Pneumococcus, also known as Streptococcus pneumoniae, are normal inhabitants of the upper respiratory tract that can cause pneumonia and ear infections. They are gram-positive cocci arranged in pairs or chains. Identification involves testing for morphology, culture characteristics like alpha-hemolysis on blood agar and bile solubility. There are over 90 serotypes classified by their polysaccharide capsule which is a major virulence factor and basis for vaccines. Pneumonia results from inhalation of the bacteria when immunity is low, often following a viral infection.

1. PNEUMOCOCCUS
Pneumococci are normal commensals of the upper respiratory tract.
They are important pathogens of pneumonia and otitis media in children.
They are reclassified as Streptococcus pneumoniae because ofits genetic relatedness to
streptococcus.
They differ from streptococci in their morphology ( diplococci),bile solubility, optochin
sensitivity and by a specific polysaccharide capsule.
PNEUMOCOCCUS
A. Morphology
Pneumococci are Gram positive,
small (1 µm diameter)
slightly elongated cocci
arranged in pairs (diplococci) within the broad ends in apposition.
Each coccus has one endbroad or rounded and other pointed (flame shaped or lanceolate
appearance)
They are capsulated and the capsule encloses each pair.
The capsule may bedemonstrated as a clear halo in India ink preparation
In old cultures, the capsule is usually lost.
They are non-motile and non-sporing.
B. Culture
- Pneumococci have complex nutritional requirement and therefore grow only in enriched
media especially supplemented with blood.
They are aerobes and facultative anaerobes and their growth is improved by 5- 10 per cent
CO2.
The optimum temperature for growth is 37°C (range25°C to 42°C) and pH 7.8 (range 6.5-
8.3).

2. On blood agar, after incubation for 18 hours, the colonies are usually small (0.5- 1 mm),
dome shaped, withan area of greenish discolouration (alpha haemolysis) around them.
On prolonged incubation, the coloniesbecome flat, with raised edges and central
umbonation(due to autolysis occuring at centre) which creates adraughtsman
appearance (concentric rings are seen whenviewed from above).
Some strains such as those of serotype 3 and 7 form large mucoid colonies due to
productionof abundant capsular polysaccharides.
Pneumococci are typically alpha haemolytic but under anaerobic conditions colonies
show beta haemolysis due to liberation of oxygen labile pneumolysin O by these bacteria.
In liquid medium such as glucose broth, pneumococci produce uniform turbidity.
These cocci readily undergoautolysis in cultures due to the action of
intracellularenzymes.
Autolysis is enhanced by bile salts andother surface active agents.
This property is helpful to differentiate it from other streptococci.
C. Biochemical Reactions
Pneumococci ferment several sugars with production of acid only.
Fermentation is tested in Hiss's serum water.
Fermentation of inslin by pneumococci is of great valueto differentiate them from
streptococci.

3. Pneumococci are soluble in bile. When 2% sodiumdeoxycholate solution is added to a
broth culture atneutral pH, the culture clears due to the lysisof the cocci.Alternatively, if a
loopful of 10% sodium deoxycholatesolution is placed on a pneumococcus colony, lysis
ofcolony occurs within a few minutes.
Bile solubility test is an important diagnostic test to differentiate pneumococcusfrom
other streptococci.
This test is based on the presenceof an autolytic enzyme amidase in the pneumococci.
Thisenzyme cleaves the bond between alanine and muramicacid in the peptidoglycan.
The amidase is activated bybile salts, resulting in lysis of the bacteria.Pneumococci are
catalase and oxidase negative.
D. Resistance
Pneumococci are delicate organisms and are destroyedat 52°C for 15 minutes.
They are more sensitive to usual antiseptics.
They are sensitive to a wide range of antibiotics including penicillins, erythromycin,
tetracycline, chloramphenicol, vancomycin and clindamycin.
Penicillin resistant strainsfirst appeared in 1967. It may be due to mutation orgene
transfer.
The drug resistance to penicillin is notdue to production of beta lactamase, but alteration
inthe penicillin binding proteins on the bacterial surface.
These strains are also resistant to multiple drugs
Pneumococci are sensitive to optochin (ethyl-hydrocuprein hydrochloride) in a
concentration of1/500,000. When a 6 mm optochin disc (5 µg) is applied ona blood agar
plate inoculated with pneumococci, a widezone (14 mm or more) of inhibition occurs on
incubation.
This is very useful test to differentiate pneumococci fromother streptococci which do not show
zone of inhibition by optochin disc.

4. E. Antigenic Structure:
The most important antigen of the pneumococcus iscapsular polysaccharide.
Other antigens are somatic M protein and a group specific cell wall carbohydrate.
1. Capsular Polysaccharide:
Capsular polysaccharide is type specific.
Since this polysaccharide is soluble in tissue and culture fluid, it diffusesinto the culture
medium or tissues and infective exudates,hence, it is also called the soluble specific
substance (SSS).
Pneumococci are classified into types based on the nature of the capsular
polysaccharide.
More than 90 serotypesare recognised.
These are named 1, 2, 3, and so on.Serological typing of pneumococcus is carried out by
three methods.
(i) Agglutination of organisms with type specific
antiserum.
(ii) Precipitation of capsular polysaccharide with
type specific antiserum.
(iii) Quellung reaction or capsule swelling reaction
was described by Neufeld (1902).
In this reaction,a suspension of pneumococci is mixed on a slidewith a drop of specific
antiserum and a loopfulof methylene blue solution. In presence of thehomologous
antiserum, the capsule around pneumococci reveals an apparent swelling,sharply
delineated and refractile under the microscope.
The Quellung test can be done eitherin sputum or in culture, and used to be a routine
bedside procedure in olden days.
2. M Protein
M protein is characteristic for each type of pneumococcus.
It is not associated with virulence and antibody to M protein is non-protective.
3. Cell Wall Carbohydrate

5. (C-Substance) Pneumococci contain a species specific carbohydrateantigen which is
named as C-substance.
It is present in all pneumococci.
The C-substance is precipitated by a abnormal protein (ß-globulin), that appears in the
acute phase sera of cases of pneumonia but disappears during convalescence.
It is also detected in blood of patients with some other illnesses.
This is known as the C-reactive protein (CRP).
It is not an antibody of C substance.
It is an 'acute phase' substance, produced in hepatocytes.
Its production is stimulated by bacterial infections,malignancies and tissue destruction.
CRP is used as anindex of activity in rheumatic fever and certain otherconditions.
CRP is routinely detected in serum by latex agglutination test.
F. Variation Pneumococcus is virulent in capsulated (smooth) form but non-capsulated
(rough) forms are avirulent. On repeatedsubculture, a smooth to rough (S-.R) variation
occurs.
R forms arise as spontaneous mutants and outgrow the parental
S forms in artificial culture.
Such R mutants are eliminated in tissues by phagocytosis.
Rough pneumococci of one serotype may be made to produce capsules of the same or
different serotypes,on treatment with DNA from the respective serotypes of
pneumococci.
This transformation was discovered byGriffith (1928) as the first instance of genetic
exchange of information in bacteria.
G. Toxins and Other Virulence Factors
Pneumococci produce an oxygen labile intracellular
haemolysin and a leucocidin.
The virulence of pneumococci is dependant upon its capsule which prevents or inhibits
phagocytosis.
Non-capsulated strains are avirulent.
The antibody to the capsular polysaccharide protects against infection.
Due to the abdundance of capsular material, the virulence of type-3 pneumococcus is
enhanced.
Pneumolysin, a toxin produced by penumococci, is another virulence factor.
It has cytotoxic and complement activating properties.
It is immunogenic H. Pathogenesis Str. pneumoniae is one of the most common bacteria
causing pneumonia, both lobar and bronchopneumonia.
It is also responsible for acute tracheobronchitis and empyema.
1. Lobar Pneumonia -
Pneumonia results only when the general resistance is lowered.

6. Common infective types of Str. pneumoniae include types 1- 12 in adults
and types 6, 14, 19 and 23 are responsible in children.
2. Bronchopneumonia-
It is almost always a secondary infection following viralinfections of the respiratory tract.
Any serotype of pneumococcus can produce bronchopneumonia.
Other causative agents responsible for bronchopneumonia include Staph. aureus, K.
pneumoniae, Str. pyogenes,H. influenzae, Fusobacterium species and Bacteroides.
3. Meningitis
It is the most serious of pneumococcal infections. Str. pneumoniae is the second most
important cause of pyogenic meningitis after Nmeningitidis.
This disease is common in children. Pneumococcus spreads from the pharynx to the
meninges via blood stream.
Other bacterialagents of pyogenic meningitis include N meningitidis,H. influenzae, Str.
agalactiae (group-B) and Listeria monocytogenes.
4.Other Infections
Pneumococcus may also produce empyema, pericarditis,otitis media, sinusitis,
conjunctivitis, peritonitis andsuppurative arthritis, usually as complications of pneumoma.
I. Epidemiology
The source of human infection is carrier and less often patient.
Pneumococci are transmitted by inhalation of contaminated dust, droplets or droplet
nuclei.
Infection usually leads to pharyngeal carriage.
Disease results only when the host resistance is lowered by factors such asrespiratory
viral infections, malnutrition etc.
Lobar pneumonia is usually a sporadic disease but epidemics may occur.
The incidence of bronchopneumonia is more when an epidemic of influenza or other viral
infections of the respiratory tract occurs.
J. Laboratory Diagnosis
1. Specimens:
Clinical samples, such as
sputum cerebrospinal fluid (CSF)
pleural exudate
blood are collected according to the site of lesion.
Blood culture is useful in pneumococcal septicaemia.

7. 2. Collection and Transport
All the specimens should be collected in sterile containers
under all aseptic conditions.
They should be processed immediately.
In case of delay, CSF specimen should never be refrigerated but kept at 37°C (H.
influenzae, another causative agent of pyogenic meningitis may die at cold temperature).
3. Direct Microscopy and Antigen Detection :
Gram staining of smear reveals a large number of polymorphs and typical organism.
In case of meningitis, presumptive diagnosis may be made by finding Gram positive
diplococci which may be intracellular as well as extracellular in CSP smear.
Capsule swelling may beobserved under microscope, when pneumococci are mixed with
type specific antisera.
Capsular polysaccharide antigen can be demonstrated by counterimmunoelectrophoresis.
This has been employed in blood, urine and cerebrospinal fluid.
Antigen may also be detected by immunochromatographic assay, latex agglutination or
coagglutination.
4. Culture
Specimen is inoculated on blood agar and incubated at 37°C for 24 hours in the presence
of 5- 10% CO2.
Typical colonies develop with a- haemolysis.
Organisms from the isolated colony are identified by Gram staining and biochemical
reactions
5. Colony Morphology and Staining
Colonies are usually small (0.5- 1 mm), with alpha haemolysis around them.
On prolonged incubation, colonies have draughtsman appearance.
On Gram staining pneumococci are Gram positive, small (1 µm diameter), diplococci
They are flameshaped or lanceolate in appearance.
The capsule may be demonstrated as a clear halo in India ink preparation

8. 6. Biochemical Reactions
Important biochemical tests are inulin fermentation and bile solubility tests.
Another test which has a great value is optochin sensitivity test.
7. Animal Pathogenicity Test
From specimens where organisms are expected to be scanty, intraperitoneal inoculation
in mice may be used.
Inoculated mice die in 24-48 hours.
Heart blood and peritoneal exudate of the animal shows pneumococci.
The test may be negative with occasional strains which are avirulent for mice (type 14
strains).
8. Antibiotic Sensitivity Test
It is especially useful in strains which are resistant.
K. Treatment
The antibiotic of choice is parenteral penicillin.
Cephalosporin is indicated in case of penicillin resistant strains.
L. Prophylaxis
Immunity is type specific and is associated with antibody to the capsular polysaccharide.
The existence of more than 90 serotypes makes prophylactic immunisation impracticable.
But, as a limited number of serotypes cause most of the pneumonia cases, vaccination is
possible.
Three pneumococcal vaccines are available.
A polysaccharide vaccine containing prevalentserotypes (23 serotypes) is administered
by a single dose injection.
Such vaccines are used only in those persons who are at enhanced risk of pneumococcal
infection.
These include persons with absent or dysfunctional spleen, nephrotic syndrome, sickle cell
anaemia, multiple myeloma, hepatic cirrhosis, diabetes mellitus and immunodeficiencies
including HIV infection.
Vaccination is contraindicated in lymphomas.
The heptavalent pneumococcal conjugate vaccine (7 serotypes) is composed of purified
polysaccharides of

9. seven serotypes conjugated to a diphtheria protein is now available for use in children
from two months to two years.
More recently, a new 13-valent vaccine that contain six additional serotypes was
approved for young children.
However, protection would de
pend on the principle thatserotypes prevalent in the community and serotypes used in the
vaccine are the same.
Pdf made by : Priyanshu verma
Reference : CP BAVEJA MICROBIOLOGY
pdf made by : Priyanshu verma
Reference : CV BAVEJA MICROBIOLOGY