Evaluation of Double-Disk Potentiation and Disk Potentiation Tests Using Dipicolinic Acid for Detection of Metallo-β-Lactamase-Producing Pseudomonas spp. and Acinetobacter spp

Abstract

Accurate detection of metallo-β-lactamase (MBL)-producing Pseudomonas spp. and Acinetobacter spp. became very important with the increasing prevalence of carbapenem-nonsusceptible clinical isolates. The performance of phenotypic MBL detection methods may depend on the types of MBL and the characteristics of the isolates. A high false-positive rate is a problem with EDTA-based MBL detection methods. We evaluated the performance of double-disk potentiation tests (DDPTs) and disk potentiation tests (DPTs) with dipicolinic acid (DPA) using 44 isolates of Pseudomonas spp. and Acinetobacter spp. producing IMP-1-like, VIM-2-like, and SIM-1 type MBLs. Also, we characterized P. aeruginosa isolates with positive imipenem (IPM)-DPA DDPT, but negative meropenem (MEM)-DPA DDPT, and determined possibility of improving a DDPT by using MacConkey agar. Among five different DDPT methods, the IPM-DPA 250-μg method showed the highest sensitivity (97.7%) and specificity (100%). Among four DPT tests, the highest sensitivity (100%) was shown by the IPM-EDTA 1,900-μg disk method, but the specificity was very low (11.4%). Five of six P. aeruginosa isolates with false-negative DDPTs with MEM-DPA 250-μg disks carried bla(IMP-6,) and the high level resistance to MEM (MIC ≥ 512 μg/ml) was reduced by the presence of phenylalanine arginine β-naphtylamide. Improvement of DDPTs was observed when MacConkey agar was used instead of Mueller-Hinton agar. In conclusion, DPA is a better MBL inhibitor than EDTA for detection of Pseudomonas spp. and Acinetobacter spp. with IMP-1-like, VIM-2-like, and SIM-1-type MBLs. In DPA DDPTs, IPM disks perform better than MEM disks when the isolates are highly resistant to MEM due to the overexpression of efflux pumps.