Optochin Susceptibility Test- Principle, Procedure, Results

Pneumococcus, also known as Streptococcus pneumoniae, is a type of bacteria that can cause various infections in humans, especially in the respiratory tract. Pneumococcus is one of the most common causes of community-acquired pneumonia, which is a lung infection that occurs outside of hospitals or other health care facilities. Pneumococcus can also cause other diseases, such as sinusitis, otitis media, meningitis, sepsis, and more.

Pneumococcus is a Gram-positive, spherical bacterium that usually appears in pairs (diplococci) under the microscope. It has a polysaccharide capsule that protects it from being recognized and destroyed by the immune system. There are more than 100 different types (serotypes) of pneumococcus, and they vary in their ability to cause disease (virulence), their prevalence in different regions of the world, and their resistance to antibiotics.

Pneumococcus can be found in the nose and throat of healthy people, who are called carriers. Carriers do not have any symptoms of infection, but they can spread the bacteria to others through respiratory droplets when they cough or sneeze. Pneumococcus can also be transmitted by touching contaminated surfaces or objects.

Pneumococcus can cause infection when it invades the tissues or blood of a susceptible person. Susceptibility depends on several factors, such as age, immune status, underlying medical conditions, and previous exposure to pneumococcus or its vaccine. Some people are more likely to get sick from pneumococcus than others, such as young children, older adults, people with chronic diseases (such as diabetes, heart disease, lung disease, or HIV), and people who have weakened immune systems (such as those who have had organ transplants, cancer treatment, or spleen removal).

Pneumococcal infections can range from mild to severe and life-threatening. The symptoms and complications depend on the type and site of infection. Some common signs and symptoms of pneumococcal infections are:

• Fever
• Cough
• Chest pain
• Shortness of breath
• Sputum production
• Headache
• Stiff neck
• Confusion
• Ear pain
• Nasal congestion
• Facial pain or swelling

Pneumococcal infections can be diagnosed by taking samples of body fluids (such as blood, sputum, cerebrospinal fluid, or middle ear fluid) and testing them for the presence of pneumococcus or its antigens. Pneumococcus can be identified by its characteristic appearance on Gram stain (lancet-shaped diplococci), its sensitivity to optochin (a chemical that inhibits its growth), and its solubility in bile (a substance that dissolves its capsule). Pneumococcus can also be typed by its serotype using specific antibodies or molecular methods.

Pneumococcal infections can be treated with antibiotics, but some strains of pneumococcus have become resistant to many commonly used drugs. Therefore, it is important to choose the appropriate antibiotic based on the local patterns of resistance and the results of susceptibility testing. In some cases, additional treatments may be needed to manage the complications of pneumococcal infections, such as oxygen therapy, fluid replacement, surgery, or intensive care.

Pneumococcal infections can be prevented by vaccination and by avoiding exposure to the bacteria. There are two types of vaccines available for pneumococcus: a polysaccharide vaccine (PPSV23) that covers 23 serotypes and a conjugate vaccine (PCV13) that covers 13 serotypes. The vaccines are recommended for different groups of people based on their age and risk factors. The vaccines can reduce the incidence and severity of pneumococcal infections, but they cannot prevent all cases or eliminate carriage of the bacteria. Therefore, it is still important to practice good hygiene measures, such as washing hands frequently, covering coughs and sneezes, and staying away from sick people.

Pneumococcus is a major public health problem worldwide, causing millions of cases and deaths every year. It is estimated that pneumococcus is responsible for about 15% of all pneumonia cases, 50% of all bacterial meningitis cases, and 40% of all sepsis cases globally. Pneumococcus also contributes to the burden of antimicrobial resistance, as it is one of the most common bacteria that develop resistance to multiple drugs. Therefore, it is essential to monitor the epidemiology, microbiology, and pathogenesis of pneumococcus and to implement effective strategies for its prevention and control.

The optochin susceptibility test is a simple and reliable method to differentiate Streptococcus pneumoniae from other α-hemolytic (viridans) Streptococci. These two groups of bacteria are Gram-positive cocci that grow on blood agar and produce a green-colored zone of incomplete hemolysis. However, they have different clinical significance and treatment options.

Streptococcus pneumoniae is a major cause of pneumonia, meningitis, otitis media, sinusitis, and septicemia in humans. It is also known as the pneumococcus or the pneumococcal bacterium. It is susceptible to a variety of antibiotics, but some strains have developed resistance to penicillin and other drugs.

Viridans Streptococci are a heterogeneous group of bacteria that are part of the normal flora of the oral cavity, upper respiratory tract, gastrointestinal tract, and genitourinary tract. They are usually harmless commensals, but they can cause opportunistic infections such as endocarditis, dental caries, abscesses, and bacteremia in immunocompromised or debilitated patients. They are generally resistant to optochin and penicillin.

Optochin is a chemical compound that inhibits the growth of Streptococcus pneumoniae by interfering with its cell membrane function. It is also known as ethylhydrocupreine hydrochloride or p-hydroxyquinoline ethyl ester. Optochin-impregnated disks are used to test the sensitivity of bacterial isolates to this compound.

The objective of the optochin susceptibility test is to identify Streptococcus pneumoniae among other α-hemolytic Streptococci by observing their reaction to optochin. The test is based on the principle that Streptococcus pneumoniae is the only known optochin-sensitive viridans Streptococcus. Hence, it shows a clear zone of inhibition around the optochin-impregnated disk; whereas other viridans Streptococci do not display a zone of inhibition around the optochin-impregnated disk.

The optochin susceptibility test is a quick and inexpensive way to confirm the presence or absence of Streptococcus pneumoniae in clinical specimens or pure cultures. It can also be used as a screening test for pneumococcal identification before performing other confirmatory tests such as bile solubility test, quellung reaction, or molecular methods. The test can help in choosing appropriate antibiotics for treating pneumococcal infections and preventing their spread.

Optochin is a chemical compound that inhibits the growth of some bacteria by interfering with their cell membrane function and ATP synthesis. Optochin is also known as ethylhydrocupreine hydrochloride or p-hydroxyquinoline ethyl ester.

The optochin susceptibility test is based on the differential sensitivity of Streptococcus pneumoniae and other α-hemolytic streptococci to optochin. Streptococcus pneumoniae is the only known optochin-sensitive viridans streptococcus. Hence, it shows a clear zone of inhibition around the optochin-impregnated disk on a blood agar plate; whereas other α-hemolytic streptococci do not display a zone of inhibition around the optochin-impregnated disk.

The optochin susceptibility test can be used to differentiate S. pneumoniae from other α-hemolytic streptococci based on this feature. However, some strains of S. pneumoniae may show intermediate or resistant reactions to optochin, so a confirmatory test such as bile solubility test may be required in such cases.

The optochin susceptibility test is simple, rapid and inexpensive. It can be performed on any α-hemolytic colony isolated from a clinical specimen or a pure culture. The test requires a 5% sheep blood agar plate, an optochin-impregnated disk and an incubator with 5 to 10% CO2 environment. The results can be obtained within 18 to 24 hours of incubation.

The optochin susceptibility test is useful for the identification of S. pneumoniae, which is an important human pathogen that causes pneumonia, meningitis, otitis media, sinusitis and septicemia. The accurate identification of S. pneumoniae is essential for the appropriate treatment and prevention of these infections.

To perform the optochin susceptibility test, you will need the following materials:

• Test organism: You will need a pure culture of alpha-hemolytic streptococci, which are Gram-positive cocci that produce a green zone of incomplete hemolysis on blood agar. You can obtain the test organism from a clinical specimen, such as sputum, blood, or cerebrospinal fluid, or from a reference strain, such as Streptococcus pneumoniae ATCC 49619 (positive control) or Streptococcus mitis ATCC 49456 (negative control).
• Culture media: You will need a 5% sheep blood agar plate as the culture medium for the test. This medium is prepared by adding 5% (v/v) of sterile defibrinated sheep blood to a molten blood agar base medium. The blood agar base contains beef heart peptone, peptone, sodium chloride, and agar. The final pH of the medium should be 7.3 ± 0.2 at 25°C.
• Optochin disks: You will need optochin-impregnated disks to test the sensitivity of the test organism to optochin. Each disk should have a diameter of 6 mm and contain 5 µg of optochin (ethylhydrocupreine hydrochloride). Optochin is a chemical that inhibits the growth of S. pneumoniae by interfering with its ATP synthesis.
• Reagents: You will need defibrinated sheep blood for preparing the blood agar medium. You will also need a McFarland standard number 0.5 suspension for adjusting the turbidity of the bacterial suspension. This suspension is prepared by mixing 9.95 mL of 1% sulfuric acid and 0.05 mL of 1% barium chloride in a clear test tube.
• Equipment: You will need an incubator that can provide an anaerobic environment with 5 to 10% CO2 at 35 ± 2°C. You will also need sterile forceps, inoculating loops or cotton swabs, test tubes, and a ruler or caliper for measuring the zone of inhibition.

These are the basic requirements for performing the optochin susceptibility test. In the next section, we will discuss the preparation of the blood agar plate and the bacterial suspension.

The culture media used for the optochin susceptibility test is a 5% Sheep Blood Agar Plate (BAP). This media is prepared by adding 5% v/v defibrinated sheep blood in a molten blood agar base medium. The blood agar base medium consists of the following components:

• Beef Heart Peptone or Meat Extracts (10.0 grams): These are sources of nitrogen, carbon, vitamins and minerals for the growth of bacteria.
• Peptone or Tryptose (10.0 grams): These are also sources of nitrogen, carbon, vitamins and minerals for the bacterial growth.
• Sodium Chloride (5.0 grams): This maintains the osmotic balance of the medium.
• Agar (15.0 grams): This is a solidifying agent that provides a firm surface for bacterial growth and diffusion of optochin.

The final pH of the blood agar base medium should be 7.3 ±0.2 at 25°C.

The sheep blood provides a source of hemoglobin, which is essential for detecting the hemolytic activity of bacteria. The hemoglobin also enhances the growth of some fastidious bacteria, such as S. pneumoniae.

The optochin susceptibility test requires a 5% sheep blood agar plate because S. pneumoniae is a fastidious organism that needs enriched media for optimal growth and hemolysis. Other types of blood, such as human or horse blood, may not produce the same results as sheep blood. Therefore, it is important to use 5% sheep blood agar plate for this test.

Blood agar plate (BAP) is a culture medium that contains 5% sheep blood in a blood agar base. It is used to grow and differentiate bacteria based on their hemolytic properties. Hemolysis is the breakdown of red blood cells by bacterial enzymes. There are three types of hemolysis: alpha (α), beta (β), and gamma (γ). Alpha-hemolysis produces a greenish zone around the colonies, beta-hemolysis produces a clear zone around the colonies, and gamma-hemolysis produces no change in the agar.

To prepare BAP, you will need the following ingredients and equipment:

• Blood agar base powder or components (beef heart peptone, peptone, sodium chloride, and agar)
• Distilled water
• Defibrinated sheep blood
• Conical flask or glass bottle
• Magnetic stirrer or manual stirrer
• Autoclave
• Petri plates
• Measuring cylinder
• Pipette

The steps for preparing BAP are as follows:

1. Measure the appropriate amount of blood agar base powder (or the media components) and mix in the water of the required volume in a conical flask (or glass bottle) according to the instruction of the manufacturing company. For example, if you want to prepare 500 mL of BAP, you will need 19.5 g of blood agar base powder and 500 mL of distilled water.
2. Stir well using a magnetic stirrer or manually and heat to boiling so that all the components and agar dissolve completely in water.
3. Autoclave the flask or bottle at 121°C and 15 lbs pressure for 15 minutes and let it cool to around 40 – 45°C.
4. Pour 5% (25 mL) v/v sterile defibrinated sheep blood into the flask with blood agar base slowly with constant stirring. Mix properly so that blood dissolves uniformly in the medium. This mixture is the blood agar.
5. In a sterile Petri plate (glass plate with 10 cm diameter), pour around 25 mL of the blood agar and let it solidify properly by leaving it at room temperature. (Store BAPs in a freeze at 4°C for use up to 2 to 4 weeks maximum)

The main reagent used for the optochin susceptibility test is the optochin-impregnated disk. This is a 6 mm disk of sterile filter paper that contains 5 μg of optochin, a chemical compound that inhibits the growth of pneumococci. Optochin is also known as ethylhydrocupreine hydrochloride, a derivative of quinine that was introduced in 1911 as a potential treatment for pneumococcal infections .

Another reagent that is required for the test is defibrinated sheep blood, which is used to prepare the blood agar plate (BAP) as the culture medium. Defibrinated sheep blood is obtained by removing the fibrin from the blood of healthy sheep by mechanical agitation or heating. The blood should be sterile and free from hemolysis.

A third reagent that is needed for the test is the McFarland standard number 0.5 suspension, which is used to adjust the turbidity of the bacterial suspension to a standard level. The McFarland standard number 0.5 suspension is prepared by mixing 0.05 mL of 1% anhydrous barium chloride (BaCl₂) solution with 9.95 mL of 1% sulfuric acid (H₂SO₄) solution in a clean and clear test tube. The resulting suspension has a turbidity equivalent to a bacterial concentration of approximately 1.5 x 10⁸ CFU/mL.

A McFarland standard suspension is a turbidity standard that is used to adjust the density of a bacterial suspension to a desired level. It is commonly used in microbiology to estimate the number of viable bacteria in a sample. The McFarland standard suspension is prepared by mixing a specific amount of barium chloride and sulfuric acid solutions, which form a white precipitate of barium sulfate. The turbidity of the suspension is compared to that of the bacterial suspension using a spectrophotometer or a nephelometer. Alternatively, the suspensions can be visually compared by holding them against a light source and a white background with black lines.

The McFarland standard number 0.5 corresponds to approximately 1.5 x 10^8^ colony forming units (CFU) per mL, which is suitable for the optochin susceptibility test. To prepare the McFarland standard number 0.5 suspension, follow these steps:

• In a clean and clear test tube, add 9.95 mL of 1% H2SO4 solution using a pipette or a graduated cylinder.
• In another clean and clear test tube, add 0.05 mL of 1% BaCl2 solution using a pipette or a graduated cylinder.
• Mix the two solutions by pouring one into the other and shaking gently. A white precipitate of barium sulfate should form immediately.
• Label the test tube as McFarland standard number 0.5 and store it in a dark place at room temperature. The suspension is stable for up to six months if not contaminated.
• Before using the suspension, shake it well and check for any signs of deterioration, such as discoloration, settling, or clumping. If any of these are observed, discard the suspension and prepare a fresh one.

To perform the optochin susceptibility test, you will need the following equipment and test organisms:

• A sterile test tube
• A sterile inoculating loop or cotton swab
• Sterile forceps
• A ruler or caliper
• A 5% sheep blood agar plate
• An optochin-impregnated disk (6 mm disk with 5 μg of optochin)
• A bacterial suspension of well isolated α-hemolytic colonies of the sample organism
• A bacterial suspension of Streptococcus pneumoniae ATCC 49619 as a positive control
• A bacterial suspension of Streptococcus mitis ATCC 49456 or Enterococcus faecalis ATCC 29212 or Streptococcus pyogenes ATCC 12384 as a negative control

The sample organism can be any Gram-positive cocci in pairs or chains that produce α-hemolysis on blood agar. The positive control is a known strain of S. pneumoniae that is sensitive to optochin. The negative control is a known strain of viridans streptococci or other Gram-positive cocci that are resistant to optochin.

The equipment and test organisms should be properly labeled and stored at appropriate conditions before use. The test tube, inoculating loop, cotton swab and forceps should be sterilized by autoclaving or flaming. The blood agar plate should be stored at 4°C and warmed to room temperature before use. The optochin disk should be stored in a sealed container at 2-8°C and protected from light. The bacterial suspensions should be prepared from fresh cultures and adjusted to 0.5 McFarland standard turbidity.

• In a sterile test tube, prepare a bacterial suspension of well isolated α-hemolytic colonies of the sample organism. Adjust the turbidity of the suspension to 0.5 McFarland standards. This can be done by comparing the suspension with a standard barium sulfate solution under a light source. Alternatively, a spectrophotometer can be used to measure the optical density of the suspension at 600 nm wavelength and adjust it to 0.08 to 0.1.
• Using a sterile inoculating loop or cotton swab, streak (or spread) the bacterial suspension in two directions (at least) over the 5% Sheep Blood Agar Plate. Make sure to cover the entire surface of the plate with a thin and even layer of bacteria.
• Let the suspension adhere to the plate and dry by leaving the plate in an upright position for about 5 to 10 minutes.
• Using sterile forceps, place an optochin antibiotic disk in the area of inoculation and gently press the antibiotic disk to ensure its adherence. The disk should be placed about 25 mm away from the edge of the plate. If using multiple antibiotic disks, place them at least 25 mm apart from each other.
• Incubate anaerobically (in 5 to 10% CO2 environment) at 35±2°C for overnight (18 to 24 hours).
• Following the incubation period, observe the formation of a zone of inhibition around the optochin antibiotic disk and measure the zone diameter using a ruler or a caliper. Record the results in millimeters.

The procedure is simple and straightforward, but it requires careful handling of the bacterial suspension and the antibiotic disk to avoid contamination and ensure accurate results. The following image shows an example of optochin susceptibility test on blood agar plates.

After incubating the blood agar plate with the optochin disk, observe the formation of a zone of inhibition around the disk and measure the diameter of the zone. The results can be interpreted as follows:

• Optochin sensitive: If the zone of inhibition is 14 mm or greater around a 6-mm disk, then the bacteria are likely to be Streptococcus pneumoniae . This is because S. pneumoniae is the only known optochin-sensitive viridans Streptococcus.
• Optochin resistant: If there is no zone of inhibition around the disk, then the bacteria are likely to be viridans Streptococci . This is because viridans Streptococci are not inhibited by optochin at low concentrations.
• Optochin intermediate: If the zone of inhibition is less than 14 mm around a 6-mm disk, then the bacteria are uncertain and need further confirmation . This is because some pneumococcal strains are optochin-resistant or intermediate, and some viridans Streptococci may be inhibited by optochin at higher concentrations.

The optochin susceptibility test is just indicative and not definitive. Additional biochemical tests, such as bile solubility test or serology, are suggested for confirming the identity of the bacteria .

The only known optochin susceptible bacteria among the α-hemolytic Streptococci is Streptococcus pneumoniae. This bacterium causes pneumonia, meningitis, otitis media, sinusitis, and other infections in humans. It is also known as pneumococcus or pneumococcal bacterium.

S. pneumoniae is sensitive to optochin because it has a thin cell wall and a large capsule that makes it vulnerable to the detergent-like action of optochin. Optochin disrupts the membrane integrity and inhibits the ATP synthase activity of S. pneumoniae, leading to cell death.

S. pneumoniae can be easily identified by its optochin susceptibility test result. If the bacterium produces a clear zone of inhibition of 14 mm or more around the optochin disk on a blood agar plate, it is considered optochin sensitive and presumptively identified as S. pneumoniae. However, some strains of S. pneumoniae may show intermediate susceptibility to optochin, which requires further confirmation by bile solubility test.

Optochin susceptibility test is a simple, rapid, and reliable method for differentiating S. pneumoniae from other α-hemolytic Streptococci. It is especially useful for clinical laboratories that do not have access to more sophisticated methods such as serological typing or molecular techniques. Optochin susceptibility test can also be used for quality control of pneumococcal vaccines and epidemiological studies of pneumococcal infections.

Optochin resistance is a rare but important feature that can affect the identification of Streptococcus pneumoniae, which is usually optochin sensitive. Optochin resistance can be caused by mutations in the genes encoding the F0F1 ATPase, a membrane-bound enzyme that pumps protons across the bacterial cell membrane and generates ATP. Optochin inhibits the activity of this enzyme and disrupts the proton gradient, leading to cell death. However, some pneumococcal strains have acquired nucleotide substitutions in the atpA or atpC genes, which code for the a-subunit or c-subunit of the F0F1 ATPase, respectively. These substitutions alter the amino acid sequence and reduce the binding affinity of optochin to the enzyme .

Optochin resistant pneumococci are usually isolated from patients with invasive diseases, such as meningitis, bacteremia, or pneumonia . They may also be associated with antibiotic resistance, especially to penicillin and macrolides . Optochin resistance does not seem to confer any selective advantage to pneumococci in terms of virulence or fitness.

The most common optochin resistant bacteria are other alpha-hemolytic streptococci, also known as viridans streptococci. These bacteria are part of the normal flora of the oral cavity and upper respiratory tract and can cause opportunistic infections in immunocompromised or debilitated patients. They are usually differentiated from S. pneumoniae by their lack of capsule, bile solubility, and autolysis. Some examples of optochin resistant viridans streptococci are:

• S. mitis group: This group includes S. mitis, S. oralis, S. sanguinis, S. gordonii, S. cristatus, and S. parasanguinis. They are associated with endocarditis, dental caries, and bacteremia.
• S. anginosus group: This group includes S. anginosus, S. intermedius, and S. constellatus. They are associated with abscesses, meningitis, and endocarditis.
• S. mutans group: This group includes S. mutans, S. sobrinus, S. cricetus, and S. ferus. They are associated with dental caries and endocarditis.
• S. salivarius group: This group includes S. salivarius, S. vestibularis, and S. thermophilus. They are associated with oral infections and endocarditis.

Other optochin resistant bacteria that may be confused with pneumococci are:

• Enterococcus faecalis: This bacterium is a gram-positive coccus that grows in pairs or chains and is part of the normal flora of the gastrointestinal tract. It is associated with urinary tract infections, endocarditis, wound infections, and bacteremia. It can be differentiated from pneumococci by its positive catalase test and growth in 6.5% NaCl.
• Streptococcus pyogenes: This bacterium is a gram-positive coccus that grows in chains and is part of the normal flora of the skin and upper respiratory tract. It is associated with pharyngitis, scarlet fever, rheumatic fever, impetigo, cellulitis, necrotizing fasciitis, and toxic shock syndrome. It can be differentiated from pneumococci by its beta-hemolysis on blood agar and positive bacitracin test.

Quality control measures are essential to ensure the accuracy and reliability of the optochin susceptibility test. The following steps should be taken to perform quality control:

• Use a pure culture of the test organism and avoid contamination.
• Use fresh and properly stored optochin disks and blood agar plates.
• Use appropriate positive and negative control strains along with the test organism. For example, Streptococcus pneumoniae ATCC 49619 as a positive control and Streptococcus mitis ATCC 49456 or Enterococcus faecalis ATCC 29212 as a negative control.
• Adjust the turbidity of the bacterial suspension to 0.5 McFarland standards using a spectrophotometer or a nephelometer.
• Inoculate the blood agar plate evenly and place the optochin disk gently on the surface of the agar.
• Incubate the plate anaerobically (in 5 to 10% CO2 environment) at 35±2°C for overnight (18 to 24 hours).
• Measure the zone of inhibition around the optochin disk using a ruler or a caliper and compare it with the standard criteria.
• Record and interpret the results correctly and report any discrepancies or errors.

The expected results for the quality control strains are:

• Streptococcus pneumoniae ATCC 49619 produces a well-defined zone of inhibition of ≥14 mm around the optochin disk. This indicates that it is optochin sensitive and presumptively identified as S. pneumoniae.
• Streptococcus mitis ATCC 49456 or Enterococcus faecalis ATCC 29212 produce no zone of inhibition around the optochin disk. This indicates that they are optochin resistant and not S. pneumoniae.

If the quality control results do not match the expected results, the test should be repeated with fresh reagents and media. If the problem persists, the source of error should be identified and corrected. Some possible sources of error are:

• Improper storage or expiration of optochin disks or blood agar plates
• Inaccurate preparation or adjustment of bacterial suspension
• Inadequate inoculation or incubation of blood agar plate
• Incorrect measurement or interpretation of zone of inhibition

• Do not pour blood in molten blood agar base if it is above 45°C, as it may damage the blood cells and affect the hemolysis pattern.
• Pour the media into petri plates in a sterile zone before the temperature of the molten media drops below 40°C; otherwise, clumps may form.
• For bacterial identification, always use 5% sheep blood agar, as other types of blood agar may give different results.
• Place the optochin disk about 25 mm away from the edge of the plate. If using multiple antibiotic disks, place them at least 25 mm apart from each other to avoid overlapping zones of inhibition.
• Incubate the plates anaerobically (in 5 to 10% CO2 environment) at 35±2°C for overnight (18 to 24 hours), as some S. pneumoniae isolates will grow poorly or not at all aerobically.
• Use a calibrated ruler or a zone reader to measure the zone diameter accurately and record it in millimeters.
• Do not interpret the results based on optochin susceptibility alone, as some S. pneumoniae strains may be optochin-resistant and some viridans streptococci may be optochin-sensitive. Confirm the identification by performing other tests such as bile solubility test, Gram stain, catalase test, and serological typing .

The optochin susceptibility test is mainly used for the identification of Streptococcus pneumoniae from other viridans streptococci. S. pneumoniae is a common cause of pneumonia, meningitis, otitis media, sinusitis, and septicemia in humans. It is also a part of the normal flora of the upper respiratory tract in some individuals. Therefore, it is important to differentiate it from other alpha-hemolytic streptococci that are usually harmless or less pathogenic.

The optochin susceptibility test is a simple, rapid, and inexpensive method that can be performed in any microbiology laboratory. It does not require any special equipment or reagents, except for the optochin-impregnated disks and the 5% sheep blood agar plates. The test can provide a presumptive identification of S. pneumoniae within 18 to 24 hours of incubation.

The optochin susceptibility test can also be used as a screening tool for detecting optochin-resistant strains of S. pneumoniae, which have been reported occasionally. Optochin resistance may be due to mutations in the genes encoding the F0F1-ATPase or the cell wall biosynthesis enzymes. Optochin-resistant strains of S. pneumoniae may have reduced susceptibility to other antibiotics, such as penicillin and erythromycin. Therefore, it is advisable to perform further tests, such as bile solubility, DNA probe, or capsular typing, to confirm the identity and susceptibility of optochin-resistant isolates.

The optochin susceptibility test can also be used for epidemiological studies of S. pneumoniae infections. For example, it can help to monitor the prevalence and distribution of optochin-resistant strains in different regions or populations. It can also help to evaluate the impact of vaccination programs on the incidence and severity of pneumococcal diseases.

The optochin susceptibility test is a simple and reliable method to differentiate S. pneumoniae from other viridans Streptococci based on their sensitivity to the optochin antibiotic. However, the test has some limitations that should be considered before interpreting the results. Some of the limitations are:

• The test requires a 5% sheep blood agar medium and a CO2 incubator for culture and incubation. These materials may not be readily available in some laboratories or settings.
• The test may not be conclusive for some strains of S. pneumoniae that show intermediate or resistant reactions to optochin. In such cases, a bile solubility test or other confirmatory tests may be needed to verify the identity of the organism.
• The test may not be able to detect some rare or novel variants of S. pneumoniae that have acquired optochin resistance through genetic mutations or horizontal gene transfer. These strains may pose a challenge for diagnosis and treatment of pneumococcal infections.
• The test is a culture-based method that requires at least 24 hours for results and interpretation. This may delay the initiation of appropriate antimicrobial therapy for patients with suspected pneumococcal infections. Rapid molecular or immunological methods may offer faster and more accurate identification of S. pneumoniae in clinical specimens.

Therefore, the optochin susceptibility test should be used as a screening tool rather than a definitive test for S. pneumoniae identification. The results should be interpreted in conjunction with other phenotypic and genotypic characteristics of the organism, as well as the clinical and epidemiological context of the infection.

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