Evaluation of different assays for

detection of -lactamase
production in S. aureus isolates
Brandi Limbago and David Lonsway, CDC,
Mary Jane Ferraro, Massachusetts General Hospital,
Tove Hjbjerg , Aalborg University Hospital
Robert Skov, Statens Serum Institut
Background
Treatment of S. aureus infections with
penicillin requires that the test can
accurately detect -lactamase production
Penicillin is preferred by many provided
the strain is -lactamase negative
Background
A recent publication by Kaase suggested that
pcr detecting the betalactamase gene was
superior
Furthermore that the different phenotypic tests
was not reliable
This study was performed to compare different
phenotypic methods with genotypic detection of
pnase production using both a challenge
collection and consecutive isolates
EUCAST - CLSI criteria
MIC - EUCAST - CLSI
S 0.125 mg/L
R> 0.125 mg/L / R 0.25 mg/L
Disk diffusion - CLSI
10 IU
R 28 mm (R<29 mm)
S 29 mm
CLSI - When MIC 0.125 mg/L or zone size 29
mm, perform an induced -lactamase test
Rare -lactamase positive isolates may not be positive by the
induced -lactamase test
Isolates
348 S. aureus
62 from CDC
Challenge set
199 from Massachusetts General Hospital (MGH)
Consecutive isolates with an MIC < 0.25 mg/L by Vitek II
87 from Aalborg University Hospital, Denmark
Presumed susceptible based on disk diffusion and cloverleaf
test
Methods
PCR for blaZ (M. Kaase et al.)
+ 5 additional primer sets for discrepant results
Microbroth dilution
Disk diffusion using penicillinG 1 and 10 U disks
Nitrocefin -lactamase
Cefinase, (BD BBL Paper Discs) and Dryslide, BD.
using induced colonies (taken from the zone edge of a 1 U
PC disk), read for up to 60 minutes.
All isolates with discrepant results tests were
repeated using colonies from the edges of penicillin,
cefoxitin and oxacillin disks, respectively
Methods
Qualitatively reading of the zone edge (G. I. C.
Ingram,1951)
cliff vs beach/fuzzy zone edge
Cloverleaf -lactamase test
5% blood agar plate, 1U penicillin disk, S. aureus
ATCC 25923 as the -lactamase negative lawn,
Test strain is streaked as a cross
for 50 isolates including all with discrepant results also on
Mueller Hinton agar using both 1U 10U disks
Cloverleaf test
Results
PCR
Initial testing
302 negative
46 positive
5 isolates gave discrepant results from phenotypic tests
PCR using additional primers and or sequencing
Samle No. BlaZ
MIC
(mg/L) Cefinase
Dry
Slide Zoneedge Cloverleaf PG 10 U
32455-09 positive 0,032 negative negative fuzzy negative 40
158390-08 positive 0,064 negative negative fuzzy negative 38
HIP11353 positive 0,032 negative negative fuzzy negative 36
159570-08 negative 0,25 positive pos sharp positive 37
H30876 negative 0,064 negative negative fuzzy wk positive 32
Results
PCR
Additional testing revealed
3 blaZ positive isolates had stop codons (N=2) or had a deletion (N=1)
Marked red
2 isolates were found blaZ positive by additianal primers and the primer
mismatch was identified
Marked blue
Samle No.
Final
BlaZ
MIC
(mg/L) Cefinase
Dry
Slide Zoneedge Cloverleaf PG 10 U
32455-09 negative 0,032 negative negative fuzzy negative 40
158390-08 negative 0,064 negative negative fuzzy negative 38
HIP11353 negative 0,032 negative negative fuzzy negative 36
159570-08 positive 0,25 positive pos sharp positive 37
H30876 positive 0,064 negative negative fuzzy wk positive 32
Results
PCR
Functional blaZ gene
303 negative
45 positive
Results
Correlation to other assays MIC
Total MGH (consecutive)
MIC blaZ functional
(mg/L) Negative Positive
0.008 2
0.016 15
0.032 180 1
0.064 90 5
0.125 15 17
0.25 1 14
0.5 4
2 2
4 1
Total 303 45
MIC blaZ functional
mg/L negative positive
0,008 1
0,016 7
0,032 105 1
0,064 57 2
0,125 11 8
0,25 1 5
4 1
Total 182 17
Sens 47 % Spec 99.7 % Sens 35 % Spec 99.5 %
blaZ vs disk diffusion (1U)
45 1 2 1 9 3 1 3 4 8 4 1 8 positive
303 6 13 27 31 48 61 57 35 12 4 8 1 negative
Total >31 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 <16
Zone size Pen 1 U disk BlaZ
17 1 1 1 2 4 4 3 1 positive
182 1 1 11 27 37 50 33 11 3 7 1 negative
Total 32 30 29 28 27 26 25 24 23 22 21 20 19 18 10
Zone size Pen 1 U disk BlaZ
S 26 mm Sens 93% , Spec 79%, S 25 mm Sens 91% , Spec 91%
S 26 mm Sens 94% , Spec 70%, S 25 mm Sens 88% , Spec 88%
blaZ Zone size Pen 10 U disk
<22 22 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 >39 Total
neg. 1 4 4 13 14 33 35 45 47 31 34 42 303
pos. 6 4 2 2 4 3 2 3 4 5 6 1 1 2 45
blaZ Zone size Pen 10 U disk
16 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 >39 Total
neg. 1 3 4 11 13 26 32 31 29 15 10 7 182
pos. 1 1 2 2 2 2 2 3 1 1 17
blaZ vs disk diffusion (10U)
Total
MGH
Sens 51 % Spec 99.7 %
Sens 59 % Spec 99.5 %
blaZ vs cefinase
blaZ cefinase, BD Dryslide
negative positive Wk positive negative positive Wk positive Total
negative 303 303 303
positive 10 28 7 5 33 7 45
blaZ cefinase, BD Dryslide
negative positive Wk positive negative positive Wk positive Total
negative 182 182 182
positive 3 14 2 15 17
Total
MGH
Sens 77 % Spec 100 %
Sens 82 % Spec 100 % Sens 88% Spec 100 %
Sens 88 % Spec 100 %
blaZ vs zone edge
blaZ PG Zone edge
sharp fuzzy Total
negative 303 303
positive 43 2 45
blaZ PG Zone edge
sharp fuzzy Total
negative 182 182
positive 15 2 17
Total
MGH
Sens 96 %
Spec 100 %
Sens 88 %
Spec 100 %
32 26 weak positive fuzzy negative negative 0,064 H30876
37 25 positive fuzzy positive positive 0,032 W53156
PG 10 U
disk
PG 1 U
disk
Cloverleaf 5%
BP
PG Zone
edge Dry Slide
Cefinase,
BD
MIC
(mg/L)
Samle
No.
blaZ vs cloverleaf
blaZ Cloverleaf
negative positive wk positive Total
negative 303 303
positive 44 1 45
blaZ Cloverleaf
negative positive wk positive Total
negative 182 182
positive 16 1 17
Total
MGH
Sens 100%
Spec 100%
Sens 100%
Spec 100%
Conclusion
The present algorithm using a cefinase/dryslide
test as a confirmatory test for lack of -lactamase
production is suboptimal due to false negative
reactions
Looking at the zone edge when performing disk
diffusion will allow labs using disk diffusion to
screen for -lactamase production with high
sensitivity
The cloverleaf test performs with higher
sensitivity and is thus better suited as a
confirmatory test
Suggestion for new algorithm
Keep present breakpoints for initial evaluation
MIC > 0.125: report as resistant
Zone < 26 mm: report as resistant
Strains with an MIC of 0.125
confirm by cloverleaf test
Strains with zones 26 mm (( 25 mm))
Look at zone edge:
If sharp report as resistant
If fuzzy confirm by cloverleaf test