Abstract

The Study

We included in the study 162 carbapenemase-producing strains of various enterobacterial species isolated from clinical samples (e.g., blood cultures, urine, sputum) and of global origin (Table 1). This collection of strains also included 46 strains that were fully susceptible to carbapenems or showed a decreased susceptibility to carbapenems as a consequence of non–carbapenemase-based mechanisms (Table 2). Antibiograms were carried out for all strains on Mueller-Hinton agar (Becton Dickinson, Le Point de Chaix, France) according to guidelines of the Clinical and Laboratory Standards Institute (13). The Carba NP (Carbapenemase Nordmann-Poirel) test was performed as follows. One calibrated loop (10 μL) of the tested strain directly recovered from the antibiogram was resuspended in a Tris-HCl 20 mmol/L lysis buffer (B-PERII, Bacterial Protein Extraction Reagent; Thermo Scientific Pierce, Rockford, IL, USA), vortexed for 1 minute and further incubated at room temperature for 30 minutes. This bacterial suspension was centrifuged at 10,000 × g at room temperature for 5 minutes. Thirty µL of the supernatant, corresponding to the enzymatic bacterial suspension, was mixed in a 96-well tray with 100 µL of a 1-mL solution made of 3 mg of imipenem monohydrate (Sigma, Saint-Quentin Fallavier, France), pH 7.8, phenol red solution, and 0.1 mmol/L ZnSO₄ (Merck Millipore, Guyancourt, France). The phenol red solution was prepared by mixing 2 mL of a phenol red (Merck Millipore) solution 0.5% (wt/vol) with 16.6 mL of distilled water. The pH value was then adjusted to 7.8 by adding drops of 1 N NaOH. A mixture of the phenol red solution and the enzymatic suspension being tested was incubated at 37°C for a maximum of 2 hours. Test results were interpreted by technicians who were blinded to the identity of the patients who gave the samples.

All strains had previously been characterized for their β-lactamase content at the molecular level. MICs of carbapenems were determined by using the Etest (AB bioMérieux, Solna, Sweden), and results were recorded according to US guidelines (Clinical and Laboratory Standards Institute), as updated in 2012 (13). The breakpoints used were those for imipenem and meropenem: susceptibility <1 μg/mL, resistance >4 µg/mL, and for ertapenem, susceptibility <0.25 μg/mL, resistance > µg/mL.

When the Carba NP test was used, the color of the wells turned from red to orange or yellow (Figure 1) for all tested strains that were producing carbapenemases (Table 1), whereas wells corresponding to bacterial extracts of isolates that did not produce carbapenemase remained red, whatever their level of carbapenem susceptibility (Table 2). The color changed from red to yellow as early as 5–10 minutes after incubation for KPC producers began. In most cases, incubation for 30 minutes was sufficient for obtaining a frank color change for carbapenemase producers. The test’s specificity and sensitivity were 100% when results were compared with those from molecular-based methods, the reference standard for identifying carbapenemase genes. All tests were performed in triplicate, giving identical and reproducible results.

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Figure 1

Representative results of the Carba NP test. The Carba NP test was performed with a noncarbapenemase producer (Escherichia coli producing the extended-spectrum β-lactamase CTX-M-15, upper panel) and with a carbapenemase producer (Klebsiella pneumoniae–producing New Delhi metallo-β-lactamase-1, lower panel) in a reaction medium without (left panel) and with (right panel) imipenem. Uninoculated wells are shown as controls. Photographs were taken after a 1.5-hour incubation.

The Carba NP test perfectly differentiates carbapenemase producers (Table 1) from strains that are carbapenem resistant due to non–carbapenemase-mediated mechanisms, such as combined mechanisms of resistance (outer-membrane permeability defect associated with overproduction of cephalosporinase and/or extended-spectrum β-lactamases) or from strains that are carbapenem susceptible but express a broad-spectrum β-lactamase without carbapenemase activity (extended-spectrum β-lactamases, plasmid and chromosome-encoded cephalosporinases) (Table 2). Interpretable positive results were obtained in <2 hours, making it possible to implement rapid containment measures to limit the spread of carbapenemase producers.

Conclusions

The Carba NP test has multiple benefits. It is inexpensive, rapid, reproducible, and highly sensitive and specific. It eliminates the need for using other techniques to identify carbapenemase producers that are time-consuming and less sensitive or specific. Using this accurate test would improve detection of patients infected or colonized with carbapenemase producers. The test has been routinely implemented in our microbiology department at Hôpital de Bicêtre and is giving excellent results (data not shown). In addition, use of the Carba NP test has greatly decreased the laboratory technicians’ workload and simplified the clinical management of potential carbapenemase producers.

This test could be used, for example, for directly testing 1) bacteria obtained from antibiograms of blood culture or 2) bacterial colonies grown on culture media before antimicrobial drug susceptibility testing (Figure 2). Further studies will evaluate its clinical value for antimicrobial drug stewardship on bacteria isolated directly from clinical samples (Figure 2). When the Carba NP test is used for that purpose, we expect that the time to detect carbapenemase producers will decrease by at least 24 hours (Figure 2).

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Figure 2

Strategy for identification of carbapenemase-producing Enterobacteriaceae. The time needed to perform the test is indicated before each test. The number of flasks indicates the degree of specialization needed to perform the test; the number of $ indicates the relative cost of each test.

The test could also be used to quickly identify carbapenem-resistant isolates from fecal specimens screened for multidrug-resistant bacteria (Figure 2). This capability would be valuable in preventing outbreaks. To determine positive and negative predictive values of the test, additional evaluations will be required with strains isolated from clinical samples screened on different types of selective media. The use of the Carba NP test may also support novel antimicrobial drug development by facilitating patient enrollment in pivotal clinical trials. Its use as a home-made test may contribute to the global surveillance network.

The Carba NP test can efficiently indicate the strains to be further tested by PCR or submitted to sequencing for a detailed identification of the carbapenemase genes. Last, the test could be used in low-income countries that are large reservoirs for carbapenemase producers (2). It offers a practical solution for detecting a main component of multidrug resistance in Enterobacteriaceae. Use of the Carba NP test will contribute to a better stewardship of carbapenems by changing the paradigm of controlling carbapenemase producers worldwide

Acknowledgments

Footnotes

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