bacteriology ,microbiology, streptococcal infections ,post streptococcal infections. acute glomerular nephritis ,ARF

2. The Streptococci
 Gram positive cocci which typically form pairs /
chains (divide only in one plane)
 Some species cause important diseases
 Many are part of the normal flora

3. Classification
A heterogeneous group which no single system
suffices to classify.
Classification is based on multiple criteria ->
1. Their Oxygen requirement
2. Hemolytic pattern on Sheep blood agar
3. Antigenic characteristics
4. Biochemical properties

4. Streptococci
O2 requirement
Aerobes, Obligate
. anaerobes
Facultative anaerobes Eg: Peptostreptococcus
1.Alpha hemolytic 2.Beta hemolytic 3. Non hemolytic
Partial hemolysis Complete hemolysis Eg: Enterococcus
spp
(Greenish discoloration) (Clear zone)

5. Beta hemolysis Alpha hemolysis

7. 1. Alpha Hemolytic streptococci
Eg: Viridans Streptococci,
(Classified by biochemical reactions)
3. Non-Hemolytic streptococci
Eg: Enterococcus spp
Nonenterococci
(Classified by biochemical reactions)

8. 2. Beta hemolytic Streptococci
Lancefield Grouping
Based on group specific “C” carbohydrate antigen
Lancefield Groups -> A – H & K – V
Group A Streptococcus
Serotyping,
Based on specific “M” Protein (80 types)
Griffith’s typing
Griffith’s types

9. S.PYOGENES
 Morphology:
 GPC in chains.( divides in only one plane)
 Non motile,non sporing
 Opt temp : 37 0 C
 Some strains of Gp A & C posses hyaluronic acid
capsules.Gp B & D : poly saccharide capsules

10.  Culture:
 BA : small ,circular , semi transparent convex colonies,
with beta haemolysis
 5 – 10 % CO2 promotes growth & haemolysis
 Liquid media : granular turbidity

11.  Biochemical Rxns:
 Ferments several sugars with acid only.
 Catalase : negative
 Not soluble in 10 % bile.
 PYR test : positive
 Ribose fermentation : negative

12.  Resistance :
 Delicate organism ,destroyed at 54 0 C for 30 minutes.
 Storage of cocci is at 4 0 C in RCM.

13. Group A Streptococcus
(Streptococcus pyogenes)
Pathophysiology ->
Bacterial virulence factors:-
Virulence factors of Streptococcus pyogenes
may be divided into two categories –
Cellular virulence factors
Extracellular virulence factors

14. Cellular virulence factors
1. Capsule ->
Most strains are capsulated
Capsule made up of Hyaluronic acid
Potent antiphagocytic factor
Helps in adherence to host tissue

15. Cellular virulence factors
2. ‘M’ Protein ->
Major virulence factor
Strains without M protein are avirulent
Present on surface as hair like projections (pilli)
Approx. 80 serotypes exist
Acts as ‘adherence factor’
Potent ‘antiphagocytic action’
Highly antigenic, specific Abs are protective
Abs to M protein cross react with cardiac tissue

16.  T protein : acid labile ,trypsin resistant .
 May be specific but many diff M types posses same T
protein.
 Demonstration : slide agglutination test using trypsin
treated whole streptococci.

17.  R protein : some types of S.pyogenes (2 , 3, 28, 48)&
some strains of B,C & G
 No relation to virulence
 MAP : non type specific M associated protein.

18. 3. Group specific cell wall ‘C’ carbohydrate Ag
->
Component of the cell wall
Forms basis of serologic grouping by Lancefield
Lancefield groups – (A – H and K – V)
Antigenic specificity lies in the amino sugar
present
Grouping may be done by ‘Co-agglutination’
tests

19.  Ag is the integral part of cellwall.
 Strep grown in Todd Hewitt broth & extracted with
HCl ( Lancefield’s acid extraction )
 formamide ( Fuller’s method)
 An enzyme produced by S.albus ( Maxted’s
method)
 Autoclaving(Rantz& Randall’s method)

20.  The extract & specific antisera allow to react in a
capillary tube .
 Precipitation occurs in minutes at the interface.
 Gel electrophoresis also used for grouping

21. Cross reacting Antigens
 Various streptococcal Ags have similarity to different
host tissues ->
 Capsular hyaluronic acid <–> human synovial fluid
 Cell wall M protein <–> myocardium
 Cytoplasmic membrane antigen <–> vascular intima
 These antigenic cross reactions may be responsible
for the non-suppurative complications of group A
Streptococcal infections.

22. Extracellular Virulence Factors
A. Toxins ->
1. Pyrogenic exotoxins (erythrogenic toxins) A, B,
C ->
Toxins produced by only strains which carry a
lysogenic phage
Pyrogenic – induce fever
Responsible for skin rashes in scarlet fever
Superantigens, responsible for streptococcal SSS
Highly antigenic, Abs are protective
Have ability to alter lymphocytic activity

23. 2. Streptolysins ->
Hemolysins of Group A streptococcus
Two types – Streptolysin ‘O’
- Streptolysin ‘S’

24. Streptolysin ‘O’
‘O’ -> means oxygen labile, hemolytic activity
observed only under anaerobic conditions
MW -> 60,000 Daltons
Active against both RBC & WBC
Antigenic -> Abs raised are called
Antistreptolysin O Abs (ASO)
Estimation of this antibody (ASO) titre helps in
the diagnosis of streptococcal disease

25. Streptolysin ‘S’
Produced in presence of serum
It is oxygen stable
Active against RBC, WBC, Platelets
Responsible for the Beta hemolysis seen around
the colonies on the surface of blood agar
Has nephrotoxic activity

26. B. Enzymes of Str.pyogenes
1. Streptokinase ->
Transforms plaminogen in plasma to plasmin
which can digest fibrin clots
Helps in Streptococcal infections by breaking
down the fibrin barrier around the lesions and
facilitating the spread of infection
Practical use – given IV for treatment of
pulmonary emboli; coronary artery/venous
thromboses

27. 2. Streptodornase (Streptococcal DNAse) ->
 De-polymerises DNA & nucleoprotein released by dead
host cells
 Responsible for the decrease in viscosity of purulent pus
 Responsible for the serous nature of exudate in
streptococcal infection
 Help in spread of lesion
 Mixture of streptokinase/streptodornase is used in
enzymatic debridement of necrotized tissues & empyemas
– facilitates early recovery of infected tissue

28. 3. Hyaluronidase ->
Degrades hyaluronic acid , which is the ground
substance of subcutaneous tissue. Also known as
‘Spreading factor’ – it facilitates the rapid spread of
Str. pyogenes in skin infection
Other organisms ->
Staphylococcus aureus
Clostridium perfringens

29. Streptococcus pyogenes infections
Pathogenesis ->
1. Source:-
a) Patients with skin lesions; respiratory
secretions of sore throat cases
b) Carriers
2. Mode of infection:-
 Direct/Indirect contact or inhalation

30. A. Diseases attributable to local infection
1. Streptococcal sore throat ->
Most common infection of small children
a sub acute nasopharyngitis with thin serous
discharge,
little fever
tendency to spread to middle ear (otitis); the
mastoid (mastoiditis) & meninges (meningitis)

31. In older children & adults –
Intense nasopharyngitis
Tonsillitis with redness, edema & purulent
exudate
High fever
DD ->
- Diphtheria, Gonococcal infection
- Infectious mononucleosis
- Adenoviral infection

32. Complications seen with sore throat ->
1. Peritonsillar abscesses (Quinsy)
2. Ludwig’s angina (massive swelling of floor of
mouth, blocking air passage)
3. Scarlet fever, if infecting strain produces
erythrogenic toxin
4. Rarely, pneumonia, usually secondary to
viral infections

33. 2. Streptococcal pyoderma
Local infection of superficial layers of skin –
impetigo
Highly communicable disease of children
Characterized by superficial blisters which
breakdown to form eroded areas covered with
pus/crusts

34. Streptococcal pyoderma contd….
Extensive infection may occur in burn cases /
wounded skin and may progress to –> cellulitis
M types -> 49, 57, 59 – 61 (Nephritogenic strains)
Most often precede Glomerulonephritis

35. B. Diseases attributable to invasion
1. Erysipelas ->
 Rapidly spreading infection of the superficial
lymphatics
 Characterized by massive edema with induration
and red discoloration & a rapidly advancing
margin

36. 2. Cellulitis ->
 Rapidly spreading infection of skin &
subcutaneous tissues
 Usually follows infection of mild trauma,
burns, wounds
 Characterized by pain, tenderness,
swelling, erythema

37. 3. Necrotizing fasciitis (Streptococcal gangrene) ->(
m types 1 & 3 forming pyrogenic exotoxin A)
 Infection of subcutaneous tissue & fascia
 Extensive & rapidly spreading necrosis of skin
/subcutaneous tissues
 Strains causing this condition – “ flesh eating
bacteria”

39. 4. Puerperal fever ->
 Infection of uterus just after delivery
 A septicemia originating in the uterus
(endometritis)
5. Sepsis ->
 Infection of traumatic / surgical wounds
 Has also been called ‘surgical scarlet fever’

40. 6. Streptococcal toxic shock syndrome ->
 Invasive infection may also be accompanied by
– streptococcal TSS
 caused by M types 1, 3, 12, 28
 Characterized by –>
• Shock,
• Bacteremia
• Respiratory & multi-organ failure

41. 7. Scarlet fever ->
 May follow severe throat / skin infection
 Rashes appearing on trunk within 24 hrs of
illness, spreading to extremities
 Streptococcal TSS & scarlet fever are overlapping
diseases

42. 8. Infective endocarditis ->
 Following sepsis
 Streptococci settle on healthy / previously
deformed heart valves
 Cause rapid destruction of heart valves
 Resulting in acute bacterial endocarditis
 Fatal cardiac failure within days / weeks

43. D. Post-streptococcal diseases
(Non-suppurative complications of Group A
Streptococcal infection)
 Complications occur 1 – 4 weeks after an episode of
acute infection
 Latent period suggests an hypersensitive / auto-
immune basis for development of complication
 Streptococci or its toxins cannot be detected from
affected tissues
Acute Glomerulonephritis
Rheumatic Fever

44. 1. Acute Glomerulonephritis
 Develops about 3 weeks after a skin infection
 Caused by nephritogenic strains (M types -> 49, 55,
59 – 61)
 Probable mechanism involved ->
1. Deposition of Ag-Ab complexes on glomerular
basement membrane
2. Initiation of Complement mediated damage to
basement membrane (Type III Hypersensitivity)
3. Resulting in glomerulonephritis

45. Acute Glomerulonephritis contd….
Condition is characterized by ->
Blood & protein in urine (hematuria
/proteinuria)
High blood pressure
Edema of neck & ankles
Low serum complement levels
Self limiting and prognosis is good

46. 2. Acute Rheumatic fever
 Occurs 1-4 weeks following Group A Streptococcal
pharyngitis (any strain)
 Probable mechanism involved ->
1. Abs formed against cell wall Ags of Streptococcus
pyogenes cross-react with cardiac tissue (due to
antigenic similarity between streptococcal cell wall
Ag & cardiac tissue)
2. Cause damage to all parts of the heart (endo, myo
& pericardium) – Type II - cytotoxic
Hypersensitivity / auto-immune disorder

47. Acute Rheumatic fever contd ….
Condition is characterized by ->
Fever
Malaise
Poly-arthritis
Inflammation of cardiac tissue - including
connective tissue degeneration of the heart
valves and inflammatory myocardial lesions

48. Acute Rheumatic fever contd ….
Condition has marked tendency for
reactivation due to recurrent Group A
streptococcal infection
Damage increases with each subsequent attack
Requires long term prophylactic Penicillin
administration for protection against repeated
attacks
Has variable prognosis

49. Comparison of Acute Rheumatic Fever and Acute
Glomerulonephritis
Features ARF AGN
1o site of infection Throat Skin or throat
Prior sensitization Essential Not required
Serotypes
responsible
Any Nephritogenic M types
49, 55, 59 – 61
Immune response Marked Moderate
Complement level Unaffected Lowered
Repeated attack Common Absent
Course Progressive or
static
Spontaneous
resolution
Prognosis Variable Good
Penicillin
prophylaxis
Essential Not indicated

51. Lab diagnosis
1. Specimen -> collection depends on type
of disease
Throat swab,
pus,
exudates, fluids
necrotic tissue –> for culture
Serum -> for serology

52. Methods:-
2. Microscopy ->
Gram’s stained smear:-
Gram positive cocci in long chains along with
pus cells
(May not be useful in case of throat swabs)
Immunofluorescent staining:-
Direct immunofluorescent staining using
specific monoclonal Abs – highly sensitive &
specific

53. 3. Culture ->
Specimen should be immediately processed
Transport media – Pike’s / Stuart’s media used
if delay in processing
Routine media used – Sheep Blood agar;
Thioglycollate medium
Incubated for 24 – 48 hrs at 37o C in 5 -10% CO2

54. Culture contd….
Identification of Isolate ->
Beta hemolytic colonies
Gram positive cocci in chains
Catalase test negative
Isolate is susceptible to bacitracin
Grouping (Lancefield) is done by Co-
agglutination test to detect ‘Group A Streptococci’

55. 4. Serology ->
Rise in titre of Abs to many Group A
Streptococcal Ags may be demonstrated.
1. Antistreptolysin O (ASO) test:-
A serological test used to detect ASO Abs
present in patient’s serum
A passive latex agglutination test
ASO titre of more than 200 units is indicative
of recent streptococcal infection

56. May also indicate an exaggerated immune
response to an earlier exposure (ARF)
Useful in the diagnosis of ARF but not in AGN
as titre is low (usually occurs as a result of skin
infection and being oxygen labile toxin, Ab will
not be produced in high titre)

57. Other serological tests ->
2. Antistreptodornase (AntiDNAse) test
3. Antihyaluronidase test
4. Antistreptokinase test

58. Treatment
Group A Streptococci are susceptible to
penicillin. Thus Penicillin G is the drug of choice
In patients allergic to penicillin, erythromycin /
cephalexin may be used
All acute infections should be treated, as
antimicrobials have no effect on already
established ARF & AGN
Long term Penicillin therapy for ARF patients to
prevent recurrence of infection

60. Streptococcus agalactiae
 It is part of the normal flora of the female genital
tract (5 – 20% of women)
 Human pathogenecity ->
1. Neonatal infections
2. Infections in adults

61. 1. Neonatal infections ->
A. Early onset disease->
Seen at birth or within 7 days of birth
Source of infection -> mothers birth canal
Predisposing factors ->
 Prolonged labor
 Premature child
 Low birth weight
Manifestations –> Septicemia, meningitis,
pneumonia

62. B. Late onset disease ->
Occurs after first week of life
Source -> hospital staff, environment
Manifestation ->
o Sepsis with /without meningitis
o Respiratory tract infection

63. B. Adult infections ->
1. In women ->
Abortion
Chorioamnionitis
Post partum sepsis
2. In debilitated individuals ->
Septicemia
Meningitis
Endocarditis

64. Lab diagnosis ->
1. Specimen -> CSF, Blood, Exudates
2. Cultured onto -> Sheep Blood Agar
3. Incubated at -> 37o C for up to 48 hrs
4. Isolate identified by ->
a) Beta hemolysis on blood agar
b) Gram stained smear – Gram positive cocci in
chains
c) Ability to hydrolyze Hippurate
d) CAMP test positive

65. Christie Atkins Munch Petersen Test
(CAMP Test)
Definition ->Test used to identify Group B
Streptococci
Principle ->Enhancement of β hemolysis of S.aureus
by a soluble protein (CAMP factor) produced by
Strep. agalactiae
Observation -> Arrow head
hemolysis is seen in-between
the two streaks of growth

66. Treatment
 Penicillin is the drug of choice

68. Classification
This group is divided into two groups ->
1. Enterococci ->
o Have Group ‘D’ specific Ag
o Differ from streptococci in biochemical
characteristics:-
Ability to survive in 40% bile
Grow in medium with 6.5% NaCl
Bile-Esculin hydrolysis test – Positive
Heat resistant (tolerate heat of 60oC for 30 min)

69. o May be non-hemolytic or alpha hemolytic
o Important species include:-
 Enterococcus faecalis
 Enterococcus faecium
 Enterococcus durans
o Part of the normal flora of the gut
o Pathogenicity -> - Urinary tract infection
- Wound infection
- Intra-abdominal abscess
- Sub-acute bacterial endocarditis

70. 2. Nonenterococci (Group D Streptococci) ->
o Also contain Group ‘D’ Ag
o Important species:-
Streptococcus bovis
Streptococcus equinus
o May be non-hemolytic or alpha hemolytic
o Streptococcus bovis -> has been isolated from
blood (septicemia) in patients with colon
carcinoma

71. Lab diagnosis
 Specimens –> Urine, pus, blood
 Microscopy -> Gram stain:
Gram positive oval cocci arranged
in pairs at an angle to each other
 Culture –>
Blood agar - non-hemolytic / α hemolytic
MacConkey’s agar - magenta pink
coloured colonies

72. Growth on Bile Esculin agar – Positive
Treatment
 Multi-drug resistance is common with
enterococci
 Enterococci are intrinsically resistant to
Cephalosporins and Aminoglycosides
 Vancomycin is drug of choice

74. The Viridans Streptococci
A heterogeneous group of streptococci which
cannot be grouped under Lancefield's
classification
Part of the normal flora of the oral cavity and
upper respiratory tract
Mostly alpha hemolytic (greenish color
‘viridans’)
Important species ->
Streptococcus mitis
Streptococcus mutans
Streptococcus sanguis
Streptococcus salivarius

75. Characteristics of viridans group ->
1. Difficulty in grouping
2. Alpha hemolytic
3. Insoluble in bile
4. Resistant to ‘Optochin’
5. Not pathogenic to mice on intraperitoneal
inoculation
6. Antibiotic susceptibility -> susceptible to
penicillin

76. Human pathogenicity
1. Dental caries ->
Str. mutans produces large amounts of slime which
adheres to teeth to form ‘plaques’
Bacteria within plaques ferment dietary starch into
acids which damage dentine resulting in caries
2. Sub-acute bacterial endocarditis ->
After dental extraction/manipulations, bacteria
enter blood stream, settle on damaged heart valves,
form vegetation and cause sub-acute endocarditis

78. Streptococcus pneumoniae
o The pneumococci are capsulated, gram positive,
lanceolate shaped diplococci which are an
important cause of sinusitis, otitis,
bronchitis, pneumonia,
bacteremia & meningitis
o They are part of the normal flora of the upper
respiratory tract in approximately 5 – 40% of
humans

79. Pathophysiology ->
Bacterial virulence factors ->
1. Capsular polysaccharide antigen –>
Major virulence factor, non-capsulated strains are
avirulent
Antiphagocytic – inhibit entrapment &
phagocytosis
Antigenic – 90 serotypes exist
In children – 6, 14, 19 & 23; In adults – 1 to 8
serotypes

80. Quellung (capsular swelling) reaction
 Mixing of capsulated pneumococci present in
sputum/suspension with type specific or polyvalent
antisera (Abs) on a glass slide results in apparent
swelling of capsules around the pneumococci
 Observed under light microscope
 Swelling is due to Ag-Ab reaction
and a change in the refractive index
of the capsular material
 Use – in direct demonstration, identification & in
typing

81. 2. Lipoteichoic acid – activates complement
and induces inflammatory cytokine
production
3. Pneumolysin – a hemolysin causes α
hemolysis & contributes to pathogenesis
4. Ig A1 Protease – enhances ability of the
organism to colonize the mucosa of URT by
destroying secretory IgA present on the
respiratory epithelia

82. Pathogenesis ->
1. Source of infection:-
Exogenous – Patients / carriers -> shed
organisms in their respiratory secretions
(40 – 70% of a population harbor pneumococci
at some time during their lifetime)
Endogenous – Part of the normal flora of URT
2. Mode of infection – Inhalation of droplet
nuclei or by direct / indirect contact

83. 3. Host predisposing factors ->
Infection & carriage is common but disease
occurs only under certain predisposing
conditions
A. Abnormalities of the Respiratory tract ->
a) Viral infections damaging surface cells
b) Abnormal accumulation of mucus – allergy
c) Bronchial obstruction – atelectasis
d) Respiratory tract injury due to irritants
disturbing its muco-ciliary function

84. Predisposing factors contd….
B. Alcohol / Drug intoxication -> results in,
i. Decreased phagocytic activity
ii. Decreased cough reflex, &
iii. Facilitates aspiration
C. Abnormal circulatory dynamics ->
i. Pulmonary congestion
ii. Heart failure

85. Predisposing factors contd….
D. Other factors ->
i. Malnutrition
ii. General debility
iii. Old age
iv. Sickle cell anemia
v. Hyposplenism
vi. Nephrosis
vii. Complement deficiency
viii.convulsions

86. 4. Sequence of events ->
a. Colonization of the upper respiratory tract
b. Whenever patient is predisposed, spread of
infection to the lower respiratory tract
c. Pneumococci produce disease through their
ability to multiply in tissues
d. Multiplication in the lungs results in
outpouring of edema fluid, WBC, RBC into
the alveoli, resulting in consolidation of
portions of the lungs

87. e. Later, mononuclear cells actively phagocytose the
bacteria and cell debris and digest them
intracellularly
f. Recovery begins between 5 – 10 days, with the
development of type specific Abs.
5. Clinical findings & complications ->
Lobar Pneumonia - Sudden onset of fever, chills &
sharp pleural pain, with expectoration of bloody or
rusty colored sputum
Mortality rate in pneumonia is high (30%) if cases
are untreated

88. Clinical findings & complications contd….
Contiguous spread may result in middle ear
infection (otitis); the mastoid (mastoiditis); the
paranasal sinuses (sinusitis)
Pus in pleural space (empyema) may require
aspiration / drainage
In 10 – 20% of patients, bacteremia occurs with
metastatic involvement of meninges (meningitis);
joints (septic arthritis) and rarely, the
endocardium (acute endocarditis)

89. Lab diagnosis
1. Specimen ->
Sputum,
Blood,
CSF,
Pus from suppurative lesions,
Synovial fluid,
Laryngeal swab (in the place of sputum in
children)

90. Lab diagnosis contd ….
2. Microscopy ->
a. Gram’s stained smear :-
Pneumococci are seen as Gram positive lancet or
flame shaped cocci in pairs along with pus cells

91. Lab diagnosis contd ….
b. Capsule demonstration:-
i. By negative staining – India Ink or Nigrosin –
capsule may be demonstrated as clear halo
ii. Quellung reaction (Capsule swelling reaction)

92. 3. Culture ->
Media used - Blood agar; Chocolate agar
Specimens are inoculated onto media and
incubated at 37o C under 5-10% CO2
Colony characteristics ->
Alpha hemolytic colonies dome shaped initially
which on further incubation acquires ‘Draughtsman’
appearance
This is due to the production of autolysins by the
streptococci which cause lysis of the older cells in
the centre

94. 4. Identification of isolate ->
a) alpha hemolytic colonies
b) Gram positive Lancet shaped cocci in pairs
c) Capsule demonstration
d) Bile solubility test –> positive
e) Optochin sensitivity test –> sensitive
f) Animal pathogenicity test –> Virulent to
mouse

95. 5. Serology ->
 Rapid diagnostic method ->
 Detection of capsular polysaccharide Ag in blood,
CSF by ->
Latex agglutination ,
Co-agglutination
ELISA
6. Antibiotic susceptibility testing ->
Should be done to determine susceptibility pattern

96. Differences between
Str. pneumoniae & Viridans Streptococci
Property Pneumococci Viridans
Streptococci
Morphology Lanceolate,
diplococci
Spherical / oval
cocci In long
chains
Capsule Present Absent / Slime
Colony Draughtsman Dome
Bile solubility Soluble Insoluble
Optochin
sensitivity
Sensitive Resistant
Animal
pathogenicity
(mouse)
Virulent Avirulent

97. Treatment
 Penicillin – drug of choice
 Tetracycline/ Erythromycin – also effective
 Penicillin resistant strains – 3rd generation
Cephalosporin are used

98. Prevention
 Immunity against Pneumococci is type specific
and associated with Antibody to capsular
polysaccharide
 A polyvalent polysaccharide vaccine containing
prevalent 23 serotypes is available and
administered by single dose injection
 Not for general use
 Only in persons at enhanced risk of
pneumococcal infection – children and elderly

100. C Reactive Protein (CRP)
Acute phase protein (Beta globulin) produced by
the liver and is found in serum
Precipitates with somatic ‘C’ antigen of
Pneumococci Therefore known as ‘C Reactive
Protein’
Not an Antibody produced as a result of
Pneumococcal infection
Its production is stimulated by bacterial
infections, inflammations, malignancies and

101. It disappears when the inflammatory reactions
subside
CRP can be detected by passive latex
agglutination and it is a routine diagnostic
procedure
CRP is used as an index of response to treatment
in Rheumatic fever and certain other conditions