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416 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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e diameter of the zone of inhibition has been correlated
to the MIC of the antibiotic from broth or agar dilution against
the infecting organism using regression analysis.
20,25,28,29
CLSI has
established interpretive criteria based on this relationship to categorize zone diameters as S, I, and R for each antibiotic against each
organism.
25,28,29
e results of disk diusion test are considered
qualitative because they only reveal the zone of inhibition and
comparative activity of an antibiotic rather than an exact MIC.
e disk diusion susceptibility test allows the simultaneous
testing of several antibiotics in a relatively easy and inexpensive
manner and provides exibility in determining the antibiotics
that will be tested for susceptibility, provided a lter paper disk
for the desired antibiotic is available. However, the major disadvantages of disk diusion include inability to generate an exact
MIC and diculty in determining the susceptibility of fastidious
or slow-growing organisms.
Antibiotic Concentration Gradient Methods
Epsilometer test. e Epsilometer test, or Etest (bioMérieux)
combines the benets of broth microdilution with the ease of disk
diusion.11 e Etest method simultaneously evaluates the activity of numerous concentrations of an antibiotic using a single
plastic strip impregnated on one side with a known, predened
concentration gradient of an antibiotic. One side of the Etest strip
is marked with a numeric scale that depicts the concentration of
antibiotic at that location on the reverse side of the test strip.
Like disk diusion, the Etest strip is applied onto a solid media
agar plate that has been inoculated with a standardized concentration of the infecting bacteria. Several Etest strips can be placed
on the same agar plate to provide the simultaneous susceptibility
testing of several antibiotics.
9,20,25
During overnight incubation,
bacteria multiply on the agar plates as the antibiotic diuses out
of the Etest strip according to the concentration gradient. Bacterial growth occurs only in areas on the agar plate in which drug
concentrations are below those required to inhibit growth. An
elliptical zone of growth inhibition forms around the Etest strip
where the MIC is read as the drug concentration where the ellipse
intersects the plastic strip (Figure18-4A and B).
20,25
Etest results are reported as the exact MIC of the infecting bacteria with the corresponding CLSI susceptibility interpretation.
e MIC results derived from the Etest correlate well with the
results obtained using other susceptibility testing methods.
e advantages of the Etest method include its ease of use and the
ability to evaluate the susceptibility of several antibiotics simultaneously as well as the fact that the results yield an exact MIC,
and the laboratory can choose the antibiotics to be tested. However, the Etest method is considerably more expensive than disk
diusion or broth microdilution methods, the results may be
reader-dependent, and testing is limited to only those antibiotics
for which an Etest strip is commercially available.
e Etest is currently used by some microbiology laboratories for the susceptibility testing of fastidious bacteria, such
as Spneumoniae, H inuenzae, and anaerobes, as well as for
testingantibiotics in which a routine susceptibility test is not
available (eg, antibiotic is not on standard broth microdilution
panels used by the hospital) and when an exact MIC result is
preferred.
9,25
9,20,25
9,20,25
(A)
(B)
FIGURE 18-4. The Etest. (A) Individual Etest strips
strip at the arrow. (Source: Reprinted with permission
Bailey and Scott’s Diagnostic
Microbiology
2017:177–204.)
Specialized Susceptibility Tests
Additional tests may be performed in the microbiology laboratory to provide further information on the activity of an antibiotic against an organism. ese specialty susceptibility tests
CHAPTER 18 • InfECTIous DIsEAsEs: BACTERIA 417
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may measure the bactericidal activity of the antibiotic (eg, MBC
testing, time-kill curves, and serum bactericidal tests [SBTs]) or
the activity of a combination of antibiotics against an infecting
organism (eg, synergy testing using the checkerboard technique
or time-kill studies). ese tests are not routinely performed in
most microbiology laboratories due to biological and technical
diculties, complexity in the interpretation of the results, and
uncertain clinical applicability.
22-24,31
Testing methods for determining bactericidal activity. Several
methods measure the direct killing activity of an antibiotic
against an organism and, if used, should be performed only for
antibiotics that are generally considered to be bactericidal. As
noted earlier, there are only a limited number of clinical circumstances in which this information may be useful. e determination of bactericidal activity may have the best clinical utility in the
treatment of infections at anatomic sites where host defenses are
minimal or absent, such as endocarditis, meningitis, and osteomyelitis, as well as in the treatment of severe and life-threatening
infections in immunocompromised patients.
9,20,22-24,31
Testing
methods that determine the bactericidal activity of an antibiotic
include the MBC test, time-kill assays, and SBTs.
20,22-24,31
e MBC is the lowest concentration of an antibacterial agent
that kills 99.9% of the bacterial inoculum, which represents a
≥3 log reduction in the original inoculum.
11,22
e methodology
for determination of the MBC has been previously described
in detail in the section on broth macrodilution because it is an
extension of that test. CLSI has developed guidelines to standardize the methodology for MBC testing.31 If the MBC is ≥32
times higher than the MIC or exceeds the achievable serum
concentrations of the antibiotic, “tolerance” may be observed.20
Tolerance occurs when a normally bactericidal antibiotic only
can inhibit the growth of bacteria based on MBC testing. MBC
testing is not routinely performed by most laboratories because
it is labor intensive with limited clinical use.
20,22,31
Time-kill studies, also known as time-kill curves, measure the
rate of bacterial killing over a specied period of time, which is
in contrast to the MBC that measures the bactericidal activity at
a single point in time following an incubation period.
20,24,31
For
time-kill studies, a standardized bacterial inoculum is placed
into test tubes containing broth with several dierent concentrations of an antibiotic (usually the MIC and multiples of the
MIC in separate tubes). Samples of the antibiotic-broth solutions are obtained at predetermined time intervals to evaluate
the number of viable bacterial colonies present over the 24-hour
incubation period.
11,20,24,31
e number of viable bacteria present at each time point are plotted over time to determine the
rate and extent of bacterial killing of the antibiotic against the
organism. A ≥3 log reduction in viable bacterial counts is representative of bactericidal activity.
11,20,24,31
Because it is labor and
resource intensive, this test is not routinely performed in many
clinical microbiology laboratories, but it is oen used in the
research setting.
e SBT, or Schlichter’s test, is similar to MIC and MBC testing, except the SBT measures the bacterial killing activity of the
patient’s serum against their infecting organism aer receiving
a dose of an antibiotic.
9,20,23,24,31-33
e methodology is similar to
determining the MIC using broth macrodilution, but dilutions
of the patient’s serum are used instead of 2-fold serial dilutions
of an antibiotic.
9,24,31-33
e patient’s serum is obtained at predened intervals before and aer a dose of an antibiotic, specically at the time of expected peak concentration and at the
time of expected trough concentration. e patient’s serum is
then serially diluted and inoculated with a standardized concentration of the infecting organism. e SBT is the highest
dilution of the patient’s serum that reduces the original standardized bacterial inoculum by ≥99.9%. e results of the SBT
are reported as a titer, which represents the number of 2-fold
serial dilutions of the patient’s serum that led to bacterial kill
ing (eg, SBT = 1:16), with a higher titer indicating better activity
against the organism.
ology standards for performance of the SBT.
20,22,24,31-33
e CLSI has developed method-
31,33
However, this
test is not routinely performed by most microbiology laboratories because of technical diculties. In addition, limited inconclusive data has been published regarding the clinical usefulness
of SBTs in guiding therapy (only a few studies in the treatment
of endocarditis, osteomyelitis, and serious infections in febrile
neutropenia).
22-24,31-33
Antimicrobial combination testing (synergy testing). In
thetreatment of bacterial infections, there are several clinical
situations in which combination antimicrobial therapy may
be used. e decision to use combination therapy is primarily based on the severity of infection, the causative organism,
and the type of infection. e potential benets of combination antibiotic therapy include (1) expanding the antimicrobial spectrum of activity, especially empiric therapy for a
life-threatening infection or for the treatment of polymicrobial
infections; (2) producing synergistic bactericidal activity with
the combination that is not observed with each agent alone,
such as the use of ampicillin and gentamicin for the treatment
of Enterococcal endocarditis; and (3) decreasing the emergence of resistant organisms, which has been observed in the
treatment of tuberculosis (TB).24 Routine antimicrobial susceptibility tests measure the activity of an antibiotic against a
particular organism. ere are several tests, however, that evaluate the eects of combination antimicrobial therapy against
an infecting organism (synergy testing), with the results being
expressed as one of three types of activity
1.
Synergy: e activity of the antimicrobial agents in com-
11,20
:
bination is signicantly greater than the additive eects of
each agent alone.
2.
Indierence: e activity of the antimicrobial agents in combination is similar to the additive eects of each agent alone.
3.
Antagonism: e activity of the antimicrobial agents in combination is less than the additive eects of each agent alone.
erefore, before two antibiotics are used together, it may be
useful to determine the eects of the combined antibiotics
against the infecting organism, especially because some antibacterial combinations may produce suboptimal eects.
Synergy testing of an antimicrobial combination can be performed using the checkerboard technique, the time-kill curve
technique, the disk diusion assay, or the Etest method, with the
checkerboard and time-kill curve techniques being most oen
418 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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20,24
used.
e checkerboard technique is performed in macrodilution tubes or microdilution plates containing serial dilutions
of the antibiotics alone and in combination. e tubes or plates
are incubated with a standardized inoculum of the infecting
bacteria for 24 hours. e eect of the antibiotic combination
is determined by comparing the MICs of the agents when used
in combination with the MICs of each agent alone. A synergistic combination displays lower MICs than when each agent is
used alone. e time-kill curve method for combination therapy
is similar to the time-kill curve method used to determine the
rate of bacterial killing of a single agent, except that two antibiotics are added to the tubes in xed concentrations. e eect
of the antibiotic combination is determined by comparing the
time-kill rates of combination therapy with the time-kill rates of
each agent alone. A synergistic combination displays 100-fold or
more killing activity than the most potent agent tested alone.
11,20
In the clinical setting, synergy testing methods are not routinely
performed due to their tedious, time-consuming methodologies,
their expense, and their limited clinical applicability in predicting clinical outcome.
11,20,24
Methods Detecting the Presence of
AntibioticResistance Mechanisms
Detection of b-Lactamase Activity
To date, more than 2,700 dierent β-lactamase enzymes have been
characterized.34 β-lactamase enzymes can be chromosomally-,
plasmid-, or transposon-mediated and may be produced constitutively or inducibly. ese enzymes cause hydrolysis of the
cyclic amide bond in the β-lactam ring and, depending on the
type of enzyme, may result in inactivation of one or numerous
β-lactam antibiotics. It is important to understand the consequences of detecting a particular β-lactamase enzyme in an
organism because certain enzymes produce resistance only to
certain antimicrobials.
Several methods detect the presence of β-lactamase
enzyme depending on the organism and type of β-lactamase
enzyme suspected. Some tests directly detect the presence of
β-lactamase activity whereas others predict the presence of certain β-lactamase enzymes (such as the inducible AmpCs or the
extended-spectrum β-lactamases [ESBLs]) based on resistance
patterns and MICs derived from routine susceptibility tests.
e assays that directly detect β-lactamase activity include
the acidimetric, iodometric, and chromogenic tests, which all
measure the presence of β-lactamase enzyme by observing a
color change based on reactions to dierent substrates.
chromogenic test is the most common test used by microbiology
laboratories because of its reliability in detecting β-lactamase
enzymes produced by many dierent bacteria.
mogenic tests use chromogenic cephalosporins (nitrocen,
cefesone, or cenase) incorporated into lter paper disks or
strips that produce a color change when they are hydrolyzed by
β-lactamase enzymes present in a clinical specimen once inoculated onto the disk or strip. Test tube assays using chromogenic
cephalosporins can also be also used. A positive reaction using
one of these direct β-lactamase tests for H inuenzae, Moraxella
20,23,34,35
20,36
e chro-
20,36
e
catarrhalis, and N gonorrhoeae predicts resistance to only penicillin, ampicillin, and amoxicillin but not to other β-lactam antibiotics that are more stable to β-lactamase enzymes. A positive
β-lactamase test for Staphylococcus spp. predicts resistance to
penicillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, and
piperacillin.
Extended-spectrum β-lactamases are plasmid-encoded
β-lactamase enzymes (eg, TEM, SHV, CTX-M) that hydrolyze
most penicillins, cephalosporins (except the cephamycins and
cefepime), and aztreonam.35 Historically, routine susceptibility tests using CLSI breakpoints did not reliably detect ESBLproducing organisms. erefore, new CLSI interpretive criteria
using lower MIC breakpoints for several cephalosporins and
aztreonam for Enterobacterales were recently introduced to better detect resistance and obviate the need for ESBL screening
and conrmatory tests (except for infection control or epidemiologic purposes). CLSI guidelines also outline criteria for
performing screening and conrmatory tests for ESBLs that
involve MIC and disk diusion screening breakpoints for
particular antibiotics using β-lactamase inhibitors.
21,23,26-28,36,37
However, false-negative results may occur with these phenotypic ESBL conrmatory tests in the presence of an AmpC
enzyme, which is not inhibited by clavulanic acid.23 Several
automated susceptibility test systems, such as Vitek 2 and the
Phoenix System, contain phenotypic ESBL detection tests that,
when used with expert system soware, are able to accurately
detect ESBLs.
37
AmpC β-lactamases are chromosomal- or plasmid-mediated
β-lactamase enzymes that hydrolyze rst-, second-, and thirdgeneration cephalosporins and cephamycins, and also display
resistance to some currently available β-lactamase-inhibitors,
such as clavulanic acid, sulbactam, and tazobactam. Several
gram-negative bacteria, such as S marcescens, P aeruginosa,
indole-positive Proteus spp., Acinetobacter spp., Citrobacter
freundii, and Enterobacter spp. (oen referred to as the SPICE or
SPACE bacteria) contain chromosomally-mediated, inducible
AmpC enzymes that, when hyperproduced, can also hydrolyze
penicillins and aztreonam in addition to the cephalosporins
and cephamycins listed previously.37 AmpC hyperproduction
can occur during the treatment of infection due to one of these
organisms, especially when a strong inducer such as ceazidime or clavulanic acid is used.35 Plasmid-mediated AmpC
enzymes have also been reported in Klebsiella spp., Proteus
mirabilis, Citrobacter koseri, and Salmonella spp., and oen display an antibiotic susceptibility prole similar to chromosomally-mediated AmpC hyperproducers.37 All SPICE and SPACE
bacteria should be assumed to be AmpC producers, so specic
detection of AmpC production is not recommended.37 However, plasmid-mediated AmpC β-lactamases can be detected
by demonstrating cephamycin hydrolysis using the AmpC
disk test, the modified Hodge test (MHT), or the threedimensional test.
37
Carbapenemase enzymes have also emerged that may be
chromosomal- (Stenotrophomonas maltophilia) or plasmidmediated (eg, Pseudomonas aeruginosa, Acinetobacter spp.,
Klebsiella pneumoniae, etc.). Several plasmid-mediated
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carbapenemase enzymes have been characterized (KPC, VIM,
OXA-4, NDM, and IMP) that hydrolyze carbapenems and most
other β-lactam antibiotics, as well as display resistance to multiple other antibiotic classes.
34,37
e modied Hodge test can
be used for carbapenemase detection on isolates with elevated
carbapenem MICs; however, it cannot dierentiate between carbapenemase types.
23,37
Because of the wide diversity of β-lactamase enzymes and
the potential limitations of phenotypic ESBL-, AmpC-, and carbapenemase detection methods, several commercial tests have
been developed to aid in the detection these enzymes, some of
which include the RAPIDEC CARBA NP (bioMérieux, Durham, NC; phenotypic, colorimetric test that detects KPC, NDM,
VIM, IMP, and OXA-48), Check-MDR assay (Wageningen, e
Netherlands; microarray that detects TEM, SHV, CTX-M, some
AmpCs, and NDM, VIM, IMP, OXA-48, and KPC), Expert CarbaR (Cepheid, Sunnyvale, CA; PCR that detects NDM, VIM,
IMP, and OXA-48), FilmArray (bioMérieux; PCR that detects
only KPC), and the Verigene (Luminex; PCR that detects CTXM, KPC, NDM, VIM, OXA-48, and IMP).
38
As mentioned earlier, the CLSI recently lowered the cephalosporin and carbapenem breakpoints for Enterobacterales
in an attempt to better identify antibiotics with predictable
ecacy against bacteria with multiple resistance mechanisms
and eliminated the recommendation to perform specialized
testing to detect ESBL-, AmpC-, or carbapenemase-mediated
resistance. However, this recommendation has gained considerable criticism from many clinicians and microbiologists
because detection of the exact mechanism of resistance is
thought to be important for both treatment and epidemiologic purposes.
37
High-Level Aminoglycoside Resistance
Aminoglycosides display relatively poor activity against Enterococcus spp. due to poor intracellular uptake (intrinsic, moder-
ate-level resistance), so they should not be used alone in the
treatment of infections due to enterococci. In addition, enterococci can acquire resistance to aminoglycosides through acquisition of genes that code for aminoglycoside-modifying enzymes
(acquired resistance), which oen leads to elevated aminoglycoside MICs (high-level aminoglycoside resistance or HLAR).23
Aminoglycosides (primarily gentamicin or streptomycin) may
be considered with ampicillin, penicillin, or vancomycin to provide synergistic bactericidal activity, especially in the treatment
of Enterococcal endocarditis or Enterococcal osteomyelitis.
However, supplemental testing should be performed to detect
the presence of HLAR, which predicts the lack of synergism
between gentamicin or streptomycin and cell wall active agents
against Enterococcus spp.
e presence of HLAR can be evaluated using agar dilution
(agar plates) or broth microdilution (wells) using high concentrations of gentamicin (500 mcg/mL) and streptomycin (2000
mcg/mL).23 e plates or wells are inoculated with a standardized suspension of the infecting Enterococcus spp. and incubated for 24 hours in ambient air.23 e growth of one or more
Enterococcus spp. colonies on the agar plate or in the broth
20,23
microdilution well demonstrates the presence of HLAR, signifying that the corresponding aminoglycoside cannot be used with
a cell-wall active agent to achieve synergistic bactericidal activity. HLAR can also be detected using a disk diusion method
in which disks containing high concentrations of gentamicin
(120 mcg) and streptomycin (300 mcg) are used.23 HLAR to
gentamicin also confers resistance to tobramycin, netilmicin,
and amikacin but not necessarily streptomycin, which should be
tested independently.23 Testing for HLAR is usually performed
only on Enterococcal isolates from infections that may require
combination bactericidal activity, such as bacteremia, endocarditis, osteomyelitis, or meningitis.
23
Tests for the Detection of MRSA, VISA, VRSA, and VRE
Several tests are available that can quickly detect or conrm the
presence of methicillin-resistant S aureus (MRSA) or vancomycin-
resistant enterococci (VRE). For the detection or conrmation
of MRSA, the cefoxitin disk diusion test, oxacillin–salt agar
screening tests, culture-based chromogenic media, rapid latex
agglutination (LA) tests, or molecular methods using real-time
PCR can be used.
e cefoxitin disk diusion test is performed using routine
CLSI procedures, with modied interpretive criteria used to
detect mecA-mediated resistance in MRSA in which MRSA is
reported for S aureus strains with a zone size of ≤21 mm.
is test has also been useful in detecting methicillin-resistance
in some coagulase-negative staphylococci.23 e oxacillin–salt
agar screening tests have been widely used for the detection of
mecA-mediated resistance MRSA, but they appear to lack sensitivity for the detection of strains that exhibit heteroresistance.23
A standard inoculum of S aureus is inoculated onto an agar plate
containing Mueller-Hinton agar supplemented with 4% sodium
chloride and 6 mcg/mL of oxacillin and incubated in ambient
air at 33°C to 35°C for 24 hours.23 e growth of more than one
colony indicates MRSA, which also confers resistance to nafcillin, oxacillin, cloxacillin, dicloxacillin, and all cephalosporins
excluding cearoline. However, this test is not recommended
for the detection of methicillin-resistance in other Staphylococ-
cus spp.
23
Selective chromogenic media are available to detect MRSA
from surveillance specimens, all of which produce a characteristic pigment in the presence of MRSA with results available
within 24 hours.23 ere are also numerous rapid commercial
LA tests to detect MRSA in clinical specimens by using highly
specic monoclonal antibodies for the detection of penicillinbinding protein (PBP) 2a (also termed PBP 2′), the protein
encoded by the mecA gene in MRSA.
Several molecular tests for the detection of MRSA are commercially available that detect the mecA resistance determinant.38 Depending on the test, they may be used for surveillance
testing (detecting colonization) or for the diagnosis of infection. Some examples of common molecular MRSA surveillance tests include the GeneOhm MRSA Assay (BD), the Xpert
MRSA (Cepheid, Sunnyvale, CA), and the LightCycler MRSA
Advanced Test (Roche Diagnostics, Indianapolis, IN), which all
are FDA-approved PCR assays for the rapid, direct detection of
23,30,39
23,28
23,39
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nasal colonization by MRSA for the prevention and control of
MRSA infection in healthcare institutions.
23,30,38,39
ese assays
can detect the presence of MRSA directly from nasal swab specimens within 2 hours using real-time PCR that couples primers
specic for mecA and the S aureus-specic gene orfX (sensitivity
93%, specicity 96%).39 Several PCR-based tests also exist for the
diagnosis of infection due to MRSA and include the GeneOhm
StaphSR Assay (BD; blood cultures), the XPert MRSA/SA BC
and SSTI tests (Cepheid; blood cultures and skin/so tissue
infections), the Verigene Gram-Positive Blood Culture Nucleic
Acid Test (Luminex), and the mecA XpressFISH (OpGen, Gaithersburg, MD), with results typically available within 1 to 2 hours
of culture positivity.
38
e CLSI reference broth microdilution method can accu-
rately detect vancomycin intermediate S aureus (VISA, MIC4 to
8 mcg/mL) and vancomycin-resistant S aureus (VRSA, MIC≥16
mcg/mL) but may not consistently detect the presence of heteroresistant VISA.23 e use of brain heart infusion (BHI) agar
plates with 6 mcg/mL of vancomycin (VRE screening plates
described below) can be considered for the detection of S aureus
strains with an MIC of≥8 mcg/mL but is not useful for VISA
strains with an MICof 4 mcg/mL.23 Lastly, the disk diusion test
is unable to accurately detect VISA strains but detects VRSA
strains mediated by vanA.
23
Current automated susceptibility testing methods, including the Vitek 2 system and the BD Phoenix, are able to accurately detect the presence of VRE.23 VRE can also be detected
using the vancomycin agar screen test, and is oen performed
on rectal swab specimens to detect carriers of VRE. A standard
inoculum of the infecting Enterococcus spp. is inoculated onto an
agar plate supplemented with BHI broth containing vancomycin
6mcg/mL and incubated in ambient air for 24 hours.
20,23
e
presence of any growth demonstrates the presence of VRE. is
test is most useful for detecting acquired vancomycin resistance
(eg,vanA or vanB) in E faecalis and E. faecium, but it is not as
useful for strains that display intrinsic resistance to vancomycin
(eg, vanC), such as E. gallinarum and E. casseliavus, in which
MICs range from 8 to 16 mcg/mL (intermediate) and growth is
variable on agar screening plates.
D-Zone Test for Detecting Inducible
ClindamycinResistance
Clindamycin resistance in staphylococci, S pneumoniae, and
β-hemolytic streptococci is typically mediated by expression
of the erm gene, which also confers resistance to macrolides,
lincosamides, and streptogramin b (called MLSb-type resistance). MLSb resistance can be either constitutive or inducible,
especially in staphylococci.
streptococci if susceptibility is performed) that are macrolide resistant but clindamycin susceptible should be evaluated for inducible clindamycin resistance using the -zone
20,23
test.
e -zone test is a disk diusion procedure in which
a 15-mcg erythromycin disk is placed 12mm (streptococci)
or 15 to 26mm (staphylococci) apart from a 2-mcg clindamycin disk on an agar plate inoculated with the infecting organ-
20,23
ism.
Ifinducible clindamycin resistance is present in the
organism, the clindamycin zone of inhibition will be attened
20,23
Staphylococci (and β-hemolytic
on the side nearest the erythromycin disk, demonstrating the
letter D in appearance. Organisms that display a attening of
the clindamycin zone are -zone test positive and should be
reported resistant to clindamycin in the nal organism susceptibility report.
Special Considerations for Fastidious, Anaerobic,
orMiscellaneous Bacteria
e susceptibility testing of fastidious bacteria (eg, H inuenzae,
N gonorrhoeae, and S pneumoniae) and anaerobes cannot be per-
formed using standard broth microdilution, disk diusion, or
automated susceptibility testing methods because these organisms require more complex growth media and environmental
conditions to support bacterial growth.
fastidious bacteria or anaerobes may require media with supple
mental nutrients, prolonged incubation times, and incubation in
atmospheres with higher CO2 concentrations.
ogy reference texts and CLSI standards have been developed to
outline specic methodologies (broth dilution, disk diusion,
and automated methods), quality control guidelines, and interpretive breakpoint criteria that should be used for the susceptibility testing of these bacteria.
21,25-29,36,40,41
e clinical signicance of anaerobes as a cause of infection
is more widely appreciated, and the susceptibility of anaerobes
to various anti-infective agents is no longer predictable.
handling and processing of biologic specimens for anaerobic
culture and susceptibility testing are extremely crucial to the
validity of the results because most anaerobic bacteria of clinical importance are intolerant to oxygen.
collected in appropriate anaerobic transport systems (commercially available vials or tubes) that contain specialized media and
atmospheric conditions to support the growth of the anaerobic
bacteria until the specimen is processed in the laboratory.40 Once
collected, the specimens should be transported to the laboratory
within minutes to hours of collection, processed for culture in
anaerobic jars or chambers in the appropriate growth media,
and incubated in anaerobic atmospheric conditions. e clinical specimens that provide the best yield for anaerobic culture
include aspirated or tissue biopsy specimens.
e identication of anaerobic bacteria by an individual
hospital laboratory may be performed using one of three methods: (1) presumptive identication based on information from
the primary growth plates, including the Gram stain results,
patterns of growth on selective or dierential media, plate and
cell morphology, and results of various rapid spot and disk
tests; (2)denitive identication based on the results of individual biochemical tests that detect the presence of preformed
enzymes found in certain anaerobes; and (3) rapid identication of anaerobes using commercially available NA detection
panels or MALDI-TOF.
40,41
Many hospital laboratories do not
have the resources for commercially available, anaerobic bacteria identication systems and rely on the rst two methods
for presumptive identication of anaerobic bacteria. If necessary, clinical isolates can be sent to a reference laboratory for
further testing.
Most clinical microbiology laboratories do not currently
offer routine susceptibility testing of anaerobic bacteria
36,40,41
e cultivation of
36,40,41
Microbiol-
9,40-43
9,40
Specimens should be
40
e
-
CHAPTER 18 • InfECTIous DIsEAsEs: BACTERIA 421
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because of the uncommon occurrence of pure anaerobic infections, the uncertain role of anaerobes in mixed infections, the
previous predictable susceptibility of anaerobic bacteria to
antibiotics, the previous lack of standardization of antimicrobial susceptibility testing of anaerobes, and the technical
diculties in performing the tests.
40-42
However, it is becoming apparent that routine antimicrobial susceptibility testing
of anaerobic bacteria is necessary due to the increasing incidence of serious infections caused by anaerobic bacteria, the
emerging resistance of anaerobic bacteria to multiple antibiotic
agents, and the poor clinical outcomes observed when ineective antibiotics are used for the treatment of infections caused
by anaerobes.
40-43
e susceptibility testing of anaerobic bacteria has undergone
numerous methodological modications and standardization
over the past several years.
41-43
e CLSI has recently published
a standard outlining the clinical situations where anaerobic susceptibility testing should be considered, the methods of susceptibility testing that should be used, when and how surveillance
susceptibility reporting should be performed, and the antibiotic
agents that should be tested for susceptibility.
42
Susceptibility testing for anaerobes should be performed
in patients with serious or life-threatening infections such
as endocarditis, brain abscess, osteomyelitis, joint infection,
refractory or recurrent bacteremia, and infection of prosthetic
devices or vascular gras.
40-42
Susceptibility testing should also
be performed in patients with persistent or recurring anaerobic
infections despite appropriate antibiotic therapy.
41,42
Lastly, susceptibility testing of anaerobic bacteria should be periodically
performed within geographic areas or individual institutions
to monitor regional susceptibility patterns of anaerobic bacteria over time.
40-42
e recommended anaerobic susceptibility testing methods
include agar dilution and broth microdilution using supplemented Brucella broth, both of which can be reliably performed by most clinical microbiology laboratories.
40-43
e
agar dilution method is the gold standard reference method
that can be used for susceptibility testing of any anaerobic
bacteria, whereas the broth microdilution method has been
validated only for antimicrobial susceptibility testing of Bac-
teroides spp. and Parabacteroides spp.
41,42
In contrast to agar
dilution, the broth microdilution method can evaluate the
susceptibility of multiple antibiotics simultaneously, and several microdilution panels are now commercially available for
routine susceptibility testing, including Anaerobe Sensititre
panel (ANO2, ermo Fisher Scientic) and Oxoid ANA MIC
Panel (ermo Fisher Scientic). egeneral methodology
for each of these tests is similar to those described previously
for aerobic bacteria. Etest strips can also be used for anaerobe
susceptibility testing, and results appear to correlate well with
agar dilution. In addition, β-lactamase testing of anaerobes
can be performed according to CLSI guidelines using chromogenic disks.
41-43
Because routine antimicrobial susceptibility of anaerobes is
not performed by all hospital microbiology laboratories or for
all anaerobic isolates, antibiotic therapy for infections caused by
anaerobes is usually selected empirically based on susceptibility
reports published by reference laboratories.
41,42
However, if susceptibility testing is performed on an individual anaerobic isolate, the results should be used to guide the anti-infective therapy
for the patient.
Lastly, several miscellaneous (oen uncommon) pathogenic
bacteria are dicult to detect or cultivate using the standard
microbiologic procedures outlined previously. ese organisms
oen pose a diagnostic dilemma because they oen require specialized testing for identication. It is beyond the scope of this
chapter to describe all specialized testing methods that are available to detect these organisms; however, an abbreviated list can
be found in Table18-10.
4,13,44-61
Methods for Reporting Susceptibility Results
Individual Isolate Susceptibility Reports
When a bacterial isolate is recovered from a clinical specimen, the identication and susceptibility results are compiled
in a report that is available electronically or via a hard copy
in the patient’s chart. e bacterial identication and antibiotic susceptibility report oen contains the following information: the patient’s name, medical record number, the date and
time of specimen collection, the source of specimen (eg, blood,
wound, urine), the bacteria that were identied (if any), and
the list of antibiotics tested for susceptibility with the MIC or
disk diusion result and CLSI interpretive category, as shown
in Figure18-5.
lection (eg, drawn peripherally versus central line) and the
time to positivity are occasionally reported. In some hospitals, the individual isolate susceptibility report may also contain information regarding the usual daily dose and cost of
antibiotics.
Once bacterial culture and susceptibility results are available,
this information should be used to change the patient’s empiric
antibiotic regimen, which usually covers a broad spectrum of
bacteria, to a more directed antibiotic regimen targeting the
infecting bacteria and antibiotic susceptibility (de-escalation).
e directed antibiotic regimen should be chosen based on clinical and economic factors, some of which include the severity of
infection, the site of infection, the activity (MIC value) of the
antibiotic against the infecting organism, the proven ecacy of
the antibiotic in the treatment of the particular infection, the
overall spectrum of activity of the antibiotic (a narrow spectrum agent is preferred), the end-organ function of the patient,
the presence of drug allergies, the route of administration (oral
versus parenteral), the daily cost of the antibiotic, and so on.
esusceptibility report provides some of the information necessary for the de-escalation of antibiotic therapy, namely, the site
of infection, the infecting organism(s), and the susceptibility of
the infecting organism(s).
As seen in the sample susceptibility report in Figure18-5,
several antibiotics may display activity against the infecting
bacteria, oen with dierent MICs. It is not always advanta
geous to choose the antibiotic with the lowest MIC against a
particular organism on a susceptibility report. As discussed earlier in this chapter, antibiotics have dierent MIC breakpoints
corresponding to S, I, and R (and SDD for select antibiotics) for
20,62
Additionally, the site of blood culture col-
-
422 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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TABLE 18-10.
CLINICAL
ORGANISM
TYPE OF
ORGANISM
FINDINGS AND
INFECTIONS
DIAGNOSTIC
METHOD POSITIVE RESULT REFERENCE
Bordetella
pertussis
(Whooping
cough)
Borrelia
burgdorferi
(Lyme
disease)
tract symptoms,
characteristic
whooping cough,
pneumonia
Erythema migrans,
pericarditis,
arthritis, neurologic
disease
Growth within 3–7 days;
more sensitive when
performed early in course of
infection
Rapid detection of B.
antibodies; should be used in
conjunction with culture due
Direct detection in 1–2
most sensitive when
performed early in course
of infection
Not routinely performed
sensitivity; long incubation
(hold cultures for up to 12 wk)
screening test
borderline or
positive, second step
within 1–2 wk) and IgG
(appears within 4–6 wk)
antibodies against B
burgdorferi
antibodies against
B burgdorferi
pertussis
days;
44
13,45,46
Brucella spp.
(can involve any
organ); spondylitis,
arthritis,
endocarditis
spirochetes; especially useful
Growth within 7 days, but
cultures should be held for
3 wk
Detects antibodies to most
Brucella spp.; titer of ≥1:160
is diagnostic in conjunction
with appropriate clinical
scenario
chronic or past brucellosis;
most useful for diagnosis of
neurobrucellosis
Detection of Brucella-
routinely available in most
laboratories
47
CHAPTER 18 • InfECTIous DIsEAsEs: BACTERIA 423
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TABLE 18-10.
CLINICAL
ORGANISM
TYPE OF
ORGANISM
FINDINGS AND
INFECTIONS
DIAGNOSTIC
METHOD POSITIVE RESULT REFERENCE
Chlamydia
pneumoniae
Clostridioides
difcile
(Pseudomembranous
colitis)
Atypical
bacteria
Anaerobic
bacteria
tract infections,
pharyngitis;
pneumonia
Pseudomembranous
colitis, diarrhea
detect organism in culture
4-fold rise in antibody titer
between paired sera (acute
and convalescent samples)
4-fold rise in antibody titer
between paired sera (acute
and convalescent samples)
or a single serum sample
1:16 or an IgG titer 1:512
Detection of C pneumoniae
DNA
growth media
test
Growth within 48 hr; most
sensitive test
test more sensitive than EIA
detection of Clostridioides
difcile tcdB gene
4,13,48
49
Coxiella
burnetti
(Q fever)
Cryptosporidium
parvum
Acute or chronic
systemic illness,
pneumonia,
hepatitis,
endocarditis
Protozoa Acute diarrhea
(self-limiting to
severe), abdominal
pain, dehydration
Glutamate
dehydrogenase
(GDH) assays
Growth in 6–14 days, organism
Ziehl-Neelsen or
Kinyoun staining
DFA using a
monoclonal antibody
against oocyst
EIA Detection of C. parvum
Detection and differentiation
Detects GDH, an enzyme
present in all Clostridioides
difcile isolates; cannot
detected by DFA
titer of 1:200
Detection of oocysts in stool
or intestinal scrapings
Detection of oocysts in stool
or intestinal scrapings
antigen in stool or intestinal
scrapings
of Cryptosporidium spp.
13,48
50
(continued)
424 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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TABLE 18-10.
CLINICAL
ORGANISM
TYPE OF
ORGANISM
FINDINGS AND
INFECTIONS
DIAGNOSTIC
METHOD POSITIVE RESULT REFERENCE
Ehrlichia spp.
myalgia, headache,
malaise, rash
(more common in
children), nausea,
vomiting, diarrhea,
leukopenia,
thrombocytopenia,
elevated hepatic
transaminases; may
be life-threatening
Entamoeba
histolytica
Giardia spp. Protozoa Acute diarrhea
Protozoa Amebiasis: intestinal
(colitis, diarrhea)
(liver abscess)
(self-limiting
to severe),
malabsorption
syndromes, lowgrade fever, chills,
abdominal pain
IFA serology 4-fold rise in antibody titer
between paired sera (acute
and convalescent samples)
Peripheral blood
smear WrightGiemsa or
Diff-Quik stain
Detection of E chaffeensis or
ova and parasites
Detection of E histolytica
Antigen detection
on fresh stool
samples
Detection and differentiation
ova and parasites
Wet preps or
stains of duodenal
material
antigen detection
assays
Detect morulae
(cytoplasmic vacuoles)
E phagocytophilum DNA
sequences
Detection of trophozoites
and cysts
antibodies with titer ≥1:200
Detection of E histolytica or
E dispar
of E histolytica or E dispar
Detection of trophozoites
Detection of trophozoites
Detection of trophozoite
48,51,52
50
50
Helicobacter
pylori
Peptic ulcer disease Positive test indicative of
the presence of organism
LA serologic tests Detect IgG antibodies
against H pylori
using monoclonal
antral biopsy
specimen
Detection of H pylori DNA
Detection of H pylori antigen
Positive test indicative of
active infection
53
CHAPTER 18 • InfECTIous DIsEAsEs: BACTERIA 425
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TABLE 18-10.
CLINICAL
ORGANISM
TYPE OF
ORGANISM
FINDINGS AND
INFECTIONS
DIAGNOSTIC
METHOD POSITIVE RESULT REFERENCE
Legionella
pneumophila
Leishmania
spp.
Atypical
bacteria
Protozoa
Pneumonia
mucocutaneous,
or visceral
(VL, kala-azar)
infection; can infect
reticuloendothelial
system
Growth in 3–5 days 4,54
specialized media
DFA staining L. pneumo phila antigen
IFA serology 4-fold rise in antibody titer
between paired sera (acute
and convalescent samples)
detection (EIA,
Detection of Legionella spp.
Giemsa staining and
light microscopy
Growth of promastigotes
(VL)
LA (VL) Detection of leishmanial
Detects Legionella
pneumophila serogroup 1
antigen only
DNA
Amastigotes within the
specimen
Detection of antileishmanial
antibodies in blood or serum
antigen in urine
55
Leptospira
spp.
Mycoplasma
hominis
Leptospirosis
(self-limiting with
fevers, chills,
myalgia, headache,
aseptic meningitis);
icteric leptospirosis
(severe form
associated with
jaundice, bleeding,
and renal failure)
Atypical
bacteria
infections, including
prostatitis, PID,
bacterial vaginosis,
urethritis; systemic
infection in
neonates and
immunocompromised
patients
Detection of Leishmania DNA
microscopy or
Growth within 6 wk
Detection of leptospiral
Detection of leptospiral
selective media
Detection of motile
leptospires
4-fold or greater rise in
agglutinating antibody titer
between paired sera (acute
and convalescent samples)
antibodies
DNA
Growth within 5 days 13,56
13,46
(continued)
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