An Overview of Streptococcal Infections

An Overview
of
STREPTOCOCCAL
INFECTIONS
By
Dr. Basil, B. C – MBBS (Nig),
Department of Chemical Pathology/Metabolic Medicine,
(Microbiology Postings)
Benue State University Teaching Hospital, Makurdi.
February 2016. 1

INTRODUCTION:
• STREPTOCOCCI are widely distributed in nature and some are
members of the normal flora while others are pathogenic
• Pathogenicity can be attributed in part to
• Infection by the organism, and
• Sensitization to them
• They elaborate a variety of extracellular substances and enzymes
• They are a large and heterogeneous group of bacteria impossible to
classify into one system
• Classification by various properties is key to understanding their
medical importance.
2

IDENTIFICATION:
• Gram positive spheres (cocci) like staphylococci
• But unlike staphylococci that appear in clusters, streptococci appear
in strips (chains) on gram stain – determined by their planes of
division
• 1µm in diameter and usually capsulated
• Facultative anaerobes (some species – microaerophilic)
• Some – Capnophilic
• For most – growth and hemolysis are aided by incubation in 10% CO2
• They are catalase negative
• Non-sporing bacteria and non-motile.
• Growth requires enriched media containing blood or serum.
3

IDENTIFICATION:
• Catalase test:
• Catalase converts H2O2 (which is used by macrophages and neutrophils) to
Water and O2
4
Catalase +ve
Staphylococci
Catalase –ve
Streptococci

CLASSIFICATION:
• Based on the hemolytic properties:
 Beta-hemolytic: - clear zone of hemolysis around the colony
 Alpha-hemolytic: - greenish discolouration of the culture medium around the
colony (partial hemolysis)
 Gamma-hemolytic: - no hemolysis of RBCs (Non-hemolytic streptococci)
• Based on antigenic characteristics of cell wall CHO (C – carbohydrate)
– Lancefield Antigens (Serologic Classification from A to V):
 Out of over 30 species of streptococci, only 5 are significant human
pathogens
 3 have Lancefield Antigens:
 Group A – (S. pyogenes),
 Group B – (S. agalactiae), and
 Group D – (Enteroccoci + Non-enterococci)
 2 have no Lancefield antigens – Lancefield Non-groupable:
 S. pnuemoiae, and
 Viridans group Streptoccoci
5

CLASSIFICATION:
• Hemolysis on Blood agar:
6

CLASSIFICATION:
• Historically, Lancefield antigens have been used as a major way of
differentiating the many streptococci though not applicable to a
number of organisms including some pathogenic species
• Identification of Streptococcal organisms require a combination of
several characteristics including:
• Antigenic composition including Lancefield antigens,
• Patterns of hemolysis,
• Biochemical reactions,
• Growth characteristics, and
• Genetic studies
7

BIOCHEMICAL REACTIONS:
Differentiation between beta-hemolytic
streptococci:
10
• Bacitracin susceptibility test
• CAMP test
• Bile Esculin Test

Bacitracin Test -
11
• Bacitracin susceptibility Test:
• Specific for S. pyogenes (Group A) – for its presumptive identification
• Principle:
• To distinguish between S. pyogenes (susceptible to B) & non group A such as S.
agalactiae (resistant to B)
• Bacitracin will inhibit the growth of Group A – S. pyogenes giving zone of inhibition
around the disk
• Procedure:
• Inoculate BAP with heavy suspension of tested organism
• Bacitracin disk (0.04 U) is applied to inoculated BAP
• After incubation, any zone of inhibition around the disk is considered as susceptible

Bacitracin Test:
12

CAMP test – (Christie Atkins Munch-Petersen)
• Principle:
• Group B streptococci produce extracellular protein (CAMP factor)
• CAMP act synergistically with staph. beta-lysin to cause lysis of RBCs
• Procedure:
• Single streak of streptococci to be tested and staph. aureus are made
perpendicular to each other
• 3 – 5mm distance was left between two streaks
• After incubation, a positive result appear as an arrowhead shaped zone of
complete hemolysis
• S. agalactiae is CAMP test positive while non-group B streptococci are
negative
13

CAMP test -
14

Bile Esculin Test -
• Differential agar (BEA) used to isolate and identify Enterococcus (group D
streptococci) and differentiate it from other streptococci
• Bile salts are the selective component, while Esculin is the differential
component
• Must be interpreted in conjunction with gram stain morphology
• Principle:
• Enterococcus hydrolyze Esculin liberating glucose (which is used up) and Esculetin.
• Esculetin react with ferric citrate in the medium to produce insoluble iron salts,
resulting in the blackening of the medium
• Many bacteria can hydrolyze Esculin, but only few can do so in the presence of bile.
15

Bile Esculin Test:
• After a maximum of 48hrs
incubation,
• Less than half darkened agar
slant – Negative result.
• Greater than half darkened
agar slant – Positive result.
16

Differentiation between alpha-hemolytic
Streptococci
• Optochin test
• Bile solubility test
• Inulin fermentation
17

Optochin test -
• Principle:
• S. pneumonia is inhibited by Optochin reagent (<5 μg/mL) giving an inhibition
zone of ≥14mm in diameter.
• Procedure:
• BAP is inoculated with the organism to be tested and an Optochin disc placed
in the center of the plate
• After incubation at 37o
C for 18hrs, carefully measure the diameter of the
inhibition zone with a ruler
• ≥14mm is positive; ≤ 13mm is negative
• S. pneumonia is positive (S); S. viridans is negative (R)
18

Optochin test -
19

Bile solubility test -
• Principle:
• S. pneumonia produce a self-lysing enzyme capable of inhibiting its growth
and this is accelerated in the presence of bile.
• Procedure:
• Add 10 parts of the broth culture of the organism to be tested to one part of
2% Na-deoxycholate (bile) in a test-tube
• Negative control is made by adding saline instead of bile to the culture
• Incubate at 37o
C for 15mins
• Observe and record your findings.
20

Bile solubility test -
• Clearing in the presence
of bile – positive;
turbidity – negative.
• S. pneumonia is soluble
in bile – positivity,
whereas
• S. viridans are insoluble
in bile – negativity.
21

Inulin fermentation -
• Useful to differentiate
Pneumococci from
other Streptcocci:
• Pneumococci ferments
inulin
22

IDENTIFICATION OF beta- and alpha-
HEMOLYTIC STREOTOCOCCI:
23
ferment
Not ferment

STREPTOCOCCAL VIRULENCE
FACTORS
24

GROUP A: Streptococcus pyogenes
METABOLISM:
• Catalase – negative
• Microaerophilic
• Beta-hemolytic – due to enzymes that destroy blood cells
• Streptolysin-O:
• Oxygen labile
• Antigenic
• Streptolysin-S:
• Oxygen stable
• Non-antigenic
26

VIRULENCE:
• M-protein (70 types) – major virulence factor
• Adherence factor
• Antiphagocytic
• Antigenic: induces antibodies which can lead to phagocytosis
• Lipoteichoic acid:
• Adherence factor
• Streptokinase - FIBRINOLYSIN
• Hyaluronidase – destroys CT and aids spread of the organism
• DNAase (Streptodornase)
• Anti-C5a peptidase – prevents C5a mediated phagocytic activity
• Protein F -
• Streptolysin O – also antigenic; (but Streptolysin S – not antigenic)
• Hence Anti-Streptolysin O (ASO) antibody titer rises in recent infections
• Skin infection does not induce ASO
27

TOXINS:
• Erythrogenic or pyrogenic toxin (produced only by lysogenized Group
A Streptococci): responsible for scarlet fever
• More than 4 serologically distinct toxins (Spe - A, B, C and F).
• Dick Test: once commonly used to confirm Scarlet Fever diagnosis.
• Some strains produce pyrogenic exotoxins which act as superantigens that
superstimulate T cell leading to release of cytokine which cause the Toxic
shock syndrome.
• Toxic shock syndrome toxin (similar to, but different from the staph
exotoxin TSST-1)
28

PATHOLOGY:
DIRECT INVASION/TOXIN:
•Pharyngitis:
• Red, Swollen tonsils and pharynx
• Purulent exudate on tonsils
• Fever
• Swollen lymph nodes
•Skin infections:
• Folliculitis, Erysipelas, pyoderma
• Cellulitis
• Impetigo
• Necrotizing fasciitis
•Scarlet fever: - fever and scarlet red rash on body
•Toxic shock syndrome
29

PATHOLOGY:
ANTIBODY MEDIATED (Delayed):
•Rheumatic fever (may follow streptococcal pharyngitis):
• Fever
• Myocarditis: heart inflammation >> Rhuematic valvular heart disease many years
later.
• Arthritis: migratory polyarthritis
• Chorea (Sydenham’s chorea or St. Vitus dance)
• Rash: Erythema marginatum
• Subcutaneous nodules: 10 – 20 yrs after infection, may develop permanent heart
valve damage
•Acute post-streptococcal Glomerulonephritits:
• Tea or coca cola coloured urine, following streptococcal skin or pharynx infection
• Follows skin or throat infection by Nephritogenic strains
32

DIAGNOSIS:
• Gram stain: - gram positive cocci in chains
• Culture on standard laboratory media: - Growth is inhibited by
bacitracin
• S. pyogenes is the only beta-hemolytic strep which is sensitive to bacitracin
• Pharyngitis: - Throat swab rapid antigen detection test (RADT) is
specific for S. pyogenes and immunologically detects group A
carbohydrate antigen.
• In children, RADT should be backed up by a throat culture due to the high
incidence of “strep throat” and moderate sensitivity to RADT.
33

TREATMENT:
• Penicillin G
• Penicillin V
• Penicillinase-resistant penicillin e.g Dicloxacillin: in skin infections,
where staphylococci could be the responsible organism
 Following rheumatic fever:
 Patients are placed on continuous prophylactic antibiotics to prevent repeat
strep throat infection that could potentially lead to repeat case of rheumatic
fever
 For invasive S. pyogenes infections, such as necrotizing fasciitis or
streptococcal toxic shock syndrome, consider adding Clindamycin.
34

GROUP B: Streptococcus agalactiae
METABOLISM:
• Facultative anaerobe
• Beta-hemolytic
• Part of normal flora:
• 25% of pregnant women carry Group B streptococci in their vagina.
• Can be transmitted to neonates during birth
35

PATHOLOGY:
• Neonatal meningitis
• Neonatal pneumonia
• Neonatal sepsis
• Sepsis in pregnant women (with secondary infection of fetus)
• Increasing incidence of infections in elderly >65yrs of age and patients
with diabetes or neurological disease: causes sepsis and pneumonia
36

DIAGNOSIS:
• Gram stain of CSF or Urine: - positive cocci in chains
• Culture of CSF, Urine or Blood
37

TREATMENT:
• Penicillin G
38

GROUP C and G Streptococcus:
• Beta-hemolytic
• S. equi, S. canis
• Associated diseases:
• Pharyngitis, pneumonia, cellulitis, pyoderma, erysipelas, impetigo, wound
infections, puerperal sepsis, neonatal sepsis, endocarditis, septic arthritis
• Treatment:
• Penicillin, vancomycin, cephalosporins, macrolides (variable susceptibility)
39

GROUP D Streptococcus:
• 2 SUB-TYPES:
• Enterococci: (recently given their own
genus because they sufficiently differ from
the streptococci)
• S. faecalis
• S. faecium
• Non-enterococci:
• S. bovis
• S.equinus
• Enterococcus:
• Gram +ve cocci
• Singly/in pairs/short chains
40
Enterococci

METABOLISM:
• Catalase – Negative
• Facultative anaerobes
• Usually Gamma-hemolytic, but maybe alpha-hemolytic
• Positive bile esculin test
41

VIRULENCE:
• Extracellular dextran helps them bind to heart valves
42

PATHOLOGY:
• Sub-acute bacterial endocarditis
• Biliary tract infections
• Urinary tract infections (especially the Enterococci)
• S. bovis is associated with colonic malignancies
43

DIAGNOSIS:
• Gram stain – positive cocci in chains
• Culture:
• Enterococci can be cultured in:
• 40% bile
• 6.5% Sodium chloride
• Non-enterococci can only grow in bile
44

TREATMENT:
• Ampicillin, sometimes combined with an aminoglycoside
• Resistant to Penicillin G
• Emerging resistance to vancomycin
• For vancomycin resistant organisms (VRE), consider Linezolid,
Daptomycin and Nitrofurantoin.
45

VIRIDANS GROUP Streptococci:
• Part of normal oral flora,
• Found in the nasopharynx and gingivial crevices
• GI tract
• Members:
• Mitis group: S. mitis, S. sanguis, S. parasanguis, S. gordonii, S. crista, S.
infantis, S. oralis, S. peroris
• Salavarius group: S. salavarius, S. vestibularis, S. thermophiles
• Mutans group: S. mutans, S. sobrinus, S. criceti, S. rattus, S. downeii, S.
macacae
• Angionosus group: S. angionosus, S. constellatus, S. intermedius
46

METABOLISM:
• Facultative anaerobes
• Mostly alpha-hemolytic; some beta- and gamma
• Resistant to Optochin
• Bile solubility - Negative
47

VIRULENCE:
• Extracellular dextran – helps them bind to heart valves
48

PATHOLOGY:
• Sub-acute bacterial endocarditis: caused by S. mitis group
• Dental caries (cavities): caused by S. mutans group
• Brain or Liver abscesses: caused by S. angionosus group
• Microaerophilic
• Found alone in pure cultures or in mixed cultures with anaerobes
49

DIAGNOSIS:
• Gram stain
• Culture – antibiotics may be added to inhibit growth of
contaminating bacteria
• Resistant to optochin
• Detection of group A streptococci by molecular methods:
PCR assay for pharyngeal specimens
• Antibody detection
• ASO titration for respiratory infections.
• Anti-DNAase B and Antihyaluronidase titration for skin
infections.
• Anti-streptokinase; Anti-M type-specific antibodies
50

TREATMENT:
• Penicillin G
• Effective doses of penicillin or erythromycin for 10 days can prevent
post-streptococcal diseases.
• Drainage and aggressive surgical debridement must be promptly
initiated in patients with serious soft tissue infections.
• Antibiotic sensitivity test is helpful for treatment of bacterial
endocarditis.
51

Streptococci Pneumoniae
(Pnuemococcus):
METABOLISM:
• Gram-positive lancet-shaped diplococci.
• Alpha-hemolytic (Pneumolysin is similar to streptolysin O).
• Form small round colonies on the plate, at first dome-shaped and
later developing a central plateau with an elevated rim.
• Facultative anaerobe
• Autolysis is enhanced in bile salt.
• Growth is enhanced by 5-10% CO2 – capnophilic.
52

VIRULENCE:
• Polysaccharide Capsule (90 serotypes):
• Protects the organism from phagocytosis
• Highly antigenic – opsonized by antibodies specific to it
• Due to the variable serotypes, surviving an infection from one serotype does
not confer immunity over the others
• Cell wall polysaccharide
• Phosphocholine
• Pneumolysin
• IgA protease, etc.
53

TOXINS:
• Pneumolysin: binds to cholesterol in host-cell membranes (but its
actual effect is unknown)
• MAJOR HOST DEFENCE MECHANISM: - Ciliated Cells of the Resp tract
and Spleen
• Loss of natural resistance may be due to:
• Abnormalities of the respiratory tract (e.g. viral RT infections).
• Alcohol or drug intoxication; abnormal circulatory dynamics.
• Patients undergone renal transplant; chronic renal diseases.
• Malnutrition, general debility, sickle cell anemia, hyposplenism or
splenectomy, nephrosis or complement deficiency
• Young children and the elderly
54

PATHOLOGY:
• Pnuemonia – pneumococcus is the most common cause of
pneumonia in adults
• Meningitis – most common cause of bacterial meningitis in adults
• Sepsis
• Otitis media (most common cause in children)
55

DIAGNOSIS:
• Gram stain: - reveals gram-positive
diplococcic
• Culture: does not grow in presence of:
• Optochin
• Bile
• Capsular polysaccharide antigen
detection
• DNA probe specific to S. pneumonia
• Virulence to mice
56
Pneumococcus Specific DNA probe

DIAGNOSIS:
• Positive Quellung test:
swelling when tested
against antiserum
containing anti-capsular
antibodies
• Quellung reaction:
technique used to
detect encapsulated
bacteria (such as S.
pneumonia and H.
influenza)
57
Quellung Antibody reaction

TREATMENT:
• Penicillin G (IM)
• Erythromycin
• Ceftriaxone
• Vaccine: made against 23 most common capsular antigens.
• Vaccinate individuals who are susceptible such as elderly or asplenic
individuals (including being functionally asplenic due to sickle cell anaemia)
• Pnuemococcal conjugate vaccine - Capsular polysaccharide + Protein Carrier
• Below 2yrs
• Heptavalent and the newer 13 valent conjugated vaccines are
effective at preventing otitis media and pneumonia.
58

CONCLUSION:
• STREPTOCOCCI are widely distributed in nature and some are
members of the normal flora while others are pathogenic
• Pathogenicity can be attributed in part to
• Infection by the organism, and
• Sensitization to them
• They elaborate a variety of extracellular substances and enzymes
• They are a large and heterogeneous group of bacteria impossible to
classify into one system
• Classification by various properties is key to understanding their
medical importance.
59

An Overview of Streptococcal Infections

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An Overview of Streptococcal Infections