Evaluation of the double-disk synergy test for New Delhi metallo-β-lactamase-1 and other...

ORIGINAL ARTICLE

Evaluation of the double‐disk synergy test for New Delhimetallo‐b‐lactamase‐1 and other metallo‐b‐lactamaseproducing gram‐negative bacteria by usingmetal‐ethylenediaminetetraacetic acid complexesMomoko Fujisaki1, Shinya Sadamoto1 and Akira Hishinuma2

1Biochemical Research Laboratories, Research and Development Division, Eiken Chemical, 143 Nogi, Nogi‐machi Shimotsuga‐gun,Tochigi, 329‐0114, Japan and 2Department of Infection Control and Clinical Laboratory Medicine, Dokkyo Medical University,Kitakobayashi 880, Mibu, Tochigi, 321‐0293, Japan

ABSTRACTNew Delhi metallo‐b‐lactamase‐1 (NDM‐1), one of the metallo‐b‐lactamases (MBLs), has beenidentified from clinical isolates worldwide. Rapid detection of NDM‐1 producers is necessary to preventtheir dissemination. Seven types of EDTA complexes were evaluated as MBL inhibitors in double‐disksynergy tests (DDSTs), resulting in detection of the first isolate of NDM‐1‐producing Escherichia coli(NDM‐1 Dok01) in Japan. NDM‐1 Dok01 was detected when EDTA magnesium disodium salttetrahydrate (Mg‐EDTA), EDTA calcium disodium salt dihydrate, EDTA cobalt disodium salttetrahydrate and EDTA copper disodium salt tetrahydrate were used as MBL inhibitors. The sensitivityand specificity of DDSTs usingMg‐EDTA for 75MBL producers and 25 non‐MBLproducers were 96.0%and 100%, respectively. These findings indicate that the DDST method using Mg‐EDTA can detectMBL‐producing strains, including NDM‐1 producers.

Key words b‐lactamases, double‐disk synergy test, gram‐negative bacteria, metallo‐b‐lactamase.

Metallo‐b‐lactamases are Ambler class B enzymes andhydrolyze broad‐spectrum b‐lactam agents, includingthird generation cephalosporins and carbapenems. Sincethe early 1990s, researchers all over the world havereported new MBL‐encoding genes in gram‐negativebacilli, most commonly Pseudomonas spp., Acinetobacterspp., and Enterobacteriaceae (1). MBL antimicrobialresistance genes are carried on mobile genetic elements,allowing transfer of the resistance genes to various strainsand species of bacteria. The MBL genes may spread

rapidly to clinically important pathogens; nosocomialoutbreaks caused by MBL‐producing K. pneumoniae havebeen reported (2).

New Delhi metallo‐b‐lactamase‐1 was first identifiedin 2008 in a single isolate of K. pneumoniae that had beenrecovered from a patient who was transferred to Swedenafter treatment in a hospital in New Delhi (3). Followingthe initial report from Sweden, NDM‐1‐producingorganisms have been isolated from clinical specimensin many countries including the UK, India, Pakistan,

CorrespondenceMomoko Fujisaki, Biochemical Research Laboratories, Research and Development Division, Eiken Chemical, 143 Nogi, Nogi‐machi Shimotsuga‐gun,

Tochigi, 329‐0114 Japan.

Tel: þ81 280 57 0718; fax: þ81 280 57 0712; email: [email protected]

Received 17 December 2012; revised 5 February 2013; accepted 14 February 2013.

List of Abbreviations: A. baumannii, Acinetobacter baumannii; ATCC, American Type Culture Collection; CAZ, ceftazidime; Ca‐EDTA, EDTA

calcium disodium salt dihydrate; C. freundii, Citrobacter freundii; Co‐EDTA, EDTA cobalt disodium salt tetrahydrate; Cu‐EDTA, EDTA copper disodium

salt tetrahydrate; DDST, double‐disk synergy test; E. cloacae, Enterobacter cloacae; E. coli, Escherichia coli; Fe‐EDTA, EDTA iron sodium salt trihydrate;

IPM, imipenem; K., Klebsiella; MBL, metallo‐b‐lactamase; Mg‐EDTA, EDTAmagnesium disodium salt tetrahydrate; MgEDTA–CAZ, double‐disk synergy

tests using 10 mg EDTA magnesium disodium salt tetrahydrate with ceftazidime; MgEDTA–IPM, double‐disk synergy tests using 10 mg EDTA

magnesium disodium salt tetrahydrate with imipenem; Mn‐EDTA, EDTA manganese disodium salt trihydrate; NDM‐1, New Delhi metallo‐b‐lactamase‐

1; P., Pseudomonas; SMA, sodium mercaptoacetic acid; Zn‐EDTA, EDTA zinc disodium salt tetrahydrate.

Microbiol Immunol 2013; 57: 346–352doi: 10.1111/1348-0421.12042

346 © 2013 The Societies and Wiley Publishing Asia Pty Ltd

USA, Canada, Belgium, France, Australia and others(4–9). NDM‐1 positive bacteria have been found not onlyin clinical specimens, but also in drinking water andseepage in New Delhi (10). The first case of a NDM‐1producing E. coli (NDM‐1 Dok01) infection in Japan wasreported in 2010 (11). This organism was isolated fromthe blood culture of a patient who had been hospitalizedin India. The complete sequence of the NDM‐1‐bearingplasmid was also reported (GenBank accession numberAP012208) (12).Rapid detection of MBL‐producing strains, including

NDM‐1 producers, is necessary to prevent their dis-semination and associated nosocomial infections.Researchers have developed several phenotypic methodsto detect MBL production. These tests include DDSTsusing 2‐mercaptoacetic acid or EDTA, combined disktests with dipicolinic acid or EDTA, Etest MBL(BioMérieux, Durham, NC, USA) and the modifiedHodge test (13–17). The DDSTs using SMAwith CAZ orIPM disks are simple methods and commonly used inclinical laboratories in Japan. However, the growth‐inhibitory zone does not enhance sufficiently when theDDST using SMA with CAZ is performed for NDM‐1Dok01. In addition, with IPM disks, the results areequivocal because the enhancement of the zone ofinhibition is only 4 mm, which researchers haveinterpreted as negative (11). Aoki et al. reported thatcalcium disodium EDTA, a metal–EDTA complex thatincorporates calcium ions into EDTA, is an effectiveinhibitor of MBL (18). The purpose of this study was toevaluate the efficacy of detection of MBL, includingNDM‐1, of DDSTs using seven kinds of metal‐EDTAcomplexes.

MATERIALS AND METHODS

Bacterial strains

NDM‐1 Dok01 was isolated at Dokkyo MedicalUniversity Hospital. K. pneumoniae ATCC BAA‐2146was used as a quality control strain that produces NDM‐1.Strains evaluated were stock cultures of known MBL‐producing strains of 46 P. aeruginosa, 7 A. baumannii, 5P. putida, 3 E. coli, 2 Achromobacter xylosoxidans, 2 E. cloacae,2 Serratia marcescens, 2 K. pneumoniae, 1 K. oxytoca, 1Citrobacter freundii, 1 Pseudomonas spp., and 1 Acinetobacterspp. Non‐MBL producing strains of 7 K. pneumoniae,1 K. oxytoca, 6 E. coli, 3 C. freundii, 4 P. aeruginosa, and 4A. baumannii were also evaluated.

Antimicrobial susceptibility testing

Minimum inhibitory concentrations were determined bythe broth microdilution method, which was performed

on Dry Plate Eiken DPD1 (Eiken Chemical, Tokyo,Japan) according to the manufacturer's instructions.

Comparison of metallo‐b‐lactamaseinhibitors

Sodium mercaptoacetic acid and seven types of metal‐EDTA complexes were used as MBL inhibitors. Metallo‐b‐lactamase SMA Eiken (SMA disk; Eiken Chemical)contains 3 mg of SMA. Ca‐EDTA, Mg‐EDTA, Co‐EDTA,Cu‐EDTA, Mn‐EDTA, Fe‐EDTA and Zn‐EDTA werepurchased from Dojindo Laboratories (Dojindo Labora-tories, Kumamoto, Japan). These seven metal‐EDTAcomplexes were dissolved in water at concentrations thatprovided maximum solubility. The concentrations ofeach metal‐EDTA complex were as follows: 480 mg/mLCa‐EDTA, 480 mg/mL Mg‐EDTA, 480 mg/mL Co‐EDTA, 600 mg/mL Cu‐EDTA, 400 mg/mL Mn‐EDTA,80 mg/mL Fe‐EDTA and 480 mg/mL Zn‐EDTA.

Phenotypic detection of metallo‐b‐lactamase‐producing strain

The DDSTs were performed as described previously (13,19). A 0.5 McFarland bacterial suspensionwas inoculatedon a Mueller Hinton agar plate (Eiken Chemical).Antimicrobial disks containing either 30 µg CAZ, 10 µgIPM, 10 µg panipenem, 10 µg meropenem, 10 µgbiapenem, 10 µg doripenem or 10 µg tebipenem (EikenChemical) were used as substrates. Two disks of anantimicrobial agent were placed at least 30 mm apart on aMueller Hinton agar plate and a blank or SMAdisk placedeither 7, 10, 15, or 20 mm from the antimicrobial disks(measured from center to center). Twenty‐five micro-liters of each metal‐EDTA solution was added to ablank disk. After incubation at 35°C for 16–18 hrs, theappearance of a �5 mm enhanced zone around theantimicrobial disk near the inhibitor disk was classified aspositive (Fig. 1).

RESULTS

Using an SMA disk and seven types of metal‐EDTA disks,DDSTs were performed for seven MBL producerscarrying NDM‐1, IMP‐1, VIM‐2 and IMP‐11 and threenon‐MBL producers carrying KPC, CTX‐M‐2 andchromosomal AmpC (Table 1). CAZ or IPM diskswere placed 15 mm from the metal‐EDTA disks and theresultant enhancement of the zone of growth inhibitionevaluated. Two NDM‐1 producers showed negativeresults when SMA disks were used. However, DDSTsusing Mg‐EDTA, Ca‐EDTA, Co‐EDTA or Cu‐EDTAwerepositive for NDM‐1 producers when IPM disks wereused. Regarding IMP‐1, VIM‐2 and IMP‐11 producers,

Double‐disk synergy test for MBL

© 2013 The Societies and Wiley Publishing Asia Pty Ltd 347

Mg‐EDTA and Cu‐EDTA inhibited all five MBLs in theDDSTs using CAZ. There were no false positive results forthe three non‐MBL producers. Because P. aeruginosa 7117was positive only when Mg‐EDTA and IPM were used,Mg‐EDTA was selected for further studies.

First, the appropriate concentration of Mg‐EDTA fordetecting MBLwhen a Mg‐EDTA disk was placed 15 mmfrom an IPM disk was evaluated. A. baumannii 7170carrying blaIMP‐1 was negative when 8 mg Mg‐EDTAdisks were used with IPM disks and positive when 10 mg

Fig. 1. DDST using inhibitor (metal‐EDTA complexes or SMA) disks with antimicrobial disks 15 mm (center to center) apart. NDM‐1

Dok01 was used as a test strain. When enhancement of growth inhibition (d ¼ a � b) is more than 5 mm, the test is judged positive.

Table 1. DDST results using SMA or seven metal EDTAs with CAZ or IPM for seven MBL producers and three non‐MBL producers

Strain

Type of

b‐lactamase

Anti-

microbial

disk

DDST result

SMA

(3 mg)

Mg‐EDTA

(12 mg)

Ca‐EDTA

(12 mg)

Co‐EDTA

(12 mg)

Mn‐EDTA

(10 mg)

Cu‐EDTA

(15 mg)

Fe‐EDTA

(2 mg)

Zn‐EDTA

(12 mg)

MIC

(µg/mL)

E. coli Dok01 NDM‐1 IPM � þ þ þ � þ � � 8

CAZ � � � � � � � � >64

K. pneumoniae ATCC

BAA‐2146

NDM‐1 IPM � þ þ þ þ þ � � 32

CAZ � � � � � � � � >64

K. pneumoniae 4635 IMP‐1 IPM þ þ � � � þ � � 2

CAZ þ þ þ þ þ þ þ � >64

P. aeruginosa 7102 VIM‐2 IPM � þ þ þ þ þ þ � >32


P. aeruginosa 7103 VIM‐2 IPM � þ þ þ þ þ þ � >32


P. aeruginosa 7116 IMP‐11 IPM � � � � � � � � >32

CAZ þ þ � � � þ � � >64

P. aeruginosa 7117 IMP‐11 IPM � þ � � � � � � >32

CAZ þ þ þ � þ þ � � >64

K. pneumoniae ATCC

BAA‐1705

KPC IPM � � � � � � � � 16

CAZ � � � � � � � � 64

E. coli 6663 CTX‐M‐2 IPM � � � � � � � � �1

CAZ � � � � � � � � �1

P. aeruginosa 7149 Chromosomal

AmpC

IPM � � � � � � � � 2

CAZ � � � � � � � � >64

þ, enhancement of growth inhibition �5 mm; �, enhancement of growth inhibition <5 mm.


M. Fujisaki et al.

Mg‐EDTA disks were used with IPM disks. Therefore, adisk content of 10 mg Mg‐EDTA was selected for thesubsequent experiments.Next, the optimal distance between antimicrobial and

Mg‐EDTA disks was evaluated. K pneumoniae ATCC BAA‐2146 was used as a positive control strain for NDM‐1producers, and A. baumannii 7170 as a weak positivecontrol strain for IMP‐1 producers. Two strains produc-ing either NDM‐1 or IMP‐1 were positive when 10 mgMg‐EDTA disks were placed 15 mm away from the IPMdisks; however, they were negative when the Mg‐EDTA

disks were placed 20 mm away from the IPM disks.Therefore, it was decided that the Mg‐EDTA and IPMdisk would be placed 15 mm apart for the subsequentexperiments.

To evaluate the efficiency of Mg‐EDTA disks, 75 stockcultures carrying the various MBL genes and 25 stockcultures carrying other b‐lactamase genes were tested byDDSTs using 10 mg MgEDTA–IPM or MgEDTA–CAZ.Positive results for MgEDTA–CAZ were obtained in 69test strains (92.0%) and for MgEDTA–IPM in 58 teststrains (77.3%) (Table 2). The results for MgEDTA–IPM

Table 2. DDST results using 10 mg Mg‐EDTA with CAZ or IPM on 75 MBL producers and 25 non‐MBL producers

Strain Type of b‐lactamase No. tested

No. of isolates with positive result evaluated by DDST

10mg Mg‐EDTA

CAZ IPM Overall

MBL producer

P. aeruginosa IMP‐1 27 27 24 27

Chromosomal AmpC þ IMP‐1 5 5 5 5

IMP‐11 4 4 2 4

VIM‐2 10 8 8 8

P. putida IMP‐1 5 5 5 5

Pseudomonas spp. IMP‐1 1 1 0 1

A. baumannii IMP‐1 2 2 2 2

Chromosomal AmpC þ IMP‐1 2 1 2 2

IMP‐2 3 3 3 3

Acinetobacter spp. IMP‐1 1 1 1 1

A. xylosoxidans IMP‐1 2 2 0 2

E. coli IMP‐1 3 3 1 3

NDM‐1 1 0 1 1

K. pneumoniae IMP‐1 2 2 0 2

NDM‐1 1 0 1 1

K. oxytoca IMP‐1 1 1 1 1

S. marcescens Chromosomal AmpC þ IMP‐1 2 2 2 2

E. cloacae Chromosomal AmpC þ IMP‐1 2 1 0 1

C. freundii Chromosomal AmpC þ IMP‐1 1 1 0 1

Total 75 69 58 72

Non‐MBL producer

P. aeruginosa Chromosomal AmpC 4 0 0 0

A. baumannii Chromosomal AmpC 1 0 0 0

Chromosomal AmpC þ CTX‐M 3 0 0 0

K. pneumoniae SHV‐26 1 0 0 0

CTX‐M‐2 3 0 0 0

CMY‐8 1 0 0 0

DHA‐1 þ CTX‐M‐9 1 0 0 0

KPC 1 0 0 0

E. coli CTX‐M‐2 2 0 0 0

CTX‐M‐9 3 0 0 0

CMY‐8 1 0 0 0

K. oxytoca DHA‐1 þ RbiA 1 0 0 0

C. freundii Chromosomal AmpC 2 0 0 0

Chromosomal AmpC þ CTX‐M 1 0 0 0

Total 25 0 0 0

Sensitivity 92.0% 77.3% 96.0%

Specificity 100% 100% 100%



and MgEDTA–CAZ were discordant for 16 MBLproducers (Table 3). There were no false positive resultsfor MgEDTA–IPM and MgEDTA–CAZ. Two P. aeruginosacarrying VIM‐2 and one E. cloacae carrying IMP‐1 hadnegative results with MgEDTA–IPM and MgEDTA–CAZ(Table 4); they were also negative by the SMA diskmethod. However, two false negative P. aeruginosa becamepositive when biapenem and doripenem were used withMg‐EDTA, and one false negative E. cloacae becamepositive when panipenem and meropenem were used assubstrates.

DISCUSSION

After NDM‐1 Dok01 was reported, two NDM‐1‐producing K. pneumoniae were identified by govern-ment‐instigated surveillance in Japan. These isolates werecollected from elderly people who had not recentlytraveled abroad and had had no contact with the Indian

subcontinent. Although NDM‐1 producers from clinicalisolates are rare in Japan, accurate screening methodsto detect them are needed to prevent their furthertransmission in both hospitals and communities.

Many clinical laboratories perform confirmatory testsfor MBL production against carbapenem‐resistantstrains (20). The DDSTusing SMA is themost convenientof the phenotypic MBL detection methods. However, thegrowth‐inhibitory zone between IPM and the SMA disksis not large enough to be classified as positive with NDM‐

1 Dok01 (11). In contrast to SMA disks, DDSTs usingIPM disks and Mg‐EDTA, Ca‐EDTA, Co‐EDTA or Cu‐EDTA detected two NDM‐1 producers. In addition, theDDSTs using Mg‐EDTA had high sensitivity (96.0%) andspecificity (100%) for 75 MBL producers and 25 non‐MBL producers. Galani et al. reported that combineddisk test with CAZ and EDTA (750 µg), and DDSTs withIPM disks 10 mm away from EDTA disks have highsensitivity (97.9–100%) and specificity (91.9–96%) in

Table 3. Strains with discordant results between MgEDTA‐IPM and MgEDTA‐CAZ by DDST

Strain Type of b‐lactamase Antimicrobial disk DDST result b: zone diameter(mm) MIC (µg/mL)

E. coli 7142 IMP‐1 IPM � 19 8

CAZ þ 8 >64

E. coli 7143 IMP‐1 IPM � 22 2

CAZ þ 11 >64

K. pneumoniae 7144 IMP‐1 IPM � 21 4

CAZ þ 0 >64

E. cloacae 7172 Chromosomal AmpC þ IMP‐1 IPM � 22 2

CAZ þ 0 >64

C. freundii 7174 Chromosomal AmpC þ IMP‐1 IPM � 17 4

CAZ þ 0 >64

A. xylosoxidans 7112 IMP‐1 IPM � 16 >32

CAZ þ 0 >64

A. xylosoxidans 7113 IMP‐1 IPM � 14 32

CAZ þ 0 >64

P. aeruginosa 7116 IMP‐11 IPM � 0 >32

CAZ þ 0 >64


CAZ þ 0 >64


CAZ þ 0 >64


CAZ þ 0 >64


CAZ þ 0 >64

Pseudomonas spp. 9995 IMP‐1 IPM � 0 64

CAZ þ 0 8

E. coli Dok01 NDM‐1 IPM þ 17 8

CAZ � 0 >64

K. pneumoniae ATCC BAA‐2146 NDM‐1 IPM þ 16 32

CAZ � 0 >64

A. baumannii 7170 Chromosomal AmpC þ IMP‐1 IPM þ 16 4

CAZ � 10 >64

þ, enhancement of growth inhibition �5 mm; �, enhancement of growth inhibition <5 mm.


M. Fujisaki et al.

Enterobacteriaceae (14). That we obtained similar sensitivi-ty and specificity demonstrates that Mg‐EDTA can beused as a MBL inhibitor.Several reports have indicated that AmpC b‐lactamase

may cause false negative results in DDSTs using SMA (20,21). Arakawa et al. also reported that some MBL‐producing gram‐negative bacilli are difficult to detect.Because they have a low level of resistance to IPM, theexpansion of the zone of inhibition is inconclusive (13).In our study, only 3 of 75 strains were false negative byboth MgEDTA–CAZ and MgEDTA–IPM; these threestrains were also false negative in DDSTs using SMA.Two false negative P. aeruginosa strains were resistant to sixcarbapenems and one false negative E. cloacae wasresistant to CAZ but susceptible to six carbapenems.Carbapenem resistance in P. aeruginosa is considered to beassociated with loss of OprD outer membrane proteinsand/or overexpression of active efflux systems incombination with strong expression of AmpC b‐lactamase (22). Furthermore, IPM induces expressionof AmpC b‐lactamase in P. aeruginosamore strongly thandoes doripenem (23). Other resistant mechanisms, suchas AmpC b‐lactamase, may cause false negative results;such additional mechanisms could mask MBL inhibitionby Mg‐EDTA. Further studies are needed to investigatethe reasons for false positives.

We found that the sensitivity of MgEDTA–CAZ ishigher than that of MgEDTA–IPM. This makes CAZpreferable to IPM as a substrate in DDSTs. However, oneIMP‐1‐producing A. baumannii and two NDM‐1‐produc-ing Enterobacteriaceae were positive when IPM was used,but negative when CAZwas used. Kim et al. have reportedthat, because these organisms have other CAZ resistantmechanisms such as ESBL and AmpC b‐lactamaseproduction, DDSTs using CAZ have difficulty detectingMBL‐producing Acinetobacter (21). Therefore, DDSTsusing Mg‐EDTA should use both IPM and CAZ disks assubstrates in order to further reduce false negative results.

False positive results reportedly also occur with MBLphenotypic methods using EDTA and IPM. It is believedthat such false positive results are attributable toincreasing membrane permeability caused by chelatingagents (24, 25) and the anti‐bacterial activity ofEDTA (19, 24, 25). DDSTs using Mg‐EDTA yielded nofalse positive results among 25 non‐MBL producers. Thedisk content of Mg‐EDTAwas 10 mg, this concentrationbeing higher than that of the EDTAwas used in previousreports. Because false positive results were confirmed forP. aeruginosa and Acinetobacter spp. by the Etest MBL andcombined disk test, DDST using Mg‐EDTA should beevaluated for specificity using non‐MBL‐producingP. aeruginosa or Acinetobacter spp.

Table 4. DDST results using Mg‐EDTA or SMA with ceftazidime or six carbapenems negative either by MgEDTA‐IPM or MgEDTA‐CAZ

Strain

Antimicrobial

disk

b: zone

diameter

(mm)

MgEDTA, 10 mg SMA, 3 mg

a: enhanced

zone (mm)

d: difference

(mm)

DDST

result

a: enhanced

zone (mm)

d: difference

(mm)

DDST

result

P. aeruginosa 7123 Ceftazidime 12 13 1 � 13 1 �Imipenem 0 3 3 � 0 0 �Panipenem 0 0 0 � 0 0 �Meropenem 0 4 4 � 0 0 �Biapenem 0 6 6 þ 0 0 �Doripenem 0 7 7 þ 1 1 �Tebipenem 0 0 0 � 0 0 �

P. aeruginosa 7124 Ceftazidime 0 0 0 � 0 0 �Imipenem 0 3 3 � 0 0 �Panipenem 0 0 0 � 0 0 �Meropenem 0 5 5 þ 2 2 �Biapenem 0 6 6 þ 0 0 �Doripenem 0 7 7 þ 3 3 �Tebipenem 0 0 0 � 0 0 �

E. cloacae 7173 Ceftazidime 0 0 0 � 0 0 �Imipenem 22 25 3 � 25 3 �Panipenem 19 25 6 þ 24 5 þMeropenem 18 23 5 þ 26 8 þBiapenem 28 28 0 � 29 1 �Doripenem 20 24 4 � 25 5 þTebipenem 20 23 3 � 24 4 �

þ, enhancement of growth inhibition (d) �5 mm; �, enhancement of growth inhibition (d) <5 mm.



In conclusion, this is the first report to evaluate severalmetal‐EDTA complexes as inhibitors of MBL. Use of Mg‐EDTA in DDSTs is the most useful phenotypic methodfor detecting MBL producers, including NDM‐1 produc-ing strains, in clinical laboratories. Because we tested onlytwo NDM‐1 producers by the Mg‐EDTA DDSTmethod,other NDM‐1 producers should be confirmed bysubsequent studies in actual clinical practice.

DISCLOSURE

M. Fujisaki and S. Sadamoto are employees of EikenChemical.

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Evaluation of the double-disk synergy test for New Delhi metallo-β-lactamase-1 and other...

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