Evaluation of Phenotypic Tests for Detection of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Strains in China

Abstract

A total of 264 nonduplicate strains of imipenem (IPM)-nonsusceptible Pseudomonas aeruginosawere isolated from hospitals in 16 different regions throughout China. These 264 IPM-nonsusceptible clinical isolates of P. aeruginosa were examined by PCR, a metallo-␤-lactamase (MBL) Etest, a double-disk synergy test (DDST), and a test using combined IPM disks supplemented with various amounts of EDTA. A total of 24 strains positive for MBLs were confirmed by PCR and DNA sequence analysis: 10 strains positive for the bla VIM-2 gene, 13 strains positive for the bla IMP-9 gene, and 1 strain positive for the bla IMP-1 gene. Real-time reverse transcriptase PCR (RT-PCR) was used to verify whether the isolates harboring MBL genes produced the enzyme and was considered the standard for evaluation of the methodology in this study. Of these 24 MBL-positive stains, 21 were confirmed as MBL-producing strains by real time RT-PCR for MBL expression and the other 3 had no expression of MBLs. The sensitivities, specificities, and positive and negative predictive values for the MBL Etest, the DDST, and the combined disk (CD) assay were evaluated. The best method for screening for MBL production in P. aeruginosa strains from China was the CD assay (IMP-EDTA) using 750 g of EDTA/disk with a breakpoint of >6 mm. In the CD assay (IPM-EDTA) with 290 g and 750 g EDTA, the zone diameter increases for VIM-2-producing P. aeruginosa isolates were greater than those for IMP-9-producing P. aeruginosa isolates (P ‫؍‬ 0.00).The worldwide spread of acquired metallo-␤-lactamases (MBLs) in clinically important pathogens, such as Pseudomonas spp., Acinetobacter spp., and members of the Enterobacteriaceae family, has become a great concern (9, 12). Increased mortality rates have been documented for patients infected with MBL-producing Pseudomonas aeruginosa, and these rates are especially due to inadequate empirical therapy (27). Most of the MBL-encoding genes reside on class 1 integrons and plasmids that usually confer high mobility to these genetic elements (8,17,22,24,26). Therefore, early detection of MBL-producing organisms is of crucial importance for prevention of their inter-and intrahospital dissemination, not only in institutions with high prevalences of such isolates but also in those in which phenotypes of resistance have never been detected. Various criteria for screening for MBL production in P. aeruginosa have been suggested (15). Currently, the most widely accepted standardized MBL functional screen is the MBL Etest (AB BioDisk, Solna, Sweden). However, due to the high cost and unavailability of Etest strips, many clinical microbiology laboratories use alternative screening methods, such as the double-disk synergy test (DDST) and the combined disk (CD) assay. Although the DDST and the CD assay are simple to perform and cheaper than the MBL Etest, they have shown discordant results, depending on the employed methodology, ␤-lactam substrates, MBL inhibitors (IMBL), and bacterial genus tested (7,11,14,15). Standardization of a phenotypic meth...

Comparison Between Phenotypic and Genotypic Methods for the Detection of Metallo Beta Lactamases Producing Pseudomonas Aeruginosa

“…[33] In the current study, IPM-EDTA DDST showed a medium degree of agreement with PCR assay 37.5% sensitivity and 100% specificity. This finding agreed with studies performed by [34,9] . However, the result of the sensitivity may have increased if the distance between the inhibitor and the substrate was 15 mm instead of 20mm as reported by.…”

Isolation and molecular characterization of New Delhi metallo-beta-lactamase-1 producing superbug in Bangladesh

“…Double-disk synergy tests (DDS) [17] and combined disk (CD) assays [18] were performed to screen MBLs producers. An increased zone diameter of ≥ 6 mm around the disk containing imipenem supplemented with EDTA compared to the disk containing imipenem alone was interpreted as MBLs production.…”

A study on metallo--lactamases producing Pseudomonas aeruginosa in water samples from various parts of Malaysia

“…Modified Hodge test detects only carbapenemase activity, which prevents the use of EDTA and therefore, does not confirm the metal dependence of the carbapenemase (Kali et al, 2013). Modified Hodge test, DDST and the CDT are easy, reliable, simple to perform and cheaper (Qu et al, 2009). The CDT is the most sensitive techniques for detecting MBL compared to DDST (Biradar and Roopa, 2015).…”