Streptococcus pneumoniae- Morphology and Cultural Characteristics, Pathogenesis, Laboratory diagnosis, Epidemiology, Prevention and control of diseases

 S. pneumoniae

• Gram positive, lanceolate shaped diplococcus - 

• Pneumococci 

•  Formerly called, Diplococcus pneumoniae 

•  Normal inhabitants of the human upper respiratory tract 

• Common cause of Pneumonia and Otitis media in children 

•  Also cause Sinusitis, Bronchitis, Bacteremia, Meningitis 

• First noticed by Louis Pasteur and Sternberg- inoculated with human saliva in rabbits and produced fatal septicemia- isolated pneumococci from the blood of infected rabbits- 

• Fraenkel and Weichselbaum established the relation between pneumonia and pneumococci

MORPHOLOGY 

-Small, slightly elongated cocci - One end broad or rounded and the other pointed 

- Flame shamed or lanceolate Occur in pairs with broad ends in apposition 

-Capsulated- a pair in a capsule- capsule best seen in material taken from exudates and maybe lost on repeated subcultures 

-Seen as more rounded in culture- Typical morphology may not occur 

- Non motile, Non spore forming 

-Gram positive-readily stained with aniline dyes

- Capsule demonstrated in India ink preparation 

CULTURAL CHARACTERISTICS 

 Complex growth requirements-Enriched media 

- Aerobes and facultative anaerobes 

- On Blood agar, at 18 hours, colonies are small (0.5 – 1 mm), dome shaped and glistening with α haemolysis (green discolouration)

- Further incubation, colonies become flat with raised edges and central umbonation -Concentric rings on surface when viewed from above - Draughtsman or carrom coin appearance 

- Large Mucoid clonies ( types 3 and 7)- abundant capsular material 

-Under anaerobic conditions, ß haemolysis in due to oxygen labile Hemolysin O

-Glucose broth – growth occurs as uniform turbidity 

- Readily undergoes autolysis due to the activity of intracellular enzymes 

-Autolysis is enhanced by bile salts, Sodium lauryl sulphate and surface active agents 

ANTIGENIC PROPERTIES

Important antigens

• ·         Capsule
• ·         Nucleoprotein antigen
• ·         ‘C’ carbohydrate antigen u C – reactive protein (CRP)

Type specific Capsular polysaccharide –diffuses into culture medium/exudates/tissues- Specific Soluble Substance (SSS)

Based on Antigenic nature of Capsular polysaccharide, pneumococci were classified into pneumonia causing  I, II, III and heterogenous IV group

IV group has 90 serotypes named 1, 2, 3 and so on

Serotyping carried out by agglutination of cocci with type specific antiserum, Precipitation of SSS with specific serum or capsule swelling reaction/Quellung reaction  demonstrated by Neufeld

Quellung reaction  (Neufeld in 1902)

Suspension of S. pneumoniae is mixed on a slide with a drop of type specific antiserum and a loopful of methylene blue - Capsule becomes swollen and refractile (scatter light)

Done directly with sputum in acute pneumonia cases- was done routinely in the past when the specific antiserum was used for treatment of pneumonia

CRP –Abnormal protein (beta globulin) which precipitates with the somatic C antigen of pneumococci-appears in acute phase of pneumonia, disappear later.

C reactive protein- common in other pathological conditions too.

Not an antibody produced in pneumococcal infections- produced in hepatocytes- stimulated by bacterial infections, inflammation, tissue destruction

Smooth to rough (S – R) variation

In the R form, cocci are non capsulated, auto agglutinable and avirulent –arise as spontaneous mutants in cultures and outgrow S forms, In tissues R mutants are eliminated by phagocytosis

Rough S. pneumoniae of one serotype may be made to produce capsules of the same or different serotype, on treatment with DNA from the respective serotypes

Transformation by Griffith (1928)- demonstration of genetic material in bacteria

 Biochemical Reactions

• ·         Catalase and oxidase negative
• ·         Ferments sugars with acid production -Fermentation tested in Hiss’s serum water **
• ·         Ferments Inulin-streptococci does not  
• ·         Pneumococci are bile soluble-of diagnostic importance
• ·      Bile solubility test: If a few drops of 10% sodium deoxycholate are added to 1 mL of an overnight broth culture, the culture clears due to lysis of cocci- due to an autolytic amidase that cleaves the bond between alanine and muramic acid in the peptidoglycan; Amidase is activated by bile salts

          

**(Hiss's serum water is used in carrying out fermentation tests with certain pathogenic organisms, such as the pneumococcus that do not grow well in the peptone–water solutions. Fermentation is indicated by the change in the indicator by the production of acid that may also coagulate the serum. Litmus milk is used for biochemical tests. Acid production, a result of the fermentation of lactose, turns the medium pink and may also coagulate it; proteolytic activity is indicated by digestion and alkali formation.)

Resistance

• ·         Pneumococci are delicate organisms.
• ·         Readily destroyed by heat- 52° C for 15 minutes and antiseptics
•    Cultures die on prolonged incubation due to accumulation of toxic peroxides- maintained by lyophilisation
• ·         Sensitive to beta lactam antibiotics initially- now resistant-resistance due to alteration in penicillin binding proteins on the bacterial surface
• ·         Pneumococci sensitive to Optochin (Ethyl hydrocuprein) sensitive- Streptococci are not

Toxins and Virulence Factors

• ·         Virulence depends on its capsule and the production of a toxin called pneumolysin
• ·         Capsular Polysaccharide (acidic & hydrophilic) - protects the cocci from phagocytosis
• ·      Enhanced Virulence of type 3 due to abundant capsular material- Non capsulated strains are avirulent - Antibody to capsular polysaccharide is protective
• ·         Pneumolysin –membrane damaging toxin produced by Pneumococci- cytotoxic and complement activating properties- virulence factor-immunogenic
• (Pneumolysin-negative mutants showed less virulence in experimental animals)
• ·         Pneumococcal Autolysins- release bacterial components in infected tissues- contribute to virulence
•  Oxygen labile hemolysin and leucocidin – not significant in virulence

Epidemiology

• ·         S. pneumoniae occurs in the throats of 50% of human population, any time 
•        Source of infection is the respiratory tract of carriers, and at times, of patients.
• ·         Transmitted by fingers or by inhalation of droplets
• ·         Dissemination is facilitated by crowding
• ·         Infection usually leads to pharyngeal carriage-- disease results when host resistance is lowered by viral infection, pulmonary congestion, stress, malnutrition, immunodeficiency or alcoholism

  Pathogenicity

• ·         Experimental infection in mice & rabbits by intraperitoneal inoculation - fatal infection – pneumococci demonstrable in peritoneal exudate and heart blood–death in 1-3 days

• ·         Colonise the human Nasopharynx – cause infection of middle ear, paranasal sinuses and respiratory tract – direct infection
• ·         Can spread through blood and lymphatic system– cause Meningitis – distant infections in the heart, peritoneum or joints
• ·         Infection is generally, endogenous in nature, can be Exogenous also

Common Pneumococcal infections 

• otitis media and sinusitis – prior respiratory infection or allergy can lead to these conditions

• common cause of lobar and bronchopneumonia –also cause acute tracheobronchitis and empyema (collection of pus in the pleural cavity)

•  Normal mucosal defense mechanisms (entrapment, cough, ciliary defense) prevent establishment of infection. But if these defense mechanisms are compromised by infections, anesthesia or chilling etc, Pneumococci can multiply and spread through lung and lymphatics- Bacteremia is common in lobar pneumonia

• Diffusion of capsular polysaccharide into the blood and tissues- cause toxemia.
•  SSS neutralized by anti-capsular antibodies produced by the patient- cause fall in temperature and general relief of symptoms
•  In adults, 50% fatalities are due to Pneumococcal bacteremia.

• Bronchopneumonia is always a secondary infection – damage to the respiratory epithelium and excessive bronchial secretions caused by the primary infection, lead to the invasion of Pneumococci to the bronchial tree- terminal event in aged and debilitated patients 

• Meningitis – most serious Pneumococcal infection
• Is a secondary infection after the primary infections like  pneumonia, otitis media, sinusitis or conjunctivitis - Occur at all ages 
• If untreated, highly fatal 
• 25% fatality even with antibiotic therapy

•  Pneumocci also cause suppurative lesions in different parts of the body- empyema, pericarditis, otitis media, sinusitis, conjunctivitis, suppurative arthritis, peritonitis , keratitis, dacryocystitis (lacrymal sacs infection)

Laboratory Diagnosis

• Clinical diagnosis of Pneumonia is easy -etiological diagnosis needs laboratory assistance- many organisms involved
• Specimen – Sputum, CSF, Blood and Urine
• Microscopy - In acute lobar pneumonia, rusty sputum contain Pneumococci in large numbers -hardly any other bacterium- Gram stain.

(Rusty sputum- sputum produced in pneumonia caused by S pneumoniae - contain bacteria, hemorrhage, mucus,  necrotic lung tissue.)

• In the pre-antibiotic era, direct serotyping with Quellung reaction followed by treatment with specific antiserum-routine process

• Culture – The sputum after homogenization, inoculated onto blood agar plates, incubated at 37^oC, under 5-10 % CO₂, overnight. In infants, laryngeal swabs may be used for culture. Gentamycin can be added to blood agar to facilitate isolation of Pneumococci.

• In acute pneumonia, the organism may be obtained from Blood culture in Glucose broth – isolation of pneumococci in blood indicates bad disease condition.

• Animal inoculation – Intraperitoneal inoculation in mice, if specimen contains scanty numbers of the organism and even if cultures are negative. Pneumococci may be demonstrated in the peritoneal exudates and heart blood. Inoculated mice die in 1-3 days.
• Test maybe negative if the strains are avirulent for mice (type 14 strain)

• In otitis media, Pneumococci demonstrated in fluid aspirated from the middle ear

• Meningitis- presumptive diagnosis from Gram stained films of CSF-Gram positive diplocoocci visible inside polymorphs and extracellularly. Diagnosis confirmed by culture. If negative in culture, SSS demonstrated in CSF by immunoprecipitation (with antisera)

• Antigen Detection  -SSS/Capsular polysaccharide -in blood, urine, CSF by Precipitation, counterimmunoelectrophoresis
• Antibody demonstrated by agglutination, precipitation, indirect haemagglutination, radioimmunoassay etc

Prophylaxis

• Immunity is type specific – antibodies against  capsular polysaccharide
• ` 90 serotypes- so complete polyvalent vaccine not practical
• A polyvalent polysaccharide vaccine with  capsular antigens of the 23 most prevalent serotypes gives 80 – 90 % protection 
• not meant for general use but for people at increased risk such as absent or dysfunctional spleen,  Sickle cell disease, chronic heart, renal, lung and celiac disease, Diabetes mellitus, HIV etc
• not recommended for children under two years of age and those with immunosuppressive therapies.

Treatment 

• Parenteral administration of Penicillin in serious cases and  Amoxycillin in mild cases (if the isolate is sensitive to penicillin)  
• Many Penicillin resistant (alteration in Penicillin Binding Protein)- strains are resistant to erythromycin and tetracycline
• For resistant isolates- Third generation cephalosporins and in life threatening illnesses with highly resistant strains, Vancomycin