Combination of modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) for phenotypic detection of carbapenemase-producing Enterobacteriaceae

Carbapenemase-resistant Enterobacteriaceae (CRE) cause many serious infections resulting in increasing treatment cost, prolonged hospitalization, and mortality rate. Reduced expression and/or mutations of porins and the presence of carbapenemase promote Enterobacteriaceae survival under carbapenem treatments. Development of accurate methods for the detection of antimicrobial resistance is required not only for therapy but also to monitor the spread of resistant bacteria or resistance genes throughout the hospital and community. In this study, we aimed to evaluate the phenotypic methods, Modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and EDTA-CIM (eCIM) for the detection of carbapenemase-producing Enterobacteriaceae (CPE). The results showed that mCIM had a sensitivity of 100% and a specificity of 100%, whereas the MHT had a sensitivity of 84.8% and a specificity of 97.8% for the 195 CRE isolates tested (105 CPE and 90 non-CPE isolates). The sensitivity of the mCIM/eCIM to detect metallo-carbapenemases in this study was 89.3% and the specificity was 98.7% as compared to the genotypic PCR detection. These findings indicate that the mCIM combined with eCIM is useful for detecting and distinguishing different types of carbapenemase in Enterobacteriaceae.


Background
Enterobacteriaceae are Gram-negative, facultatively anaerobic, non-spore-forming rods and one of the most prevalent causes of invasive infections and nosocomial infections [1]. Infections caused by Enterobacteriaceae are often difficult to treat due to the high antimicrobial resistance among clinical isolates [2]. Carbapenems (imipenem, ertapenem, meropenem, and doripenem) are considered as "antibiotics of last-resort" in the treatment of critically ill patients with a variety of bacterial infections due to their broad spectrum among β-lactam antibiotics and relative resistance to hydrolysis by most β-lactamases [3,4]. However, carbapenem-resistant Enterobacteriaceae (CRE) have emerged and become a public health threat worldwide in the past decade [5].
While carbapenems enter bacterial cells via specific porins, the reduced expression and/or mutation of porins plays a critical role in the resistance to carbapenems [6,7]. Moreover, the presence of carbapenemase genes on conjugative plasmids for the hydrolysis of carbapenems to promote Enterobacteriaceae survival under antibiotic treatments is associated with the rapid emergence of CRE [8]. β-lactamases are categorized according to sequence homology into four molecular classes: A, B, C and D [9], whereas carbapenemases are the members of class A, B, and D β-lactamases [10]. Moreover, based on the participation of divalent cations in enzyme activation, carbapenemases are segregated into non-metallo-βlactamases (zinc-independent serine carbapenemases, classes A and D) and metallo-β-lactamases (MBLs, zincdependent, class B) [11]. Therefore, EDTA or dipicolinic acid can serve as chelators to block class B carbapenemases activity by binding zinc [12]. MBL genes such as bla NDM , bla IMP , bla VIM , and non-metallo-carbapenemase genes, bla OXA (class D) and bla KPC (class A), have been frequently reported in CRE, called carbapenemaseproducing Enterobacteriaceae (CPE) [8,[13][14][15].
The characterization of underlying mechanisms leading to carbapenem resistance of clinical isolates is not undertaken by most clinical microbiology laboratories for therapeutic decision-making; however, understanding if an isolate is CPE has significant epidemiological implications for monitoring local epidemiology and also lead to more effective treatment of infections caused by CPE (e.g. ceftazidime-avibactam or meropenem-vaborbactam, which have activity against KPC-producer) [16].
The Modified Hodge test (MHT) is the first Clinical & Laboratory Standards Institute (CLSI) recommended growth-based carbapenemase detection test in 2009 with high level of sensitivity and specificity in detecting carbapenemases [17,18]. Currently, the modified carbapenem inactivation method (mCIM) has been reported to accurately identify carbapenemases but cannot distinguish between serine and metallo-carbapenemases [19,20]. Therefore, a further modification to mCIM with the addition of EDTA (eCIM) has been endorsed in the CLSI M100-S28 supplement in 2018 to specifically identify metallo-carbapenemases [19,20]. In this study, we aimed to evaluate the phenotypic detection methods MHT, mCIM, and eCIM for detecting CPE.

Sensitivity and specificity of mCIM and eCIM for the detection of CPE
The distribution of carbapenemase genes and results of phenotypic detection of carbapenemases in our CRE isolates were shown in Table 2. The MHT, mCIM, and   eCIM tests were replicated by two independent investigators to ensure reproducibility, and the results showed full reproducibility of these tests. Importantly, two isolates (1 K. pneumoniae and 1 K. aerogenes) with a falsepositive result on MHT showed a negative result on mCIM. Moreover, 16 isolates with a false-negative result on MHT were mCIM positive ( Table 2). The sensitivity and specificity for the mCIM to detect carbapenemase were both 100% in this study. The sensitivity for the mCIM combined with eCIM to detect different classes of carbapenemases was shown in Table 3. Twenty-five metallo-carbapenemase producers showed positive results by eCIM. However, a total of 3 isolates escaped the detection of metallo-carbapenemase,   including 2 E. coli isolates having both bla NDM and bla OXA-48 , and 1 K. pneumoniae having bla IMP . One out of 77 isolates characterized to carry the serine carbapenemase genes showed positive results by eCIM ( Table  3). The sensitivity of the eCIM observed in this study was 89.3% (25/28) and the specificity was 98.7% (76/77) as compared to the genotype (Table 3). mCIM/eCIM assay is designed to simultaneously detect and distinguish the types of carbapenemase. Sfeir et al. showed that the sensitivity and specificity of eCIM was both 100%, in the presence of 5 mM EDTA [21]. Our results showed that the sensitivity and specificity for the mCIM/eCIM to detect MBLs is 89.3 and 98.7%, respectively (Table 3). K. pneumoniae isolate 456, an IMP-8 producer, showed a false-negative result by mCIM/ eCIM (Table 4). Our sequencing data identified a wildtype bla IMP-8 in isolate 456. However, isolate 456 showed low resistance to carbapenems (Table 4). Minimum inhibitory concentrations (MICs) of isolate 456 to imipenem, ertapenem, and meropenem were 0.5, 2, and ≤ 0.25, respectively (Table 4). E. coli isolates 488 and 492 containing bla OXA-48 /bla NDM-5 also showed falsenegative results by mCIM/eCIM (Table 4).

Discussion
The first CLSI recommended growth-based carbapenemase detection test was the MHT in 2009. Here, we showed that MHT had excellent sensitivity for the detection of bla OXA-48 (95.1%) and bla KPC (100%) (nonmetallo-carbapenemase genes) (Table 1). However, the MHT has some limitations, notably insensitivity for the detection of MBL enzymes [22]. Our results (Table 1) are consistent with previous studies which showed a high rate of false-negative MHT results with MBLproducers [19,23,24]. Moreover, MHT results are often difficult to interpret, and false-positive results are observed for isolates producing ESBL or AmpC βlactamase with porin loss [25].
The overall sensitivity and specificity of MHT in this study for the detection of CPE was 84.8 and 97.8%, respectively (Table 1). However, 16 isolates with a falsenegative result on MHT were mCIM positive ( Table 2). Our results showed that the sensitivity of mCIM to detect MBLs is 100% and the mCIM is more accurate compared to MHT. mCIM/eCIM assay is designed to simultaneously detect and distinguish the different types of carbapenemase. Previous studies showed that the sensitivity and specificity of eCIM was both 100%, in the presence of 5 mM EDTA [21]. However, the number of E. coli and K. pneumoniae isolates used to evaluate mCIM/eCIM assay for identifying CPE in Sfeir's report was limited [21]. In this study, 50 E. coli (32 carbapenemase producers) and 118 K. pneumoniae (64 carbapenemase producers) were enrolled to evaluate mCIM/eCIM for detecting CPE. Our results showed that the sensitivity and specificity for the mCIM/eCIM to detect MBLs is 89.3 and 98.7%, respectively (Table 3). Therefore, the inconsistency of sensitivity and specificity for the mCIM/eCIM assay to detect MBLs between our results and Sfeir's findings might be caused by the number of tested isolates. In this study, we used PCR targeting carbapenemase genes as the gold standard to evaluate the performance of phenotypic tests for identifying carbapenemase producers. This might be a limitation as new or truncated carbapenemase genes might not be identified consistently with the phenotype.
K. pneumoniae isolate 456 (bla IMP-8 positive) showed a false-negative result by mCIM/eCIM (Table 4). Although wild-type bla IMP-8 was identified in isolate 456, the expression level of bla IMP-8 in isolate 456 remains to be determined. Isolate 456 showed low resistance to carbapenems (Table 4). In addition, no additional carbapenemase genes (bla GES , bla IMI , bla SME , bla SPM , bla SIM , bla DIM , and bla GIM ) were detected in isolate 456. Therefore, it is worth investigating whether the carbapenem resistance level of bacteria is associated with the accuracy of mCIM/eCIM to detect carbapenemase. E. coli isolates 488 and 492 having bla OXA-48 /bla NDM-5 showed false-negative results for MBL detection by mCIM/eCIM (Table 4). These results suggest the expression of both An isolate is positive for metallo-carbapenemase production when the eCIM zone size increases by ≥5 mm compared to the zone size observed for the mCIM and is considered negative for a metallo-carbapenemase if the increase in zone size is < 4 mm IPM imipenem, ETP ertapenem, MEM meropenem carbapenemase genes can cause the misidentification of MBLs by mCIM/eCIM. E. coli isolate 514, which carried only the serine carbapenemase genes (bla OXA-48 ), showed false-positive results by eCIM based on the PCR targeting carbapenemase genes as the gold standard in this study (Table 4). However, we could not rule out the possibility of a novel carbapenemase gene in isolate 514, and therefore, an entire genome analysis of isolate 514 is worth investigating in the future.

Conclusion
Understanding the mechanism(s) causing carbapenem resistance of Enterobacteriaceae has important clinical implications and results in different prevention measurements and individualized antibiotic therapy. In this study, our results indicate that the phenotypic detection, mCIM combined with eCIM, showed high sensitivity and specificity to detect carbapenemase-producing Enterobacteriaceae, compared with MHT.

Identification of carbapenem-resistant Enterobacteriaceae isolates
Enterobacteriaceae isolates were recovered in En Chu Kong hospital, 2011 to 2019. These isolates were identified in the clinical laboratory by colony morphology, Gram stain, biochemical tests, and the Vitek 2 system (bioMérieux, Marcy l′Etoile, France) according to the manufacturer's recommendations. Susceptibility to thirdgeneration cephalosporins (ceftazidime or ceftriaxone, 30 μg/disc, BD BBL™ Sensi-Disc™, Sparks, MD, USA) for Enterobacteriaceae isolates were determined by the disk diffusion method on Mueller-Hinton (MH) agar plates (Bio-Rad, Marne la Coquette, France) based on the CLSI guidelines (M100-S30) [26]. Third-generation cephalosporin -resistant isolates were further tested for their susceptibility to carbapenems, including imipenem, ertapenem, meropenem, and doripenem (10 μg/disc, BD BBL™, USA). MICs to imipenem, ertapenem, and meropenem were further determined by Vitek 2 using the AST-N322 card according to the manufacturer's instructions. A total of 419 CRE isolates were identified and stored at − 80°C in tryptic soy broth (TSB) containing 20% glycerol (v/v) until used.

Carbapenemase gene detection
Bacterial genomic DNA was isolated from bacteria grown overnight at 37°C in 3 mL LB broth. Bacterial culture was centrifuged for 1 min at 12,000 rpm, and the supernatant was removed. Crude DNA extracts were obtained by suspending the pellet in 300 μL distilled water and boiling at 95°C for 10 min, followed by centrifugation at 12,000 rpm for 5 min. The supernatant containing DNA was transferred to a new eppendorf tube, and the DNA samples were stored at 4°C until testing. PCR targeting carbapenemase genes was used as the standard to assess the performance of phenotypic tests. Therefore, PCR amplification for the detection of βlactamase genes (bla KPC, bla NDM, bla IMP, bla VIM, bla OXA-48, bla GES , bla IMI , bla SME , bla SPM , bla SIM , bla DIM , and bla GIM ) was carried out on a iCycler iQ5 real time PCR system (Bio-Rad, USA) with the HotStar PCR SuperMix (Gene-DireX, USA). Primers and PCR procedures used in βlactamase genes detection were described in previous studies [15,[27][28][29]. The PCR products were analyzed by electrophoresis with 2% agarose gels in 0.5× Tris-borate-EDTA (TBE) buffer. The gels were stained with Novel Juice (GeneDireX, USA), and the PCR products were visualized with UV light. Clinical K. pneumoniae isolates harboring bla KPC, bla NDM, bla IMP, bla VIM, and bla OXA-48 were used as PCR positive controls. In accessibility to have strains with bla GES , bla IMI , bla SME , bla SPM , bla SIM , bla DIM , or bla GIM as our PCR controls. The PCR products with relevant expected size were purified and verified by sequencing.
Phenotypic detection of carbapenemase-producing Enterobacteriaceae MHT, mCIM, and eCIM were performed on CRE isolates according to the CLSI guidelines to detect the presence of carbapenemase [20]. The mCIM and eCIM procedures are illustrated in Fig. 1a. In brief, a 1-μL loopful of bacteria was resuspended in a 2-mL tube of TSB. Another 1-μL loopful of bacteria was resuspended in a 2-mL tube of TSB supplemented with EDTA (Thermo Fisher Scientific, Carlsbad, CA, USA) at a final concentration of 5 mM (add 20 μL of 0.5 M EDTA to 2 mL of TSB). A meropenem disk was placed in each tube, and the tubes were incubated at 35°C for 4 h ± 15 min. Subsequently, the disks were removed and applied to MH agar plates freshly plated with a 0.5 McFarland suspension of a carbapenem-susceptible E. coli ATCC 25922 strain (Fig. 1a). The plates were incubated at 35°C for 16 to 20 h and the mCIM and eCIM results were interpreted as previously described [21,30]. The mCIM is considered negative if the zone size is ≥19 mm, positive if the zone size is 6 to 15 mm, or intermediate (defined as positive) if pinpoint colonies are present within a 16-to 18-mm zone [21,30]. An isolate is positive for metallo-carbapenemase production when the eCIM zone size increases by ≥5 mm compared to the zone size observed for the mCIM and is considered negative for a metallo-carbapenemase if the increase in zone size is < 4 mm [21,30]. According to CLSI guidelines [20], K. pneumoniae ATCC BAA-1706 (carbapenemase negative), K. pneumoniae ATCC BAA-1705 (bla KPC positive), and K. pneumoniae ATCC BAA-2146 (bla NDM positive) were used as internal controls for mCIM and eCIM tests. The MHT, mCIM and eCIM tests were replicated by two independent investigators to ensure reproducibility.