Rapid detection of metallo-␤-lactamase (MBL)-producing gram-negative bacilli is necessary to prevent their dissemination. The method using a disk with imipenem plus 750 g of EDTA differentiated all MBL-producing pseudomonads, and the sensitivity and specificity for acinetobacters were 95.7 and 91.0%, respectively. The imipenem-EDTA disks were stable for 12 and 16 weeks at 4 and ؊20°C, respectively.An increasing prevalence of carbapenem resistance mediated by acquired metallo-␤-lactamases (MBLs) is being reported, particularly for Pseudomonas aeruginosa clinical isolates in several countries (4, 6, 8, 9, 11-14, 17, 18). In Korea, approximately 10 and 50% of imipenem resistance in P. aeruginosa (8) and Acinetobacter spp. (19), respectively, are due to MBL production. The resistance may spread rapidly to various species of gram-negative bacilli, as the MBL genes reside in mobile gene cassettes inserted in integrons (3). The rapid detection of MBL-positive gram-negative bacilli is necessary to aid infection control and to prevent their dissemination (5). A PCR method was simple to use in detecting MBL-producing isolates initially (16), but it became more difficult with the increased number of types of MBLs.MBL activity is inhibited by chelating agents. Double-disk synergy tests using a ceftazidime disk and a 2-mercaptopropionic acid disk (1), or an imipenem disk and an EDTA disk (7), have been reported as a simple method to detect MBL-producing clinical isolates. However, occasional adjustment of the distance between the two disks is required to obtain optimal results (1, 7), as is the case with the double-disk test for the detection of extended-spectrum ␤-lactamase-producing isolates (2). For the phenotypic confirmation of extended-spectrum-␤-lactamase-producing isolates, inhibition zones are compared by using both ceftazidime and cefotaxime disks with and without clavulanic acid (10). The aim of this study was to determine the feasibility of using an imipenem disk with added EDTA to confirm MBL-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp.The MBL-producing gram-negative bacilli used in this study were 102 isolates of P. aeruginosa, 14 of Pseudomonas putida, 20 of Acinetobacter baumannii, and 3 of Acinetobacter genomospecies 3. All of the isolates were VIM-2 MBL producers except for one isolate of P. aeruginosa and five isolates of acinetobacters, which were IMP-1 MBL producers. MBL genes were detected by PCR, and MBL production was detected by the imipenem-EDTA double-disk synergy test as described previously (8). Imipenem-resistant or -intermediate but non-carbapenemase-producing isolates were included for comparison.Test organisms were inoculated onto plates of Mueller-Hinton agar (Becton Dickinson, Cockeysville, Md.) as recommended by the National Committee for Clinical Laboratory Standards (10). A 0.5 M EDTA solution was prepared by dissolving 186.1 g of disodium EDTA ⅐ 2H 2 O (Junsei Chemical, Tokyo, Japan) in 1,000 ml of distilled water and adjusting it to pH 8.0 by using NaOH. The m...

Gram-negative bacilli with acquired metallo-␤-lactamase (MBL) production have been increasingly reported in some countries, necessitating their detection. The aim of this study was to evaluate the performance of the Hodge test and those of the imipenem (IPM)-EDTA, ceftazidime (CAZ)-mercaptopropionic acid (MPA), and CAZ-sodium mercaptoacetic acid (SMA) double-disk synergy tests (DDSTs). The efficiencies of testing CAZ-resistant and IPM-nonsusceptible isolates were also compared. Strains used for the evaluation were known IMP-1 and VIM-2 MBL-producing isolates and consecutive and CAZ-nonsusceptible isolates of pseudomonads and acinetobacters. The performance of the Hodge test was improved by addition of zinc sulfate (140 g/disk) to an IPM disk. In DDSTs, EDTA (ca. 1,900 g) disks were better at detecting MBL-producing strains among pseudomonads, while MPA (3 l) and SMA (3 mg) disks performed better for acinetobacters. EDTA (ca. 750 g)-plus-SMA (ca. 2 mg) disks performed better than EDTA, MPA, or SMA disks with both organisms. CAZ-SMA DDSTs failed to detect 22 of 80 (28%) MBL-producing acinetobacters. In conclusion, use of an IPM disk and an EDTA (750 g)-plus-SMA (2 mg) disk improves performance, and testing IPMnonsusceptible isolates rather than CAZ-resistant isolates could reduce screening work. Further evaluation of the test is required for the detection of other types of MBL-producing gram-negative bacilli.

No abstract

Carbapenem resistance mediated by acquired carbapenemase genes has been increasingly reported, particularly for clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. Of 1,234 nonduplicate isolates of carbapenem-resistant Pseudomonas spp. and Acinetobacter spp. isolated at a tertiary-care hospital in Seoul, Korea, 211 (17%) were positive for metallo-␤-lactamase (MBL). Of these, 204 (96%) had either the bla IMP-1 or bla VIM-2 allele. In addition, seven Acinetobacter baumannii isolates were found to have a novel MBL gene, which was designated bla SIM-1 . The SIM-1 protein has a pI of 7.2, is a new member of subclass B1, and exhibits 64 to 69% identity with the IMP-type MBLs, which are its closest relatives. All SIM-1-producing isolates exhibited relatively low imipenem and meropenem MICs (8 to 16 g/ml) and had a multidrug resistance phenotype. Expression of the cloned bla SIM-1 gene in Escherichia coli revealed that the encoded enzyme is capable of hydrolyzing a broad array of ␤-lactams, including penicillins, narrow-to expanded-spectrum cephalosporins, and carbapenems. The bla SIM-1 gene was carried on a gene cassette inserted into a class 1 integron, which included three additional cassettes (arr-3, catB3, and aadA1). The strains were isolated from sputum and urine specimens from patients with pneumonia and urinary tract infections, respectively. All patients had various underlying diseases. Pulsed-field gel electrophoresis of SmaI-digested genomic DNAs showed that the strains belonged to two different clonal lineages, indicating that horizontal transfer of this gene had occurred and suggesting the possibility of further spread of resistance in the future.Gram-negative bacilli have a propensity to develop and acquire resistance to multiple antimicrobials. A significant increase in the prevalence of multidrug-resistant gram-negative bacilli, even among isolates recovered at admission, was reported (20). Carbapenems have been the most successful ␤-lactam antibiotics in evading bacterial resistance (14). However, carbapenem resistance, mediated by acquired carbapenemase genes, has been increasingly reported, particularly for clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. (7). Although some enzymes of molecular classes A and D can hydrolyze carbapenems, metallo-␤-lactamases (MBLs) are the most prevalent acquired carbapenemases (5,14,18,24,27,28).Of acquired MBLs, the IMP-and VIM-type enzymes are the most common and exhibit a worldwide distribution (7,8,19,24,26). Recently, however, two additional types, SPM and GIM, have been reported for P. aeruginosa isolates from Brazil and Germany, respectively (2, 22). In Korea, a prevalence of both IMP-1-and VIM-2-type MBLs has been reported (10, 12). Here we report the detection of a new acquired MBL in clinical isolates of Acinetobacter baumannii from Korea. MATERIALS AND METHODSBacterial strains. A total of 1,234 nonduplicate imipenem-resistant clinical isolates of Pseudomonas spp. and Acinetobacter spp. isolated in 2003-2004 at a tertiary-ca...

We investigated the phenotypic and genetic properties of metallo-␤-lactamase-producing Pseudomonas isolates collected at a tertiary-care hospital in Korea since 1995. The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates reached 16% in 1997, when 9% of the resistant organisms were found to produce VIM-2 ␤-lactamase, a class B enzyme previously found only in P. aeruginosa isolates from Europe. VIM-2-producing isolates of Pseudomonas putida were also detected. Resistance was transferable from both these species to P. aeruginosa PAO4089Rp by filter mating, although the resistance determinant could not be found on any detectable plasmid. Serotyping showed that many of the VIM-2-producing P. aeruginosa isolates belonged to serotypes O:11 and O:12, and pulsed-field gel electrophoresis of XbaI-digested genomic DNA revealed that many had identical profiles, whereas the P. putida isolates were diverse. Sequencing showed that the bla VIM-2 genes resided as cassettes in class 1 integrons. In contrast to previous VIM-encoding integrons, the integron sequenced from a P. aeruginosa isolate had bla VIM located downstream of a variant of aacA4. bla VIM also lay in a class 1 integron in a representative P. putida strain, but the organization of this integron was different from that sequenced from the P. aeruginosa strain. In conclusion, the metallo-␤-lactamase produced by these imipenem-resistant Pseudomonas isolates was VIM-2, and the accumulation of producers reflected clonal dissemination as well as horizontal spread. Strict measures are required in order to control a further spread of resistance.Carbapenems such as imipenem are stable to most ␤-lactamases. Nevertheless, the first isolate of Pseudomonas aeruginosa with transferable imipenem resistance due to a metallo-␤-lactamase production was reported in Japan in 1991 (26). The enzyme produced by this isolate almost certainly was IMP-1, which subsequently has been repeatedly found in Japan, not only in P. aeruginosa and Acinetobacter baumannii (23), but also in Serratia marcescens (14) and other members of Enterobacteriaceae (5). Rasmussen and Bush (18) predicted that the increasing use of carbapenems would select for more carbapenem-hydrolyzing organism on a wider scale, and this prediction is now proving correct. Several close relatives of IMP-1 have been reported during the past 3 years: IMP-2 from an isolate of A. baumannii in Italy (19), IMP-3 (MET-1) from Shigella flexneri in Japan (6), and IMP-4 from Acinetobacter and Citrobacter spp. from Hong Kong, China (3).Descriptions of IMP-5 to IMP-8 enzymes are understood to be in press. IMP-1 to IMP-4 have at least 80% homology to each other, but in 1999, a new type of acquired metallo-␤-lactamase, VIM-1, was reported to have been found in an isolate of P. aeruginosa collected in Italy (8). Soon afterwards, isolates of P. aeruginosa with a related enzyme, VIM-2, were reported in France (16,17). Since then, outbreaks of VIM ␤-lactamaseproducing P. aeruginosa have been reported in Greece (25) and Italy (...