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.

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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...

Some of the previously reported clinical isolates of Elizabethkingia meningoseptica may be later named species of Elizabethkingia. We determined the accuracy of species identification (with two matrix-assisted laser desorption ionizationtime of flight mass spectrometry [MALDI-TOF MS] systems and the Vitek 2 GN card), relative prevalence of three Elizabethkingia spp. in clinical specimens, and antimicrobial susceptibility of the species identified by 16S rRNA gene sequencing. Specimens for culture were collected from patients in a university hospital in Seoul, South Korea, between 2009 and 2015. All 3 Elizabethkingia spp. were detected in patients; among the 86 isolates identified by 16S rRNA gene sequencing, 17 (19.8%) were E. meningoseptica, 18 (20.9%) were Elizabethkingia miricola, and 51 (59.3%) were Elizabethkingia anophelis. Only the MALDI-TOF Vitek MS system with an amended database correctly identified all of the isolates. The majority (76.7%) of the isolates were from the lower respiratory tract, and 8 (9.3%) were from blood. Over 90% of E. meningoseptica and E. anophelis isolates were susceptible to piperacillin-tazobactam and rifampin. In contrast, all E. miricola isolates were susceptible to fluoroquinolones except ciprofloxacin. Further studies are urgently needed to determine the optimal antimicrobial agents for the treatment of infections due to each individual Elizabethkingia species.KEYWORDS Elizabethkingia meningoseptica, Elizabethkingia miricola, Elizabethkingia anophelis, antimicrobial susceptibility, 16S rRNA gene sequencing E lizabethkingia species are aerobic, nonmotile, oxidase-positive, indole-positive, Gram-negative bacilli that do not ferment glucose. Elizabethkingia spp. can be found frequently in soil, freshwater, salt water, and in hospital environments (1). However, they do not normally exist in the human body. Elizabethkingia meningoseptica (formerly Chryseobacterium meningosepticum) has been a well-known human pathogen since its first description in a case of neonatal meningitis by Elizabeth O. King in 1959 (2). This organism was reported to cause various invasive infections in immunocompromised hosts and to be associated with nosocomial infections and outbreaks in intensive care units (ICUs) (3-5). It has been considered that the incidence of E. meningoseptica bacteremia has increased over the last decade (6). Two new species of Elizabethkingia, Elizabethkingia miricola and Elizabethkingia anophelis, were proposed in 2003 and 2011, respectively (7-9). Therefore, some of the previously reported clinical isolates of E. meningoseptica may be later named species of Elizabethkingia. The first