Bum Sik Chin, et al. β-lactam antibiotics. Particularly, the emergence of ac- quired metallo-β-lactamase (MBL) among major gram- negative pathogens [Pseudomonas aeruginosa (PA), Aci- netobacter spp., Enterobacteriaceae] is a matter of particular concern on account of their rapid spread and increasing di- versity/number of species involved.1 However, limited clin- ical data are available regarding to the clinical significance of infections caused by these strains. Several studies have demonstrated that MBL producer (MP) acquisition is asso- ciated with increased mortality in PA infection.2-5 However, carbapenem susceptible strains are included in MBL non- producer (MNP) in most studies, thus revealing higher mortality in the MBL producing group. It is well known that acquisition of carbapenem resistance is associated with increased mortality in PA and Acinetobacter baumannii (AB) infections.6,7 Therefore, to characterize the MBL pro- duction itself rather than carbapenem resistance, we com- pared MBL producing PA/AB and imipenem-nonsuscepti- ble MNPs (INMNP) isolated from respiratory and urinary tracts which are common sites of clinical infections and sources of nosocomial spread. The microbiology laboratory database was reviewed, and we selected imipenem-nonsusceptible PA and AB which had been isolated from sputum or urine for the purpose of microbiologic diagnosis irrespective of hospitalization be- tween January 2007 and June 2007 in a single 2,000-bed tertiary hospital in Seoul, Republic of Korea. Clinical data for the source patients of the isolates were collected by a retrospective analysis of the electronic medical records, and patients were excluded from the analysis if medical records prior to the isolation were not sufficient (e.g., transfer from another hospital). Patients without available follow-up data after discharge were also excluded from the analysis. Iso- lates referred from outpatient departments (OPD) were in- cluded if sufficient medical history was available. All the isolates that had been related with both clinical infections or colonization of the respiratory or urinary tracts were con- sidered eligible for the study. Only the first isolate from each patient was included in the analysis. The PA/AB strains were identified using conventional techniques and/or ATB 32 GN system (BioMérieux, Mar- cy-l’Étoile, France).8 The antimicrobial susceptibility was determined using a disk-diffusion method. Results were in- terpreted using the guidelines of the Clinical and Laborato- ry Standards Institute.9 Among the imipenem-nonsusceptible isolates, carbapen- emase production was screened by the imipenem disk Hodge (cloverleaf) test, using MacConkey agar instead of the pre- viously used Mueller-Hinton agar.10 MBL production was screened by the double-disk synergy test using an imipen- em disk and an EDTA (750 μg) plus sodium mercaptoace- tic acid (SMA, 2 mg) disk on Mueller-Hinton agar with 10 mm distance from the edge to the edge of the disk.10 Com- mercial imipenem disks and media (Becton-Dickinson, Sparks, MD, USA) were used for these tests, while the EDTA-SMA disks were prepared from commercially avail- able chemicals (Sigma Chemical, St. Louis, MO, USA). The blaIMP-1, blaVIM-2 and blaSIM-1 alleles were detected by polymerase chain reaction (PCR) among the screening pos- itive isolates as described previously11 and the primers used were: IMP1-F 5’-CAT GGT TTG GTG GTT CTT GT-3’, IMP1-R 5’-ATA ATT TGG CGG ACT TTG GC-3’, VIM2- F 5’-ATG TTC AAA CTT TTG AGT AAG-3’, VIM2-R 5’-CTA CTC AAC GAC TGA GCG-3’, SIM1-F 5’-TAC AAG GGA TTC GGC ATC G-3’ and SIM1-R 5’-TAA TGG CCT GTT CCC ATG TG-3’. To perform the pulsed field gel electrophoresis (PFGE), genomic DNA of MBL-producing PA and AB was digested with XbaI and SmaI, respectively, as suggested by the man- ufacturer. Fragments were separated for 20 hours at 6 V/cm at 11°C using a CHEF-DR II system (Bio-Rad, Hercules, CA, USA). ProMega-Markers Lambda Ladders G3011 (Promega, WI, USA) was used for molecular mass marker. To identify risk factors contributing to MBL producer ac- quisition, we reviewed age, gender, Charlson index, APA- CHE II score, underlying illness, the entire hospital stay pe- riod including multiple admissions within six months prior to isolation, intensive care unit (ICU) stay, ventilator care, and antibiotics exposure prior to isolation such as piperacillin/ tazobactam, cefoperazone/sulbactam, aztreonam, fluoroqui- nolones, aminoglycosides, glycopeptides, ceftazidime, and carbapenems. ICU stay, ventilator care and antibiotic expo- sure were included when each factor lasted more than 48 hours within 28 days prior to isolation. Neutropenia (abso- lute neutrophil count < 1,000 mm3), immunosuppression (administration of cytostatic agent or glucocorticoid equiv- alent or more potent than methylprednisolone 20 mg per day for more than 14 days), and antineoplastic treatment within 3 months prior to the isolation were also investigat- ed. To assess the outcome, 28-day case-fatality after isola- tion and in hospital case-fatality were analyzed. Statistical analyses were performed by SAS version 9.1.3 (SAS Insti- tute, Inc., Cary, NC, USA). All tests were two-tailed, and a p value < 0.05 was considered significant. 352 Yonsei Med J http://www.eymj.org Volume 52 Number 2 March 2011