Suitability of Carbapenem Inactivation Method (CIM) for Detection of IMP Metallo-β-Lactamase-Producing Enterobacteriaceae

Abstract

Carbapenem-resistant Enterobacteriaceae (CRE) strains have become globally distributed in the past decade, resulting in concern over the control of hospital infections and antimicrobial therapies (1, 2). The majority of CRE isolates are carbapenemase-producing Enterobacteriaceae (CPE) strains, so early detection of CPE strains is essential for providing optimal antimicrobial therapies and preventing horizontal transmission. In 2015, van der Zwaluw et al. reported the carbapenem inactivation method (CIM), a new method for detecting carbapenemase producers with high sensitivity and specificity (3). However, it was unclear whether CIM can identify IMP producers with a low carbapenem MIC or non-CPE strains that are highly carbapenem resistant. In this study, we evaluated whether CIM can identify CPE strains independently of their carbapenem MICs.Were used 233 CPE and 51 non-CPE strains isolated in general hospitals across Japan from 2012 to 2016 and stocked in our laboratory. The strains were collected from blood, sputum, wounds, urine, and feces. Their antibiotic susceptibilities were determined by the agar dilution method in accordance with the recommendations of the CLSI (4). The CPE strains included 191 IMP, 20 KPC, 17 NDM, and 5 OXA-48-like (OXA-48 and OXA-244) producers. The non-CPE strains included 31 extended-spectrum beta-lactamase (CTX-M, SHV, and TEM) producers and 5 plasmid-mediated AmpC ␤-lactamase (DHA, CMY, and CFE) producers. They were identified by DNA sequence amplification as previously described (5-10). Of the non-CPE strains, 13 highly carbapenem-resistant strains were identified as producing cephalosporinases and lacking porin function by SDS-PAGE, DNA sequencing, and quantitative reverse transcription-PCR (11).The CIM was conducted as previously described (3). The isolates were cultured on Mueller-Hinton agar (MHA) plates. A full 10-l inoculation loop of each strain was suspended in 400 l of sterile distilled water, and a 10-g meropenem (MEM) susceptibilitytesting disk (E-DF85; EIKEN) was immersed in the solution. After incubation at 35°C for 2 h, the disk was removed and placed on an MHA plate inoculated with a 0.5 McFarland standard of Escherichia coli strain ATCC 25922 with a sterile cotton swab. Finally, the plate was incubated overnight at 35°C and the inhibition zone around each disk was measured. Inhibition circles Ͻ10 mm in diameter were judged to indicate CIM positivity.The CIM showed a sensitivity of 100% (233/233) for CPE strains and a specificity of 96.1% (49/51) for non-CPE strains (Table 1). The MICs of MEM for the 191 IMP producers ranged from 0.125 to 32 g/ml (MIC 50 ϭ 0.5 g/ml, MIC 90 ϭ 2 g/ml), and those of imipenem (IPM) ranged from 0.06 to 8 g/ml (MIC 50 ϭ 0.125 g/ml, MIC 90 ϭ 0.5

High prevalence of extended-spectrum β-lactamase (blaCTX-M-15) and New Delhi metallo-β-lactamase-1 (NDM-1) genes among high-level carbapenem resistance Klebsiella pneumonia: an alarm for our health system

“…Using PCR, 40 (74%) of these isolates were found to contain a carbapenemaseencoding gene. These results of CIM are consistent with other findings stated by some other researches [14,[15][16][17][18][19]. With respect to our research, CIM for the first time in Iran was found to be very efficient and low-cost in the recognition of carbapenemaseproducing K. pneumoniae isolates.…”

“…MIC results for five antibiotics Cp, IMP, MP, CAZ, and CRO showed a high level of resistance (MIC≥4). The phenotypic confirmatory test showed that among the 54 isolates, 54 (100%) were ESBL producers (14). The PCR test of the ESBLproducing isolates showed that TEM 24 (44.4%) isolates were positive for the blaTEM gene, 40 (80.1%) positive for the blaCTX-M groups, and 30 (55.6%) isolates had the blaSHV gene.…”

“…BlaNDM1, blaOXA-48 and blaKPC were identified in carbapenem-resistant isolates, 22 (%40.7), 10 (%18.5), and 7 (%12.9) respectively. [14]. In this situation, with increasing high-level resistant isolates, early detection of CPK strains is critical.…”

“…Finally, incubation of the plate was done at 35°C for 24 h. The results of the test were surveyed after overnight incubation: The inhibition zone in plates around each disk was measured. Plates with inhibition circles <10 mm in diameter were considered to indicate CIM positivity [13][14][15].…”

Evaluation of the carbapenem inactivation method (CIM) for detecting carbapenemase activity in enterobacteria

“…Recently, several publications on the CIM assay have been published, reinforcing the usefulness and improvements of this phenotypic method [40][41][42]. In order to improve the…”