BACKGROUND: Plasmid-mediated AmpC beta-lactamases (PABLs) have been detected in the strains of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Salmonella spp. PABLs may be difficult to detect and might interfere in the therapeutic and infection-control processes. Although several PABL-detection methods based on phenotypes have been reported, the Clinical and Laboratory Standards Institute currently does not recommend a routine detection method for PABLs. The aim of this study is to compare the performances of 3 phenotypic PABL detection methods. METHODS: Total 276 non-duplicated clinical isolates of E. coli (N=97), K. pneumoniae (N=136), and P. mirabilis (N=43) were collected from 14 hospitals in Korea between April and June 2007 in a non-consecutive and non-random manner. Multiplex PCR was performed to detect the PABL genes. Further, 3 phenotypic detection methods-cephamycin-Hodge test, Tris-EDTA (TE) disk test, and combination-disk test with 3-aminophenylboronic acid (BA)-were performed using cefoxitin and cefotetan disks. RESULTS: PABL genes were detected by multiplex PCR in 122/276 isolates, including 14/97 E. coli, 105/136 K. pneumoniae, and 3/43 P. mirabilis isolates. The combination-disk test with BA showed higher sensitivity (98.4%), specificity (92.2%), and efficiency (96.3%) than the cephamycin-Hodge (76.2%, 96.1%, and 88.6%, respectively) and the TE-disk (80.3%, 91.6%, and 87.9%, respectively) tests. CONCLUSIONS: The combination-disk test with BA is a simple, efficient, and interpretable test that can be applicable in clinical laboratories involved in the detection of PABLs in clinical isolates of E. coli, K. pneumoniae, and P. mirabilis.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*analysis ; Cefotetan/pharmacology ; Cefoxitin/pharmacology ; Disk Diffusion Antimicrobial Tests/*methods ; Escherichia coli/genetics/*isolation & purification ; Humans ; Klebsiella pneumoniae/genetics/*isolation & purification ; Phenotype ; Plasmids ; Proteus mirabilis/genetics/*isolation & purification ; Sensitivity and Specificity ; beta-Lactamases/*analysis

BACKGROUND: We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-beta-lactamase (MBL)-producing Pseudomonas spp. METHODS: A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum beta-lactamase [GES]-5, 9 New Delhi metallo-beta-lactamase [NDM]-1, 5 Verona integron-encoded metallo-beta-lactamase [VIM]-2, 3 imipenem-hydrolyzing beta-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain. RESULTS: Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively. CONCLUSIONS: Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.
Bacterial Proteins/antagonists & inhibitors/*metabolism ; Boronic Acids/chemistry/pharmacology ; Disk Diffusion Antimicrobial Tests/*methods ; Edetic Acid/chemistry/pharmacology ; Enterobacteriaceae/drug effects/*enzymology ; Enterobacteriaceae Infections/diagnosis ; Humans ; Pseudomonas/drug effects/*enzymology ; Pseudomonas Infections/diagnosis ; Sensitivity and Specificity ; beta-Lactamases/chemistry/*metabolism

Production of extended-spectrum beta-lactamase (ESBL) is one of the most important resistance mechanisms that hamper the antimicrobial treatment of infections caused by Enterobacteriaceae. ESBLs are classified into several groups according to their amino-acid sequence homology. While TEM and SHV enzymes were the most common ESBLs in the 1990s, CTX-M enzymes have spread rapidly among Enterobacteriaceae in the past decade. In addition, some epidemiological studies showed that organisms producing CTX-M enzymes had become increasingly prevalent in the community setting in certain areas in the world. Several novel enzymes with hydrolyzing activity against oxyimino-cephalosporins, albeit with additional enzymatic characteristics different from those of original TEM and SHV ESBLs (e.g., inhibitor-resistance), have been discovered and pose a problem on the definition of ESBLs. Although several methods to detect the production of ESBL are available in clinical laboratories, existence of other factors contributing resistance against beta-lactams, e.g., inducible production of Amp-C beta-lactamase by some species of Enterobacteriaceae, or inhibitor-resistance in some ESBLs may hinder the detection of ESBLs with these methods. Carbapenems are stable against hydrolyzing activity of ESBLs and are regarded as the drug of choice for the treatment of infections caused by ESBL-producing Enterobacteriaceae. Although several other antimicrobial agents, such as fluoroquinolones and cephamycins, may have some role in the treatment of mild infections due to those organisms, clinical data that warrant the use of antimicrobial agents other than carbapenems in the treatment of serious infections due to those organisms are scarce for now.
Anti-Bacterial Agents/*pharmacology/therapeutic use ; Carbapenems/pharmacology/therapeutic use ; Disk Diffusion Antimicrobial Tests ; Enterobacteriaceae/drug effects/*enzymology/genetics ; Enterobacteriaceae Infections/*drug therapy/microbiology ; Fluoroquinolones/pharmacology/therapeutic use ; Humans ; Microbial Sensitivity Tests/methods ; beta-Lactamases/*biosynthesis/metabolism ; beta-Lactams/*pharmacology/therapeutic use