A Combined Disk Test for Direct Differentiation of Carbapenemase- Producing Enterobacteriaceae in Surveillance Rectal Swabs

Carbapenemase-producing Enterobacteriaceae (CPE) are rapidly spreading worldwide. Early detection of fecal CPE carriers is essential for effective infection control. Here, we evaluated the performance of a meropenem combined disk test (CDT) for rap- idly differentiating CPE isolates directly from rectal swabs. The screening method was applied for 189 rectal swabs from hospitalized patients at high risk for CPE carriage. Swabs were suspended in 1 ml saline and cultured for confluent growth onto a Mac- Conkey agar plate with a meropenem (MER) disk alone, a MER disk plus phenyl boronic acid (PBA), a MER disk plus EDTA, and a MER disk plus PBA and EDTA. An inhibition zone of <25 mm around the MER disk alone indicated carriage of carbapenem- resistant organisms. Furthermore, >5-mm differences in the inhibition zone between MER disks without and with the inhibitors (PBA, EDTA, or both) were considered positive results for detecting Klebsiella pneumoniae carbapenemase (KPC), metallolactamase (MBL), or both carbapenemases, respectively. For comparison, rectal suspensions were tested using MacConkey plates with ertapenem (MacERT) disks and PCR (PCR-S) for carbapenemase genes. Of the 189 samples, 97 were genotypically confirmed as CPE positive by one of the three protocols tested. The CDT, MacERT disks, and PCR-S assays exhibited sensitivities of 94.8%, 96.9%, and 94.8% and specificities of 100%, 98.9%, and 100%, respectively, for detecting CPE-positive swabs. More- over, the CDT correctly differentiated the production of KPC, MBL, or both carbapenemases in 78 of the 97 (80.4%) CPE-positive rectal swabs. Our results demonstrate that the CDT may provide a simple and inexpensive method for detecting and differ- entiating the carbapenemase type within a single day without requiring further testing and additional delay, supporting the timely implementation of infection control measures.

The ongoing dissemination of carbapenemase-producing Enterobacteriaceae (CPE) represents a significant public health issue in many regions of the world. Early detection and infection control strategies are key factors for successfully restricting the further spread of CPE (1–3). Toward this goal, surveillance cultures have been applied effectively as part of the multifaceted nationwide interventional strategies contributing to a decrease in the CPE prevalence in countries where it is highly endemic (4). Furthermore, the prompt identification of CPE carriers at the hospital level may guide the recognition of cases brought into the hospital and may allow the early cohorting of colonized patients, thus assisting in the control of hospital epidemics (5–8). In addition, the identification of an individual patient’s carriage status provides information about patients at risk for CPE infections who should be monitored closely, and it facilitates the administration of appropriate empirical treatments (9, 10).

Several approaches have been developed for rectal screening. In-house prepared protocols include selective agar plates with impregnated carbapenems or with carbapenem disks (11– 13). These approaches have been applied with or without initial broth enrichment, which causes an additional delay, while its performance still remains controversial (12, 13). Several chro- mogenic media, such as CHROMagar KPC and Colorex KPC (CHROMagar, Paris, France) (11, 14–16), Brilliance CRE (Oxoid, Basingstoke, United Kingdom) (14, 17), and chromID Carba (bioMérieux, Marcy l’Etoile, France) (16, 18), have also been used for the active screening of carbapenem-resistant En- terobacteriaceae (CRE) or CPE. In addition, the specific culture medium Supercarba was developed and evaluated for the detection of CPE (14, 19), while the nonspecific chromogenic medium chromID extended-spectrum-lactamase (ESBL) (bioMérieux) has been applied for CRE detection (16, 20). These culture-based techniques exhibited variable performance characteristics; while they detect carbapenem-resistant organisms, they cannot differentiate the carbapenemase type present in the same day, requiring subsequent phenotypic or molecular testing of isolated colonies, with a further delay of at least 1 day (21). Rapid and specific PCR techniques have also been used to directly identify the carbapenemase type (15, 22–25); how- ever, they do not yield isolates for susceptibility testing, they are not widely applicable, and they are relatively costly.

We have previously shown that a meropenem combined disk test (CDT) provides a simple and accurate phenotypic method for the specific differentiation of Enterobacteriaceae isolates possessing Klebsiella pneumoniae carbapenemases (KPCs), metallolactamases (MBLs), or both carbapenemase types (26). The aim of the present study was to evaluate this method as an easy and low- cost approach for the rapid detection of CPE and the differentiation of carbapenemase types directly from rectal swabs.