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STREPTOCOCCUS PNEUMONIAE

-BACTERIAL PNEUMONIA

-LAYA K PILLAI

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HISTORY

-First discovered by Pasteur in 1881-Confused with other causes of pneumonia until discovery of gram stain in 1884-More than 80 serotypes by 1940

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INTRODUCTION

• Also known as pneumococci• Gram positive • Lanceolate shaped diplococci• Possess a specific polysaccharide capsule• Normal inhabitants of human upper respiratory tract• Causes pneumonia, otitis media in children, sinusitis, meningitis etc

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MORPHOLOGY

-Typically small (1µm)-Elongated cocci with one end broad and other end pointed presenting a lanceolate appearance-They are capsulated enclosing each pair-Non motile and Non sporing-Readily stained with aniline dyes-capsules may be demonstrated as clear halo in India ink preparations

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CULTURAL CHARACTERISTICS

• Grow only in enriched media• They are aerobes and facultative anaerobes• Optimum temperature 37 ⁰C (25-42) and ph of 7.8 (6.5-8.3)• On blood agar, after incubation for 18 hrs, the colonies are dome shaped

and glistening with an area of green discolouration(alpha hemolysis) around them resembling streptococcus viridans. On further incubation they become flat with raised edges and central umbonation (Draughtsman or carom coin appearance)• Under anaerobic conditions, colonies on blood agar are surrounded by a

zone of beta hemolysin O.• In liquid media such as glucose broth, growth occurs as uniform turbidity

the cocci readily undergoes autolysis which is enhanced by bile salts etc

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S.Pneumoniae colonies

‘Draughtsman’ appearance

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BIOCHEMICAL REACTIONS

• They ferment several sugars, forming only acid . Fermentation is tested in Hiss’s serum sugars.• S. pneumonia are catalase and oxidase negative.• Fermentation of inulin is a useful test for differentiating them from

streptococci, as the latter do not ferment it.

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Bile solubility test

• S. pneumonia are bile soluble.• If a few drops of 10% Na deoxycholate soln are added to 1ml of an

overnight broth culture, the culture clears due to lysis of cocci . Bile solubility is a constant property of S. pneumoniae and hence is of diagnostic importance.• Test should be done at neutral ph using deoxycholate and live young cell

in saline suspension• Principle: bile solubility is due to presence of an autolytic amidase that

cleaves the bond between alanine and muramic acid in the peptidoglycan. The amidase activated by surface active agents like bile or bile salts.

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Results of the bile solubility test are shown for two different strains of bacteria. For strain 1, a slight decrease in turbidity is observed in the tube containing the bile salts (2nd from left), but the contents are almost as turbid as the control tube (far left); therefore, strain 1 is not S. pneumoniae. For strain 2, all turbidity in the tube containing the bile salts (far right) has cleared, indicating that the cells have lysed, in contrast to the control tube (2nd from right), which remains turbid; therefore, strain 2 is S. pneumoniae.

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RESISTANCE

• Readily destroyed by heat (thermal death point 52 ⁰C for 15 min) and antibiotics.

• In culture they die on prolonged incubation due to accumulation of toxic peroxides. Strains may be maintained on semi solid blood agar or by lyophilisation.

• Sensitive to most antibiotics beta lactams being the drug of choice.

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Optochin sensitivity

• Sensitvity of S.pnuemoniae to optochin (ethyl hydrocuprein) is useful in differentiating them from streptococci.

• When a disc impregnated with optochin is applied on a plate of blood agar inoculated with S.pneumonia , a wide zone of inhibition appears on incubation .

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ANTIGENIC PROPERTIES

CAPSULE : The most important antigen of S.pneumonia is the type specific capsular polysaccharide. As this polysaccharide diffuses into the culture medium or infective exudates and tissues , it is also called “specific soluble substance” (SSS)

S.pneumonia are classified based on antigenic nature of capsular polysaccharide .

More than 90 serotypes are recognized named 1,2,3 .. etc

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SEROTYPING based on capsular antigens may be carried out by:

1. Agglutination of cocci with type specific serum

2. Precipitation of the SSS with specific serum

3. By capsule swelling or “QUELLUNG REACTION” described by Neufeld. Here a suspension of S.pneumonia is mixed on a slide with a drop of type specific antiserum and a loopful of methylene blue soln. In the presence of homologous antiserum, the capsule becomes apparently swollen, sharply delineated and refractile. The quellung reaction test can be done directly with sputum from pneumonia cases.

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Contd…

4. PCR based tests : These test have shown higher sensitivity in detection of infection esp in meningitis as it can detect the presence of a small number of the specific DNA sequences of bacteria which cannot be cultured by conventional methods due to administration of prior antibiotics or because of a smaller bacterial load in body fluids.

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OTHER ANTIGENS A nucleoprotein deep inside the cell and a somatic ‘C’ carbohydrate antigen both of which are species specific.

‘C’ ANTIGEN OF BACTERIAL CELL WALL AND CRP: An abnormal protein (beta globulin) called C reactive protein (CRP) that precipitates with the somatic ‘C’ antigen of S.pneumonia appears in the acute phase sera of pneumonia but disappears during convalescence. It is not an antibody but an ‘acute phase’ substance produced in hepatocytes. Its production is stimulated by bacterial infections, inflammation, malignancy. It disappears when the infection subsides.

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VARIATION

• On repeated subculture, the bacteria undergo smooth-to-rough (S-R) variation. R form are rough and non capsulated, auto agglutinable and avirulent. R forms arise as spontaneous mutants and outgrow the parental S forms in artificial culture while in tissues such R mutants are phagocytosed.

• R forms derived from the capsulated cells of one serotypes can be made to produce capsules of same or diff serotypes on treatment with DNA of respective serotypes (Griffith Transformation).

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TOXINS AND VIRULENCE FACTORSThe virulence of S.pneumonia depends on :

• The capsular polysaccharide, because of its acidic and hydrophilic properties, the cocci protected from phagocytosis. But they are susceptible to ‘surface phagocytosis’, being engulfed against a firm surface such as clot or epithelium.

The enhanced virulence of type3 S.pneumonia is due to abundance of capsular material

• Pneumolysin: a membrane damaging toxin produced by the cocci has cytotoxic and complement activating properties. Pneumolysin –ve mutants show reduced virulence.

• Autolysin: these helps in releasing bacterial components and toxins like pnemolysin and thus contribute to virulence

• Oxygen labile hemolysin and leucocidin – weak , produce no virulence

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PNEMOCOCCUS CAUSE MULTIORGAN DISEASE

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DISEASES CAUSED BY PNEUMOCOCCI

NON-INVASIVE DISEASES INVASIVE DISEASES

BACTEREMIA • MENINGITIS (CNS)• ENDOCARDITIS (CVS)• PERITONITIS (body cavity)• SEPTIC ARTHRITIS • OTERS (appendicitis,

salpingitis, soft tissue infections)

• SINUSITIS• OTITIS MEDIA (middle ear)• PNEUMINIA (lungs)

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• S.pneumonia are one of the most common bacteria causing community acquired pneumonia (CAP) (both lobar and bronchopneumonia.)• They also cause acute tracheobronchitis and empyema.• Aspiration of nasopharyngeal secretions containing the bacteria is the

common event.• Normal mucosal defense mechanism such as entrapment, expulsion

and cough reflex, aided by the ciliary escalator effect, prevent the establishment of infection.• But when normal defenses are compromised by viral infection.• The bacteria penetrate the bronchial mucosa and spread through lung

along peribronchial tissues and lymphatics

PNEUMONIA

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Contd…..

• Bacteremia is common during early stage of lobar pneumonia• Toxemia is due to diffusion of capsular polysaccharide into blood and

tissues.• Bronchopneumoniae is almost always secondary infection• The damage to respiratory epithelium by primary infection facilitate

the invasion of the cocci the bronchial tree

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• Meningitis : most serious of pneumococcal infections. It is usually secondary to infections like pneumonia, otitis media etc

Pneumococcal meningitis is an infection of the covering of the brain and spinal cord. Symptoms include: Stiff neck Fever Headache• Suppurative lesions : empyema, pericarditis, otitis media, sinusitis,

conjunctivitis, suppurative arthritis and peritonitis.

• They are also responsible for ocular infections like keratitis, dacrocystitis.

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• Bacteremia (blood stream infection) pneumococcal bacteria can invade the bloodstream, causing bacteremia, and the tissues and fluids surrounding the brain and spinal cord, causing meningitis which may be fatal.Pneumococcus is the most common cause of bloodstream infections. Symptoms include: Fever Chills Low alertness

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• Much of the clinical severity of pneumococcal disease is due to the activation of the complement pathways and cytokine release, which induce a significant inflammatory response. S. pneumoniae cell wall components, along with the pneumococcal capsule, activate the alternative complement pathway; antibodies to the cell wall polysaccharides activate the classic complement pathway. Cell wall proteins, autolysin, and DNA released from bacterial breakdown all contribute to the production of cytokines, inducing further inflammation.

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EPIDEMIOLOGY

• S.pneumonia colonise in nasopharynx. They are transmitted by contaminated droplets.• Dissemination is facilitated by crowding.• Disease results only when host resistance is lowered by viral infections,

pulmonary congestion, stress, malnutrition, immunodeficiency• Splenectomy and sickle cell disease are important predisposing factors.• Type 3 is the most virulent • In India, lobar pneumonia is usually a sporadic diseasebut epidemics

may occur in closed communities.• They affect the two extreme age groups more often. And seasonally

winter

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• Children at increased risk for pneumococcal disease include those Who have certain illnesses (sickle cell disease, HIV infection, or chronic heart or lung conditions) With cochlear implants or cerebrospinal fluid (CSF) leaks (escape of the fluid that surrounds the brain and spinal cord)Adults 65 years or older are at increased risk for pneumococcal disease.

• adults increased risk for pneumococcal disease, including those: With chronic illnesses (lung, heart, liver, or kidney disease; asthma; diabetes; or alcoholism) With conditions that weaken the immune system (HIV/AIDS, cancer, or damaged/absent spleen)• TransmissionPneumococcal bacteria spread from person-to-person by direct contact with respiratory secretions, like saliva or mucus (exogenous) . Many people, especially children, have the bacteria in their nose or throat at one time or another without being ill. When immune system is impaired the symptoms of infection will manifest (endogenous).

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LAB DIAGNOSIS

1. SPECIMEN: sputum, blood for culture, CSF and urine are used for antigen detection

2. MICROSCOPY: In pneumonia sputum examintn by gram stain. In otitis media fluid aspirated from middle ear. In meningitis CSF examnatn by gram staining.

3. CULTURE: sputum is inoculated on blood agar and incubated at 37⁰C under 5-10% CO₂. Blood culture in glucose broth.

4. MOUSE INOCULATION: In specimen where the cocci are scanty, intraperitoneal inoculation in mice.

5. ANTIGENIC DETECTION: By demonstrating the SSS in CSF by pptn with antisera or the latex agglutination test.

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Contd…

6. BIOMARKERS: CRP testing by passive agglutination using latex particles coated with anti-CRP antibody is a routine Dx procedure. Procalcitonin is another biomarker which is elevated in invasive pneumococcal disease.

7. MOLECULAR METHODS: PCR-based methods.

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PROPHYLAXIS• Immunity is type specific and associated with antibodies to capsular

polysaccharide.

• A polyvalent polysaccharide vaccine (PPV) representing capsular antigens of 23 most prevalent serotypes has been stated to give 80-90% protection.

• Not meant for general use but only for prone cases like enhanced risk of infection, dysfunctnal spleen, sickle cell disease, celiac disease, DM, liver diseases, HIV and for lymphoreticular malignancies.

• A 7 valent conjugate vaccine now available for children from 2 months to 2 yrs old.

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TREATMENT

• Antibiotic of choice is parenteral penicillin in severe cases and amoxicillin in milder ones• Many resistant strains originated which caused problems in

treatment.• The mode of resistance is not production of beta lactamase, but

alteration in penicillin binding protein on bacterial surface.• In such cases, a third generation cephalosporin is indicated.

Vancomycin is to be reserved for life threatening illness with highly resistant strains.

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SELECTION OF APPROPRITE ANTIBIOTICS DEPENDS ON DIAGNOSIS!!If susceptible:• penicillin• ampicillin• amoxicillin

PENICILLIN RESISTANT:• cephalosporins III (e.g., cefotaxime, ceftriaxone)

• ALTERNATIVES:• vancomycin• chloramphenicol

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PACE

• The Pneumococcal Awareness Council of Experts (PACE) is a project of the Sabin Vaccine Institute and is composed of global experts in infectious diseases and vaccines. Established in December 2006, The Council seeks to raise awareness among policymakers and aims to secure global commitments to prevent pneumococcal disease, a leading infectious killer of children and adults worldwide. The Council works in collaboration and partnership with countries, NGOs.

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