Antimicrobial sensitivity testing is done to provide testing of bacterial pathogen to set of available antibiotics to determine its susceptibility profile and so predict the effectiveness of the antibiotic regimen and secondly to assess the effectiveness of the treatment regimen [1]. 

Purposes

• To guide the clinician in selecting the best antibiotic agent for an individual patient

• To control the use of antibiotics in clinical practice

• To accumulate epidemiological information on the resistance of microorganisms of public health importance within the community

• To reveal the changing trends in the local isolates

• Improved patient outcomes

• Lower mortality rates 

• Less diagnostic procedures ordered including Laboratory tests and Diagnostic  imaging procedures 

• Shortened  ICU stays 

• Decreased ventilator days 

• Fewer prescriptions  for inadequate antibiotic therapy 

• Shortened length of stay 

• Lower total cost of hospitalization             

Testing Methods 

• Three general methods are available to detect and evaluate antimicrobial susceptibility

• Methods that directly measure the activity of one or more antimicrobial agents against a bacterial isolate 

• Methods that directly detect the presence of a specific resistance mechanism in a bacterial isolate 

• Special methods that measure complex antimicrobial organism interactions

• The method used depends on factors such as clinical need, accuracy, and convenience. Given the complexities of antimicrobial resistance patterns, a laboratory may commonly use methods from more than one category

Definitions of Susceptibility Testing Interpretive Categories

• Susceptible (S) Indicates that the antimicrobial agent in question may be an appropriate choice for treating the infection caused by the organism. Bacterial resistance is absent or at a clinically insignificant level. 

• Resistant (R) Indicates that the antimicrobial agent in question may not be an appropriate choice for treatment, either because the organism is not inhibited with serum-achievable levels of the drug or because the test result highly correlates with a resistance mechanism that indicates questionable successful treatment

• Intermediate (I) Indicates a number of possibilities, including: 

• The potential utility of the antimicrobial agent in body sites where it may be concentrated (e.g., the urinary tract) or if high concentrations of the drug are used

• Possible effectiveness of the antimicrobial agent against the isolate, but possibly less so than against a susceptible isolate 

• Use as an interpretive safety margin to prevent relatively small changes in test results from leading to major swings in interpretive category (e.g., resistant to susceptible or vice versa) 

Standardization of Antimicrobial Sensitivity Testing (AST) Methods

• Growth medium (typically a Mueller-Hinton base) 

• Bacterial inoculum size 

• pH 

• Cation concentration 

• Blood and serum supplements 

• Thymidine content 

• Incubation atmosphere 

• Incubation temperature 

• Incubation duration 

• Antimicrobial concentrations

Media

Best Medium – MHA (Mueller Hinton Agar) 

• Shows acceptable batch to batch reproducibility for susceptibility testing 

• Low in sulphonamide, trimethoprim and tetracycline inhibitors 

• Gives satisfactory growth of most non fastidious pathogens

• A large body of data and experience has been collected concerning susceptibility tests performed with this medium

MHA (Mueller Hinton Agar)

• Cool the medium to 45–50 ⁰C and pour into the plates. Allow to a depth of approximately 4 mm

• A 9-cm plate requires approximately 25 ml of medium

• When the agar has solidified, for 10–30 minutes at 35 ⁰C by placing them in the upright position in the incubator with the lids tilted

• If it is not to be used immediately, the agar medium can be stored in a refrigerator (2 to 8degree C) for 2 weeks

• A supply of cotton wool swabs on wooden applicator sticks should be prepared [2]

• They can be sterilized in tins, culture tubes, or on paper, either in the autoclave or by dry heat

Inoculum

• Inoculum is usually maded from broth culture that has been incubated for 4 to 6 hours

• The density of the suspension is adjusted to approximately 10⁸ colony-forming units(CFUs) per milliliter by comparing its turbidity to a McFarland 0.5  BaSO4 standard

• Commonly, the degree of cloudiness in the broth is compared with the standard, visualizing the two against a white background on which black lines have been drawn

• Nephelometers may be used to determine turbidity 

• Turbidity standard is checked by spectrophotometer – 625nm – absorbance should be 0.008 to 0.10

• Seal the tubes containing the Mcfarland standards and store in dark at RT

• The standard turbidity should be mixed throroughly every time before use

• Check the density monthly and replace it monthly

Ph

• Tests are performed using media for which the  pH  has been standardized at physiologic levels.

• Media pH should be between 7.2 and 7.4 at room temperature.

• The pH of broth media may be tested directly with a pH electrode, and agar media may be tested by macerating enough of  the agar so that the tip of the electrode can be submerged, by allowing a portion of agar to solidify around the electrode, or by using a properly calibrated surface electrode

Cation Concentration

• Agar and broth media varygreatly in the concentration of divalent cations [3].

• By convention, antibiotic susceptibility testing is done under physiologic conditions.

• MHA has very low concentrations of divalent cations, but is adjusted to physiologic concentrations (20 to 35 mg/L Mg2+              and 50 to 100 mg/L Ca2+) during production

Serum

• Antibiotics differ greatly in the degree to which they bind to proteins. In the bloodstream, free antibiotic is in equilibrium with serum protein-bound antibiotic

• Free and protein-bound antibiotic can be measured, but it is not clear which is the more useful result

• The CLSI method does not include added serum because of the difficulty in standardization of the product and uncertainty about how to interpret the results

Environmental Conditions

• Antibiotic susceptibility tests are routinely incubated in ambient air at 35°C. Agar or broth is incubated in an ambient air incubator.

• Incubation times vary depending on the test system used. The recommended incubation time for conventional disk diffusion systems is 16 to 18 hours, while that for dilution tests is longer at 16 to 20 hours

• However, tests to determine the MICs of staphylococci for oxacillin and vancomycin, and the MIC of enterococci for vancomycin should be incubated a full 24 hours [4]

Selection of Antimicrobial Agents for Testing

• The antimicrobial agents chosen for testing against a particular bacterial isolate are referred to as the antimicrobial battery or panel

• A laboratory may use different testing batteries, but the content and application of each battery are based on specific criteria

• Final decision should not be made independently by the laboratory; input from the medical staff (particularly infectious diseases specialists) and pharmacists (e.g., a Pharmacy and Therapeutics committee) is imperative

Specific Criteria

• Organism Identification or Group: certain antimicrobials were developed specifically for use against particular organisms, but not against others (e.g., ceftazidime for use against Pseudomonas aeruginosa but not against Staphylococcus aureus); such agents should be included only in the appropriate battery. 

• Acquired Resistance Patterns Common to Local Microbial Flora.

• Depending on the testing method, some agents do not reliably detect resistance and should not be included in the battery.

• Site of Infection.

• Availability of Antimicrobial Agents in the Formulary

• CLSI Grouping of Recommended Antimicrobial Agents for Testing and    Reporting 

Antibiotic Stock Solutions

• Buy commercial pure source of antibiotics

• Don’t use injectable solutions 

• Accurate weighing of powders is must 

• Standard strains of stock cultures should be used to evaluate the stock solution After preparing the stock solution, make 5 ml aliquots and should be frozen.

• Calculation of stock solution

1000 * V * C  =  W

P

Dried Filter Paper Discs

• Whatmann no.1 filter paper is made to form a disc size of 6mm

• Keep in petridish and sterilize in a hot air oven

• With the help of antibiotic delivery loop which has a 20G wire with diameter of 2mm 

• The antibiotic is delivered (0.005ml) 

Storage of discs

• Refrigerate at 2 to 8 deg C 

• Beta lactam class drugs should be frozen 

• The drugs should be kept outside at Room Temperature 1 to 2 hours before work 

• The dispensing apparatus which is used to deliver the drugs should also be refrigerated 

• Check the expiry of drugs 

AST Types (figure-1)

• Qualitative: For the testing of isolates from “healthy” patients with intact immune defenses. – For such as uncomplicated urinary tract infections

• Quantitaive: In the treatment of serious infections such as endocarditis or osteomyelitis. – For infections in high-risk patient groups such as immune compromised patients (e.g. transplant patients).  Those who are critically ill

Disk Diffusion Method

It is the Simplest and most convenient method widely used everywhere and Developed by Kirby, Sherrris, Bauer and Turk in 1966.  Types are Kirby Bauer method  and Stokes method

Figure 1: Types of Antimicrobial Sensitivity Testing

Figure 2: Kirby Bauer Method

Figure 3: Broth Dilution Method

Figure 4: Broth Dilution Method

Figure 5: Agar Dilution Method

Figure 6: Epsilometer Test

Figure 7: Vitek 2

Kirby Bauer Method

Application of Discs

• 150 mm plate - 12 discs 

• 100 mm plate - 6 discs 

• Drug should not be relocated 

• Distance from the lid edge - 15mm 

• Distance between two drug from center to center - 24mm

• Inoculum - disc placement - incubation (only 15 minutes delay is acceptable each) [5]

Procedure

• To prepare the inoculum from the primary culture plate, touch the top of the colony 3 to 5, of similar appearance, of the organism to be tested

• Transfer this growth to a tube of saline. Compare the tube with the turbidity standard and of the test suspension to that of the standard by adding more bacteria or more sterile saline

• Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess inoculum by pressing and rotating the swab firmly against the side of the tube above the level of the liquid

• Streak the swab all over the surface of the medium three times, rotating the plate through an angle of 60⁰ after each application.Finally, pass the swab round the edge of the agar surface

• Leave the inoculum to for a few minutes at room temperature with the lid closed. They may be placed on the inoculated plates using sterile forceps, a template, a sterile needle tip and antibiotic disc dispenser

• The plates should be placed in an incubator within 30 minutes of preparation. Temperatures above 35 degree celsius invalidate results   for oxacillin/methicillin. Disks should not be moved after diffusion

Interpretation/Results

A dark background and reflected light are used to examine disk diffusion plates. The plate is situated so that a ruler or caliper can be used to measure the inhibition zone diameters for each antimicrobial agent.In instances not involving swarming organisms or the testing of sulfonamides and trimethoprim, hazes of growth that occur in more obvious inhibition zones should not be ignored.  Interpretation should be done after 16 to 18 hours, Confluent lawn of growth, Zones of inhibition - uniformly circular and read with reflected light but For MRSA - read in transmitted light. When proteus is tested the thin veil of swarming growth after the zone of inhibition should be ignored.

Stokes Method

• Built in controls against many variables and provide dependable results 

• A standard sensitive strain of the bacterium is inoculated in the middle third of the culture plate

• The test bacterium is inoculated in the upper and lower third of the plate 

• Antibiotic discs are placed between the standard and test inocula so that zones of inhibition formed around each disc are composed of standard and test bacteria

• The results are reported as Susceptible, Intermediate , Resistant

Dilution Methods

In this, the minimum concentration of antimicrobial to inhibit or to kill the microorganism is determined. MIC is the lowest concentration of antimicrobial that will inhibit the visible growth of an organism in an ideal growth condition while MBC is the least concentration of the test antibiotic which will completely kill the bacteria tested. It is of two types, Broth dilution method and Agar dilution method 

Broth Dilution Method

The Mueller-Hinton preparation is the standard medium used for most broth dilution testing, and conditions in the medium (e.g., pH, cation concentration, thymidine content) are well controlled by commercial manufacturers. Broth dilution testing is divided into two general categories: microdilution and macrodilution.  The principle of each test is the same; the only difference is the volume of broth in which the test is performed.  For microdilution testing, the total broth volume is 0.05 to 0.1 mL; for macrodilution testing, the broth volumes are usually 1 mL or greater. Because most susceptibility test batteries require testing of several antibiotics at several different concentrations, the smaller volume used in microdilution allows this to be conveniently accomplished in a single microtiter tray   

Broth dilution

• Media: cation adjusted MH broth with a pH of 7.2 to 7.4 

• For H.influenzae – HTM 

• For TMP-SMX – Thymidine free medium 

• For oxacillin Resistance – MH broth with 2% NaCl 

Working Antibiotic Solution

• Take original stock solution 

• Prepare stock dilutions of the antibiotic of concentrations 1000 and 100ug/ml 

• Arrange two rows of 12 sterile 7.5*1.3cm capped tubes in the rack 

• Take a 30ml universal screw capped bottle 

• Add 8ml broth and required antibiotic concentration and mix the contents

• Take 2ml + 2ml and put it in first tube in both rows  Remaining 4ml broth - add 4ml fresh broth

• Transfer 2ml+2ml and put in second tube 

• Continue this dilution upto 11 tubes 

• 12th tube acts as control. 

Inoculation

• Inoculation of 1st row with one drop of overnight broth culture 1 in 1000 dilutions 

• 2nd row with control with known sensitivity organisms 

• Final inoculum = 5 * 10⁵ cfu/ml 

• Incubate at 37degC for 16 to 18 hours 

• Inoculate another tube with 2ml broth and keep at 4degC in a refrigerator overnight to be used as standard for determinattion of complete inhibition 

Interpretation

• Positive control tube – turbidity 

• Negative control tube – clear

• MIC end point is read

Agar Dilution Method

• 1ml concentration of the drug + 24 ml of MHA 

• Inoculum – make 1:10 dilution of 0.5Mcfarland standard inoculum which delivers 10⁷ cfu/ml

• Using pipette or calibrated loop , deliver 0.001 ml on the surface of the agar giving the final inoculum of 10⁴ cfu /spot

• Inoculate a control plate

Interpretation

• Examine the drug free control growth of the test organism for viability and purity 

• Place the plate on a dark background and examine them for the lowest concentration that inhibits visible growth 

• A single colony of a faint haze is not recorded as growth

Epsilometer Test

• An exponential gradient testing methodology 

• A predefined stable antimicrobial gradient is present on a thin inert carrier strip 

• Following incubation, the E strip releases drug and a symmetrical inhibition ellipse is produced 

• MIC = intersection of the inhibitory zone edge and the calibrated carrier strip

Automated Antimicrobial Susceptibility Test Systems

• Vitek 2 systems (bioMérieux, Inc., Durham, NC)

• The MicroScan WalkAway system (Beckman Coulter, Inc., Brea, CA)

• The Phoenix system (BD Microbiology Systems, Sparks, MD)

Vitek 2

• AST inoculum is automatically introduced by a filling tube into a miniaturized, plastic, 64-well, closed card containing specified concentrations of antibiotics.

• Cards are incubated in a temperature-controlled compartment. 

• Optical readings are performed every 15 minutes to measure the light transmitted through each well, including a growth control well.

• Algorithmic analysis of the growth kinetics in each well is performed by the system’s software to derive the MIC data. 

• The MIC results are validated with the Advanced Expert System (AES) software, a category interpretation is assigned, and the organism’s antimicrobial resistance patterns are reported. 

• Resistance detection is enhanced with the sophisticated AES software, which can recognize and report resistance patterns using MICs. In summary, this system facilitates standardized susceptibility testing in a closed environment with validated results and recognition of an organism’s antimicrobial resistance mechanism in 6 to 8 hours for most clinically relevant bacteria

MicroScan WalkAway System

• It uses the broth microdilution panel format manually inoculated with a multiprong device. Inoculated panels are placed in an incubator-reader unit, where they are incubated for the required time, and then the growth patterns are automatically read and interpreted. Depending on the microdilution tray used, bacterial growth may be detected using spectrophotometry or fluorometry

• Spectrophotometric analyzed panels require overnight incubation, and the growth patterns may be read manually 

• Fluorometric analysis is based on the degradation of fluorogenic substrates by viable bacteria. The fluorogenic approach can provide susceptibility results in 3.5 to 5.5 hours. Either full dilution schemes or breakpoint panels are available

• In addition to speed and facilitation of workflow, the automated systems provide increasingly powerful computer-based data management that can be used to evaluate the accuracy of results, manage larger databases, and interface with the pharmacy to improve and advance the utility of antimicrobial susceptibility testing data

Phoenix System

The Phoenix system provides a convenient, albeit manual, gravity-based inoculation process. Growth is monitored in an automated fashion based on a redox indicator system, with results available in 8 to 12 hours. Supplemental testing (e.g., confirmatory extended-spectrum beta-lactamase [ESBL] test for E. coli) is included in each panel, reducing the need for additional or repeat testing. Interpretation of results is augmented by a rules-based data management expert system.

1. Wikipedia. "Antibiotic sensitivity testing." Available at: https://en.wikipedia.org/wiki/Antibiotic_sensitivity_testing (Accessed on 16 June 2022).

2. MedlinePlus. "Antibiotic sensitivity test." Available at: https://medlineplus.gov/lab-tests/antibiotic-sensitivity-test/ (Accessed on 17 June 2022).

3. Healthline. "Sensitivity analysis." Available at: https://www.healthline.com/health/sensitivity-analysis (Accessed on 18 June 2022).

4. Testing.com. "Antibiotic susceptibility testing." Available at:https://www.testing.com/tests/antibiotic-susceptibi lity-testing/ (Accessed on 19 June 2022).

5. TMCC. "Antimicrobial susceptibility testing." Available at: https://www.tmcc.edu/microbiology-resource-center /lab-protocols/antimicrobial-susceptibility-testing (Accessed on 17 June 2022).