SXT Testing: Principle, Procedure, and Results

A throat swab grows beta-hemolytic colonies, and the question is the one that matters most for a sore throat: is this group A strep, the kind that causes strep throat and can lead to rheumatic fever? Full Lancefield grouping takes time and reagents. Two small antibiotic disks, bacitracin and SXT, placed on the plate overnight, give a fast presumptive answer. Reading the two zones together is what separates group A from group B from the rest.

SXT (trimethoprim-sulfamethoxazole) susceptibility testing helps separate group A and group B streptococci from other beta-hemolytic streptococci. Group A and group B streptococci are resistant to SXT, whereas the other beta-hemolytic groups are susceptible. Combined with bacitracin testing, SXT helps tell group A and group B apart from each other as well.

Adding SXT to 5% sheep blood agar suppresses the normal flora of the pharynx. This makes it easier to recover group A streptococci, especially Streptococcus pyogenes, and group B streptococci from throat swabs.

Principle of SXT Test

Sulfamethoxazole and trimethoprim are two antibiotics of the category co-trimoxazole. These antibiotics act synergistically (working together to give greater effect) to disrupt the folic acid metabolism of bacteria. So, they are mostly given together.

An SXT disc usually has 1.25 µg of trimethoprim and 23.75 µg of sulfamethoxazole, which is approximately in the ratio of 1:19. When the disc is placed on the surface of sheep blood agar, a clear zone around the disc is observed, which means the bacteria is susceptible to SXT. If the growth of bacteria is up to the edge of the disc, the bacteria is considered resistant to SXT. SXT and bacitracin susceptibility testing are done together by placing a disc of bacitracin and SXT on the plate at least 4 cm apart.

Figure: SXT-Bacitracin test for Streptococcus pyogenes. Bacitracin is on the left (S) and SXT on the right (R)

Materials Required for SXT Test

1. 5% defibrinated blood agar plate
2. Inoculating loop
3. Bacterial sample
4. SXT disc
5. Bacitracin disc

Procedure of SXT Test

The procedure of SXT susceptibility testing is as follows:

1. Inoculate the bacterial sample in continuous streaking in a 5% defibrinated (removal of fibrin) blood agar plate using a sterile inoculating loop.
2. Then carefully place the SXT antibiotic disc at the center of the inoculated blood agar plate under the aseptic environment.
3. Incubate the plate at 37⁰C overnight in the atmosphere with 5% CO₂.
4. Observe the plate.
5. Read the zone around the SXT disc. A clear zone of inhibition means the organism is susceptible to SXT. Growth right up to the edge of the disc, with no clear zone, means the organism is resistant. Group A and group B streptococci are both resistant to SXT, so they grow up to the disc. Other beta-hemolytic streptococci, such as groups C, F, and G, are susceptible and show a clear zone.

Alternative method using SXT blood agar

Figure: SXT Blood agar with heavy growth of beta hemolytic streptococci

The alternative method to perform SXT susceptibility testing is using the 5% sheep blood agar with SXT. The procedure for the alternative method is as follows:

1. In the aseptic environment, use a sterile inoculation loop to streak bacterial inoculum in the agar plate with SXT blood agar. The agar should have a smooth surface, without visible droplets of surface moisture.
2. Incubate the agar plate at 37⁰C overnight in aerobic conditions with 5% CO₂.
3. Observe the plate for isolated colonies with some form of hemolysis.

Note: Further Gram staining, biochemical tests, and bacitracin sensitivity testing are necessary to confirm isolated bacteria species.

To perform SXT and Bacitracin Test Together

The procedure for performing SXT and bacitracin antibiotic susceptibility testing together is as follows:

1. Using a sterile inoculating loop, perform continuous streaking on the 5% blood agar plate with the bacterial sample.
2. With the help of sterile forceps, place the SXT disc in the inoculated plate, slightly off center.
3. Then place the bacitracin disc 4 cm apart from the SXT disc.
4. Incubate the plate at 37⁰C overnight in the atmosphere with 5% CO₂.
5. Observe the plate after incubation.

Result of SXT Test

Reactions of Beta-hemolytic Streptococci to Bacitracin and SXT

The possible result of SXT and Bacitracin test together

1. Susceptible to both and susceptible to SXT but resistant to bacitracin: Clear zone around the discs sensitive to both antibiotics. The clear zone around SXT but growth around the bacitracin disc suggests susceptibility to SXT and resistance to bacitracin. These results are possible in groups C, F and G streptococci.
2. Resistant to both: Growth around both discs. For example, group B streptococci like Streptococcus agalactiae.
3. Resistant to SXT and susceptible to bacitracin: Growth at the edge of SXT and clear zone around bacitracin. For example, group A streptococci like Streptococcus pyogenes.

How SXT, bacitracin, and CAMP fit together

SXT is rarely read on its own. It is part of a small set of disk and plate tests used to presumptively group beta-hemolytic streptococci before definitive grouping. The three that work together are bacitracin, SXT, and the CAMP test.

Bacitracin presumptively identifies group A. Streptococcus pyogenes (group A) is susceptible to the small amount of bacitracin in a Taxo A disk, while most other beta-hemolytic streptococci are resistant. On its own it is imperfect, because up to about 10% of group A strains can be resistant and a few group C and G strains can be susceptible, which is why it is paired with SXT.

SXT separates the groups by resistance. Group A and group B are both resistant to SXT, while groups C, F, and G are susceptible. So SXT alone cannot tell A from B, but it does separate A and B together from the others.

Reading bacitracin and SXT together is what makes the pair useful:

CAMP test and hippurate confirm group B. Because group A and group B are both SXT-resistant, and bacitracin separates A from B, a bacitracin-resistant and SXT-resistant strain that is presumptively group B is confirmed by a positive CAMP test and hippurate hydrolysis, which are the standard group B markers.

The combined bacitracin and SXT test is about 94% accurate for sorting isolates into group A, group B, or not-A-not-B. It is presumptive only. Definitive identification uses Lancefield grouping, or biochemical, molecular, or mass spectrometry methods.

For the bacitracin test in full, including its procedure and zone interpretation, see the separate article on the bacitracin susceptibility test.

Quality Control

Antibiotic susceptibility testing quality control requires checking the antibiotic disc with quality control strains. According to CLSI, the quality control strain for SXT-1.25/23.75 µg is Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923.

The quality control for SXT blood agar requires examining plates for product deterioration and culturing the quality control strains under CLSI guidelines.

1. Streptococcus pyogenes ATCC 19615 and ATCC 49117, Streptococcus agalactiae ATCC 13013– fair to heavy growth (positive control),
2. Streptococcus mitis ATCC 13813– no growth (negative control), and
3. Neisseria subflava ATCC 14799 and Neisseria sicca ATCC 9913– Partial inhibition (negative control).

References

1. Leboffe, M. J., and Pierce, B. E. (2021). A Photographic Atlas for the Microbiology Laboratory (5th ed.). Morton Publishing.
2. Tille, P. M. (2022). Bailey and Scott's Diagnostic Microbiology (15th ed.). Elsevier.
3. Clinical and Laboratory Standards Institute. (2024). Performance Standards for Antimicrobial Susceptibility Testing (M100, 34th ed.). CLSI.