly virulent clonal variant of a common serotype emerged in the early 1980s. Mortality 30-70%.

c. Necrotizing fasciitis is a deep-seated infection of the subcutaneous tissue that progressively destroys fascia and fat but spares the skin and muscle. Most begin at the site of trivial or inapparent trauma. Patient experiences local pain, fever and systemic toxicity (toxins in bloodstream) associated with shock and organ failure. The disease is often fatal unless promptly recognized and aggressively treated (surgical debridement and antibiotics). Diagnosis difficult.

d. invasive infections without hypotension: bacteremia or focal infections

 

  1. nonsuppurative sequelae: acute rheumatic fever and acute glomerulonephritis. These are "diseases" not infections because there are no viable strep at the sites.

**Two serious postinfection sequelae made identification and prompt treatment of group A strep infections of critical importance:

These occur 1-3 weeks after acute streptococcal illness;

?cause of these sequelae

 

Acute glomerulonephritis - acute inflammation of the renal glomeruli - follows infections caused by a limited number of M types of group A strep. Many nephritogenic strains belong to M type 12.

occurs 1 to 4 weeks after the onset of an acute pyogenic infection, usually of the pharynx or skin.

The pathogenesis of acute glomerulonephritis is believed to be due to an immune complex reaction.

Antigens from the cytoplasmic membrane of the bacterial cell may cross-react with the basement membrane of the glomerulus of the kidney, precipitating the immune complex reaction.

Evidence: streptococcal antigens, immunoglobulin and C3 have been demonstrated in glomerular lesions.

Rheumatic fever may follow pharyngeal infection only with almost any type of group A strep, and it occurs 2 - 5 weeks following the infection. <1% of pharyngitis cases lead to rheumatic fever.

characterized by heart muscle damage and fever; is often accompanied by tissue destruction of heart valves as well as inflammation of the joints.

The pathogenesis of rheumatic fever is obscure. Possibilities include:

a. Streptolysin O - shown to be cardiotoxic;

b. streptococcal proteinase injected IV causes subendocardial lesions (as do other unrelated proteases);

c. cell wall fragments, composed of group-specific carbohydrate and peptidoglycan, can cause cardiac and joint lesions.

  1. Immunologic cross-reactions between streptococcal antigens and tissue antigens of cardiac muscle. VACCINE CONCERNS.

Cross-reactions between group A carbohydrate (C substance) and a structural glycoprotein of heart valves. Patients with rheumatic fever have high levels of serum antibodies to the group A carbohydrate. These ?cross-reactive serum antibodies may contribute to the heart valve damage associated with rheumatic fever.

Cross reactions between M antigens and heart antigens have also been reported.

 

Pathogenesis:

Group A strep is an extracellular pathogen. Once ingested by phagocytes, it is rapidly killed. Thus its antiphagocytic properties (hyaluronic acid capsule and M protein) play a critical role in virulence.

In addition, the organism combats phagocytic clearance by producing streptolysin S which is leukotoxic.

 

Virulence determinants/Surface antigens

**a. M antigens. Protein ags - >80 immunologic types

distributed on the bacterial surface as a fibrillar structure (fimbriae)

antiphagocytic - interferes with C deposition

anti-M antibodies are protective; they neutralize this antiphagocytic antigen.

coiled-coil structure, with a highly variable distal amino terminus and a conserved C terminus.

Strains that are M+ may lose their M antigens, particularly in the lab. When such M- strains are passed through mice, they frequently revert to M+.

Certain M types are more associated with skin infections and glomerulonephritis and others are associated with throat infections and rheumatic fever. M typing can help trace virulent strains through a population (molecular epidemiology).

**b. Capsules - hyaluronic acid capsule which is expressed through the log phase of growth, but disappears rapidly as the organisms reach stationary phase. Highly encapsulated strains form mucoid, watery colonies. Because it is chemically indistinguishable from the hyaluronate of mammalian connective tissue, the production of the hyaluronic acid capsule does not elicit the formation of antibodies - not immunogenic.

Upon repeated culture in the lab, mucoid strains may lose their capsule.

  1. Lipoteichoic acid (glycerophosphate) is involved the attachment and colonization of streptococci on mucosal surfaces.
  2. F protein binds fibronectin and promotes streptococcal adherence.
  3. Protein G - receptor for the Fc portion of IgG.

f. C5a peptidase - enzyme that inactivates the chemotactic complement fragment C5a.

g. Group specific C carbohydrate antigens (C substance). In group A, it is a rhamnose backbone with N-acetyl glucosamine side chains.

h. Miscellaneous cell wall proteins:

T antigens are useful to serotype M- strains. Abs not protective.

R proteins have no known clinical or pathogenic importance.

**Bacterial adhesins: lipoteichoic acid, F protein, M protein, hyaluronic acid

 

Group A strep extracellular virulence determinants:

**1. Streptococcal pyrogenic exotoxins (erythrogenic toxins):

3 immunologically distinct forms (pyrogenic exotoxins A, B, and C).

SpeA and SpeC are phage-encoded proteins

SpeA is the most potent

protein superantigens similar to the enterotoxins of staph. Biological effects in animals include pyrogenicity and enhancement of the toxic action of gram-negative endotoxins. Stimulate T cells to proliferate.

Scarlet fever occurs as a complication of pyogenic streptococcal disease when the infecting strain produces erythrogenic toxin in a susceptible patient (usually a child). Antibodies to the erythrogenic toxin afford protection against scarlet fever, but not pharyngitis.

Recently there has been a reemergence of exotoxin A-producing strains with increasing episodes of septic shock (streptococcal toxic shock syndrome).

2. streptolysins S and O - beta hemolysins

Two types of beta hemolysin:

Streptolysin O is reversibly inactivated by oxygen and is therefore demonstrable on subsurface colonies.

It elicits antibodies that neutralize its hemolytic action. Because most strains of group A strep produce streptolysin O, patients recovering from streptococcal disease usually have antistreptolysin O antibodies in their serum. Both toxins can injure the membranes of cells other than RBCs.

Streptolysin O, like staphylococcal alpha toxin, forms ring-like pores in the lipid bilayer of membranes, allowing leakage of cellular contents.

Streptolysin S is oxygen stable and is responsible for surface colony hemolysis.

Streptolysin S is largely cell bound; leukotoxic activity.

 

3. Spreading factors - responsible for spread of strep from mucosa to surrounding tissue

extracellular products that can dissolve the matrix of connective tissue and clotted material accumulated as a result of host inflammatory responses. Spreading factors include the following:

a. Streptokinases - streptococcal fibrinolysin. Catalyzes the conversion of plasminogen to plasmin. Plasmin digest fibrin and other serum factors important in the formation of blood clots. Two types: streptokinase A and B differ antigenically. Elicit anti-streptokinase abs in the course of most diseases caused by group A strep.

b. Nucleases (DNAse; streptodornase) - At the site of infection the host's inflammatory response results in the accumulation of nuclear exudate from dead or injured white blood cells. Nucleases depolymerize the highly viscous DNA that accumulates in thick pus, facilitating the spread of trapped streptococci. The released purine and pyrimidine bases can also be used nutritionally by the strep.

Nuclease enzymes do not penetrate the plasma membranes of living mammalian cells.

c. Hyaluronidase - lytic effects on ground substance of connective tissue.

d. Proteinase - ?role in destroying M protein and other extracellular proteins such as streptolysin O and streptokinase. When the purified proteinase is injected into susceptible animals, it causes extensive lesions in the connective tissue of the heart.

 

Immunity

Only anti-M antibodies protect the host against invasive streptococcal disease.

Since there are more than 80 serologic types of M protein, we never become immune to group A strep infections. Antibodies produced in a patient to a type-specific M protein confer long-lasting immunity against infection from the same streptococcal strain.

Only a few M types are nephritogenic, there are rarely repeat attacks of glomerulonephritis.

 

Lab diagnosis

a. bacitracin disks to ID group A strep - 95% accurate

b. anti-streptolysin O test measures abs against streptolysin O to obtain evidence for recent streptococcal infection. (2-4 wks after acute infection)

c. can also measure abs to streptokinase, hyaluronidase, or DNase to see evidence of recent strep infection.

d. Presumptive diagnosis of group A strep infection - throat culture swab may contain sufficient numbers of microorganisms. The swab is treated with nitrous acid which extracts streptococcal antigens. The extracted ag is added to coagglutination group A antibody reagent (latex particles coated with antibodies to the C-specific Group A antigen). A positive test is represented by an agglutination reaction.

The test can yield results in 30 min after swabbing the throat. Negative assay must be confirmed by negative throat culture since this rapid assay is less sensitive (but faster!) than culture.

Treatment

very susceptible to the bactericidal effects of penicillin

important to treat early in the course of acute streptococcal pharyngitis to depress the patient's ab response so that the risk of rheumatic heart disease is reduced. Secondary abic choices: Em, clindamycin.

Because of the many different streptococcal types, a vaccine utilizing all the type-specific M proteins has proved impractical.

 

 

GROUP B STREPTOCOCCI - also beta hemolytic - S. agalactiae.

Commensals found in the oral cavity, intestinal tract, and vagina. Carriage rate 15-40%

During the past 2 decades, GBS has become the most common cause of meningitis and septicemia in newborn babies. The annual incidence of GBS infection in the US is ~1.8 cases per 1000 live births. Factors conducive to GBS infection include premature labor, prolonged rupture of placental membranes, and birth weight less than 3 pounds.

Two forms of GBS disease in neonates:

a. early onset disease (bacteremia, pneumonia, meningitis) - from vertical transmission of GBS from mother to infant in utero or during passage through the birth canal. It occurs <6 days of delivery and has a high mortality rate as the result of sepsis or pneumonia. May be caused by any capsule type.

 

  1. Late onset (up to 3-4 months after birth) disease may be acquired from mothers, hospital personnel, or contacts outside the hospital.

Meningitis is the most frequent complication of late onset disease, and it is nearly always caused by capsule type III strains.

Meningitis is the leading cause of GBS-related deaths. Approximately half of the survivors suffer nerve damage, learning disabilities, deafness, seizure disorders, or mental retardation.

Virulence = Capsule

9 different capsular serotypes - principal determinants of virulence.

In common with other bacteria that cause meningitis (menningococci, E.coli K1), sialic acid is an important component of the polysaccharide capsule. Abs to the capsule are protective against infection.

Affected infants have inadequate levels of maternally transmitted abs to the capsule. Capsular polysaccharide is the focus of a vaccine study to elicit abs in women of child-bearing age. Since polysaccharides are poor immunogens, efforts are directed toward production of protein-polysaccharide conjugate vaccines.

There are also two C proteins on the surface of the organism called alpha and beta C proteins. Antibodies to these proteins also are protective (in experimental animals), even against encapsulated strains.

Pregnant women who are culture-positive and high-risk are now treated with antibiotics prior to delivery.

 

 

Alpha hemolytic streptococci (the viridans group)

Viridans strep do not react with Lancefield grouping sera - no C substance.

Variable hemolysis; there are many species that are called viridans strep.

Classification is based more on culture characteristics rather than serologic analysis. Example: S. salivarius, S. mitis, S. mutans, and S. sanguis.

Alpha strep colonize the respiratory tract and they are a major component of the oral flora in humans; they are seen in virtually every throat culture.

These organisms have a low degree of pathogenicity particularly compared to pneumococci (also alpha hemolytic).

S. mutans is the causative agent of tooth decay. Adheres to tooth surface via extracellular glucan polymer.

Their principal significance in infectious diseases is subacute bacterial endocarditis. Necessary predisposition of the patient: endocardial surface is damaged by rheumatic fever or congenital heart disease.

When alpha strep seed the bloodstream (from the throat and around the teeth), they may get trapped on the surface of a tiny fibrin clots where the endocardial surface is damaged. This may initiate endocarditis in patients with abnormal valves. Vegetations form on the heart valves where the organisms grow in large colonies embedded in a fibrinous, acellular exudate. Many of the strains are drug-resistant.

This is a subacute infection unlike acute infection caused by staph. Difficult to diagnose and difficult to treat.

 

 

Nonhemolytic strep

low pathogenicity for man

commensals in the intestinal tract (their niche).

Group D strep

Best known: Enterococcus faecalis and E. faecium.

especially hardy

resistant to 62C for 30 min

grow at pH 9.6 in 6.5% NaCl

grow at temperatures ranging from 10 to 45C

resistant to bile - grow well in bowel and gall bladder.

 

These organisms cause an estimated 5 to 15 percent of bacterial endocarditis, and are also a frequent cause of UTI, particularly in hospitalized patients. Many strains in clinical samples are highly resistant to antibiotics, making the treatment of enterococcal endocarditis especially difficult.

Vancomycin resistance is becoming more prevalent among these organisms. Vancomycin resistance Enterococcus (VRE) in the US first appeared in 1988. Nosocomial infections due to VRE: 1989-1993, 0.03%-7.9%, resepctively.

 

Streptococcus pneumoniae

gram-positive, lancet-shaped diplococci.

encapsulated

non-motile

alpha hemolytic

no Lancefield group antigen

humans - only reservoir

lactic acid bacteria: facultative anaerobes that derive their energy primarily from fermentation of carbohydrates to lactic acid (similar to other Strep)

very fastidious in their cultural requirements

some strains need an elevated level of CO2 for initial isolation.

Pneumococci decolorize with aging - autolytic enzymes -> cell lysis. Autolysis accelerated by surface-active agents like bile or detergents that activate an amidase that splits the tetrapeptide from muramic acid in PG.

 

Clinical

primary cause of community-acquired pneumonia

important cause of otitis media (inflammation of the middle ear), sinusitis, and bacteremia and meningitis in infants and children.

Between 30% and 70% of normal human adults carry one or more capsular types of pneumococci in their respiratory tracts (reservoir). Thus, pneumococcal infections are acquired endogenously through lowered host resistance (rather than exogenously by direct contact).

Predisposing factors: age (old or very young), underlying illness, and viral infections of the upper respiratory tract

**Pneumococcal pneumonia develops most often during the course of viral infections of the upper RT when the resulting increase in mucous secretions in the nose and pharynx enhance the likelihood of their aspiration.

Causes pneumonia that is particularly serious in infants, those over age 55, or patients with chronic complicating illness.

Because many healthy humans carry pneumococci in their throats, demonstration of the organism in sputum or a pharyngeal culture does not provide conclusive evidence of pneumococcal infection. Recovery of the organism from the patient's blood, however, is diagnostic.

Virulence factors:

1. Capsule. There are >90 serologic types of pneumococci recognized, based on their capsular polysaccharides; antiphagocytic.

80-90% of bacteremic infections in the US are caused by 23 of the 90 recognized capsule types. Type 3 strains cause the highest fatality rate in humans. Six different capsular serotypes account for more than half of all pneumococcal infections, including otitis media, in children. Eight groups cause most childhood disease in the world.

mucoid colonies; as the culture ages, the centers of the colonies autolyze first, giving rise to forms resembling checkers.

That the capsule is important for virulence of the pneumococcus is based on several observations:

a. Only encapsulated strains are pathogenic for man and animals.

b. Abs to a specific polysaccharide produces a high level of resistance to infection with the homologous serotype organism.

c. Treatment of animals with an enzyme that depolymerizes type 3 capsular polysaccharide protects them against type 3 organisms.

d. Virulence studies with variants:

highly encapsulated (most virulent) > small capsule variants > unencapsulated variants (least vitulent) (Griffith exp).

 

Free soluble polysaccharide produced during infection promotes further bacterial invasion by diffusing into tissue fluids and neutralizing ab there.

The pneumococcus can lose its capsule by serial passage in the laboratory particularly in the presence of anti-capsular antibodies. Passage of a strain through a mouse at frequent intervals maintains maximal virulence.

2. Pneumolysin - cytoplasmic hemolysin; released by autolysins. Cytolytic for eucaryotic cells with cholesterol in membrane.

Toxic to human respiratory epithelium in organ culture; causes ciliary slowing, changes in cell ultrastructure, and disruption of epithelial integrity. Pneumolysin-negative mutant less virulent for mice.

3. Neuraminidase - cleaves terminal N-acetyl-neuraminic acid from cell-surface glycolipids and glycoproteins. ?role in disease.

4. Adherence - adheres to N-acetylglucosamine-galactose moiety of cell surface glycolipids.

5. Proteases - cleave immunoglobulins

 

Immunity

Type-specific immunity to pneumococcal infection is long-lasting; presence of anticapsular abs - enhance phagocytosis and are protective.

A vaccine containing 23 capsular ags available - for elderly, children >2 yrs of age, immunocompromised, those w/ pulmonary disease, and persons in epidemic-prone areas such as military bases. It is not effective for children under two years of age.

New: conjugate vaccine under development: ~8 common capsule serotypes.

New vaccine candidate: PspA = pneumococcal surface protein A. Found in virtually all pneumo strains. Abs to PspA protect against experimental pneumococcal infections, even by encapsulated strains. Analogous to C proteins of Group B strep.

 

Treatment

penicillin, but there is now emerging resistance (15-25% of strains) not beta-lactamase mediated (altered PBPs).

Although pneumococcal diseases usually respond to antimicrobial therapy, the case fatality rate of optimally-treated bacteremic infection among those over 50 years of age and in individuals with underlying systemic illness exceeds 25%.