Improving screening for carbapenemase producing organisms: Evaluation of two
chromogenic media and confirmatory disc diffusion assays
G.L. Vanstone, A Lyons, A Morkowska, A Williams, R Smith & I. Balakrishnan.
Royal Free London NHS Foundation Trust.
RESULTS
Introduction
Conclusions
Screening Media
• Carbapenemases can confer resistance
susceptibility to all β-lactam antibiotics.
or
reduced
• Treatment options are extremely limited and as many acquired
carbapenemases are plasmid-mediated there is potential for
spread between strains, species and genera.
• Screening of high-risk patients is now recommended, but a
standardised methodology has not been established. The Royal
Free London NHS Foundation Trust implemented screening of ITU
patients and high risk groups in April 2013 using a method based
on selective growth in nutrient broth containing imipenem which is
sub-cultured onto CLED agar seeded with an imipenem disc.
• Various commercial culture based and nucleic acid amplification
technology (NAAT) methods are now available for the detection of
carbapenemases. Here, we evaluate two chromogenic media
(BrillianceTM CRE [Oxoid] and chromID CARBA [BioMerieux]), and
confirmatory assays KPC/MβL Confirm Kit and KPC/MβL in P.
aeruginosa/ Acinetobacter Confirm Kit (Rosco Diagnostica) for
the detection of carbapenemase producing bacteria.
Brilliance CRE
Isolate # Carbapenemase
Clinical Isolates
1
VIM
2
VIM
3
VIM
4
VIM
5
VIM
6
VIM
7
KPC
8
KPC
9
KPC
10
11
12
13
14
15
OXA-48
OXA-48
NDM-1
NDM-1
IMP
Control strains
VIM
16
KPC
17
OXA-48
18
NDM-1
chromID CARBA
ID
Growth
Conc
Growth
Conc
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
E. coli
K. pneumoniae
K. pneumoniae
K. pneumoniae
+
+
+
+
+
+
+
+
+
1 x 101
1 x 102
1 x 101
1 x 101
1 x 102
1 x 101
1 x 103
1 x 101
1 x 101
+
+
+
+
+
+
+
+
+
1 x 101
1 x 101
1 x 101
1 x 101
1 x 103
1 x 101
1 x 101
1 x 101
1 x 101
E. coli
K. pneumoniae
E. coli
E. coli
P. aeruginosa
+
+
+
+
N/A
1 x 102
1 x 101
1 x 101
1 x 103
+
+
+
+
+
1 x 105
1 x 103
1 x 101
1 x 101
1 x 102
+
1 x 101
+
1 x 101
+
1 x 101
+
1 x 101
+
1 x 102
+
1 x 102
+
1 x 101
+
1 x 101
P. aeruginosa NCTC 13437
K. pneumoniae NCTC
13438
K. pneumoniae NCTC
13442
K. pneumoniae NCTC
13443
3/5 AmpC positive isolates, 1/5 ESBL
positive isolate and 1 P. aeruginosa strain
with porin loss grew on both media types.
These isolates were shown to have
reduced carbapenem sensitivity.
When used for direct screening from
routine rectal swabs, organisms other
than Enterobacteriaceae and
Pseudomonas were able to grow on both
plate types. Those most commonly
isolated were Candida spp. and
Enterococcus spp. With experience,
colony morphology was used to disregard
these isolates; alternatively MALDI-ToF
identification direct from the chromogenic
plate was performed.
Table 1: Summary of results from carbapenemase positive isolates when grown on Brilliance CRE and chromID CARBA
media. Conc = lowest concentration in cfu/ml at which growth was detected.
The two chromogenic media (BrillianceTM CRE [Oxoid] and chromID CARBA
[BioMerieux]) were challenged with:
• 18 carbapenemase producing isolates, including 4 NCTC control strains (KPC n
= 1, VIM n = 1, NDM-1 n = 1 and OXA-48 n =1), and 14 fully characterised clinical
isolates (KPC n = 3, VIM n = 6, NDM-1 n = 2, IMP n = 1, and OXA-48 n = 2). 100 µl
of concentrations between 1x 101 cfu/ml and 1x109 cfu/ml were tested on the
media.
• 30 carbapenemase negative isolates, including 5 AmpC positive, 5 ESBL
positive and 20 carbapenem sensitive isolates. 100 µl of concentrations between
1x 101 cfu/ml and 1x109 cfu/ml were tested on the media.
All isolates were also tested by the relevant Rosco confirmation assay (KPC/MβL
Confirm Kit for Enterobacteriaceae and KPC/MβL in P. aeruginosa/Acinetobacter
Confirm Kit for P. aeruginosa and Acinetobacter isolates).
• Both kits are disc diffusion assays that utilise discs of Meropenem 10 µg (MRP10),
Meropenem 10 µg + Phenylboronic Acid (MRPBO), Meropenem 10 µg + Cloxacillin
High (MPCXH), Dipicolonic acid (DPA) (Pseudomonas/Acinetobacter kit only),
Imipenem 10 µg (IMI10) (Pseudomonas/Acinetobacter kit only) and Meropenem 10
µg + Dipicolinic acid combination disc (MRPDP) (KPC/MβL Confirm Kit only).
Phenylboronic acid inhibits both AmpC and KPC, Cloxacillin inhibits AmpC alone
and Dipicolonic acid inhibits MßL so a comparison of zone sizes around the
different discs allows for carbapenemase detection.
• Rosco confirmatory assays were performed following Manufacturers’ kit inserts
other than CLED agar was used (in place of McConkey) for the KPC/MβL in P.
aeruginosa/Acinetobacter Confirm Kit.
• To aid OXA-48 detection, Temocillin MIC of all isolates was determined by Etest.
In addition, both media types were evaluated for direct screening from routine
screening samples (rectal swabs) and results were compared to our current laboratory
screening protocol.
KPC/MβL Confirm Kit
• Zone sizes were easy to read and all MßL positive, KPC
positive and carbapenemase negative isolates were
correctly identified.
•
When used in combination with in-house Temocillin AST,
this method highlighted the three OXA-48 positive
isolates as ‘possible carbapenemase producers’.
1
•
•
We found that the requirement for the zone sizes around
the MRP10, MRPBO and MPCXH to all be within 2 mm of
each other too stringent, and sometimes gave results that
could not be interpreted as they did not correspond to
either KPC negative or KPC positive. When results were
interpreted using a difference of 3 mm or less, all of the
KPC negative isolates were correctly identified.
If synergy was present between the MRP10 disc and the
DPA and if the area of growth inhibition was large, the
zone size around the MRP10 disc was difficult to read
making comparison to MRPBO and MPCXH difficult.
This could make identification isolates that have both
MßL and KPC type carbapenemases difficult.
2
3a
3b
3a
KPC/MβL in P. aeruginosa/Acinetobacter Kit
• All MßL positive isolates were correctly identified. No
KPC positive P. aeruginosa or Acinetobacter isolates
were included in this study.
4
• Carbapenemase
negative
P.
aeruginosa
and
Enterobacteriaceae isolates with reduced susceptibility to
carbapenems (eg. AmpC with porin loss) were able to grow
on both media types, highlighting the need to perform
confirmatory testing on all isolates that grow on selective
media.
• When used for direct screening, isolates other than
Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp.
(such as Enterococcus spp. and Candida spp.) were able
to grow on the chromogenic media. However, morphology
and/or MALDI-ToF identification can be used to quickly and
easily identify these isolates.
•The Rosco confirmatory assays performed well on the fully
characterised isolates and gave no false-negative results
when used in combination with in-house Temocillin AST to
predict the presence of OXA-48. If Temocillin AST is
unavailable, the KPC/MßL and OXA-48 Confirm kit can
used in place of the KPC/MßL Confirm kit.
Confirmatory Disc Diffusion Assays
Methods
• Brilliance CRE Chromogenic media failed to detect one of
the three OXA-48 positive isolates included in this study.
OXA-48 carbapenemases can be associated with only
slightly reduced susceptibility to carbapenems, which can
make them difficult to detect using selective media.
5
•The Rosco confirmatory assays are unable to distinguish
between different MβL types (NDM, VIM, IMP etc.), making
further analysis and reference laboratory confirmation
necessary for outbreak investigation and/or typing. In
addition, suspect OXA-48 positive isolates must also be
confirmed by either reference laboratory referral or an
alternative method such as NAAT.
• Direct screening on ChromID CARBA followed by
identification by MALDI-ToF and confirmatory testing using
KPC/MβL
Confirm
and
KPC/MβL
in
P.
aeruginosa/Acinetobacter Confirm kits has now been
implemented into diagnostic service at The Royal Free
London NHS Foundation Trust. To allow further evaluation
of the kits, all isolates that are tested by the Rosco
confirmatory assays are sent to the Antibiotic Resistance
Monitoring and Reference Laboratory, Public Health
England for further investigation.
References/Acknowledgements
Figure 1: Images illustrating disc diffusion assay results 1) E. coli VIM+ showing zone around MRPDP
> 5mm in comparison to MRP, 2) K. pneumoniae KPC+ showing zone around MRPBO > 4mm in
comparison to MRPCX, 3a&3b) E. coli OXA-48+ showing no difference in zone sizes but high temocillin
MIC of >1024, 4) E. coli carbapenemase negative, 5) P. aeruginosa VIM+ showing synergy between
DPA and imipenem/meropenem.
•The Department of Medical Microbiology, The Royal Free London NHS Foundation Trust
•Contact: [email protected]