Bacitracin Susceptibility Test- Principle, Procedure, Results, Uses

Bacitracin susceptibility test is a simple and rapid method to differentiate group A streptococci (GAS) from other beta-hemolytic streptococci (BHS) based on their sensitivity to a low concentration of bacitracin, an antibiotic produced by Bacillus subtilis. It is also used to distinguish micrococci from staphylococci based on their susceptibility to bacitracin.

Bacitracin is a polypeptide antibiotic that inhibits the synthesis of bacterial cell wall by interfering with the transport of peptidoglycan precursors across the cytoplasmic membrane. GAS are highly susceptible to bacitracin and show a clear zone of inhibition around a bacitracin disk, whereas other BHS are resistant and show no zone of inhibition. Micrococci are also susceptible to bacitracin, while staphylococci are resistant.

Bacitracin susceptibility test is performed by placing a filter paper disk impregnated with 0.04 units of bacitracin on a blood agar plate inoculated with the test organism. After incubation, the presence or absence of a zone of inhibition around the disk is observed and measured. A zone of inhibition of 10 mm or more indicates susceptibility, while no zone or a zone of less than 10 mm indicates resistance.

Bacitracin susceptibility test is useful for the presumptive identification of GAS, which are the most common cause of pharyngitis, impetigo, cellulitis, necrotizing fasciitis, and rheumatic fever. It can also help in the identification of micrococci, which are opportunistic pathogens that can cause endocarditis, septicemia, and wound infections.

Bacitracin susceptibility test is not a definitive test and should be confirmed by other methods such as serological grouping or molecular typing. It also has some limitations such as false-positive results due to light inoculum or old media, false-negative results due to different concentrations of bacitracin or different media, and variable results due to some strains of GAS, BHS, micrococci, and staphylococci that show intermediate susceptibility to bacitracin.

The main objectives of Bacitracin susceptibility test are:

• To differentiate Group A beta-hemolytic Streptococci (primarily S. pyogenes) from other beta-hemolytic Streptococci. Group A Streptococci are responsible for various infections such as pharyngitis, scarlet fever, rheumatic fever, and impetigo. They can be distinguished from other Streptococci by their sensitivity to Bacitracin, which inhibits their growth on blood agar plates.
• To observe the pattern of antibiotic susceptibility of different organisms against Bacitracin. Bacitracin is a polypeptide antibiotic that interferes with the peptidoglycan synthesis in bacteria. It is effective against Gram-positive bacteria, especially those that produce beta-lactamases. However, some bacteria may develop resistance to Bacitracin by modifying their cell wall or by producing enzymes that degrade the antibiotic. Therefore, it is important to monitor the susceptibility of bacteria to Bacitracin and other antibiotics for optimal treatment of infections.
• To identify Micrococci and Rothia from coagulase-negative Staphylococci. Micrococci and Rothia are Gram-positive cocci that are catalase-positive and coagulase-negative. They can be confused with Staphylococci, which have similar characteristics. However, Micrococci and Rothia are susceptible to Bacitracin, while Staphylococci are resistant. This can help in the identification and classification of these bacteria.

Bacitracin is a polypeptide antibiotic that inhibits the synthesis of bacterial cell walls by interfering with the transport of peptidoglycan precursors across the cytoplasmic membrane. Bacitracin susceptibility test discs are filter paper discs impregnated with 0.04 units of bacitracin, which is a low concentration that can differentiate group A streptococci (primarily S. pyogenes) from other beta-hemolytic streptococci. Group A streptococci are sensitive to bacitracin and show a zone of inhibition around the disc, whereas other beta-hemolytic streptococci are resistant and grow up to the edge of the disc. The test is based on the diffusion of bacitracin from the disc into the agar medium and the interaction of bacitracin with the bacterial cells in the vicinity of the disc. The test result is evaluated by measuring the diameter of the zone of inhibition around the disc after incubation. A zone of inhibition greater than 10 mm indicates susceptibility, while no zone of inhibition indicates resistance. Bacitracin susceptibility test can also be used to distinguish staphylococci (resistant) from micrococci (susceptible), as well as some other gram-positive cocci. However, bacitracin susceptibility test is not a definitive test and should be confirmed by serological testing or other methods.

Bacitracin susceptibility test is used to test the effect of a small amount of bacitracin (0.04 or 0.05 units) on different microorganisms, especially gram-positive cocci. The test can help to differentiate:

• Group A beta-hemolytic streptococci (primarily Streptococcus pyogenes) from other beta-hemolytic streptococci. Group A streptococci are usually susceptible to bacitracin, while other groups are usually resistant. However, some Lancefield groups C and G streptococci may also show susceptibility to bacitracin, so further confirmation by serological testing or PYR test is recommended.
• Staphylococci species (resistant) from micrococci (susceptible). Bacitracin susceptibility test can be used for penicillin-susceptible or “sticky” colonies of gram-positive cocci in clusters that are catalase-positive and coagulase-negative from invasive-site specimens. Lemon yellow-colored colonies are not tested as those are presumed to be micrococci.
• Haemophilus influenzae (resistant) from other upper respiratory microbiota (susceptible). Bacitracin susceptibility test can be used on chocolate agar plates inoculated with direct sputum samples to improve the detection of H. influenzae.

Media

• Blood agar or
• Mueller Hinton Agar

Supplies

• Bacitracin 0.04-unit discs
• Sterile forceps
• Swabs
• Broth for inoculation

Bacitracin susceptibility test discs are filter paper discs impregnated with 0.04 units of Bacitracin. The impregnated disks are then placed on the agar where they allow the antimicrobial to diffuse through the medium and inhibit the growth of the organism. The concentration of Bacitracin used in this test is different from the sensitivity disks (10 U) that are used for other purposes. The disks should be stored in a desiccant to maintain a moisture level of less than 2%, ensuring long term stability.

Two different procedures are available for the Bacitracin Susceptibility test using two different nutrient media. The testing can be done with both pure cultures of an organism obtained via subculture or directly from the clinical samples. Procedures for two different methods of Bacitracin susceptibility test are given below:

Hebert’s method using Blood Agar plates

1. Prepare a 0.1 McFarland suspension of the organism from the overnight culture of the organism.
2. Inoculate different sections of the blood agar plate with the suspension, resulting in a lawn culture. Each of the sections should be inoculated in one particular direction, and the area of inoculation should allow a separation of 10 mm between each disk to be placed on the plate.
3. After about 10 minutes for the drying, place the disks on the agar with sterile forceps. Tap the disks gently with a sterile stick to ensure adherence.
4. Incubate the plates at 35-37°C for 24 hours in 5% to 10% carbon dioxide (CO2) for streptococci differentiation.
5. After incubation, observe and measure the zone of inhibition around the disks. Further confirmation can be obtained by serological testing.

Mueller Hinton Agar method

1. Prepare a 0.5 McFarland suspension of the organism from the overnight culture of the organism.
2. Inoculate the MHA plates with the suspension with sterile swabs to form bacterial lawns on the agar.
3. After about 10 minutes of drying, place the antibiotic disks on the agar plates with sterile forceps, allowing a distance of about 10 mm between the disks.
4. Tap the disks gently with a sterile stick to ensure adherence of the disk to the plates.
5. Incubate the inverted plates for 24 hours at 35-37°C in ambient air for staphylococci differentiation.
6. Observe and measure the zone of inhibition around the disks. Further confirmation can be obtained by serological testing.

The result of the bacitracin susceptibility test is based on the presence or absence of a zone of inhibition around the disk after incubation. The zone of inhibition is measured in millimeters (mm) and compared with the standard criteria for interpretation. The following table summarizes the interpretation of the test results for different groups of organisms:

A susceptible result indicates that the organism is inhibited by a low concentration of bacitracin and can be presumptively identified as Group A beta-hemolytic Streptococci or Micrococci. A resistant result indicates that the organism is not inhibited by bacitracin and can be presumptively identified as other beta-hemolytic Streptococci or coagulase-negative Staphylococci.

However, the bacitracin susceptibility test is not a definitive test and should be confirmed by other methods such as serological testing, biochemical testing, or molecular testing. Some exceptions and variations may occur in the test results due to factors such as inoculum size, disk potency, medium composition, incubation time, and temperature. Therefore, it is important to follow the standard protocol and quality control measures for performing and interpreting the test.

Bacitracin susceptibility test has several uses in microbiology, such as:

• It is used for the presumptive identification and differentiation of Group A beta-hemolytic Streptococci (primarily S. pyogenes - susceptible) from other beta-hemolytic Streptococci. This is important because Group A Streptococci are responsible for various infections such as pharyngitis, scarlet fever, rheumatic fever, and necrotizing fasciitis.
• It is also used to distinguish staphylococci species (resistant) from micrococci (susceptible) in clusters of penicillin-susceptible or “sticky” Gram-positive cocci that are catalase-positive and coagulase-negative from invasive-site specimens. This is useful because micrococci are usually harmless commensals, while staphylococci can cause serious infections such as endocarditis, osteomyelitis, and septicemia.
• It can also be used to observe the pattern of antibiotic susceptibility of different organisms against Bacitracin. This can help in selecting the appropriate antibiotic therapy for the treatment of infections caused by these organisms.

• Bacitracin susceptibility test should not be performed on old or dried blood agar plates, as the diffusion of the antibiotic may be reduced, leading to false-negative results.
• Bacitracin susceptibility test should not be used as the sole criterion for the identification of group A streptococci, as some strains of Lancefield groups C and G streptococci may also show susceptibility to bacitracin. Serological testing should be performed for further confirmation.
• Bacitracin susceptibility test should not be used with high concentrations of bacitracin (10 U or higher), as different zones of inhibition may be observed with different concentrations of the antibiotic. Differential disks (0.04 U) should be used instead of sensitivity disks (10 U).
• Bacitracin susceptibility test should not be used with light inoculum, as it may result in a false zone of inhibition. A sufficient inoculum resulting in confluent growth should be used.
• Bacitracin susceptibility test may not be reliable for the differentiation of staphylococci from micrococci, as some micrococci may produce zones of inhibition smaller than 10 mm if incubated for less than 24 hours or if Mueller Hinton agar is used. Other tests, such as catalase and coagulase tests, should be performed for further confirmation.

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