Antimicrobial Susceptibility Testing (AST): Introduction, Methods, Procedu

Antimicrobial Susceptibility Testing (AST): Introduction, Methods, Procedure and Result Interpretation

Antimicrobial Susceptibility Testing (AST)

Antimicrobial Susceptibility Testing (AST) is a laboratory method for determining the susceptibility of organisms to therapy with antibiotics. Antimicrobial Susceptibility Testing (AST) is usually carried out to determine which antibiotic / antimicrobial agent will be most successful in treating a microbial infection in vivo.

Antimicrobial Susceptibility Testing Methods

Disc diffusion method e.g. Kirby-Bauer and Stokes method
Dilution method

Tube dilution
Agar dilution
Disc diffusion and Dilution method e.g. Epsilometer Test or E-Test

Preparation of the inoculum

The routinely used method is the turbidity standard (0.5 McFarland).

Emulsify 2-3 colonies in sterile saline matching the turbidity standard.

Kirby-Bauer Method (Disc Diffusion Test)

Commonly used method for determining the antibiotic susceptibility of a bacteria.

Test Requirements

Pure cultures of the organism isolated from the clinical specimen
Muller Hinton Agar
Antibiotic Disks
Turbidity Standard
Swabs

Antimicrobial Susceptibility Testing Procedure

Mostly Muller Hinton agar( MHA) is used in this antibiotic susceptibility test.
Emulsify 2-3 colonies in sterile saline matching the turbidity that standard (0.5 McFarland).
Place a sterile cotton swab in the bacterial suspension and remove the excess fluid by pressing and rotating the cotton against the inside of the tube above the fluid level.
The swab is streaked in three directions over the surface of the MHA to obtain uniform growth.
Allow the plates to dry for 10-15 minutes.
Using sterile forceps or a suitable disk dispenser, place paper disks impregnated with a fixed concentration of an antibiotic, on the surface agar plates having distance disc to disc 25 mm while plate border to disk 15 mm.
Incubate the plates at 37°C for 24 hours.
Following overnight incubation, measure the diameter of the zone of inhibition in millimeters (mm) around each disc.

Result Interpretation of Susceptibility Testing

Using a standard table of antibiotic susceptibilities, determine if the strain is resistant, intermediate, or susceptible to the antibiotics tested.

Introduction of Stokes disc diffusion method

Stokes disc diffusion method as shown above picture. Antibiotic Sensitivity Testing (AST) is a determination of the least amount of an antimicrobial chemotherapeutic agent that will inhibit the growth of microorganisms in vitro and it is achieved by following methods 1. Disk diffusion method: A. Kirby Bauer method, B. Stokes method 2. MIC: A. Broth dilution method B. Agar dilution method and 3. E-test. Stokes disc diffusion method varies from Kirby Bauer disc diffusion in the use of both control and test strain on the same plate. A set of standard strains are used as control strains depending on the organism to be tested. The control strains are E. coli NCTC 10414 for testing coliform bacilli from the urinary tract, Pseudomonas aeruginosa NCTC 10662 against aminoglycosides. Comparative disc diffusion techniques based on Stokes method is still in wide use in a developed country like the UK, to determine antibiotic susceptibility and therefore it is also useful in developing countries.

Stokes disc diffusion method is of two types-

Stokes disc diffusion method and
Modified Stokes disc diffusion method

Stokes disc diffusion method (conventional): The test organism is inoculated on the central one-third and controls on the upper and lower thirds of the plate.

Modified Stokes disc diffusion method: In this method, the test bacterium is inoculated over the upper and lower thirds of the plate and control on the central one-third as shown below the video.

Principle of Stokes Disc Diffusion Method

In this Stokes disc diffusion method discs are applied between the standard and test inocula, so that zone inhibition (ZOI) formed around each disc are composed of standard and test bacteria. The diffusion of antibiotic takes place and thus the susceptibility of those organisms to the antibiotics are known by measuring zone size.

Requirements for the test

MHA plate
Antimicrobial discs
Sterile cotton swabs
Sterile forceps
Control strains depending on the bacterium tested-S. aureus NCTC 6571 or E. coli NCTC 10414 or P. aeruginosa NCTC 10662
Bunsen burner
Inoculating loop
O.5 McFarland Std. or Mc Farland Densitometer
Incubator
Wickerham Card

Procedure of Stokes Disc Diffusion Method

Take 3-5 well-isolated colonies of the same morphological type of both test and control strains. Transfer the colonies into a tube containing 4 to 5 ml of a tryptic soy broth.
Incubate the broth culture at 37°C until it achieves the turbidity of the 0.5 McFarland standard and it usually takes 2 to 6 hours.
Adjust the turbidity of the actively growing broth culture with sterile saline or tryptic soy broth when high but in low turbidity further, incubate to obtain turbidity optically comparable to that of the 0.5 McFarland standard. This standard is equivalent to a suspension containing approximately 1 to 2 x 108 CFU/ml for E.coli ATCC 25922.
To perform this step properly, either read inoculum in McFarland densitometer or, if done visually, adequate light is needed to visually compare the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines called Wickerham card.
Dip sterile cotton swabs into each of the adjusted suspensions (within 15 minutes after adjusting the turbidity).
Rotate the swabs several times and pressed firmly on the inside wall of the tube above the fluid level and this will remove excess inoculum from the swab.
Dry the inoculation plates with the lid open so that there should be free droplets of moisture on the surface.
Apply the control culture in two bands on either of the plates leaving a central band uninoculated with the help of sterile swabs.
Apply the test organism in the central portion without touching either side and this is applicable for conventional Stokes disc diffusion method whereas modified Stokes disc diffusion method reverse of steps 9 and 10.
Apply the antibiotics discs with forceps on the line between test and control organisms and press gently to ensure even contact with the medium. Note: there should be a minimum distance of 2 cm between two discs.
Incubate the plates overnight aerobically at 35-37 °C.

Result Interpretation of Stokes Disc Diffusion Method

Measure the radius of the inhibition zone from the edge of the disc to the edge of the zone as shown above image-

Sensitive (S)– The zone size of the test strain is larger than or equal to the control strain. If the zone size of the test bacterium is smaller than that of the control, the difference between the two should not be more than 3 mm.

Intermediate(I)– zone size of the test strain is > 2 mm but smaller than the control by > 3 mm.

Resistant(R)– The zone size of the test strain is smaller than 2 mm.

Advantages of Stokes method

The control strain and test strain can be checked on the same plate.
More reliable for the quality testing of discs.
Both control and test organism is the same environment i.e. the effect of variation of an environmental condition like temperature, time affect both simultaneously thus minimizing error
Errors due to using too heavy or light inocula will be detected.
The strips with multiple Antibiotics can be tested on a single plate.

Disadvantages of Stokes disc diffusion method

Stokes disc diffusion technique is not as highly standardized as the Kirby Bauer technique and is used in laboratories particularly when the exact amount of antimicrobial in a disc cannot be guaranteed due to difficulties in obtaining discs and storing them correctly or when the other conditions required for the Kirby-Bauer technique cannot be met.
A low number of antibiotics are tested in a plate.

Notes

Four discs can be accommodated on an 85 mm circular plate.
Extremes in inoculum density must be avoided. Never use undiluted overnight broth cultures or other un-standardized inocula for streaking plates.
For inoculation, a rotatory plating method can also be used wherein the control strain is applied on the outer periphery and the test strain is applied in the central portion. In such a method, six discs can be put on an 85 mm circular plate.

Dilution method

Used to determine the minimal concentration of the antimicrobial agent to inhibit or kill microorganisms.

Methods: It can be achieved by following methods-

Tube dilution methods
Agar dilution method

Minimum inhibitory concentration (MIC): The lowest concentration of antimicrobial agent that inhibits the growth of organisms.

Minimum bactericidal concentration (MBC): Lowest concentration of an antimicrobial that kills organisms isolated from the patient.

Tube dilution methods

In this method, sterile Muller Hinton broth (MHB) is used.
Two folds dilution of antibiotics in the broth i.e. 2µg/ml, 4µg/ml, 8µg/ml, 16µg/ml, and so on is made.
Then broth culture (0. 1 ml ) of test organism is added to the prepared dilutions.

Method

There are two types of dilution methods by which it can be achieved and they are-

a) Micro-dilution method

It is performed in 96 well microtiter plates.

About 0.1 ml total broth volume is used.

b) Macro-dilution method

Test tubes for this test are used.
About 1 ml total broth volume is used in which two-fold dilution of antibiotics is made.

Incubation

In both dilution methods, after adding the test organism, the tubes are incubated at 37°C for 24 hours.

Result Interpretation

The minimum inhibitory concentration (MIC) of antibiotics is determined in µg/ml.

Minimum inhibitory concentration (MIC): Minimum concentration of antibiotic that inhibits the growth of bacteria i.e. a clear broth.

Minimal bactericidal concentration (MBC): The minimal bactericidal concentration can be determined by sub-culturing all tubes showing no visible turbidity. The tube with the highest dilution that fails to yield growth on the subculture plate contains the MBC of antibiotics for the test strain.

Agar dilution method

Serial dilution of antibiotics is prepared in agar and poured into plates.

Epsilometer test or E-test

It is a quantitative assay for determining the Minimum Inhibitory Concentration (MIC) of antimicrobial agents against microorganisms and for detecting the resistance mechanisms.
MIC Test Strip are paper strips with special features that are impregnated with a predefined concentration gradient of antibiotics.
On one side of the strip is indicated a MIC scale in μg/ml and a code that identifies the antimicrobial agent.
The exponential gradient of the antimicrobial agent is immediately transferred to the agar matrix.
After 18 hours of incubation or longer, an asymmetrical inhibition ellipse centered along the strip is formed.
The MIC is read directly from the scale in terms of μg/ml at the point where the edge of the inhibition ellipse intersects the MIC Test Strip as shown above picture.

Epsilometer test or E-test Procedue

Take 24-48 hours old broth (Liquid) culture of bacteria to be tested.
Place a sterile cotton swab in the bacterial suspension and remove the excess fluid by pressing and rotating the cotton against the inside of the tube above the fluid level.
The swab is streaked in three directions over the surface of the Mueller-Hinton agar to obtain uniform growth. A final sweep is made around the rim of the agar.
Allow the plates to dry for five minutes.
With the help of sterile forceps apply E-test strips at equal distance on inoculated Muller Hilton agar plate.
Incubate the plates at 37°C for 24 hours.
Following overnight incubation, an inhibition ellipse is produced.
The edge of the ellipse corresponding to the antibiotic concentration on the scale indicates the MIC.

Result Interpretation

MIC: the point where the edge of the inhibition ellipse intersects the MIC Test Strip i.e. 0.75 μg/ml.

Advantages

Simple
Active
Reliable

Factors Influencing Antimicrobial Susceptibility Testing

pH: The pH of each batch of MHA should be checked when the medium is prepared. The exact method used will depend largely on the type of equipment available in the laboratory. The agar medium should have a pH between 7.2 and 7.4 at room temperature after gelling. If the pH is too low, certain drugs will appear to lose potency (e.g. aminoglycosides, quinolones, and macrolides), while other agents may appear to have excessive activity (e.g. tetracyclines). If the pH is too high, the opposite effects can be expected.

Moisture: If, just before use, excess surface moisture is present, the plates should be placed in an incubator (35°C) or a laminar flow hood at room temperature with lids ajar until excess surface moisture is lost by evaporation (usually 10 to 30 minutes). The surface should be moist, but no droplets of moisture should be apparent on the surface of the medium or on the Petri dish covers when the plates are inoculated.

Effects of Thymidine or Thymine: Media containing excessive amounts of thymidine or thymine can reverse the inhibitory effect of sulfonamides and trimethoprim, thus yielding smaller and less distinct zones, or even no zone at all, which may result in false-resistance reports. MHA that is as low in thymidine content as possible should be used. To evaluate a new lot of MHA, E. faecalis ATCC 29212, or alternatively, E. faecalis ATCC 33186, should be tested with trimethoprim/sulfamethoxazole( Co-trimoxazole) disks. Satisfactory media will provide essentially clear, distinct zones of inhibition 20 mm or greater in diameter. Unsatisfactory media will produce no zone of inhibition, growth within the zone, or a zone of less than 20 mm.

Effects of Variation in Divalent Cations: Variation in divalent cations, principally magnesium and calcium, will affect the results of aminoglycoside and tetracycline tests with Pseudomonas aeruginosa strains. Excessive cation content will reduce zone sizes, whereas low cation content may result in unacceptably large zones of inhibition. Excess zinc ions may reduce zone sizes of carbapenems. Performance tests with each lot of MHA must conform to the control limits.

Testing strains that fail to grow satisfactorily: Only aerobic or facultative bacteria that grow well on unsupplemented MHA should be tested on that medium. Certain fastidious bacteria such as Haemophilus species, Neisseria gonorrhoeae, Streptococcus pneumoniae, and viridans and ß-hemolytic streptococci do not grow sufficiently on unsupplemented MHA. These organisms require supplements or different media to grow, and they should be tested on the media described in separate sections.

Key Notes on Antimicrobial Susceptibility Testing (AST)

Ideal antibiotic therapy is based on the determination of the etiological agent and its relevant antibiotic sensitivity.
Empiric treatment is often started before laboratory microbiological reports are available when treatment should not be delayed due to the seriousness of the disease.
Genotypic AST Methods: PCR, DNA microarray and DNA chips, and loop-mediated isothermal amplification (LAMP) are some of the genotypic techniques for the detection of antibiotic resistance.
Emerging Methods for AST: Microfluidics-based diagnostics are one of the most promising emerging tools for AST. Microfluidics is an evolving field characterized by the manipulation of fluids in micro-volume, thereby offering portability, cost-effectiveness, multiplexing, reproducibility, and a controllable environment in an in vitro system.
The newly developed MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) is a more straightforward and cost-effective modulation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) used for both AST and MIC determination.
Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) are Institutions those AST guidelines followed globally.

References

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627445/
https://en.wikipedia.org/wiki/Antibiotic_sensitivity_testing
https://www.acmicrob.com/microbiology/a-comprehensive-review-
https://www.intechopen.com/books/latest-research-into-quality-
https://link.springer.com/article/10.1007/s10096-017-3089-2
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Testing; Twentieth Informal Supplement M100-S20. Volume 30 Number 1, Clinical and Laboratory Standards Institute, Villanova, PA. 2010.
Centers for Disease Control and Prevention. Manual for the laboratory Detection of Antimicrobial Resistance among Community-acquired Bacterial Pathogens of Public Health Concern in the Developing World. World Health Organization.WHO/CDS/ CSR/ EPH.2002.15
Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier

[14231 visitors]

universe84a