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Antimicrobial Susceptibility Testing

Editor: Bradley N. Bragg Updated: 5/27/2024 12:50:44 PM


The majority of infectious diseases are bacterial in origin. With the discovery of laboratory methods to grow these microorganisms using an appropriate growth medium known as “culture,” determining the sensitivity and resistance of specific pathogens to a wide range of antimicrobial agents is necessary so clinicians can immediately institute proper treatment regimens.[1] Antimicrobial susceptibility testing is a laboratory procedure performed by medical technologists (clinical laboratory scientists) to identify which antimicrobial regimen is effective for individual patients. On a larger scale, the testing aids in evaluating treatment services provided by hospitals, clinics, and national programs to control and prevent infectious diseases. Recently, researchers have had to implement continuous surveillance activities for resistance patterns due to the mutations in bacterial deoxyribonucleic acid.[2][3]

Clinical laboratories currently employ several methods depending on their laboratory test menu. These approaches include the disk diffusion and minimum inhibitory concentration (MIC) methods. Commercial systems are also available across health centers and hospital facilities, utilizing phenotypic and genotypic characterization of bacterial resistance.[4] While routine antimicrobial susceptibility testing for gram-positive (eg, Staphylococcus aureus) and gram-negative bacteria (eg, Pseudomonas aeruginosa) are commonly available in peripheral laboratories, drug susceptibility testing for Mycobacterium tuberculosis is usually carried out within more complex facilities like reference laboratories. Despite the differences in the techniques for susceptibility tests, all laboratories are critical in each step of the sampling and testing process so that test results are obtained with consistently high levels of accuracy and reliability.[5]

Specimen Collection

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Specimen Collection

Specimen requirements for routine susceptibility testing using the disk diffusion method and MIC method are similar to the guidelines for collecting samples for bacterial culture since a certain number of well-isolated colonies (usually 3 to 5) grown from a culture is necessary to prepare a suspension of inoculum. The usual culture and sensitivity test specimens are blood, urine, cerebrospinal fluid, sputum, wound, stool, and other body fluids and discharge.[6]

Special susceptibility tests via commercial systems may not always require bacterial colonies from culture because they can detect resistance to antimicrobial drugs by employing molecular techniques. An example is the Xpert MTB/Rif assay, which directly determines sensitivity or resistance to rifampicin from sputum specimens.[7]


Both disk diffusion and MIC methods employ the phenotypic identification of susceptibility and, therefore, require the following process:

  1. Preparation of a standardized inoculum from a bacterial culture:
    • Choosing well-isolated colonies
    • Creating a bacterial suspension (inoculum)
    • Standardizing the bacterial suspension using McFarland standards
  2. Dilution of bacterial suspension (only for the MIC method)
  3. Inoculation of bacterial suspension to one of the following:
    • A particular growth medium (eg, Mueller Hinton Agar, MHA for disk diffusion)
    • A MIC panel
  4. Addition of antimicrobial disks (only for disk diffusion)
  5. Incubation of plates (disk diffusion) or panels for MIC  
  6. Measuring the zone of inhibition or reading the MIC panel
  7. Interpretation of antimicrobial susce­ptibility testing results [5]

Commercial systems have their laboratory procedures according to the manufacturer’s guidelines. Generally, the direct colony suspension method is used for preparing inoculum from colonies grown within 18 to 24 hours, while the growth method can be used by incubating the inoculated broth (with fast-growing bacteria) within 2 to 6 hours. The usual McFarland standard for the turbidity of the inoculum is 0.5.[8]

Dilution of bacterial suspension (commonly 1:20) for MIC must occur within 15 minutes after making the standard inoculum. Saline can be used as a diluent for a small amount of inoculum to create a concentration of 5 x 10 colony-forming units (CFU)/milliliter. As the inoculum is carefully poured over the panel tray and transferred to the panel prongs, the final concentration is expected to be relatively the same. Before inoculating the bacterial suspension into the growth medium, ensure the inoculum is not excessive. This check is accomplished by pressing the swab on the sides of the bacterial suspension tube before inoculating to the Mueller-Hinton agar plate. Inoculate the Mueller-Hinton agar plate with the swab by starting from the top and carefully swabbing from side to side down to the bottom. This step is performed 3 times after each plate rotation (usually 60°) to cover the whole plate.

The MIC method delivers the inoculum to each well via panel prongs. These panel prongs containing inoculum must be pressed on all sides and at the center, ensuring that the right volume of bacterial suspension transfers to each well, which is approximately 0.1 milliliters.[9] The addition of antimicrobial disks on inoculated Mueller-Hinton agar plates can be completed manually using sterile forceps, placing each disk within equal distances from other disks. An example of a recommendation states that a 150-millimeter diameter plate is best applied with only 12 antimicrobial disks. Each disk is pressed towards the surface of the agar to ensure that disk displacement will not occur during incubation.[10]

Incubation of the inoculated Mueller-Hinton agar plates with disks must take place considering the type of pathogen. Commonly, non-fastidious pathogens are incubated at 35 °C for about 16 to 18 hours at ambient temperature.  Other organisms require longer times (eg, 24 hours).[11] Fastidious pathogens such as Haemophilus and Neisseria spp require 16 to 18 and 20 to 24 hours, respectively.[5] The inoculated panel is incubated using the MIC method with the same temperature and incubation time requirements. Additionally, panels are recommended by a plastic seal or in a plastic bag to prevent the panels from dehydration since each well contains a relatively minimal amount of bacterial suspension.[12]

Integral to susceptibility testing is the implementation of a robust quality assurance program. Quality assurance ensures that testing conditions and test systems performance are acceptable, limits variance in operator performance, and helps identify random errors. For susceptibility testing, measures are in place to ensure that media and reagents are of good quality, equipment is serviced correctly, staff is competent at performing testing, routine quality control (QC) testing is within acceptable limits, and patient results are reviewed before finalization.[13]

QC strains are used for QC testing. These isolates have disk zones or MIC ranges established across several laboratories. These isolates are tested periodically (daily or weekly) to ensure that testing conditions, media, and reagents are acceptable. Results that are out of the acceptable QC range (ie, zones or MICs outside the acceptable range) may result from identifiable errors (eg, issues with the QC strain or supplies) or random.[14]

A random error of up to 5% is acce­ptable. If encountere­d, laboratories should promptly redo quality control testing. When the error is resolve­d, regular testing can resume­. However, persiste­nt errors require imme­diate action to identify the root cause­, and patient results should not be re­ported until resolved, which may involve­ testing new quality control strains, evaluating additional mate­rials, or consulting with commercial antimicrobial susceptibility te­sting equipment manufacturers. Laboratories may ne­ed to switch to alternative te­sting methods until the issue is fixe­d.[10]

Accurate antimicrobial susce­ptibility testing is crucial, and staff competency plays a vital role­.[15] Technologists should demonstrate proficie­ncy in performing and interpreting the testing, ide­ntifying and confirming atypical results, reporting appropriate age­nts according to guidelines, notifying rele­vant parties about unusual findings, and consulting clinicians for specialized te­sting requests.[16]

Revie­wing the antimicrobial susce­ptibility testing profile for eve­ry organism tested before­ reporting is essential. QC te­sting cannot guarantee the­ accuracy of each result for a patient’s isolate­. Potential errors include mixe­d cultures, bacterial misidentification, and issue­s with specific antimicrobial agent-organism combinations.[17] The laboratory technologist or director should review antimicrobial susceptibility testing results to ensure the following: bacterial identification and testing results correlate, appropriate drugs are reported, results of agents within the same class correlate, and results are consistent with previous results.[18]

Laboratories that pe­rform antimicrobial susceptibility testing must participate in proficie­ncy testing programs. These programs asse­ss the accuracy and consistency of testing me­thods. Proficiency testing helps pre­vent errors and ensure­s high-quality results, which is crucial given the global issue­ of antimicrobial resistance.[19] By comparing results with othe­r labs and following guidelines from organizations like the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST), proficie­ncy testing programs maintain quality assurance standards, safeguarding patie­nt care and reducing the­ risk of incorrect antimicrobial susceptibility test findings.[20] Participation in the­se programs is mandated by the Centers for Medicare & Medicaid Services (CMS) under Clinical Laboratory Improvement Amendments (CLIA) re­gulations.


Susceptibility testing for antimicrobials is necessary for patients who raise suspicion of infection based on history and physical exam. Antibacterial agents are then used to detect sensitivity or resistance from bacteria. Although this review aims primarily towards susceptibility testing for bacterial pathogens, antifungal susceptibility tests also exist to address fungal infection (eg, CandidaAspergillus spp). Furthermore, antiviral susceptibility tests are also available (eg, influenza) via molecular technologies, including sequencing analysis such as Sanger and pyrosequencing methods.[21]

Potential Diagnosis

A unique impact of antimicrobial susceptibility testing on patient care is identifying the specific diagnosis and targeting the particular etiologic agent causing the disease. No 2 patients have the same care plan, especially if they have the same signs and symptoms but different treatment regimens because the same causative organism can have different resistance patterns.[22] For example, 2 patients may present with an ordinary strain of Staphylococcus aureus vs methicillin-resistant Staphylococcus aureus (MRSA). Another common occurrence is in patients with drug-susceptible and drug-resistant tuberculosis.

Normal and Critical Findings

Antimicrobial susceptibility testing results are reported using interpretive criteria called clinical breakpoints. Essentially, a clinical breakpoint represents a value (such as MIC or zone diameter) that helps differentiate between organisms inhibited effectively or killed by a given antimicrobial, administered according to standard dosing (classified as susceptible), and those expected to be unaffected or resistant.[23] 

For disk diffusion, the zone of inhibition is measured using a dedicated caliper that correctly measures the diameter by the edges of the inhibition zone. Reading each set of wells for an antibiotic drug is for MIC panels. MIC determination is by either a clear or slight whiteness on the well. The results of the inhibition zones and MIC breakpoints are reported using either the terms “susceptible” or “resistant” based on the set cut-off range for zone diameter in the nearest whole millimeter and microgram per milliliter, respectively. 

MIC results depend on the interaction between the antimicrobial agent and the organism and test conditions. These conditions include pH and ion concentrations of testing media, the temperature at which the test system is incubated, the incubation atmosphere, the number of organisms used in testing, and the length of time the system incubates. The CLSI and the EUCAST developed expert-approved guidelines on breakpoints for reporting the results of these methods.[5][24]

Interfering Factors

Several factors affect the result of the susceptibility testing, which covers the whole sampling, testing, and reporting procedures. Any deviation from the standard antimicrobial susceptibility testing procedure can significantly impact succeeding areas of laboratory workflow, which could later affect patient diagnosis, treatment, and management. The laboratory workflow support systems require strict monitoring, and laboratory personnel should be well-trained and competent to perform the procedure.[25]

For instance, poor specimen quality is the first sign leading to an erroneous result. A perfectly carried out inoculation to the Mueller-Hinton agar plate using mixed colonies yields unsatisfactory results. Poor standardization of bacterial suspension and a “longer depth of agar” could yield misleading endpoints. Supervision for laboratory staff is necessary to prevent wasting time and resources.[26]

Purchasing poor-quality MIC panels can lead to “dehydrated wells” or “mixed wells.” Completing the procedure without personal protective equipment can increase the incidence of laboratory-acquired infections.[27] Inadequacy and lack of supplies will extend turn-around time, decreasing laboratory productivity and delaying patient therapy.[28] Completing the testing for antibiotics that do not align with the hospital’s formulary makes laboratory service available yet ineffective.[29]


Inconsistencies in the antimicrobial susceptibility testing results must be investigated. No results should be released when quality control measures are unsatisfactory. Releasing inaccurate drug susceptibility or resistance results can inflict more harm to the patients, leading to severe clinical conditions and poor prognosis. A consequence of delivering false results can lead to inappropriate treatment plans that might cause further mutations to infectious organisms, exposing the patients and the community to a higher risk.[30]

Patient Safety and Education

Patients should be informed about antimicrobial susceptibility testing, indications, requirements, and clinical use for managing cases.[31] Clinicians are encouraged to disseminate correct information about the test. However, the antimicrobial susceptibility testing results must be interpreted between the patient and the treating clinician to facilitate compliance with the prescribed medications and prevent self-medication.[32] With the rise of antimicrobial resistance, testing requires an emphasis on medical, laboratory, and nursing staff, patients and their family members, and the whole community, leading to a unified approach.[33]

Clinical Significance

When antimicrobial susceptibility results are available, clinicians can develop treatment regimens for each patient. Prescribed medications of appropriate antibiotics need individualization for each patient diagnosed with an infectious disease. Resistance to primary drugs requires more antimicrobial stewardship, including prudent use of second-line drugs.[22]



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