Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Clinical Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Bacteriology

Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-Producing Enterobacteriaceae from Bacterial Colonies and Directly from Positive Blood Cultures

Hoon Seok Kim, Jung Ok Kim, Ji Eun Lee, Kang Gyun Park, Hae Kyung Lee, Soo-Young Kim, Sun-Joon Min, Juhyeon Kim, Yeon-Joon Park
Nathan A. Ledeboer, Editor
Hoon Seok Kim
aDepartment of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Hoon Seok Kim
Jung Ok Kim
aDepartment of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ji Eun Lee
aDepartment of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kang Gyun Park
bDepartment of Laboratory Medicine, Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hae Kyung Lee
aDepartment of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Soo-Young Kim
aDepartment of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sun-Joon Min
cDepartment of Chemical & Molecular Engineering/Applied Chemistry, Hanyang University, Ansan, Gyeonggi-do, Republic of Korea
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Juhyeon Kim
dDepartment of Chemistry, Korea University, Seoul, Republic of Korea
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yeon-Joon Park
aDepartment of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nathan A. Ledeboer
Medical College of Wisconsin
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JCM.01026-19
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Rapid and accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) is critical for appropriate treatment and infection control. We compared a rapid fluorogenic assay using a carbapenem-based fluorogenic probe with other phenotypic assays: modified carbapenem inactivation method (mCIM), Carba NP test (CNP), and carbapenemase inhibition test (CIT). A total of 217 characterized isolates of Enterobacteriaceae were included as follows: 63 CPE; 48 non-carbapenemase-producing carbapenem-resistant Enterobacteriaceae (non-CP-CRE); 53 extended-spectrum β-lactamase producers; and 53 third-generation-cephalosporin-susceptible isolates. The fluorogenic assay using bacterial colonies (Fluore-C) was conducted by lysing the isolates followed by centrifugation and mixing the supernatant with fluorogenic probe. In addition, for the fluorogenic assay using spiked blood culture bottles (Fluore-Direct), pellets were obtained via the saponin preparation method, which can directly identify the pathogens using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The fluorescence signal was measured over 50 min using a fluorometer. The fluorescent signal of CPE was significantly higher than that of non-CPE in both Fluore-C (median relative fluorescence units [RFU] [range], 5,814 [240 to 32,009] versus 804 [36 to 2,480], respectively; P < 0.0001) and Fluore-Direct (median RFU [range], 10,355 [1,689 to 31,463] versus 1,068 [428 to 2,155], respectively; P < 0.0001) tests. Overall, positive and negative percent agreements of Fluore-C, mCIM, CNP, CIT, and Fluore-Direct were 100% and 98.7%, 98.3% and 97.5%, 88.1% and 100%, 96.4% and 98.7%, and 98.3% and 98.1%, respectively. The relatively lower positive percent agreement (PPA) of CNP was mainly observed in OXA-type CPE. The fluorogenic assay showed excellent performance with bacterial colonies and also directly from positive blood cultures. We included many non-CP-CRE isolates for strict evaluation. The fluorogenic assay will be a useful tool for clinical microbiology laboratories.

INTRODUCTION

Carbapenems are the last resort to control infections caused by Gram-negative bacteria. Currently, however, the emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) are a threat to global public health (1–3). Both carbapenemase-producing Enterobacteriaceae (CPE) and non-carbapenemase-producing CRE (non-CP-CRE) are associated with significant mortality, and CPE bacteremia is particularly associated with unfavorable patient outcomes compared with non-CP-CRE bacteremia (4). Moreover, the carbapenemase genes located in CPE can be easily spread by mobile elements (5). Therefore, it is important to distinguish CPE and non-CP-CRE as early as possible.

Laboratories use molecular or phenotypic techniques to detect carbapenemases. A molecular assay takes only a few hours to differentiate the genotypes but is expensive and requires specialized personnel. The phenotypic methods, such as the carbapenemase inhibition test (CIT) (6), carbapenem inactivation method (CIM) (7), and modified carbapenem inactivation method (mCIM) (8), take at least 18 to 24 h, which delays detection. The Carba NP test (CNP) requires only 2 h to obtain results (9), but its sensitivity for OXA-48-like carbapenemases is significantly low (10). In addition, the subjective interpretation of the color changes can be problematic.

Here, we introduce a fast and accurate method using a fluorogenic probe. Fluorogenic β-lactamase probes have attracted considerable attention because of their high sensitivity, operational simplicity, and relatively low costs (11–16). Moreover, several studies have utilized fluorescent probes that specifically detect carbapenemase-producing bacteria (17–20). They used the carbapenem moiety as a substrate for carbapenemases and utilized boron-dipyrromethene (BODIPY) or umbelliferone as a fluorophore. Recently, we developed a novel carbapenem-based fluorogenic probe consisting of a carbapenem moiety with umbelliferone connected by an active linker (21). We used benzyl ether to not only induce a cascade reaction with our probe upon enzymatic hydrolysis but also facilitate its binding with active sites of different types of carbapenemases for the detection of a wide range of CPE.

In this study, we evaluated the performance of the rapid fluorogenic assay using a carbapenem-based fluorogenic probe by comparing it with other phenotypic assays (mCIM, CNP, and CIT). In addition, as bacteremia is one of the most important causes of mortality and morbidity worldwide (22), we also investigated the performance of the fluorogenic assay with the pellet derived from positive blood culture, which can be used for direct identification with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and antimicrobial susceptibility testing with Vitek 2 (bioMérieux, Marcy l’Etoile, France) (23). To the best of our knowledge, this is the first demonstration of a fluorescence assay for the detection of carbapenemases not only from bacterial isolates but also directly from positive blood cultures.

MATERIALS AND METHODS

Bacterial isolates.A total of 217 previously characterized isolates of Enterobacteriaceae were included in this study, including 63 CPE isolates with the following carbapenemase genotypes: KPC (n = 36), GES (n = 3), NDM (n = 9), VIM (n = 5), IMP (n = 1), OXA (n = 6), KPC plus OXA (n = 2), and NDM plus OXA (n = 1). The other isolates included 154 non-CPE isolates including 48 non-CP-CRE, 53 extended-spectrum-β-lactamase (ESBL) producers, and 53 third-generation-cephalosporin-susceptible isolates (nonproducers). The isolates were collected from various clinical samples obtained from multiple Korean hospitals, and three CPE coproducing multiple carbapenemases were generous gifts from Patrice Nordmann (University of Fribourg, Switzerland). All isolates were stored at −70°C and were thawed and subcultured on a blood agar plate before testing. The MICs of imipenem, meropenem, and ertapenem were obtained using the broth microdilution method according to the CLSI guideline (24). Isolates were previously characterized by PCR amplification and sequencing of carbapenemase genes, including KPC, NDM, VIM, IMP, OXA, and GES, as previously described (25, 26). The presence of the CTX-M gene was investigated by multiplex PCR (27). Detailed characteristics of the isolates are listed in Table S1 in the supplemental material.

Fluorogenic probe.The novel fluorogenic probe 1 ((4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid; C27H25NO8; molecular weight 491.50), which is composed of a carbapenem moiety, hydroxybenzyl ether linker, and umbelliferone fluorophore, was synthesized in several steps starting from the key intermediate 2 (21) (Fig. 1). When the β-lactam ring of probe 1 was selectively hydrolyzed by carbapenemase, the cascade reaction occurred rapidly to release hydroxybenzyl alcohol 3 and an anionic form of umbelliferone 4, which is responsible for high fluorescence. The 1 mM stock solution of synthetic probe 1 was prepared using 5% (vol/vol) methanol–phosphate-buffered saline (PBS) as a solvent.

FIG 1
  • Open in new tab
  • Download powerpoint
FIG 1

Carbapenemase-specific fluorogenic probe 1 and its hydrolysis by carbapenemase (21).

PBS (pH 7.4) buffer plays a crucial role in the fluorescence assay. First, this buffer minimized the hydrolysis of probe 1, which was rapidly decomposed under basic conditions to generate umbelliferone and affect background fluorescence signal. Second, the buffer maintained a consistent pH (around 7.4) during the enzymatic reaction. Third, the buffer ensured that umbelliferone (generated by the enzymatic reaction) existed in an anionic form (deprotonated form), which induced fluorescence emission at 465 nm upon excitation at 360 nm. Our probe showed a strong absorbance at 325 nm in the beginning (red), but a high absorbance signal at 360 nm after treatment with NDM-1 (black) (Fig. S1). Based on this result, our fluorescence assay was performed at 360 nm.

Fluorogenic assay using bacterial colonies (Fluore-C).The fluorogenic assay was initiated by lysing a 2-μl loopful of bacterial isolates with 150 μl of B-PER II (bacterial protein extraction reagent; Thermo Scientific Pierce, Rockford, IL, USA). After vortexing for 1 min, the mixture was left at room temperature for 30 min and centrifuged at 10,000 × g for 5 min. The supernatant (30 μl) was mixed with 100 μl PBS and 13 μl of fluorogenic probe in a flat-bottom 96-well microplate (Greiner Bio-One GmbH, Germany). Subsequently, the fluorescent signal was measured (λex = 360 nm/λem = 465 nm) over 50 min using a fluorometer (Infinite F200pro; Tecan Group Ltd.). The fluorescent signal was calculated by subtracting the value at 0 min from the value at 50 min because our analysis was based on the fluorescence generated by the probe degraded by carbapenemase. For quality control, in every run, we included Escherichia coli ATCC 25922 as a negative control and umbelliferone (Sigma-Aldrich, St. Louis, MO, USA) as a fluorescence control that continuously emitted fluorescence regardless of the presence of carbapenemase.

Fluorogenic assay directly from positive blood culture (Fluore-Direct).The mixture of 1 ml (1.5 × 106 CFU/ml) of each strain and 4 ml of sheep blood was inoculated into Bactec aerobic/F blood culture bottles, and incubated via the Bactec FX (Becton, Dickinson, Franklin Lakes, NJ, USA) blood culture system. After the culture bottle was flagged positive, pellets were obtained using the saponin method with some modifications (23). In detail, 1 ml of culture-positive blood was added to 200 μl of 2% saponin (Sigma-Aldrich) solution and mixed well. After centrifugation at 13,000 × g for 1 min, the supernatant was removed, and the pellet was suspended in 1 ml of sterilized distilled water. The washing steps (from centrifugation to suspension) were repeated twice. Finally, the pellet was suspended in 100 μl of B-PER II. After vortexing for 1 min, it was left at room temperature for 30 min and centrifuged at 10,000 × g for 5 min. A 30-μl amount of supernatant was mixed with 100 μl PBS and 13 μl of fluorogenic probe. The fluorescence was measured in the same way as the Fluore-C assay.

Other phenotypic methods.The mCIM test was performed according to the CLSI guidelines (28) using a 10-μg meropenem disk (Becton, Dickinson).

The CNP was conducted as described in the CLSI guidelines (28) except for one modification as reported previously (29), in which we used another tube, c, which did not contain zinc sulfate. Solutions A and B were prepared as described in the CLSI guidelines. Solution C was prepared similarly to solution B except that no zinc sulfate was added to solution A. Therefore, the following solutions were used: solution A, solution B (solution A plus 6 mg/ml imipenem), and solution C (solution A without zinc plus 6 mg/ml imipenem). A 1-μl loopful of the test isolates was resuspended in 100 μl B-PER II in each of the three 1.5-ml microtubes, a, b, and c. After vortexing the tubes for 5 s, we added 100 μl of solution A to tube a, solution B to tube b, and solution C to tube c. The tubes were vortexed well, incubated at 35°C, and read every 30 min for 2 h. The test result was interpreted as follows: a carbapenemase producer was detected if the color of tubes b and c changed from red to yellow while the color of tube a remained red, an Ambler class B carbapenemase producer was detected if the color of tube b changed from red to yellow while the color of tubes a and c remained red, no carbapenemase was detected if the color of all tubes remained red, and the test was not interpretable if the color of tube a changed to yellow or the color of any tube changed to orange.

The CIT was performed as described previously (6, 30) using a 10-μg meropenem disk (Becton, Dickinson) and 10 μl of three different β-lactamase inhibitors: 60 mg/ml phenylboronic acid (PBA) (Sigma), 0.2 M EDTA (Sigma), and 75 mg/ml cloxacillin (CLX) (Sigma). An 0.5 McFarland suspension of the test isolate was prepared and inoculated into a Mueller-Hinton agar (MHA) plate. Four meropenem disks were placed on the plate at least 24 mm apart, and subsequently 10 μl of each inhibitor solution was added onto the meropenem disk. After incubation for 18 h at 35°C, the diameter of the growth-inhibitory zone around each meropenem disk containing inhibitor was compared with that around the meropenem disk alone. An increase of ≥5 mm in zone diameter was considered positive for each inhibitor. The test result was interpreted as follows: an Ambler class A carbapenemase was detected if positive only for PBA synergy, an Ambler class B carbapenemase was detected if positive only for EDTA synergy, and no carbapenemase was detected if the test was negative for all inhibitors or positive for both PBA and CLX synergy. However, the OXA-type CPE were not included in calculations of CIT performance, as there was no specific inhibitor.

Comparative evaluation.A consensus positive was defined as a positive result from at least three assays among five phenotypic assays: mCIM, CNP, CIT, Fluore-C, and Fluore-Direct. A consensus negative was defined as a negative result from at least three of the five assays. As we defined consensus results as described above, the estimates were called positive percent agreement (PPA) and negative percent agreement (NPA), rather than sensitivity and specificity.

Statistical analysis.The MedCalc statistical software version 18.9 (MedCalc Software bvba, Ostend, Belgium) was used to calculate the Cohen kappa coefficient (κ) and the Spearman correlation coefficient (ρ) and to determine the cutoff value of the fluorogenic assay using receiver operating characteristic (ROC) curve analysis. The Mann-Whitney U test and F-test were used for statistical analysis of fluorescent signals. A P value of <0.05 was considered statistically significant.

Ethics statement.This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital, Seoul, South Korea (KC17SCSI0569). No informed consent was needed as no personal information was used in this study.

RESULTS

Overall performance.Phenotypic assays (Fluore-C, mCIM, CNP, CIT, and Fluore-Direct) were conducted with 63 CPE and 154 non-CPE. The overall PPAs and NPAs of Fluore-C, mCIM, CNP, CIT, and Fluore-Direct were 100% and 98.7%, 98.3% and 97.5%, 88.1% and 100%, 96.4% and 98.7%, and 98.3% and 98.1%, respectively (Table 1). The results of phenotypic assays for the detection of CPE according to the genotypes and carbapenem MICs of the isolates are summarized in Table 2. The characteristics of 18 isolates which showed discrepant results between phenotypic assays are presented in Table 3. Three isolates (one OXA producer and two GES producers) were determined as consensus negative, even though they were CPE. Another OXA producer was not subject to any consensus result because it showed neither three positive nor three negative results.

View this table:
  • View inline
  • View popup
  • Download powerpoint
TABLE 1

Performance of fluorogenic assays, mCIM, CIT, and CNPc

View this table:
  • View inline
  • View popup
  • Download powerpoint
TABLE 2

Results of MICs, fluorogenic assays, mCIM, CNP, and CIT in 63 CPE and 154 non-CPE isolatesb

View this table:
  • View inline
  • View popup
  • Download powerpoint
TABLE 3

Characteristics of isolates showing discrepant results between phenotypic assaysa

Fluorogenic assay using bacterial colonies (Fluore-C).The PPA, NPA, and κ of Fluore-C were 100%, 98.7%, and 0.977, respectively (Table 1). The Fluore-C detected all isolates determined consensus positive. One isolate of the non-CPE and one of the GES producers showed false-positive results (Table 3).

The areas under the curve (AUCs) were the largest at 50-min fluorescent signal difference, and the value was 0.958 with Fluore-C. The cutoff value for positivity was determined as 1,971 relative fluorescence units (RFU) (see Fig. S2 in the supplemental material) according to the literature for optimal point (31). The distribution of fluorescence was plotted for each carbapenemase or group in Fig. 2A. The fluorescent signal of CPE was significantly higher than that of non-CPE (median RFU [range], 5,814 [240 to 32,009] versus 804 [36 to 2,480], respectively; P < 0.0001, Mann-Whitney U test). The differences in fluorescent signal between subgroups were as follows: KPC versus non-CPE, median RFU (range), 5,709 (3,024 to 8,677) versus 804 (36 to 2,480), respectively (P < 0.0001, Mann-Whitney U test), and OXA versus non-CPE, median RFU (range), 3,223 (240 to 7,282) versus 804 (36 to 2,480), respectively (P = 0.0966, Mann-Whitney U test).

FIG 2
  • Open in new tab
  • Download powerpoint
FIG 2

Distribution of fluorescent signals in fluorogenic assays: Fluore-C (A) and Fluore-Direct (B). Abbreviations: RFU, relative fluorescence units; Fluore-C, fluorogenic assay using bacterial colonies; Fluore-Direct, fluorogenic assay directly from positive blood culture; nonproducer, third-generation-cephalosporin-susceptible isolates.

Correlation between fluorescent signal and imipenem, meropenem, and ertapenem MICs.The correlation between fluorescent signal and exponents of MICs was investigated in 63 CPE, which showed a weak correlation (Spearman’s correlation coefficient [ρ], 0.261, 0.232, and 0.123 for imipenem, meropenem, and ertapenem, respectively; P values, 0.0385, 0.0677, and 0.3349, respectively). In addition, we categorized CPE into three groups based on the results of Fluore-C and CNP and distributed them according to MICs (Fig. 3). Although the carbapenem MICs were broadly distributed in the three groups, they were higher in the group testing positive for both Fluore-C and CNP than the group positive for only Fluore-C.

FIG 3
  • Open in new tab
  • Download powerpoint
FIG 3

Frequencies of CPE categorized with Fluore-C and CNP according to MICs of imipenem (A), meropenem (B), and ertapenem (C). Abbreviations: CPE, carbapenemase-producing Enterobacteriaceae; Fluore-C, fluorogenic assay using bacterial colonies; CNP, Carba NP test.

Modified carbapenem inactivation method (mCIM).The PPA, NPA, and κ of mCIM were 98.3%, 97.5%, and 0.943, respectively (Table 1). All the 36 KPC-producing, 15 MBL-producing, and 3 coproducing isolates yielded positive results. However, a false-negative result was observed in a GES producer and there were four indeterminate results among ESBL producers (Table 3) with an inhibitory zone diameter of 18 mm, which were similar after the retest.

Carba NP (CNP) test.The PPA, NPA, and κ of CNP were 88.1%, 100%, and 0.915, respectively (Table 1). All the 36 KPC-producing, 9 NDM-producing, and 3 coproducing isolates showed positive results. The CNP did not give false-positive results at all, but a total of seven false-negative results were found in 1 GES, 2 VIM, 1 IMP, and 3 OXA producers (Table 3).

Carbapenemase inhibition test (CIT).The PPA, NPA, and κ of CNP were 96.4%, 98.7%, and 0.951, respectively (Table 1). By using carbapenemase inhibitors, we detected the carbapenemase types of the isolates. The CIT showed two false-negative results among isolates harboring VIM and IMP. Class A results were observed in 100% (36/36) of KPC producers, 67% (2/3) of GES producers, and 100% (2/2) of KPC-OXA coproducers. However, the result of one GES producer was classified as false positive because the consensus result was negative. Class B results were observed in 100% (9/9) of NDM producers, 80% (4/5) of VIM producers, none (0/1) of the IMP producers, and 100% (1/1) of NDM-OXA coproducers. A false-positive class A result was found in non-CP-CRE (Table 3). The six OXA producers were not included in the evaluation of this assay.

Fluorogenic assay directly from positive blood culture (Fluore-Direct).The PPA, NPA, and κ of Fluore-Direct were 98.3%, 98.1%, and 0.954, respectively (Table 1). A single false-negative result was observed with a VIM-producing Citrobacter freundii isolate. Three false positives were found in one OXA producer and two GES producers. As the consensus results were negative, their results were classified as false positives (Table 3).

For Fluore-Direct, the AUC at 50-min fluorescent signal difference was 0.999 with a positive cutoff value of 2,155 RFU (Fig. S2). The distribution of fluorescence values was plotted in Fig. 2B. The Fluore-Direct test also showed a significant difference in fluorescent signals between CPE and non-CPE (median RFU [range], 10,355 [1,689 to 31,463] versus 1,068 [428 to 2,155], respectively; P < 0.0001, Mann-Whitney U test). The differences in fluorescent signal between subgroups were as follows: KPC versus non-CPE, median RFU (range), 11,532 (4,597 to 25,968) versus 1,068 (428 to 2,155), respectively (P < 0.0001, Mann-Whitney U test), and OXA versus non-CPE, median RFU (range), 5,669 (3,335 to 8,242) versus 1,068 (428 to 2,155), respectively (P < 0.0001, Mann-Whitney U test).

DISCUSSION

In this study, we evaluated a novel fluorogenic probe 1 comprising a partial structure of carbapenem conjugated with hydroxybenzyloxy umbelliferone. We compared the assay with other phenotypic assays such as mCIM, CNP, and CIT. The Fluore-C fluorogenic assay using bacterial colonies showed 100% PPA by detecting 59 CPE within 90 min. During our study, a fluorescent probe with a similar carbapenem/umbelliferone structure was reported by another research group (20). It showed good selectivity for MBL but was a poor substrate for KPC and OXA carbapenemases. We presumed that the presence of umbelliferone as a sole leaving group might diminish the sensitivity of their probe to KPC- and OXA-type carbapenemases.

In our study, however, our probe 1, containing hydroxybenzyl ether as an active linker between the carbapenem moiety and fluorophore umbelliferone, showed excellent performance in detecting KPC as well as MBL. The cutoff (1,971 RFU) of Fluore-C was determined by the ROC curve, and the fluorescent signal of CPE was significantly higher than that of non-CPE. As shown in Fig. 2A, the fluorescent signals of MBL were remarkably high, and those of KPC and OXA were also high enough to distinguish from the non-CPE group.

In contrast to a previous fluorogenic probe for third-generation cephalosporins, which showed a positive correlation with cefotaxime and ceftazidime MICs (32), the fluorescent signal of CPE showed weak correlations (ρ = 0.261, 0.232, and 0.123 for imipenem, meropenem, and ertapenem, respectively) with the exponents of MICs of carbapenems. In addition, the distribution of carbapenem MICs was broad among the three categories (Fig. 3) because the carbapenem resistance of CPE was attributed to other resistance mechanisms such as AmpC β-lactamase overproduction, efflux, and porin loss in addition to carbapenemase production contributing to increasing carbapenem MICs (33).

All discrepant results (despite retest) between phenotypic assays using characterized isolates are listed in Table 3. In class A carbapenemases, all KPC producers were detected in all phenotypic tests. For the three GES producers, although two of them were defined as negative, while CNP and mCIM did not detect any of them, Fluore-C, Fluore-Direct, and CIT detected two, three, and two isolates, respectively. This finding was partly in line with a previous study (34) where the sensitivity for GES-4 producers was 0% in CNP and 17% in CIM but 100% in mCIM. In another study involving 22 GES-6 producers, the CIM showed a sensitivity of 50% (35). This phenomenon could be related to the low hydrolytic properties or enzyme types of GES producers. Although GES-type carbapenemase producers are still rare, further studies with a larger number of strains including various GES enzyme types are necessary to elucidate the performance of phenotypic assays for GES producers.

The CLSI guideline stated that the CNP showed >90% sensitivity and mCIM showed >99% sensitivity when detecting class B carbapenemases, including NDM, VIM, and IMP, among Enterobacteriaceae isolates (28). Also, recent studies showed that the sensitivity for VIM producers was 85.7 to 100% with CNP, 71.4 to 100% with CIM, and 100% with mCIM (10, 36, 37) and the sensitivity for IMP producers was 93.8 to 100% with CNP, 93.8 to 100% with CIM, and 100% with mCIM (34, 38–40). Our results also coincided with these results. All nine NDM producers were detected in all phenotypic tests. The fluorogenic assay and mCIM detected all five VIM producers, but CNP and CIT missed two and one, respectively. In addition, although only a single IMP producer was included in our study, it was detected by fluorogenic assay and mCIM but not by CNP and CIT.

For OXA producers, the fluorogenic assay and mCIM showed higher PPA (100%) than CNP (25%). The low ability of the CNP to detect OXA producers is now well known (10, 36, 37, 41). In contrast, the mCIM detects OXA producers well as demonstrated by the CLSI and previous studies (8, 28, 42). Likewise, in our study, among the six OXA producers, five (83%) were detected by mCIM, four (67%) were detected by fluorogenic assay, and one (17%) was detected by CNP.

In addition, we conducted a fluorogenic assay using bacterial pellets obtained directly from positive blood cultures. Using the positive blood culture directly saves time by avoiding subcultures on solid medium. The Fluore-Direct showed high PPA and NPA (98.3% and 98.1%, respectively), suggesting that the fluorogenic assay can be used to detect CPE not only among bacterial isolates but also directly from positive blood cultures. Interestingly, the distribution of fluorescence signals among 36 KPC producers was broad in Fluore-Direct but narrow in Fluore-C (standard deviation, 5,491 RFU versus 1,485 RFU, respectively; P < 0.0001, F-test) for unexplained reasons.

Using the mCIM method, we obtained four indeterminate results among 53 ESBL producers. Their inhibitory zone diameters were 18 mm even after retest. With Fluore-C, we had one positive result of a non-CP-CRE isolate. The above five isolates were subjected to additional PCR to confirm rare carbapenemases such as AIM, GIM, SPM, SIM, DIM, and BIC (25), which tested negative (data not shown). In addition, with the CIT method, a single non-CP-CRE, Enterobacter cloacae, showed a positive result indicating class A carbapenemase. We performed another PCR to identify possible NmcA-type carbapenemase in E. cloacae (43). The PCR result was negative (data not shown), indicating a false synergy with PBA despite the absence of carbapenemase. We considered these false positives lacking in carbapenemase. Further investigation using whole-genome sequencing may be helpful in the future.

At the very beginning of this study, we performed mCIM using bacterial isolates in excess of a 1-μl loopful, and nearly half of non-CP-CRE showed indeterminate results (data not shown). They tested negative when reevaluated with an adequate 1-μl loopful of bacterial isolates, suggesting that adequate amounts of bacterial colonies are needed for the mCIM test as indicated in the CLSI guidelines.

Recently, new or modified methods such as CIM-plus, SMA-mCIM, and meropenem hydrolysis assays using MALDI-TOF MS have been continuously introduced for fast and accurate CPE detection (39–41, 44). However, in many laboratories, CNP is still the most commonly used method for rapid detection of CPE. We here demonstrated that the fluorogenic assay has significant advantages over the CNP. First, the fluorogenic assay showed higher PPA than CNP, especially for OXA producers. Second, it can be interpreted objectively according to the cutoff based on fluorescent signals. Last, the measurement of fluorescence takes only 90 min, which is shorter than CNP, and positive blood culture samples can also be tested without subculture. Although the performance of the CNP test was good for the detection of CPE in positive blood cultures, it required 3 h of incubation after the blood culture tested positive in addition to mechanical lysis via vigorous agitation using microbead tubes and vortexing for 30 min (45). Therefore, the fluorogenic assay with higher PPA than CNP especially for the OXA type can be used to detect CPE within 90 min not only from bacterial colonies but also from positive blood cultures.

In conclusion, we evaluated a novel fluorogenic assay using carbapenem-specific fluorogenic probes with 217 characterized Enterobacteriaceae isolates. We included many non-CP-CRE to evaluate the performance with high stringency. Nonetheless, the novel fluorogenic assay showed good agreement compared with mCIM, CNP, and CIT, and the results can be obtained within 90 min. Moreover, this novel fluorogenic assay can be used directly to test positive blood cultures with the pellet which can also be used for identification with MALDI-TOF MS and the antimicrobial susceptibility test and is, therefore, very useful for the clinical microbiology laboratory.

ACKNOWLEDGMENTS

This work was supported by grants of the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant numbers HI17C1037 and HI16C0443).

No potential conflicts of interest relevant to this article were reported.

FOOTNOTES

    • Received 25 June 2019.
    • Returned for modification 18 July 2019.
    • Accepted 23 October 2019.
    • Accepted manuscript posted online 30 October 2019.
  • Supplemental material is available online only.

  • Copyright © 2019 American Society for Microbiology.

All Rights Reserved.

REFERENCES

  1. 1.↵
    1. Nordmann P,
    2. Naas T,
    3. Poirel L
    . 2011. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 17:1791–1798. doi:10.3201/eid1710.110655.
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. Canton R,
    2. Akova M,
    3. Carmeli Y,
    4. Giske CG,
    5. Glupczynski Y,
    6. Gniadkowski M,
    7. Livermore DM,
    8. Miriagou V,
    9. Naas T,
    10. Rossolini GM,
    11. Samuelsen O,
    12. Seifert H,
    13. Woodford N,
    14. Nordmann P
    . 2012. Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe. Clin Microbiol Infect 18:413–431. doi:10.1111/j.1469-0691.2012.03821.x.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Logan LK,
    2. Weinstein RA
    . 2017. The epidemiology of carbapenem-resistant Enterobacteriaceae: the impact and evolution of a global menace. J Infect Dis 215:S28–S36. doi:10.1093/infdis/jiw282.
    OpenUrlCrossRef
  4. 4.↵
    1. Tamma PD,
    2. Goodman KE,
    3. Harris AD,
    4. Tekle T,
    5. Roberts A,
    6. Taiwo A,
    7. Simner PJ
    . 2017. Comparing the outcomes of patients with carbapenemase-producing and non-carbapenemase-producing carbapenem-resistant Enterobacteriaceae bacteremia. Clin Infect Dis 64:257–264. doi:10.1093/cid/ciw741.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Queenan AM,
    2. Bush K
    . 2007. Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev 20:440–458. doi:10.1128/CMR.00001-07.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Giske CG,
    2. Gezelius L,
    3. Samuelsen O,
    4. Warner M,
    5. Sundsfjord A,
    6. Woodford N
    . 2011. A sensitive and specific phenotypic assay for detection of metallo-beta-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin. Clin Microbiol Infect 17:552–556. doi:10.1111/j.1469-0691.2010.03294.x.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. van der Zwaluw K,
    2. de Haan A,
    3. Pluister GN,
    4. Bootsma HJ,
    5. de Neeling AJ,
    6. Schouls LM
    . 2015. The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods. PLoS One 10:e0123690. doi:10.1371/journal.pone.0123690.
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Pierce VM,
    2. Simner PJ,
    3. Lonsway DR,
    4. Roe-Carpenter DE,
    5. Johnson JK,
    6. Brasso WB,
    7. Bobenchik AM,
    8. Lockett ZC,
    9. Charnot-Katsikas A,
    10. Ferraro MJ,
    11. Thomson RB, Jr,
    12. Jenkins SG,
    13. Limbago BM,
    14. Das S
    . 2017. Modified carbapenem inactivation method for phenotypic detection of carbapenemase production among Enterobacteriaceae. J Clin Microbiol 55:2321–2333. doi:10.1128/JCM.00193-17.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Nordmann P,
    2. Poirel L,
    3. Dortet L
    . 2012. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 18:1503–1507. doi:10.3201/eid1809.120355.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Tamma PD,
    2. Opene BN,
    3. Gluck A,
    4. Chambers KK,
    5. Carroll KC,
    6. Simner PJ
    . 2017. Comparison of 11 phenotypic assays for accurate detection of carbapenemase-producing Enterobacteriaceae. J Clin Microbiol 55:1046–1055. doi:10.1128/JCM.02338-16.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Gao W,
    2. Xing B,
    3. Tsien RY,
    4. Rao J
    . 2003. Novel fluorogenic substrates for imaging beta-lactamase gene expression. J Am Chem Soc 125:11146–11147. doi:10.1021/ja036126o.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    1. Rukavishnikov A,
    2. Gee KR,
    3. Johnson I,
    4. Corry S
    . 2011. Fluorogenic cephalosporin substrates for beta-lactamase TEM-1. Anal Biochem 419:9–16. doi:10.1016/j.ab.2011.07.020.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Xie H,
    2. Mire J,
    3. Kong Y,
    4. Chang M,
    5. Hassounah HA,
    6. Thornton CN,
    7. Sacchettini JC,
    8. Cirillo JD,
    9. Rao J
    . 2012. Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe. Nat Chem 4:802–809. doi:10.1038/nchem.1435.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Zhang J,
    2. Shen Y,
    3. May SL,
    4. Nelson DC,
    5. Li S
    . 2012. Ratiometric fluorescence detection of pathogenic bacteria resistant to broad-spectrum beta-lactam antibiotics. Angew Chem Int Ed Engl 51:1865–1868. doi:10.1002/anie.201107810.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Thai HB,
    2. Yu JK,
    3. Park BS,
    4. Park YJ,
    5. Min SJ,
    6. Ahn DR
    . 2016. A fluorogenic substrate of beta-lactamases and its potential as a probe to detect the bacteria resistant to the third-generation oxyimino-cephalosporins. Biosens Bioelectron 77:1026–1031. doi:10.1016/j.bios.2015.10.081.
    OpenUrlCrossRef
  16. 16.↵
    1. Mao W,
    2. Qian X,
    3. Zhang J,
    4. Xia L,
    5. Xie H
    . 2017. Specific detection of extended-spectrum beta-lactamase activities with a ratiometric fluorescent probe. Chembiochem 18:1990–1994. doi:10.1002/cbic.201700447.
    OpenUrlCrossRef
  17. 17.↵
    1. Shi H,
    2. Cheng Y,
    3. Lee KH,
    4. Luo RF,
    5. Banaei N,
    6. Rao J
    . 2014. Engineering the stereochemistry of cephalosporin for specific detection of pathogenic carbapenemase-expressing bacteria. Angew Chem Int Ed Engl 53:8113–8116. doi:10.1002/anie.201402012.
    OpenUrlCrossRef
  18. 18.↵
    1. Mao W,
    2. Xia L,
    3. Xie H
    . 2017. Detection of carbapenemase-producing organisms with a carbapenem-based fluorogenic probe. Angew Chem Int Ed Engl 56:4468–4472. doi:10.1002/anie.201612495.
    OpenUrlCrossRef
  19. 19.↵
    1. Song A,
    2. Cheng Y,
    3. Xie J,
    4. Banaei N,
    5. Rao J
    . 2017. Intramolecular substitution uncages fluorogenic probes for detection of metallo-carbapenemase-expressing bacteria. Chem Sci 8:7669–7674. doi:10.1039/c7sc02416a.
    OpenUrlCrossRef
  20. 20.↵
    1. Mao W,
    2. Wang Y,
    3. Qian X,
    4. Xia L,
    5. Xie H
    . 2019. A carbapenem-based off-on fluorescent probe for specific detection of metallo-beta-lactamase activities. Chembiochem 20:511–515. doi:10.1002/cbic.201800126.
    OpenUrlCrossRef
  21. 21.↵
    1. Sun-Joon M
    . 27 May 2019. Probes for detecting carbapenem-resistance bacteria in a sample and uses thereof. Republic of Korea patent 10-2019-0061630.
  22. 22.↵
    1. Farina C,
    2. Arena F,
    3. Casprini P,
    4. Cichero P,
    5. Clementi M,
    6. Cosentino M,
    7. Degl’Innocenti R,
    8. Giani T,
    9. Luzzaro F,
    10. Mattei R,
    11. Mauri C,
    12. Nardone M,
    13. Rossolini GM,
    14. Serna Ortega PA,
    15. Vailati F
    . 2015. Direct identification of microorganisms from positive blood cultures using the lysis-filtration technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): a multicentre study. New Microbiol 38:245–250.
    OpenUrl
  23. 23.↵
    1. Lee JE,
    2. Jo SJ,
    3. Park KG,
    4. Suk HS,
    5. Ha SI,
    6. Shin JS,
    7. Park YJ
    . 2018. Evaluation of modified saponin preparation method for the direct identification and antimicrobial susceptibility testing from positive blood culture. J Microbiol Methods 154:118–123. doi:10.1016/j.mimet.2018.10.004.
    OpenUrlCrossRef
  24. 24.↵
    CLSI. 2015. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-10th ed. CLSI M07-A10. CLSI, Wayne, PA.
  25. 25.↵
    1. Poirel L,
    2. Walsh TR,
    3. Cuvillier V,
    4. Nordmann P
    . 2011. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 70:119–123. doi:10.1016/j.diagmicrobio.2010.12.002.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Poirel L,
    2. Le Thomas I,
    3. Naas T,
    4. Karim A,
    5. Nordmann P
    . 2000. Biochemical sequence analyses of GES-1, a novel class A extended-spectrum beta-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrob Agents Chemother 44:622–632. doi:10.1128/aac.44.3.622-632.2000.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Woodford N,
    2. Fagan EJ,
    3. Ellington MJ
    . 2006. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. J Antimicrob Chemother 57:154–155. doi:10.1093/jac/dki412.
    OpenUrlCrossRefPubMedWeb of Science
  28. 28.↵
    CLSI. 2019. Performance standards for antimicrobial susceptibility testing, 29th ed. CLSI supplement M100. CLSI, Wayne, PA.
  29. 29.↵
    1. Dortet L,
    2. Poirel L,
    3. Nordmann P
    . 2012. Rapid identification of carbapenemase types in Enterobacteriaceae and Pseudomonas spp. by using a biochemical test. Antimicrob Agents Chemother 56:6437–6440. doi:10.1128/AAC.01395-12.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Tsakris A,
    2. Themeli-Digalaki K,
    3. Poulou A,
    4. Vrioni G,
    5. Voulgari E,
    6. Koumaki V,
    7. Agodi A,
    8. Pournaras S,
    9. Sofianou D
    . 2011. Comparative evaluation of combined-disk tests using different boronic acid compounds for detection of Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae clinical isolates. J Clin Microbiol 49:2804–2809. doi:10.1128/JCM.00666-11.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Perkins NJ,
    2. Schisterman EF
    . 2006. The inconsistency of “optimal” cutpoints obtained using two criteria based on the receiver operating characteristic curve. Am J Epidemiol 163:670–675. doi:10.1093/aje/kwj063.
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    1. Park MJ,
    2. Park YJ,
    3. Oh EJ,
    4. Chang J,
    5. Kim Y,
    6. Yu J,
    7. Park KG,
    8. Ahn DR
    . 2016. Performance of a novel fluorogenic chimeric analog for the detection of third-generation cephalosporin resistant bacteria. J Microbiol Methods 131:161–165. doi:10.1016/j.mimet.2016.10.016.
    OpenUrlCrossRef
  33. 33.↵
    1. Fernandez L,
    2. Hancock RE
    . 2012. Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance. Clin Microbiol Rev 25:661–681. doi:10.1128/CMR.00043-12.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Kuchibiro T,
    2. Komatsu M,
    3. Yamasaki K,
    4. Nakamura T,
    5. Nishio H,
    6. Nishi I,
    7. Kimura K,
    8. Niki M,
    9. Ono T,
    10. Sueyoshi N,
    11. Kita M,
    12. Kida K,
    13. Ohama M,
    14. Satoh K,
    15. Toda H,
    16. Mizutani T,
    17. Fukuda N,
    18. Sawa K,
    19. Nakai I,
    20. Kofuku T,
    21. Orita T,
    22. Watari H,
    23. Shimura S,
    24. Fukuda S,
    25. Nakamura A,
    26. Wada Y
    . 2018. Evaluation of the modified carbapenem inactivation method for the detection of carbapenemase-producing Enterobacteriaceae. J Infect Chemother 24:262–266. doi:10.1016/j.jiac.2017.11.010.
    OpenUrlCrossRef
  35. 35.↵
    1. Aguirre-Quiñonero A,
    2. Cano ME,
    3. Gamal D,
    4. Calvo J,
    5. Martínez-Martínez L
    . 2017. Evaluation of the carbapenem inactivation method (CIM) for detecting carbapenemase activity in enterobacteria. Diagn Microbiol Infect Dis 88:214–218. doi:10.1016/j.diagmicrobio.2017.03.009.
    OpenUrlCrossRef
  36. 36.↵
    1. Tijet N,
    2. Patel SN,
    3. Melano RG
    . 2016. Detection of carbapenemase activity in Enterobacteriaceae: comparison of the carbapenem inactivation method versus the Carba NP test. J Antimicrob Chemother 71:274–276. doi:10.1093/jac/dkv283.
    OpenUrlCrossRefPubMed
  37. 37.↵
    1. Gauthier L,
    2. Bonnin RA,
    3. Dortet L,
    4. Naas T
    . 2017. Retrospective and prospective evaluation of the carbapenem inactivation method for the detection of carbapenemase-producing Enterobacteriaceae. PLoS One 12:e0170769. doi:10.1371/journal.pone.0170769.
    OpenUrlCrossRef
  38. 38.↵
    1. Yamada K,
    2. Kashiwa M,
    3. Arai K,
    4. Nagano N,
    5. Saito R
    . 2016. Comparison of the modified-Hodge test, Carba NP test, and carbapenem inactivation method as screening methods for carbapenemase-producing Enterobacteriaceae. J Microbiol Methods 128:48–51. doi:10.1016/j.mimet.2016.06.019.
    OpenUrlCrossRef
  39. 39.↵
    1. Zhou M,
    2. Wang D,
    3. Kudinha T,
    4. Yang Q,
    5. Yu S,
    6. Xu YC
    . 2018. Comparative evaluation of four phenotypic methods for detection of class A and B carbapenemase-producing Enterobacteriaceae in China. J Clin Microbiol 56:e00395-18. doi:10.1128/JCM.00395-18.
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    1. Yamada K,
    2. Kashiwa M,
    3. Arai K,
    4. Nagano N,
    5. Saito R
    . 2017. Evaluation of the modified carbapenem inactivation method and sodium mercaptoacetate-combination method for the detection of metallo-beta-lactamase production by carbapenemase-producing Enterobacteriaceae. J Microbiol Methods 132:112–115. doi:10.1016/j.mimet.2016.11.013.
    OpenUrlCrossRef
  41. 41.↵
    1. Papagiannitsis CC,
    2. Studentova V,
    3. Izdebski R,
    4. Oikonomou O,
    5. Pfeifer Y,
    6. Petinaki E,
    7. Hrabak J
    . 2015. Matrix-assisted laser desorption ionization-time of flight mass spectrometry meropenem hydrolysis assay with NH4HCO3, a reliable tool for direct detection of carbapenemase activity. J Clin Microbiol 53:1731–1735. doi:10.1128/JCM.03094-14.
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    1. Miller SA,
    2. Hindler JA,
    3. Chengcuenca A,
    4. Humphries RM
    . 2017. Use of ancillary carbapenemase tests to improve specificity of phenotypic definitions for carbapenemase-producing Enterobacteriaceae. J Clin Microbiol 55:1827–1836. doi:10.1128/JCM.00157-17.
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    1. Pasteran F,
    2. Mendez T,
    3. Guerriero L,
    4. Rapoport M,
    5. Corso A
    . 2009. Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae. J Clin Microbiol 47:1631–1639. doi:10.1128/JCM.00130-09.
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Camelena F,
    2. Cointe A,
    3. Mathy V,
    4. Hobson C,
    5. Doit C,
    6. Bercot B,
    7. Decre D,
    8. Podglajen I,
    9. Dortet L,
    10. Monjault A,
    11. Bidet P,
    12. Bonacorsi S,
    13. Birgy A
    . 2018. Within-a-day detection and rapid characterization of carbapenemase by use of a new carbapenem inactivation method-based test, CIMplus J Clin Microbiol 56:e00137-18. doi:10.1128/JCM.00137-18.
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    1. Dortet L,
    2. Brechard L,
    3. Poirel L,
    4. Nordmann P
    . 2014. Rapid detection of carbapenemase-producing Enterobacteriaceae from blood cultures. Clin Microbiol Infect 20:340–344. doi:10.1111/1469-0691.12318.
    OpenUrlCrossRefPubMed
PreviousNext
Back to top
Download PDF
Citation Tools
Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-Producing Enterobacteriaceae from Bacterial Colonies and Directly from Positive Blood Cultures
Hoon Seok Kim, Jung Ok Kim, Ji Eun Lee, Kang Gyun Park, Hae Kyung Lee, Soo-Young Kim, Sun-Joon Min, Juhyeon Kim, Yeon-Joon Park
Journal of Clinical Microbiology Dec 2019, 58 (1) e01026-19; DOI: 10.1128/JCM.01026-19

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Clinical Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-Producing Enterobacteriaceae from Bacterial Colonies and Directly from Positive Blood Cultures
(Your Name) has forwarded a page to you from Journal of Clinical Microbiology
(Your Name) thought you would be interested in this article in Journal of Clinical Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-Producing Enterobacteriaceae from Bacterial Colonies and Directly from Positive Blood Cultures
Hoon Seok Kim, Jung Ok Kim, Ji Eun Lee, Kang Gyun Park, Hae Kyung Lee, Soo-Young Kim, Sun-Joon Min, Juhyeon Kim, Yeon-Joon Park
Journal of Clinical Microbiology Dec 2019, 58 (1) e01026-19; DOI: 10.1128/JCM.01026-19
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

fluorogenic assay
carbapenemase-producing Enterobacteriaceae
positive blood culture
direct
fluorogenic probe

Related Articles

Cited By...

About

  • About JCM
  • Editor in Chief
  • Board of Editors
  • Editor Conflicts of Interest
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Resources for Clinical Microbiologists
  • Ethics
  • Contact Us

Follow #JClinMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

 

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0095-1137; Online ISSN: 1098-660X