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155（ 13 ）Evaluation of the modified sodium mercaptoacetic aciddouble disk synergy test for detecting the metalloβ-lactamase producing Enterobacteriaceae
Daisuke Sakanashi1, Makoto Kawachi2, Yuki Uozumi2, Mitsuru Nishio3,
Yuki Hara4, Hiroyuki Suematsu1, Mao Hagihara1, Yuka Yamagishi1,5and Hiroshige Mikamo1,5,*
1Department of Infection Control and Prevention,
Aichi Medical University Hospital2
Department of Clinical Laboratory, Konan Kosei Hospital3
Department of Clinical Laboratory, Komaki City Hospital
4
Department of Clinical Laboratory,
Japanese Red Cross Nagoya Daini Hospital
5
Department of Clinical Infectious Diseases,
Aichi Medical University Hospital(Received for publication December 5, 2016)

Background: Sodium mercaptoacetic acid double disk synergy test (SMA-DDSTs)
is frequently used in Japan to detect metallo-β-lactamase (MBL) producer easily. In
this study, we evaluated SMA-DDST at the points of the antimicrobial disk, and thedistance between SMA and an antimicrobial disk.
Methods: Forty-one clinical isolates ceftazidime (CAZ)-resistant Enterobacteriaceae
and 12 reference strains were tested. Those isolates included 17, 19, 4 and 13 of only
IMP-, IMP with other classes of β-lactamases-, New Delhi MBL with other classesof β-lactamases- and non-MBL-producers, respectively. SMA-DDSTs wereperformed with CAZ, imipenem (IPM) and meropenem (MEPM)-containing diskswith the distance between SMA and antimicrobial disk at 11 mm or 16 mm (centerto-center).
Results: The sensitivities were 27/40 (68%), 17/40 (43%), 35/40 (88%), 29/40(73%), 25/40 (63%), and 39/40 (98%) for 16 mm CAZ-SMA-DDST, 16 mm IPMSMA-DDST, 16 mm MEPM-SMA-DDST, 11 mm CAZ-SMA-DDST, 11 mm IPMSMA-DDST, and 11 mm MEPM-SMA-DDST, respectively. CAZ and MEPM disk
showed the highest sensitivity for only MBL-producing isolates and MBL withcoproducing other classes of β-lactamases-producing isolates, respectively. Among
any of antimicrobials used in this study, 11 mm SMA-DDSTs showed higher
sensitivity than those 16 mm. The speciﬁcity was 100% for all SMA-DDSTs.
* Corresponding author156（ 14 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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However, the ambiguous false-expansion of growth inhibition zone was induced in
11 mm CAZ-SMA-DDST for non-MBL-producing isolates.
Conclusion: This study suggested that the combination of 16 mm CAZ-SMA-DDST
and 11 mm MEPM-SMA-DDST is suitable for screening MBL-producing isolates.

Introduction
In recent years, an increasing number of carbapenemase producers, which hydrolyze and areresistant to carbapenem antimicrobials, a last resort for the treatment of infections, have been ofgreat concern in the world1). In Japan, metallo-β-lactamase (MBL) producers, especially IMP
MBL producers, are most frequently detected2). Tests for MBL producers include genetic testingsuch as polymerase chain reaction (PCR) assay and loop-mediated isothermal ampliﬁcation
assay, phenotyping based on inhibition of MBL activity by EDTA, thiol compounds, etc., anddouble disk synergy test (DDST)3).
In Japan, sodium mercaptoacetic acid (SMA)-containing disks for DDSTs (SMA-DDSTs),
which are commercially available and require no special device or operation, are widely used inclinical laboratory testing because of their high economic efﬁciency and convenience. According
to the package inserts of metallo-β-lactamase SMA Eiken (SMA Eiken)(SMA 3 mg/disk, Eiken
Chemical, Tokyo, Japan), the test can perform at a center-to-center distance of 15 to 20 mm between the SMA Eiken and ceftazidime (CAZ) or imipenem (IPM) disks with CAZ-resistant bacteria as target.
More recently, SMA-DDST were reported to show false-negative results for NDM and VIM
MBL producers, leading to further validation of optimal conditions for SMA-DDST4, 5). At present, Japan Nosocomial Infection Surveillance and some studies have reported the modiﬁed SMADDST that detection sensitivity can be enhanced6, 7, 8, 9).
The main changes compared to original were at using meropenem (MEPM) disk together
with CAZ disk, and a shorter center-to-center distance of 11 mm between the SMA and antimicrobial disks. As for speciﬁcity, to the best of our knowledge, including the above-mentioned report, no false-positive case has been reported regardless of the distance between the disks and anantimicrobial. However, we occasionally had observed ambiguous expansion of growth inhibitionzone in modiﬁed SMA-DDST for non-MBL producers. Thus, we believed that a reevaluation ofthe SMA-DDST is essential. Therefore, this study aimed to determine the best condition for thedetection of MBL-producing Enterobacteriaceae among SMA-DDSTs.

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157（ 15 ）Materials and Methods
Strains used and antimicrobial susceptibility testing
The test collection included 53 well-characterized Enterobacteriaceae isolates meeting thespeciﬁed in the package inserts of SMA Eiken (i.e., CAZ-resistant). Forty-one of them were collected from 10 hospitals in Aichi prefecture Japan, during 2011 to 2016, while the other 12 were
ATCC or NCTC reference strains. Identiﬁcation of isolates was performed using MALDI Biotyper (Bruker Daltonics). The β-lactamase productions were conﬁrmed by previously published
PCR analysis methods10, 11, 12). The minimum inhibitory concentration (MIC) was measured basedon CLSI criteria by broth microdilution method13) (Table 1).
SMA-DDSTs
SMA-DDSTs were performed as follows: After each test strain adjusted to McFarland standard No. 0.5 was applied with a sterile cotton swab to the overall surface of Mueller Hinton agar
(Becton, Dickinson and Company, Tokyo, Japan) in three directions and the surface became dry,metallo-β-lactamase SMA Eiken (SMA 3 mg/disk, Eiken Chemical, Tokyo, Japan) and antimicrobial disks (CAZ 30 µg/disk or IPM 10 μg/disk or MEPM 10 μg/disk) (Eiken Chemical, Tokyo,Japan) were placed as shown in Fig. 1. The center-to-center distance between SMA and antimicrobial disk was kept at 16 mm (16 mm SMA-DDST) or 11 mm (11 mm SMA-DDST). Differences in inhibition zones were measured after an 18-h incubation at 35°C. When the no inhibitionzone was observed around an antimicrobial disk, the inhibition zone diameter was measured as
6 mm that was the disk diameter. According to the package insert for SMA Eiken, the test strain
was considered MBL-positive when the clear growth inhibition zone of the antimicrobial disk
placed close to the SMA was at least 5 mm greater than that of the antimicrobial disk alone.
Inter-facility validation using K. pneumoniae ATCC 700603
At 4 facilities (A, B, C, and D) preserving Klebsiella pneumoniae ATCC 700603 (SHV-18producer) in Aichi prefecture, 15 clinical microbiology technicians (a, b, c, d, e, f, g, h, i, j, k, l,m, n, and o) who had performed SMA-DDSTs in daily practice were asked to carry out CAZSMA-DDSTs at inter-disk distances of 11 and 16 mm (center-to-center), blinded to the origin anddrug susceptibility of the test bacteria, and inhibition ring measurement and MBL production assessment were individually investigated.

Results
SMA-DDSTs
Table 2 shows the performance of SMA-DDSTs. The sensitivities were 29/40 (73%), 25/40

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70―3(a); NCTC 13476, (b); ATCC BAA-2146, (c); ATCC BAA-200, (d); ATCC 51983, (e); ATCC 700603, (f); ATCC BAA-201, (g); ATCC BAA-198, (h); ATCC 51503,(i); ATCC BAA-1705 and BAA-1900, (j); ATCC BAA-1144, (k); ATCC BAA-2355.

Table 1. Distribution of antimicrobial MICs for the metallo-β-lactamase (MBL)-producing and non-producing isolates in this study

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70―3159（ 17 ）Fig. 1. Disk placement position and interpretation criteria of sodium
mercaptoacetic acid (SMA)-double disk synergy test in this study

The test strain was considered as metallo-β-lactamase positive when (b) – (a) ≥ 5 mm.

Table 2. The performance of sodium mercaptoacetic acid (SMA)-double disk synergy test(DDST) with the center-to-center distance between SMA and antimicrobial disk at 16 mm or
11 mm

The boldface with underline indicates the highest sensitivity in each β-lactamase types.
MBL indicates metallo-β-lactamase.

(63%), 39/40 (98%), 27/40 (68%), 17/40 (43%), and 35/40 (88%) for 11 mm CAZ-SMA-DDST,
11 mm IPM-SMA-DDST, 11 mm MEPM-SMA-DDST, 16 mm CAZ-SMA-DDST, 16 mm IPMSMA-DDST, and 16 mm MEPM-SMA-DDST, respectively. CAZ-SMA-DDSTs detected only
IMP-producing isolates with high sensitivity, but the tests were inadequate for the isolates that
coproducing other classes of β-lactamases (Table 2 and Fig. 2). An IMP-producing Klebsiella160（ 18 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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Fig. 2. Unclear expansion of inhibition zone that includes both false negative and non-speciﬁc wereobserved in ceftazidime (CAZ)-sodium mercaptoacetic acid (SMA)-double disk synergy test(a)Klebsiella pneumoniae producing IMP with CTX-M (clinical isolate)(b)

Citrobacter braakii producing NDM with CTX-M (clinical isolate)June 2017

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Fig. 2. Continued(c)Escherichia coli producing SHV-4 (ATCC BAA-200)(d)K. pneumoniae producing SHV-18 (ATCC 700603)

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70―3Fig. 2. Continued(e)K. pneumoniae producing KPC-2 (ATCC BAA-1705)(f )K. pneumoniae producing KPC-3 (ATCC BAA-1900).
Expansion of inhibition zone by the effect of SMA is indicated arrows.

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70―3163（ 21 ）oxytoca that showed low-level carbapenem MIC (both IPM and MEPM MIC; ≤1 μg/mL) was detected only CAZ-SMA-DDSTs. The 16 mm MEPM-SMA-DDST detected IMP-producing isolates with high sensitivity, but the test was inadequate for NDM-producing isolates. The 11 mm
MEPM-SMA-DDST improved this point and showed the best sensitivity. Among any of antimicrobials used in this study, 11 mm SMA-DDSTs showed higher sensitivity than those 16 mm. The
speciﬁcity was 100% for all SMA-DDST. However, ambiguous expansion of growth inhibitionzone was observed for Escherichia coli ATCC BAA-200 (SHV-4 producer), K. pneumoniae
ATCC 700603 (SHV-18 producer), K. pneumoniae ATCC BAA-1705 (KPC-2 producer) and K.
pneumoniae ATCC BAA-1900 (KPC-3 producer) in 11 mm CAZ-SMA-DDST (Fig. 2).
Inter-facility validation using K. pneumoniae ATCC 700603
Measurement results are shown in Table 3. At a CAZ-SMA distance of 16 mm, expansion ofgrowth inhibition zone ranged from 0 to 2 mm with a mean of 0.6 mm, showing MBL-negative
results for all operators. At a CAZ-SMA distance of 11 mm, on the other hand, expansion ofgrowth inhibition zone ranged from 1 to 6 mm with a mean of 2.9 mm, showing a false MBL-positive result for one operator (g).

Discussion
In this study, we reevaluated SMA-DDST at the points of the antimicrobial disk, and the distance between SMA and an antimicrobial disk.
At ﬁrst, the antimicrobial disk, our result demonstrated that CAZ and MEPM disk showed
the highest sensitivity for only MBL-producing isolates and coproducing other classes ofβ-lactamases, respectively. Recently in Japan, IMP-6-producing isolates has increased2). They aremostly acquired high activity against MEPM but not at IPM, and coproducing extended-spectrum
β-lactamases (ESBLs)14). This fact suggested that CAZ- and IPM-SMA-DDST reduce their performance, and MEPM-SMA-DDST increase. Presently, the package insert of metallo-β-lactamase
SMA Eiken recommends that use with CAZ or IPM disk. However, we strongly considered thatusing MEPM disk in SMA-DDST is necessary.
Second, the distance between SMA and an antimicrobial disk, among any of antimicrobials
used in this study, 11 mm SMA-DDSTs showed higher sensitivity than those 16 mm. These results
were supports the ﬁndings of a previous evaluation8, 9). Furthermore, our result obtained as a newknowledge that the ambiguous false-expansion of growth inhibition zone was induced in 11 mm
CAZ-SMA-DDST for SHV- or KPC-producing isolates. As stated above, CAZ-SMA-DDST wassuitable for only IMP-producing isolates, and in particular for IMP-producing isolates that often
showed low-level carbapenem resistance. In our results, both 11 mm and 16 mm CAZ-SMADDSTs showed 100% sensitivity for only IMP-producing isolates. Therefore, we considered that

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Table 3. Results of CAZ-SMA-DDSTs for Klebsiella pneumoniae ATCC 700603 among 15
operators in 4 facilities

CAZ-SMA-DDSTs not necessary to shorten the distance between SMA and CAZ.
In conclusion, our result demonstrated that the combination of 16 mm CAZ-SMA-DDST and
11 mm MEPM-SMA-DDST is suitable for screening MBL-producing isolates. One limitation ofthis study is that the MBL-producing isolates except IMP or NDM have not been studied. Thus,
our result may not be applicable to MBL-producing isolates from other types than IMP or NDM.
However, we strongly believed that this method was useful in IMP-endemic areas such as ourcountry, Japan.
Acknowledgments
This study was supported by the Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare, Japan (H28-Shinkou-Ippan-003). All of the authors thank allof the clinical microbiology technicians that assisted with the provision of data for this project.

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70―3165（ 23 ）Conﬂict of interest
Hiroshige Mikamo received Research funding from Sumitomo Dainippon Pharma Co., Ltd.,Taisho Toyama Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Pﬁzer Co., Ltd., Astellas
Pharma Inc., MSD K.K., Toyama Chemical Co., Ltd., Takeda Pharmaceutical Co., Ltd., MeijiSeika Pharma Co., Ltd., Shionogi & Co., Ltd., Boehringer Ingelheim Co., Ltd., Taisho Pharmaceutical Co., Ltd., KYORIN Pharmaceutical Co., Ltd., Bayer Yakuhin Ltd.; Consulting fee/honorarium from Sumitomo Dainippon Pharma Co., Ltd., Taisho Toyama Pharmaceutical Co., Ltd.,
Daiichi Sankyo Co., Ltd., Pﬁzer Co., Ltd., MSD K.K., Astellas Pharma Inc., Meiji Seika PharmaCo., Ltd., MIYARISAN Pharmaceutical Co., Ltd.; Leadership position/Advisory role fromToyama Chemical Co., Ltd. The other authors declare that they have no conﬂict of interest.

References
1) Nordmann P, Naas T, Poirel L: Global spread of carbapenemase-producing Enterobacteriaceae.
Emerg Infect Dis. 2011; 17: 1791–8.
2) Yano H, Ogawa M, Endo S, et al.: High frequency of IMP-6 among clinical isolates of metalloβ-lactamase-producing Escherichia coli in Japan. Antimicrob Agents Chemother. 2012; 56: 4554–5.
3) Arakawa Y, Shibata N, Shibayama K, et al.: Convenient test for screening metallo-β-lactamaseproducing Gram-negative bacteria by using thiol compounds. J Clin Microbiol. 2000; 38: 40–3.
4) Miki K, Takegawa H, Etoh M, et al.: First outbreak report of VIM-1 metallo-β-lactamase producing Pseudomonas aeruginosa in Japan. J Jpn Assoc Infect Dis. 2010; 84: 721–6.
5) Ishii Y: Characteristics of New Delhi metallo-β-lactamase (NDM-1), a novel carbapenem resistance factor. Jpn J Chemother. 2010; 58: 639–43.
6) Japan Nosocomial Infection Surveillance: http://www.nih-janis.jp/material/material/NDM-1%20
SMA20101007.pdf (accessed 31 January 2017).
7) Japan Nosocomial Infection Surveillance: http://www.nih-janis.jp/material/material/modiﬁed%20
SMA-disk%20method.pdf (accessed 31 January 2017).
8) Hattori T, Kawamura K, Arakawa Y: Comparison of test methods for detecting metalloβ-lactamase-producing Gram-negative bacteria. Jpn J Infect Dis. 2013; 66: 512–8.
9) Kitanaka H, Sasano M, Yokoyama S, et al.: Invasive infection caused by carbapenem-resistantAcinetobacter soli, Japan. Emerg Infect Dis. 2014; 20: 1574.
10) Monstein HJ, Östholm-Balkhed Å, Nilsson MV, et al.: Multiplex PCR ampliﬁcation assay for thedetection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae. Acta Pathol Microbiol
Immunol Scand. 2007; 115: 1400–8.
11) Pérez-Pérez FJ, Hanson ND: Detection of plasmid-mediated AmpC β-lactamase genes in clinicalisolates by using multiplex PCR. J Clin Microbiol. 2002; 40: 2153–62.
12) Sakanashi D, Miyazaki N, Suzuki T, et al.: Screening multiplex PCR for six important plasmidmediated carbapenemase genes. Jpn J Med Technol. 2015; 64: 460–7.
13) Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twenty-fourth informational supplement. CLSI: 2014; M100–S24.
14) Yano H, Kuga A, Okamoto R, et al.: Plasmid-encoded metallo-β-lactamase (IMP-6) conferring resistance to carbapenems, especially meropenem. Antimicrob Agents Chemother. 2001; 45: 1343–8.