Journal of Microbiological Methods 108 (2015) 45–48

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Evaluation of a simple phenotypic method for the detection of carbapenemase-producing Enterobacteriaceae Ryoichi Saito a,⁎, Saho Koyano b, Misato Dorin a, Yoshimi Higurashi b, Yoshiki Misawa b, Noriyuki Nagano c, Takamasa Kaneko d, Kyoji Moriya b a

Department of Microbiology and Immunology, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Tokyo, Japan Department of Infection Control and Prevention, The University of Tokyo Hospital, Tokyo, Japan Medical Microbiology Laboratory, Funabashi Municipal Medical Center, Chiba, Japan d Kanto Chemical Co., Inc., Tokyo, Japan b c

a r t i c l e

i n f o

Article history: Received 27 August 2014 Received in revised form 16 November 2014 Accepted 16 November 2014 Available online 20 November 2014

a b s t r a c t We investigated the performance of a phenotypic test, the Carbapenemase Detection Set (MAST-CDS), for the identification of carbapenemase-producing Enterobacteriaceae. Our results indicated that MAST-CDS is rapid, easily performed, simple to interpret, and highly sensitive for the identification of carbapenemase producers, particularly imipenemase producers. © 2014 Elsevier B.V. All rights reserved.

Keywords: Carbapenemase Enterobacteriaceae Imipenemase Klebsiella pneumoniae carbapenemase New Delhi metallo-β-lactamase Oxacillinase-48-like

1. Introduction Resistance to carbapenems in Gram-negative bacteria, including Enterobacteriaceae, is an increasingly serious problem globally, since the clinical utility of these antimicrobials is compromised (Hawkey and Jones, 2009). This resistance is facilitated by carbapenemases, such as Amber class A (Klebsiella pneumoniae carbapenemase [KPC] enzymes), class B (metallo β-lactamases [MBLs]; imipenemase [IMP], Verona integron-encoded MBL [VIM], and New Delhi MBL [NDM] enzymes), and class D (oxacillinase [OXA]-48-like enzymes) β-lactamases. The genes encoding these enzymes are mainly carried on transferable plasmids that also harbor resistance genes against various antibiotics, including quinolone and aminoglycosides (Koyano et al., 2013). Therefore, it is crucial for infection control procedures and epidemiological

⁎ Corresponding author at: Department of Microbiology and Immunology, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Tel./fax: +81 3 5803 5368. E-mail addresses: [email protected] (R. Saito), [email protected] (S. Koyano), [email protected] (M. Dorin), [email protected] (Y. Higurashi), [email protected] (Y. Misawa), [email protected] (N. Nagano), [email protected] (T. Kaneko), [email protected] (K. Moriya).

http://dx.doi.org/10.1016/j.mimet.2014.11.008 0167-7012/© 2014 Elsevier B.V. All rights reserved.

investigations that carbapenemase-producing Enterobacteriaceae be identified accurately. To date, some phenotypic confirmation methods have been described for the identification of carbapenemase-producing Enterobacteriaceae, such as Enterobacter spp., Escherichia spp., Klebsiella spp., and Serratia spp. (Birgy et al., 2012; Tsakris et al., 2010; van Dijk et al., 2014). Recently, the Carbapenemase Detection Set (MAST-CDS; Mast Group, Merseyside, UK), which is based on the hydrolysis of carbapenemases with inhibitors against MBL, KPC, and AmpC enzymatic activity, has become commercially available. Although its performance has been evaluated by Doyle et al. (2012), this test has been shown to be only 40% sensitive for IMP producers. Moreover, IMP producers have been reported more frequently in southern Europe and Asia and are the predominant MBLs in Japan (Ito et al., 1995; Nordmann et al., 2011); the additional data derived from studies of a large number of IMP producers have increased the usefulness of this test. We here investigated the performance of the commercially available phenotypic test MAST-CDS for the identification and presumptive characterization of carbapenemase-producing Enterobacteriaceae, including a large number of IMP producers, and compared its performance with that of other phenotypic confirmation tests, viz., the modified Hodge test (MHT) for carbapenemases, the combination test using dipicolinic

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R. Saito et al. / Journal of Microbiological Methods 108 (2015) 45–48

acid (CT-DPA) for MBLs, and the combination test using clavulanic acid (CT-CVA) for extended-spectrum β-lactamases (ESBLs). 2. Materials and methods 2.1. Bacterial strains A total of 38 carbapenemase-producing Enterobacteriaceae, including 2 reference strains, viz., K. pneumoniae ATCC BAA-1705 and K. pneumoniae ATCC BAA-2146, which had been already confirmed to harbor the genotypes blaKPC, blaIMP, blaVIM, blaNDM, and blaOXA-48-like by previously published methods (Dallenne et al., 2010; Poirel et al., 2004; Saito et al., 2014), was used in this study. The test strains included 2 KPC producers (2 K. pneumoniae), 29 IMP producers (6 Citrobacter freundii, 20 Enterobacter cloacae, 1 Klebsiella oxytoca and 2 Serratia marcescens), 4 NDM producers (2 Escherichia coli and 2 K. pneumoniae), and 3 OXA-48-like producers (1 E. coli and 2 K. pneumoniae); these are listed in Table 1. Furthermore, 36 carbapenemase-non-producing Enterobacteriaceae (9 ESBL producers, 20 ampicillinase C [AmpC] producers, and 7 β-lactamases non-producers), which had been confirmed phenotypically using the AmpC and ESBL Detection Set (Mast Group), were also included (Table 1).

and aminophenylboronic acid 600 μg as a KPC inhibitor), and disk D (meropenem 10 μg and cloxacillin 750 μg as an AmpC inhibitor) were placed on Mueller-Hinton agar plates on which the test strains had been inoculated. Plates were incubated at 37 °C for 24 h, after which the zone difference, ≥ 5 mm for disk B and ≥ 4 mm for disk C, as compared to disk A, was interpreted as indicating MBL- and KPCproducing bacteria, respectively. Moreover, a zone difference of both ≥4 mm for disk C and ≥5 mm for disk D, as compared to disk A, was interpreted as indicating AmpC producers with porin loss. MAST-CDS is not designed to identify OXA-48-like producers. MHT was performed for the confirmation of carbapenemase producers as recommended in the CLSI guidelines (CLSI, 2011). CT-DPA was performed with broth microdilution methods, using imipenem and dipicolinic acid (400 μg/mL), an MBL inhibitor; a ≥8-fold decrease in the MIC caused by the presence of the inhibitor was taken as indicating MBL-positive bacteria. For confirmation of ESBL producers, CT-CVA was performed as recommended in the CLSI guidelines (CLSI, 2011). The AmpC and ESBL Detection Set (Mast Group) was also implemented according to manufacturer's instructions to identify ESBL and AmpC producers phenotypically.

3. Results 2.2. Antimicrobial susceptibility testing The minimum inhibitory concentration (MIC) of imipenem, meropenem, ceftazidime, and cefotaxime was determined by broth microdilution methods, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2011). 2.3. Phenotypic β-lactamase testing MAST-CDS was performed according to manufacturer's instructions. Briefly, disk A (meropenem 10 μg), disk B (meropenem 10 μg and dipicolinic acid 1000 μg as a MBL inhibitor), disk C (meropenem 10 μg

Among the carbapenemase producers investigated in this study, the MIC ranges of imipenem and meropenem of IMP producers were extremely broad (from 2 to N 8 mg/L; Table 1). Moreover, although OXA-48-like producers showed low-level resistance to imipenem and meropenem (both 2 mg/L), KPC and NDM producers demonstrated high-level resistance to meropenem (MIC N8 mg/L) in particular. Of the 36 carbapenemase non-producers, 56% (5/9) of ESBL producers, 15% (3/20) of AmpC producers, and 86% (6/7) of carbapenemase, ESBL, and AmpC non-producers were not susceptible to imipenem (MICs of 2–4 mg/L), while all these strains were susceptible to meropenem (Table 1).

Table 1 Distribution of carbapenem MICs for carbapenemase-producing and non-producing Enterobacteriaceae. β-lactamase type (no.)a

Species

Carbapenemase

MIC (mg/L) and no. of isolates Imipenem ≤1

KPC (2) IMP (29)

NDM (4) OXA-48 (3) ESBL (9)

AmpC (20)

Carbapenemase, ESBL and AmpC negative (7)

a

Klebsiella pneumoniae Citrobacter freundii Enterobacter cloacae Klebsiella oxytoca Serratia marcescens Serratia marcescens Escherichia coli Klebsiella pneumoniae Escherichia coli Klebsiella pneumoniae Escherichia coli Klebsiella pneumoniae Proteus mirabilis Citrobacter freundii Escherichia coli Klebsiella pneumoniae Morganella morganii Providencia stuartii Serratia marcescens Morganella morganii Proteus mirabilis Proteus penneri Proteus vulgaris

KPC-2 IMP-1 IMP-1 IMP-1 IMP-1 IMP-11 NDM-1 NDM-1 OXA-48-like OXA-48-like NDb ND ND ND ND ND ND ND ND ND ND ND ND

Meropenem

2

4

8

N8

1 8 1

2 8

2 2

2 1 2

≤1

2

4

8

N8

4

2 7 1

1 5

2 3 4

1

1

1

1 1 1

1 1

2 2

1 2

1 2

3 1 4

1

5 10 1 2 1 1 1 3 1 1

1

3 1 5 5 10 1 2 1 1 1 4 1 1

KPC, Klebsiella pneumoniae carbapenemase; IMP, imipenemase; NDM, New Delhi metallo β-lactamase; OXA, oxacillinase; ESBL, extended-spectrum β-lactamases; AmpC, ampicillinase C. b ND, not detected.

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Table 2 Performance of four phenotypic tests for carbapenemase producing and non-producing Enterobacteriaceae. Test

Detection of β-lactamase

No. of positive isolates/no. of total isolatesa Carbapenemase producer

MAST-CDS MHT CT-DPA CT-CVA a b c

KPC, MBL KPC, MBL, OXA-48-like MBL ESBL

Sensitivityc (%)

Specificity (%)

91 87 88 91

100 100 100 100

Carbapenemase non-producer

KPC

IMP

NDM

OXA-48-like

ESBL

Otherb

2/2 2/2 0/2 1/2

26/29 28/29 25/29 0/29

4/4 0/4 4/4 0/4

0/3 3/3 0/3 0/3

0/9 0/9 0/9 9/9

0/27 0/27 0/27 0/27

KPC, Klebsiella pneumoniae carbapenemase; IMP, imipenemase; NDM, New Delhi metallo β-lactamase; OXA, oxacillinase; ESBL, extended-spectrum β-lactamases. Carbapenemase and ESBL-nonproducing Enterobacteriaceae. Sensitivity of carbapenemases targeted by each test.

The performance results of the 4 phenotypic tests investigated are shown in Table 2. MAST-CDS, MHT, and CT-CVA could detect KPC producers with a sensitivity of 100% (2/2), 100% (2/2), and 50% (1/2), respectively. MAST-CDS, MHT, and CT-DPA could detect MBL producers with a sensitivity of 91% (30/33), 85% (28/33), and 88% (29/33), respectively. Some IMP-1 producers were not identified by MAST-CDS, MHT, and CT-DPA tests, and these strains showed low-level MICs to imipenem and meropenem (MIC range: 2–4 mg/L). Upon MAST-CDS, all these strains demonstrated a 3–4-mm zone difference between disk A and disk B. Moreover, among these 3 tests, MHT did not identify 4 NDM-1producing strains. In contrast, 3 OXA-48-like producers were identified only with MHT. Of the 36 carbapenemase non-producers, 9 ESBL producers were only identified by CT-CVA, which is the CLSI-recommended phenotypic confirmatory test for these bacteria (CLSI, 2011). The sensitivity of MAST-CDS, MHT, and CT-DPA in all carbapenemase producers tested was 84% (32/38), 87% (33/38), and 74% (29/38), respectively. However, in terms of the carbapenemases targeted by each test, the sensitivity was 91% (32/35) for MAST-CDS, 87% (33/38) for MHT, and 88% (29/33) for CT-DPA (Table 2). Moreover, the sensitivity of CT-CVA for detecting ESBL producers, including 2 KPCs, was 91% (10/11). In contrast, the specificity of the 4 phenotypic tests used in this study was all 100%.

4. Discussion Currently, carbapenemases are mainly characterized using molecular methods; however, these methods cannot be performed routinely in the clinical laboratory, given the cost and workload entailed. Therefore, rapid and easy identification and presumptive characterization of carbapenemase-producing Enterobacteriaceae are required. A previous study showed that the sensitivity of MAST-CDS for KPC (98%) and NDM (100%) producers is higher than that for IMP (40%) and VIM (53%) producers (Doyle et al., 2012). In this study, the sensitivity of MAST-CDS for the identification of IMP producers (90%; 26/29) was much higher than that previously reported. Thus, our results demonstrated that the dipicolinic acid of MBL inhibitors used in MAST-CDS may have different effects on distinct types of IMP enzymes. Moreover, it is challenging to identify strains with low-level MICs (range: 2–4 mg/L) to imipenem and meropenem, suggesting that different methods, such as MHT and genetic testing, may be necessary for the accurate identification of such stains. Moreover, although MHT detects carbapenemase-producing strains, including OXA-48-like β-lactamase producers, it is known that it does not accurately identify NDM producers (Doyle et al., 2012; Nordmann et al., 2011); our results also supported this notion (Doyle et al., 2012). Furthermore, the MICs for ceftazidime and cefotaxime did not decrease in the presence of clavulanic acid in 1 of 2 KPC-2 producers (data not shown). This may be due to the low efficiency of clavulanic acid in inhibiting the KPC enzyme.

The results of our present study demonstrated that, although the sensitivity of MHT, the test recommended by the CLSI guidelines, was higher than that of MAST-CDS and CT-DPA in all carbapenemase producers, including OXA-48-like producers, MAST-CDS demonstrated the highest sensitivity among the 3 tests in strains producing specific carbapenemases (KPC, NDM, and IPM). Furthermore, although MHT could not phenotypically characterize the carbapenemases involved, we suggest that MAST-CDS is suitable for routine analysis of presumptive characterization of carbapenemase-producing Enterobacteriaceae. We anticipate the possibility that a strategy employing screening media for carbapenemase producers, such as chromogenic media (Wilkinson et al., 2012) in addition to MAST-CDS, would lead to further reduction of the time required for the identification of carbapenemaseproducing Enterobacteriaceae; further assessments over and above the routine procedure will be required when using clinical specimens. 5. Conclusion Although only few KPC, NDM-1, and OXA-48-like producers were tested in this study, our results indicated that MAST-CDS is rapid, easily performed, simple to interpret, and highly sensitive for the identification of carbapenemase producers, particularly IMP producers. Therefore, we propose that MAST-CDS be accepted as a phenotypic test for the identification and presumptive characterization of carbapenemase-producing Enterobacteriaceae, although other methods, such as genetic testing, may be required for the confirmation of MBL producers. Conflict of interest All authors declare that they have no conflicts of interest. Acknowledgments We would like to express our gratitude to Prof. Kosuke Haruki who provided NDM-1-producing Enterobacteriaceae. We would like to thank Kanto Chemical (Tokyo, Japan) for supplying the Carbapenemase Detection Set as well as the AmpC and ESBL Detection Set. This work was presented in part at the 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2014. References Birgy, A., Bidet, P., Genel, N., Doit, C., Decre, D., Arlet, G., Bingen, E., 2012. Phenotypic screening of carbapenemases and associated β-lactamases in carbapenem-resistant Enterobacteriaceae. J. Clin. Microbiol. 50, 1295–1302. CLSI, 2011. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement M100-S21. CLSI, Wayne, PA, USA. Dallenne, C., Da Costa, A., Decre, D., Favier, C., Arlet, G., 2010. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother. 65, 490–495. Doyle, D., Peirano, G., Lascols, C., Lloyd, T., Church, D.L., Pitout, J.D.D., 2012. Laboratory detection of Enterobacteriaceae that produce carbapenemases. J. Clin. Microbiol. 50, 3877–3880.

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