1

Detection of IMP metallo-β-lactamase in carbapenem-non-susceptible 1

Enterobacteriaceae and glucose non-fermenting Gram-negative rods by 2

immunochromatography assay 3

4

Shigeyuki Notake1, 2, Mari Matsuda1, Kiyoko Tamai2, Hideji Yanagisawa2, 5

Keiichi Hiramatsu1 and Ken Kikuchi1* 6

7

Department of Infection Control Science, Faculty of Medicine, Juntendo University, 8

2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan1 and Miroku Medical Laboratory Inc., 9

659-2 Innai, Saku, Nagano 384-2201, Japan2. 10

11

Running title: Detection of IMP by immunochromatography 12

13

*Corresponding author. Mailing address: Ken Kikuchi, M.D., Ph.D. 14

Department of Infection Control Science, Faculty of Medicine, Juntendo University, 15

2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 16

17

Phone: 81-3-3813-3111 ext. 3822 18

Fax: 81-3-5684-7830 19

E-mail: kikuti@juntendo.ac.jp20

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00234-13 JCM Accepts, published online ahead of print on 27 March 2013

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ABSTRACT 21

Metallo-β-lactamases (MBLs) are transmissible carbapenemases of increasing 22

prevalence in Gram-negative bacteria among healthcare facilities worldwide. Further 23

spread of these carbapenem-resistant bacteria relies on clinical microbiological 24

laboratories correctly identifying and classifying the MBLs. In this study, we evaluated a 25

simple and rapid detection method of IMP, the most prevalent metallo-β-lactamase 26

(MBL) in Japan, we evaluated a simple and rapid detection method of IMP, the most 27

prevalent metallo-β-lactamase (MBL) in Japan, using an immunochromatography (IC) 28

assay for 181 carbapenem-non-susceptible (non-susceptible to imipenem or meropenem: 29

CNS) strains comprising 74 IMP-producing and 33 IMP-non-producing strains of 30

glucose non-fermenting Gram-negative rods (NFGNR), as well as 64 IMP-producing and 31

10 IMP-non-producing Enterobacteriaceae strains. The IC assay results were compared 32

to those from the double-disc synergy test (DDST), the MBL Etest, and the modified 33

Hodge test (MHT, only for Enterobacteriaceae). IMP type was confirmed by specific 34

PCR and direct sequencing. IC assay could detect all the IMP-type MBL including IMP-1, 35

2, 6, 7, 10, 11, 19, 20, 22, and 40-42 (new type) with 100% specificity and sensitivity 36

against all strains tested. Although sensitivity and specificity of the DDST and MHT was 37

equivalent to that for the IC assay, the positive MBL Etest was only 87% for NFGNR and 38

31% for Enterobacteriaceae due to the low MIC of imipenem causing an indeterminate 39

evaluation. These results indicated that the IC assay could be a useful alternative to PCR 40

for IMP-MBL detection screening. 41

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INTRODUCTION 43

The recent worldwide emergence and dissemination of carbapenemase-producing 44

Gram-negative rods (GNR) that are resistant to carbapenems is a significant concern with 45

respect to patient care and infection control strategies (1). The transmissible 46

carbapenemases are divided into three different classes: class A (serine carbapenemases, 47

such as KPC), class B (metallo-β-lactamase: MBL, such as IMP, VIM, NDM), and class 48

D (OXA carbapenemases, such as OXA-23, OXA-48) (1, 2). Rapid and adequate 49

detection of carbapenemases is very important for appropriate antimicrobial 50

chemotherapies and infection control measures. Various phenotypic confirmation tests 51

for detecting carbapenemases have been performed including inhibition tests of 52

carbapenemase activity, the modified Hodge test (MHT), and detection of carbapenem 53

hydrolysis (1-8). However, there are no complete assays available to confirm and specify 54

carbapenemases correctly because carbapenemase-producing bacteria, notably 55

Enterobacteriaceae, show variable carbapenem MIC distribution (even under 56

breakpoint) and sometimes have carbapenemase-independent mechanisms, such as 57

reduced permeability by porin alternations, efflux pumping, and hyperproduction of class 58

C β-lactamases (e.g. AmpC) or extended-spectrum β-lactamases (ESBLs) that operate 59

with or without carbapenemase activity (1-4). Moreover, phenotypic assays cannot 60

specify types within each class of carbapenemases, such as IMP, VIM, NDM, SIM, and 61

GIM in MBL (1-4). Therefore, molecular confirmation of carbapenemases is 62

recommended for suspected carbapenemase-producing strains (1-4). However, although 63

molecular detection methods such as PCR and sequencing of carbapenemase genes are 64

reliable for confirmation of carbapenemases, it is difficult to perform such tests in routine 65

clinical microbiology laboratories because of the skill level required, higher cost, and 66

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requirement for special equipment (1-4). A simple and rapid alternative method is thus 67

needed to confirm carbapenemase presence in bacteria. 68

In Japan, IMP-MBL is the most prevalent transmissible carbapenemase, and 69

especially members of the IMP-1 group (9, 10), while KPC is quite rare and OXA-48 has 70

not been reported (11). The first IMP-MBL was described in Pseudomonas aeruginosa in 71

Japan (12), and is now found worldwide in glucose non-fermenting Gram-negative rods 72

(NFGNR) other than P. aeruginosa and Enterobacteriaceae (1-4, 8, 13). Recently, Kitao 73

et al. (14) developed an immunochromatography (IC) assay for the production of 74

IMP-MBL in P. aeruginosa and Acinetobacter. This assay is easy to perform, rapid (≤ 20 75

min required), requires no special equipment, and detects the 24 established IMP types. In 76

addition it shows excellent correlation with PCR results. In countries like Japan, wherein 77

IMP-MBL is the most prevalent mechanism of carbapenem resistance, this assay 78

provides a useful alternative to PCR for classifying MBLs in clinical microbiology 79

laboratories. Since it is uncertain that this system can detect IMP-MBL in 80

Enterobacteriaceae, we evaluated the usefulness of this IC assay in 81

carbapenem-non-susceptible (CNS) Enterobacteriaceae and NFGNR strains with the 82

MIC of imipenem (IPM) or meropenem (MEM) > 1 μg/ml. 83

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MATERIALS AND METHODS 85

Strains 86

A total of 181 CNS strains including Pseudomonas aeruginosa MRY 06-352 87

(producing IMP-1), and Serratia marcescens MRY 06-353 (producing IMP-19) as 88

IMP-positive controls provided by Dr. Y. Arakawa (National Infectious Disease Institute, 89

Japan) were used. These included 74 NFGNR (Pseudomonas aeruginosa, Pseudomonas 90

spp., Acinetobacter spp., and Achromobacter xylosoxidans) and 64 Enterobacteriaceae 91

(Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Klebsiella spp., 92

Providencia rettgeri, and Serratia marcescens) strains that produce IMP-MBL. An 93

additional 43 CNS, but carbapenemase-negative, strains (33 NFGNR and 10 94

Enterobacteriaceae) were also used as negative controls. The 179 strains, excluding the 95

IMP-positive controls, were collected in Miroku Medical Laboratory from 2001 to 2012. 96

Each strain was species-identified by MicroScan Breakpoint Combo Panel Type 6.23J 97

(Siemens Healthcare Diagnostics, Tarrytown, NY, USA), and all the NFGNR strains were 98

re-identified using matrix-assisted laser desorption ionization-time-of-flight mass 99

spectrometry (MALDI-TOF MS, Microflex LT, Bruker Daltonik GmbH, Leipzig, 100

Germany) with MALDI Biotyper software (version 3.0, Bruker Daltonik) (15, 16). 101

Identification of Pseudomonas spp. other than P. aeruginosa and Acinetobacter spp., was 102

confirmed by 16S rRNA gene, rpoB, or gyrB sequence (17, 18). Strains used are listed in 103

Table 1, while the clinical sources and the place and date of isolation are presented in the 104

supplement material. 105

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MIC determination 107

MICs of IPM and MEM were determined by the broth microdilution method 108

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according to the CLSI M07-A9 guideline (19) and the supplement M100-S21 (20). CNS 109

was designated as intermediate or resistant to IPM or MEM (MIC > 1 μg/ml). 110

111

Phenotypic detection of MBL 112

Double disk synergy tests (DDST) with sodium mercaptoacetate (SMA; 113

metallo-β-lactamase SMA 'Eiken'; Eiken Chemical Co., Ltd., Tokyo, Japan) were 114

performed according to the manufacturer's instructions based on the method described 115

previously (21). A McFarland 0.5 standard suspension of each test strain was inoculated 116

on Mueller-Hinton agar (Nippon Beckton-Dickinson, Fukushima, Japan). Two 117

commercial Kirby-Bauer (KB) disks (Nippon Beckton-Dickinson) containing 30 μg of 118

ceftazidime (CAZ) or 10 μg IPM were placed on the plate and an SMA disk was placed at 119

a distance of 10 mm (edge to edge). Each agar plate was incubated at 35°C overnight. The 120

presence of a synergistic inhibition zone of CAZ or IPM (≥5 mm of enlargement with the 121

SMA disk side) was interpreted as positive. The MBL Etest and MHT were performed 122

according to a previous report (22) and the CLSI guideline (20). 123

124

Determination of IMP-MBL genes 125

Screening of carbapenemase genes was carried out by PCR as described previously 126

(9, 23). Strains carrying transmissible carbapenemases other than IMP (VIM, SIM, GIM, 127

AIM, DIM, GIM, NDM, KPC, BIC, and OXA-48) were excluded from this study. If 128

blaIMP was positive by screening PCR, blaIMP types were determined using each 129

IMP-specific PCR to amplify the whole length of blaIMP, and direct sequencing was 130

performed. Primers used are listed in Table 2. PCR was performed in 50-μl reaction 131

mixtures that comprised 2.5 U of Ex Taq DNA polymerase (Takara Bio Inc., Shiga, 132

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Japan), 0.2 mM dNTP, 25 pmol of each primer, and 2 μl of DNA template. PCR 133

conditions were as follows: initial denaturation at 95°C for 10 min, followed by 30 cycles 134

of denaturation at 95°C for 30 sec, annealing at 62°C for 1 min and DNA extension at 135

72°C for 1 min, with final extension at 72°C for 10 min. PCR products were visualized 136

under UV light exposure after 1% agarose gel electrophoresis with ethidium bromide. 137

Amplicons obtained from each PCR were sequenced using M13F and M13R primers, the 138

BigDye Terminator v3.1 Cycle Sequencing Kit (ABI, Carlsbad, CA), and an ABI 139

sequence analyzer 3730XL (ABI). Each type of IMP was determined by BLAST search 140

and data based on all the blaIMP described previously 141

(http://www.lahey.org/studies/other.asp#Table1, 24). 142

143

Detection of IMP-MBL by immunochromatography 144

The IMP-MBL IC assay kit (Quick Chaser® IMP) was kindly provided by Mizuho 145

Medy Co., Ltd. (Saga, Japan), and used according to the manufacturer’s instruction, 146

based on a previous report (14). Briefly, fresh cultured colonies were dispensed into 700 147

μl of extraction reagent solution with nonionic detergent at McFarland 4.0 standard. After 148

vigorous vortexing, 3 drops of this suspension were applied onto the sample area of the 149

IC assay test plate. These plates were incubated for 15 min at room temperature and 150

results were interpreted visually (14). 151

152

Statistical analysis 153

Receiver operating characteristic (ROC) curves and the area under the ROC curve 154

with its standard error were used to analyze the IC assay, DDST, and MHT method results, 155

using PCR results for IMP as the gold standard. Statistically significant differences were 156

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evaluated by comparing 95% confidence intervals of the corresponding areas. The 157

statistical analysis was performed using STATA 11.0 (StataCorp LP, College Station, 158

TX). 159

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RESULTS 161

Species identification of test strains and determination of IMP-type 162

Identification of all the strains of A. xylosoxidans and P. aeruginosa was identical 163

between MicroScan and MALDI-TOF MS. Both IMP-producing and 164

IMP-non-producing Pseudomonas spp. strains were identified as P. putida or P. moteilii, 165

which belonged to the Pseudomonas putida group by MALDI-TOF MS, but were 166

distinguished from any species in this group by 16S rRNA, rpoB, gyrB sequence, 167

suggesting a new species. The seventeen IMP-producing Acinetobacter strains were 168

identified as ten A. pittii, three Acinetobacter genomospecies 13, three A. soli, and one A. 169

johsonii by rpoB sequence (18), whereas the ten non-IMP-producing Acinetobacter were 170

A. baumannii (Table 1). 171

The most prevalent IMP was IMP-1, comprising 48 of 74 (64%) NFGNR and 49 of 64 172

(76%) Enterobacteriaceae strains. In this study, three new IMP-type MBLs were 173

discovered and designated as IMP-40, IMP-41, and IMP-42. IMP-40 is closely related to 174

IMP-10, with a nucleotide alteration in the T206C of blaIMP-10 causing F69S amino acid 175

substitution according to the standard numbering scheme of MBL (25). IMP-41 is similar 176

to IMP-11, with a G145T nucleotide change causing a V49F amino acid substitution. 177

IMP-42 shows a G45R amino acid substitution of IMP-1, by the G133A nucleotide 178

change. IMP-40, IMP-41, and IMP-42 were found in two P. aeruginosa, one P. 179

aeruginosa, and two A. soli strains, respectively (Table 1 and Supplemental material). 180

One A. pittii and one E. coli strain showed a C294T nucleotide change in blaIMP-1 and a 181

C306T change in blaIMP-6, respectively, but neither base change altered the amino acid 182

sequence (Table 1). 183

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Detection of IMP in CNS-NFGNR by three different methods 185

Table 3 shows the results of three IMP detection methods in CNS-NFGNR. The IC 186

assay detected all IMP-MBL regardless of IMP type including the new IMPs, IMP-40, 187

IMP-41, and IMP-42, with 100% sensitivity and specificity (area under the curve of 188

ROC: 1.000). DDST also showed good sensitivity and specificity at 96% and 98%, 189

respectively. The areas under the curve of ROC for the IC assay and DDST were not 190

statistically different. However, there were fewer positive detections with the MBL Etest 191

(87%) compared to the IC assay and DDST because some strains showed a low MIC of 192

IPM and four tests (5%) were be evaluated as "indeterminate" due to the MIC being out of 193

range. Moreover, the MBL Etest showed negative results for three strains of 194

IMP-1-producing P. aeruginosa, one IMP-1-producing A. xylosoxidans and one 195

IMP-11-Acinetobacter genomospecies 13. The MBL Etest results were excluded for 196

statistical analysis because of data including "indeterminate". 197

198

Detection of IMP in CNS-Enterobacteriaceae by four different methods 199

Table 4 details results for the four IMP detection methods in CNS-Enterobacteriaceae. 200

In these bacteria, the IC assay also showed 100% sensitivity and specificity (area under 201

the curve of ROC: 1.000), as did DDST and MHT at 95% and 96%, respectively, and the 202

areas under the curve were not statistically different among these three detection methods. 203

In comparison, the MBL Etest was inadequate for 68% of IMP-producing 204

CNS-Enterobacteriaceae, because 59% of these strains showed low susceptibility to IMP 205

within the Etest range (≤4 μg/ml). In addition, the area under the curve of ROC could not 206

be calculated for the MBL Etest results because some data were deemed "indeterminate". 207

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DISCUSSIONS 208

Many types of carbapenemase-producing Gram-negative rods have emerged 209

worldwide with various carbapenemases types disseminated among countries and 210

continents. IMP-MBL is one of the most prevalent carbapenemases in Asia, Europe, and 211

some areas of the North and South America, and Australia (13). Currently, IMP-MBL has 212

spread through most Enterobacteriaceae including E. coli and K. pneumoniae, both of 213

which are prevalent in community-acquired and healthcare-associated infections (1-5, 214

13). Since some IMP-producing Enterobacteriaceae strains, especially, E. coli and K. 215

pneumoniae, show low-level resistance or even sensitivity to carbapenems, the CLSI 216

breakpoints of carbapenems in Enterobacteriaceae have changed since June 2010, as 217

follows: IPM (S ≤ 4 μg/ml, I = 8 μg/ml, R ≥ 16 μg/ml,) and MEM (S ≤ 4 μg/ml, I = 8 218

μg/ml, R ≥ 16 μg/ml) have moved to (S ≤ 1 μg/ml, I = 2 μg/ml, R ≥ 4 μg/ml), and (S ≤ 1 219

μg/ml, I = 2 μg/ml, R ≥ 4 μg/ml) (3, 19). Moreover, EUCAST has established 220

epidemiological cut-off values (ECOFFs) that discriminate wild-type isolates lacking any 221

carbapenem-resistance mechanisms from those possessing resistance as IPM (1-4 μg/ml) 222

and MEM (0.125-0.25 μg/ml), in addition to carbapenem clinical breakpoints (2). If a 223

strain of Enterobacteriaceae for which carbapenem MICs are below ECOFFs is detected 224

and becomes prevalent, clinical microbiology laboratory staff should consider 225

surveillance of carbapenemase-producing strains (2). Such surveillance by routine 226

clinical microbiological laboratories ideally requires a rapid, reliable, inexpensive, and 227

simple detection method of IMP. The IC assay evaluated in this study showed excellent 228

sensitivity (100%) and specificity (100%) across MIC ranges (area under the curve of 229

ROC: 1.000). Current phenotypic detection of MBL in clinical laboratories uses testing 230

by DDST or MHT, and the IC assay results in this study are statistically comparable with 231

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those acquired using these two tests by ROC analysis. However, both DDST and MHT 232

require overnight culture (> 16 h), compared with only 20 min for the IC assay. The 233

DDST is also not recommended by the Dutch Working Party on the Detection of Highly 234

Resistant Microorganisms because the sensitivity depends on the optimal distance 235

between the disks, and this cannot be predicted (4). In our results, the MBL Etest was 236

inappropriate for detecting IMP, especially in Enterobacteriaceae, mainly because of the 237

low MIC of IPM. These results confirm similar studies previously described (26-28). 238

Moreover, the MBL Etest had five false-negative results involving four IMP-1 and one 239

IMP-11-producing strains. In addition, Laraki (29) described that even 10 mM EDTA 240

could not inhibit the enzyme activity of IMP-1 isolated from one Japanese strain. Poor 241

inactivation of IMP by EDTA might account for such false-negative results of the MBL 242

Etest. The IC assay detected all IMP types including 3 new types as IMP-40, 41, and 42, 243

and did not react with VIM-2 in two P. aeruginosa strains, NDM-1 in one K. pneumoniae 244

isolate, OXA-23 in one A. baumannii strain and KPC-2 in K. pneumoniae ATCC 245

BAA-1705 (data not shown). Two monoclonal antibodies designated as 4C9-C/F6 and 246

4E7-C/F6 are used in this IC assay system (14). Since 4C9-C/F6 and 4E7-C/F6 recognize 247

the highly conserved amino acids 124-130 (H2 region) and 134-140 (S6 region) in IMP 248

(14, 24), and since the H2 and S6 regions of the new IMP types, IMP-40, 41, and 42, are 249

well conserved, the IC assay can detect such IMP types. Thus, the IC assay might also be 250

used for detecting emerging types of IMP that retain the original H2 and S6 regions. The 251

cost of an IC assay is also comparable to other conventional or molecular detection 252

methods of MBL at ¥1,000 per test. 253

In conclusion, the IC assay is considered an alternative and suitable method to detect 254

IMP in routine clinical microbiology testing including IMP surveillance with advantages 255

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in cost, processing time, reliability, convenience, and no requirement for special 256

equipment. In countries like Japan where IMP-MBL is highly prevalent, and in areas of 257

newly developed MBL endemics, IC assay testing would be quite useful for the early 258

detection and control of nosocomial and community spreads of bacteria with IMP-MBL. 259

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ACKNOWLEDGMENTS 261

We thank Dr. Y. Arakawa for providing the IMP-positive control strains. We also thank 262

Mr. T. Mizutani for assistance with the statistical analysis. This study was supported in 263

part by a Grant-in-Aide (S0991013) from Ministry of Education, Culture, Sport, Science, 264

and Technology, Japan (MEXT) for the Foundation of Strategic Research Projects in 265

Private Universities. 266

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