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Antimicrobial Susceptibility Testing

Authors

Marlon L. Bayot1; Bradley N. Bragg2.

Affiliations
1 Adventist University of the Philippines-College of Medicine; Cavite State University-Med Tech Dept.
2 Mayo Clinic Florida

Last Update: October 17, 2021.

Introduction
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 becomes necessary so that healthcare providers can
immediately institute proper treatment regimens to their patients.[1]

Antimicrobial susceptibility testing (AST) is a laboratory procedure performed by medical technologists (clinical
laboratory scientists) to identify which antimicrobial regimen is specifically effective for individual patients. On a
larger scale, it aids in the evaluation of treatment services provided by hospitals, clinics, and national programs for the
control and prevention of infectious diseases.  Recently, researchers have had to implement continuous surveillance
activities for resistance patterns due to the mutations in bacterial DNA.[2][3]

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

Specimen Collection
Specimen requirements for routine susceptibility testing using the disk diffusion method and minimum inhibitory
concentration (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 specimens sent for culture and sensitivity tests are blood, urine, cerebrospinal fluid, sputum,
wound, stool, and other body fluids and discharge.[4]

Special susceptibility tests via commercial systems may not always require bacterial colonies from culture
because they can detect resistance to certain antimicrobial drugs by employing molecular techniques for detecting
resistant genes.  An example would be the Xpert MTB/Rif assay which determines sensitivity or resistance to
rifampicin directly from sputum specimens.[5]

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

Preparation of a standardized inoculum from a bacterial culture:

Choosing well-isolated colonies

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Creating a bacterial suspension (inoculum)

Standardizing the bacterial suspension using McFarland standards

Dilution of bacterial suspension (only for MIC method)

Inoculation of bacterial suspension to one of the following:

A particular growth medium (e.g., Mueller Hinton Agar, MHA for disk diffusion)

A MIC panel

Addition of antimicrobial disks (only for disk diffusion)

Incubation of plates (disk diffusion) or panels (MIC)

Measuring the zone of inhibition or reading MIC panel

Interpretation of AST results

Commercial systems have their own sets of laboratory procedures that should be followed 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.

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) per 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 the inoculation of bacterial suspension in a growth medium, for instance, MHA, check that there must not be
an excessive amount of inoculum.  This check is accomplished by pressing the swab on the sides of the bacterial
suspension tube before inoculating it to the MHA plate.  Inoculate the MHA plate with the swab by starting from the
top, carefully swabbing from side to side down to the bottom of the plate.  This step is done three times after each
rotation of the plate (usually 60 degrees) to cover the whole MHA plate with evenly swabbed inoculum.

In the MIC method, the inoculum is delivered 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 ml.

The addition of antimicrobial disks on inoculated MHA plates can be done 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 can be best applied with only 12 antimicrobial disks.  Each disk must be pressed towards the surface of
the agar to ensure that disk displacement will not occur during incubation.

Incubation of the inoculated MHA 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 in ambient temperature.  Other organisms
require longer times (e.g., 24 hours).  Fastidious pathogens such as Haemophilus and Neisseria spp. require 16 to 18
and 20 to 24 hours, respectively.  In the MIC method, the inoculated panel can be incubated using the same
temperature and incubation time requirements.  Additionally, panels are recommended to be covered by a plastic seal
or be contained in a plastic bag to prevent the panels from dehydration since each well contains a relatively minimal
amount of bacterial suspension.

Indications
Susceptibility testing for antimicrobials is necessary for patients who raise suspicion of infection with specific
pathogens based on disease manifestation and clinical correlation.  Antibacterial agents are then used to detect
sensitivity or resistance from bacteria.  Although the purpose of this review is primarily towards the susceptibility
testing for bacterial pathogens, it is important to note that antifungal susceptibility tests also exist for addressing

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fungal infection (e.g., Candida, Aspergillus spp.).  Furthermore, antiviral susceptibility tests are also available (e.g.,
influenza) via molecular technologies including sequencing analysis such as Sanger and pyrosequencing methods.[6]

Potential Diagnosis
 A unique impact of AST on patient management is the identification of the specific diagnosis, and additionally,
targeting the particular etiologic agent causing the disease.  No two patients can be managed similarly, especially if
they have the same signs and symptoms (disease manifestation) but with different treatment regimens because the
same causative organism can have different resistance patterns. For example, two patients may present with an
ordinary strain of Staphylococcus aureus vs. methicillin-resistant Staphylococcus aureus (MRSA); and another
example would be patients with drug-susceptible (DS-TB) and drug-resistant tuberculosis (DR-TB).

Normal and Critical Findings

For disk diffusion, measuring the zone of inhibition is done by using a dedicated caliper.  Correctly measure the
diameter by the edges of the inhibition zone.  For MIC panels, reading each set of wells for an antibiotic drug is done. 
MIC determination is by either a clear or slight whiteness on the well.  Reporting the results of the inhibition zones
and MIC breakpoints is made 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.  The Clinical Laboratory
Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)
developed expert-approved guidelines on breakpoints for reporting results of these methods (e.g., CLSI M100-
ED29:2019 Performance Standards for Antimicrobial Susceptibility Testing, EUCAST Clinical breakpoints for
bacteria).[7][8]

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 AST procedure can significantly impact succeeding areas of laboratory
workflow which in turn could later affect patient diagnosis, treatment, and management.  Support systems of the
laboratory workflow require strict monitoring, and laboratory personnel should be well trained and competent enough
to perform the procedure.

For instance, "poor specimen quality" can be the first sign leading to an erroneous result.  A perfectly carried out
inoculation to MHA plate using “mixed colonies” will turn out unsatisfactory results.  Poor standardization of
bacterial suspension and a "longer depth of agar" could yield misleading endpoints.  Supportive supervision for
laboratory staff is necessary, to prevent wasting time and resources.

Purchasing poor quality MIC panels can lead to "dehydrated wells" or "mixed wells."  Doing the procedure without
personal protective equipment (PPE) can increase the incidence of laboratory-acquired infections.  Inadequacy and
lack of supplies needed to perform AST will extend turn-around time and therefore, decrease laboratory productivity
and delay patient therapy.  Providing AST on drugs that do not align with the hospital’s formulary makes laboratory
service available yet ineffective.

Complications
Inconsistencies in the AST results must be investigated and acted upon immediately.  No results should be released
when quality control measures are not satisfactory.  Releasing inaccurate drug susceptibility or resistance results can
inflict more harm to the patients, leading to severe clinical conditions and poor prognosis.  A worse consequence in
delivering false AST results can result in wrong treatment management plans which might cause further mutations to
these infectious organisms, exposing the patients and the community to a higher risk.[9]

Patient Safety and Education

Patients should be adequately informed about the AST and its indications, patient requirements, and its clinical use for
patient management.  Healthcare providers such as physicians, laboratory personnel, nurses, and pharmacologists are
encouraged to disseminate correct information about the test.  However, interpretation of the AST results must take
place between the patient and the physician to facilitate good compliance with the prescribed medications and to

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prevent self-medication. With the rise of antimicrobial resistance, the importance of AST requires an emphasis on
medical, laboratory, and nursing staff, as well as patients and their family members, and the whole community leading
to a unified approach.[10][11][12]

Clinical Significance
Once antimicrobial susceptibility results become available, treatment regimens for each patient can be developed by
healthcare providers.  Prescribed medications of appropriate antibiotics need individualization for each patient
diagnosed with an infectious disease.  Moreover, resistance from primary drugs will require a higher level of
antimicrobial stewardship, including prudent use of second-line drugs.

Review Questions

Access free multiple choice questions on this topic.

Comment on this article.

References
1. Lagier JC, Edouard S, Pagnier I, Mediannikov O, Drancourt M, Raoult D. Current and past strategies for bacterial
culture in clinical microbiology. Clin Microbiol Rev. 2015 Jan;28(1):208-36. [PMC free article: PMC4284306]
[PubMed: 25567228]
2. Sawatzky P, Liu G, Dillon JA, Allen V, Lefebvre B, Hoang L, Tyrrell G, Van Caeseele P, Levett P, Martin I.
Quality Assurance for Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae in Canada, 2003 to 2012. J
Clin Microbiol. 2015 Nov;53(11):3646-9. [PMC free article: PMC4609721] [PubMed: 26338862]
3. Graham DR, Dixon RE, Hughes JM, Thornsberry C. Disk diffusion antimicrobial susceptibility testing for clinical
and epidemiologic purposes. Am J Infect Control. 1985 Dec;13(6):241-9. [PubMed: 3936382]
4. Coorevits L, Boelens J, Claeys G. Direct susceptibility testing by disk diffusion on clinical samples: a rapid and
accurate tool for antibiotic stewardship. Eur J Clin Microbiol Infect Dis. 2015 Jun;34(6):1207-12. [PMC free
article: PMC4426127] [PubMed: 25698312]
5. Xie YL, Chakravorty S, Armstrong DT, Hall SL, Via LE, Song T, Yuan X, Mo X, Zhu H, Xu P, Gao Q, Lee M,
Lee J, Smith LE, Chen RY, Joh JS, Cho Y, Liu X, Ruan X, Liang L, Dharan N, Cho SN, Barry CE, Ellner JJ,
Dorman SE, Alland D. Evaluation of a Rapid Molecular Drug-Susceptibility Test for Tuberculosis. N Engl J Med.
2017 Sep 14;377(11):1043-1054. [PMC free article: PMC5727572] [PubMed: 28902596]
6. Santos DA, Barros ME, Hamdan JS. Establishing a method of inoculum preparation for susceptibility testing of
Trichophyton rubrum and Trichophyton mentagrophytes. J Clin Microbiol. 2006 Jan;44(1):98-101. [PMC free
article: PMC1351939] [PubMed: 16390955]
7. Kassim A, Omuse G, Premji Z, Revathi G. Comparison of Clinical Laboratory Standards Institute and European
Committee on Antimicrobial Susceptibility Testing guidelines for the interpretation of antibiotic susceptibility at a
University teaching hospital in Nairobi, Kenya: a cross-sectional study. Ann Clin Microbiol Antimicrob. 2016
Apr 11;15:21. [PMC free article: PMC4827198] [PubMed: 27068515]
8. Sahu C, Jain V, Mishra P, Prasad KN. Clinical and laboratory standards institute versus European committee for
antimicrobial susceptibility testing guidelines for interpretation of carbapenem antimicrobial susceptibility results
for Escherichia coli in urinary tract infection (UTI). J Lab Physicians. 2018 Jul-Sep;10(3):289-293. [PMC free
article: PMC6052810] [PubMed: 30078964]
9. Zaman SB, Hussain MA, Nye R, Mehta V, Mamun KT, Hossain N. A Review on Antibiotic Resistance: Alarm
Bells are Ringing. Cureus. 2017 Jun 28;9(6):e1403. [PMC free article: PMC5573035] [PubMed: 28852600]
10. Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. P T. 2015 Apr;40(4):277-83. [PMC free
article: PMC4378521] [PubMed: 25859123]
11. Aslam B, Wang W, Arshad MI, Khurshid M, Muzammil S, Rasool MH, Nisar MA, Alvi RF, Aslam MA, Qamar
MU, Salamat MKF, Baloch Z. Antibiotic resistance: a rundown of a global crisis. Infect Drug Resist.
2018;11:1645-1658. [PMC free article: PMC6188119] [PubMed: 30349322]
12. Rather IA, Kim BC, Bajpai VK, Park YH. Self-medication and antibiotic resistance: Crisis, current challenges,
and prevention. Saudi J Biol Sci. 2017 May;24(4):808-812. [PMC free article: PMC5415144] [PubMed:
28490950]
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