Metallo-β-Lactamase Detection: Comparative Evaluation of Double-Disk Synergy versus Combined Disk Tests for IMP-, GIM-, SIM-, SPM-, or VIM-Producing Isolates

Abstract

The emergence of metallo-␤-lactamase (MBL)-producing isolates is a challenge to routine microbiology laboratories, since there are no standardized methods for detecting such isolates. The aim of this study was to evaluate the accuracy of different phenotypic methods to detect MBL production among Pseudomonas spp., Acinetobacter spp., and enterobacterial isolates, including GIM, IMP, SIM, SPM, and VIM variants. A total of 46 genetically unrelated Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter sp., and enterobacterial strains producing distinct MBLs were tested. Nineteen strains were included as negative controls. The inhibition of bacterial growth and ␤-lactam hydrolysis caused by MBL inhibitors (IMBL) also were evaluated. The isolates were tested for MBL production by both a double-disk synergy test (DDST) and a combined disk assay (CD) using imipenem and ceftazidime as substrates in combination with distinct IMBL. One hundred percent sensitivity and specificity were achieved by DDST using 2-mercaptopropionic acid in combination with ceftazidime and imipenem for the detection of MBL production among P. aeruginosa and Acinetobacter species isolates, respectively. The CD test showed the same results for detecting MBL-producing enterobacteria by combining imipenem and EDTA, with a 5.0-mm-breakpoint increase in the size of the inhibition zone. Our results indicate that both phenotypic methods to detect MBL-producing isolates should be based on the genera to be tested, regardless of the enzyme produced by such isolates, as well as on the local prevalence of MBL producers.Since the early 1990s, new metallo-␤-lactamase (MBL)-encoding genes have been reported all over the world in clinically important pathogens, such as Pseudomonas spp., Acinetobacter spp., and members of the Enterobacteriaceae family (19,27,35,40). The emergence of MBL-encoding genes is worrisome, since they usually are carried by mobile genetic structures with great ability to spread (3,5,18,24,36). Moreover, increased mortality rates have been documented for patients infected with MBL-producing Pseudomonas aeruginosa, especially due to inadequate empirical therapy (39). Therefore, early detection of MBL-producing organisms is crucial to establish appropriate antimicrobial therapy and to prevent their inter-and intrahospital dissemination (10, 35).Several phenotypic methods based on MBL inhibition by EDTA or thiol-based compounds have been published. Although they are simple to perform and cheaper than genotypic methods, they have shown discordant results depending on the employed methodology, ␤-lactam substrates, MBL inhibitors (IMBL), and bacterial genus tested (11,14,17,21,26). In addition, SPM-, GIM-, and SIM-producing pathogens rarely have been evaluated by these studies.The high diversity and prevalence of MBL-producing P.aeruginosa, Acinetobacter spp., and Enterobacteriaceae isolates have motivated the search for an accurate MBL screening test. The aim of this study was to evaluate the accuracy of the double-disk synergy test (DDST) ...

Detection of SPM and IMP metallo-β-lactamases in clinical specimens of Pseudomonas aeruginosa from a Brazilian public tertiary hospital

“…9 However, we can wonder how much longer these bacteria will persist susceptible to these remaining drugs if this selective pressure goes on. Comparing different phenotypic tests to detect MBL, we found that ceftazidime disk and mercaptopropionic acid association, in a double disk synergy test, was the most sensitive and specific assay, as it was reported by Picão et al 7 We observed that all other combinations of substrata and inhibitors (imipenem and mercaptopropionic acid; imipenem and mercaptoacetic acid; imipenem and EDTA; ceftazidime and mercaptoacetic acid; ceftazidime and EDTA) showed maximum specificities (100%), but low sensitivity to these combinations (zero to 71.4%), restricting their utilization. In spite of the high cost, MBL E-test strips can be employed to detect the MBL-producing P. aeruginosa due its high sensibility and specificity.…”

“…Colistin-only susceptible P. aeruginosa is a relatively contemporary issue, once carbapenems remained as effective therapeutic choice until the carbapenemases emergence on the last two decades. 7,8,14 Investigating MBL-encoding genes, we found isolates carrying bla SPM and bla IMP genes that together correspond to 29.8% of evaluated samples. In Brazil, the dissemination of P. aeruginosa carrying bla SPM gene has been reported throughout from several regions, 15,16 and SPM is the prevalent MBL among the isolates, although other enzymes have also been described, but with low frequency.…”

“…MICs were determined (in duplicate) by microdilution broth method for imipenem (ABL), meropenem (Astra Zeneca) and ceftazidime (NovaFarma); quality controls were performed with Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 strains. MBL phenotypic detection was evaluated by the double-disk synergy test (DDST) with imipenem or ceftazidime disks (Oxoid) as substrata, and mercaptopropionic acid (Sigma-Aldrich), mercaptoacetic acid (Sigma-Aldrich), or ethylenediamine tetra-acetic acid (EDTA, Sigma-Aldrich) as MBL inhibitors, according to Picão et al 7 Genotypic detection targeting to the bla SPM and bla IMP genes was carried out by polymerase chain reaction (PCR), according to protocols published elsewhere. 9 Positive and negative quality control strains were included to assess the quality of phenotypic and genotypic assays.…”

“…Early identification of MBL in clinical specimens is important to provide correct antimicrobial therapy guidance, once appropriate therapy implementation can nullify the worst prognostic of MBL producing P. aeruginosa. 3 When detected, it is possible to avoid the dissemination of such strains among patients [7][8][9] and to avoid the MBL genetic determinants spread to different species of Gram-negative bacteria, as Enterobacteriaceae. 6 The contention of MBL producing P. aeruginosa is highly recommended, because patient-to-patient transmission may lead these strains to be endemic in such institution 3 or among different hospitals.…”

Prevalence of ESBL and MBL Producing Pseudomonas aeruginosa in a Tertiary Care Hospital in Tirunelveli

“…Detection of MBL producing isolates by PCR is expensive, requires specialized technicians and instruments, and more importantly, is able to detect only previously described MBL-encoding genes (Picão et al, 2008). The development of simple screening tests to detect acquired MBL production is a crucial step towards large scale monitoring of these emerging resistant determinants (Manoharan et al, 2010).…”