Bacitracin and SXT Susceptibility Tests- Principle, Procedure and Results Interpretation

 

Introduction

Bacitracin susceptibility test and SXT susceptibility test are two different types of tests used to determine the susceptibility of bacteria to certain antibiotics.

The Bacitracin susceptibility test is used to determine if a bacterium is susceptible to the antibiotic bacitracin. It involves streaking a pure culture of the bacterium on an agar plate containing bacitracin. If the bacterium is susceptible to bacitracin, there will be a clear zone around the streaked area where the antibiotic has inhibited bacterial growth.

The SXT susceptibility test, also known as the co-trimoxazole susceptibility test, is used to determine if a bacterium is susceptible to the antibiotics trimethoprim and sulfamethoxazole (SXT). It involves testing the bacteria's ability to grow in the presence of various concentrations of the antibiotic combination. The minimum inhibitory concentration (MIC) is determined by observing the lowest concentration of the antibiotic that prevents bacterial growth.

Principle

Susceptibility to low concentrations of the polypeptide antibiotic bacitracin and to the combination sulfonamide– 7trimethoprim–sulfamethoxazole (SXT) provides an easy and inexpensive method for the presumptive identification of both group A and group B β-hemolytic streptococci. Although still used in many laboratories, this test has largely been supplanted by reliable and relatively inexpensive serologic procedures, and more recently by mass spectrometry.

Group A streptococci are susceptible to relatively low concentrations of bacitracin and are resistant to SXT. Group B streptococci are resistant to both antibiotics. Other β-hemolytic streptococci show varying susceptibility to bacitracin, but these organisms are usually susceptible to SXT. Therefore, the performance of the SXT test along with the bacitracin test increases the sensitivity and predictive value of the bacitracin test.

Reagents

1. Sheep blood agar plate
2. Taxo “A” bacitracin differential disks (0.04 units/disk)
3. SXT disks (trimethoprim–sulfamethoxazole, 1.25 μg/23.75 μg)

Quality Control

A. Bacitracin S, SXT R: Group A Streptococcus

B. Bacitracin R, SXT R: Group B Streptococcus

C. Bacitracin S or R, SXT S: β-Hemolytic Streptococcus, groups C, F, or G

Test Procedure

1. Pick three to four isolated colonies of the β-hemolytic Streptococcus, and streak the inoculum down the center of half of a blood agar plate.
2. Using a sterile swab or a bacteriologic loop, spread the inoculum as a lawn over the entire half of the plate.
3. Aseptically place a Taxo “A” bacitracin disk and an SXT disk on the inoculated area. Make sure that the disks are spaced evenly. Using flamed forceps, gently tap down the disks so that they adhere to the agar surface.
4. Incubate the plate in ambient air at 35°C.

Results Interpretation

1. Susceptible (S): Any zone around either of the disks

2. Resistant (R): Growth up to the edge of the disk

Bacitracin SXT Identification

Bacitracin	SXT	Identification
Sensitive	Resistant	Presumptive Group A b-hemolytic Streptococci
Resistant	Resistant	Presumptive Group B b-hemolytic Streptococci
Sensitive/Resistant	Sensitive	Not Group A or B b-hemolytic Streptococci

 

3. A reporting recommendation is: “β-hemolytic streptococci, presumptive group A, by bacitracin/ SXT,” or “β-hemolytic streptococci, presumptively not group A by bacitracin/SXT.”

4. Because these tests are generally performed on throat isolates, for which group A streptococci are being sought, the presumptive group B is generally not reported.

Limitations of Test

1. Only β-hemolytic streptococci should be tested, because many α-hemolytic streptococci (including pneumococci) are susceptible to low concentrations of bacitracin.
2. No data are available to indicate that zones of inhibition should be measured. Interpretation of SXT susceptibility may be difficult, because the organisms may grow slightly before total inhibition of growth occurs.
3. The lawn of bacterial inoculum should be confluent. Too light an inoculum will cause non–group A streptococci to appear susceptible to bacitracin.

Reference

1. Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott’s Diagnostic Microbiology, 12th Ed., Mosby Elsevier.
2. Winn et al. Koneman’s Color Atlas and Text book of Diagnistic Microbiology. 6th Ed., Lippincott Williams & Wilkins, Wolters Kluwer.