MICROBIOLOGY LECTURE 5 – Genus StreptococcusNotes from LectureUSTMED ’07 Sec C – AsM

Generalities

1. Gram positive cocci that grow in pairs or chains2. Widely distributed in nature3. 27 recognized species 4. Some are members of the normal human flora5. Others are associated with important human

disease attributable to infection by streptococci and to sensitization to them

6. The most important of the human pathogens:a. Streptococcus pyogenes (Group A)b. Streptococcus agalactiae (Group B)c. Enterococcus faecalis(Streptococcus faecalis)

(Group D)d. Streptococcus pneumoniaee. some of the oral streptococci

7. Elaborates a variety of extracellular substances and enzymes

8. Streptococci cause a wide variety of infections:a. Streptococcus pyogenes (Group A) streptococci-

leading bacterial cause of pharyngitis and cellulitis; inciting factor of two important immunologic diseases: acute rheumatic fever and acute glomerunephritis

b. Streptococcus agalactiae (Group B streptococci)- leading cause of neonatal sepsis and meningitis

c. Enterococcus faecalis – important cause of hospital acquired urinary tract infections and endocarditis

d. Viridans group streptococci – most common cause of endocarditis

e. Streptococcus bovis – may also cause endocarditis

9. Most species are facultative anaerobes; maybe strictly anaerobic to capnophilic

10. Have complex nutritional requirements necessitating blood or serum enriched medium for isolation

11. Carbohydrates are fermented with the production of lactic acid but not gas

12. Catalase negative13. Can be classified as to:

a. clinical presentation(pyogenic,oral,enteric)b. serological properties (Lancefield grouping A-H,

K-V) based on the antigenic composition of cell wall carbohydrates

serogroups A,B,C,D and G – most commonly found associated with human disease

c. Hemolytic patterns in blood agar plates Beta hemolysis – clear zone of

hemolysis around the colony as a result of complete lysis of the red blood cells

Alpha hemolysis – zone of partial hemolysis with a greenish discoloration of the medium

Gamma hemolysis – no color change or lysis of red blood cells ; nonhemolytic

d. Biochemical (physiological) properties Sugar fermentation reactions Tests for the presence of enzymes Tests for susceptibility or resistance to

certain chemical agentse. antigenic specificity of the capsular

polysaccharide

Beta hemolysis on 5% sheep blood agar

There is complete lysis of RBC surrounding the colony that can be seen macroscopically

There is a clear colorless zone around a streptococcal colony in

which the RBC have undergone complete destruction Alpha hemolysis on 5% sheep blood agar

an indistinct zone of partial lysis of red blood cells causing a green to greenish brown discoloration of the medium immediately surrounding the colony

Alpha hemolysis showing partially hemolyzed RBC immediately surrounding the colonoy

Gamma hemolysis on 5% sheep blood agar

Gamma denotes lack of hemolysis; the RBC surrounding the colonies are intact

Macroscopically there is no apparent hemolytic activity or discoloration produced by the colony

CLASSIFICATION OF STREPTOCOCCI

1. Group A streptococcus – Streptococcus pyogenesa. contains the group A antigenb. beta hemolyticc. main human pathogen associated with

local or systemic invasion and poststreptococcal immunologic disorders

d. PYR positive(hydrolysis of L-pyrrolidonyl-2-napthylamide)

e. usually susceptible to bacitracin

2. Group B streptococcus – Streptococcus agalactiaea. contains the group B antigenb. members of the normal flora of the female

genital tractc. an important cause of neonatal sepsis and

meningitisd. beta hemolytic e. hydrolyzes sodium hippuratef. gives a positive response to CAMP test

3. Groups C and G streptococcia. occur sometimes in the nasopharynxb. may cause sinusitis, bacteremia or

endocarditisc. beta hemolyticd. identified by reactions with specific

antisera for groups C or G

4. Group D streptococcia. enterococcal – Enterococcus

faecium Enterococcus durans

Part of the normal enteric flora Usually nonhemolytic; occasionally

alpha hemolytic PYR positive Grow in the presence of bile

Hydrolyze esculin (bile esculin positive)

Grow in 6.5% NaCl resistant to penicillin G Some strains are vancomycin

resistant

b. Non-enterococcal – Streptococcus bovis Part of the enteric flora Occasionally cause endocarditis Sometimes cause bacteremia in

patients with colon carcinoma Nonhemolytic and PYR negative Grow in the presence of bile Hydrolyze esculin (bile esculin-

positive) Do not grow in 6.5% NaCl Sensitive to penicillin

5. Streptococcus anginosus or Streptococcus milleri, Streptococcus intermedius, Streptococcus constellatus

a. part of the normal florab. may be beta, alpha or nonhemolyticc. includes:

Beta hemolytic streptococci that form minute colonies (<0.5 mm in diameter) and react with groups A,C or G antisera

All beta hemolytic group F streptococci

Voges Proskauer test positive Those that are group A are PYR

negative May be classified as Viridans

streptococci

6. Group N streptococci a. rarely found in human disease statesb. produce normal coagulation (souring )

of milk

7. Groups E,F,G,H and K-U streptococci- occur primarily in animals

8. Streptococcus pneumoniaea. alpha hemolyticb. growth inhibited by optochin

(ethylhydrocupreine hydrochloride)c. colonies are bile soluble

9. Viridans streptococci – Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguis (Group H), Streptococcus mutans

a. typically alpha hemolytic ; may be nonhemolytic

b. growth not inhibited by optochinc. colonies are not soluble

bile(deoxycholate)d. most prevalent members of the normal

flora of the upper respiratory tract e. important for the healthy state of the

mucous membranesf. may reach the blood stream due to

traumag. a principal cause of endocarditis on

abnormal heart valvesh. Streptococcus mutans – synthesizes

large polysaccharides (dextrans and levans) from sucrose which may lead to dental caries

10. Nutritionally variant streptococci or pyridoxal-dependent streptococci – Streptococcus defectives, Streptococcus adjacens

a. require pyridoxal or cysteine for growth on blood agar

b. grow as satellite colonies around colonies of staphylococci and other bacteria

c. usually alpha hemolytic; may be nonhemolytic

d. part of the normal flora

e. occasionally cause bacteremia or endocarditis

f. can be found in brain abscesses and other infections

11. Peptostreptococcusa. grow only under anaerobic or

microaerophilic conditionsb. variably produce hemolysisc. part of the normal flora of the mouth,

upper respiratory tract, bowel and female genital tract

d. often participate in mixed anaerobic infections in the abdomen, pelvis, lung or brain

Antigenic Structure of Group A Streptococcus

1. Capsule - hyaluronic acid; nonimmunogenic;

antiphagocytic

2. Cell walla. group specific carbohydrates of group A – is a

dimer of N-acetylglucosamine and rhamnose

b. type specific protein antigensi. M protein

o a major antigen associated with virulent streptococci

o located at the end of the hairlike fimbriae that are anchored in the cell wall and extending through the capsule

o antiphagocytico anticomplimentary

ii. T or trypsin resistant proteino With M protein – an important

epidemiological marker of group A strains

o no relationship to virulenceiii. R protein

c. Other surface antigensi. F protein or fibronectin binding protein

o Has a receptor for fibronectino Major adhesin for bacterial attachment

to the epithelial cells of the pharynx and skin

ii. Lipoteichoic acido Lipid moiety is implicated to binding to

fibronectin.o Possibly mediates adherence to

epithelial cells

Antigenic structure of Group B Streptococcus

PATHOGENESIS

Group A streptococci (Streptococcus pyogenes) cause disease by three mechanisms:

- pyogenic inflammation – induced locally at the site of the organisms in tissue

- exotoxin production – can cause widespread systemic symptoms in areas of the body where there are no organisms

- immunologic – occurs when antibody against a component of the organism cross-reacts with normal tissue or forms immune complexes that damage normal tissue

Inflammation-related enzymes produced by Group A Streptococcus (Streptococcus pyogenes)1. hyaluronidase(spreading factor)

- degrades hyaluronic acid which is the ground substance of the connective tissue

- Facilitates spreading of the microorganisms- Antigenic – specific antibodies are found in the

serum after infection with hyaluronidase producing organisms

2. streptokinase(fibrinolysin)- Transforms plasminogen of human plasma into

plasmin, an active proteolytic enzyme that digests fibrin and other proteins

- Given intravenously for treatment of pulmonary emboli and of coronary artery and venous thromboses

3. streptodornase (streptococcal deoxyribonuclease)- Depolymerizes DNA in exudates or necrotic

tissue- With streptokinase – used in enzymatic

debridement; Helps liquefy exudates and facilitates removal of pus and necrotic tissue

- Antibody to Dnase develops after streptococcal skin infection (normal limit –100 units)

Toxins and hemolysins produced by Group A streptococci (Streptococcus pyogenes)

1. Streptococcal pyrogenic toxins

- Three antigenically distinct toxins

a. Exotoxin C– classic erythrogenic toxin causes the rash in scarlet

fever Produced only by strains

lysogenized by a bacteriophage carrying the gene for the toxin

Dick test – The injection of a skin test dose of erythrogenic toxin gives a positive result (an erythematous reaction in the skin of nonimmune persons who lack antitoxins)

Schultz Charlton reaction – antitoxin injected into the skin of a patient with scarlet fever causes localized blanching as a result of neutralization of erythrogenic toxin

Dick test – positive – erythema surrounding the injection site

Schultz Charlton reaction. Convalescent serum from a scarlet fever patient blanches the rash of a patient with scarlet fever

b. Exotoxin B a cysteine protease that

rapidly destroys tissue and is produced by strains that cause necrotizing fasciitis

c. Exotoxin A May cause streptococcal toxic

shock syndrome

2. Hemolysins

a. Streptolysin S oxygen stable,

nonimmunogenic cell bound hemolysin capable of lysing eryhtrocytes, leukocytes and platelets

stimulate release of lysosomal contents after engulfment

responsible for the hemolytic zones around streptococcal colonies growing on the surface of blood agar

not antigenic

b. Streptolysin O a protein that is hemolytically

active in the reduced state responsible for the hemolysis

seen when growth is in cuts deep into the medium in blood agar

antigenic – antibodies are formed against streptolysin O following infection with streptococci that produce streptolysin O

ASO serum titer in excess of 160-200 units – suggests:

Recent infection with streptococci

Persistently high antibody levels dueto an exaggerated immune response to an earlier exposure in a hypersensitive person

Pathogenesis of Streptococcus agalactiae (Group B streptococci)

a. is based on the ability of the organism to induce an inflammatory response

b. no cytotoxic exotoxins are producedc. role of enzymes in the pathogenesis of

infection is unknown – deoxyribonucleases, hyaluronidase, neuraminidase, proteases, hiuppurase and hemolysins

d. no evidence for any immunologically induced disease

e. has a polysaccharide capsule – antiphagocytic

f. anticapsular antibody is protective CLINICAL FINDINGS

Types of diseases produced by Streptococcus pyogenes1. Pyogenic diseases

a. pharyngitisb. cellulitis and erysipelas

c. impetigo(pyoderma)2. Toxigenic diseases

a. scarlet feverb. toxic shock syndrome

3. Immunologic diseasesa. rheumatic feverb. acute glomerunephritis

1a. Pharyngitis1. Streptococcus pyogenes is the major cause of

bacterial pharyngitis2. A disease of children 5-15 years3. Spread by person to person by respiratory

droplets

4. Characterized by sore throat, fever, malaise, headache and nausea

5. Posterior pharynx erythematous with an exudate; cervical lymphadenopathy present

6. Can result to complications (tonsillar abscesses, mastoiditis, septicemia, osteomyelitis, rheumatic fever)

1b. Cellulitis1. Cardinal features –

erythema, swelling, heat and pain

2. Erythema may be pink or red but lacks the intense, fiery red or salmon colored appearance of erysipelas.

3. Initiated by infection through a small break in the skin

4. Can invade the subcutaneous tissue and advance rapidly through lymphatics --à septicemia

1b. Erysipelas1. Characteristic appearance – bright red or

salmon red painful confluent erythema in a “butterfly” distribution involving the nasal eminence, cheeks, and nose with abrupt borders along the nasolabial folds

2. Erythema increases over a course of 3-6 days and usually resolves in 7-10 days

3. Erysipelas usually occur on the face, although any skin surface such as the leg, can be affected.

4. Note the sharp line of demarcation and bright red color, features that distinguish it from cellulitis

1c. Impetigo1. A superficial infection

that usually begins as small vesicles progressing to weeping lesions with amber crust and slightly cloudy purulent exudate.

2. Serotypes implicated – M types 2,49, 55 & 57

3. May result to nephritis as a complication

2a. Scarlet fever1. The primary site of the infection is usually the

pharynx, with the distinctive rash resulting from an erythrogenic toxin produced by the streptococcus.

2. The rash appears within 2 days after the onset of the sore throat and disappears in 6-10 days.

Left – scarlet feverRight – measles

2b. Streptococcal toxic shock syndrome (also called toxic shock like syndrome)

1. Characterized by hypotension, diffuse erythroderma, hypoalbuminemia and multiorgan failure(kidney, lungs, liver, heart)

2. serotypes implicated – M1,M3 or M183. due to the production of pyrogenic exotoxins –

exotoxin A

desquamation of skin occurs 10-14

days after infection at sites that were

erythematous during the initial

phase

3a. Rheumatic fever1. most serious sequelae of hemolytic

streptococcal infection because it results in damage to heart muscle and valves

2. occurs 2 weeks after a group A streptococcal infection usually a pharyngitis

3. results in a systemic inflammatory process involving the connective tissue, heart, joints, and CNS

4. Characterized by fever, migratory polyarthritis, and carditis

5. Due to an immunologic reaction between cross-reacting antibodies to certain streptococcal M proteins and antigens of joint and heart tissue

6. Treat promptly with penicillin which is continued prophylactically to prevent recurrence and increased damage

Revised Jones Criteria for the Diagnosis of Rheumatic Fever

The diagnosis of rheumatic fever is highly likely if supported by evidence of a preceding group A streptococcal infection and the presence of two major manifestations or one major and two minor manifestations.

Supporting evidence of antecedent group A streptococcal infection

Positive throat culturePositive streptococcal antigen testElevated or rising streptococcal antibody titer

Major Manifestations

CarditisPolyarthritisChoreaErythema marginatumSubcutaneous nodules

Minor manifestations

Clinical findings: arthalgia, feverLaboratory findings

Elevated acute phase reactants (erythrocyte sedimentation rate, C-reactive protein)

Prolonged PR interval on elevtrocardiography

3b. Acute glomerulonephritis1. Typically occurs 2-3 weeks after streptococcal

skin infections with M types 2,4,12 or 49 (most frequent)

2. More frequent after skin infections than after pharyngitis

3. Characterized by hypertension, edema of the face(especially periorbital edema) and ankles, & “smoky” urine

4. Complete recovery; reinfection with streptococci rarely leads to recurrence.

5. Initiated by deposition of soluble streptococcal antigen-antibody complexes and complement on the glomerular basement membrane – lumpy-bumpy pattern on immunofluorescence

6. Can be prevented by early eradication of nephritogenic streptococci from skin colonization sites

Diseases produced by Streptococcus agalactiae

Neonatal Group B streptococcal disease

LABORATORY DIAGNOSIS

I. Streptococcus pyogenes

1. Microscopy – Gram stain

Gram stain of streptococci in a positive broth culture Gram positive

cocci in chains

2. Cultures

Colonies of group A streptococci on 5% sheep

blood agar small colonies with

a wide zone of beta hemolysis

Group A StreptococcusStreptococcus selective agar – contains sulfamethoxazoleAnd trimethoprim which inhibits the growth of nongroupA betahemolytic streptococci, staphylococci, viridansStreptococci and gram negative bacilli

Left – blood agar plate; right – streptococcus selective agar

PYR TestThe presence of an aminopeptidase enzyme that degrades the substrate is a 10 minute presumptive test for Group A streptococci (beta hemolytic) and Aerococcus, Enterococcusand Gemella(alpha or nonhemolytic)

left(+) colorless; right (+) red color

Bacitracin susceptibility test

An alternative to PYR test for the presumptive identification of Group A beta hemolytic streptococci 0.04 units of bacitracin disk is placed on an inoculum of the microorganism on sheep blood agar

Positive test – zone of inhibition

II. Streptococcus agalactiae - Group B beta hemolytic streptococcus

1. Microscopy- Gram stain of vaginal secretionsGram positive cocci in pairs,suggestive of Streptococcus agalactiae which colonize the genitourinary tract of women2. culture

Colonies of Group B streptococcus on 5% sheep blood agarColonies are larger than other beta hemolytic streptococci. Hemolytic zone surrounding the colony is smaller.Tests

1. Hippurate hydrolysis test

Incubate a suspension of themicroorganism for 2 hours at35 C in a hippurate solution.Add ninhydrin(indicator)

Hydrolysis of sodium hippurateleads to the formation of glycine and sodium benzoate.

Deamination of glycine –purple color

2. CAMP test (Christie,Atkins, Munich-Peterson) - An alternative to hippurate hydrolysis

Demonstrates the arrowhead shaped enhancement of betahemolysis that occurs when the hemolytic beta toxin producedby Staphylococcus aureus acts synergistically with the CAMP factor

III. Group D streptococci

1. enterococcus

Colonies of Enterococcus spp. on 5% sheep blood agarColonies are raised, white to gray white ranging from 0.5 to 1.5 mm. In size and are usually nonhemolytic

Tests

1. Bile esculin and 6.5% NaCl

- Bile esculin slant(left) – indicates that the microorganism can grow in the presence of bile and hydrolyze esculin.

2. 6.5% NaCl broth(right)- growth is indicated by turbidity and change in the indicator from pink to yellow

3. PYR Test

- The presence of an aminopeptidase enzyme that degrades the substrate PYR(L-pyrrolidonyl-B napthylamide) is a 10 minute presumptive test for group A streptococci(beta Hemolytic) and Aerococcus, Enterococcus and Gemella(alpha or nonhemolytic)

IV. Viridans streptococci

- Gram stain of a viridans streptococcus species in Blood culture broth appear in long chains especially when recovered from a blood culture broth

- Optochin susceptibility test. A paper disk containing optochin (ethylhydrocupreine Hydrochloride) is applied to the surface of an inoculated 5% sheep blood agar plate.

No zone of inhibition- Identification of viridans streptococci with

conventional biochemical reactions. Definitive identification requires several substrates including Bile esculin, arginine decarboxylase, 6.5% NaCl, lactose, Mannitol, raffinose, sorbitol, arabinose, inulin, sucrose and Esculin.

TREATMENT1. All group A streptococci are susceptible to

penicillin G.- mild - oral penicillin V

- if allergic: erythromycin or its derivatives (azithromycin)

2. Endocarditis caused by Viridans streptococci is curable by prolonged penicillin treatment

3. Enterococcal endocarditis – eradicated only by a penicillin or vancomycin combined with an aminoglycoside

4. Vancomycin resistant enterococci – linezolid (Zyvox) and quinupristin/dalforpristin(Synercid)

5. Nonterococcal Streptococcus bovis:penicillin G6. Group B streptococcal infections- penicillin G or

ampicillin in combination with an aminoglycoside

7. Peptostreptococci – penicillin G

PREVENTION1. Rheumatic fever can be prevented by prompt

treatment of Group A streptococcal pharyngitis with penicillin.

2. Penicillin prophylaxis for acute rheumatic fever patients to prevent recurrence of the disease; not needed in acute glomerulonephritis

3. In patients with damaged heart valves who undergo invasive dental procedures, endocarditis can be prevented by using amoxicillin perioperatively.

4. In patients with damaged heart valves who undergo gastrointestinal or urinary tract procedures, endocarditis caused by enterococcus can be prevented by using ampicillin and gentamicin perioperatively.

5. Neonatal sepsis caused by group B streptococci can be prevented by administration of parenteral ampicillin perinatally to women who experience prolonged (longer than 18 hours) rupture of membranes, whose labor begins before 37 weeks gestation or who have a fever at the time of labor.

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