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

bAccurate detection of metallo-␤-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-g 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-g disk method, but the specificity was very low (11.4%). Five of six P. aeruginosa isolates with false-negative DDPTs with MEM-DPA 250-g disks carried bla IMP-6, and the high level resistance to MEM (MIC > 512 g/ml) was reduced by the presence of phenylalanine arginine ␤-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. C arbapenems are often used as "antibiotics of last resort" when patients with infections become gravely ill or are suspected of harboring resistant bacteria (14). However, carbapenem-resistant Gram-negative bacilli have been increasingly reported worldwide. In Korea, carbapenem-resistant isolates of Enterobacteriaceae remains very rare, but during the period of 2001 to 2009, imipenem (IPM)-resistant Pseudomonas aeruginosa and Acinetobacter spp. increased from 17 to 26% and from 5 to 51%, respectively (10). A significant proportion of the resistance in P. aeruginosa is due to the production of VIM-2-like and IMP-1-like metallo-␤-lactamase (MBL), whereas in Acinetobacter spp. the majority are due to the production of OXA-type carbapenemase, although VIM-2-like, IMP-1-like, and SIM-1 enzyme-producing isolates have also been detected (8).Various inhibitor-based methods are commonly used to detect MBLs, but these techniques are not highly sensitive or highly specific. A disk potentiation test (DPT) is also called a combined disk test. A DPT using imipenem (IPM) and EDTA (IPM-EDTA DPT) showed a high false-positive rate with Acinetobacter spp. due to growth inhibition by EDTA alone (20). A double-disk potentiation test (DDPT) is also known as a double-disk synergy test. To reduce false-positive results from t...

Low prevalence of metallo-beta-lactamase in Pseudomonas aeruginosa isolated from a tertiary burn care center in Tehran

“…The main mechanism of resistance in carbapenems is the production of carbapenemase by gramnegative bacteria. Ethylenediaminetetraacetic acid [EDTA]) and some MBL inhibitor like dipicokinic acid, [10][11][12][13][14][15][16], nevertheless the specificity and sensitivity of these methods can be variable. 4,6,7 The first isolate of MBL producer P. aeruginosa was reported in Japan in 1991 and since it has been reported worldwide.…”

A combined disc method with resazurin agar plate assay for early phenotypic screening of KPC, MBL and OXA-48 carbapenemases among Enterobacteriaceae

“…and Acinetobacter spp. producing IMP-1-like, VIM-2-like and SIM-1-type MBLs (Yong et al 2012). Giske et al (2011) found that DPA synergy test with MER as a substrate had 100% in both sensitivity and specificity, which had superior performance in detection of MBL producers compared with EDTA.…”

Antimicrobial Profile and Phenotypic Metallo-β-Lactamase Detection of Acinetobacter baumannii Isolated From Clinical and Environmental Specimens

“…More recently, new methods for phenotypic detecting carbapenemase producing isolates have been described based on expansion of inhibition zone [19,20]. They have used other materials like mercaptoacetate and dipicolinic acid which shows statistical and practical benefits.…”