Microbiology seminar

1. STREPTOCOCCUS PNEUMONIAE
-BACTERIAL PNEUMONIA
-LAYA K PILLAI
July 20, 2015 1

2. 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
July 20, 2015 2

3. 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
July 20, 2015 3

4. 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
July 20, 2015 4

5. July 20, 2015 5

6. 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
July 20, 2015 6

7. S.Pneumoniae colonies
‘Draughtsman’
appearance
July 20, 2015 7

8. 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.
July 20, 2015 8

9. 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.
July 20, 2015 9

10. 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.July 20, 2015 10

11. 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.
July 20, 2015 11

12. 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 .
July 20, 2015 12

13. July 20, 2015 13

14. 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
July 20, 2015 14

15. 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.
July 20, 2015 15

16. July 20, 2015 16

17. July 20, 2015 17

18. July 20, 2015 18

19. 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.
July 20, 2015 19

20. 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.
July 20, 2015 20

21. 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).
July 20, 2015 21

22. TOXINS AND VIRULENCE FACTORS
The 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
July 20, 2015 22

23. July 20, 2015 23

24. PNEMOCOCCUS CAUSE MULTIORGAN DISEASE
July 20, 2015 24

25. 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)
July 20, 2015 25

26. • 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
July 20, 2015 26

27. 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
July 20, 2015 27

28. July 20, 2015 28

29. July 20, 2015 29

30. July 20, 2015 30

31. • 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.
July 20, 2015 31

32. • 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
July 20, 2015 32

33. • 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.
July 20, 2015 33

34. 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
July 20, 2015 34

35. • 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)
• Transmission
Pneumococcal 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).
July 20, 2015 35

36. 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.
July 20, 2015 36

37. 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.
July 20, 2015 37

38. 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.July 20, 2015 38

39. 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.
July 20, 2015 39

40. SELECTION OF APPROPRITE ANTIBIOTICS DEPENDS ON DIAGNOSIS!!
If susceptible:
• penicillin
• ampicillin
• amoxicillin
PENICILLIN RESISTANT:
• cephalosporins III (e.g., cefotaxime, ceftriaxone)
• ALTERNATIVES:
• vancomycin
• chloramphenicol
July 20, 2015 40

41. 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.
July 20, 2015 41

42. July 20, 2015 42

43. July 20, 2015 43

44. July 20, 2015 44