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QUESTIONS AND PREFACE
ANSWERS
on Antimicrobial Of all the laboratory examinations performed daily by clinical

Susceptibility
microbiologists, in vitro susceptibility testing is of particular clinical
importance as an aid for selecting the most appropriate antimicrobial
therapy for individual patients, monitoring the evolution of microbial
Testing of Bacteria resistance, and updating empirical therapeutic strategies.

and Fungi The methodology for in vitro antibacterial testing and the criteria for
interpretation are well established. Antibacterial susceptibility testing is
routinely performed in microbiology laboratories worldwide. Methods
for in vitro antifungal testing and criteria for interpretation have been
This brochure is intended to provide developed more recently and are similar in concept to antibacterial
testing. Susceptibility testing of certain antiviral agents (e.g. anti-HIV
succinct answers to common agents) is also established but the methods and concepts are quite
questions about the performance of different. The scope of this brochure will be limited to a discussion of
in vitro antimicrobial susceptibility antibacterial and antifungal susceptibility testing.

testing of bacteria and fungi and the This brochure explains basic facts concerning the relevance and
procedures of in vitro susceptibility testing. It provides information on
value of the results in guiding the essential elements required to perform and utilize susceptibility
antimicrobial therapy. testing as a tool for optimizing anti-infectious therapy.

This brochure was compiled with the

Prof. John TURNIDGE
help of John Turnidge and Clinical Director of Microbiology and Infectious Diseases
Jan Bell, SA Pathology, Adelaide, and
Jan BELL
South Australia. Unit Head, Antimicrobials and Multi-Resistant Organisms
SA Pathology, Women’s and Children’s Hospital
Adelaide, South Australia

1
 1. What is in vitro susceptibility the empirical therapy and/or indicate appropriate alternative agents
for treatment. Alternative agents may be required when resistance
testing? is detected or the patient experiences an adverse reaction to the
empirical agent. Often, it is possible to identify appropriate agents
Susceptibility testing measures the level at which a particular for oral step-down therapy or narrower spectrum agents likely to
antimicrobial inhibits the growth of a specific microbial strain. be as effective as the broader empirical therapy.
A variety of laboratory methods can be used to measure the
A second important purpose of routine in vitro susceptibility testing is
in vitro susceptibility of microbial pathogens to antimicrobial
to monitor the evolution of bacterial and fungal resistance. This
agents. Methods should be standardized based on international
role requires periodic statistical analysis of the accumulated levels of
standards such as EUCAST, CLSI and ISO 20774 for antibacterials
resistance per species, type of specimen, and patient, in order to
(antifungal standard under development).
guide the initial empiric choice of antimicrobial therapy while awaiting
“Sensitivity” is a widely used term and is essentially synonymous laboratory test results. The pattern of antimicrobial resistance by
with susceptibility in the context of susceptibility testing. ward, healthcare establishment, region or country guides empiric
antibiotic therapy choices and antibiotic formulary decisions.
Detailed statistical analysis enables the detection of outbreaks,
especially in the hospital or long-term care setting, caused by multi-
resistant bacteria, which justify investigation and appropriate infection
2. Why perform control intervention. The detection of a new resistance pattern or
in vitro susceptibility testing? a large number of patients infected with multi-resistant bacterial
strains at one time and in the same place may indicate the need
for implementation or change of infection control practices.
The goal of in vitro antimicrobial susceptibility testing is to assess
the activity of an antimicrobial agent on a bacterial or fungal Data from routine antimicrobial susceptibility testing performed in
strain in order to predict the likelihood of in vivo efficacy of clinical microbiology laboratories therefore influences the therapeutic
antimicrobial therapy when the antimicrobial is administered to decisions for current and future patients.
patients.
The main purpose of routine in vitro susceptibility testing in the
clinical microbiology laboratory is to guide physicians in selecting
antimicrobial therapy for treatment of individual patients. The DUAL PURPOSE OF SUSCEPTIBILITY TESTING:
susceptibility testing is performed on bacterial and fungal strains
isolated from individual patients and presumed to be the etiology Individual (to guide the selection
of their infection. The physician utilizes the susceptibility test result and modification of antimicrobial therapy)
along with other available clinical information (e.g. site of infection, and
severity of infection, immune status of patient, co-morbidities, etc.) Epidemiological
to select the optimal therapeutic agent for that particular patient’s
infection. Usually the susceptibility test results become available
after initiation of empirical antimicrobial therapy. When this occurs,
the susceptibility test results serve to confirm the appropriateness of

2 3
 3. When should a susceptibility
test be performed?
In general, assuming that standardized testing methodologies have Sometimes microbiologists cannot definitely determine if
been developed, susceptibility testing is indicated for microorganisms susceptibility testing is required, without obtaining the clinical
causing infections warranting antimicrobial therapy when the information that only a clinician can provide.
susceptibility cannot be reliably predicted based on the known
For example, a commensal bacterium (e.g. Staphylococcus
characteristics of the organism.
epidermidis) is occasionally isolated from sterile site cultures
In vitro susceptibility testing methodology is well established for (e.g. blood, joint fluid, cerebrospinal fluid) due to inadequate
bacteria and is considered a routine part of the culture process decontamination of the skin during specimen collection.
(Clinical Laboratory Standards Institute (CLSI), 2009 and European
Susceptibility testing should not be performed on probable
Committee for Antimicrobial Susceptibility Testing (EUCAST), 2000,
contaminants. However, the same S. epidermidis can cause a true
2003). In vitro susceptibility testing is usually performed each time
bloodstream infection in an immuno-compromised patient or an
bacteria considered to be responsible for a patient’s infection are
infection at a specific body site (e. g. prosthetic joint, cerebrospinal
isolated from a clinical specimen.
fluid shunt) in which case, susceptibility testing should be
Susceptibility testing for yeast species is less commonly performed performed.
and is not available for all species. There are published reference
Clinical symptoms can also be a determining factor when deciding
methods (CLSI and EUCAST) and some commercial products are
whether to perform susceptibility tests (e.g. diagnosis of urinary
available. Each laboratory will determine the need for routine testing
tract infection with a low bacterial count).
of yeast isolates from clinical specimens based on the need of the
patient population. Reference methodology for in vitro testing of
mould species is under development and currently only available
from specialised mycology laboratories. Establishing the need for susceptibility testing
requires a close working relationship
When the same species is isolated from specimens taken from
different body sites (e.g. urine and blood) or from multiple specimens
between microbiologists and clinicians.
from the same body site (e.g. multiple blood culture bottles are
positive), the laboratory may elect not to perform susceptibility
testing on all of the isolates from the patient.

Susceptibility testing should not be routinely

performed on organisms that are part
of the normal bacterial flora and usually not
considered pathogenic.

4 5
 4. Can susceptibility and/or
resistance of bacteria to an
antibiotic be predicted?
Each antibiotic is characterized by a natural spectrum of antibacterial Acquired resistance is a characteristic specific to some strains,
activity. This spectrum is the list of bacterial species which, in their within a naturally susceptible bacterial species, in which the
naïve (wild-type) state, have their growth inhibited at a concentra- genotype has been modified by gene mutation or gene
tion known to predict efficacy in vivo. These bacterial species are acquisition. Contrary to natural resistance, acquired
said to be naturally susceptible to this antibiotic. Bacterial species resistances are evolutionary and their frequency is often
which are not included in this spectrum are said to be naturally dependent on the amount of exposure to antibiotics. Given the
(intrinsically) resistant. evolution of acquired resistances, the natural activity spectrum
is no longer sufficient to guide the choice of antibiotic therapy
for numerous species. Acquired resistance results from a
Natural resistance is a stable characteristic of all strains of mutation in the microbial chromosome or the acquisition of
the same bacterial species. It occurs as a result of the extra-chromosomal DNA. In bacteria, the spread of resistance
microorganism’s genetic composition. This intrinsic resistance mechanisms occurs through vertical transmission (parent to
means that the antimicrobial agent is unlikely to ever be useful daughter cells) of inherited mutations from previous genera-
to treat an infection due to strains of this species. Knowledge tions as well as horizontal spread of mobile genetic elements
of naturally resistant species enables prediction of the likely such as plasmids (moving between cells and often between
inactivity of a molecule in relation to the identified or probable different species of bacteria).
bacterial pathogen. It sometimes constitutes an identification
Acquisition of antimicrobial resistance mechanisms can render
aid as some species can be characterized by their natural
previously useful antimicrobial agents useless for treating most
resistance.
strains of the species (e.g. penicillin and Staphylococcus aureus).
EXAMPLES : Therefore, susceptibility testing becomes essential for the
• Natural resistance of Klebsiella pneumoniae to aminopenicillins (ampicillin, detection of acquired resistance.
amoxicillin) due to a β-lactamase (mostly SHV-1).
• Natural resistance of Proteus mirabilis to tetracyclines and colistin (due to
natural targets with reduced binding ability).
NATURAL RESISTANCE:
permanent characteristic of the species, which is
known and predictable.
ACQUIRED RESISTANCE:
characteristic of some bacterial strains, which is
evolutionary, unpredictable and justifies the need
for susceptibility testing.

6 7
 Examples of natural resistance (adapted from Livermore DM et al., 2001)

Pseudomonas
Enterobacteriaceae Streptococci Enterococci Staphylococci
aeruginosa
Antibiotics
Enterobacter
E. coli Klebsiella
Serratia

Penicillin G

Aminopenicillin

Aminopenicillin
+ beta-lactamase inhibitor

Cephalosporins: C1G
C2G
C3G

Aztreonam

macrolides

Glycopeptides

Colistin

= Natural resistance

C1G : 1st generation cephalosporins

C2G : 2nd generation cephalosporins
C3G : 3rd generation cephalosporins

8 9
 5. What is an antibiotic clinical 6. How is the susceptibility to an
spectrum? antimicrobial measured?
In order to take into account the evolution of acquired resistances Most susceptibility testing is growth based. It involves exposing a
and therefore provide clinicians with useful information when pure culture of a microorganism to a range of concentrations of
choosing empiric antibiotic therapy, the concept of clinical spectrum an antimicrobial agent and observing the presence or absence of
complements that of the natural spectrum. growth after a period of incubation. The results can vary widely
depending on the conditions of testing. It is therefore imperative
The clinical spectrum is defined for each antibiotic and in some
to use standardized methods.
jurisdictions is included in the technical package insert which is
approved during the registration of antibiotics. This spectrum is When performing in vitro susceptibility testing, technical factors
initially defined by clinical breakpoints, which are devised by must be controlled by rigorous standardization of all the analysis
integrating microbiological data (Minimum Inhibitory Concentra- stages (purity and density of the bacterial inoculum, medium
tions – MICs, and wild-type MIC distributions), pharmacokinetics/ composition, reagents, incubation conditions, reading method and
pharmacodynamics, and clinical outcome data. Regulators may biological and clinical criteria for interpretation of these results).
also define susceptible species as only those species for which the Detailed and continuously updated international recommendations
clinical activity of the product has been demonstrated. Strains other are available, such as those compiled by the CLSI and EUCAST.
than those defined by the regulator may still be susceptible to an Quality control procedures for evaluating analytical accuracy and
antimicrobial agent at the same clinical breakpoints. The treating precision must also be applied regularly in order to guarantee the
clinician then takes responsibility when using that agent to treat quality of the susceptibility test.
infections caused by such strains. Broth dilution was one of the earliest antimicrobial susceptibility
The clinical spectrum is regularly revised to take into account the testing (AST) methods. Originally performed in test tubes (macro-
evolution of acquired resistances. The prevalence of resistance may broth dilution), it has been miniaturized into microtiter plates (micro-
vary geographically and with time for selected species and local broth dilution). Two-fold dilutions of the antimicrobial are made in a
information on resistance is desirable, particularly when treating nutrient broth and then each well is inoculated with a standardized
severe infections. number of microorganisms. As defined by standardized methodol-
ogy, the plates are incubated at a defined temperature for a defined
period of time. The lowest concentration of antimicrobial with no
visible growth is the minimum inhibitory concentration (MIC).
CLINICAL SPECTRUM OF ACTIVITY: Modern technology has allowed miniaturization and automation of
• useful to guide empiric antibiotic therapy broth dilution methodology which has reduced the time to results.
In general, for routine bacterial susceptibility testing, results are
• depends on the clinical breakpoints available in several hours to a day.
and frequency of resistance
and the in vivo activity of the antibiotic Automation increases precision, minimizes
operator error, and provides traceability to
AST methods.

10 11
 Antimicrobial gradient diffusion is another form of AST in which a Routine Laboratory methods for MIC determination
concentration gradient is established in an agar medium onto which
a standardized suspension of microorganism is inoculated. This Time
Principle to results
method has the capability to generate a MIC value across an extensive
range of dilutions for a wide range of organism/antimicrobial
Bacterial growth
combinations. measured
according to 18 hrs
The disk diffusion method involves placing antimicrobial impreg- an antibiotic
nated disks onto an agar surface that has been inoculated with a concentration
standardized suspension of microorganism. After a defined period gradient
Gradient diffusion

MANUAL METHODS
of incubation, the zone of no growth around each disk is measured
and interpreted based on published interpretive criteria, which
have been developed by comparison with MIC methods.

Bacterial growth
measured

7. What is a MIC and how

ATB™ strip according 18 hrs
to 2 or several

is it used? antibiotic
concentrations

The basic measurement of the susceptibility of a microorganism

to an antimicrobial agent is based on the determination of the
Microtiter plate
minimum inhibitory concentration (MIC).
Bacterial growth
The MIC is defined as the lowest concentration of a range of measured
SEMI-AUTOMATED OR AUTOMATED METHODS

antibiotic dilutions that inhibits visible growth of bacteria within according to one 4 hrs
a defined period of time. (4 hrs) to
or 2 antibiotic 18 hrs
The MIC is a measure of antimicrobial potency. It is the concentrations
(18 hrs)
fundamental reference value that enables a range of antimicro- ATB - rapid ATB
bial activity to be established for different microbial species, and
to which all other testing methods are compared. Each microbial
species will have a unique MIC distribution in the naïve or
wild-type state (EUCAST website), i.e. not all members of a species
will have exactly the same wild-type MIC. Kinetic
Microtiter plate analysis 4 hrs
Various laboratory techniques enable the MIC value to be measured to
of bacterial 18 hrs
or estimated semi-quantitatively in routine use (see opposite). growth

Using the MIC, a tested strain can then be categorized according

to its susceptibility for the antimicrobial being tested. This strain is
said to be Susceptible (S), Intermediate (I) or Resistant (R) to
VITEK® 2 cards
the antibacterial or antifungal agent.

12 13
 8. What are clinical breakpoints? the zone diameters generated by the standardized disk diffusion
method on large numbers of relevant strains, using sophisticated
statistical techniques.
In general, two antimicrobial concentrations, known as «break-
points» or interpretive criteria determine three categories:
Susceptible (S), Intermediate (I), and Resistant (R). Below (or equal
to) one concentration, the clinical isolate is categorized as S, above
(or equal to) the second concentration, it is categorized as R, and
between these two concentrations, it may be categorized as I.
9. What do categories S, I, R mean?
For some newer antimicrobials, where resistance is very rare or un- For a given antimicrobial, a bacterial or yeast strain is classified
reported, the categories may be Susceptible and Non-susceptible. according to the following criteria.
Although the term “breakpoint” has been used in a wide range of Susceptible (S)
contexts, it should be reserved for the values determined by the Susceptible means that the infection caused by that strain is highly
methods described below. likely to respond to treatment, at the site from which the strain
Breakpoints are developed through a detailed examination of MIC was isolated, with the usual antimicrobial regimen for that type
data and distributions, resistance data and mechanisms, pharmaco- of infection.
kinetic and pharmacodynamic properties of the antimicrobial Intermediate (I)
and available data on clinical outcome (by MIC of the infecting Intermediate means that the infection is likely to respond to higher
strain if possible). They are regularly reviewed and revised as dosing regimens (where possible) or because the antimicrobial is
appropriate when new information becomes available in one or concentrated at the site of infection. It is also a buffer category to
more of these data sources. ensure day-to-day test variation does not result in a resistant isolate
Breakpoints are used by clinical microbiology laboratories to being categorized as susceptible or vice versa.
categorize and report clinical isolates as S, I or R, which will then Resistant (R)
assist physicians in selection of antimicrobial therapy. Resistant means that the infection caused by that strain is unlikely
AST interpretive category classifications are based on the in vitro to respond to treatment with any regimen of the antimicrobial agent.
response of an organism to an antimicrobial agent at levels Susceptible-dose dependent (SDD)
correlated to blood or tissue levels attainable with the usually The “susceptible-dose dependent” category implies clinical efficacy
prescribed doses of that agent. The intention is to correlate clinical when a higher than normal dosage of a drug is used and maximal
breakpoints with the likely performance of the drug when used blood level achieved. This is currently reserved for certain antifungal
to treat an infected patient. drugs (e.g. fluconazole)
Breakpoints are of two types: Nonsusceptible (NS)
• MIC breakpoints, applied to broth or agar-based methods, This category is used for organisms that currently have only a
including the automated methods such as VITEK 2. susceptible interpretive category, but not an intermediate or
• zone diameter breakpoints, used to categorize test results for resistant interpretive category. It is often given to new antimicrobial
the disk diffusion method. Disk diffusion breakpoints compare agents for which few or no resistant clinical isolates have yet been
the MICs determined using a reference MIC testing method with encountered.

14 15
 10. What criteria are used to select Often laboratories report only a selection of the antimicrobials
tested, those that are most appropriate or commonly used.
the antibiotics to be tested? This selected reporting is often preferred because it assists in
“antimicrobial stewardship” by guiding the prescriber to the most
The selection of the antimicrobial agents to be tested must be carefully appropriate agents for the treatment of the infection and
determined depending on the microbial species and their natural withholding results for agents which may be effective but are
resistance, local epidemiology of acquired resistances, the site of unnecessarily broad or are only used as reserve agents.
infection and local therapeutic options (formulary).
For example, the laboratory may choose ”cascade reporting”, e.g.
Each laboratory selects which antimicrobials are appropriate to test to withhold the results of a third-generation cephalosporin and a
and report for each organism isolated. carbapenem for a strain of Escherichia coli which is susceptible to
earlier generations of cephalosporins and beta-lactamase inhibitor
• The antibiotics tested are those of therapeutic value for the type
combinations, or to modify the report to include additional agents
of infection and the body site from which the specimen has been
when certain resistances are detected.
collected.
• Due to the importance of acquired resistance, it is sometimes
necessary to test antibiotics which serve as resistance «markers»,
i.e. which are useful to detect resistance mechanisms.
EXAMPLE: Ertapenem is an excellent carbapenemase resistance marker for Entero-
bacteriaceae. 12. What is antibiotic equivalence?
Equivalence is the prediction of in vivo activity for one antimicrobial
based on results obtained by testing another, related antimicrobial
The choice of antibiotics to be tested is made agent. In this case, only a category result (S, I, R) can be reported.
in relation to their therapeutic value and their
EXAMPLE: Equivalence between erythromycin which is tested and other macrolides
usefulness to detect resistance mechanisms. (e.g. azythromycin and clarithromicin) which are not tested. The category (S, I, or R)
result for the other antimicrobials can be predicted from that obtained for erythromycin.

It is possible to test a restricted number

11. How are susceptibility results of antibiotics without limiting therapeutic
reported? possibilities.

Susceptibility testing results are usually reported as S, I, or R for

each antimicrobial tested for that isolate. Most laboratories will also
include the actual MIC value, as it provides critical information that
assists clinicians in making therapeutic decisions.

16 17
 13. Why do some patients with
susceptible isolates fail therapy?
A MIC is an in vitro measurement (a laboratory assay) that When multiple reports of the correlation of therapeutic outcome
provides an estimate of antimicrobial potency. It does not take with in vitro susceptibility are examined (Rex J and Pfaller MA 2002),
into account host factors, especially the kinetics of the agent in an a pattern referred to as the “90-60 rule”, or natural response rate,
individual host, which are just as important as susceptibility test emerges. This 90-60 rule observes that infections due to susceptible
results in determining the outcome of treatment. Therefore, the isolates respond to appropriate therapy approximately 90% of the
susceptibility of a microorganism in vitro does not always assure time, whereas infections due to resistant isolates (or infections
successful therapy. As a corollary, resistance defined in vitro often, treated with inappropriate antibiotics) respond less than 60% of
but not always, predicts therapeutic failure. the time. Although there are some important exceptions, this rule
is relatively robust and holds whether the outcome measurement
is clinical response, bacteriological response, or mortality.

Antimicrobial Drugs: why do they sometimes fail?

PK and PD
Metabolism and Elimination
PROPERTIES OF
Tissue penetration
ANTIMICROBIAL
Method of action
Adverse event profile

Immune System In Vitro

Underlying diseases
HOST FACTORS Testing PATHOGEN
Barrier status
Foreign bodies MIC & SIR
Species
Virulence factors
Resistance
mechanisms
CNS
Intravascular
Pulmonary SITE OF INFECTION
Need for surgical
intervention

18 19
 14. How do bacteria acquire Modification of the
antimicrobial target resulting
resistance? in reduced binding.

The genetic mechanism of acquired resistance can be: EXAMPLE: Modification of Penicillin-
Binding Proteins (PBPs) of:
• The mutation of a gene involved in the mode of action of - oxacillin-resistant Staphylococcus
the antimicrobial that results in an alteration in the molecule aureus (known as MRSA*).
- penicillin-resistant S. pneumoniae.
that is the target of the antimicrobial. Most often, this type of
resistance mutation results in reduced antimicrobial binding to * methicillin-resistant Staphylococcus aureus

the target. This mechanism is the most commonly observed

for the following antibiotics: quinolones, rifampin, fusidic acid,
fosfomycin, antituberculosis drugs, and sometimes cephalosporins.
EXAMPLE: Resistance to quinolones by modification of DNA gyrase in
Enterobacteriaceae.

• Acquisition of resistance genes transferred from a strain Impermeability of the

belonging to an identical or different species, usually on a bacterial outer membrane
mobile genetic element such as a plasmid. Some antibiotics by alteration or quantitative
are particularly affected by this mechanism: ß-lactams, decrease of porins.
aminoglycosides, tetracyclines, chloramphenicol, sulfonamides. EXAMPLE: Imipenem-resistant
EXAMPLE: Resistance to ampicillin in E. coli and Proteus mirabilis. Pseudomonas aeruginosa.

The biochemical mechanism of resistance can be due to:

Production by the bacteria of Efflux mechanism: expulsion

enzymes inactivating the of the molecule by active
antibiotic. transport.

EXAMPLE: Penicillinase in staphylococci, EXAMPLE: Tetracycline-resistant

extended spectrum ß-lactamase (ESBL) staphylococci.
in Enterobacteriaceae.

20 21
 15. Can antimicrobials «induce» 17. What is cross-resistance or
resistance? associated resistance?
Antimicrobials do not cause resistance, but may allow resistant Cross-resistance is a resistance mechanism that affects an entire
mutants to proliferate by eliminating susceptible microorganisms. class or subclass of antibiotics.
This is known as selection pressure. EXAMPLES:
• For Streptococci, resistance to 14- and 15-membered macrolides can be
The increase in the frequency of resistant strains is often linked to predicted by testing erythromycin.
increased use of a specific antimicrobial. • Resistance to oxacillin in Staphylococci confers in vivo resistance to almost
all ß-lactams.

In certain cases, a resistance mechanism can affect antibiotics

from different classes.
EXAMPLE: Resistance due to impermeability to tetracyclines also affects
chloramphenicol and trimethoprim.

16. Which methods enable Resistances are said to be associated when several resistant
mechanisms involving different antibiotic classes frequently
resistance mechanisms to be occur together. Associated resistance is often plasmid-mediated,
demonstrated in vitro? and in Gram-negative bacteria can often be encoded by genes
strung together inside an integron.
For the moment, only certain techniques enable the direct detection EXAMPLE: Resistance to oxacillin in staphylococci is often associated with
resistance to quinolones, aminoglycosides, macrolides and tetracyclines.
of biochemical mechanisms (example: detection of ß-lactamase by
hydrolysis of the indictor ß-lactam nitrocefin) or genetic determinants
of resistance (example: detection of the mecA gene responsible for
staphylococcal resistance to oxacillin).
Susceptibility test results can suggest the presence of a resistance
mechanism.

22 23
 18. Why is it necessary to interpret EXAMPLES:
• Strains of Staphylococcus aureus that test Resistant to methicillin or oxacillin
susceptibility test results? (MRSA) may test Susceptible in vitro to other beta-lactams, especially cepha-
losporins. However, in vivo data have demonstrated a high level of treatment
failures of MRSA infections with beta-lactam therapy. Therefore the interpretation
The rapid evolution of acquired resistance mechanisms by clinically is changed to “Resistant” for all beta-lactams regardless of the actual MIC value.
significant bacteria and the sometimes weak expression of these • A strain of S. aureus resistant to erythromycin can test in vitro as Susceptible to
resistance characteristics may require the use of tests in addition clindamycin and as positive by an Inducible Clindamycin test. Treatment with
to those routinely performed. The goal of the additional tests is clindamycin can result in the selection of resistant mutants that cause inactivation
of the antibiotic and treatment failure. For strains of S.aureus testing positive
to avoid categorizing bacteria as susceptible when they express with the Inducible Clindamycin test, the Susceptible result for clindamycin must
only low-level resistance in vitro but are likely to cause therapeutic be modified to Resistant or a standard comment must be attached to the report
failure in vivo. alerting the prescriber of this possibility.

To avoid this, susceptibility testing must be interpreted to discern “Interpretive reading” can also be enhanced by the use of expert
even a weakly expressed resistance mechanism (by comparing systems that are capable of examining resistance profiles and making
results for each antibiotic). Therefore, with appropriate interpretation, predictions about other agents not tested or their likely therapeutic
a strain initially testing as susceptible will be categorized as I or R. efficacy (see Question 19).

Through the judicious choice of antibiotics

tested, the interpretation of susceptibility test
results can help detect resistance weakly
expressed in vitro.
Interpretive procedure

Knowledge
of resistance
mechanisms

Determination
Resistance phenotype of probable
Result given
observed in vitro resistance
mechanism

Additional Validation/
tests if required Modification

24 25
 19. What is the role of an Expert
System?
Validation of a susceptibility test result requires a comprehensive indicating a rare phenotype in a given context
knowledge of resistance mechanisms and antibiotic activity. An Expert The regular update of information constituting the knowledge-
System is a software package designed to help integrate this knowledge base is essential. The genetic make-up of the predominant
and automatically interpret susceptibility tests, check results and strains varies over time and from one geographical location
suggest the necessary corrections. to another.
The Expert System contributes to the reliability of the result by: EXAMPLES:
• MRSA strains have been resistant to gentamicin for a very long time, but
ensuring consistency between the susceptibility test result this association is less true today due to the emergence of community-
and bacterial identification associated strains, e.g. USA300, which are gentamicin susceptible.
• Vancomycin-resistant enterococci are frequent in the USA but are uncommon
EXAMPLE: Klebsiella pneumoniae - ampicillin S = improbable phenotype. in Europe.
identifying improbable or impossible resistance phenotypes screening for important resistance mechanisms
EXAMPLE: E. coli - cefazolin S - cefotaxime R = impossible phenotype. EXAMPLE: Detection of carbapenemases in Gram-negative bacteria or
heterogeneous vancomycin resistance in Staphylococcus aureus.
detecting insufficiently expressed resistances
EXAMPLE: Detection of a third-generation susceptible Enterobacter cloacae
and correction of S results to R, or appending a standard comment to a report
for third-generation cephalosporins.

The Expert System: a tool for interpretation of susceptibility

test results

Knowledge
base

Deduction of Result
Resistance phenotype probable resistance checked and
observed in vitro mechanism modified
Inference
Engine

26 27
 20. Some current resistance issues What is the probability of infection by Enterobacteriaceae
with extended spectrum β-lactamase (ESBL) strains?
In patients from the community, the frequency of this type
Are bacteria responsible for community-acquired urinary of multi-resistant bacteria (i.e. with acquired resistance to
tract infections affected by antibiotic resistance? numerous antibiotics) used to be linked to a previous hospital
stay. These bacteria were mainly found in hospitals, where
Although bacterial resistance is more frequent in hospitals
their multi-resistance gives them a selective advantage.
than in the community, bacteria that are most often found in
Generally transmitted from one patient to another in the
community-acquired pathologies, such as E. coli, can acquire
same healthcare unit (hospital, clinic, nursing home, etc.), they
antibiotic resistance.
were and still are responsible for nosocomial infections.
For example, in 2005 the level of acquired resistance of E.coli to antibiotics
frequently used for the treatment of urinary tract infections were: In recent years, however, ESBL-producing E. coli have begun to
emerge in community strains worldwide (Oteo J et al., 2010).
Ampicillin 49% These strains carry ß-lactamases of the CTX-M type, rather
Fluoroquinolones 23% than the more common TEM or SHV type found in hospital-
Trimethoprim-sulfamethoxazole 27% associated strains. One clone in particular, O25:H4-ST131,
Source: TRUST 2007 encoding CTX-M-15, seems to have spread widely across the
world (Nicolas-Chanoine M-H et al, 2008). Not unexpectedly,
they are most frequently isolated from urine. Many strains are
What is Community-associated MRSA? resistant to multiple anti-microbial agents and are challenging
for outpatient management. They are adding to the overall
The first reported cases of CA-MRSA began to appear in the
burden of ESBLs in hospitals because they require the same
mid-1980s in Australia and New Zealand, and in the mid-1990s
infection control interventions.
in the United States, the United Kingdom, Europe and Canada.
These cases were notable because they involved people who
had not been exposed to a healthcare setting. Why is it essential to check the susceptibility of
S. pneumoniae to antibiotics and notably to penicillin G?
This increase in the incidence of MRSA infection has been
associated with the recognition of new MRSA clones known For over 3 decades, pneumococcal non-susceptibility to
as community-associated MRSA (CA-MRSA). CA-MRSA strains penicillin G has continuously increased in many countries
infect a different group of patients, cause different clinical (less than 1% in 1985, 10-50% in the mid 2000’s (Linares J
syndromes, and differ in antimicrobial susceptibility patterns et al., 2010). This resistance is often associated with resist-
compared to healthcare-associated MRSA (HA-MRSA) strains, ance to other antibiotics (e.g. tetracyclines, macrolides).
CA-MRSA strains can spread rapidly among healthy people This emerging resistance impacts empirical antibiotic therapy
in the community and are now a frequent cause of infections for acute otitis media, sinusitis and bronchopulmonary
in healthcare environments as well. The clinical spectrum of infections, as well as meningitis which are often caused by
infectious syndromes associated with CA-MRSA strains ranges S. pneumoniae. In some cases, resistance of S. pneumoniae
from a commensal state to severe, overwhelming infections, to penicillin G may indicate resistance to other ß-lactam anti-
especially skin and pulmonary. (David MZ and Daum RS 2010). biotics, making determination of susceptibility necessary.

28 29
 Is there any problem with resistance to antifungal agents?
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et al., 2006). These species are resistant or less susceptible to Testing of Yeasts. Approved Standard–Third Edition; M27-A3, 2008, CLSI, Wayne,
some commonly used antifungal agents. With this change in PA. n Clinical and Laboratory Standards Institute: Reference Method for Broth
epidemiology as well as an increased choice in available Dilution Antifungal Susceptibility Testing of Yeasts. Third Informational Supplement;
antifungal agents from which to choose, antifungal M27-S3, 2008, CLSI, Wayne, PA. n Clinical and Laboratory Standards Institute:
susceptibility testing is becoming more of a necessity, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous
especially for the commonly isolated yeasts. Fungi. Approved Standard–Second Edition; M38-A2, 2008, CLSI, Wayne, PA.
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Conclusion (ESCMID). Determination of minimum inhibitory concentrations (MICs) of antibac-
terial agents by agar dilution. Clin Microbiol Infect, 2000; 6:509-15. n European
Antibiotic susceptibility testing, at the interface between the Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society
clinical diagnosis and the therapeutic decision, is a key element of Clinical Microbiology and Infectious Diseases (ESCMID). Determination of
essential for guiding both microbiologically-documented and minimum inhibitory concentrations (MICs) of antibacterial agents by broth dilution.
empiric antibiotic therapy. Not only is the susceptibility test result Clin Microbiol Infect, 2003; 9:1-7. n EUCAST definitions of clinical breakpoints
and epidemiological cut-off values. 2010 At: http://www.srga.org/Eucastwt/
of immediate interest for the clinician to guide selection of
eucastdefinitions.htm. n EUCAST disk diffusion test. 2010. At: http://www.
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eucast.org/eucast_disk_ diffusion_test/ n Liñares J, Ardanuy C, Pallares R, Fenoll A.
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relationship between the microbiologist and the clinician more
n Nicolas-Chanoine M-H, Blanco J, Leflon-Guibout V, et al. Intercontinental
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30 Infect Dis 2002; 35:982–9. 31
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