Special Methods for

Detecting Antimicrobial
Resistance

OUTLINE
Special tests to detect resistance in
Enterococci
Staphylococci
Pneumococci
Enterobacteriaceae*
Other organisms

SCREENING TESTS
Screening tests detects specific resistance
mechanism or phenotype in certain
bacterial strains
characterize an organisms susceptibility or
resistance to an antimicrobial agent
These tests do not provide an MIC of the
antimicrobial agent
some have sufficient sensitivity and
specificity that confirmation of the result is
unnecessary
some results can be reported without
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additional testing.

SCREENING TESTS (2)

Special tests for detecting antibacterial resistance

SCREENING TESTS (3)

Special tests for detecting antibacterial resistance

Enterococci

Tests to detect resistance in

Enterococci

HLAR
Pen/Vanco + Gent/Streptomycin for
treatment of serious invasive enterococcal
infections
These agents act synergistically to
enhance killing
Aminoglycosides cannot be used as single
agents for therapy due to poor activity
against enterococci
Intrinsic, moderate level resistance (MICs
range from 8 to 256 mg/ml)
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Tests to detect resistance in

Enterococci (2)
HLAR
Aminoglycoside resistance usually
corresponds to MICs that are significantly
above the concentration normally tested
in routine susceptibility tests, e.g., 2,000
g/ml for streptomycin and 500 g.ml
for gentamicin and is designated HLR
(HLAR)
For routine AST: Pen and Vanco, and high
level Gent and Streptomycin
Gentamicin , tobramycin, amikacin
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Streptomycin

Tests to Detect Resistance in

Enterococci (3)

Vancomycin Resistance
CLSI MIC for Vanco: 4 g/ml (S), 8 to 16
g/ml (I), 32 g/ml (R)
Three most common phenotypes of Vancomycin
resistance:
High-level, MICs 64 g/ml + Teicoplanin
resistance, MICs, 16 g/ml (VanA
phenotype)
Moderate- to high-level vancomycin
resistance, MICs, 16 to 512 g/ml , most
commonly without teicoplanin resistance
(VanB phenotype)
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Intrinsic low-level resistance associated
with

Tests to Detect Resistance in

Enterococci (4)
Both VanA and VanB phenotypes are
most common in E. faecalis, E.
faecium, E. casseliflavus, and E.
gallinarum
VanD, VanE, VanG, VanL genotypes
have been found but are rare

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Tests to Detect Resistance in

Enterococci (5)
Screening methods for detecting vancomycin and
high level aminoglycoside resistance in
enterococci

REFER TO CLSI M100-S25 TABLE 3F & 3I

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Tests to Detect Resistance in

Enterococci (6)
VRE: Vancomycin agar screen test

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Staphylococcus
aureus

Tests to Detect Resistance in

S. aureusCLSI M100-S25

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Tests to Detect Resistance in

S. aureus (2)

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Tests to Detect Resistance in

S. aureus (3)
Oxacillin Resistance & mecA-mediated
Ox resistance:
MRSA still commonly used even though
methicillin is no longer available for
treatment
Most resistance to Oxacillin in
Staphylococci are mediated by the mecA
gene
mecA directs the production of a
supplemental penicillin binding protein
(PBP2a) during bacterial cell replication
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Tests to Detect Resistance in

S. aureus (4)
Resistance is expressed either
homogeneously or heterogeneously
Homogeneous nearly all bacterial cell
progeny express the resistance phenotype
easier to detect with standard AST
methods
Heterogenous only a fraction of the
progeny population express resistance
(e.g. 1 in 1M cells)
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Tests to Detect Resistance in

S. aureus (5)

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Tests to Detect Resistance in

S. aureus (6)
Report: Methicillin-resistant S. aureus
isolated (MRSA
Oxacillin resistant staphylococci are
resistant to all -lactam agents; other
beta-lactam agents should be reported as
(R) or should not be reported
mecA positive staphylococci are (R) to
oxacillin (not cefoxitin); other -lactams
except those with anti-MRSA activity
should be reported as (R)
*Cefoxitin is used as a surrogate for mecAmediated

oxacillin resistance

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Tests to Detect Resistance in

S. aureus (7) Slide agglutination
test for S. aureus ID

Commercial
Methods: Slide
agglutination test for
PBP2a (MRSA)
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Tests to Detect Resistance in

S. aureus (8)
MRSA: Oxacillin salt screen agar test

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Tests to Detect Resistance in

S. aureus (9)

MRSA: Commercial Chromogenic agars

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Tests to Detect Resistance in

S. aureus (10)
Reporting:
Methicillin-resistant S. aureus (MRSA)
isolated
No Methicillin-resistant S. aureus
isolated
MRSA +: Report Oxacillin as resistant
Notify clinician and infection control
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Tests to Detect Resistance in

S. aureus (11)
Penicillin Resistance and -Lactamase
Penicillin (R) strains of Staph produce lactamase
Some -lactamase producing
staphylococcal isolates test susceptible to
penicillin (MIC 0.12 g/ml or zone
diameters 29mm)
Staphylococcal -lactamase is readily
inducible

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Tests to Detect Resistance in

S. aureus (12)
Tests for detection of -lactamase
production
Penicillin disk diffusion zone edge
test
Nitrocefin-based test (disk,
commercially available)
Perform these tests for isolate that
test (S) to Penicillin using routine AST
method
CLSI Interpretive criteria: 29
25 mm or

Tests to Detect Resistance in

S. aureus (13)
Penicillin zone edge test

More sensitive
than Nitrocefin
in detecting lactamase
production in S.
aureus
Sharp zone edge (cliff) =
-lactamase positive
Resistant to penicillin,
aminocarboxy-, and
ureidopenicillins
REFER TO CLSI M100-S25 TABLE 3D

Fuzzy zone edge

(beach) =
-lactamase negative

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Tests to Detect Resistance in

S. aureus (14)

Nitrocefin -lactamase test

Chromogenic method

Yellow (-)
Red (+)
Positive within 5
minutes to 1 hour

For CoNS including S.

lugdunensis:
use induced growth as
inoculum for testing
(growth taken from the zone
margin surrounding a
penicillin or cefoxitin disk test
on MHA plate after 16-18
hours incubation)

Commercial method,
follow manufacturers
direction

Report: -lactamase
positive
Resistant
to Penicillin
REFER
TO CLSI M100-S25
TABLE 3D
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Tests to Detect Resistance in

S. aureus (15)
Test for inducible clindamycin
resistance (ICR)

Clindamycin (lincosamide); Erythromycin

(macrolide)
Mechanisms of action and resistance for
Erythromycin and Clindamycin are similar
M type mechanism confers resistance to
macrolide only
MSLB type mechanism confers resistance
to macrolides, lincosamide and
streptogramin B agents mediated by an
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erm gene (usually ermA or ermC)

Tests to Detect Resistance in

S. aureus (16)

Test for inducible clindamycin

resistance (ICR)
In Staph, (MSLB) resistance is either
constitutive or inducible
S. aureus: Erythromycin (R), Clinda (S) ->
test for ICR
Broth microdilution method: 4 g of
erythromycin and 0.5 g of clindamycin/ml
in same well
35C; 1824 h
ICR (+): Any growth in well
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Tests to Detect Resistance in

S. aureus (17)
S. aureus
& S.Inducible
lugdunensis
D-Test
Test
for
clindamycin
Use Erythromycin and Clindamycin disks 15-26 mm apart to
detect inducible clindamycin
resistance
(ICR)resistance
D-shape = Clinda Resistance

20
20 mm
mm

For beta-streptococci place

disks 12-15 mm apart

12 mm
E

No D shape = Clinda susceptible

CC

Tests to Detect Resistance in

S. aureus (18)
Reporting of ICR + strains
Report as Clindamycin resistant
Include comment:
This isolate is presumed to be resistant
based on detection of inducible clindamycin
resistance.
REFER TO CLSI M100-S25 TABLE 3G

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Tests to Detect Resistance in

S. aureus (19)
Vancomycin Resistance

CLSI vancomycin interpretive categories for S.

aureus:
2 g/ml (S), 4 to 8 g/ml (I), 16 g/ml (R)
Interpretive categories for CoNS including S.
lugdunensis:
4 g/ml (S) 8 to 16 g/ml (I); and 32 g/ml
(R)
Vancomycin resistant S. aureus VRSA
MICs range from 32 to 1,024 g/ml
Vancomycin-intermediate S. aureus VISA
due to changes in the cell wall, not the van gene
CoNS (VISS) due to cell wall changes;
32 no van

Coagulase-negative
Staphylococcus

Tests to Detect Resistance in

CoNS

Fox disk:
24mm = mecA +
25mm = mecA -

Broth microdilution:
>4 g/ml = mecA +
4 g/ml= mecA -

-lactamase test:
If test is negative, use induced growth as inoculum for
testing
(growth taken from the zone margin surrounding a penicillin
or cefoxitin disk test on MHA plate after 16-18 hours
incubation)
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Streptococcus
pneumoniae

Detection of Penicillin Resistance in

Streptococcus pneumoniae
Use Mueller-Hinton + 5% sheep blood agar
Incubate in CO2 instead of air
Report actual MIC
Report both Pen & Cefotaxime or
Ceftriaxone & Mero MICs for S. pneumo
from CSF

Oxacillin 19 mm = Pen R, I or
S
Always confirm with MIC

Other Organisms
- lactamase test

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Thank you

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