Non-beta-lactam inhibitor-based methods were evaluated for detecting plasmid-mediated AmpC ␤-lactamases in Klebsiella spp., Escherichia coli, and Proteus mirabilis. Using CLSI methodology and disks containing cefotetan alone and in combination with 400 g of boronic acid, 9 of 10 positive control strains and 54 of 55 AmpC-PCR-positive clinical isolates were detected. Importantly 71% and 40% of these clinical isolates were susceptible by routine testing to ceftriaxone and ceftazidime, respectively. Boronic acid disks also enhanced detection of expanded-spectrum ␤-lactamases in AmpC producers.Pai et al. recently demonstrated a high rate of clinical failure among patients who were infected in the bloodstream with AmpC-␤-lactamase-producing Klebsiella pneumoniae and who received initial antimicrobial therapy, especially cephalosporin treatment (21). Of eight patients who were infected with organisms harboring DHA-1-like or CMY-1-like ␤-lactamases and who received initial and definitive therapy with cefotaxime or ceftazidime and no imipenem, six died. In contrast, 12 of 14 patients who were infected with organisms harboring one of the same two ␤-lactamases but who received imipenem as definitive therapy were cured. Clearly detection of AmpCproducing organisms is important to ensure effective therapeutic intervention and optimal clinical outcome. Imported ampC genes have also been found on the plasmids of other organisms that usually do not harbor these genes, such as Klebsiella oxytoca, Escherichia coli, and Proteus mirabilis (24). Some organisms may harbor plasmid-mediated expanded-spectrum ␤-lactamases (ESBLs) and AmpC ␤-lactamases.Currently, no guidelines for detection of plasmid-mediated AmpC-producing organisms or organisms harboring multiple ␤-lactamases are available. A study was designed to detect AmpC ␤-lactamase-producing isolates of Klebsiella spp., E. coli, and P. mirabilis by methods that closely resemble the phenotypic confirmatory tests for ESBLs (4). Two novel methods that use boronic acid, a known class-C enzyme inhibitor (7), were first tested against positive and negative control strains and then applied as confirmatory tests to clinical isolates.Positive control strains produced the following plasmid-mediated AmpC ␤-lactamases: MOX-1, LAT-2, DHA-1, DHA-2, ACC-1, MIR-1, ACT-1, FOX-1, and FOX-5b. This group included one or more enzymes from each of six families of plasmid-mediated AmpC ␤-lactamases recently selected by Perez-Perez and Hanson, who developed a PCR technique to identify family-specific ampC genes (23). Strains used as negative controls included an OMP F(-) strain, an OMP C(-) strain, and strains that produced the following ␤-lactamases: CTX-M-5, CTX-M-9, TEM-3, TEM-12, SHV-2, SHV-5, SHV-18 (K. pneumoniae ATCC 700603), OXA-3, and KPC-2. A K1-hyperproducing strain of K. oxytoca was also tested.Clinical isolates were recovered at the McGuire Veterans Affairs Medical Center and identified by the Vitek and the API 20E systems (bioMerieux Vitek, Hazelwood, Mo.). Isolates were screened for cef...

Hospital formulary restriction of clindamycin is an effective way to decrease the number of infections due to C. difficile. It can also lead to a return in clindamycin susceptibility among isolates and can effect cost savings to the hospital.

AmpC beta-lactamases are cephalosporinases that confer resistance to a wide variety of β-lactam drugs and that may thereby create serious therapeutic problems. Although reported with increasing frequency, the true rate of occurrence of AmpC beta-lactamases inEscherichia coli, Klebsiella pneumoniae, andProteus mirabilis remains unknown. We tested a total of 1,286 consecutive, nonrepeat isolates of these three species and found that, overall, 45 (3.5%) yielded a cefoxitin zone diameter less than 18 mm (screen positive) and that 16 (1.2%) demonstrated AmpC bands by isoelectric focusing. Based on the species, of 683 E. coli, 371 K. pneumoniae, and 232 P. mirabilisisolates tested, 13 (1.9%), 28 (7.6%), and 4 (1.7%), respectively, demonstrated decreased zone diameters and 11 (1.6%), 4 (1.1%), and 1 (0.4%), respectively, demonstrated AmpC bands. Cefoxitin resistance was transferred for all but 8 (E. coli) of the 16 AmpC producers. We also describe a three-dimensional extract test, which was used to detect phenotypically isolates that harbor AmpC beta-lactamase. Of the 45 cefoxitin-resistant isolates, the three-dimensional extract test accurately identified all 16 AmpC producers and 28 of 29 (97%) isolates as non-AmpC producers. Interestingly, most (86%) isolates in the latter group were K. pneumoniae isolates. These data confirm that, at our institution, E. coli, K. pneumoniae, and P. mirabilis harbor plasmid-mediated AmpC enzymes.

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We tested 190 Klebsiella pneumoniae bloodstream isolates recovered from 189 patients in 30 U.S. hospitals in 23 states to determine the occurrence of extended-spectrum ␤-lactamase (ESBL) and AmpC ␤-lactamase producers. Based on growth inhibition by clavulanic acid by disk and MIC test methods, 18 (9.5%) of the isolates produced ESBLs. Although the disk diffusion method with standard breakpoints identified 28 cefoxitin-nonsusceptible isolates, only 5 (18%) of these were confirmed as AmpC producers. Of two AmpC confirmatory tests, the three-dimensional extract test was easier to perform than was the double-disk approximation test using a novel inhibitor, Syn2190. Three of the five AmpC producers carried the bla FOX-5 gene, while the other two isolates harbored the bla ACT-1 gene. All AmpC genes were transferable. In vitro susceptibility testing with standard inocula showed that all five AmpC-producing strains were susceptible to cefepime, imipenem, and ertapenem but that with a high inoculum, more of these strains were susceptible to the carbapenems than to cefepime. All but 1 of 14 screen-positive AmpC nonproducers (and ESBL nonproducers) were susceptible to ceftriaxone and cefepime at the standard inoculum as were 6 of 6 isolates that were randomly selected and tested with a high inoculum. These results indicate that (i) a significant number of K. pneumoniae bloodstream isolates harbor ESBL or AmpC ␤-lactamases, (ii) confirmatory tests are necessary to identify true AmpC producers, and (iii) in vitro, carbapenems are active against AmpC-producing strains of K. pneumoniae.