BACKGROUND: Accurate and rapid detection of extended-spectrum beta-lactamases (ESBLs) is important in guiding proper antimicrobial therapy for infected patients. We evaluated the performance of MicroScan NegCombo Type 44 panel (Dade Behring, USA), which was developed to confirm ESBL-producing Enterobacteriaceae using ceftazidime/clavulanate and cefotaxime/clavulanate. METHODS: From August 30 to September 20, 2007, 206 non-duplicate clinical isolates, including 106 Escherichia coli, 81 Klebsiella pneumoniae, 11 Klebsiella oxytoca, and 8 Proteus mirabilis were subcultured and tested with Type 32 and Type 44 panels. The results were compared with those of the CLSI phenotypic confirmatory test (CLSI-PCT) and disk approximation test (DAT). Isolates not susceptible to cefotetan or flagged as "Possible ESBL, unable to interpret confirm test (Possible ESBL)" on Type 44 panel were tested with boronic acid disks to confirm AmpC beta-lactamases (AmpC) production. RESULTS: Of the 206 isolates tested, 44 (21.4%) produced ESBL by CLSI-PCT or DAT, including 27 E. coli, 14 K. pneumoniae, 2 K. oxytoca, and 1 P. mirabilis. Thirty-eight isolates flagged as "Confirmed ESBL" on Type 44 panel were all confirmed as ESBL-producers. Of 14 K. pneumoniae flagged as "Possible ESBL", 6 were confirmed as ESBL and AmpC co-producers and 8 as AmpC-producers. CONCLUSIONS: Type 44 panel showed an excellent performance in detecting ESBL-producing E. coli, Klebsiella spp., and P. mirabilis. When flagged as "Confirmed ESBL", no other confirmatory test was necessary to report as ESBL; however, "Possible ESBL" required a differential test for AmpC production.
Bacterial Proteins/*biosynthesis ; Cefotetan/pharmacology ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Bacterial ; Escherichia coli/*enzymology/isolation & purification ; Humans ; Klebsiella/*enzymology/isolation & purification ; Proteus mirabilis/*enzymology/isolation & purification ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; beta-Lactamases/*biosynthesis

BACKGROUND: The aim of this study was to compare the BD Phoenix (Beckton Dickinson Diagnostic Systems, USA) extended-spectrum beta-lactamase (ESBL) test with the Clinical and Laboratory Standards Institute (CLSI) ESBL phenotypic confirmatory test by disk diffusion (CLSI ESBL test) in Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Proteus mirabilis. METHODS: We tested 224 clinical isolates of E. coli, K. pneumoniae, K. oxytoca and P. mirabilis during May 2006 to March 2007. These isolates were examined by the Phoenix and the CLSI ESBL tests simultaneously. For the isolates showing discordant results between the two tests, boronic acid disk test was performed to differentiate AmpC beta-lactamase and ESBL. RESULTS: Among the 224 clinical isolates, 75 and 79 isolates were positive for ESBL by CLSI ESBL test and Phoenix test, respectively. Having detected 4 more isolates as ESBL-producers, Phoenix test showed a 98.2% agreement with a 100% sensitivity and 97.3% specificity compared with CLSI ESBL test. Among the four false positive isolates, three were AmpC-positive but ESBL-negative. CONCLUSIONS: The BD Phoenix ESBL test was sensitive and specific, and can be used as a rapid and reliable method to detect ESBL production in E. coli, Klebsiella species, and P. mirabilis.
Automation ; Bacterial Proteins/classification/*metabolism ; Disk Diffusion Antimicrobial Tests ; Escherichia coli/drug effects/*enzymology/isolation & purification ; Humans ; Klebsiella/*enzymology ; Klebsiella oxytoca/drug effects/enzymology/isolation & purification ; Klebsiella pneumoniae/drug effects/enzymology/isolation & purification ; *Microbial Sensitivity Tests ; Proteus mirabilis/drug effects/*enzymology/isolation & purification ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; beta-Lactamases/classification/*metabolism

We investigated the occurrence and genetic basis of AmpC beta-lactamase (AmpC)-mediated antibiotic resistance, by examining Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a university hospital, from 2007 to 2010. The ampC genes were detected by multiplex AmpC PCR, and AmpC-positive strains were subjected to DNA sequencing. Extended-spectrum beta-lactamase (ESBL) production was assessed using the ESBL disk test based on the utilization of boronic acid. Carbapenem-resistant isolates were further investigated by the modified Hodge test, a carbapenemase inhibition test and SDS-PAGE experiments. AmpC expression was detected in 1.6% of E. coli (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, 7.2% of K. pneumoniae (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, and 2.5% of P. mirabilis (8 CMY-2 and 1 CMY-1) isolates. Of the 198 acquired AmpC producers, 58 isolates (29.3%) also produced an ESBL enzyme. Among the acquired AmpC-producing K. pneumoniae isolates, the minimum inhibitory concentration (MIC) MIC50/MIC90 values for cefoxitin, cefotaxime, cefepime, imipenem, and meropenem were >32/>32, 16/>32, 1/16, 0.25/0.5, and <0.125/0.125 microg/mL, respectively. The MIC values for carbapenem were > or =2 microg/mL for 2 K. pneumoniae isolates, both of which carried the blaDHA-1 gene with a loss of OmpK36 expression, but were negative for carbapenemase production. The acquisition of AmpC-mediated resistance in K. pneumoniae isolates increased, as did the proportion of AmpC and ESBL co-producers among the hospital isolates. The accurate identification of isolates producing AmpCs and ESBLs may aid in infection control and will assist physicians in selecting an appropriate antibiotic regimen.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics ; DNA, Bacterial/genetics ; Enterobacteriaceae Infections/*epidemiology/*microbiology ; Escherichia coli/drug effects/enzymology/isolation & purification ; Hospitals, University/statistics & numerical data ; Humans ; Klebsiella pneumoniae/drug effects/enzymology/isolation & purification/*physiology ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Proteus mirabilis/drug effects/enzymology/isolation & purification ; Republic of Korea/epidemiology ; beta-Lactamases/*genetics