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ANSWERSon Antimicrobial
Of all the laboratory examinations performed daily by clinical
microbiologists,
in vitro susceptibility testing is of particular clinical importance as an aid for selecting the most appropriate antimicrobial
Testing of Bacteria
therapy for individual patients, monitoring the evolution of microbial resistance, and updating empirical therapeutic strategies.
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
succinct answers to common
testing. Susceptibility testing of certain antiviral agents (e.g. anti-HIV 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
value of the results in guiding
procedures of
in vitro susceptibility testing. It provides information on 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,
South Australia.
Jan BELLUnit Head, Antimicrobials and Multi-Resistant OrganismsSA Pathology, Women's and Children's HospitalAdelaide, South Australia
1. What is
in vitro susceptibility
the empirical therapy and/or indicate appropriate alternative agents
for treatment. Alternative agents may be required when resistance 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-
2. Why perform
resistant bacteria, which justify investigation and appropriate infection 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.
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,
severity of infection, immune status of patient, co-morbidities, etc.)
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
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 characteristics of the organism.
For example, a commensal bacterium (e.g.
Staphylococcus 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 Committee for Antimicrobial Susceptibility Testing (EUCAST), 2000,
Susceptibility testing should not be performed on probable
2003).
In vitro susceptibility testing is usually performed each time
contaminants. However, the same
S. epidermidis can cause a true
bacteria considered to be responsible for a patient's infection are
bloodstream infection in an immuno-compromised patient or an
isolated from a clinical specimen.
infection at a specific body site (e. g. prosthetic joint, cerebrospinal fluid shunt) in which case, susceptibility testing should be
Susceptibility testing for yeast species is less commonly performed
and is not available for all species. There are published reference methods (CLSI and EUCAST) and some commercial products are
Clinical symptoms can also be a determining factor when deciding
available. Each laboratory will determine the need for routine testing
whether to perform susceptibility tests (e.g. diagnosis of urinary
of yeast isolates from clinical specimens based on the need of the
tract infection with a low bacterial count).
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. 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
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
dif erent species of bacteria).
bacterial pathogen. It sometimes constitutes an identification aid as some species can be characterized by their natural
Acquisition of antimicrobial resistance mechanisms can render
previously useful antimicrobial agents useless for treating most strains of the species (e.g. penicillin and
Staphylococcus aureus).
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.
Examples of natural resistance (adapted from Livermore DM et al., 2001)
+ beta-lactamase inhibitor
Cephalosporins: C1G
= Natural resistance
C1G : 1st generation cephalosporins C2G : 2nd generation cephalosporinsC3G : 3rd generation cephalosporins
5. What is an antibiotic clinical
6. How is the susceptibility to an
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
The clinical spectrum is defined for each antibiotic and in some
depending on the conditions of testing. It is therefore imperative
jurisdictions is included in the technical package insert which is
to use standardized methods.
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.
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
method has the capability to generate a MIC value across an extensive
range of dilutions for a wide range of organism/antimicrobial
Bacterial growth
The disk diffusion method involves placing antimicrobial impreg-
nated disks onto an agar surface that has been inoculated with a
standardized suspension of microorganism. After a defined period
Gradient diffusion
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
7. What is a MIC and how
The basic measurement of the susceptibility of a microorganism to an antimicrobial agent is based on the determination of the minimum inhibitory concentration (MIC).
The MIC is defined as the lowest concentration of a range of
Bacterial growth
antibiotic dilutions that inhibits visible growth of bacteria within
according to one 4 hrs
a defined period of time.
The MIC is a measure of antimicrobial potency. It is the
fundamental reference value that enables a range of antimicro-
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.
Various laboratory techniques enable the MIC value to be measured
or estimated semi-quantitatively in routine use (see opposite).
Using the MIC, a tested strain can then be categorized according to its susceptibility for the antimicrobial being tested. This strain is
SEMI-AUTOMATED OR AUTOMATED METHODS
said to be Susceptible (S), Intermediate (I) or Resistant (R) to
the antibacterial or antifungal agent.
8. What are clinical breakpoints?
the zone diameters generated by the standardized disk diffusion method on large numbers of relevant strains, using sophisticated
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
9. What do categories S, I, R mean?
between these two concentrations, it may be categorized as I.
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
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
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
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
10. What criteria are used to select
Often laboratories report only a selection of the antimicrobials
the antibiotics to be tested?
tested, those that are most appropriate or commonly used. 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
• The antibiotics tested are those of therapeutic value for the type
earlier generations of cephalosporins and beta-lactamase inhibitor
of infection and the body site from which the specimen has been
combinations, or to modify the report to include additional agents
when certain resistances are detected.
• 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-
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 usefulness to detect resistance mechanisms.
EXAMPLE: Equivalence between erythromycin which is tested and other macrolides (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
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.
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 defi ned 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?
Metabolism and Elimination
Tissue penetration
Adverse event profi le
Underlying diseases
Virulence factors
SITE OF INFECTION
Need for surgical
14. How do bacteria acquire
Modification of the
antimicrobial target resulting 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*).
that is the target of the antimicrobial. Most often, this type of
- penicillin-resistant S. pneumoniae.
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.
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
EXAMPLE: Penicillinase in staphylococci,
EXAMPLE: Tetracycline-resistant
extended spectrum ß-lactamase (ESBL)
15. Can antimicrobials «induce»
17. What is cross-resistance or
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
resistance mechanisms to be
mechanisms involving different antibiotic classes frequently 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
of biochemical mechanisms (example: detection of ß-lactamase by
resistance to quinolones, aminoglycosides, macrolides and tetracyclines.
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.
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.
signifi cant 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
to avoid categorizing bacteria as susceptible when they express
of the antibiotic and treatment failure. For strains of S.aureus testing positive with the Inducible Clindamycin test, the Susceptible result for clindamycin must
only low-level resistance in vitro but are likely to cause therapeutic
be modifi ed 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 profi les 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.
effi cacy (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
Resistance phenotype
observed in vitro
tests if required
19. What is the role of an Expert
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.
The Expert System contributes to the reliability of the result by:
• 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 identifi cation
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.
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
detecting insuffi ciently expressed resistances
heterogeneous vancomycin resistance in Staphylococcus aureus.
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
Resistance phenotype
probable resistance
observed in vitro
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
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).
These strains carry ß-lactamases of the CTX-M type, rather
Fluoroquinolones
than the more common TEM or SHV type found in hospital-
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
The first reported cases of CA-MRSA began to appear in the
for outpatient management. They are adding to the overall
mid-1980s in Australia and New Zealand, and in the mid-1990s
burden of ESBLs in hospitals because they require the same
in the United States, the United Kingdom, Europe and Canada.
infection control interventions.
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
This increase in the incidence of MRSA infection has been
S. pneumoniae to antibiotics and notably to penicillin G?
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.
Is there any problem with resistance to antifungal agents?
Patient characteristics, antifungal prophylaxis, and other factors appear to have contributed to a change in the spectrum of
n Clinical and Laboratory Standards Institute: Performance Standards for
invasive fungal pathogens. Infections with more resistant
Antimicrobial Disk Susceptibility Tests - Tenth Edition; M2-A10, 2009, CLSI, Wayne,
species of Candida (e. g. C. glabrata, C. krusei), and Aspergillus
PA. n Clinical and Laboratory Standards Institute: Methods for Dilution Antimicrobial
(e.g. A. terreus) as well as non-Aspergillus moulds appear to
Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard–
be on the rise, at least among certain populations. (Pfaller MA
Eighth Edition; M7-A7, 2009, CLSI, Wayne, PA. n Clinical and Laboratory Standards Institute: Reference Method for Broth Dilution Antifungal Susceptibility
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. n Clinical and Laboratory Standards Institute: Analysis and Presentation of Cumulative Antimicrobial Susceptibility Test Dta; Approved Guideline–Second Edition; M39-A2, 2005, CLSI, Wayne, PA. n Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Disk Testing. Twentieth Informational Supplement; M100-A7, 2010, CLSI, Wayne, PA. n David MZ and Daum RS. Community-Associated Methicillin-Resistant Staphylococcus aureus: Epidemiology and Clinical Consequences of an Emerging Epidemic. Clin Microbiol Rev, 2010; 23:616-687. n European Committee for Antimicrobial Susceptibility Testing
(EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (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
of immediate interest for the clinician to guide selection of
and epidemiological cut-off values. 2010 At: http://www.srga.org/Eucastwt/
antimicrobial therapy, but it also plays a role as an epidemiological
eucastdefinitions.htm. n EUCAST disk diffusion test. 2010. At: http://www. eucast.org/eucast_disk_ diffusion_test/ n Liñares J, Ardanuy C, Pallares R, Fenoll A.
surveillance tool for local bacterial and fungal resistance patterns.
Changes in antimicrobial resistance, serotypes and genotypes in Streptococcus pneu-
The evolution of resistance, as well as the development of new
moniae over a 30-year period. Clin Microbiol Infect 2010; 16:402-10. n Livermore
antibiotics and laboratory techniques make a close working
DM, Winstanley TG, Shannon KP. Interpretive reading: recognizing the unusual and
relationship between the microbiologist and the clinician more
inferring resistance mechanisms from resistance phenotypes. JAC 2001;48:87-102 n Nicolas-Chanoine M-H, Blanco J, Leflon-Guibout V, et al. Intercontinental
necessary now than ever before.
emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. J Antimicrob Chemother 2008; 61:273-81. n Oteo J, Pérez-Vásquz M, Campos J. Extended-spectrum beta-lactamase producing Escherichia coli : changing epidemiology and clinical impact. Curr Opin Infect Dis 2010; 23:320-6. n Pfaller MA, Pappas PG, Wingard JR. Invasive Fungal Pathogens: Current Epidemiological Trends. CID 2006; 43:S3–14. n Rex J and Pfaller MA. Has Antifungal Susceptibility Testing Come of Age? Clin
Infect Dis 2002; 35:982–9.
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For which strategies of suicide prevention is there evidence of effectiveness? ABSTRACT This Health Evidence Network (HEN) synthesis report notes that suicide rates among adolescents and young adults have increased considerably over the last decades. In addition, many widely-used suicide prevention programmes have never been scientifically assessed, thus making it uncertain which are effective. Due to the limited evidence and the heterogeneity of the interventions, it is not possible to determine if onesingle intervention was more effective than another. A broad array of suicide preventive interventionsaddressing different risk factors at various levels will be required. HEN, initiated and coordinated by the WHO Regional Office for Europe, is an information service for public health and health care decision-makers in the WHO European Region. Other interested parties mightalso benefit from HEN. This HEN evidence report is a commissioned work and the contents are the responsibility of the authors.They do not necessarily reflect the official policies of WHO/Europe. The reports were subjectedto international review, managed by the HEN team. When referencing this report, please use the following attribution: Guo B, Harstall C (2004) For which strategies of suicide prevention is there evidence of effectiveness?Copenhagen, WHO Regional Office for Europe (Health Evidence Network report;, accessed 15 July 2004).
Journal of Chinese Medicine • Number 91 • October 2009 The treatment of Elevated FSH Levels with Chinese Medicine The treatment of Elevated FSH Levels with Chinese Medicine Follicle stimulating hormone (FSH) levels are routinely tested during biomedical investigations into female fertility. An appropriately low FSH level is frequently required by fertility clinics as an entry requirement for women wishing to receive assisted reproductive technology (ART) treatment such as in vitro fertilisation (IVF). This article