Identification of Plasmid-Mediated AmpC -Lactamases in Escherichia coli, Klebsiella spp., and Proteus Species Can Potentially Improve Reporting of Cephalosporin Susceptibility Testing Results

The goal of this study was to determine if the interpretations of extended-spectrum and advanced-spectrum cephalosporins (ESCs and ASCs, respectively) for isolates of Enterobacteriaceae would be impacted by the results of aminophenylboronic acid (APBA) testing. Fifty-three isolates of Escherichia coli , 21 Klebsiella species, and 6 Proteus species that were resistant to at least one ESC were tested by disk diffusion with ceftazidime and cefotetan disks with and without APBA. Ceftazidime disks with and without clavulanic acid (CLAV) were also tested to conﬁrm extended-spectrum (cid:1) -lactamase (ESBL) carriage. Twenty-nine (36.3%) isolates were only APBA test positive, 27 were only CLAV test positive, 2 were positive with both substrates, and 22 were negative with both substrates. Thirteen (41.9%) of the 31 APBA-test-positive isolates (all E. coli ) tested susceptible to cefotaxime, ceftriaxone, or ceftazidime. Since clinical data suggest that AmpC-producing isolates should be reported as resistant to all ESCs, APBA testing can be helpful in identifying such organisms. Screening for AmpC-producing organisms using nonsusceptibility to cefoxitin and amoxicillin-clavulanate was less speciﬁc than APBA testing; it identiﬁed ESBL as well as AmpC-producing organisms. Only 18 of 31 APBA-positive isolates were positive by PCR for an AmpC (cid:1) -lactamase gene. Thus, testing with APBA could improve the accuracy of reporting ESCs, especially for E. coli . However, results of APBA and CLAV testing did not correlate well for isolates containing both AmpC (cid:1) -lactamases and ESBLs. Thus, additional data are needed before formal recommendations can be made on changing the reporting of ASC test results. screened for ESBL production by disk diffusion with both CAZ and CTX, with and without CLAV. Thus, in this study we screened only with disks containing CAZ and CAZ plus CLAV to ensure that the ESBL type was consistent with previous results. All ESBL results were consistent). Plates were incubated for 18 to 20 h in ambient air at 35°C. Zone diameter differences of (cid:1) 5 mm between the CTT or CAZ disks without APBA and those supplemented with APBA were considered positive for AmpC (cid:2) -lactamases, as previously described (9, 12, 33). control strains for APBA and PCR testing are listed above. E. 25922. ESBL CAZ CLAV, CLSI Molecular characterization of (cid:1) -lactamase genes. Organisms were tested for of six families of plasmid-mediated AmpC (cid:2) -lactamase genes using the multiplex PCR assay by Perez-Perez and This included the genes within the following families: ACC, CIT (including LAT-1 to LAT-4, CMY-2 to CMY-7, and BIL-1), DHA (DHA-1 and DHA-2), EBC (including MIR-1T and ACT-1), FOX (including FOX-1 to FOX-5b), and MOX (including MOX-1, MOX-2, CMY-1, and CMY-8 to CMY-11). Positive- and negative-control organisms were included in each aIEF. Analytical isoelectric focusing (aIEF) was performed to determine the pIs of ß-lactamases from isolates, as previously described (22, 34). Brieﬂy, elec-trophoresis was performed on precast polyacrylamide gels, either pH 3 to pH (Bio-Rad Inc.) or pH 3.5 to 9.5