Antimicrobial Susceptibility Testing - Primer For Clinicians

Antimicrobial Susceptibility Testing: A Primer for Clinicians (printer-frie...
5/14/2010 9:05 AM
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From Pharmacotherapy
Kristi M. Kuper, Pharm.D.; Deborah M. Boles, M.S.; John F. Mohr, Pharm.D.; Audrey Wanger, Ph.D.
Posted: 11/17/2009; Pharmacotherapy. 2009;29(11):1326-1343. © 2009 Pharmacotherapy Publications
Abstract and Introduction
Abstract
Appropriate use of antimicrobials in health care continues to be a challenge. Reliable and reproducible antimicrobial
susceptibility testing methods are necessary to provide the clinician with valuable information that can be translated into
positive clinical outcomes at the bedside. However, there are nuances with these testing methods that, if unrecognized, could
lead to misinterpretation of results and inappropriate antibiotic selection. This primer describes the common antimicrobial
susceptibility tests used in the clinical microbiology laboratory and reviews how subtle differences in testing methods and
technique can influence reported results. Clinicians who have a thorough understanding of qualitative and quantitative
methods, automated susceptibility testing systems, and commonly used screening and confirmatory tests for antibiotic-
resistant organisms can strengthen institutional antibiotic stewardship programs and improve patient outcomes.
Introduction
In January 2007, the Infectious Diseases Society of America, in conjunction with the Society for Healthcare Epidemiology,
released guidelines for developing an institutional program to enhance antimicrobial stewardship.[1] These guidelines
underscore the need for accurate and reproducible identification of microorganisms and antibiotic susceptibilities. This is
integral to the care of patients with infectious diseases and plays a critical role in antimicrobial stewardship and epidemiologic
investigations.
The clinician who has an advanced knowledge of antimicrobials, coupled with an understanding of commonly used
microbiology testing methods, can be effective in improving antimicrobial utilization and optimizing patient care. These skills
are particularly relevant today and can help combat increased antimicrobial resistance in the hospital and community settings.
The purposes of this review are to help pharmacists gain a basic understanding of common antimicrobial susceptibility tests
used in the clinical microbiology laboratory, and to demonstrate how differences in testing can influence therapeutic choices
for treating infectious diseases.
Basic Principles of Antimicrobial Susceptibility Testing
The purpose of performing antimicrobial susceptibility testing is to assist clinicians with the selection of appropriate targeted
antibiotic therapy in order to optimize clinical outcomes. Infection-related and overall mortality is reduced when patients are
treated expeditiously with an antibiotic to which the organism is susceptible.[2]
Two basic methods of antimicrobial susceptibility testing are available to laboratories: qualitative and quantitative. Disk
diffusion (also known as the Kirby-Bauer method) is a qualitative method of susceptibility testing that may be prone to some
degree of error depending on the drug and organism being tested.[3-7] However, it is an acceptable option for testing among
patients with uncomplicated urinary tract infections or other less severe infections where eradication is augmented by the
patient's immune system. Because of the high spontaneous cure rates for some mild infections, an organism that is truly
resistant yet reported as susceptible by the microbiology laboratory may not always be clinically relevant.[8] Broth microdilution
and agar dilution methodologies are considered quantitative because they can measure the minimum inhibitory concentration
(MIC). The MIC is defined as the lowest concentration of an antibiotic that inhibits visible growth of a microorganism. Both
quantitative methods are considered the reference methods for susceptibility testing because of their high levels of
reproducibility.
For convenience, most clinical laboratories use an automated system supplemented with one or more manual methods of
susceptibility testing. Automated systems must provide susceptibility results that are consistent with a reference method.
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Antimicrobial Susceptibility Testing: A Primer for Clinicians (printer-frie... 5/14/2010 9:05 AM
Regardless of the testing methodology selected, the results are correlated with a set of standardized interpretations known as
breakpoints. The term "breakpoint" can be confusing, as it may refer to one derived from microbiologic, clinical, or
pharmacokinetic-pharmacodynamic data. Microbiologic breakpoints refer to the MIC for an antibiotic that distinguishes
wild-type bacterial populations from those that have either selected or acquired resistance mechanisms. These breakpoints
are derived from moderate-to-large numbers of in vitro MIC tests. A clinical breakpoint is derived from the antimicrobial MICs
for the infecting organisms isolated in prospective clinical trials. Susceptibility in this context correlates with a high likelihood of
clinical success. Finally, a pharmacokinetic-pharmacodynamic breakpoint predictive of microbiologic effects is derived from
human or animal data that are modeled by using statistical or mathematic techniques such as a Monte Carlo simulation.[9]
Breakpoints for new antibiotics are approved by the United States Food and Drug Administration (FDA) based on composite
data from the breakpoint methodologies described previously and are not frequently altered after the product is released to
market. A recent inventory of antibacterial drug labels indicated that among more than 100 currently approved drug labels, over
70 contained breakpoints that were outdated.[10] Breakpoints may not be available for older drugs (e.g., polymyxin or colistin
for Enterobacteriaceae) or organisms that rarely cause human disease such as Flavobacterium species.
Ongoing surveillance and laboratory process standardization is governed by a nonprofit global standards developing
organization known as the Clinical and Laboratory Standards Institute (CLSI), previously known as the National Committee for
Clinical Laboratory Standards (NCCLS). The CLSI promotes the development and use of voluntary consensus standards and
guidelines within the health care community and publishes a series of reference manuals that serve as useful guides for
antimicrobial susceptibility testing.[11, 12] These manuals are referenced by a number-letter combination that represents the
subject matter and the version. Two manuals of interest to the pharmacist include the M-39 and the M-100. The M-39 is
updated every 3 years (current version is M-39A3) and provides recommendations for antibiogram preparation. The M-100 is
updated yearly (current version is S19) and contains organism-specific breakpoint reference tables for disk diffusion and MIC
testing. The breakpoints published in the M-100 by the CLSI are for both branded and generic antibiotics against different
microorganisms and incorporate in vitro microbiologic information, human and animal pharmacokinetic-pharmacodynamic data,
and clinical and bacteriologic outcomes from clinical studies.[9] These breakpoints may be discordant with the FDA-approved
values (Table 1),[12-18] as well as the values set by the European Union Committee on Antimicrobial Susceptibility Testing
(known as EUCAST).
Table 1. Examples of Discrepancies Between United States Food and Drug Administration and Clinical
and Laboratory Standards Institute Minimum Inhibitory Concentration Breakpoints[12-18]
MIC Breakpoints (μg/ml)
FDA Approved CLSI Approved
Organism Drug Susceptible Intermediate Resistant Susceptible Intermediate Resistant
Streptococcus
Ceftriaxone
pneumoniae
Nonmeningitis ≤ 0.5 1 ≥ 2a ≤1 2 ≥4
Meningitis ≤ 0.5 1 ≥ 2a ≤ 0.5 1 ≥2
Enterococcus sp Daptomycin ≤ 4b —b —b ≤ 4b —b —b
Enterobacteriaceae Tigecycline ≤2 4 ≥8 None
c c
Doripenem ≤ 0.5 — — None
Colistin or
Acinetobacter sp None ≤2 — ≥4
polymixin
FDA = U.S. Food and Drug Administration; CLSI = Clinical and Laboratory Standards Institute; MIC =
minimum inhibitory concentration.
a
The package insert for ceftriaxone does not make a distinction between nonmeningeal and
meningeal isolates of S. pneumoniae.
b
The FDA-approved breakpoint in the daptomycin package insert is for vancomycin-susceptible
Enterococcus faecalis strains only, but there is no FDA-approved breakpoint for vancomycin-
susceptible Enterococcus faecium strains. The CLSI-approved breakpoint can be applied to all
Enterococcus species.
c
The FDA approved only a susceptible classification because of the absence of resistant isolates in
clinical trials. Drugs with an MIC (or disk diffusion test) that suggests nonsusceptibility of isolates
should undergo additional testing.
Qualitative Testing
Disk Diffusion
The principle behind the disk diffusion method is that antibiotic molecules diffuse out from a disk into the agar, creating a
dynamically changing gradient of antibiotic concentrations while the organism being tested starts to divide and growth
progresses toward the critical mass.[19] The zone edge is where the concentration of antibiotic begins to inhibit the organism
reaching an overwhelming cell mass. At this point, the density of cells is high enough to absorb antibiotic in the immediate
vicinity, thus maintaining concentrations at subinhibitory levels, and enabling the test organism to grow. For most rapidly
growing aerobic and facultative anaerobic bacteria, the critical time it takes for the organism to absorb the antibiotic varies from
3-6 hours, but interpretation by the microbiologist generally occurs between 18 and 24 hours. For some slow-growing
organisms, such as Bacteroides fragilis, the gradient is formed before the organism reaches its critical mass, resulting in false
susceptibility.[20]
The standardized testing process described here allows reproducible and consistent results. Before testing, the
microbiologist will prepare a fresh agar plate by using an inoculum that contains a predefined organism density. The inoculum
is prepared by suspending isolated bacterial colonies (taken from an 18-24-hr agar plate) in broth or saline to a turbidity
matching a 0.5 McFarland standard (1 x 108 colony-forming units/ml). This standard is used to measure the turbidity of the
bacteria and ensure a certain number of colony-forming units in the inoculum.[12] The standard concentration of the solution is
important because an inoculum that is too high in colony-forming units can result in falsely smaller zone sizes (i.e., false
resistance), whereas too low of an inoculum can result in falsely larger zone sizes (i.e., false susceptibility).[21, 22]
The inoculum suspension should be used within 15 minutes of preparation. This is particularly important for fastidious
organisms (e.g., Neisseria species, Haemophilus influenzae, and β-hemolytic streptococci) that lose their viability rapidly,
resulting in a low inoculum. A sterile cotton swab is dipped into the suspension and pressed firmly on the inside of the tube to
remove excess liquid. The entire dried surface of the agar plate is inoculated by streaking the surface in three directions. After
each streak, the plate is rotated 60 degrees to obtain an even distribution of the inoculum. After streaking, the plate should be
dried for no more than 15 minutes. Once the agar plate is completely dry, antibiotic disks are applied either manually or with a
dispensing apparatus. For most organisms, no more than 12 disks should be placed on a 150-mm agar plate or 5 disks on a
90-mm plate. It is best to place disks that have predictably small zone sizes (e.g., gentamicin) next to disks that give
predictably larger zones of inhibition (e.g., cephalosporins) so that zone overlap can be prevented.[23]
Optimally, disks should be positioned at a distance of 30 mm apart, and no closer than 24 mm apart when measured center to
center, to minimize inhibition zone overlap.[23] Most dispenser devices are self-tamping (disks are tapped or pressed onto the
agar surface) but may need to be pressed down to make immediate and complete contact with the agar surface. Once in
contact with the agar, the disk cannot be moved because the antibiotics diffuse rapidly once the disk comes into contact with
the agar. The microbiologist's technique is important because movement can disrupt the process integrity and lead to
inaccurate results.
Agar plates are incubated in an inverted position (agar side up) under conditions appropriate for the test organism. The
incubation period is 16-18 hours for rapidly growing aerobic bacteria, and longer for fastidious organisms or special resistance
conditions. When detecting vancomycin or oxacillin resistance in Staphylococcus aureus, plates need to be kept for a
minimum of 24 hours per CLSI testing procedures.[23] After incubation, the agar plate is examined to determine if a
semiconfluent and even "lawn" of growth has been obtained before reading the plate. If individual colonies are seen, the
inoculum is too light and the test should be repeated since the large zone sizes are inaccurate. The same holds true for an
excessively heavy inoculum causing overly small zones with very hazy edges.[22] Either outcome could result in false readings.
If the lawn of growth is satisfactory, the zone diameter is read to the nearest millimeter by using a ruler or sliding calipers. The
zone margin is identified as the area where no obvious visible growth is seen by the naked eye, unless otherwise specified.
Zone diameters on Mueller-Hinton agar (without blood supplements) are read from the back of the plate, whereas blood-
containing agars are read from the surface to ensure that the zone of inhibition is read accurately. The line of demarcation for
this zone should be clear; faint growth or microcolonies detectable only with a magnifying glass or by tilting the plate should be
ignored. Again, inappropriate technique could lead to erroneous susceptibility interpretation.
The main advantages of disk diffusion testing are simplicity, inexpensive equipment, and the cost-effective and flexible choice
of antibiotics for testing. Disadvantages include the inability to obtain an actual MIC and the additional time required by the
technologist for test preparation and providing an interpretation of manually determined zone sizes based on standards.
Commercial zone readers (e.g., BioMICV;[3] Giles Scientific, Inc., Santa Barbara, CA) are available to simplify this process.
Certain antibiotics can be problematic to test with the disk diffusion method because of the specific physiochemical properties
of the molecules. Vancomycin, colistin, and macrolides have higher molecular weights and therefore diffuse very slowly in
agar.[22] The limited diffusion and poorly resolved concentration gradient around these disks result in only a few millimeters of
difference in zone sizes between susceptible and resistant strains, which could result in a potentially ambiguous reading.
Results can also be influenced by the positioning of the light source on the plate. Most plates are read with use of reflected
light, with the exception of linezolid, oxacillin, and vancomycin for both S. aureus and Enterococcus species. In these cases,
the zones should be measured by using transmitted light in order to ensure an accurate measurement of zone diameter. If
results are unexpected or borderline, another method of testing may be required or the test repeated for confirmation.
Quantitative Testing
Minimum Inhibitory Concentration
In critical infections such as bacteremia and endocarditis, accurate quantitative determination of the exact MIC value may be
useful for therapy guidance.[24, 25] In these situations, an organism with an MIC of 0.016 μg/ml to an antimicrobial may have a
significantly different therapeutic implication from that of the same organism-antibiotic combination that is susceptible with an
MIC of 1 μg/ml. The pharmacodynamics of the drug and the infection site factor into this assessment. For example, a
vancomycin MIC of 1.5 μg/ml or greater was found to be independently associated with treatment failure in patients with
methicillin-resistant S. aureus (MRSA) bacteremia (adjusted risk ratio 2.6, 95% confidence interval 1.3-5.4, p=0.01).[26]
Interpreted, this means that patients with bacterial infections caused by MRSA strains that have an antibiotic MIC of 1.5 or 2
μg/ml may fail therapy even though the isolate is considered to be susceptible based on current breakpoints. The clinician who
uses the reported antibiotic MIC for the organism coupled with pharmacokinetic-pharmacodynamic data of the antibiotic can
customize therapy to improve clinical outcomes and reduce mortality.[27-30]
Although all of these processes may seem straightforward, MICs can vary based on the end point, type of media, and
conditions of the incubation period. For example, end points for bacteriostatic drugs should be read at 80% inhibition
(significant reduction in growth) and bactericidal drugs at 100% inhibition (complete inhibition). If a bacteriostatic drug is
evaluated at 100% inhibition (as opposed to 80% inhibition), the MIC reported will be falsely elevated and may result in the
organism being categorized as resistant, instead of susceptible. In addition, capnophilic (carbon dioxide "loving") organisms
such as pneumococci, streptococci, gonococci, and Haemophilus species are incubated in 5% carbon dioxide and MICs will
vary based on this carbon dioxide level.[31, 32]
Most testing methods used in the hospital laboratory are correlated with either broth microdilution, broth macrodilution, or agar
dilution methods. Both broth methods and agar dilution are quantitative tests that are predominantly performed in reference or
research laboratories. These tests are considered reference methods because they are used to gauge the accuracy of other
testing methods. These tests are not easily automated and require the laboratory to prepare stock solutions of antimicrobial
agents that are then dispensed into test tubes (macrodilution), multiple well plates (microdilution; Figure 1), or an agar medium.
Each tube, well, or agar plate contains a differing standard concentration of antibiotic that is inoculated with the specimen. The
MIC can then be determined based on the level of visible growth. The broth dilution and agar dilution methods can be used to
verify the accuracy of an automated instrument should a discrepancy arise.
Figure 1. Broth microdilution plate contains standard dilutions of eight bacterial organisms in each row (denoted by
letters A-H). Each column of wells (denoted by numbers 1-11; 12 is a sterile control) contains a standard antibiotic
concentration that doubles when moving from right to left (e.g., row 11 = 0.06 μg/ml, row 10 = 0.12 μg/ml, row 9 = 0.25
μg/ml, etc.). The minimum inhibitory concentration (MIC) is determined by the first well where there is no visible growth.
For example, the MIC of this antibiotic for organism A is 4 μg/ml, and the MIC for organism B is 8 μg/ml.
Agar dilution should not be confused with newer colorimetric agar tests. Colorimetric agars contain certain chemicals that
cause a color change when select organisms are present. These tests are also referred to as agar screening methodologies.
Such agar-based testing is outside the scope of this review.
Etest
Etest (bioMérieux, Durham, NC) was approved by the FDA in 1991. It is a manual method of exact MIC testing that uses a
gradient technique combining the principles of both disk diffusion and agar dilution. However, Etest differs from disk diffusion
and is applicable for use with a wide range of fastidious and nonfastidious aerobic and anaerobic organisms, with varying
growth rates, critical times for antibiotic absorption, and testing procedures. Etest offers convenience for hospital laboratories
and is commonly used to complement other routine automated laboratory antimicrobial susceptibility testing methods.
Etest uses a preformed and predefined gradient of varying antibiotic concentrations immobilized in a dry format onto the
surface of a plastic strip. The concentration of the gradient is calibrated across a continuous MIC range covering 15 2-fold
dilutions. When applied to the surface of an inoculated agar plate, the antibiotic on the plastic strip is transferred to the agar in
the form of a stable continuous gradient directly beneath and in the immediate vicinity of the strip. The stability of this antibiotic
gradient is maintained for up to 20 hours. After incubation, a parabolic-shaped inhibition zone centered alongside the test strip
is visible. The MIC is read at the point where the growth or inhibition margin of the organism intersects the edge of the
calibrated strip (Figure 2).[33] This point can be easily identified for most organisms. Using a magnifying glass and/or tilting the
plate can be helpful to visualize microcolonies and hazes or other colonies within the ellipse of inhibition.
Figure 2. This Etest strip contains graduated concentrations of ampicillin ranging from 0.016 μg/ml (not shown) to 256
μg/ml placed on an agar plate growing Escherichia coli. Since the intersection of the growth-inhibition margin lies
between two minimum inhibitory concentrations (MICs)—0.38 and 0.5 μg/ml—the test is interpreted at the highest value
(0.5 μg/ml). This organism is defined as susceptible since the MIC lies below the breakpoint of 8 μg/ml or lower.
The key advantages of Etest are that it uses a stable gradient, it is a convenient agar-based method, and higher inocula can be
used to detect antibiotic resistance. Furthermore, molecular properties of the antibiotic do not affect the performance of Etest
since the gradient is preformed and not dependent on diffusion. Thus, the Etest gradient can accommodate large molecules,
such as vancomycin, and lipophilic compounds, such as amphotericin, that would otherwise be a limitation for disk diffusion.[33]
Although the Etest complements many of the laboratory's functions, it is not used as the sole method of testing. An Etest is
not available for all antimicrobials. Test setup can be labor intensive, and there is a propensity for error in interpretation if the
point of intersection with the strip does not appear to be well marginated or microcolonies growing in the inhibition area are
missed on visual inspection.
Etest is currently FDA approved for a variety of antibiotics for clinical testing of aerobes, anaerobes, pneumococci,
streptococci, Haemophilus species, and gonococci.[34] Laboratories may choose to use an Etest to confirm equivocal results
with their routine systems or test organisms with distinctive resistance mechanisms that can be difficult to detect with standard
methods. Etest may be helpful in detecting resistance in organisms that are highly inoculum dependent, as they can be falsely
reported as susceptible in some automated systems.[35]
Automated Systems
Before the 1970s, labor-intensive manual susceptibility testing was the dominant method. In 1974, the first automated system
known as the Autobac I disk elution system was introduced by Pfizer Diagnostics.[36] Now, approximately 83% of clinical
laboratories report using an automated instrument for primary susceptibility testing.[37]
Several automated systems are available in the United States. They include Phoenix (Becton Dickinson, Franklin Lakes, NJ),
Vitek (bioMérieux), MicroScan WalkAway (Siemens Healthcare Diagnostics, Tarrytown, NY), and Sensititre (Trek Diagnostics,
Cleveland, OH). Most systems contain a computerized algorithm for interpreting results and identifying inconsistencies
between organism identification and susceptibility. These automated systems use commercially available panels that contain
added growth factors to speed organism growth, thereby providing more rapid results compared with traditional methods.
They also use sophisticated software to analyze the growth rates and determine the antibiotic MIC for the organism by using
specialized decision technology. These systems do not read the actual MIC, but rather they use a sophisticated rules system
to determine whether to adjust to a higher or lower MIC such that the results fit into a doubling dilution result (e.g., 1, 2, 4, 8
μg/ml, etc.). Although the general process of identification is similar, there are differences among each system.
Vitek and Vitek 2
The Vitek was first developed in the 1960s. It uses small plastic cards containing multiple wells filled with either biochemical
substrates or antibiotic dilutions. Cards can be used for both identification and susceptibility testing for most aerobic
gram-positive and gram-negative organisms. The system uses a manual filler sealer module to inoculate the cards with the
organism. After inoculation, a reader incubator module uses a single wavelength of light to measure turbidity and color
changes in the card wells. Robotics technology is used to move the cards every 15 minutes to different reading areas. The
Vitek then uses regression analysis to calculate the MIC.[38]
The Vitek 2 system is the second generation of Vitek and offers a more sophisticated model of data analysis as well as a fully
automated process for card identification, organism suspension dilution, and card filling. Once these steps are complete, the
Vitek 2 seals the cards into a chamber to prevent contamination during processing. The cards are then loaded into the reader
incubator, which ejects them at the end of testing. The Vitek 2 uses colorimetric technology with use of three wavelengths of
light to provide broad profiles for the most clinically significant organisms. The data analysis is performed by using algorithms
to look at a variety of parameters and test conditions to ensure accurate results and early detection of resistance mechanisms
through the use of proprietary software.[38] The Vitek 2 Compact offers a condensed version of the Vitek 2; although it
requires more technician involvement before reading the cards, the compact system is desirable for smaller hospitals or
clinics with limited space. Species identification with these systems is complete in an average of 3 hours with rapid methods,
but may take up to 5.7 hours for slow-growing organisms that require colorimetric testing methodology. Susceptibility results
may take up to 15 hours, with a mean of about 9 hours.[39, 40]
MicroScan WalkAway
The MicroScan offers a choice of an overnight panel or the more rapid identification-susceptibility panel that uses fluorescent
technology. In contrast to the Vitek system, the MicroScan panels are the size of conventional microdilution trays. Depending
on the laboratory's preference, either a combination identification-susceptibility panel or separate identification and
susceptibility panels may be used. The WalkAway system consists of an incubator-reading unit that can read either as a
conventional panel or a fluorescent rapid-read panel. Panels are manually inoculated by using a proprietary device known as
the RENOK (rehydrator-inoculator device). Once the panels are placed into the incubator-reader unit,[41] the remainder of the
process is fully automated. A bar code reader identifies each panel, incubates it for the appropriate amount of time, and
moves the panels to the reading position. Organism identification can be determined within an average of 2.5 hours by rapid
methods but may take 6-18 hours with use of conventional testing methodologies. Final results for susceptibilities take an
average of 20 hours, with a range of 16.8-27.8 hours depending on type of organism.[40, 41]
Phoenix
This system uses an optimized colorimetric oxidation-reduction indicator for susceptibility testing and a variety of fluorometric
and colorimetric indicators for bacterial identification. The panels are inoculated manually and require a specific organism
dilution for accuracy. If the inoculation is incorrect, the system will automatically reject the panel from the incubation and
reading phase. After inoculation, the panels are loaded into the incubator-reader module, and all subsequent steps are fully
automated. The Phoenix database then analyzes the kinetic measurements of bioreactivity within individual wells. The average
time to identifi-cation with the Phoenix system is 4.3 hours. The time to final susceptibility results ranges from 7.5-16 hours,
with a mean of 10.5 hours.[40, 42] Although all the systems offer some basic epidemiologic software and data for antibiogram
production, the Phoenix database allows for greater flexibility and data analysis.[42]
Sensititre
The Sensititre susceptibility system is a version of the broth dilution method and can provide both in vitro qualitative and
quantitative susceptibility results in a dried plate format. Isolated colonies for testing are diluted according to a predefined
standard and then used to inoculate a 96-well microdilution plate. A fluorescent precursor may be added at the same time as
the test organism. Inoculated panels are sealed with adhesive film to prevent evaporation and then incubated at 34-36°C for
18-24 hours. During incubation, bacterial surface enzymes will cleave key bonds on the precursor and trigger the release of
fluorophores, which emit fluorescence. Unlike the Vitek and Phoenix systems, the contents of the wells can then be examined
manually for bacterial growth. However, Sensititre can also be read with an automated reading system that is able to detect the
fluorescence. The amount of fluorescence detected is directly related to the activity of bacterial surface enzymes and,
therefore, to the presence of bacterial growth and resistance to the antibiotic. Additional interpretations of susceptibility results
are performed by the software using rules derived from FDA standards.[43]
Advantages and Disadvantages
One major advantage of automated susceptibility methodologies is a reduction in labor.[44-46] Another advantage is that these
systems can provide faster reporting of susceptibility results, potentially leading to the earlier initiation of appropriate antibiotic
therapy. Although the reduction in labor requirements and faster reporting are significant advantages to using automation in the
microbiology laboratory, definite disadvantages exist.
First, all automated systems require the manual step of preparing an inoculum. This step is critical to the accuracy of the
results because over- or underinoculating or mixing cultures can result in false or misleading results. Second, all automated
systems can test only a limited range of organisms. Slow-growing or fastidious organisms must still be tested by alternative
methods. Mucoid Pseudomonas aeruginosa isolated from patients with cystic fibrosis also presents a challenge for
automated systems because of the thick cell wall caused by exopolysaccharide-alginate and its slow growth. In one study,
antibiotic susceptibilities for 498 strains from patients with cystic fibrosis (one third of which were mucoid) were performed by
using Vitek and MicroScan WalkAway systems, and results were compared with those of a reference broth microdilution
method. "Very major" error rates (i.e., erroneously reported as susceptible, yet determined to be resistant by a reference
method) were 17% and 10.4-12.4%, respectively. (See Agreement Among Methods section for a detailed description of
types of errors associated with testing.) The authors concluded that these commercial systems performed poorly for cystic
fibrosis isolates in contrast to high result correlations that had been reported previously with reference methods and Etest.[47]
Panel and Card Selection
All of the automated systems offer several panel choices for the susceptibility portion of the testing. Panels and cards are
separated into gram-positive, gram-negative, and fungal panels (for select systems).[48] Each manufacturer's Web site
provides examples of the potential panels and cards. The choice of panel should be aligned with the hospital's antimicrobial
formulary. Pharmacy personnel play an important role in panel selection by ensuring that formulary antibiotics are reported on
the culture and sensitivity reports.
Depending on the system, separate cards may need to be purchased for organism identification, as well as gram-positive and
gram-negative susceptibilities. The identification panel may be used alone when susceptibilities are not clinically significant.
For example, Lactobacillus and Pasteurella species are not tested because approved susceptibilities are lacking, and
Staphylococcus saprophyticus will only be identified because susceptibilities will not change the course of therapy.
Interchangeability between panels is limited by several factors. Whenever a new panel or card is used, a series of quality
assurance tests must be performed before starting the new panel; this may take an average of 20-30 days and limits the
number of times that panels can be changed. Most microbiology laboratories purchase supplies in bulk to obtain better pricing.
In an effort to reduce cost, the laboratory may need to finish the existing panel supply before proceeding to a new one. This
can be a challenge when changes are made to the antibiotic formulary. If the new antibiotic is not on the susceptibility panel,
then manual tests must be conducted to verify susceptibilities to the new agents and the results for nonformulary antibiotics
need to be suppressed. Customized testing panels can be developed for a specific institution based on their specific
formulary or to increase the number of dilutions tested to provide a better range of MICs. However, this may not be a
cost-effective strategy, especially for institutions with dynamic formularies. Finally, testing space on the antibiotic susceptibility
cards is not infinite, and therefore not all MICs can be tested. Generally, the range of MICs tested are doubling dilutions (e.g.,
0.25, 0.5, 1, 2 μg/ml). In some cases, only the breakpoint is reported (e.g., MIC ≤ 2 μg/ml).
Special Considerations in Antimicrobial Susceptibility Testing
Fastidious Organisms
As their category implies, most fastidious organisms do not grow well enough in automated antimicrobial testing systems and
require some type of supplementation (e.g., blood). Disk diffusion was initially developed for susceptibility testing of rapidly
growing aerobic bacteria, including enterococci, staphylococci, Enterobacteriaceae, and P. aeruginosa. Modifications for
performing disk diffusion testing with certain fastidious organisms such as H. influenzae, Neisseria gonorrhea,
Streptococcus pneumoniae, and other Streptococcus species have been described by CLSI and include using agar that is
enriched with glucose, yeast extract, and other substances. Modifications for broth microdilution can also be used for testing
fastidious organisms, although results must be read manually and not by automated instruments.[49] Automated testing
methodologies can be developed to correlate results that are obtained with CLSI recommendations for testing of fastidious
organisms.
Because of the stability of the gradient, Etest can also be used for testing very slow-growing fastidious organisms, as
previously mentioned. Clinical situations often warrant susceptibility testing of rarely encountered, unusual, and/or opportunistic
pathogens for which no CLSI guidelines are available. In these cases, scientific references can be used to guide the choice of
appropriate media, incubation conditions, and antibiotics to test. Because of the lack of guidelines, accompanying
interpretations may be difficult to find, so the MIC value is reported without interpretation. These situations should be handled
on an individual basis in consultation with an infectious diseases physician. Factors that may influence the course of action
include the organism(s), site of infection, pharmacokinetics of the antibiotic, and previous experience with a similar antibiotic
for treating the same condition.
Anaerobes
Most hospital laboratories do not routinely perform anaerobic susceptibilities and must send the sample out to a reference
laboratory if requested. Indications for anaerobic susceptibility testing include the management of patients with serious
infections requiring long-term therapy and selection of appropriate therapy for organisms that are not predictably susceptible.
The CLSI recommends that agar dilution (known as the Wadsworth method) be used as the reference method for anaerobic
susceptibility testing.[50] A broth microdilution method is also approved but is limited to rapidly growing anaerobes
(Bacteroides species). Another disadvantage is that the method is only described for a limited number of antimicrobials, and
the panels are not commercially available. Because of these limitations, performing susceptibility testing on obligate
anaerobes is not a standard procedure.[51] However, due to the increasing resistance rates reported among some anaerobes
in recent years, the CLSI recommends susceptibility testing for clinically significant anaerobes (e.g., B. fragilis, Prevotella
species, and Fusobacterium species) and creating an antibiogram.[52-55] The Etest can be used for testing individual isolates
as well as batch-testing for an antibiogram.
Mycobacteria
Because of minimal equipment requirements for acid-fast bacilli smears and their ability to produce rapid results, almost all
laboratories have the capacity to conduct these tests. Further identification and susceptibility testing of mycobacteria are
generally performed in larger institutions or reference laboratories with the equipment necessary to further process these
organisms. A negative pressure room and a sterile hood are required, along with a specialized set of materials and techniques
specific to the selection and testing of mycobacteria. Clinical laboratories that test mycobacteria use an automated or
semiautomated broth-based system for growth as well as susceptibility of Mycobacterium tuberculosis. Susceptibility testing
of rapidly growing mycobacteria can be performed by either broth microdilution or by Etest.[56, 57]
Specialized Screening and Confirmatory Tests for Resistant Organisms
Microbiologists can use certain antibiotics to screen for key resistance patterns among common bacteria. Screening tests may
differ from traditional susceptibility methods in some cases because the antibiotic used to detect the resistance may be
incongruous with the antibiotic selected for treatment. Most screening tests used in the clinical laboratory are designed to
detect methicillin-, oxacillin-, or vancomycin-resistant gram-positive organisms (most commonly MRSA, vancomycin-resistant
Enterococcus, and β-lactamase-producing gram-negative organisms).
There are several methods that can be used to detect oxacillin resistance among S. aureus. In addition to disk diffusion or
broth microdilution, other methodologies used include the oxacillin salt agar screen and the cefoxitin disk screen.[12] A latex
agglutination test for penicillin-binding protein 2a is also available, but this is not commonly used in the traditional hospital
microbiology laboratory because of the ease of other methods and cost. All methods vary in terms of their specificity and
sensitivity, and the results can vary depending on strain type.[58, 59] The oxacillin salt agar screen test uses Mueller-Hinton agar
supplemented with 4% sodium chloride and oxacillin 6 μg/ml inoculated with a 0.5 McFarland suspension of the organism. The
plates are incubated for 24 hours at a maximum temperature of 35°C. Testing at higher temperatures may interfere with the
detection of methicillin resistance.[23] The appearance of more than one colony indicates resistance. This screen has a high
sensitivity for detecting resistant strains, but sensitivity decreases when very heteroresistant strains are tested or when the MIC
is borderline.[59, 60] Recently, the cefoxitin disk screen has been recognized as a better predictor of methicillin resistance than
many other classic methods because cefoxitin is a more potent inducer of mecA than are penicillins.[61] The substitution of a
cefoxitin disk for an oxacillin disk results in an easier test for the microbiologist to read and has similar sensitivity and
specificity to detect oxacillin resistance in Staphylococcus species. The incubation time required for testing S. aureus and
Staphylococcus lugdunensis is only 16 hours,[23] but some studies have shown that results can be achieved even earlier.[62]
In addition, the cefoxitin disk screen has equal sensitivity but improved specificity in coagulase-negative Staphylococcus
species. The disadvantage of this test is that it may falsely detect resistance in some mecA-negative strains and may fail to
detect resistance in some strains of mecA-positive Staphylococcus simulans.[63]
Brain-heart infusion agar supplemented with vancomycin 6 μg/ml can be used to screen for vancomycin-resistance in S.
aureus isolates with a vancomycin MIC of 4 μg/ml or more.[64] Organisms that grow on the agar should be suspected as
possible vancomycin-intermediate or vancomyc-inresistant S. aureus and confirmed by using a validated MIC method such as
broth dilution, agar dilution, or Etest, or through approved antimicrobial susceptibility testing.[65] Screening and detection of
heterogeneous vancomycin-intermediate S. aureus presents a significant challenge in the clinical laboratory. Although
methods exist in research, there is no standardized method for identifying heterogeneous vancomycin-intermediate S. aureus
consistently in the clinical microbiology laboratory.[66]
Brain-heart infusion agar supplemented with vancomycin 6 μg/ml can also be used as a screen for vancomycin resistance in
enterococci.[67] Plates are inoculated, incubated, and interpreted in the same manner as the MRSA screen agar plates
described previously. Additional screening agars are also available for testing for high-level aminoglycoside resistance in
enterococci by using brain-heart infusion agar supplemented with gentamicin 500 μg/ml and streptomycin 2000 μg/ml. The
presence of high-level aminoglycoside resistance indicates a lack of synergistic effect when an amino-glycoside is combined
with a cell-wall inhibitor. Gentamicin high-level resistance is associated with high-level resistance to other aminoglycosides,
such as tobramycin, netilmicin, amikacin, and kanamycin. Streptomycin resistance occurs by means of a separate mechanism.
This is the justification for testing both gentamicin and streptomycin for high-level resistance in enterococci.[68]
Some strains of macrolide-resistant S. aureus and coagulase-negative Staphylococcus species have a transferable
resistance mechanism known as macrolide-lincosamide-streptogramin B (MLSB) resistance. This inducible resistance can
result in clindamycin treatment failures,[69, 70] but it can be detected by using a disk approximation test known as the double
disk diffusion, which is frequently referred to as the D-test. A 2-μg clindamycin disk is placed 15-26 mm away from the edge of
a 15-μg erythromycin disk on Mueller-Hinton agar. After incubation, when inducible resistance is not present, the zone of
inhibition around the clindamycin disk will appear as a concentric circle. However, if the edge of the clindamycin zone closest
to the erythromycin disk is flattened (thus causing the D shape), then the isolate is reported as clindamycin resistant (Figure
3).[12] Although the CLSI recommends a distance of up to 26 mm, one recent study found that using a 22-mm distance was
associated with inaccuracies, but placing the disks 15 mm apart resulted in 100% sensitivity and specificity.[71]
Figure 3. Positive double disk diffusion test (D-Test) shows induction of clindamycin (CC) resistance by erythromycin
(E) in this methicillin-resistant Staphylococcus aureus isolate. This is indicated by the blunting of the clindamycin zone of
inhibition, which appears as a D shape.
One of the most frequently used screening tests detects β-lactamases, of which the most common among gram-negative
bacteria is the enzyme known as TEM-1. This enzyme causes ampicillin and penicillin resistance in H. influenzae and N.
gonorrhea.[72] Most hospitals will perform a β-lactamase screening test for these two organisms because these β-lactamases
may produce low-level resistance that may go undetected. There are three categories of β-lactamase tests: colorimetric,
acidimetric, and iodometric. In the hospital microbiology laboratory, the colorimetric method is the most commonly used and
involves sticks or disks impregnated with certain compounds such as nitrocefin or cephalosporin-pyridinium-2-azo-
p-dimethylaniline that produce a color change when the β-lactam is hydrolyzed. Acidometric and iodometric tests are referred
to as linked detection systems because more than one chemical reaction must occur to produce an interpretive result.[73]
These tests are typically performed by using penicillin as a substrate and therefore may only detect enzymes that hydrolyze
penicillins.[74] These β-lactamase tests also may be performed on Moraxella catarrhalis. However, because β-lactamase-
positive M. catarrhalis is so frequent, it is safe to assume resistance will be present, and treatment with a β-lactam-enzyme
inhibitor combination will be necessary.
Hospital microbiology laboratories also have the ability to detect extended-spectrum β-lactamases (ESBLs). Although ESBLs
have been identified in many organisms,[75] CLSI procedures for screening and confirmation of ESBLs exist only for
Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, and clinically relevant Proteus mirabilis strains (e.g., a
bacteremic isolate).[12] Screening for ESBLs in E. coli, K. pneumoniae, or K. oxytoca involves performing a preliminary
susceptibility test against cefpodoxime, ceftazidime, aztreonam, cefotaxime, or ceftriaxone. The process is similar for P.
mirabilis but does not involve the use of aztreonam or ceftriaxone. If the results suggest the presence of an ESBL (based on
CLSI procedure), a phenotypic confirmatory test is performed by using either disk diffusion or broth microdilution. The disk
diffusion confirmatory test involves placing a 30-μg ceftazidime disk and a 30-μg cefotaxime disk, both alone and in
conjunction with clavulanic acid 10 μg, on a Mueller-Hinton agar plate. If the difference between the zone diameter for the
single antibiotic versus the combination with clavulanic acid is 5 mm or greater, then the organism is classified as
ESBL-positive. For broth microdilution, the ESBL test is positive if a three or more 2-fold concentration decrease in the MIC
occurs between the original single antibiotic (e.g., ceftazidime or cefotaxime) and the combination of the reference antibiotic
with clavulanic acid (e.g., going from 8 μg/ml with a single antibiotic to 1 μg/ml or less with the inhibitor present).[12] Etest strips
are also available that contain cefotaxime or ceftazidime on one end of the strip and cefotaxime or ceftazidime plus clavulanic
acid on the other end of the strip (Figure 4).[33] The interpretation for the Etest ESBL test is the same as that for broth
microdilution. Several automated systems can also be used to detect ESBLs. Early and appropriate detection of
ESBL-producing organisms allows for better customization of therapy, which can lead to improvements in morbidity and
mortality.[76]
Figure 4. Confirmatory extended-spectrum β-lactamase (ESBL) with use of the Etest. The strip on the left contains
cefotaxime + clavulanic acid (CTL) and cefotaxime (CT). The strip on the right contains ceftazidime + clavulanic acid
(TZL) and ceftazidime (TZ). The organism is ESBL-positive based on the deformation of the CT ellipse, but may also be
determined by the CT and TZ minimum inhibitory concentrations (MICs) and the CT:CTL MIC ratio or the TZ:TZL MIC
ratio.[33]
In addition to ESBLs, there are several β-lactamase-producing enzymes that may also affect interpretation and antibiotic
selection. One of the most common is the AmpC type β-lactamases. The AmpC β-lactamases are commonly isolated from
extended-spectrum, cephalosporin-resistant, gram-negative bacteria such as Serratia, Enterobacter, and Citrobacter
species.[77] The AmpC enzymes are inhibitor-resistant β-lactamases. They are resistant to clavulanic acid and other
β-lactamase inhibitors but susceptible to cloxacillin. High level of expression of AmpC may prevent recognition of an ESBL
because of this resistance to clavulanic acid, which is used to detect the presence of ESBLs.[77] The presence of AmpC
ultimately could lead to a false-negative ESBL screen. One possible solution is to include cefepime as an ESBL screening
agent because high-level AmpC expression is minimally affected with this drug. There is also a commercially available Etest
for the detection of AmpC, but it is not FDA approved. The Etest contains a double-sided strip whereby one side contains a
cephamycin (e.g., cefotetan or cefoxitin) alone and the other side contains a cephamycin with cloxacillin. A positive test for
AmpC is noted if the cephamycin MIC decreases by three or more 2-fold dilutions in the presence of cloxacillin or a
deformation of the inhibition ellipses around the Etest.[78, 79]
Organisms with increased carbapenem MICs are of great concern. The metallo-β-lactamases are carbapenem-hydrolyzing
β-lactamases that emerged in Japan in the 1990s.[80] These enzymes effectively hydrolyze both β-lactam antibiotics and
carbapenems except aztreonam. Although the clinical prevalence of these enzymes is low, they have been reported in
Enterobacter and Pseudomonas species. Because of difficulty of detection, a screening method for metallo-β-lactamases
can be used when an increase in the MIC of carbapenems is observed.[81, 82] The metallo-β-lactamase enzymes are zinc
mediated and can be repressed by using ethylenediaminetetraacetic acid (EDTA). A commercially available Etest may be
used for metallo-β-lactamase detection, but it is not FDA approved. One half of the Etest strip is impregnated with imipenem
and the other half contains imipenem plus EDTA. A reduction in the MIC of imipenem of three or more 2-fold dilutions in the
presence of EDTA represents a positive metallo-β-lactamase organism.[82, 83]
Carbapenemase-producing Enterobacteriaceae (CPE), such as K. pneumoniae, are endemic in the northeastern part of the
United States [84] and present a new challenge in antimicrobial susceptibility testing and breakpoint reporting. They can be
missed by automated testing systems[85] and may require manual testing. Only recently have formalized screening and
confirmatory standards been developed by the CLSI.[12] For screening purposes, a zone of inhibition of 19-21 mm for an
ertapenem 10-μg disk or a zone of 16-21 mm for a meropenem 10-μg disk suggests a possible CPE. No interpretative
standards exist for imipenem since it performs poorly as a screen for carbapenemases. Broth microdilution using imipenem,
meropenem, and ertapenem 1 μg/ml can also be used as a screen. An MIC of 2-4 μg/ml for imipenem and meropenem or an
MIC of 2 μg/ml for ertapenem may indicate the presence of a carbapenemase despite being considered susceptible based
on standard interpretations. To confirm the presence of a CPE, a modified Hodge test must be performed. This test involves
streaking CPE-positive and -negative reference strains along with the clinical isolate onto a Mueller-Hinton agar plate that has
already been inoculated with a reference strain of E. coli. An ertapenem disk is placed in the center. If the test is read as
positive, the CLSI suggests that the actual carbapenem MIC be reported along with a comment indicating that the isolate
demonstrates carbapenemase production and that the clinical efficacy of treating these organisms with carbapenems has not
been established. If the test is negative, the CLSI recommends interpreting the carbapenem MICs by using current
interpretative breakpoints.
Interpretation
On completion of antimicrobial susceptibility testing and determination of the MIC or zone diameter, the organism may be
classified into one of three interpretative categories: susceptible, intermediate, or resistant. There is an inverse relationship
between results and the interpretative categories when conducting MIC testing versus disk diffusion testing. Organisms with an
MIC less than (or equal to) a certain concentration (μg/ml) or a zone diameter greater than (or equal to) a specific millimeter
measurement are considered to be susceptible and will likely respond to treatment with a standard antibiotic dosage as
indicated. Organisms are considered to have intermediate susceptibility to an antibiotic when the MICs approach the top end
of the usually achievable serum concentrations for a standard dose or when the zone diameter falls within a certain range. The
intermediate category also serves as a buffer zone to help prevent major categoric errors caused by slight changes in the
zone sizes due to the influence of technical variables. Antibiotics used for the treatment of intermediately susceptible
organisms may achieve clinical success when the antibiotic being used concentrates at the site of infection (e.g.,
fluoroquinolones for urinary tract infections) or when a higher than normal dosage is used (e.g., β-lactams). If the MIC is
greater than (or equal to) or the zone diameter is less than (or equal to) the resistant breakpoint, this implies that the infection is
not likely to respond to therapy because the physiologic concentrations required to overcome resistance would cause toxicity
in humans.
There are some caveats to these interpretative categories. For some organisms, such as P. aeruginosa, there are no
interpretative categories for disk diffusion tests, only MIC tests. For S. pneumoniae, breakpoints vary depending on the
source of the isolate. For example, S. pneumoniae with a penicillin MIC of 1 μg/ml in a patient with meningitis would be
defined as resistant since it is above the defined resistant breakpoint of 0.12 μg/ml or greater. The same isolate from a
nonmeningeal source, such as sputum, would be considered susceptible because the MIC fell below the susceptible
breakpoint of 2 μg/ml or lower for nonmeningitis isolates. This differentiation by site of infection also exists with β-lactam-
β-lactamase inhibitors and intravenous cephalosporins. Some antibiotic-organism combinations only have a susceptible and
nonsusceptible or susceptible and resistant categorization. For example, the susceptibility breakpoint for doripenem tested
against Enterobacteriaceae is 0.5 μg/ml or lower, whereas any MIC above this is only granted a non-susceptible classification.
For piperacillintazobactam and P. aeruginosa, there is no intermediate interpretative standard, only susceptible (MIC ≤ 64/4
μg/ml) or resistant (MIC ≥ 128/4 μg/ml).[17, 86]
Agreement among Methods
Because of the impracticality of reference methods for routine susceptibility testing in the clinical microbiology laboratory,
automated susceptibility testing methods and the Etest have become the microbiologist's workhorse tools. However, these
nonreference method testing systems must show that the results obtained with the test system agree with the results obtained
from a reference method. The FDA sets standards to ensure new devices and new drugs to be added to the panels of the
automated systems are substantially equivalent to a reference method for determining susceptibility testing. The reference
methods for susceptibility testing are broth dilution and agar dilution.[87]
When conducting these studies (frequently referred to as a 510[k]), the testing device is being evaluated to determine if the
results are the same as those of a proven reference method. A testing system is said to have "essential agreement" when the
device under evaluation has an MIC within ± one 2-fold dilution compared with the reference method MIC determination.
"Categorical agreement" is when the interpretive results (susceptible, intermediate, or resistant) between a new device under
evaluation and a standard reference method are the same, by using FDA interpretive criteria as presented in the
FDA-approved pharmaceutical antimicrobial agent package insert. It is possible for there to be essential agreement with
categorical disagreement. When this occurs, it is considered a "minor error" and frequently results in an intermediate
susceptibility with one method and susceptible or resistant with another method. For drugs without an intermediate category,
there cannot be minor errors. A "major error" occurs when the testing method determines that the organism is resistant, but the
reference method determines that the organism is susceptible. Finally, a "very major error" occurs when the converse is
true—the testing method indicates the organism is susceptible but the reference method indicates resistant ( Table 2 ).
Table 2. Agreement Categories According to Reference Method versus Test Method Results
Reference Method Result Test Method Result Agreement Category
Resistant Resistant Agreement

Intermediate Minor error
Susceptible Very major error
Intermediate Resistant Minor error
Intermediate Agreement
Susceptible Minor error
Susceptible Resistant Major error
Intermediate Minor error
Susceptible Agreement
The FDA has issued guidance that outlines acceptable performance when susceptibility testing is conducted by methods
other than a reference method.[87] First, the percentage categorical and essential agreement must be greater than 89.9%.
Categorical agreement less than 90% may be acceptable if there is very good essential agreement, with the majority of the
discrepancies as minor discrepancies. In addition, the major error rate must be less than 3% of all the susceptible organisms
tested. Finally, the very major error rate based on the number of resistant organisms tested must include an upper 95%
confidence limit for the true very major error rate of 7.5% or less, and the lower 95% confidence limit for the true very major
error rate of 1.5% or less. If 100 resistant organisms were tested, the very major error rate should not be greater than 2/100
(95% confidence interval 0.24-7.04).
Two studies demonstrate these concepts for gram-negative and gram-positive organisms. In the first study, the investigators
tested 101 bacteremic isolates of MRSA recovered from 2002-2006.[88] Each isolate was tested by using both the broth
microdilution and agar dilution methods, and the results were compared with those of Etests performed on two different
brands of Mueller-Hinton agar plates. Vancomycin MIC results from the Etests were consistently 1-2-fold higher than the MICs
determined with the reference methods. In the second study, the investigators evaluated the accuracy of three automated
testing systems and tested the susceptibility of five broad-spectrum β-lactams against 100 strains of P. aeruginosa.[89]
Although the rate of major errors was minimal, the investigators noted that two of the systems produced minor error rates of
8-32% for cefepime and that all three systems had minor error rates when testing aztreonam. Clinically, these discrepancies
could manifest as lower-than-expected susceptibility rates on a hospital antibiogram. If there are sudden sharp declines in
susceptibility of P. aeruginosa to cefepime, confirmatory testing of a subset of isolates using another testing method may be
prudent.
Clinical Practice Application
Patients infected with an organism that is considered susceptible to an antibiotic do not respond 100% of the time; conversely,
patients who are infected with an organism that is resistant to an antibiotic do not universally fail treatment. Infections caused
by susceptible isolates respond to appropriate therapy approximately 90% of the time, whereas infections caused by resistant
isolates actually respond to the inappropriate antibiotic about 60% of the time—the "90-60" rule.[8] Either the susceptibility
methodologies or patient factors likely explain the variability seen in patient outcomes based solely on susceptibility results.
Thus, the interpretation should be used as part of the antibiotic therapy selection process. An understanding of the antibiotic's
pharmacokinetic properties, the antibiotic's MIC for the infecting organism, and the pharmacodynamic relationship between the
two can help boost the patient's outcome to the 90% clinical success rate.
Although there are hundreds of in vitro microbiology tests that can be performed in the hospital microbiology laboratory,
maintaining a working knowledge of common clinical laboratory tests enables pharmacists to become valuable resources in
helping to translate bench results into bedside application. For example, many practitioners interpret the results of culture and
susceptibility tests at "face value" and do not understand the discordance between laboratory tests and clinical interpretation.
Some practitioners associate a lower MIC with increased efficacy. However, this is erroneous because each organism-drug
combination varies and the MIC does not reflect pharmacodynamics or patient-specific factors such as renal function, site of
infection, or volume of distribution. Pharmacists can translate these objective measurements into meaningful decisions for
clinicians who deliver patient care.
Collective susceptibility testing results, compiled as an antibiogram, allow hospitals to use local susceptibility data to guide
empiric therapy before culture and susceptibility results. This enables the clinician to preliminarily "predict" the susceptibility of
the infecting organism and ensure the early, appropriate therapy associated with improved patient outcomes.[90-92] The ability
to recognize erroneous results in an antibiogram is important. As previously mentioned, there are certain known limitations with
both automated and manual systems that can influence results. Under-standing these discrepancies can help institutions
distinguish between true resistance issues and laboratory testing errors. Advances in rapid diagnostic testing will undoubtedly
influence antibiotic utilization and ultimately patient outcomes. The higher cost of the tests and laboratory resources may be
offset by reductions in unnecessary drug therapies.
The benefits of applied microbiology principles in the clinical pharmacy setting are numerous and are summarized as follows:
Results interpretation
Detecting antibiogram inaccuracies
Translating how changes in CLSI or FDA breakpoints affect antibiotic susceptibility trending
Recognizing the influence of human factors on test results
Recognizing organism-antibiotic testing mismatches
Assisting in the selection of antimicrobial susceptibility testing panels based on formulary
Clinical therapeutics
Pharmacodynamic dosing based on patientlevel MIC data

Antimicrobial stewardship
Translating susceptibilities into empiric therapy guidelines
Educating prescribers to optimize antimicrobial selection
Reducing patient morbidity and mortality
Financial savings
Researching alternative sources for manual susceptibility testing from pharmaceutical manufacturers
Reducing total health care resource consumption through early and appropriate antimicrobial therapy
Conclusion
Maintaining an active knowledge base of the various antimicrobial susceptibility testing methods and their limitations can be
challenging. Qualitative testing methods are helpful and easily reproduced in the clinical laboratory setting with minimal
investment in equipment and personnel. However, errors in specimen preparation, disk placement, and zone interpretation can
lead to inaccurate results. Quantitative methods are more accurate and can provide specific MICs that can be used to optimize
the pharmacodynamics of antibiotic therapy. Agar and broth dilution methods are the most accurate, but are difficult to perform
in a busy hospital microbiology laboratory. The development of Etests and the deployment of automated testing systems in
most hospital laboratories have allowed for faster result interpretation and earlier initiation of appropriate therapy. However,
clinicians must be aware of the potential for major and minor errors that can occur between these systems.
Numerous screening tests exist and can help the clinician identify key resistance issues secondary to the presence of certain
enzymes. These tests are increasingly valuable as resistance patterns continue to emerge. The development of
molecular-based technologies will allow for faster identification of select resistant organisms, thus shortening the duration of
time to appropriate, targeted therapy. Clinicians who have a thorough understanding of the strengths and weaknesses of these
various testing methodologies can successfully apply this information to benefit both individual patient care and global
antibiotic stewardship within their institution.
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Authors and Disclosures

Kristi M. Kuper, Pharm.D., Deborah M. Boles, M.S., John F. Mohr, Pharm.D., and Audrey Wanger, Ph.D.
Department of Clinical Affairs, Cardinal Health, Houston, Texas (Dr. Kuper); the Department of Pharmacy, Lowell General
Hospital, Lowell, Massachusetts (Dr. Boles); Cubist Pharmaceuticals, Lexington, Massachusetts (Dr. Mohr); and the
Department of Pathology, University of Texas Medical School, Houston, Texas (Dr. Wanger).
For questions or comments, contact Kristi M. Kuper, Pharm.D., BCPS, Department of Clinical Affairs, Cardinal Health, 1330
Enclave Parkway, Houston, TX 77077; e-mail: kristine.kuper@cardinalhealth.com.
For reprints, visit http://www.atypon-link.com/PPI/loi/phco.
Acknowledgments
The authors would like to thank Sheila Maness, M.T., for assisting with content development and Allison Krug, M.P.H., of Artemis Biomedical
Communications, LLC, for assistance with editing the final manuscript.
Pharmacotherapy. 2009;29(11):1326-1343. © 2009 Pharmacotherapy Publications

Antimicrobial Susceptibility Testing - Primer For Clinicians

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Antimicrobial Susceptibility Testing: A Primer for Clinicians (printer-frie...

Basic Principles of Antimicrobial Susceptibility Testing

MIC Breakpoints (μg/ml)

FDA Approved CLSI Approved

Organism Drug Susceptible Intermediate Resistant Susceptible Intermediate Resistant

Minimum Inhibitory Concentration

Vitek and Vitek 2

Advantages and Disadvantages

Panel and Card Selection

Special Considerations in Antimicrobial Susceptibility Testing

Specialized Screening and Confirmatory Tests for Resistant Organisms

Agreement among Methods

Reference Method Result Test Method Result Agreement Category

Resistant Resistant Agreement

Clinical Practice Application

Detecting antibiogram inaccuracies

Pharmacodynamic dosing based on patientlevel MIC data

April 19-22, 2008.

Authors and Disclosures

For reprints, visit http://www.atypon-link.com/PPI/loi/phco.

Pharmacotherapy. 2009;29(11):1326-1343. © 2009 Pharmacotherapy Publications

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