Streptococcus pneumoniae under microscope

Streptococcus pneumoniae under microscope DEFAULT

Microbiology Diagnosis -- Streptococcus pneumoniae


MICROBIOLOGY DIAGNOSIS:

Streptococcus pneumoniae bacteria are gram-positive cocci arranged in chains and pairs (diplococci) on microscopic examination. A green, α-hemolytic, zone surrounds S. pneumoniae colonies on blood-agar plates. Pneumococci can be differentiated from other catalase-negative viridans streptococci by their susceptibility to Optochin and solubility in bile salts. Molecular methods for detection of S. pneumoniae, many of which are PCR-based, can also be used. 1, 2

COLONY MORPHOLOGY:

Streptococcus pneumoniae colonies vary in appearance depending on the degree of encapsulation of the organism. Heavily encapsulated strains can have large colonies, several millimeters in diameter, which appear gray and very mucoid, while less heavily encapsulated organisms usually have smaller colonies. 1

Pneumococci produce pneumolysin, which breaks down hemoglobin into a green pigment that can be observed as a large green zone surrounding S. pneumoniae colonies growing on blood-agar plates. This property is still termed α-hemolysis even though lysis of red blood cells is not responsible for it; green or yellow-green zones can also be seen around S. pneumoniae colonies growing on chocolate agar, a medium in which all red blood cells are lysed during preparation. 2

The opacity variance of S. pneumoniae colonies appears to reflect differences in the organism's pathogenesis and virulence. Animal model studies showed that transparent variants are more able to colonize the nasopharynx of infant rats, while opaque variants are more virulent in the intraperitoneal model of infection. The organism spontaneously alternates between transparent and opaque phases, each of which has different structural characteristics. For example, transparent variants are associated with increased amounts of cell-wall-associated teichoic acid, while opaque variants are associated with enhanced production of capsular polysaccharide. 2, 3

OPTOCHIN SUSCEPTIBILITY TEST:1, 2

Ethyl hydrocupreine hydrochloride (Optochin) is a quinine derivative that is used to differentiate pneumococci from other viridans streptococci, with a sensitivity of greater than 95%, because of its ability to selectively inhibit the growth of S. pneumoniae on blood agar plates at very low concentrations (≤5�g/mL).

The Optochin test is performed on a blood-agar medium using a disk diffusion principle. A few well-isolated colonies of the organism in question are streaked onto a blood-agar plate and a filter paper disk, impregnated with Optochin, is placed in the streaked area. The plate is incubated and examined after 18 to 24 hours. Pneumococci surrounding the disk are lysed, due to changes in surface tension, creating an inhibition zone (Figure 5).

An inhibition zone of 14 mm or more, around a 6-mm disk, allows for identification of the viridans streptococcus in question as Streptococcus pneumoniae. If the inhibition zone is less than 14 mm, further testing (bile solubility or serology) is indicated for the identification of S. pneumoniae.

The diagnostic reliance on a minimum diameter of the inhibition zone is due to the discovery of Optochin-resistant pneumococcal strains, and the ability of some non-pneumococcal viridans streptococci to show small zones of inhibition on the Optochin test.

BILE SOLUBILITY TEST: 1

The addition of bile salts, such as sodium deoxycholate, accelerates the natural lytic reaction observed in pneumococcal cultures by increasing the activation of autolytic enzymes produced by Streptococcus pneumoniae.

The bile solubility test is performed by adding a bile-salt solution to an established broth or blood-agar culture of the organism in question. A positive result in broth culture is obtained by noting visible clearing of the culture's turbidity, as compared to a control tube, after addition of the bile salt solution and re-incubation for up to 3 hours. On blood-agar plates, bile-soluble pneumococcal colonies "disappear" leaving behind their green zone of α-hemolysis, after placing a drop or two of the bile-salt solution on the colony and re-incubating the plate for 30 minutes.

Additional testing, by serology, may occasionally be necessary as only 86% of pneumococcal strains lyse completely with the addition of bile salts.

AUTOPSY FINDINGS

FINAL DIAGNOSIS




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Chapter 8: Identification and Characterization of Streptococcus pneumoniae

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S. pneumoniae may occur intracellularly or extracellularly as gram-positive lanceolate diplococci, but can also occur as single cocci or in short chains of cocci. S. pneumoniae is a fastidious bacterium, growing best at 35-37°C with ~5% CO2 (or in a candle-jar). It is usually cultured on media that contain blood, but can also grow on a chocolate agar plate (CAP). On a blood agar plate (BAP), colonies of S. pneumoniae appear as small, grey, moist (sometimes mucoidal), colonies and characteristically produce a zone of alpha-hemolysis (green) (Figure 1). The alpha-hemolytic property differentiates this organism from many species, but not from the commensal alpha-hemolytic (viridans) streptococci. Differentiating pneumococci from viridans streptococci is difficult as young pneumococcal colonies appear raised, similar to viridans streptococci. However, once the pneumococcal culture ages 24-48 hours, the colonies become flattened, and the central portion becomes depressed, which does not occur with viridans streptococci (Figure 2). A microscope (30-50X) or a 3X hand lens can also be a useful tool in differentiating pneumococci from viridans streptococci. Prior to identification and characterization testing procedures, isolates should always be inspected for purity of growth and a single colony should be re-streaked, when necessary, to obtain a pure culture. For the following identification and characterization procedures, it is essential to test alpha-hemolytic colonies that are less than a day old, typically grown overnight at 35-37°C with ~5% CO2 (or in a candle-jar).

The following specialized tests are used to identify colonies on a BAP that resemble pneumococci (Figure 3). S. pneumoniae can be identified using Gram stain, catalase, and optochin tests simultaneously, with bile solubility as a confirmatory test. If these tests indicate that the isolate is S. pneumoniae, serological tests to identify the serotype can be performed. This sequence of testing is an efficient way to save costly serotyping reagents and time. Additional methods for identification and characterization of S. pneumoniae using molecular tools are described in Chapter 10: PCR Methods and Chapter 12: Molecular Methods. See additional protocols used for streptococcal species identification and updates to existing methods.

Biosafety Level 2 (BSL-2) practices are required for work involving isolates of S. pneumoniae, as this organism presents a potential hazard to laboratory personnel and the surrounding working environment. Please refer to Chapter 4: Biosafety in order to follow the guidelines that have been established for laboratorians working in BSL-2 facilities as many of the tests described in this chapter require opening plates with live cultures and are often performed outside of a biosafety cabinet (BSC).

Figure 1 is a picture showing S. pneumoniae colonies with a surrounding green zone of alpha-hemolysis on a blood agar plate (BAP).

Figure 1. S. pneumoniae colonies with a surrounding green zone of alpha-hemolysis (black arrow) on a BAP

Figure 2 is a picture showing S. pneumoniae colonies have a flattened and depressed center after 24-48 hours of growth on a blood agar plate (BAP), whereas the viridans streptococci retain a raised center.

Figure 2. S. pneumoniae colonies have a flattened and depressed center after 24-48 hours of growth on a BAP, whereas the viridans streptococci retain a raised center

  1. Catalase testCatalase is the enzyme that breaks down hydrogen peroxide (H2O2) into H2O and O2. The oxygen is given off as bubbles in the liquid. The catalase test is primarily used to differentiate between gram-positive cocci. Members of the genus Staphylococcus are catalase-positive, and members of the genera Streptococcus and Enterococcus are catalase-negative.
    1. Performing the catalase test
      1. Grow the isolate(s) to be tested for 18-24 hours on a BAP at 35-37°C with ~5% CO2 (or in a candle-jar).
      2. From overnight growth on the BAP, use a disposable loop to carefully remove a colony and place it on a glass slide.
        • Do not transfer any of the blood agar to the slide as erythrocytes in the blood agar will cause a false-positive reaction.
      3. Add 1.0 ml of 3% H2O2 to the slide and mix with the bacteria.
        • H2O2 can be obtained from a commercial drug store.
        • After initially opening, store H2O2 at 4°C in a tightly closed bottle as it will slowly lose potency once opened.
      4. Observe the bacterial suspension on the slide immediately for vigorous bubbling.
      5. It is essential to use a known positive and negative quality control (QC) strain. A Staphylococcus spp. strain can be used for a positive control and a known S. pneumoniae strain or any other streptococcal spp., i.e., S. pyogenes can be used for a negative control.
    2. Reading the catalase test results
      • The absence of bubbling from a transferred colony indicates a negative test.
      • Any bubbling from a transferred colony indicates a positive test (Figure 4).
    3. Troubleshooting
      • False positives will result from transfer of red blood cells so take care when picking colonies from the BAP for this test.
    4. Quality control
      • It is essential to use a known positive and negative QC strain as described in the procedure. Opened bottles should be checked against a known catalase positive organism every 6 months.
      Figure 4 is a picture showing negative and positive catalase test results. The absence of bubbling from a transferred colony indicates a negative test. All streptococci are catalase-negative.

      Figure 4. Negative and positive catalase test results. The absence of bubbling from a transferred colony indicates a negative test. All streptococci are catalase-negative.

  2. Optochin testS. pneumoniae strains are sensitive to the chemical optochin (ethylhydrocupreine hydrochloride). Optochin sensitivity allows for the presumptive identification of alpha-hemolytic streptococci as S. pneumoniae, although some pneumococcal strains are optochin-resistant. Other alpha-hemolytic streptococcal species are optochin-resistant.
    1. Performing the optochin testOptochin (P) disks (6 mm, 5 µg) can be obtained from a commercial vendor. Optochin disks are often called “P disks” and many commercial versions are labeled with a capital “P”. If a commercial source of P disks is not available, a 1:4000 solution of ethylhydrocupreine hydrochloride can be applied to sterile 6 mm filter paper disks.
      1. Grow the strain(s) to be tested for 18-24 hours on a BAP at 35-37°C with ~5% CO2 (or in a candle-jar).
      2. Use a disposable loop to remove an isolated colony from the overnight culture on the BAP and streak onto one half of a BAP.
        • Two different isolates can be tested on the same plate, but care must be taken to ensure that the cultures do not overlap.
      3. Place a P disk within the streaked area of the plate and incubate the BAP overnight at 35-37°C with ~5% CO2 (or in a candle-jar).
      4. Observe the growth on the BAP near the P disk and measure the zone of inhibition, if applicable.
    2. Reading the optochin test results
      • Using a 6 mm, 5 µg disk, a zone of inhibition of 14 mm or greater indicates sensitivity and allows for presumptive identification of pneumococci (Figure 5).
      • Zones of inhibition should be measured from the top surface of the plate with the top removed.
      • Use either calipers or a ruler with a handle attached for these measurements. Measure the diameter of the zone holding the ruler over the center of the surface of the disk when measuring the zone of inhibition. In the case of an isolate completely resistant to optochin, the diameter of the disk (6 mm) should be recorded.
    3. Troubleshooting
      • A smaller zone of inhibition (< 14 mm) or no zone of inhibition indicates that the bile solubility test is required. It is important to remember that pneumococci are sometimes optochin-resistant.
    4. Quality control
      • Each new lot of optochin disks should be tested with positive and negative controls. The growth of S. pneumoniae strain ATCC 49619 is inhibited by optochin and growth of S. mitis strain ATCC 49456 is not inhibited by optochin.
      Figure 5 is a picture showing the optochin test for S. pneumoniae using optochin disks. The strain on the left is resistant to optochin with no zone of inhibition, and therefore is not a pneumococcus. The strain on the right is susceptible to optochin and is S. pneumoniae.

      Figure 5. Optochin test for S. pneumoniae using optochin disks. The strain on the left is resistant to optochin with no zone of inhibition, and therefore is not a pneumococcus. The strain on the right is susceptible to optochin and is S. pneumoniae.

  3. Bile solubility testThe bile (sodium deoxycholate) solubility test distinguishes S. pneumoniae from all other alpha-hemolytic streptococci. S. pneumoniae is bile soluble whereas all other alpha-hemolytic streptococci are bile resistant. Sodium deoxycholate (2% in water) will lyse the pneumococcal cell wall.
    1. Preparation of 2% sodium deoxycholate (bile salt) solution
      1. Dissolve 2 g of sodium deoxycholate into 100 ml sterile distilled water.
    2. Performing the bile solubility test
      1. Grow the isolate(s) to be tested for 18-24 hours on a BAP at 35-37°C with ~5% CO2 (or in a candle-jar).
      2. Add bacterial growth from the overnight BAP to 1.0 ml of 0.85% saline to achieve turbidity in the range of a 0.5-1.0 McFarland standard.
      3. Divide the cell suspension equally into 2 tubes (0.5 ml per tube).
      4. Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one tube. Add 0.5 ml of 0.85% saline to the other tube. Mix each tube well.
      5. Incubate the tubes at 35-37°C in CO2.
      6. Vortex the tubes.
      7. Observe the tubes for any clearing of turbidity after 10 minutes. Continue to incubate the tubes for up to 2 hours at 35-37°C in CO2 if negative after 10 minutes. Observe again for clearing.
    3. Reading the bile solubility test results
      • A clearing of the turbidity in the bile tube but not in the saline control tube indicates a positive test (Figure 6).
    4. Troubleshooting
      • Partial clearing (partial solubility) is not considered positive for pneumococcal identification. Partially soluble strains that have optochin zones of inhibition of less than 14 mm are not considered pneumococci.
    5. Quality control
      • Each new lot of sodium deoxycholate should be tested with positive and negative QC strains. S. pneumoniae strain ATCC 49619 can be used as a positive control and S. mitis strain ATCC 49456 can be used as a negative control.
      Figure 6 is a picture showing results of the bile solubility test for two different strains of bacteria. For strain 1, a slight decrease in turbidity is observed in the tube containing the bile salts, but the contents are almost as turbid as the control tube; therefore, strain 1 is not S. pneumoniae. For strain 2, all turbidity in the tube containing the bile salts has cleared, indicating that the cells have lysed, in contrast to the control tube, which remains turbid; therefore, strain 2 is S. pneumoniae.

      Figure 6. 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.

  4. Commercial test kits for identificationSeveral commercial identification systems that use slide agglutination tests are available for identification of colony growth from a BAP as S. pneumoniae. These identification tests use suspensions of latex beads with rabbit antibody specific for S. pneumoniae capsular antigens. Visible agglutination occurs when the S. pneumoniae capsular antigen reacts with the antibody-coated latex beads. The manufacturer’s instructions should be followed precisely when using these kits. These kits should be regularly subjected to QC using a non-pneumococcal streptococcal species, since they can become cross-reactive with prolonged storage.
  5. Determining S. pneumoniae capsular serotypes using serologic methodsAlthough serotyping of pneumococci is not usually necessary for a clinical response, capsular serotype determination is a critical component of successful pneumococcal disease surveillance efforts. Effective current multivalent vaccines target combinations of key serotypes. Determination of serotype distributions associated with disease in certain regions provides information regarding the potential usefulness of applying existing vaccines and is also critical for assessing vaccine impact.Serotype distribution can be determined by culture of the organism followed by serological determination of the capsular type by latex agglutination and the quellung reaction. Many laboratories have opted to use simpler and less expensive methods of deducing capsular serotypes through the use of specific PCR reactions (see Chapter 10: PCR Methods and PCR Deduction of Pneumococcal Serotypes for specific PCR protocols).
  6. Latex agglutinationThe standard quellung reaction test for serotyping pneumococci can be labor-intensive and time-consuming, and requires a certain level of experience to be performed competently. An agglutination method using anti-rabbit IgG-coated latex particles sensitized to pooled and select individual serotype-specific antisera (PCV7 serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F) for serogrouping/serotyping S. pneumoniae has been developed and kits are commercially available. The latex agglutination method is simpler and faster than the quellung reaction, but is only intended for partial serotyping as it can only narrow the identification down to a group or pool of serotypes. Then the quellung reaction should be performed using individual serotype-specific antisera for each serotype in the group or pool to identify the serotype.
    Performing latex agglutination testing
    1. Grow the isolate(s) to be tested for 18-24 hours on a BAP at 35-37°C with ~5% CO2 (or in a candle-jar).
    2. From overnight growth on a BAP, use a sterile loop to prepare a light to moderate cell suspension (approximately equal to a 0.5 McFarland density standard) in 0.5 ml of 0.85% saline.
    3. On a glass slide or reaction card, add 10 µl (1 droplet) of the latex reagent and 10 µl of the cell suspension. Mix the two suspensions together.
    4. After 10-30 seconds, observe the latex agglutination reaction at an angle with oblique lighting.
    Reading the latex agglutination results
    • A positive reaction is indicated by agglutination (cells clumping together) appearing within 5-10 seconds.
    • A negative reaction is indicated by no agglutination appearing within 5-10 seconds.
    Troubleshooting
    • The latex agglutination reaction should be examined within 5-10 seconds. If the reaction time exceeds 30 seconds, false positive reactions may occur.
    Quality control
    • Each lot of latex suspension should be tested for positive agglutination reactions using S. pneumoniae reference strains with known capsular serotypes.
  7. Quellung reactionFor proper quellung-based serotyping, a high quality microscope is required. A positive quellung or Neufeld reaction is the result of the binding of the capsular polysaccharide of pneumococci with type specific antibody contained in the typing antiserum. Pneumococcal typing sera are commercially available as pooled, group, or serotype-specific (see https://www.ssi.dk/English.aspxexternal icon). It is recommended to initially test with pooled antisera in succession until a positive reaction is observed. Typing should then proceed by testing with individual group and serotype-specific antisera included in the antisera pool that gave a positive reaction to determine the serogroup and serotype. An antigen-antibody reaction causes a change in the refractive index of the capsule so that it appears “swollen” and more visible. After the addition of a counter stain (methylene blue), the pneumococcal cells stain dark blue and are surrounded by a sharply demarcated halo which represents the outer edge of the capsule. The light transmitted through the capsule appears brighter than either the pneumococcal cell or the background. Single cells, pairs, chains, and even clumps of cells may have positive quellung reactions.
    Performing the quellung reaction
    1. Grow the isolate(s) to be tested for 18-24 hours on a BAP at 35-37°C with ~5% CO2 (or in a candle-jar).
    2. From overnight growth on the BAP, use a sterile loop to prepare a light to moderate cell suspension (approximately equal to a 0.5 McFarland density standard) in 0.5 ml of 0.85% saline.
      • Optimum quellung reactions can be observed when there are 25-50 cells visible in a microscopic field at 1000X magnification.
    3. Dispense equal amounts of antiserum (5 µl) and methylene blue (5 µl) onto a microscope slide. Add approximately 0.2-1.0 µl of the diluted cell suspension and mix all three with a pipette tip.
    4. Cover the suspension with a 22 mm2 square cover-slip and incubate at room temperature (25°C) for 10-15 minutes.
      • Do not allow the fluid on the slide to dry.
    5. Examine the slide at 1000X using an oil immersion lens.
    6. Begin testing with pooled antisera. Once a positive reaction is obtained, proceed with individual group and serotype-specific antisera included in the pooled antisera that gave the positive reaction to determine the serogroup and serotype.
    Reading the quellung reaction results
    • A positive quellung reaction is observed when the capsule appears as a sharply demarcated halo around the dark blue stained cell (Figure 7).
    • A negative quelling reaction is observed when there is no appearance of a clear, enlarged halo surrounding the stained cell.
    Troubleshooting
    • In some instances, observing a positive reaction can be difficult. Prepare and read all quellung reactions on the same day that the cell suspension is prepared.
    • When reading the reactions, look for free floating single or paired cells.
    • Agglutination (cells clumping together) is NOT a positive quellung reaction.
    • If a quellung reaction is not observed in any of the antisera pools, the strain may be non-typeable, but identification of the strain as S. pneumoniae should be confirmed by optochin susceptibility and bile solubility testing.
    Quality control
    • Each lot of antisera received should be tested for positive quellung reactions using S. pneumoniae reference strains with known capsular serotypes.
    Figure 7 is a picture showing that in a positive quellung reaction, the capsule appears as an enlarged clear halo surrounding the dark blue stained cell.

    Figure 7. In a positive quellung reaction, the capsule appears as an enlarged clear halo surrounding the dark blue stained cell

  8. Determining S. pneumoniae capsular serotypes using PCR-based methodsThe high cost of antisera, subjectivity in interpretation, and technical expertise requirements associated with these serologic methods have resulted in the development of PCR-based serotyping systems. PCR-based serotyping has the potential to overcome some of the difficulties associated with serologic testing and assays for direct detection of serotypes from clinical specimens are a valuable aid in surveillance, particularly in situations where culture is insensitive.Conventional PCR-based methods have been developed to determine the serotypes of S. pneumoniae specimens. Conventional multiplex PCR assays are available for detecting 40 of the 93 S. pneumoniae serotypes. Schemes for testing based on the strains that are historically and/or currently circulating in specific regions are listed in Chapter 10: PCR Methods. Published real-time PCR serotyping assays are also available for many common pneumococcal serotypes.
  9. General methods for genotyping S. pneumoniaeThe continued study of the “seroepidemiology” of pneumococcal disease and carriage isolates is important for understanding selective effects upon regional population structures of this species. Trends in pneumococcal carriage and disease epidemiology are influenced by selective factors in the environment, such as the use of antimicrobial drugs and the introduction of conjugate vaccines. Understanding questions related to long-term effects of such pressures on the pneumococcal population require precise isolate characterization using molecular methods to characterize the strains at the genetic level. Chapter 12: Molecular Methods describes some of the most common typing methodologies used to differentiate S. pneumoniae and includes pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and the use of more variable loci such as the penicillin binding protein (pbp) genes and the pneumococcal surface protein (pspA) gene.

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Last updated on June 17th, 2021

Streptococcus pneumoniae (pneumococci) is a part of the normal nasopharyngeal and oropharyngeal flora. It is an important etiological agent of upper and lower respiratory tract infections (URTI and LRTI), bacteremia, and septicemia. Streptococcus pneumoniae is also associated with otitis media, sinusitis, meningitis, and endocarditis.

Lanceolate diplococci. Image source: microbiologyinpictures.com

Pneumococci are Gram positive lancet shaped diplococci (intracellularly or extracellularly), non-motile, and encapsulated. They occur in pairs with the broader end opposed, hence called Gram positive diplococci. S. pneumoniae is a fastidious bacterium, which grows best at  at 35-37°C with ~5% CO2 (or in a candle-jar).

Contents

Laboratory diagnosis

Laboratory diagnosis of Streptococcus pneumoniae infection is based on finding characteristics shape of the organism in the sample, characteristic colony morphology, biochemical reactions, susceptibility to certain diagnostic discs, and latex agglutination test.

Sample

Specimens used for the laboratory diagnosis of Streptococcus pneumoniae of may be

  • Specimens from respiratory tract: Sputum, lung aspirate, pleural fluid
  • Body fluids e.g., Blood/ cerebrospinal fluid
  • Exudates from the joint, middle ear, other sites

Sample Collection

  1. Sputum:
    Collect > 1.0 ml expectorated sputum in a sterile screw-capped container.
  2. Lung aspirate/ pleural fluid
    Collect > 1.0 ml by percutaneous needle aspiration in a sterile screw-capped tube.
  3. Blood:
    Clean the venipuncture site with 70% alcohol and iodine, allow it to evaporate and collect blood aseptically in a culture broth with an anticoagulant. In case of adults, collect 5-10 ml blood in culture bottle, for children < 12 year old, collect 1.5-2.0 ml blood. Mix the blood and broth by rotating gently to avoid clotting.
  4. Cerebrospinal fluid (CSF):
    Clean the skin over L3-L4 inter-space with 70% alcohol and iodine. Collect > 1.0 ml CSF in a sterile screw-capped tube. Keep the CSF in an incubator at 35-37 degree centigrade, if it is not processed immediately.
  5. Exudates from joints/middle ear
    Collect > 1.0 ml by aspiration in a sterile screw-capped tube or add directly in a culture broth used for blood culture.

Note: DO NOT REFRIGERATE THE SAMPLE 

Sample Transport

Streptococcus pneumoniae is a fastidious bacteria. Care must be taken during transport of specimen. Specimens must be transported promptly to the laboratory preferably within 1-2 hours.

Blood can only be transported after collecting in a culture broth containing appropriate anticoagulant. The inoculated medium can be held at room temperature (20°C– 25°C) for 4 – 6 hours before incubation at 37 °C. The samples during transportation should be protected from extremes of temperature (less than 18°C, more than 30 °C) and direct sunlight.

Culture and  identification during suspected Streptococcus pneumoniae infection

Flow chart for identification and characterization of a S. pneumoniae isolate
  1. Microscopy and Staining 

  • Perform Gram staining of the sample (sputum/CSF)
  • Gram staining shows Gram positive lanceolate shaped diplococci
gram positive cocci streptococcus

2. Culture and Sensitivity 

Colony morphology

  • Colonies on blood agar plate are small (0.5 mm), round, transluscent or mucoid with alpha-hemolysis (A green discolouration of the agar around the colonies). Alpha-hemolytic property differentiates S. pneumoniae  from many species, but not from the commensal alpha-hemolytic (viridans) streptococci.
  • Young alpha-hemolytic colonies appear raised, and in 24 – 48 hours colonies are flattened with depressed centre and is called draughtsman colony. It is due to partial autolysis (these colonies are tentatively identified as Pneumococci).
  • Streptococcus viridans also produces alpha-hemolytic colonies but does not produce draughstman colony.

Alpha-hemolytic colonies are further identified by the following confirmatory tests.

Identification of Streptococcus pneumoniae by biochemical reactions.

Mnemonics: “Streptococcus pneumoniae is a BOSS” i.e. Bile Soluble, Optochin Sensitive

A. Optochin test (6 mm disc with 5µg).

  • Inoculate blood agar plate with suspected alpha-hemolytic isolates.
  • Apply commercially available optochin discs on the streaked blood agar plate
  • Incubate plates at 37°C with 5-10% CO2 for 18-24 hours.

Observe the zone of growth inhibition around the disc and interpret as:

  • A zone size > 14mm indicates susceptibility which is diagnostic of Streptococcus pneumoniae.
  • alpha-haemolytic colonies with zone of inhibition between 9 and 13 mm should be tested for bile solubility.

B. Bile solubility test.

  • alpha-haemolytic colonies showing zone of inhibition around optochin disc between  9 to 13 mm should be tested for bile solubility.
  • Prepare 1.0 ml of saline suspension of the organism from blood agar plate. The turbidity of the suspension should be equivalent to 0.5 McFarland standard.
Bile solubility test: Streptococcus pneumonie colonies are lysed by bile
  • Inoculate 0.5 ml of the suspension into two tubes.
  • Add an equal amount (0.5 ml) of 2% sodium deoxycholate in one tube marked as test and 0.5ml of saline into the second tube marked as control.
  • Shake gently and incubate the tubes at 37°C for 2 hours.

Reading

  • Positive reaction: Clearing of the tube or loss in turbidity in the presence of deoxycholate due to disruption of cells.
  • Negative reaction: Persistence of turbidity.

Note:  This test can also be done directly onto the colony and the colony is lysed by the addition of bile solution.

Interpretation of Optochin and Bile solubility test

  • Zone inhibition of growth around optochin  14mm is definitely Pneumococci.
  • A definite inhibition zone around optochin disc < 14mm and if the isolate is bile soluble, the isolate  is considered as Streptococcus pneumoniae (If not bile soluble, it is not Streptococcus pneumoniae).
  • Strains with < 14 mm zone of inhibition to optochin or no zone at all and the isolate is not bile soluble it is not pneumococci. It is probably Streptococcus viridans.

Antimicrobial susceptibility

  • Perform antimicrobial susceptibility test against a selected group of antimicrobials by disk-diffusion method

Reporting of results: Streptococcus pneumoniae isolated and resistance patterns with tested antibiotics

3. Detection of the antigen

C-carbohydrate antigen of the Streptococcus pneumoniae can be detected in the urine (Read:Pneumococcal Urinary Antigen Testing (UAT): Principle, Procedure and Results ) for the diagnosis of pneumonia and in CSF for the diagnosis of pneumococcal meningitis.

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Streptococcus pneumoniae (page 1)

(This chapter has 4 pages)

© Kenneth Todar, PhD

Introduction

Streptococcus pneumoniae
is a normal inhabitant of the human upper respiratory tract. The bacterium can cause pneumonia, usually of the lobar type, paranasal sinusitis and otitis media, or meningitis, which is usually secondary to one of the former infections. It also causes osteomyelitis, septic arthritis, endocarditis, peritonitis, cellulitis and brain abscesses. Streptococcus pneumoniae is currently the leading cause of invasive bacterial disease in children and the elderly. Streptococcus pneumoniae is known in medical microbiology as the pneumococcus, referring to its morphology and its consistent involvement in pneumococcal pneumonia.

Pneumonia is a disease of the lung that is caused by a variety of bacteria including Streptococcus, Staphylococcus, Pseudomonas, Haemophilus, Chlamydia and Mycoplasma, several viruses, and certain fungi and protozoans. The disease may be divided into two forms, bronchial pneumonia and lobar pneumonia. Bronchial pneumonia is most prevalent in infants, young children and aged adults. It is caused by various bacteria, including Streptococcus pneumoniae. Bronchial pneumonia involves the alveoli contiguous to the larger bronchioles of the bronchial tree. Lobar pneumonia is more prone to occur in younger adults. A majority (more than 80%) of the cases of lobar pneumonia are caused by Streptococcus pneumoniae. Lobar pneumonia involves all of a single lobe of the lungs (although more than one lobe may be involved), wherein the entire area of involvement tends to become a consolidated mass, in contrast to the spongy texture of normal lung tissue.

Bacteriology

Streptococcus pneumoniae cells are Gram-positive, lancet-shaped cocci (elongated cocci with a slightly pointed outer curvature). Usually, they are seen as pairs of cocci (diplococci), but they may also occur singly and in short chains. When cultured on blood agar, they are alpha hemolytic. Individual cells are between 0.5 and 1.25 micrometers in diameter. They do not form spores, and they are nonmotile. Like other streptococci, they lack catalase and ferment glucose to lactic acid. Unlike other streptococci, they do not display an M protein, they hydrolyze inulin, and their cell wall composition is characteristic both in terms of their peptidoglycan and their teichoic acid.


Gram Stain of a film of sputum from a case of lobar pneumonia. CDC.

Cultivation

Streptococcus pneumoniae is a fastidious bacterium, growing best in 5% carbon dioxide. Nearly 20% of fresh clinical isolates require fully anaerobic conditions. In all cases, growth requires a source of catalase (e.g. blood) to neutralize the large amount of hydrogen peroxide produced by the bacteria.  In complex media containing blood, at 37°C,  the bacterium has a doubling time of 20-30 minutes.

On agar, pneumococci grow as  glistening colonies, about 1 mm in diameter. Two serotypes, types 3 and 37, are mucoid. Pneumococci spontaneously undergo a genetically determined, phase variation from opaque to transparent colonies at a rate of 1 in 105 . The transparent colony type is adapted to colonization of the nasopharynx, whereas the opaque variant is suited for survival in blood. The chemical basis for the difference in colony appearance is not known, but significant difference in surface protein expression between the two types has been shown.

Streptococcus pneumoniae is a fermentative aerotolerant anaerobe. It is usually cultured in media that contain blood. On blood agar, colonies characteristically produce a zone of alpha (green) hemolysis, which differentiates S. pneumoniae from the group A (beta hemolytic) streptococcus, but not from commensal alpha hemolytic (viridans) streptococci which are co-inhabitants of the upper respiratory tract. Special tests such as inulin fermentation, bile solubility, and optochin (an antibiotic) sensitivity must be routinely employed to differentiate the pneumococcus from Streptococcus viridans.


Streptococcus pneumoniae Gram-stain of blood broth culture. CDC.

Streptococcus pneumoniae is a very fragile bacterium and contains within itself the enzymatic ability to disrupt and to disintegrate the cells. The enzyme responsible is called an autolysin. The physiological role of this autolysin is to cause the culture to undergo a characteristic autolysis that kills the entire culture when grown to stationary phase. Virtually all clinical isolates of pneumococci harbor this autolysin and undergo lysis usually beginning between 18-24 hours after initiation of growth under optimal conditions. Autolysis is consistent with changes in colony morphology. Colonies initially appear with a plateau-type morphology, then start to collapse in the centers when autolysis begins.

Identification

The minimum criteria for identification and distinction of pneumococci from other streptococci are bile or optochin sensitivity, Gram-positive staining, and hemolytic activity. Pneumococci cause alpha hemolysis on agar containing horse, human, rabbit and sheep erythrocytes. Under anaerobic conditions they switch to beta hemolysis caused by an oxygen-labile hemolysin. Typically, pneumococci form a 16-mm zone of inhibition around a 5 mg optochin disc, and undergo lysis by bile salts (e.g. deoxycholate). Addition of a few drops of 10% deoxycholate at 37°C lyses the entire culture in minutes. The ability of deoxycholate to dissolve the cell wall, depends upon the presence of the autolytic enzyme, LytA. Virtually all clinical isolates of pneumococci harbor the autolysin and undergo deoxycholate lysis.


Streptococcus pneumoniae A mucoid strain on blood agar showing alpha hemolysis (green zone surrounding colonies). Note the zone of inhibition around a filter paper disc impregnated with optochin. Viridans streptococci are not inhibited by optochin.  

Serotyping

The quellung reaction (swelling reaction) forms the basis of serotyping and relies on the swelling of the capsule upon binding of homologous antibody. The test consists of mixing a loopful of colony with equal quantity of specific antiserum and then examining microscopically at 1000X for capsular swelling. Although generally highly specific, cross-reactivity has been observed between capsular types 2 and 5, 3 and 8, 7 and 18, 13 and 30, and with E. coli, Klebsiella, H. influenzae Type b, and certain viridans streptococci.


Streptococcus pneumoniae Quellung (capsular swelling) reaction can be used to demonstrate the presence of a specific capsular type of the bacterium.




chapter continued

Next Page

Sours: http://textbookofbacteriology.net/S.pneumoniae.html

Under microscope pneumoniae streptococcus

Fluorescence Imaging of Streptococcus pneumoniae with the Helix pomatia agglutinin (HPA) As a Potential, Rapid Diagnostic Tool

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Amazing Electron Microscope Images

I did not see the whip and. Never knew where the blow would be, and the Lady either waited until I relaxed, then quickly inflicted a series of blows, forcing me to grit my teeth. The blows alternated, strong and weak, I trembled, my body was as if on fire.

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Everything is just fine. Well, maybe you just want to open your legs a little, otherwise it's a very modest dress, Sergey smiled and threw the light fabric off my wife's knees. Between her tightly clenched thighs with soft, smooth skin, there was a beautiful view of the lower abdomen with a bush.

Of soft dark hair.



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