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Evaluation of the MASTDISCS combi Carba plus fordifferentiation of carbapenemase-producing
Enterobacteriaceae
Satomi Takei1, Ayako Nakamura1, Toshihiro Takahashi1,
Takaaki Kawakami1, Mai Sano1, Masayoshi Chonan1,
Shigeki Misawa1, Takashi Horii1, Satoshi Hirayama2,
Takashi Miida2 and Akimichi Ohsaka1,3
1

Department of Clinical Laboratory, Juntendo University Hospital, Tokyo, Japan2
Department of Clinical Laboratory Medicine,Juntendo University Graduate School of Medicine, Tokyo, Japan3
Department of Transfusion Medicine and Stem Cell Regulation,Juntendo University Graduate School of Medicine(Received for publication February 17, 2020)

The MASTDISCS combi Carba plus is a set of inhibitor combination discs thatdifferentiates carbapenemases from AmpC β-lactamases. We evaluated theperformance of this disc set in discriminating β-lactamase genotypes using 36
clinical and 5 reference strains, as well as discriminating clinical isolates using
phenotypic assays. The results for all Enterobacteriaceae strains producing 21
carbapenemases (16 clinical isolates and 5 reference strains), including 14 IMP-1,1 VIM-1, 2 NDM, 2 OXA-48, and 2 KPC carbapenemases, using the MASTDISCS
combi Carba plus were consistent with the genotypes identiﬁed by multiplex PCR.
Sixteen carbapenemase-producing Enterobacteriaceae clinical isolates were positivefor carbapenemase production according to the modiﬁed carbapenem inactivation
methodology, and 13 MBL-producing strains were positive for carbapenemase
production according to the sodium mercaptoacetic acid double disc synergy test.
Twenty-one of the AmpC β-lactamase- and ESBL-producing strains were identiﬁed
as AmpC- or non-carbapenemase-producing Enterobacteriaceae using theMASTDISCS combi Carba plus. Ten AmpC β-lactamase-producing clinical strains
were all positive for the aminophenyl boronic acid double disc synergy test. Overall,
the MASTDISCS combi Carba plus allowed easy identiﬁcation and successful
typing of major carbapenemases using routine microbiology methods.
Corresponding author: Satomi Takei, Tel: ＋81–3–3813–3111 (ext. 5186), E-mail: stakei@juntendo.ac.jp (S. Takei)

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Introduction
Carbapenemase-producing Enterobacteriaceae (CPE) are resistant to carbapenems andbroad-spectrum β-lactam antibiotics, and detection of CPE in clinical specimens has increased
over the last decade1,2). CPE have been detected among species including Escherichia coli,
Klebsiella spp., and Enterobacter spp. These organisms are major causes of communityassociated and healthcare-associated infections, which pose a threat especially to immunocompromised patients3).
There are limited antimicrobial therapeutic options for patients with infections caused byCPE. To control infections, rapid detection and prevention of the spread of CPE in healthcare settings are important1). Among the four classes of β-lactamases deﬁned by the Ambler classiﬁcation
system, the carbapenemases produced by CPE belong to class A (Klebsiella pneumoniae
carbapenemases [KPC]), class B (metallo-β-lactamases [MBL], e.g., IMP, VIM, and NDM), orclass D (OXA-48-like carbapenemases).
The Clinical and Laboratory Standards Institute (CLSI) recommends the modiﬁed carbapenem inactivation method (mCIM) for screening carbapenemase production4). This method can beused to screen almost all carbapenemases, but it cannot classify every type. Although the double
disc synergy test (DDST) using speciﬁc inhibitors is commonly based on phenotypic assays for
identifying β-lactamases, β-lactamases must be examined individually according to their presumed CPE type5,6). Determination of carbapenemase genes using PCR is also performed in somereference microbiology laboratories.
MASTDISCS combi Carba plus is a set of inhibitor combination discs used to diﬀerentiate
KPC, MBL, OXA, and AmpC β-lactamases using ﬁve discs that contain speciﬁc inhibitors ofeach β-lactamase7). The purpose of this study was to evaluate the performance of the MASTDISCS combi Carba plus for identifying carbapenemases in Enterobacteriaceae clinical isolates.

Materials and Methods
Bacterial strains
In total, 36 drug-resistant clinical isolates, including 10 Enterobacter spp., 9 Escherichia
coli, 10 Klebsiella spp., 5 Citrobacter spp., 1 Leclercia sp., and 1 Serratia spp., were analyzed
from 2012 to 2017 at the Juntendo University Hospital. The CPE included IMP-1, NDM, KPC,
and OXA-48, based on PCR, and the non-CPE included AmpC β-lactamase and extendedspectrum β-lactamase (ESBL), as determined by phenotypic methods in a routine microbiology
laboratory. Five carbapenemase-producing organisms, E. coli BAA-2469, K. pneumoniae NCTC
13442, K. pneumoniae NCTC 13440, K. pneumoniae NCTC 13438, and K. pneumoniae NCTC

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13809, were used as positive controls. All clinical isolates were identiﬁed by the MALDI Biotyper (Bruker Daltonics, Billerica, MA, USA). An antimicrobial susceptibility test was conductedusing the MicroScan 96 Plus with the NENC1J panel (Beckman Coulter, Brea, CA, USA), andthe minimum inhibitory concentrations (MICs) were determined based on the CLSI criteria8).
MASTDISCS combi Carba plus
The MASTDISCS combi Carba plus (Mast Group, Liverpool, UK) was used according tothe manufacturerʼs instructions as follows. A 0.5 McFarland inoculum was prepared and inoculated on Mueller–Hinton II agar plates (Nippon Becton Dickinson, Tokyo, Japan). The MASTDISCS combi Carba plus consists of ﬁve discs (A–E) containing the follow drugs: A, 10 μg faropenem; B, 10 μg faropenem with an MBL inhibitor disc; C, 10 μg faropenem with a KPC inhibitor
disc; D, 10 μg faropenem with an AmpC inhibitor disc; and E, 30 μg temocillin with an MBL inhibitor disc. Each disc was placed on the Mueller–Hinton II agar plates, ensuring suﬃcient distance between the discs, and incubated at 35°C for 18–24 h. The diameter (in mm) of the zone ofinhibition was measured. Discs showing no zone of inhibition were assigned a diameter of 6 mm.
A ﬂowchart depicting the identiﬁcation of carbapenemases using the MASTDISCS combi Carbaplus is shown in Fig. 1. The diameter of the zone of inhibition on the penem disc (A) was compared with that on each penem plus inhibitor disc (B, C, or D). If disc B only showed a diameter
diﬀerence ≥5 mm compared with disc A (the diﬀerences for discs C vs. A and D vs. A should be＜5 mm), the strain was regarded as an MBL producer. If disc C only showed a diameter diﬀerence ≥5 mm compared with disc A (the diﬀerences for discs B vs. A and D vs. A should be＜5 mm), the strain was regarded as a KPC producer. If discs C and D both showed signiﬁcant diFig. 1. Flowchart for identiﬁcation of carbapenemase using MASTDISCS Combi Carba plusaEach millimeter indicates zone diameter or zone diﬀerence between discs.

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ameter diﬀerences (≥5 mm) compared with disc A (B vs. A should be ＜4 mm), the strain was regarded as a AmpC β-lactamase producer with porin loss (impermeability). If disc E showed azone of inhibition diameter diﬀerence of ＜10 mm, and insuﬃcient diﬀerence was obtained
among discs A, B, C, and D, the strain was regarded as an OXA-48 producer. If an equivocal result was generated but resistance on disc A was shown, the organism was considered to express a
carbapenemase enzyme.
Phenotypic conﬁrmation of β-lactamase production
A phenotypic assay to detect β-lactamases was performed using the mCIM, recommended
by the CLSI4), and DDSTs. DDSTs were performed using a commercially available sodium mercaptoacetic acid (SMA) disc (metallo-β-lactamase SMA Eiken; Eiken Chemical, Tokyo, Japan) as
an inhibitor of MBL (SMA-DDST), and 50 mg/mL 3-aminophenyl boronic acid (APBA; Tokyo
Chemical Industry, Tokyo, Japan) was used as the inhibitor of AmpC β-lactamases (APBADDST). The SMA-DDST was performed using both imipenem and ceftazidime discs (SensiDisc; Nippon Becton Dickinson) according to the manufacturerʼs instructions. If SMA with imipenem and/or with ceftazidime was positive, it was interpreted as SMA-DDST positive. ABPADDST was performed using ceftazidime and cefmetazole discs (Sensi-Disc; Nippon BectonDickinson) with or without 300 μg APBA (6 μL 50 mg/mL solution) according to a previously reported method6).
PCR detection of β-lactamase genes
Bacterial DNA extraction and PCR of carbapenemase genes were performed using the Cica
Geneus DNA Extraction Reagent and Cica Geneus Carbapenemase Genotype Detection Kit(Kanto Chemical, Tokyo, Japan), respectively, according to the manufacturerʼs instructions. The
following carbapenemase genes were ampliﬁed by multiplex PCR: blaNDM, blaKPC, blaOXA-48,blaGES, blaIMP-1, and blaVIM. AmpC β-lactamase and ESBL genes were detected using multiplex
PCR using a previously published method9,10). PCR was performed using the TaKaRa PCR Thermal Cycler Dice® Gradient (TaKaRa Bio, Kusatsu, Japan). The ampliﬁed PCR products were electrophoresed on 1% agarose gels (Agarose S; FUJIFILM Wako Pure Chemical, Osaka, Japan) using
the Mupid-exU (TaKaRa Bio, Kusatsu, Japan) at 100 V for 40 min. The gels were stained for 30 min
using GelRed Nucleic Acid Stain (Biotium, Fremont, CA, USA) and visualized using UV light.

Results
The β-lactamase genotypes, MASTDISCS combi Carba plus, mCIM, and DDST for 36
strains from clinical specimens and ﬁve reference strains are shown in Table 1. Among the 36

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73―249（ 15 ）clinical strains, 16 contained carbapenemase genes, as determined by PCR (IMP-1 in 14, NDM in1, and OXA-48 carbapenemase in 1). Two strains contained genes encoding the following plasmid-mediated AmpC β-lactamases (DHA in one and CIT in one). Ten strains contained genes encoding CTX-M-9 β-lactamases. The results of the MASTDISCS combi Carba plus, mCIM, andDDST were compared with the β-lactamase genotypes. The types of carbapenemase-positive
cases according to the MASTDISCS combi Carba plus are shown in Fig 2. Number of strains
identiﬁed as MBL producers by the MASTDISCS combi Carba plus were 14 strains and all werepositive for the mCIM tests. Of these 14 strains, 13 had a positive result on the SMA-DDST
using either the imipenem or ceftazidime disc, but one K. pneumoniae was negative for the SMADDST, which is the IMP-1 types. One OXA-48 type strain was identiﬁed as a OXA-48 producer
by the MASTDISCS combi Carba plus and had a positive result on the mCIM.
Of the 36 strains, 20 had no detectable carbapenemase genes that include the DHA type andCIT type identiﬁed as an AmpC producer and non-CPE by the MASTDISCS combi Carba plus,
respectively. Ten CTX-M-9-producing strains were identiﬁed as non-CPE by the MASTDISCScombi Carba plus. Of the remaining eight strains with no detectable carbapenemase or AmpC
β-lactamase genes, ﬁve were identiﬁed as AmpC producers and three as non-CPE by the MASTDISCS combi Carba plus. These three non-CPE strains were positive according to APBA-DDST.
All ﬁve carbapenemase-producing reference strains showed agreement between the MASTDISCS combi Carba plus results and the β-lactamase genotypes.

Discussion
The MASTDISCS combi Carba plus is a unique disc set that diﬀerentiates the three majorcarbapenemase types and AmpC β-lactamases based on phenotype. It is easy to perform thismethod using the same procedure as the disc diﬀusion method.
We evaluated the performance of this disc set for identifying carbapenemases produced by
41 clinical and reference Enterobacteriaceae strains. Fourteen clinical and 5 reference strains
which were identiﬁed as MBL producers by the MASTDISCS combi Carba plus, all were consistent with the carbapenemase genotypes identiﬁed by PCR. Twenty CPE strains including MBL,OXA-48, and KPC-producing strains were correctly diﬀerentiated using the MASTDISCS combi
Carba plus. Identiﬁcation of carbapenemases from Enterobacteriaceae using the MASTDISCS
combi Carba plus has been described previously7), and our results were very similar to those ofthis report. Evaluation of inhibitor combination discs using meropenem as the inhibitor, similar tothe MASTDISCS combi Carba plus, for identifying CPE has been reported previously11〜13).
However, meropenem-based disc sets are less sensitive for detecting CPE strains, showing low
meropenem MIC values. The MASTDISCS combi Carba plus uses faropenem, which is moreTeble 1. Results of MASTDISCS combi carba plus and phenotypic assay for detction of β-lactamase of clinical and reference strains50（ 16 ）THE JAPANESE JOURNAL OF ANTIBIOTICS
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73―251（ 17 ）Fig. 2. Representative results of MASTDISCS Combi Carba plus. (2-1) IMP-1type, (2-2) KPC
type and (2-3) OXA-48 typesensitive for detecting carbapenemase production14).
We compared the results of mCIM and SMA-DDST with those of the MASTDISCS combiCarba plus for detection of carbapenemase producers. The mCIM was able to identify carbapenemase production from all strains containing carbapenemase genes. One of E. cloacae and one ofK. pneumoniae harboring IMP-1 were mCIM-positive, but not SMA-DDST. The E. cloacae
strain showed large zone of inhibition with disc A of the MASTDISCS combi Carba plus, possessing lost the drug-resistant gene. Another strain of K. pneumoniae might be highly resistant tocarbapenems, with the expansion of zone by SMA could not be clearly observed. Fourteen MBL
producers were identiﬁed by SMA-DDST using imipenem and ceftazidime discs. The ﬁrst reportof IMP-1-producing Enterobacteriaceae isolated from Serratia marcescens was in Japan15). Because the hydrolytic activity of this β-lactamase was lower toward imipenem than ceftazidime, it
was considered a false-negative for imipenem. All strains containing AmpC β-lactamase andESBL genes were identiﬁed as AmpC producers or non-CPE strains by the MASTDISCS combi
Carba plus. The APBA-DDST identiﬁed 10 AmpC β-lactamase producers, but AmpC
β-lactamase genes were detected in only two of these strains. In this study, resistance mechanisms
among these eight strains remain to be identiﬁed. We suspected that chromosomal AmpC
β-lactamase might be involved.
Our results showed that the MASTDISCS combi Carba plus is useful for accurately diﬀerentiating carbapenemase types, as well as evaluating their production. There were some limitations to this study. The KPC carbapenemase-producing strains have been tested only against reference strains, and the IMP carbapenemase-producing strain was not tested for the IMP-6 type,which has recently increased in prevalence in Japan16). Further studies are therefore required toevaluate the performance of this disc set with a larger number of KPC and IMP-6 type CPEstrains. GES-type producer is relatively rare CPE in Japan, but has not been evaluated in thisstudy17). Therefore, it is also necessary to evaluate the detection and diﬀerentiation of this type ofCPE in the future.

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In conclusion, the MASTDISCS combi Carba plus enables convenient screening and diﬀerentiation of the major carbapenemases, and this method provides useful information for antimicrobial therapies and control of infections. However, in routine microbiology laboratory, it is important that MASTDISCS combi Carba plus is rational use for diﬀerential test for CPE by theﬁrst-line test such as mCIM, although it lacks speed.
Conﬂicts of interest
There is no conﬂict of interest.
Acknowledgements
We would like to thank Takamasa Kaneko (Kanto Chemical) for providing the reference
strains.

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