Development and Validation of a Novel Anaerobic Carbapenem Inactivation Method (Ana-CIM) for the Detection of Carbapenemase Production in Bacteroides fragilis

Abstract

Antibiotic resistance, particularly to carbapenems, is of increasing concern in Bacteroides fragilis. Carbapenem resistance in B. fragilis is most often mediated by the activation of chromosomally encoded metallo-β-lactamase cfiA by the presence of an upstream insertion sequence (IS). While traditional phenotypic susceptibility methods and molecular tests to detect carbapenem resistance in B. fragilis exist, they are not available in most clinical microbiology laboratory settings. Here, we describe the development of the anaerobic carbapenem inactivation method (Ana-CIM) for predicting carbapenemase production in B. fragilis based off the principles of the well-established modified carbapenem inactivation method (mCIM) for Enterobacterales and Pseudomonas aeruginosa. We also present the clinical validation and reproducibility of the Ana-CIM at three clinical laboratory sites (with 60 clinical isolates, 45% ertapenem resistant). Compared to ertapenem susceptibility by Etest interpreted by CLSI M100 Ed30, the Ana-CIM accurately detected carbapenem resistance in B. fragilis with categorical agreement (CA) of 87% (52/60) and 0% (0/21) very major error (VME), 11% (4/36) major error (ME), and 7% (4/60) minor error (mE) rates across all sites. Additionally, the Ana-CIM demonstrated high reproducibility with 5 clinical and 3 quality control (QC) isolates tested in triplicate with 3 commercial Mueller-Hinton media across all sites, with 93% (604/648) of replicates within a 2-mm zone size of the mode for each isolate. We conclude that the Ana-CIM can be readily deployed in clinical laboratories at a low cost for detection of carbapenemase-mediated resistance in B. fragilis.

Keywords: Bacteroides fragilis; anaerobes; antimicrobial susceptibility testing; carbapenem resistance; carbapenemase production.

FIG 1

Overview of study workflow. After initial assay development at WU, a pilot study collection containing 20 isolates of B. fragilis from WU was tested by three clinical laboratories to evaluate the assay protocol. Quality control (QC) isolates were tested at all sites at least daily. Next, for the clinical validation study, 35 clinical isolates originating from all three sites were tested by the Ana-CIM at all sites. Finally, a reproducibility study was performed on a total of eight (five test and three QC) isolates. These eight isolates were tested in triplicate on three different days with three different media by all three sites for a total of 81 replicates per isolate. Abbreviations: Ana-CIM, anaerobic carbapenem inactivation method; MC, Mayo Clinic; NCH, Nationwide Children’s Hospital; QC, quality control; WU, Washington University School of Medicine.

FIG 2

Ana-CIM zone size reading guide. Representative carbapenemase positive (A, C, and D) and negative (B) clinical isolates are shown with confluent growth up to the disk (A) or microcolonies within the zone of inhibition (C and D). Consistent with the Clinical and Laboratory Standards Institute (CLSI) M100 Ed30, colonies within the zone are read as growth (6 mm). Solid black lines represent measurement, and measured zone size is listed at the bottom of each panel in millimeters.

FIG 3

Ana-CIM zone size distribution of clinical isolates tested at all sites. Zone sizes for Ana-CIM testing for each clinical validation study isolate are shown. Results for Nationwide Children’s Hospital (NCH) (orange triangles), Mayo Clinic (MC) (green squares), and Washington University School of Medicine (WU) (blue circles) are shown. The isolates labeled in red along the x axis tested resistant by the ertapenem Etest, while the isolates in black tested susceptible. Lab-developed Ana-CIM interpretive criteria are shown as red solid lines, and CLSI mCIM interpretive criteria for Enterobacterales are shown in dashed gray lines.

FIG 4

Distribution of Ana-CIM zone sizes for reproducibility study isolates. Histograms include all 81 Ana-CIM zone-of-inhibition measurements for each of the 5 test isolates (A to E) and the quality control (QC) isolates (F to H) in the reproducibility study. Each isolate was tested in triplicate on 3 different commercially available Mueller-Hinton agar plates on 3 different days at the 3 clinical laboratories (27 zone measurements from each site for each of the isolates for a total of 81 replicates for each isolate).