Systematic Bacteriology
STREPTOCOCCUS PYOGENES
Morphology: It is 0.5 to 1 μm in diameter and arranged in chain. Chain formation is due to cocci dividing in one plane only and failure of daughter cell to separate completely. The length of a chain depends upon medium in which organism is grown. It is usually encapsulated, non-sporing and non-motile.
When capsule is present it is composed of hyaluronic acid (Group A and Group C).
Streptococcus Group B and Group C show polysaccharide capsule.
Cultural character: Streptococcus pyogenes is aerobic and facultative anaerobes with optimum temperature of growth being 37°C.
Enriched media with whole blood, serum, ascitic fluid or glucose favors rapid growth.
a. Fluid media: Serum broth, 24 hours after culture shows granular growth with powdery deposits. There is no pellicle formation.
b. Blood agar: After 24 hours’ incubation colony is small, 0.5 to 1 mm (pin point colonies), circular, transparent, low convex with area of hemolysis. Strains with capsules produce mucoid colonies. Virulent strains produce matted colonies (granular). A virulent strain produces glossy colonies (Fig. 23.2).
Selective media: Blood agar medium having 1:500,000 crystal violet may be used as selective medium (Fig. 23.3).
TABLE 23.1: Diseases caused by streptococci of various groups
Group Disease caused Habitat A Majority of human
streptococcal diseases Man B Mastitis in cows,
post-natal infections in Cow, human human and sepsis in genital tract newly born
C Diseases in various Various animals animals. Mild respiratory and upper infections in human. respiratory tract
of humans.
D Infections of urogenital Milk products, tract in humans, intestines of endocarditis and humans and wound infections. animals E Diseases in pigs and Pigs and cows
cows
F Respiratory infections Upper respiratory in humans and tract of humans endocarditis
G Mild respiratory Upper respiratory infections in humans tract of humans Genital tract infections and dogs in dogs
H Endocarditis Upper respiratory tract of humans K Endocarditis Upper respiratory
tract of humans L Genital tract infections Dogs
in dogs
M Genital tract infections Dogs in dogs
N Genital tract infections Dogs in dogs
O Endocarditis Milk products
P Not known Chicken, pigs
Q Not known Human intestines
R Not known Human intestines
S Not known Human intestines
U Not known Animals
V Not known Animals
Fig. 23.2: Streptococcus pyogenes growth of blood agar medium
Fig. 23.3: Crystal violet blood agar plate—selective medium for Streptococcus pyogenes
Biochemical reactions: It ferments lactose, glucose, salicin, sorbitol, maltose, dextrin, etc.
producing acid but no gas. It is catalase negative. It does not liquefy gelatin and is not soluble in 10 percent bile. It hydrolysis pyrrolidonyl naphthylamide (PYR test), producing red colours. It does not ferment ribose.
Resistance: It is easily destroyed by heat at 56°C for 80 minutes. It can survive in dust for several weeks if protected from sunlight. It is resistant to crystal violet. It is susceptible to sulfonamide, etc. Streptococcus pyogenes is highly sensitive to bacitracin and this property is used in rapid identification of group A hemolytic strep-tococci.
Antigenic structure: Hemolytic streptococci possess a group specific polysaccharide C and three type specific protein antigen M, T, R and nucleoproteins (Fig. 23.4).
Polysaccharide C antigen: It confers serological specificity and hence it has 20 Lancefield groups. Human strain belongs to group A and bovine strain belongs to group B.
This antigen is integral part of cell wall and so it has to be extracted for grouping by precipi-tation with specific antigen. Extraction can be done by:
1. Hydrochloric acid (Lancefield’s acid extraction method).
2. Formamide (Fuller’s method).
3. An enzyme produced by Streptomyces albus (Maxted method).
4. Autoclaving (Rantz and Randall’s method).
M antigen: M protein besides determining type specificity also acts as virulence factor by inhibiting phagocytosis. Antibody to M protein is protective. M protein is alcohol soluble and is destroyed by trypsin. Over 65 proteins types have been recognized in group A.
T antigen: It is acid and heat labile but trypsin resistant. It is demonstrated by agglutination with the specific antisera. Antibodies against the antigen are not protective.
It has no relationship to virulence of strep-tococci. It is obtained from streptococci by proteolytic digestion (otherwise rapidly des-troy M proteins). It permits differentiation of certain types. Other types share the same T substance.
R antigen: Streptococcus pyogenes (serotypes 2, 3, 28, 48) and in some strain of group B and C, this R protein antigen is found. It has no relation to virulence.
Fimbria are hair like consisting of M protein which are covered by lipotecoic acid and are present in streptococci group A important to attach streptococci to epithelium.
Nucleoproteins: Extraction of nucleoproteins of streptococci by treatment with weak alkali yields mixture of proteins and other substances called P substances which probably make up most of streptococcal cell body.
Other factors: M associated protein (MAP) is identified. Some M types of Streptococcus pyogenes produce serum opacity factor.
Toxin Production
1. Hemolysins: It is filterable toxic substance. It is of two types:
a. Streptolysin O demonstrable in deep colonies which is oxygen labile, heat labile, strongly antigenic, important in contributing to virulence (intravenous injection into animal has specific cardiotoxic activity) and it is inhibited by cholesterol.
b. Streptolysin S is oxygen labile, non-antigenic, inhibited non-specifically by serum lipoprotein and may be neph-rotoxic.
Fig. 23.4: Antigenic structure of Streptococcus pyogenes
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157
2. Erythrogenic toxin is filterable and heat stable toxin and is of three types: A, B and C. In small dose if injected intradermally causes erythema in susceptible persons. Adminis-tration of larger amount causes generalized rash, fever and malaise. It is erythrogenic toxin and is associated with pathogenesis of scarlet fever.
The production of erythrogenic toxin A and C is dependent on lysogenization of streptococci with certain temperate bacteriophage. B type is chromosomal.
3. Streptokinase (fibrinolysin) is produced mostly by strain of group A, C and G. It is heat stable and antigenic. It promotes lysis of human fibrin clots by activating a plasma precursor. Fibrinolysin appears to play a part in streptococcal infection by breaking down fibrin barrier around lesion and spreading infection.
4. Deoxyribonucleases (streptodornase) causes depolymerization of DNA. It helps to liquefy thick pus. Four distinct streptodornase A, B, C and D have been recognized of which B is more antigenic in man.
5. Diphosphopyricine nucleotidase (DPNase) is antigenic and is specifically neutralized by antibody in convalescent sera. It is believed to be leukotoxic. It acts on the coenzyme DPN and liberates nicotinamide from the molecule.
6. Hyaluronidase is a spreading factor present in culture filtrate of Streptococcus pyogenes.
It breaks down hyaluronic acid of the tissue.
This favors spread of infection along intercellular space. Streptococci possess a hyaluronic acid capsule and also elaborate hyaluronidase, self destructive process. But strains that produce hyaluronidase in large amount (M type 4, 22) are non-capsulated and hence no hyaluronic acid.
7. Protease is intracellular enzyme produced at acidic pH in cultures grown at 37°C. It destroys M type specific protein and also inhibits production of fibrinolysin and hyaluronidase. It is produced in areas of inflammation. The biological significance of this enzyme is not known.
Amylase, esterase are also produced but it is not known whether they play any role in natural infection or not.
Pathogenecity: Streptococcus pyogenes is more invasive and produces septicemia readily.
There is a tendency to spread locally along lymphatics and through bloodstream.
1. Respiratory infection: Throat is the primary site of invasion causing sore throat. It may be localized in tonsils (tonsillitis) or may involve pharynx (pharyngitis).
Scarlet fever is caused by a strain pro-ducing erythrogenic toxin. This accounts for characteristic erythematous rash. This is uncommon in the tropics and does not occur in India.
From throat streptococci may spread to the surrounding tissue causing otitis media, mastoiditis, Ludwig angina and suppura-tive adenitis. It may cause meningitis.
Bronchopneumonia may occur when Strep-tococcus pyogenes acts as secondary invader, e.g. influenza. Sometimes empyema may result.
2. Skin infection: It may cause suppurative infection of skin, e.g. wound, burns, lymp-hangitis and cellulitis. Infection of abrasion may lead to fatal septicemia.
Apart from this it may cause erysepelas and impetigo. Erysipelas is a diffuse infec-tion involving superficial lymphatic. The involved skin becomes red, swollen and indurated. It is found in old patients. In impetigo Streptococcus pyogenes of group A type 4, 25 are involved. It is seen in young children. Impetigo may lead to glomerulo-nephritis in children of tropics but do not often lead to rheumatic fever.
3. Genital tract: Streptococcus pyogenes is important cause of puerperal sepsis. The source of infection is nasopharynx of doctors, nurses and attendants, etc.
4. Other infections like abscess of organs (brain, lungs, liver, kidney) may occur. It may cause septicemia and pyemia.
5. Non-suppurative complications: They are:
i. Acute rheumatic fever.
ii. Acute glomerulonephritis.
The pathogenesis of these conditions is not clearly understood. They require 1 to 3 weeks after acute infection. No organism is detected when sequelae sets in. Comparison of acute rheumatic fever and acute glomerulonephritis is shown in Table 23.2.
Acute rheumatic fever: It is a systemic non-suppurative inflammatory condition charac-terized by fever, pancarditis, migratory poly-arthritis, sometimes chorea and subcutaneous nodules. Usually disease is preceded by an attack of streptococcal sore throat, septic tonsil-litis or respiratory tract infection.
The hypersensitivity to streptococci or its products is suggested in pathogenesis of rheumatic fever because of:
a. Absence of organism in the lesion.
b. Latent period between attack of sore throat and onset of rheumatic disease.
c. Similarity of disease produced experi-mentally in rabbit.
It is suggested that lesion of rheumatic fever may be the result of hypersensitivity to some streptococcal component produced by repeated attacks. Rheumatic fever has marked tendency to be reactivated by recurrent streptococcal infections, whereas nephritis does not have this characteristic. It has also been suggested that there may be an element of anti-immunity involved and antigenic cross reaction between streptococci and heart tissue also has been demonstrated.
Acute glomerulonephritis: It is caused by only few nephritogenic strain of group A (4, 12, 49, 2, 52, 57), type 12 being the most common. The most important antigen is probably in streptococcal protoplast membrane. The immunity in streptococcal infection is type specific and so acute glomerulonephritis is non-recurring condition. Not only sore throat but pyoderma or impetigo may also lead to nephritis. It is self-limited episode that resolves without any permanent damage.
Pathogenesis may be due to antigenic cross reaction, between glomerular antigens and some component of nephritogenic streptococci.
More often it may be immune complex disease.
Laboratory Diagnosis 1. Hematological investigations:
a. Total leukocyte count may show consi-derable increase.
b. Differential leukocyte counts show in-crease in neutrophil count. Polymorph neutrophil may constitute more than 80 percent.
c. Erythrocyte sedimentation rate (ESR) is raised especially in rheumatic disease.
It is done to estimate the activity of disease.
2. Bacteriological method: Most important spe-cimens are throat swab, nasopharyngeal swab, pus swab, sputum, cerebrospinal fluid, blood, etc.
a. Smear from above material after Gram’s straining, show Gram positive cocci arranged in chains.
b. Culture: Specimen is cultured on blood agar or crystal violet blood agar media with loop. After overnight incubation at 37°C, colonies are studied. These are small (pin point); raised colonies show-ing beta hemolysis. Hemolytic strepto-cocci are grouped further by Lancefield technique. A rapid presumptive iden-tification of group ‘A’ streptococci can be made by performing bacitracin sensi-tivity test (Fig. 23.5). For rapid diagnosis swab or pus specimen are cultured in broth and after 2 to 3 hours smears are stained with fluorescein labelled group A antiserum.
c. Serological test: The titer of antistreptolysin O in a patient serum in dilution above 1 : 200 is an indication of strepto-coccal infection. It is also useful for detecting asymptomatic carriers of Streptococcus pyogenes. A titer above 286 of antistreptolysin O is suggestive of rheumatic activity. A use in antibody titer to other streptococcal antigens may be estimated, e.g. antiDNAase, antihyaluronidase (especially in skin
TABLE 23.2: Comparison of rheumatic fever and glomerulonephritis
Acute Acute
rheumatic fever glomerulonephritis Site of infection Throat Throat or skin Prior sensitization Essential Not necessary
Serotype of Any Nephritogenic only
Streptococcus pyogenes (12, 49, 2, 52, 55, 57, 4) Immune response Marked Moderate
Complement levels Unaffected Lowered Hereditary tendency Present Not known Repeated attack Common Absent Penicillin prophylaxis Essential Not indicated
Course Progressive Spontaneous
Prognosis Variable Good
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infections), antistreptokinase, antiM type specific antibodies, etc. Strepto-zyme test is useful in detection of antibodies to antigen and enzymes.
3. Skin test is known by the name Dick test.
Dick test: It is done to find out susceptibility of a person to scarlet fever. 0.2 ml erythrogenic toxin is injected intradermally on the forearm and same amount of heated inactivated toxin on the other forearm. A bright red rash appears within 6 hours and becomes maximum in 24 hours and there-after this fades away. Control forearm does not show any reaction. A positive reaction means no immunity to scarlet fever. A negative reaction means immunity.
4. Schultz-Charlton reaction: Erythrogen anti-toxin is injected intradermally in a patient with scarlatinal rash. There is local blanching of rash.
Treatment: Streptococcus pyogenes is sensitive to penicillin, sulfonamide and several other antibiotics. However, penicillin is the drug of choice. Antitoxic serum was used to be administered effectively in scarlet fever.
GROUP B
Streptococcus agalactiae is responsible for mastitis in cow. It may be present in human throat and vagina as commensal. It can be iden-tified by CAMP (Christie-Atkins-Munch-Petersen) reaction.
On the basis of type specific capsular antigens they are divided into 4 groups, ia, ib,
Fig. 23.5: Streptococcus pyogenes showing bacitracin and pencillin sensitivity
ii and iii. All the strains of type ia and ib are from human source.
It is rarely pathogenic in man. Sometimes they may cause puerperal infection, septicemia, meningitis and ulcerative endocarditis, etc.
GROUP C
Streptococcus equisimilis is isolated from horses and cows. It may produce streptolysin O and fibrinolysin. The organisms have been isolated from puerperal infection, cellulitis, wound and scarlet fever.
GROUP D
Group D streptococci can be divided into enterococci, e.g. Enterococcus fecalis (Fig. 23.6), Enterococcus fecium, Enterococcus durans, Enterococcus avium and so on. Enterococcus fecalis
DISEASES CAUSED BY GROUP ‘B’
STREPTOCOCCI Newborn
• Pneumonia
• Meningitis
• Respiratory diseases
• Osteomyelitis Adult
• Endocarditis
• Septicemia
• Meningitis
• Arthritis
• Wound sepsis
• Pyoderma
Fig. 23.6: Slanetz and Bartley medium Enterococcus fecalis
is the most common of all above. It ferments mannitol with gas production, VP positive with PYR test positive and can be grown on blood tellurite producing black colonies.
Enterococci fecalis may cause urinary tract infection, wound infection, infective endocar-ditis, biliary tract infection, peritonitis, sup-purative abdominal lesions and septicemia.
Important characteristics of enterococci are:
• Normal flora of lower intestinal tract and vagina.
• Can grow in 6.5 percent sodium chloride, 40 percent bile and at 45°C.
• On sheep blood agar may produce alpha, beta hemolysis or may be non-hemolytic
• Tiny, deep pink colonies appear ontoo.
MacConkey agar medium.
• Most strains are resistant to penicillin, sulfonamide and also to cephalosporin, gentamycin, streptomycin, etc.
• Survive heat upto 60°C for 30 minutes.
• Non-motile, Gram positive cocci, non-cap-sulated and may be arranged in pairs or short chain.
• PYR test positive.
Cultural Characters
a. Fluid media: It shows uniform turbidity after 24 hour incubation at 37°C.
b. Blood agar: Colony is little bigger than Streptococcus pyogenes, circular, raised, low convex and emulsifies easily. Most of the strains are non-hemolytic.
c. MacConkey agar medium: Colonies are tiny, and deep pink in color.
Biochemical reactions: It ferments mannitol, sucrose, sorbitol, aesculin and grows on tellurite blood agar producing black colonies.
Resistance: It grows at pH 9.6 in presence of 6.5 percent sodium chloride, and also in presence of 40 percent bile. It survives at 60°C temperature for 30 minutes.
Pathogenicity: It invades tissues and may produce pyogenic lesions, e.g. cystitis, pyelitis, vaginitis, cervicitis, puerperal sepsis and subacute bacterial endocarditis.
Non-enterococci, e.g. Streptococcus bovis, Streptococcus equinus, Streptococcus avium, etc
may cause urinary tract infection, endocarditis, septicemia. They may be non- hemolytic and susceptible to penicillin.
Group F, G, H and O may occur as com-mensal in the throat.
ALPHA HEMOLYTIC STREPTOCOCCI