Antibiotic Susceptibility Testing

Objective

The test determines the susceptibility of a microbial species against different antibiotic agents.

Principle

The introduction of various antimicrobials for treating variety of infections showed the necessity of performing antimicrobial susceptibility testing as a routine procedure in all microbiology laboratories. In laboratories it can be made available by using antibiotic disk which will diffuse slowly into the medium where the suspected organism is grown. The basic principle of the antibiotic susceptibility testing has been used in microbiology laboratories over 80 years. Various chemical agents such as antiseptics, disinfectants, and antibiotics are employed to combat with the microbial growth. Among these, antibiotics are generally defined as the substances produced by the microorganism such as Penicillium, which has the ability to kill or inhibit the growth of other microorganisms, mainly bacteria. Antimicrobial susceptibility tests (ASTs) basically measures the ability of an antibiotic or other antimicrobial agent to inhibit the invitro microbial growth.

There are many different procedures that microbiologists use to study the effects of various antimicrobial agents in treating an infection caused by different microorganisms.

Mueller Hinton Agar is considered as best for the routine susceptibility testing since it is has batch-to-batch reproducibility, low concentration of inhibitors of sulphonamide, trimethoprim and tetracyclines and produce satisfactory results for most of the non-fastidious pathogens. Fastidious organisms which require specific growth supplements need different media to grow for studying the susceptibility patterns.

The Kirby Bauer test is a qualitative assay whereby disks of filter paper are impregnated with a single concentration of different antibiotics or any chemicals that will diffuse from the disk into the agar. The selected antibiotic disks are placed on the surface of an agar plate which has already been inoculated with test bacteria. During the incubation period, the antibiotics/chemicals diffuse outward from the disks into the agar. This will create a concentration gradient in the agar which depends on the solubility of the chemical and its molecular size. The absence of growth of the organism around the antibiotic disks indicates that, the respected organism is susceptible to that antibiotic and the presence of growth around the antibiotic disk indicates the organism is resistant to that particular antibiotic. This area of no growth around the disk is known as a zone of inhibition, which is uniformly circular with a confluent lawn of growth in the media.

The diameters of the zone of inhibition are measured (including disk) using a metric scale or a sliding caliper. The measured zone diameter can be compared with a standard chart for obtaining the susceptible and resistant values. There are zone of intermediate resistance which means that the antibiotic may not be sufficient enough to eradicate the organism from the body.

Examples of Antibiotic Sensitivity Testing Methods

1. DILUTION METHODS

The Broth dilution method involves subjecting the isolate to a series of concentrations of antimicrobial agents in a broth environment.   Microdilution testing uses about 0.05 to 0.1 ml total broth volume and can be conveniently performed in a microtiter format.  Macrodilution testing uses broth volumes at about 1.0 ml in standard test tubes.  For both of these broth dilution methods, the lowest concentration at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the minimal inhibitory concentration or MIC.  The MIC is thus the minumum concentration of the antibiotic that will inhibit this particular isolate.  The test is only valid if the positive control shows growth and the negative control shows no growth.

A procedure similar to broth dilution is agar dilution.  Agar dilution method follows the principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is still inhibited. 

On this agar plate, a bacterial isolate is tested for resistance to each of twelve different antibiotics.  The clear zones around each disc are the zones of inhibition that indicate the extent of the test organism’s inability to survive in the presence of the test antibiotic. (A)The disk shows a large zone of inhibition; whereas (B) shows no zone of inhibition, indicating resistance of the isolate to the test antibiotic 

Presence of zone of inhibition is not automatically interpreted as susceptibility to the antibiotic; the zone width has to be measured and compared against a reference standard which contains measurement ranges and their equivalent qualitative categories of susceptible, intermediately susceptible or resistant.

For example, this E.coli isolate on the right has a zone of inhibition of 10.1mm around ampicillin (AM); since the zone diameter interpretation chart is as follows:
Resistant: 13mm or less
Intermediate: 14-16 mm
Susceptible: 17 mm or more
This particular E.coli isolate is read as resistant to ampicillin.

2. DISK DIFFUSION METHOD

Because of convenience, efficiency and cost, the disk diffusion method is probably the most widely used method for determining antimicrobial resistance in private veterinary clinics. 

A growth medium, usually Mueller-Hinton agar, is first evenly seeded throughout the plate with the isolate of interest that has been diluted at a standard concentration (approximately 1 to 2 x 10⁸ colony forming units per ml).  Commercially prepared disks, each of which are pre-impregnated with a standard concentration of a particular antibiotic, are then evenly dispensed and lightly pressed onto the agar surface.  The test antibiotic immediately begins to diffuse outward from the disks, creating a gradient of antibiotic concentration in the agar such that the highest concentration is found close to the disk with decreasing concentrations further away from the disk. After an overnight incubation, the bacterial growth around each disc is observed.  If the test isolate is susceptible to a particular antibiotic, a clear area of “no growth” will be observed around that particular disk.

The zone around an antibiotic disk that has no growth is referred to as the zone of inhibition since this approximates the minimum antibiotic concentration sufficient to prevent growth of the test isolate.  This zone is then measured in mm and compared to a standard interpretation chart used to categorize the isolate as susceptible, intermediately susceptible or resistant.  MIC measurement cannot be determined from this qualitative test, which simply classifies the isolate as susceptible, intermediate or resistant.

3. E-TEST

E-test (AB Biodisk, Solna, Sweden) is a commercially available test that utilizes a plastic test strip impregnated with a gradually decreasing concentration of a particular antibiotic.  The strip also displays a numerical scale that corresponds to the antibiotic concentration contained therein. This method provides for a convenient quantitative test of antibiotic resistance of a clinical isolate.  However, a separate strip is needed for each antibiotic, and therefore the cost of this method can be high.

4. AUTOMATED ANTIMICROBIAL SUSCEPTIBILITY TESTING SYSTEMS

Several commercial systems have been developed that provide conveniently prepared and formatted microdilution panels as well as instrumentation and automated reading of plates.  These methods are intended to reduce technical errors and lengthy preparation times.

Most automated antimicrobial susceptibility testing systems provide automated inoculation, reading and interpretation.  These systems have the advantage of being rapid (some results can be generated within hours) and convenient, but one major limitation for most laboratories is the cost entailed in initial purchase, operation and maintenance of the machinery.  Some examples of these include: Vitek System (bioMerieux, France), Walk-Away System (Dade International, Sacramento, Calif.), Sensititre ARIS (Trek Diagnostic Systems, East Grinstead, UK), Avantage Test System (Abbott Laboratories,  Irving, Texas), Micronaut (Merlin, Bornheim-Hesel, Germany), Phoenix  (BD Biosciences, Maryland) and many more. 

5. MECHANISM-SPECIFIC TESTS

Resistance may also be established through tests that directly detect the presence of a particular resistance mechanism.  For example, beta lactamase detection can be accomplished using an assay such as the chromogenic cephalosporinase test (Cefinase disk by BD Microbiology Systems, Cockeysville, MD and BBL DrySlide Nitrocefin, Becton Dickinson, Sparks, MD) and detection for chloramphenicol modifying enzyme chloramphenicol acetyltransferase (CAT) may utilize commercial colorimetric assays such as a CAT reagent kit (Remel, Lenexa, Kansas).

Materials Required

1. Petriplate containing microbial culture(For example, Escherichia coli)
2.  Inoculation loop
3. Bunsen burner
4. Saline solution
5. McFarland solution
6. MHA plate
7. Cotton swab
8. Antibiotic disks(Streptomycin (S), Ciprofloxacin (CIP), Chloramphenicol (C), Doxycycline (D), Penicillin G (P), Gentamycin (G)
9. Tooth pick
10. Incubator
11. Ruler
     

Procedure

1. Select a pure culture plate of one of the organisms to be tested.
2. Aseptically emulsify a colony from the plate in the sterile saline solution. Mix it thoroughly to ensure that no solid material from the colony is visible in the saline solution.
3. Repeat until the turbidity of the saline solution visually match that of the standard turbidity.
4. Take a sterile swab and dip it into the broth culture of organism.
5. Gently squeeze the swab against the inside of the tube in order to remove excess fluid in the swab.
6. Take a sterile Mueller-Hinton agar (MHA) plate or a nutrient agar (NA) plate.
7. Use the swab with the test organism to streak a MHA plate or a NA plate for a lawn of growth.
8. After the streaking is complete, allow the plate to dry for 5 minutes.
9. Antibiotic discs can be placed on the surface of the agar using sterilized forceps.
10. Gently press the discs onto the surface of the agar using flame sterilized forceps or inoculation loop.
11. Carefully invert the inoculated plates and incubate for 24 hours at 37° C.
12. After incubation, use a metric ruler to measure the diameter of the zone of inhibition for each antibiotic used.
13. Compare the measurement obtained from the individual antibiotics with the standard table to determine the sensitivity zone.
14. Compare the measurement obtained from the individual antibiotics to the standard table to determine whether the tested bacterial species is sensitive or resistant to the tested antibiotic.