BACTERIOLOGY

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Within-a-Day Detection and Rapid Characterization of

Carbapenemase by Use of a New Carbapenem Inactivation
Method-Based Test, CIMplus
François Caméléna,a,c Aurélie Cointe,a,b Vincent Mathy,b Claire Hobson,a Catherine Doit,a,b Béatrice Bercot,a,c
Dominique Decré,d,e Isabelle Podglajen,f,g Laurent Dortet,h,i Audrey Monjault,b Philippe Bidet,a,b Stéphane Bonacorsi,a,b
André Birgya,b

a IAME, UMR 1137, INSERM, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
b Service de Microbiologie, Hôpital Robert Debré, AP-HP, Paris, France
c Service de Microbiologie, Hôpital Saint-Louis, AP-HP, Paris, France
d Laboratoire de Bactériologie, Groupe Hospitalier des Hôpitaux Universitaires de l'Est Parisien, AP-HP, Paris, France
e Université Pierre et Marie Curie, Paris, France
f Service de Microbiologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France
g Université Paris Descartes, Paris, France
h Service de Bactériologie-Hygiène, Hôpital Bicêtre, AP-HP, Le Kremlin-Bicêtre, France
i
Centre National de Référence Associés de la Résistances aux Antibiotiques, Entérobactéries Productrices de
Carbapénémase, Le Kremlin-Bicêtre, France

ABSTRACT The dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a

major threat to public health. Rapid and accurate detection of CPE is essential for
initiating appropriate antimicrobial treatment and establishing infection control mea-
sures. The carbapenem inactivation method (CIM), which has good sensitivity and
specificity but a detection time of 20 h, was recently described. In this study, we
evaluated the performances of a new version, the CIMplus test, which allows detec-
tion of carbapenemases in 8 h and characterization of carbapenemase classes, ac-

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cording to the Ambler classification, in 20 h. A panel of 110 carbapenem-resistant
Enterobacteriaceae strains, including 92 CPE strains (with NDM, VIM, IMP, KPC, GES,
OXA-48, and OXA-48-like enzymes), was used to evaluate test performance. Carbap-
enemase activity was detected at 8 h and 20 h. Characterization of carbapenemase
classes, using specific inhibitors, was possible in 20 h. The CIMplus test had sensitivi-
ties of 95.7% and 97.8% at 8 h and 20 h, respectively, and a specificity of 94.4%, in-
dependent of the culture duration. Using a decision algorithm, this test was success-
ful in identifying the carbapenemase class for 98.9% of tested CPE isolates (87/88 Received 23 January 2018 Returned for
isolates). In total, the characterization was correct for 100%, 96.9%, and 100% of Am- modification 14 February 2018 Accepted 30
May 2018
bler class A, B, and D isolates, respectively. Therefore, this test allows detection of
Accepted manuscript posted online 27
carbapenemase activity in 8 h and characterization of carbapenemase classes, ac- June 2018
cording to the Ambler classification, in 20 h. The CIMplus test represents a simple, Citation Caméléna F, Cointe A, Mathy V,
affordable, easy-to-read, and accurate tool that can be used without any specific Hobson C, Doit C, Bercot B, Decré D, Podglajen
I, Dortet L, Monjault A, Bidet P, Bonacorsi S,
equipment. Birgy A. 2018. Within-a-day detection and rapid
characterization of carbapenemase by use of a
KEYWORDS carbapenemase, EDTA, Enterobacteriaceae, NDM, OXA-48, new carbapenem inactivation method-based
carbapenemase inactivation method, mCIM, phenotypic characterization, phenotypic test, CIMplus. J Clin Microbiol 56:e00137-18.
https://doi.org/10.1128/JCM.00137-18.
detection, phenylboronic acid
Editor Karen C. Carroll, Johns Hopkins
University School of Medicine
Copyright © 2018 American Society for

T he worldwide emergence and spread of carbapenemase producers represent a

clinical challenge and a public health threat (1). Carbapenemase enzymes are
clustered in different classes that define their hydrolytic profiles; VIM, NDM, and IMP
Microbiology. All Rights Reserved.
Address correspondence to Stéphane
Bonacorsi, stephane.bonacorsi@aphp.fr.
belong to the Ambler class B metallo-␤-lactamases (MBLs), Klebsiella pneumoniae

September 2018 Volume 56 Issue 9 e00137-18 Journal of Clinical Microbiology jcm.asm.org 1

Caméléna et al. Journal of Clinical Microbiology

carbapenemase (KPC) and GES belong to class A, and OXA-48 and OXA-48-like belong
to class D (2). These profiles are associated with resistance to carbapenems and to most
␤-lactam antibiotics (except for aztreonam for class B and third-generation cephalo-
sporins for class D).
The mechanisms of carbapenem resistance are the production of carbapenemase,
the association with extended-spectrum ␤-lactamases (ESBLs), and/or the production of
an AmpC ␤-lactamase combined with decreased membrane permeability. It is impor-
tant to differentiate between these different resistance mechanisms to implement
infection control measures (3).
To detect carbapenemase production, phenotype-based assays have been devel-
oped, including growth-based assays (4–6), biochemical tests (7), immunochromato-
genic assays (8), and carbapenem hydrolysis assays using matrix-assisted laser desorp-
tion ionization–time of flight mass spectrometry (9). Some authors suggest that these
tests may have low sensitivity to detect OXA-48-producing strains, however, and only
a few methods are able to classify these enzymes according to the Ambler classification
(4, 8, 10). Although molecular methods remain the gold standard, they are costly,
limited by the targets used specifically in the test, and not accessible to all microbiology
laboratories throughout the world (11).
Recently, a simple detection test, the carbapenem inactivation method (CIM) test,
has been described; it has shown excellent performance in many centers, similar to the
performance of commercial tests but with a lower cost (6, 12–15). A modified version
of this test (mCIM) appears in the Clinical and Laboratory Standards Institute (CLSI)
recommendations for the detection of carbapenemase in Enterobacteriaceae (16, 17).
Recently, the CLSI approved a modification of the mCIM, called the eCIM, that uses
EDTA inhibition to detect class B carbapenemases (18). Lastly, the SMA-mCIM, a variant
of the mCIM test, uses sodium mercaptoacetate (SMA) as an inhibitor to detect MBL
enzymes (19).
The CIM test and its variants enable the detection of carbapenemases with optimal
performance within 18 to 24 h. This lengthy delay affects the implementation of
infection control measures and, in some cases, the prescription of appropriate antibi-
otic therapy for patients. Detection in 8 h was suggested by Van der Zwaluw et al., but
the delay required for the reading and interpretation of the test results remains
debated (6, 12, 14).

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In this study, we describe a CIMplus version of the CIM test. With our test, carbap-
enemase activity can be detected within 8 h, including 2 h of incubation and 6 h of
culture. Furthermore, we can identify the type of carbapenemase, according to the
Ambler classification, in 20 h. The performance of our test is equivalent to that of the
original test, which uses overnight cultures. Thus, the CIMplus test represents a valuable
tool for guiding antibiotic therapy, epidemiological studies, and infection control
procedures.

MATERIALS AND METHODS

Bacterial isolates tested. To evaluate the performance of the CIMplus test, we analyzed 110
Enterobacteriaceae strains with decreased susceptibility (intermediate or resistant) to at least one
carbapenem (ertapenem, imipenem, or meropenem). This characteristic was defined by measuring the
inhibition zone diameter or by determining the MICs of the carbapenems, with interpretation according
to EUCAST recommendations (20). This strain collection was composed of 92 carbapenemase-producing
Enterobacteriaceae (CPE) isolates (47 Klebsiella sp. isolates, 26 Escherichia coli isolates, 6 Enterobacter sp.
isolates, 6 Citrobacter sp. isolates, and 7 Proteus sp. isolates) and 18 non-carbapenemase-producing
Enterobacteriaceae (non-CPE) isolates (9 K. pneumoniae isolates, 3 E. coli isolates, 4 Enterobacter sp.
isolates, and 2 Citrobacter freundii isolates) from five French Hospitals and was partially described in
previous studies (4, 21–23). The collection included isolates carrying the following carbapenemase genes:
17 isolates carrying blaKPC, 1 isolate carrying blaGES, 25 isolates carrying blaNDM, 8 isolates carrying blaVIM,
1 isolate carrying blaIMP, 32 isolates carrying blaOXA-48, 5 isolates carrying blaOXA-181, 1 isolate carrying
blaOXA-244, and 2 isolates carrying both blaNDM and blaOXA-181. Table S1 in the supplemental material
provides specific information for each isolate, including the carbapenem resistance mechanisms, the
carbapenem MICs, and the ␤-lactamase genes. The carbapenem MICs (imipenem, meropenem, and
ertapenem) were determined by using the Etest (bioMérieux, Marcy l’Etoile, France) and were interpreted
according to EUCAST guidelines (20).

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Rapid Detection and Characterization of Carbapenemases Journal of Clinical Microbiology

FIG 1 Strategy to identify the type of carbapenemase by using meropenem disks (10 ␮g) with or without
a specific carbapenemase inhibitor (PBA for class A enzymes and EDTA for class B enzymes).

CIMplus testing. Isolates were subcultured from frozen stock to tryptic soy agar (TSA). The protocol
used in our study to detect the presence of carbapenemases was similar to the standard CIM protocol
(6). Briefly, a 10-␮g meropenem disk (Bio-Rad, Marne La Coquette, France) was added to 400 ␮l of
distilled water containing a 10-␮l loopful of the tested strain, and the mixture was incubated for 2 h at
35°C. Simultaneously, a Mueller-Hinton agar (MHA) plate was streaked with the susceptible E. coli ATCC
25922 reference strain from a 0.5 McFarland standard inoculum and incubated at 35°C. This early
incubation allowed the start of E. coli growth.
At the same time, to characterize the type of carbapenemase, 2 other suspensions were prepared,
using the same protocol as for carbapenemase detection, to which were added inhibitors, i.e., either 0.05
M EDTA (Sigma-Aldrich, St. Louis, MO) or 20 mg/ml phenylboronic acid (PBA) (Sigma-Aldrich). PBA was
first dissolved in dimethyl sulfoxide (DMSO) and then diluted in sterile water, with a final concentration
of DMSO of 3.5%. After 2 h, the three meropenem disks were removed from the bacterial suspensions
(with water, EDTA, or PBA) and placed on preincubated MHA plates, which were then reincubated at

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35°C. Inhibition zones were evaluated 6 h (for the detection test) and 18 h (for the detection and
characterization tests) after placement of the disks.
We considered that, if the tested isolate produced a carbapenemase, then the susceptible strain
would grow to contact the disk. In contrast, if the tested isolate had decreased susceptibility to
carbapenems without carbapenemase activity, then meropenem would be incompletely hydrolyzed and
would retain activity. Similarly, in the characterization test, if the carbapenemase activity were inhibited
by one of the inhibitors, then meropenem would retain activity and an inhibition zone around the disk
would be observed. An inhibition zone corresponds to a visible diameter around the disk (⬎6 mm). When
a carbapenemase is produced, the susceptible strain grows to contact the disk and therefore no diameter
is visible.
In cases with positive carbapenemase detection, the characterization of the carbapenemase class was
interpreted as follows. (i) If the susceptible E. coli strain grows to contact the EDTA-incubated disk and
the PBA-incubated disk, then the tested isolate is neither a class A nor a class B carbapenemase producer
and thus is very likely to be a class D carbapenemase producer. (ii) If the susceptible E. coli strain grows
with an inhibition zone around the EDTA-incubated disk and grows to contact the PBA-incubated disk,
then the tested isolate is a class B carbapenemase producer. (iii) If the susceptible E. coli strain grows to
contact the EDTA-incubated disk and grows with an inhibition zone around the PBA-incubated disk, then
the tested isolate is a class A carbapenemase producer. (iv) If the susceptible E. coli strain grows with
inhibition zones around the EDTA- and PBA-impregnated disks, then the tested isolate is considered to
be a class B carbapenemase producer. Some class B carbapenemase-producing strains (17/34 strains)
were also inhibited by PBA; those strains are presented in Table S1. The decision algorithm is presented
in Fig. 1. All of the tests were performed in the same clinical microbiology laboratory at Robert Debré
Hospital.

RESULTS
The CIMplus test was evaluated with 110 carbapenem-resistant Enterobacteriaceae
isolates, to detect carbapenemase activity at 8 h and 20 h and to characterize the
carbapenemase type in 20 h. Eighty-eight (95.7%) of the 92 CPE isolates were CMIplus

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Caméléna et al. Journal of Clinical Microbiology

TABLE 1 Performance of the CIMplus test in detecting carbapenemase activity and identifying the carbapenemase class of CPE isolates
Detection of carbapenemase activity (n ⴝ 110) Characterization of carbapenemase type (n ⴝ 88)a
Time of detection and No. No. Total Sensitivity Specificity No. well No. Total Categorical
bacteria tested positive negative no. (%) (%) characterizedb mischaracterizedb no. agreement (%)
8h
Class A CPE 18 0 18 100
Class B CPE 30 4 34 88.2
Class D CPE 38 0 38 100
Class B and D CPE 2 0 2 100
Any class of CPE 88 4 92 95.7
Non-CPE 1 17 18
All isolates 89 21 110 95.7 94.4

20 h
Class A CPE 18 0 18 100 18 0 18 100
Class B CPE 32 2 34 94.1 31 1 32 96.9
Class D CPE 38 0 38 100 38 0 38 100
Class B and D CPE 2 0 2 100
Any class of CPE 90 2 92 97.8 87 1 88 98.9
Non-CPE 1 17 18
All isolates 91 19 110 97.8 94.4
aAllCPE isolates that tested positive with the CIMplus test in 18 h except for the 2 isolates that coproduced 2 carbapenemases.
bThe algorithm described in Fig. 1 was used for categorization.

positive at 8 h, and 90 (97.8%) of 92 were positive at 20 h (Table 1). All carbapenemase

nonproducers (n ⫽ 18) except 1 isolate were CIMplus negative at 8 h and 20 h, as
expected. The false-positive non-CPE isolate was a K. pneumoniae strain containing
CTX-M-15, SHV-11, and TEM-1 ␤-lactamases, which are associated with possibly de-
creased membrane permeability. Based on these results, the detection performance of
the CIMplus test had sensitivities of 95.7% and 97.8% at 8 h and 20 h, respectively, and
a specificity of 94.4%, independent of the culture duration (Table 1). All of the class A
and D isolates were well detected by the CIMplus test at 8 h and 20 h. The CIMplus test
failed to detect 4 of 34 class B CPE isolates at 8 h (2 strains with VIM enzymes and 2 with
NDM enzymes), and 2 of 34 isolates remained negative at 20 h (2 hypermucoid
VIM-producing K. pneumoniae strains). Phenotypic and genotypic methods were re-
peated to confirm the presence of carbapenemase enzymes in the 2 latter isolates. All

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CIMplus-positive isolates grew to contact the disks, whereas negative isolates had
median inhibition zone diameters of 18 mm (range, 16 to 20 mm) and 21 mm (range,
20 to 25 mm) at 8 h (Fig. 2) and 20 h, respectively.
Characterization of the carbapenemase type was performed with 88 strains, equiv-
alent to the CIMplus-positive isolates but excluding 2 strains with double carbapen-
emases. The interpretation of the carbapenemase type was made according to the
decision algorithm presented in Fig. 1. Among those 88 strains, 98.9% (87/88 strains)
were well characterized, with median inhibition zones of 17 mm and 22 mm for the
PBA- and EDTA-impregnated disks, respectively. In total, characterization was correct
for 100%, 96.9%, and 100% of class A, B, and D isolates, respectively (Table 1). The only
misclassified isolate was an IMP-1-producing K. pneumoniae strain that was classified as
class D instead of class B (Table S1). These results were confirmed in a second
experiment with 2 different readers.

DISCUSSION
In this work, we present and evaluate the performance of the CIMplus test. We can
conclude that it has high sensitivity in the detection of CPE, comparable to that of the
original CIM test and the CLSI-approved mCIM test (6, 12, 16, 17). The CIMplus test can
detect carbapenemase activity in isolates producing KPC, GES, NDM, VIM, IMP, OXA-48,
and OXA-48-like enzymes.
The CIM test results are considered to be interpretable in 8 h, although some
authors discuss this point (6, 12, 14). When the susceptible E. coli culture duration is
increased with the preincubation step, then the performance of the CIMplus test at 8 h for

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Rapid Detection and Characterization of Carbapenemases Journal of Clinical Microbiology

FIG 2 Examples of results for different carbapenemase classes identified with the CIMplus test at 8-h readings. (A)
RD30 (KPC). (B) RD7 (NDM). (C) RD4 (OXA-48). (D) SL1 (absence of carbapenemase). The culture of E. coli ATCC 25922
grows to contact the meropenem disks with the CPE isolates (A, B, and C), whereas an inhibition zone diameter is
detected with the carbapenemase nonproducer (D).

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carbapenemase detection is satisfactory. Reading and interpretation difficulties have
not been reported, allowing complete detection within 1 work day (Fig. 2). Only 2
hypermucoid K. pneumoniae strains were not detected at 8 h or 20 h, as they were
impossible to suspend in water; this phenomenon was reported previously (13). Also,
2 strains were not detected at 8 h but were detected at 20 h, probably due to partial
hydrolysis of the meropenem disk causing a delay in the growth of the susceptible E.
coli strain to contact the disk. The false-positive non-CPE strain was identified as a K.
pneumoniae strain containing, among others, the blaCTX-M-15 gene. We hypothesize that
this result is due to low carbapenemase activity of ESBL enzymes (CTX-M), as described
previously (24).
As presented in the algorithm (Fig. 1), we observed satisfactory performance of the
CIMplus test in identifying the type of carbapenemase within 20 h. Indeed, we obtained
correct characterization for 98.9% of tested CPE isolates (87/88 isolates) and categorical
agreements between 96.9% and 100%, depending on the enzyme class (Table 1). All of
the carbapenemase types were well characterized except for 1 IMP-1-producing K.
pneumoniae isolate, which was classified as class D instead of class B. To understand this
difference, we conducted further experiments. When the EDTA concentration was
increased 3-fold, an inhibition zone was observed (data not presented), confirming that
this enzyme could be inhibited when a high concentration of EDTA was used. However,
nonspecific inhibition was detected for non-class B carbapenemases at this higher
concentration. Because IMP and GES carbapenemase-producing Enterobacteriaceae
strains are scarce in France, we could test only 1 strain per type; therefore, our results
should be confirmed with a larger number of strains.

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Caméléna et al. Journal of Clinical Microbiology

The CIMplus test allows detection and characterization of the carbapenemase type,
with a cost of €0.53 per tested isolate; this cost includes the meropenem disks, the
MHA plates, the PBA, the DMSO, and the EDTA. The latter three reagents are stable at
ambient temperature and therefore can be kept for several years. Moreover, this test is
accessible to laboratories throughout the world, as it requires only basic laboratory
materials.
The test we evaluated in this study has many advantages but also some limitations.
Here carbapenemase detection was possible in 8 h, but some authors have described
faster phenotypic tests (10, 21). The test also requires many bacteria; therefore, the
initiation of our experiment required a rich culture. Moreover, characterization of the
carbapenemase type was not possible for 2 E. coli strains that simultaneously produced
2 carbapenemases from different classes. Results for characterization of isolates that
produce multiple carbapenemases depend on the classes of the enzymes they produce.
This issue has been raised for other phenotype-based characterization tests (10). Finally,
this test was evaluated with isolates from various geographical origins, but no typing
method was used. Therefore, we cannot exclude genetic links between some isolates.
Furthermore, we tested only the major carbapenemase types (NDM, VIM, IMP, KPC, GES,
and OXA-48/OXA-48-like) and a limited number of carbapenemase nonproducers. The
performance of the test should be evaluated using different carbapenemase enzymes
and a larger number of non-CPE isolates. The last item could help increase the
specificity of the CIMplus test, as demonstrated in previous studies (6, 12–15). In
conclusion, the CIMplus test represents a simple, affordable, accessible, and accurate
technique that requires only basic laboratory equipment. It allows the detection and
characterization of carbapenemase classes among CPEs.

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/JCM
.00137-18.
SUPPLEMENTAL FILE 1, XLSX file, 0.1 MB.

ACKNOWLEDGMENTS
This study was financed with internal funds.
We declare that we have no conflict of interest.

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