ANTIMICROBIAL

SUSCEPTIBILITY TESTING
Clinical and Laboratory Perspectives

PIONEERING DIAGNOSTICS
INTRODUCTION

The World Health Organization (WHO) has declared antimicrobial AST must also:
resistance as a major global health threat due to the misuse and
• Promote optimal antibiotic use – the range of antibiotics
overuse of broad-spectrum antibiotics driving the emergence of
reported should allow for optimal treatment in accordance with
multi-drug resistant and pan resistant “superbugs”.
local antimicrobial stewardship policies.
The WHO Global Action Plan on Antimicrobial Resistance1 identifies
the need to increase investment in diagnostic tools capable of • Provide cumulative data - that is organism and infection site
informing healthcare practitioners of the susceptibility of pathogens related for the generation of local antibiograms to allow
to available antibiotics. Given this key objective, antimicrobial appropriate empirical antibiotic selection.
susceptibility testing (AST) clearly plays an important role in not This practical guide provides a step-by-step approach to AST
only ascertaining the best possible antimicrobial option for patient and antibiotic therapy selection, and will focus on:
treatment but also ensuring the detection of antimicrobial resistance
• The role of AST in the clinical setting and selection of initial
mechanisms in clinical isolates.
empiric therapy based upon clinical and preliminary laboratory
Therefore, AST must be: test data and cumulative antibiograms.
• Accurate – standardized test methods capable of detecting a • Basic concepts of AST, and laboratory methods for
wide range of different antimicrobial resistance mechanisms antimicrobial susceptibility and resistance testing.
need to be used by clinical microbiology laboratories.
• Interpretation of AST results for clinical treatment and the
• Timely – results should be generated within a relevant timeframe
collection and application of cumulative AST data for wider
to allow optimal antibiotic prescribing practices.
clinical applications such as antibiograms and Antimicrobial
• Reliably reported – results reported as susceptible or resistant Stewardship Programs (ASPs).
need to apply international Minimum Inhibitory Concentration
(MIC) breakpoints, be selectively reported and include
interpretative comments.

The content of this booklet has been written by:

„ Professor David L. PATERSON, „ Narelle GEORGE
NUS Saw Swee Hock School of Public Health (Joint) Supervising Scientist, Microbiology, Pathology
NUS Yong Loo Lin School of Public Health (Joint) Queensland, Central Laboratory, Queensland Public
Director, ADVANCE-ID Health and Scientific Services, Queensland Health,
University of Queensland Faculty of Medicine (Honorary) Herston, Queensland, Australia

We wish to thank them for sharing their valuable knowledge and expertise on Antimicrobial Susceptibility Testing from both a laboratory and a clinical perspective, and for their
dedicated involvement in this booklet.
CONTENTS

PRE-AST POST-AST
CLINICAL DECISION-MAKING PROCESS CLINICAL AND LABORATORY PERSPECTIVES
1. What is AST?����������������������������������������������������������������������������������������������������������� 8 1. Interpretation of AST results and reports��������������������������������������������������� 44
2. What are the clinical benefits of performing AST?������������������������������������� 9 2. Use of cumulative laboratory AST data – the antibiogram������������������� 50
3. How does the clinical laboratory support antimicrobial selection?��� 12 3. Antimicrobial stewardship and diagnostic stewardship������������������������ 52
4. What are the types and mechanisms of antimicrobial resistance?���� 14
4.1. What is intrinsic antimicrobial resistance?������������������������������������������� 14 CONCLUSIONS AND FUTURE PERSPECTIVES������������������ 56
4.2. What is acquired antimicrobial resistance?������������������������������������������ 14
LIST OF ABBREVIATIONS���������������������������������������������������� 57
4.3. What are the main mechanisms of antimicrobial resistance
acquisition?����������������������������������������������������������������������������������������������������� 15 REFERENCES���������������������������������������������������������������������� 58

AST
BASIC CONCEPTS AND METHODS
1. What are the basic concepts of AST?���������������������������������������������������������� 20
1.1. Minimum Inhibitory Concentration (MIC)�������������������������������������������� 20
1.2. Clinical breakpoints�������������������������������������������������������������������������������������� 22
2. What AST methods are used in the laboratory?��������������������������������������� 28
2.1. Phenotypic methods - manual������������������������������������������������������������������ 29
2.2. Phenotypic methods - semi/fully automated systems��������������������� 32
2.3. Phenotypic methods - fast AST����������������������������������������������������������������� 34
2.4. Detection of antimicrobial resistance genes or mechanisms��������� 36

4 5
 PRE-AST
CLINICAL DECISION-MAKING
PROCESS
 PRE-AST - CLINICAL DECISION-MAKING PROCESS

1 WHAT IS AST? Once AST results become available, the clinician is able either to confirm
the initial empiric therapy or provide alternate antibiotic options (targeted
Antimicrobial susceptibility testing (AST) is an in vitro diagnostic test
therapy) (Figure 1). Furthermore, the results can facilitate antimicrobial
performed in the microbiology laboratory that measures the ability of an
stewardship by directing therapy towards an alternative agent, which may
antimicrobial agent to inhibit the growth of a microorganism.
be less toxic or more narrow-spectrum (effective against only a limited
Standardized test methods using a range of different antimicrobial agents range of organisms). More cost-effective antimicrobial options can also be
are used depending upon the type of microbe and the site of infection. chosen from the list of agents to which the infecting organism is susceptible.
Additional phenotypic and/or genotypic techniques may be utilized to
There are also benefits of AST which extend beyond the immediate
determine the specific resistance mechanism involved.
patient. AST allows detection of clinically significant resistant organisms
A laboratory report listing results for both susceptible and resistant which are important for infection control, e.g., methicillin-resistant
microorganisms is issued 12-72 hours post collection of the clinical sample, Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE),
depending on the AST method used. extended spectrum beta-lactamases (ESBL), carbapenemase-producing
Enterobacterales (CPE). Other longer-term benefits include compiling
„ A susceptible result indicates a high likelihood of therapeutic success
cumulative AST data for use in local / national / international epidemiological
at a standard dosing regimen.
data sources or antibiograms (see page 50 for more details).
„ Whereas a resistant result indicates a high likelihood of therapeutic
failure even at an elevated exposure.
2 WHAT ARE THE CLINICAL BENEFITS OF
Although fast susceptibility testing and gene detection may give more PERFORMING AST?
rapid results, routine phenotypic AST using conventional methods is usually Without AST, antimicrobial therapy used by clinicians would only be an
required to provide a range of possible antibiotic options for treatment. “educated guess”.
In fact, in seriously ill patients requiring immediate initiation of
Should AST be performed on all clinical samples? antimicrobials, the initial doses are always given in the absence of AST
Simply put, there is no need for AST if there is not an infection results. In this situation, clinicians use their knowledge of several factors in
which needs to be treated. Unnecessary testing is not just a waste of order to determine a rational empiric antimicrobial prescription:
money but may also “encourage” unnecessary antibiotic prescribing „ The individual’s recent history of antibiotic use and antibiotic allergies,
because medical staff are trained to respond to laboratory results. „ Prior colonization or infection with antimicrobial resistant organisms,
Unnecessary antibiotic prescribing can lead to adverse impacts on the „ Local cumulative antibiograms developed at hospital level,
microbiome without providing any clinical benefit, and also contribute
„ Current infection outbreaks of antimicrobial resistant organisms.
to the spread of antimicrobial resistance.
Some examples where samples should not be sent to the microbiology AST measures the ability of an antimicrobial to inhibit the
laboratory for processing include: growth of a microorganism.
ƒ Urine from patients without symptoms, The ability of AST methods to detect antimicrobial
ƒ Central venous line tips, from lines removed because they are not resistance and reduce the time to test results is critical.
needed any longer,
ƒ Swabs of ulcers without surrounding cellulitis.

8 9
 PRE-AST - CLINICAL DECISION-MAKING PROCESS

Figure 1. Place of AST in the patient workflow

Source: bioMerieux

ANTIBIOTIC THERAPY ANTIBIOTIC THERAPY ANTIBIOTIC THERAPY

INITIATION OPTIMIZATION DISCONTINUATION
SAMPLES

ID AST

Patient Consolidated AST data Biomarkers Pathogen Antimicrobial susceptibility Biomarkers

history Local antibiograms and outbreaks identification (ID) testing (AST)

EMPIRIC THERAPY TARGETED THERAPY MONITORING THERAPY

OPTIMIZE THERAPEUTIC EFFICACY WHILE MINIMIZING ADVERSE DRUG EVENTS

CASE STUDY 1. Outcome

A 24-year-old woman returns from a trip to visit family on a Pacific island. The wound swab grew a pure growth of Staphylococcus aureus,
She is previously healthy. While visiting, she gets a «band tattoo» around with resistance to cephalexin and clindamycin but susceptibility to
her upper right arm. Unfortunately, the tattoo site becomes infected with trimethoprim-sulfamethoxazole. The general practitioner recalled the
redness and discharge of pus. She develops a fever and becomes fatigued. patient who had not improved after 48 hours of cephalexin therapy.
Gram stain of a swab of the pus shows gram-positive cocci in clusters and
ÎBased on the AST results, the general practitioner changed
pairs (Figure 2).
the therapy to trimethoprim-sulfamethoxazole with rapid
Why is AST important in this case? resolution of the infection.
Administration of antibiotics is indicated for this wound infection related Figure 2. Gram stain showing gram-positive cocci in clusters and
to the recent tattoo. On the Gram stain, the appearance of the gram- pairs
positive cocci in clusters is suggestive evidence that Staphylococci are
Source: Pathology Queensland, reproduced with permission.
the primary pathogen causing the infection.
Her general practitioner could use cephalexin because that has been an
effective therapy for Staphylococcus aureus and Streptococcus pyogenes
for many years. However, Staphylococcus aureus may be resistant to
cephalexin. MRSA strains are resistant to beta-lactam antibiotics like
anti-staphylococcal penicillins (such as (flu)cloxacillin) and to most
cephalosporins (such as cephalexin).
AST is necessary both to detect resistance such as presence of MRSA
in which cephalexin would not be effective and also to determine if
orally administered options such as clindamycin or trimethoprim-
sulfamethoxazole are active.

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 PRE-AST - CLINICAL DECISION-MAKING PROCESS

3 HOW DOES THE CLINICAL LABORATORY „ Matrix-assisted laser desorption/ionization time-of-flight

SUPPORT ANTIMICROBIAL SELECTION? mass spectrometry (MALDI-TOF MS): allows accurate organism
identification to the species level within minutes compared to hours
To determine the most appropriate antimicrobial for the treatment of a
using conventional technology.
specific clinical infection, isolation and identification (ID) of the suspected
„ Antigen detection kits: for direct detection of a specific microorganism
pathogen is required followed by performance and interpretation of a range
in a clinical sample in less than 1 hour (e.g. urinary antigen detection kits
of antimicrobial susceptibility tests (AST).
for Legionella pneumophila and Streptococcus pneumoniae).
Clinical samples for ID/AST testing, including positive blood cultures, are „ Rapid molecular methods: for direct detection of certain organisms
routinely processed using traditional in vitro selective and non-selective and antimicrobial resistance genes within 1 to 4 hours.
agar media, inoculated and incubated aerobically/anaerobically for „ Chromogenic culture media: for direct detection of multi-drug
24-48 hours. Potential pathogens are identified to the species level and resistant organisms in clinical samples within 18-24 hours.
conventional AST is then performed. After a further incubation period of up
to 18 hours or more, the AST report is issued. Therefore, AST results may not Figure 3 shows the various laboratory steps in the processing of a clinical
be available until up to 72 hours post-initiation of empiric therapy. specimen for isolation, identification and AST of bacterial pathogens with
indication of turn-around times for the generation of clinically useful
However, a number of laboratory tests can provide important preliminary
information that can impact antimicrobial selection.
information to support the selection of the most appropriate antimicrobial
therapy until conventional AST results are available. These tests include:
Rapid laboratory tests such as microscopic appearance,
„ Microscopic examination (Gram stain): provides information about organism identification, detection of specific microbial
the types of microorganisms that may be present. The choice of empiric antigens and antimicrobial resistance genes can support the
antibiotics will be different depending on the site of infection and antibiotic selection process before AST results are available.
whether gram-positive or gram-negative bacteria are seen.

Figure 3. Laboratory processing of clinical samples

Source: Pathology Queensland, reproduced with permission

RESULT RESULT CHROMOGENIC

WITHIN WITHIN CULTURE
4-8 HOURS 24 HOURS SPECIFIC MDR

GRAM STAIN
MALDI-TOF MS
AG DETECTION PATIENT
SPECIMEN EMPIRIC SPECIMEN
CULTURE ANTIBIOTIC

RESULT
PATHOGEN RESULT DIRECT
WITHIN
IDENTIFICATION WITHIN MOLECULAR
48-72
AST 4 HOURS TESTING
HOURS

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 PRE-AST - CLINICAL DECISION-MAKING PROCESS

4 WHAT ARE THE TYPES AND MECHANISMS OF Figure 4. Intrinsic (Natural) resistance versus Acquired resistance
ANTIMICROBIAL RESISTANCE? Source: bioMérieux
All antimicrobials have a known clinical spectrum of activity that is
determined during the initial clinical studies prior to the release of the All Susceptible (S) All Resistant (R) Mixture of (R) & (S)
antimicrobial for clinical use. The product information for antimicrobial
agents often lists the genus/species occurring in clinical trials that
are susceptible to the specific antimicrobial. However, susceptibility
may change over time with the development of resistance against the
antimicrobial following its clinical use.
Natural resistance Acquired resistance
Conversely, organisms against which the antimicrobial has no activity are • In their wild-type state • Results from a gene mutation
not included in the list of susceptible organisms. The lack of activity may be • Characteristic of a bacterial or gene acquisition
due to the presence of intrinsic resistance mechanisms. species and predictable • Unpredictable

4.1. WHAT IS INTRINSIC ANTIMICROBIAL RESISTANCE?

Intrinsic (or “natural”) resistance genes form part of the genome in Taken into consideration for the
Justifies the need for AST
all strains belonging to a particular species. AST directed at the detection empiric antibiotic decision
of this resistance is generally unnecessary. However, failure to detect
resistance in this group when expected should prompt repeated testing,
particularly to confirm the organism identification. Reporting of intrinsic 4.3. WHAT ARE THE MAIN MECHANISMS OF
resistance is variable, with some laboratories reporting all agents affected ANTIMICROBIAL RESISTANCE ACQUISITION?
as “R” despite how they “test”, others suppressing result reporting, or yet Horizontal gene transfer (gene acquisition)
others reporting “as tested” and adding comments, e.g. natural resistance 1. Conjugation is the main mechanism of horizontal gene transfer. It
of Pseudomonas aeruginosa to beta-lactams due to the presence of beta- occurs when genes encoding antibiotic resistance are acquired from
lactamases. other bacteria, usually via plasmids – circular elements of DNA that can
4.2. WHAT IS ACQUIRED ANTIMICROBIAL RESISTANCE? be transferred not just to the same species but also to other genera.
Resistance to almost all classes of antimicrobials can be transferred on
A more common form of antimicrobial resistance (AMR) occurs via plasmids.
gene acquisition or gene mutation where a normally susceptible 2. Transformation occurs via direct adsorption of DNA from the external
strain becomes resistant to a particular antimicrobial after initially being environment. This mechanism is most prevalent in Streptococcus
susceptible. pneumoniae with respect to acquisition of beta-lactam resistance genes.
ÎFor example, the acquisition of genes encoding extended-spectrum 3. Transduction occurs where DNA encoding resistance genes (often small
beta-lactamases (ESBLs, such as CTX-M) by Klebsiella pneumoniae plasmids) are incorporated into bacteriophage heads. New cells acquire
or Escherichia coli. The ESBLs typically cause resistance to penicillins, the DNA through direct injection of genetic material via phage binding.
first, second and third generation cephalosporins and monobactams. This mechanism is most prevalent in those species with high rates of
lysogeny such as staphylococci.

14 15
 PRE-AST - CLINICAL DECISION-MAKING PROCESS

Figure 5. The three mechanisms of horizontal gene acquisition2 Figure 7. Three mechanisms of action against antimicrobial
Reproduced from Liu Y, et al. Microorganisms 2020;8(8):1211. CC BY 4.0 encoded by resistance genes3
A Conjugation B Transformation C Transduction Reproduced with permission from Todars Online Textbook of Bacteriology
https://textbookofbacteriology.net/resantimicrobial_3.html
Free DNA
Plasmid Bacterial DNA Efflux pump
Donor
Donor

Plasmid
Recipient Recipient Antibiotic
degrading
Antibiotic enzyme
Vertical gene transfer (and gene mutation)
Antibiotic
Unlike plasmid acquisition of genes, gene mutation only allows vertical gene Antibiotic
resistance
transfer within the population from parent to daughter cells during genes
replication. Spontaneous gene mutations on the bacterial chromosome
can also confer resistance to antimicrobials. Antibiotic
Antibiotic
Figure 6. Vertical gene transmission altering
Source: bioMérieux
enzyme

Resistance
1. Efflux pumps are high-affinity reverse transport systems located in the membrane that
transport the antibiotic out of the cell. This is the mechanism of resistance to tetracycline.
2. Enzymatic alteration of an antibiotic in a way that it loses its activity. In the case of
streptomycin, the antibiotic is chemically modified so that it will no longer bind to the
ribosome to block protein synthesis.
3. Enzymatic degradation of an antibiotic, thereby inactivating it. For example, the
penicillinases are a group of beta-lactamase enzymes that cleave the beta-lactam ring
of the penicillin molecule.

Intrinsic antimicrobial resistance is a stable part of the

Five main mechanisms of resistance against antimicrobials genome in all strains belonging to a particular species.
Antimicrobial resistance may also be acquired from other
„ Enzymatic degradation of the agent, as in beta-lactamase.
bacteria, usually via plasmids.
„ Enzymatic alteration of a side chain of the antimicrobial, as seen with
phosphorylation of aminoglycoside antimicrobials. Antimicrobial resistance mechanisms include enzymatic
degradation, efflux pump to remove antimicrobials and
„ Changes to the structure of outer membrane porins to restrict entry
outer membrane porin changes to restrict antimicrobial
into the cell (e.g. carbapenem resistance in Pseudomonas spp.).
penetration of the bacterial cells.
„ Up-regulation of efflux pumps to remove antimicrobials from inside the
cell (e.g. fluoroquinolones, macrolides and other antimicrobial classes as
seen in gram-positive organisms including Streptococcus spp.).
„ Target site modification, whereby bacteria alter the antibiotic’s binding
site, reducing the drug’s effectiveness (e.g. Streptococcus pneumoniae
modifies its penicillin-binding proteins to penicillin).
16 17
 AST
BASIC CONCEPTS AND METHODS
 AST - BASIC CONCEPTS AND METHODS

1 WHAT ARE THE BASIC CONCEPTS OF AST? The relationship of MIC to MBC is a reflection of the different modes of
antimicrobial activity.
1.1. MINIMUM INHIBITORY CONCENTRATION (MIC)
„ For bactericidal antibiotics (e.g. beta-lactams), the MIC and MBC
The basic unit of measure for AST is the MIC or Minimal will be the same or within one doubling dilution.
Inhibitory Concentration. „ For bacteriostatic agents (e.g. macrolides), the MIC will be lower
It is defined as the lowest concentration of an antibiotic than the MBC indicating that the antibiotic needs to be given at higher
that is required to inhibit visible in vitro microbial growth doses to affect microbial viability.
after overnight incubation.4
Figure 8. Minimum Inhibitory Concentration (MIC) and Minimum
MICs are used to measure the susceptibility of a pathogen to an
Bactericidal Concentration (MBC)6
antimicrobial to aid in the selection of the most appropriate antimicrobial
Adapted from Microbe Online
therapy.
MIC
„ A low MIC indicates susceptibility to low concentrations of the
antimicrobial.
„ A high MIC indicates potential resistance to the antimicrobial.
To determine the MIC using the Broth Microdilution method (BMD), a series
of doubling dilutions of the antibiotic in a chemically defined broth (e.g.
cation-adjusted Mueller-Hinton broth [MHB]) is inoculated with a standard 0 0.25 0.5 1 2 4 8

concentration
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
dilution of the test organism. After incubation at 35°C for 18-24 hours, the

Drug
No drug
broth is examined for turbidity as an indicator of growth or resistance to the control
Microbial growth after overnight incubation on drug-free agar
antibiotic.5 The MIC is the first concentration that shows no turbidity
or growth (Figure 8).
In addition to the MIC, the Minimum Bactericidal Concentration (MBC)
Drug-free
agar

can also be determined by subculturing each tube in the dilution series

onto non-selective agar and examining for microbial growth after overnight
incubation. The MBC is defined as the lowest concentration of an antibiotic
required to inhibit microbial growth after subculture onto antibiotic- MBC
free media4 (i.e. the minimum concentration required to kill a specific
bacterium). Only the BMD methods allow both the MIC and MBC of an
antimicrobial to be measured in a single test format (Figure 8). The aim of susceptibility testing and MIC measurement
is to predict the likely success or failure of a chosen
therapy and determine effective antibiotic dosing.

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 AST - BASIC CONCEPTS AND METHODS

1.2. CLINICAL BREAKPOINTS Where there are clear differences in the distribution between the two
groups then the susceptible (S) and resistant (R) breakpoints can be
The exact MIC is not routinely required to manage the majority of set. The breakpoint MIC is the MIC that separates sensitive and resistant
infections except for certain clinical conditions (e.g. meningitis and strains (Figure 9). Isolates with no known mechanisms that fall between
endocarditis), where precise dosing is required to achieve therapeutic levels, the two lie in the intermediate (I) zone.
or in the case of multi-drug resistant organisms where the antimicrobial
choices are limited. Usually, clinicians only need to know whether the MIC Another attribute of the breakpoint is correspondence to achievable serum
is low enough to render the bacterium susceptible to the antibiotic or high drug concentrations using standard human doses (except for select urine,
enough to indicate microbial resistance and therefore therapeutic failure, central nervous system, cerebro-spinal fluid breakpoints).9
and this is determined using clinical breakpoints. Where breakpoints are not clear cut, ECVs/ECOFFs offer an alternative,
until more exact clinical breakpoints can be established. Lack of breakpoints
Breakpoints are MIC values or zone diameters* that is particularly problematic when the clinician needs to treat with a non-
allow the organism to be categorized as susceptible (S), calibrated antimicrobial. The ECV/ECOFF value provides some guidance as
susceptible dose dependent (SDD), intermediate (I), to the potential for eradication based on the MIC alone.
resistant (R) or non-susceptible (NS) to a particular
antibiotic. Figure 9. Clinical breakpoints and epidemiological cut-off10
Reproduced with permission from Tascini C, et al. Ital J Med. 2016;10:289-200. CC BY-NC 4.0
Breakpoints vary according not only to the antimicrobial
but also to the organism. Number
of isolates
Clinical
EVC/ECOFF
* A zone diameter refers to the inhibition zone, i.e. the area around an antibiotic disc on an Breakpoint
agar plate where bacterial growth has been suppressed (for more details see section 2.1.4).
Susceptible Resistant
International organizations such as the Clinical & Laboratory Standards
Acquired or mutational resistance
Institute (CLSI)5 and the European Committee on Antimicrobial
Susceptibility Testing (EUCAST)7, as well as national organizations such as Wild type

the U.S. FDA8, are responsible for the development of breakpoint tables
that define susceptible and resistant MICs for antibiotics against a range of MIC

commonly encountered bacteria of medical importance. S I R

How are breakpoints determined?

To determine clinical breakpoints for a given bug/drug combination, various In certain cases, breakpoints cannot be set:
data are combined:
„ where they divide a “wild type” distribution,
„ analysis of MIC distributions for a large number of strains of the
„ where the organism is known to be a poor target for that antibiotic,
same species against a single antibiotic. This will include organisms
with no known resistance as well as those for which the resistance „ where there is insufficient clinical evidence that the organism is a good
mechanism is known, target for that antibiotic.
„ the epidemiological cut-off value (ECV for CLSI or ECOFF for Although CLSI and EUCAST use the same definitions for Susceptible and
EUCAST) that segregates natural resistance versus acquired resistance, Resistant, the definition for the Intermediate category is less clear-cut as
„ pharmacokinetic (how the drug moves through the body) and shown in Table 1 (page 24).
pharmacodynamic (how the drug affects the bacteria) data,
„ clinical outcome data from patients treated for the same drug/bug
combination. This helps to ensure that the breakpoints are based on
actual therapeutic success rates and not just in vitro susceptibility.
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 AST - BASIC CONCEPTS AND METHODS

Table 1. EUCAST and CLSI breakpoint definitions5,7,11,12 From a clinical point of view, the susceptible and resistant categories can be
clearly understood, but reporting of the intermediate category has resulted in
Adapted from Clinical and Laboratory Standards Institute: Performance standard for Antimicrobial
Susceptibility Testing, M100-S34, 2024; EUCAST Breakpoint tables for interpretation of MICs clinicians avoiding use of these antibiotics. This prompted EUCAST to amend
and zone diameters, version 13.1, valid from 2023-06-29: https://www.eucast.org/fileadmin/ its intermediate category definition to encourage use of these agents, in
src/media/PDFs/EUCAST_files/Breakpoint_tables/v_13.1_Breakpoint_Tables; EUCAST New
Definitions of S, I and R from 2019: https://www.eucast.org/newsiand order to limit the use of more broad-spectrum agents against a background
of increasing multi-drug resistance.11 Furthermore, in 2019, EUCAST also
EUCAST CLSI
added a new category for Area of Technical Uncertainty (ATU).12
S - Susceptible, standard dosing regimen: A microorganism is categorised as For newer antimicrobial agents or organisms such as Streptococcus pyogenes,
"Susceptible, standard dosing regimen", when there is a high likelihood of therapeutic
success using a standard dosing regimen of the agent.
for which no resistance to penicillin has been described, CLSI recommends
that the results be interpreted as S for susceptible or NS for non-susceptible.

R - Resistant: A microorganism is categorised as «Resistant» when there is a high likelihood Both EUCAST and CLSI provide information relating to the dosing regimens
of therapeutic failure even when there is increased exposure. to which their breakpoints are calibrated. EUCAST has added meningitis
MIC breakpoints for some drug/bug combinations to ensure accurate
reporting by laboratories.7
I - Susceptible, increased exposure*: I - Intermediate: a buffer zone for technical
a microorganism is categorised as uncertainty. Thus, when interpreting AST results from the laboratory, clinicians need
«Susceptible, increased exposure*» when
I^ - Intermediate category was added to
to be aware of which standard AST method is used by their local
there is a high likelihood of therapeutic microbiology laboratory.
success because exposure to the agent is highlight those antimicrobial agents that
concentrate in urine and the likelihood
increased by adjusting the dosing regimen
of treatment success when the agent is While most laboratories will standardize on either CLSI or EUCAST
or by its concentration at the site of
prescribed for uncomplicated urinary tract breakpoints for susceptibility testing, many will use a combination of both
infection.
infections. to cover reporting for a wider range of organisms that may not be included
*Exposure is a function of how the mode SDD - Susceptible dose-dependent: in the guideline they normally follow.
of administration, dose, dosing interval,
use of a high dosage to treat the site of
infusion time, as well as distribution and
infection, resulting in higher antibiotic N.B. In the US, laboratories may also use FDA breakpoints, and may not
excretion of the antimicrobial agent will
influence the infecting organism at the site exposure and likelihood of clinical efficacy. be able to use CLSI or EUCAST breakpoints if they are not FDA-cleared on
of infection their systems.

Breakpoints that allow the organism to be categorized as

susceptible, susceptible dose dependent, intermediate,
NS - Non-susceptible: a category used resistant or non-susceptible to a particular antibiotic form
for isolates for which only a susceptible the basis of all laboratory AST methods and reporting.
breakpoint is designated because of the
absence or rare occurrence of resistant Determination and publication of clinical breakpoints is
strains. Isolates with MICs above the
susceptible breakpoint are reported as non
largely the responsibility of international organizations
susceptible. such as CLSI, EUCAST and the FDA, though local/country-
specific guidance may also exist.

ATU - Area of Technical Uncertainty:

uncertain interpretation of antimicrobial
susceptibility testing (AST) results.

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 AST - BASIC CONCEPTS AND METHODS

CASE STUDY 2. Outcome

A 65-year-old man develops headache and neck stiffness. The emergency The microbiologist calls the intensive care unit doctor caring for the
room doctor recognizes this as possible meningitis and therefore patient and interprets the MIC values - taking care to use the breakpoints
performs a lumbar puncture. The cerebrospinal fluid has 450 white blood for penicillin and ceftriaxone for Streptococcus pneumoniae causing
cells per microliter, 95% of which are neutrophils. Gram-positive cocci are meningitis.
seen in the Gram stain.
ÎAs a result of applying the appropriate breakpoints, the
Why is AST important in this case? patient is treated with high-dose ceftriaxone and eventually
makes a full recovery.
Meningitis is a life-threatening infection. The Gram stain shows
numerous neutrophils and gram-positive cocci in pairs or short chains, more Figure 10. MIC to penicillin between 1 and 2 mg/L
characteristic of Streptococcus pneumoniae, so bacterial meningitis
Source: bioMérieux
is likely. High doses of intravenous penicillin or ceftriaxone are the
antibiotics of choice for pneumococcal meningitis. However, resistance
to penicillin and/or ceftriaxone may occur.
Antibiotics may not penetrate the blood-brain barrier very well so it is
important to assist in the optimization of therapy by not just performing
disc susceptibility but also performing MICs for penicillin and ceftriaxone.
Different breakpoints are applied for susceptibility when Streptococcus
pneumoniae causes meningitis as compared to when it causes pneumonia.
Penicillin MIC results are high (Figure 10). MICs above 0.06 mg/L are
interpreted as resistant in cases of meningitis.

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 AST - BASIC CONCEPTS AND METHODS

2 WHAT AST METHODS ARE USED IN THE 2.1. PHENOTYPIC METHODS - MANUAL
LABORATORY? 2.1.1 Broth Microdilution (BMD)
A variety of diagnostic methods are available in the laboratory to determine
both antimicrobial susceptibility (AST) and antimicrobial resistance (AMR) Broth dilution techniques are the most widely used AST methods in both
detection (Figure 11). Reference Laboratories (e.g. EUCAST, CLSI)7,13 and in the routine clinical
laboratory, because they allow for determination of the MIC.
„ AST methods are based on detection of the microbial phenotype.
The original broth macrodilution tube technique used as the reference
„ AMR methods are largely based on detection of the genotype.
method for routine MIC determinations was tube-based, but for improved ease-
of-use has now been miniaturized into the 96-well microtiter plate format (broth
Figure 11. Overview of laboratory methods available for microdilution BMD). This allows multiple antibiotics to be tested on each row
antimicrobial susceptibility and antimicrobial resistance testing of the plate (e.g. 12 antibiotic MICs using 8-fold dilutions per plate). A number
Source: bioMérieux of automated microtiter-based BMD formats are commercialized, which reduce
processing and reporting times (see section 2.2). However, whilst these provide
standardized, ready-to-use panels for AST, laboratories are limited to the range of
ANTIMICROBIAL SUSCEPTIBILITY TESTING antibiotics that are included on each panel type.
& ANTIMICROBIAL RESISTANCE TESTING
2.1.2 Agar Dilution Methods
Similar to the broth microdilution methods, agar dilution involves the
addition of antimicrobial dilutions to the AST agar medium (usually Mueller
GENOTYPIC PHENOTYPIC Hinton agar MHA). Standardized bacterial inocula are then applied to
TESTING TESTING
the agar plate using multipoint application techniques. This test format
allows 30-100 organisms to be tested on each agar depending on the plate
POLYMERASE dimensions (Figure 12). After overnight incubation, plates are examined for
CHAIN REACTION the presence of growth. The MIC is determined from the agar plate showing
(PCR) MANUAL AUTOMATED
the lowest concentration of antimicrobial that inhibits growth.
While these methods allow multiple organisms to be tested at the same time
WHOLE GENOME
SEQUENCING
AGAR/BROTH
AUTOMATED against a single antimicrobial agent or generation of data that can be used
MICRODILUTION
(WGS) BROTH DILUTION to look at MIC distribution within a population of organisms belonging to the
same genus/species, they remain manual and are therefore not widely used
GRADIENT today. However, both CLSI and EUCAST recommend use of this technique for
DIFFUSION
testing a limited range of antimicrobials (e.g. fosfomycin MIC breakpoints in
FAST AST
Escherichia coli are calibrated using agar dilution or with anaerobes using
DISK DIFFUSION
fastidious anaerobe agar (FAA MH) as defined by EUCAST).14

Figure 12. Agar dilution techniques

CHROMOGENIC Source: Pathology Queensland, used with permission
MEDIA

RAPID TEST
(e.g. Carba test)

28 29
 AST - BASIC CONCEPTS AND METHODS

2.1.3 MIC Gradient Diffusion Method 2.1.4 Disc Diffusion

The MIC gradient diffusion method utilizes both antibiotic dilution Disc diffusion is an alternative AST method to MIC that uses Mueller-Hinton
and agar diffusion technique in a combined assay to measure the MIC agar (MHA), the international standard medium for AST. For fastidious
of a clinical isolate to a given antimicrobial. A plastic/paper strip with organisms that require additional growth factors, supplementation of MHA
a single antibiotic at increasing concentration is applied to one side of with blood is required.15,16 The disc diffusion technique involves placing an
the strip with a corresponding concentration scale on the upper surface antibiotic impregnated disc with a single concentration of the antibiotic
(Figure 13). After overnight incubation, the MIC is determined at the point onto MHA inoculated with a lawn of the test isolate. Multiple antibiotic
of intersection between the zone of bacterial inhibition and the MIC scale on discs can be tested on the same agar plate making this a highly inexpensive
the strip. Where the intersection falls between a set of MIC values, the MIC and flexible test method (Figure 14). After overnight incubation, zones of
endpoint is reported as the higher of the two. Multiple strips can be tested growth inhibition are measured. Susceptible/Resistant zone diameters are
on a single agar plate depending on size but the cost per strip generally determined based on zone diameters from Disc/MIC calibrations published
precludes laboratories from testing more than 2-3 antimicrobials per by Reference Laboratories (i.e. CLSI/EUCAST).5,7
isolate. Commercially available strips include ETEST® (bioMérieux) and
MTS (Liofilchem®).
2.1.5 Chromogenic Agar
Microbiology laboratories routinely incorporate chromogenic media into
Specialized antimicrobial combination strips have been developed to their routine testing protocols for the detection of bacteria that possess
allow detection of specific antimicrobial resistance phenotypes including specific antimicrobial resistance mechanisms (Figure 15). The advantage
ESBLs, ampC and carbapenemase production in gram-negative bacteria15 of chromogenic media containing selective agents is that clinical specimens
(Table 2). can be plated directly onto the media allowing detection of significant
These dual strips have a single antimicrobial at one end with the same resistant phenotypes such as MRSA, VRE, ESBLs and CPE within 24
antimicrobial plus an enzymatic inhibitor at the other end. A positive result hours.16 Although growth on chromogenic media does not replace the need
is indicated when the MIC of the antimicrobial inhibitor combination is for routine AST on isolates, the ability to rapidly detect resistance within 24
equal to or greater than the antibiotic on its own. hours is significant for Infection Control allowing early identification of
carrier states and isolation of positive patients, and thereby restricting
Table 2. Antibiotic and beta-lactamase inhibitor combinations potential spread of AMR to other patients.
used in MIC gradient diffusion strips (ETEST®) to detect beta-
lactamases responsible for antibiotic resistance Figure 13. Gradient Figure 14. Disk Figure 15.
Source: bioMérieux diffusion method diffusion technique Chromogenic media
Source: bioMérieux Source: Queensland Pathology, Source: bioMérieux
Antibiotic/Antibiotic Inhibitor
Resistance mechanism used with permission
combination
Cefotaxime / cefotaxime clavulanate
Extended spectrum beta-lactamases
Ceftazidime / ceftazidime clavulanate
(ESBL)
Cefepime / cefepime clavulanate

AmpC cephalosporinase Cefotetan / cefotetan cloxacillin

Imipenem / Imipenem EDTA

Metallo beta-lactamases
Meropenem / Meropenem EDTA

30 31
 AST - BASIC CONCEPTS AND METHODS

2.2. PHENOTYPIC METHODS - SEMI/FULLY AUTOMATED Figure 16. VITEK® 2 instrument and ID/AST cards
SYSTEMS Source: bioMérieux image library

Automated systems for bacterial identification and AST, based on

the broth microdilution (BMD) technique, are now widely used in most
routine clinical microbiology laboratories, not only to reduce the time
to reporting of susceptibility test data, but also to increase efficiency by
allowing standardization of the test process.
For the routine microbiology laboratory, testing MICs around the susceptible
and resistant breakpoints is more cost-effective than full microbroth
dilution, allowing a wider range of antimicrobials to be tested against all
isolates. By miniaturizing the test format and utilizing optical arrays to
detect subtle changes in growth/color of test panel wells, automated
systems such as the VITEK® 2 (bioMérieux), Phoenix™ (BD), Microscan
WalkAwayplus (Beckman Coulter) and Sensititre™ (ThermoFisher) Figure 17. BD Phoenix™ M50 instrument and panels
utilize rapid AST technology on defined AST panels to provide susceptibility Used with permission from BD
results between 4 and 18 hours (Figures 16 and 17).16,17
In addition to streamlining the AST workflow, automated systems also
facilitate an improved data management process. MICs and interpreted
test results can be directly interfaced to the Laboratory Information System
(LIS), reducing technical time required for data entry and thus reducing the
rate of transcription errors. Additionally, computerized data capture within
these systems allows the storage and retrieval of cumulative AST data
which can then be linked to local antibiogram and antibiotic stewardship
programs.
Whilst panel customization is possible, the number of antibiotics that can
be included on each panel is limited. For high throughput laboratories,
separate urine and non-urine panels can be used to offset the limited range
of antimicrobials per panel. Additionally, when new antimicrobials become
available it may be 3 to 5 years before these are available for routine use
on the automated systems. In this situation, where the new agents are not
available for inclusion on extended AST panels for testing of multi-drug
resistant (MDR) gram-negatives, laboratories will augment their automated
systems with other methods such as gradient diffusion assays to provide
susceptibility data for newer agents.

32 33
 AST - BASIC CONCEPTS AND METHODS

2.3. PHENOTYPIC METHODS - FAST AST 2.3.2 Automated Fast AST

Antimicrobial susceptibility testing (AST) of bloodstream isolates is critical A number of recent developments are providing laboratories with
for optimal management of patients with bacterial infections, and in automated options for fast AST results. These have been most frequently
particular, with sepsis. developed for use on positive blood culture broths.

While traditional AST results are available only after 48–72 hours, new One such example (VITEK® REVEAL™, bioMérieux) uses colorimetric
techniques that enable rapid AST to be performed directly on positive sensors to detect volatile compounds released by bacteria as they grow
blood cultures reduce the time-to-result to 4-8 hours and have (Figure 19). By regularly monitoring color changes in the sensors, which
significant potential to improve the clinical outcomes for patients with correlate with microbial growth in different concentrations of antibiotics,
sepsis.18-20 a MIC can be rapidly determined within 6 hours on average.19

2.3.1 Rapid Disc AST Other methods, for example, ACCELERATE PHENO® (Accelerate
Diagnostics, Inc.) assess growth and cell morphology changes in the
In an effort to further reduce the time from initiation of empiric therapy to presence or absence of antibiotics using live-cell imaging and in this way
AST result, many laboratories perform direct disc susceptibility testing can also rapidly determine an MIC from positive blood culture broths
on positive blood culture broths. As the ability to control the bacterial (Figure 20).23 Other commercialized Fast AST systems include dRAST™
inoculum is not possible, the reliability and reproducibility of susceptibility (Quantamatrix), ASTar® (Q-Linea) and QuickMIC® (Gradientech).
results is variable, however the ability to detect resistance has been shown
to have a positive clinical impact when a change in therapy is indicated. Other technologies are in development which could rapidly perform AST
from respiratory samples in patients with pneumonia.
EUCAST has recently developed a Rapid Antimicrobial Susceptibility Test
(RAST) directly from positive blood cultures against a range of selected Figure 19. VITEK® REVEAL™
antibiotic discs (Figure 18).21 Interpretation of susceptibility results is Source: bioMérieux
possible within 4-8 hours reducing the reporting from the usual 18 hours.
However, it is the direct detection of antimicrobial resistance in
positive clinical samples within 1-2 hours that will provide the greatest
impact in the area of RAST.

Figure 18. Rapid Antimicrobial Susceptibility Test (RAST). Readings

at 4 (A), 6 (B) and 18 h (C) of the disk diffusion testing directly
from blood culture bottles22
Reproduced with permission from Martins A, et al. J Glob Antimicrob Resist. 2020:22:637–642.

Figure 20. ACCELERATE PHENO® system

Used with permission from Accelerate Diagnostics, Inc.

34 35
 AST - BASIC CONCEPTS AND METHODS

2.4. DETECTION OF ANTIMICROBIAL RESISTANCE GENES Figure 21. Example of colorimetric detection test
OR MECHANISMS Source: bioMérieux (extracted from Carbapenem Resistance booklet)

Unlike traditional antimicrobial susceptibility methods described above

that detect the antimicrobial phenotype, antimicrobial resistance
testing is based upon the detection of antimicrobial resistance genes Broth Broth Broth Broth
or mechanisms within the microbial genome. + +
meropenem meropenem
From a clinical perspective:
„ the phenotypic susceptibility profile allows the clinician to select the
appropriate antibiotic dosage for treatment, Non-carbapenemase Carbapenemase
producer producer
„ whereas detection of the antimicrobial resistance provides guidance
on which antibiotic(s) should not be prescribed. Disc potentiation tests are also widely used for demonstrating the
presence of enzymic resistance particularly when the resistance may
Both techniques are useful and important for optimal patient management:
only be weakly expressed. In these assays, the zones of growth inhibition
while genotypic methods are often faster and important for the first
are measured in the presence or absence of specific enzyme inducers or
hours of patient management, phenotypic methods are necessary to
inhibitors.
select the long-term targeted therapy.
ÎFor example, inducible clindamycin resistance in Streptococcus spp.
2.4.1 Rapid methods for the detection of specific AMR
and Staphylococcus spp. can be detected by placing an erythromycin
mechanisms disc in close proximity to a clindamycin disc (Figure 22).
A number of rapid methods are used in the laboratory to confirm the
presence of specific resistance genes or mechanisms when these are Figure 22. Positive D-test for Inducible Clindamycin Resistance in
phenotypically suggested by the AST profile. Staphylococcus aureus24
Enzymatic test reaction: antimicrobial resistance derived from enzymic Reproduced with permission from Prabhu K, Rao S, Rao V. J Lab Physicians 2011;3(1):25-27
breakdown of the antimicrobial can be detected using colorimetric detection
tests.16 These utilize enzyme substrates with chromogens attached that are
released in the presence of the enzyme, resulting in a change in color of the Disc potentiation tests are also used to
test format. detect inducible resistance mechanisms. In
this instance, one antimicrobial will induce
increased production of a chromosomal
ÎFor example, beta-lactamase production in Staphylococcus aureus, enzyme resulting in a flattening of zones of
Neisseria gonorrhoeae or Haemophilus influenzae can be detected within growth inhibition to another antimicrobial at
the interface between the two discs (D-test).
an hour on a test strip impregnated with a chromogenic cephalosporin.

ÎAnother example is the the RAPIDEC® CARBA NP (bioMérieux)

or Carba-NP test that utilize a color change to a pH indicator dye,
due to acid production arising from carbapenemase degradation of
meropenem in CPE (Figure 21).

36 37
 AST - BASIC CONCEPTS AND METHODS

2.4.2 Molecular Antimicrobial Resistance Gene Detection As shown in Table 3, the beta-lactamase gene markers only allow
Most methods of detection of antimicrobial resistance genes use “translation” to beta-lactam antibiotics that are potential substrates for
polymerase chain reaction (PCR). With some sample types the enzymes encoded. No comment can be specifically made about non-
(e.g., respiratory samples, synovial fluid or cerebrospinal fluid), rapid beta-lactam antibiotic classes such as polymyxins, fluoroquinolones,
molecular methods can detect certain antimicrobial resistance genes tetracyclines or aminoglycosides.
directly from clinical specimens. However, where bacterial burden is low 2.4.3 Whole Genome Sequencing
(e.g., in blood), sufficient DNA is only available in incubated samples or from
Whole genome sequencing holds the promise of providing much more
isolated colonies.
information than is available from PCR-based methods.
The major advantage of rapid molecular tests for clinicians is speed.
ÎFor example, organism identity, clonal relationship with other bacteria
However, PCR-based methods cannot indicate whether the gene detected is
previously isolated from the same facility, presence of virulence genes
actually expressed in relevant amounts, nor can they typically link the gene
and detection of the full range of resistance genes are all possible.
detected and the organism from which it was detected. This is potentially
relevant when the mecA gene is detected and it is unclear if it arose from The cost of genome sequencing has also reduced dramatically compared
Staphylococcus aureus or a coagulase-negative Staphylococcus. to prior decades making it more attractive as a practical consideration.
In addition, with rapid molecular methods, it is important to be able to However, timeliness remains an issue given that specialized bioinformatic
“translate” the results into information that can be readily used by resources are still needed in most circumstances. Rapid, low-read
prescribers. Table 3 shows an example of how this could be utilized. sequencing is becoming closer to clinical utility, but issues remain, such as
reproducibility and ability to detect low numbers of organisms, for example
Table 3. Knowledge of the presence of beta-lactamase genes can in patients with bloodstream infections.
be useful in determining which beta-lactam antibiotics could be
used for therapy and which should be avoided25
Adapted from Wright H, Bonomo RA, Paterson DL. Clin Microbiol Infect. 2017;23(10):704-712

Resistance Antibiotics that should not be Antibiotics that can be used

Gene used
CTX-M Cefazolin, ceftriaxone, ceftazidime Meropenem, ertapenem
Meropenem, piperacillin- Ceftazidime-avibactam,
KPC tazobactam, ceftriaxone, cefazolin, imipenem-relebactam,
ceftazidime meropenem-vaborbactam
Imipenem-relebactam,
meropenem, piperacillin-
OXA-48 Ceftazidime-avibactam
tazobactam, ceftriaxone, cefazolin,
ceftazidime
Ceftazidime-avibactam, imipenem- Ceftazidime-avibactam
relebactam, meropenem- combined with aztreonam,
NDM vaborbactam, meropenem, cefiderocol,
piperacillin-tazobactam, Aztreonam-avibactam (mainly
ceftriaxone, cefazolin, ceftazidime in Europe)

38 39
 AST - BASIC CONCEPTS AND METHODS

Figure 23. Microbiology laboratory workflow and timeline

from positive blood culture to bacterial identification (ID)
and antimicrobial susceptibility testing (AST)18,19,26
Adapted from Banerjee R, et al. Front Med. 2021;8:63531; Tibbetts R, et al. J Clin Microbiol. 2022;60(6):
e0009822; Wenzler EE, et al. Pharmacotherapy 2023;43(4):264-278

GRADIENT DISK BROTH

STRIP DIFFUSION MICRODILUTION

POSITIVE SUBCULTURING / COLONY ORGANISM ID /

BLOOD CULTURE INCUBATION ISOLATION PHENOTYPIC AST

CONVENTIONAL
ID/AST
AUTOMATED MANUAL

DAY 1 DAY 2 DAY 3

AUTOMATED MANUAL
RAPID
ID/AST

RAPID FAST AST DISK

ORGANISM ID DIFFUSION

40 41
 POST-AST
CLINICAL AND LABORATORY
PERSPECTIVES
 POST-AST - CLINICAL AND LABORATORY PERSPECTIVES

1 INTERPRETATION OF AST RESULTS AND Table 4. Example of CLSI testing recommendations for
REPORTS antimicrobial testing and reporting in Staphylococcus species5
Selection of antibiotics for testing and reporting Adapted from Clinical and Laboratory Standards Institute: Performance Standard for
Antimicrobial Susceptibility Testing, M100-Ed 34, 2024, CLSI
Each clinical laboratory needs to test antibiotics that are the most relevant
Group B
to the range of clinical pathogens isolated, the site of infection and the local Group C
Group A Test and report Group U
formulary of the healthcare facility. selectively
Test and report
Antimicrobials Test and Report selectively Test and
CLSI produces tables of recommended antimicrobials to be tested when resistant
routinely on all for multidrug report for urine
to same Antimi-
against gram-positive and gram-negative pathogens as well as fastidious isolates
crobial class as
resistant isolates only
strains
microorganisms (Table 4).5 Additionally, urine-only drugs such as Group A
nitrofurantoin, trimethoprim and fosfomycin are selectively applied only Penicillin
for AST testing of urinary isolates. Not all antibiotics listed as suitable for Flucloxacillin /
testing by CLSI will be tested. The selection of antibiotics will be determined Cefoxitin
by the antibiotics available locally for clinical use, the range of antimicrobials Erythromycin
provided on commercial AST systems and antibiotic combinations that Clindamycin
allow detection of AMR phenotypes of relevance to local Infection Control Trimethoprim
guidelines. sulphameth-
oxazole
Each laboratory will have a defined set of antimicrobials that they test Vancomycin
and report for all isolates, followed by a range of second tier agents that Tetracycline
are reported if resistance to the initial set of agents is detected. This is
Rifamicin
known as selective antibiotic reporting.
Linezolid
Î Selective antibiotic reporting or suppression of AST results
Daptomycin
is common practice (i.e. not all the antibiotics tested against
Ciprofloxacin
a particular bacterial pathogen are reported). Only those
antimicrobials relevant to the organism and the site of infection are Nitrofurantoin
reported. For fully susceptible microorganisms, only the first-line Trimethoprim
narrow-spectrum antibiotics will be reported. Where resistance is
detected, additional second tier agents will then be released. The The aim of selective antimicrobial reporting is to
latter is often referred to as cascade reporting (e.g. where isolates ensure good antibiotic stewardship, whereby the broad-
are resistant to first/second generation cephalosporins, then third spectrum agents are reported only for more resistant
generation cephalosporins such as ceftriaxone or ceftazidime will be microorganisms.
reported instead if they test susceptible).
Additionally, the range of antimicrobials reported should
aim to include narrow-spectrum agents if susceptible, at
least one oral and one intravenous option, and at least one
agent for patients that may have penicillin allergy.

44 45
 POST-AST - CLINICAL AND LABORATORY PERSPECTIVES

Î Comments are frequently applied to reports to guide the Role of Expert Systems in AST Platforms
clinician in the interpretation of AST results. Manual scientific review of AST results prior to reporting is an essential
This is particularly important where MIC results fall between quality activity for all clinical microbiology laboratories. This process can
susceptible and resistant reporting categories. Guidance in the be labor intensive and subject to human error.
interpretation of intermediate results is required particularly
where the intermediate MIC reflects a susceptible increased In addition to reduced turn-around times to AST results, systems such as the
exposure result as defined by EUCAST. VITEK® 2 and Phoenix™ have computerized Expert Systems which can
The comment needs to indicate that this drug can potentially rapidly screen susceptibility profiles for typical and atypical results. This process
be used as long as adequate dosing regimens are applied and involves the analysis of AST profiles against comprehensive databases consisting
concentration at the site of infection is likely to occur. of phenotypes generated by microbes with known antimicrobial resistance
mechanisms (Figure 24). This allows the AST results for different organisms to
„ Test-related comments are also added to provide clarity around be validated for accuracy ensuring that potential errors in test results, important
the potential accuracy of test results. This may be necessary resistance phenotypes or unusual MIC results are detected consistently.27
when an MIC result is reported for a drug/bug combination for
which neither EUCAST nor CLSI provide clinical breakpoints or in The VITEK® 2 Advanced Expert System (AES) has an extensive
situations where the test method recommended in the standard knowledge base that compares the AST MIC profile (phenotype) of the
is not available/used by the testing laboratory (e.g. agar dilution test isolate against a wide range of MIC distributions for different AMR
for fosfomycin testing). phenotypes (over 3,000 individual profiles are available for screening).
„ Additional comments that provide treatment information are In this phenotype mapping process, MIC results within a specific class of
included to assist the clinical team in ongoing case management antimicrobial (e.g. cephalosporins) are reviewed to screen for the presence
(e.g. with Staphylococcus aureus, use of a single oral agent such as of potential resistance mechanisms. AMR associated with different classes
ciprofloxacin, rifampicin or fusidic acid may result in development of antimicrobials (e.g. beta-lactams and aminoglycosides) can be detected
of resistance). within the same isolate. This process is referred to as Biological Validation.

Figure 24. Workflow of an Expert system in AST context

Source: Pathology Queensland, reproduced with permission

EXPERT KNOWLEDGE
DATABASE

REPORT
KNOWN WITH AMR
PHENOTYPE COMMENTS

ORGANISM ID
+ EXPERT ANALYSIS PROGRAM
AST MIC VALUES

FLAG ADD
ATYPICAL HOLD FOR CORRECTIONS
PHENOTYPE FURTHER
TESTING REPORT

46 47
 POST-AST - CLINICAL AND LABORATORY PERSPECTIVES

The Biological Validation process can also flag unusual or impossible For laboratories which do not have access to computerized Expert Systems,
phenotypes. EUCAST has published a series of Expert rules that can be applied. These
are divided into various categories based on intrinsic resistances by
Î For example, for Staphylococcus aureus that show resistance to
organism, exceptional phenotypes that require additional testing and
the glycopeptide antimicrobial teicoplanin but susceptibility to
interpretative rules that cover inferred resistance mechanisms from AST
vancomycin.
results.28,29
Additionally, Expert Systems contribute to the validation of AST results by
The ability of Expert Systems to accurately detect clinically relevant
identifying the need for Therapeutic Corrections where antimicrobial
resistance mechanisms particularly beta-lactamase resistance (e.g. ESBL,
resistance for a given organism may be insufficiently expressed or
carbapenemase) ensures not only that the appropriate antibiotic therapy can
incorrectly reported as susceptible.
be prescribed, but also that appropriate Infection Control can be initiated
Î For example, the AES will recommend a change to MIC via identification and isolation of patients to minimize nosocomial spread.
interpretation from susceptible to resistant where intrinsic
There are however limitations to Expert Systems. Of most significance is
resistance has not been detected to ensure consistency in reporting
the quality of the knowledge databases used and the need to keep these
between organism identification and AST result.
up-to-date with newly and rapidly emerging resistance phenotypes.
e.g. Enterobacter cloacae where ampicillin is reported as Additionally, clinically relevant antibiotics for the detection of specific
susceptible when it should be resistant. resistance phenotypes may not be on the test panel and differentiation
of phenotypes generated by mixed genotypes (e.g. ESBL plus ampC) may
Both Biological Validation and Therapeutic Corrections can be applied to result in incorrect analysis.
the phenotype of a single test isolate. The AES will recommend a change
to MIC interpretations from susceptible to resistant where a specific Expert Systems ensure detection of typical and unusual
resistance phenotype is detected. AMR phenotypes.
Î For example, ESBL-producing Escherichia coli where changes in
Expert System flag results for technical review thereby
the interpretation of the third generation cephalosporins including
facilitating the work required of skilled laboratory staff.
cefotaxime/ceftazidime from susceptible to resistant will be
proposed even though the MICs may fall within the susceptible However, Expert System databases must be kept up-to-
category. date with newly/rapidly emerging AMR mechanisms.

48 49
 POST-AST - CLINICAL AND LABORATORY PERSPECTIVES

2 USE OF CUMULATIVE LABORATORY AST DATA Local cumulative antibiograms are used by antimicrobial stewardship
– THE ANTIBIOGRAM programs to develop local empirical antimicrobial treatment guidelines
and to guide decisions regarding empirical antimicrobial treatment and
Cumulative antibiograms are useful tools for detecting and monitoring
antimicrobial formulary.
local trends in the prevalence of antimicrobial resistance. They help
guide clinicians and pharmacists in selecting the most appropriate Î For example, a hospital microbiology laboratory may produce
empiric antimicrobial treatment while pending microbiology culture and an antibiogram on an annual basis for the entire hospital or for a
susceptibility results. They can be developed at hospital level, or for specific specific intensive care unit.
units (e.g. burn units) or specimen sources (e.g. urine cultures), where local On the other hand, a community-based microbiology laboratory
organism epidemiology/resistance can differ significantly from the overall may produce an antibiogram for general practitioners comprising
hospital data. cumulative results from outpatient urine samples or from a specific
nursing home.
A cumulative antibiogram is a table summarizing the
percent of individual bacterial pathogens susceptible to Cumulative antibiograms can be a useful aid to prescribers,
different antimicrobial agents for a specific setting and pharmacists and infection control teams, who can use this epidemiologic
time period.30 data to “rule out” certain antibiotics for certain conditions. This may change
over time. The actual antibiotic prescribed to a patient should take into
A cumulative antibiogram is guided by specific rules (CLSI M3930 or local consideration individual factors such as allergies, renal function, pregnancy
recommendations) including deduplication of data, minimum number of or breast-feeding and known colonization or prior infection with resistant
isolates, and can be filtered according to location, specimen or patient organisms.
criteria.
Antibiogram results with an “*” on the report should be interpreted with
One of the goals is to use antibiograms to guide empirical therapy of caution, particularly when insufficient data is available to allow accurate
initial infections whether the causative organism is unknown or known but interpretation. Clinicians should be encouraged to discuss such results with
susceptibility is unknown. the microbiologist to determine the optimal interpretation and appropriate
antibiotic therapy.

Figure 25. Example of a cumulative antibiogram

Source: bioMérieux
Urine isolates
ABC HOSPITAL ADULT INPATIENTS
Uncomplicated

Nitrofurantoin
ANTIMICROBIAL SUSCEPTIBILITIES (%) Trimeth/Sulfa
Ciprofloxacin
complicated

Levofloxacin
Piperacillin/
Amoxicillin/

Vancomycin
Meropenem
Tazobactam
Clavulanate

Ceftriaxone
Cefazolin-

Cefazolin-
Ampicillin

Cefepime
isolates

Linezolid
# urine

Nafcillin

1st ISOLATE ONLY

CALENDAR YEAR: 2021

Enterococcus faecalis 168 100 0 0 0 0 0 100 100 99

Positive
Gram-

Enterococcus faecium 44 7 0 0 0 0 0 7 93 20
Staphylococcus aureus 39 0 49 49 49 95 100 100 100
Citrobacter species 25* 0 0 85 0 0 82 100 100 100 100 93 70
Enterobacter cloacae 41 0 0 67 0 0 57 89 100 100 100 87 25
Escherichia coli 575 49 82 96 86 67 90 92 100 74 75 74 98
Gram-Negative

Klebsiella aerogenes 31 0 0 71 0 0 68 100 100 100 100 100 21

Klebsiella oxytoca 16* 0 80 73 13 13 80 87 100 93 100 87 67 ≥ 90%
Klebsiella pneumoniae 222 0 87 88 86 74 88 88 100 88 95 83 38 70-90%
Proteus mirabilis 88 86 98 99 93 2 95 98 100 82 82 83 0 ≤ 70%
Pseudomonas aeruginosa 61 0 0 83 0 0 0 88 86 64 58 0 0 ≤ 30 isolates

50 51
 POST-AST - CLINICAL AND LABORATORY PERSPECTIVES

3 ANTIMICROBIAL STEWARDSHIP AND Diagnostic stewardship is performed in the microbiology laboratory

DIAGNOSTIC STEWARDSHIP (“the right test”) and is a complementary concept to antimicrobial
stewardship on the clinical side (“the right interpretation”).31-33 Diagnostic
Antimicrobial stewardship (AMS) is now commonplace in recognition
stewardship promotes the judicious use of diagnostic tests to initiate
of its role in optimizing clinical outcomes related to use of antimicrobials.
appropriate antibiotic therapy, and aims to avoid the excessive use of broad-
spectrum antibiotics. To avoid overdiagnosis and excessive healthcare
While the core members of an antimicrobial stewardship team costs, correct interpretation of test results is essential. Clinical assessment
are typically physicians and pharmacists trained in infectious of signs and symptoms combined with knowledge of the local epidemiology
diseases, microbiologists play a key role in these programs. are critical for diagnostic stewardship, and enable correct interpretation of
microbiological results.
Antimicrobial stewardship programs vary in their scope, but typically utilize
institutional guidelines for antimicrobial use. The inclusion of antimicrobials Current major barriers to the implementation of diagnostic stewardship are
in guidelines depends on a variety of factors such as clinical trial data, a lack of resources, a lack of trained personnel and, most importantly, a
cost, cumulative susceptibilities (such as obtained from an antibiogram) lack of knowledge. The lack of resources allocated to diagnostic stewardship
and potential for “collateral damage” whether that be predilection for is often due to the lack of awareness of the impact that diagnostic tests
Clostridioides difficile infection or potential for selection of antimicrobial can have on clinical decision-making and the optimization of appropriate
resistance. antimicrobial prescribing.

Diagnostic stewardship means ordering the right tests

Most antimicrobial stewardship programs undertake some
for the right patient at the right time to inform optimal
review of antimicrobial prescriptions and correlate these with
clinical care and positively impact patient outcomes.32
microbiology reports, including AST reports.
The full potential of diagnostic stewardship cannot be
This may encourage prescribers to “streamline” antibiotic therapy from a reached unless it is integrated in an AMS approach.
broad-spectrum empiric choice to a targeted choice based on AST results.
Î For example, vancomycin and piperacillin-tazobactam may have
Both antimicrobial stewardship and diagnostic stewardship require the
been commenced empirically for a seriously ill individual with sepsis, constitution and collaboration of multidisciplinary teams who can help
but when microbiology results show Streptococcus pyogenes, the establish clear criteria for ordering diagnostic tests and acquiring the
antibiotic choice may be de-escalated to penicillin alone. appropriate technologies. Clinical microbiology laboratories should integrate
diagnostic stewardship as a core activity to ensure successful AMS and
Conversely, antimicrobial stewardship review may show a need to escalate infection control (Figure 26). Moreover, information technology (IT)
antibiotic therapy. systems now play a crucial role in antimicrobial stewardship programs,
Î For example, microbiology reports may reveal the presence of enabling timely and accurate data tracking, monitoring, and analysis.
Klebsiella pneumoniae resistant to meropenem, necessitating These systems help healthcare facilities to identify patterns of antibiotic
a change in empiric therapy from meropenem to ceftazidime- use and track resistance trends. Clinical decision support systems
avibactam. (CDSS) facilitate decision-making by healthcare professionals regarding
appropriate antimicrobial therapy.

Both antimicrobial stewardship and diagnostic

stewardship have demonstrated their clinical utility
in optimizing patient outcomes, and microbiology
laboratories play a key role in both endeavors.

52 53
 POST-AST - CLINICAL AND LABORATORY PERSPECTIVES

Figure 26. The continuum from diagnostic stewardship to antimicrobial

stewardship across the diagnostic pathway33
Reproduced with permission from Zakhour J, et al. International Journal of Antimicrobial Agents 2023;2:106816

Physicians Microbiologists Nurses/Adjunct staff Microbiologists Laboratory staff Physicians

Perform thorough Recommend Ensure adequate Determine sample Ensure proper Provide real-time
history and clinical diagnostic tests sampling adequacy for testing processing and clinical feedback
exam Counsel on novel Ensure proper Recommend preservation of to guide sample
Determine optimal diagnostics sample labelling and alternative/additional samples processing and testing
sample source Assess sample transportation diagnostic tools Avoid contamination of Establish institutional
Determine pre-test appropriateness Report additional Reject damaged/ samples criteria for sample
probability clinical data unsealed samples appropriateness
Choose adequate test

1 - Pre-analytical phase 2 - Analytical phase

DIAGNOSTIC STEWARDSHIP

ANTIMICROBIAL STEWARDSHIP

3 - Post-analytical phase 4 - Antimicrobial stewardship

Laboratory staff Microbiologists Physicians Physicians Pharmacists Nurses
Ensure timely Modify reporting to Collaborate with Choose appropriate Determine Ensure proper and
reporting of results indicate colonization microbiologists to treatment while appropriateness of timely administration
Integrate Electronic Selectively report ensure proper analysis considering local provided treatment of treatment
Medical Record (EMR) susceptibility results of results susceptibility patterns Provide adequate Provide additional
into result reporting Properly analyze Correlate results to Reassess regularly and dose adjustment clinical input
results and correlate to clinical data to ensure correlate to clinical input and adverse events Report possible
pretest probability and a proper diagnosis De-escalate to narrow surveillance adverse events
clinical input spectrum or oral therapy
Recommend additional when possible
testing/novel Discontinue therapy
diagnostics if possible when infectious etiology
is unlikely

54 55
 LIST OF ABBREVIATIONS
AES Advanced Expert system
AMR Antimicrobial resistance
AMS Antimicrobial stewardship
ASP Antimicrobial stewardship program
AST Antimicrobial susceptibility testing
CONCLUSIONS AND FUTURE ATP Adenosine triphosphate
ATU Area of technical uncertainty
PERSPECTIVES BMD Broth microdilution
CDSS Clinical decision support systems
CLSI Clinical and Laboratory Standards Institute
AST is an essential tool in the fight against multi-drug resistant
CPE Carbapenemase-producing Enterobacterales
bacteria. Conventional phenotypic methods remain the mainstay of current
CTX-M Cefotaximase M type
AST in the clinical setting. Rapid disc and automated broth microdilution
(BMD) methods can generate useful AST data within 4-8 hours and 4-18 DNA Desoxyribose nucleic acid
hours respectively, but time-to-reporting of AST results needs to be further ECOFF/ECV Epidemiological cut-off value
reduced. EDTA Ethylenediaminetetraacetic acid
ESBL Extended spectrum beta-lactamase
Although rapid molecular technology is capable of detecting antimicrobial EUCAST European Committee for Antimicrobial Susceptibility Testing
resistance in bacteria within a short time period, resistance in vivo can FAA Fastidious anaerobe agar
only be inferred from the presence of resistance genes, and additional FACS Fluorescence-activated cell sorting
phenotypic testing is required to confirm this in vitro and to provide possible FDA Food and Drug Administration
alternative therapeutic options. I Intermediate
Newer technologies, e.g. microfluidics, fluorescence-activated cell sorting ID Identification
(FACS), adenosine triphosphate (ATP) bioluminescence testing, that IT Information technology
allow the real-time assessment of drug/bug interactions are under KPC Klebsiella pneumoniae carbapenemase
investigation but are not at the stage of commercialization.34 Nevertheless, LIS Laboratory information system
a better understanding of antimicrobial usage and its impact on bacterial MALDI-TOF MS Matrix-assisted laser desorption/ionization time-of-flight
resistance rates based on local cumulative AST data will help to ensure mass spectrometry
antimicrobial therapy is directed to the best possible patient outcomes, MBC Minimal bactericidal concentration
whilst reducing selection pressure for development of multi-drug resistance. MDR Multidrug-resistant
MIC Minimal inhibitory concentration
MHA Mueller Hinton agar
MHB Mueller Hinton broth
MRSA Methicillin-resistant Staphylococcus aureus
NDM New Delhi metallo-beta-lactamase
NS Non-susceptible
OXA-48 Oxacillinase-48
PCR Polymerase chain reaction
R Resistant
RAST Rapid antimicrobial susceptibility testing
S Susceptible
SDD Susceptible dose-dependent
VRE Vancomycin-resistant Enterococcus
WHO World Health Organization
56 57
 REFERENCES

REFERENCES
1. World Health Organization (WHO). Global Action Plan on Antimicrobial Resistance, 2015. 19. Tibbetts R, George S, Burwell R, et al. Performance of the Reveal Rapid Antibiotic
https://www.who.int/publications/i/item/9789241509763 Accessed 15/06/2024. Susceptibility Testing System on Gram-negative Blood Cultures at a Large Urban Hospital.
2. Liu Y, Tong Z, Shiet J, et al. Correlation between Exogenous Compounds and the Horizontal J Clin Microbiol. 2022;60(6):e0009822. doi: 10.1128/jcm.00098-22.
Transfer of Plasmid-Borne Antibiotic Resistance Genes. Microorganisms 2020;8(8):1211. 20. Couchot J, Fournier D, Bour M, et al. Evaluation of the Reveal® rapid AST system to assess
doi: 10.3390/microorganisms8081211. the susceptibility of Pseudomonas aeruginosa from blood cultures. Eur J Clin Microbiol
3. Todars Online Textbook of Bacteriology https://textbookofbacteriology.net/
Infect Dis. 2023;42(3):359-363. doi: 10.1007/s10096-023-04556-2.
resantimicrobial_3.html Accessed 15/06/2024.
4. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob 21. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European
Chemother. 2001;48:Suppl 1:5-16. doi: 10.1093/jac/48.suppl_1.5. Society of Clinical Microbiology and Infectious Diseases (ESCMID). Rapid AST in
5. Clinical and Laboratory Standards Institute: Performance Standard for Antimicrobial blood cultures, 2022. https://www.eucast.org/rapid_ast_in_bloodcultures Accessed
Susceptibility Testing, M-100 34th Edition, 2024. https://clsi.org/standards/products/ 15/06/2024.
microbiology/documents/m100/ Accessed 15/06/2024. 22. Martins A, Wink P, Pereira D, et al. Rapid antimicrobial susceptibility of Enterobacteriaceae
6. Microbe Online. https://microbeonline.com/minimum-inhibitory-concentration-and- by disk diffusion directly from blood culture bottles using the EUCAST RAST breakpoints. J
minimum-bactericidal-concentration-mbc/ Accessed 15/06/2024. Glob Antimicrob Resist. 2020:22;637-642. doi: 10.1016/j.jgar.2020.05.015.
7. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the 23. Bannerjee R, Komarow L, Kirk A, et al. Randomized Trial Evaluating Clinical Impact of RAPid
European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Clinical
breakpoints and dosing of antibiotics. https://www.eucast.org/clinical_breakpoints IDentification and Susceptibility Testing for Gram-negative Bacteremia: RAPIDS-GN. Clin.
Accessed 15/06/2024. Infect Dis. 2021;73(1):e39-e46. doi: 10.1093/cid/ciaa528.
8. U.S. Food and Drug Administration. Antimicrobial Susceptibility Test (AST) Systems - 24. Prabhu K, Rao S, Rao V. Inducible clindamycin resistance in Staphylococcus aureus isolated
Class II Special Controls Guidance for Industry and FDA. Guidance Documents (Medical from clinical samples. J Lab Physicians 2011;3(1):25-27. doi: 10.4103/0974-2727.78558
Devices and Radiation Emitting Products) Web site. Published 2018. https://www.fda. 25. Wright H, Bonomo RA, Paterson DL. New agents for the treatment of infections with
gov/medical-devices/guidance-documents-medical-devices-and-radiation-emitting- Gram negative bacteria: Restoring the miracle or false dawn? Clin Microbiol Infect.
products/antimicrobial-susceptibility-test-ast-systems-class-ii-special-controls- 2017;23(10):704-712. doi: 10.1016/j.cmi.2017.09.001.
guidance-industry-and-fda Accessed 15/06/2024.
26. Wenzler E, Maximos M, Asempa TE, et al. Antimicrobial susceptibility testing: An updated
9. Levison ME and Levison JH. Pharmacokinetics and Pharmacodynamics of Antibacterial
Agents. Infect Dis Clin North Am. 2009;23(4): 791–vii. doi: 10.1016/j.idc.2009.06.008. primer for clinicians in the era of antimicrobial resistance: Insights from the Society of
10. Tascini C, Sozio E, Viaggi B, Meini S. Reading and understanding an antibiogram. Ital J Med. Infectious Diseases Pharmacists. Pharmacotherapy. 2023;43(4):264-278. doi: 10.1002/
2016;10:289-200. doi: 10.4081/itjm.2016.794. phar.2781.
11. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European 27. Winstanley T and Courvalin P. Expert Systems in Clinical Microbiology. Clin Microbiol Rev.
Society of Clinical Microbiology and Infectious Diseases (ESCMID). EUCAST New Definitions 2011;24(3): 515–556. doi: 10.1128/CMR.00061-10.
of S, I and R from 2019. https://www.eucast.org/newsiandr. Accessed 15/06/2024. 28. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European
12. European Committee on Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Expected Resistance
Society of Clinical MIcrobiology and Infectious Diseases (ESCMID). Area of Technical
Phenotypes, version 1.2 January 2023. https://www.eucast.org/expert_rules_and_
Uncertainty (ATU) in antimicrobial susceptibility testing, version 9.0, 2019. http:// www.
eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_ tables/Area_of_ expected_phenotypes/expected_phenotypes Accessed 15/06/2024.
Technical_Uncertainty_-_guidance_2019-1.pdf Accessed 15/06/2024. 29. Livermore DM, Winstanley TG, Shannon KP. Interpretive reading: recognizing the
13. Clinical and Laboratory Standards Institute: Methods for Dilution Antimicrobial unusual and inferring resistance mechanisms from resistance phenotypes. J Antimicrob
Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard M7 12th Edition, Chemother. 2001;48:87-102. doi: 10.1093/jac/48.suppl_1.87.
2024. https://clsi.org/standards/products/microbiology/documents/m07/ Accessed 30. Clinical and Laboratory Standards Institute: Analysis and Presentation of Cumulative
15/06/2024. Antimicrobial Susceptibility Test Data; Approved Guideline, 5th edition; M39-A5,
14. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European 2022, CLSI, Wayne, PA. M39 5th Edition, 2022. https://clsi.org/standards/products/
Society of Clinical Microbiology and Infectious Diseases (ESCMID). Determination of
microbiology/documents/m39/ Accessed 15/06/2024.
minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. Clin
Microbiol Infect. 2000;6:509-15. doi: 10.1046/j.1469-0691.2000.00142.x. 31. Morgan DJ, Malani P, Diekema DJ, et al. Diagnostic Stewardship-Leveraging the Laboratory
15. Clinical and Laboratory Standards Institute: Methods for Antimicrobial Dilution and to Improve Antimicrobial Use. J Am Med Assoc. 2017;318(7):607-608. doi: 10.1001/
Disk Susceptibility Tests of infrequently isolated or Fastidious Bacteria M45 3rd Edition, jama.2017.8531.
2015. https://clsi.org/standards/products/microbiology/documents/m45/ Accessed 32. Curran EJ, Lutgring JD, Kabbani S, et al. Advancing Diagnostic Stewardship for Healthcare-
15/06/2024. Associated Infections, Antibiotic Resistance, and Sepsis. Clin Infect Dis. 2022;74(4):723-
16. Gajic I, Kabic J, Kabic D, et al. Antimicrobial Susceptibility Testing: A Comprehensive Review 728. doi: 10.1093/cid/ciab672.
of Currently Used Methods. Antibiotics 2022;11:427. doi: 10.3390/antibiotics11040427. 33. Zakhour J, Haddad SF, Kerbage A, et al. Diagnostic stewardship in infectious diseases:
17. Jorgensen JH and Ferraro MJ. Antimicrobial Susceptibility Testing: A review of
a continuum of antimicrobial stewardship in the fight against antimicrobial resistance.
General Principles and Contemporary Practices. Clin Infect Dis. 2009;49:1749. doi:
10.1086/647952. Int J Antimicrob Agents. 2023;62(1):106816. doi: 10.1016/j.ijantimicag.2023.106816.
18. Banerjee R, Humphries R. Rapid Antimicrobial Susceptibility Testing Methods for Blood 34. Salam A, Al-Amin Y, Pawar JS, et al. Conventional methods and future trends in
Cultures and Their Clinical Impact. Front Med (Lausanne). 2021;8:635831. doi: 10.3389/ antimicrobial susceptibility testing. Saudi J Biol Sci. 2023;30(3):103582. doi: 10.1016/j.
fmed.2021.635831. sjbs.2023.103582.
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