Despite the discovery of novel ␤-lactamases such as extended-spectrum ␤-lactamases (ESBLs), imported AmpC, and carbapenem-hydrolyzing ␤-lactamases at least a decade ago, there remains a low level of awareness of their importance and how to detect them. There is a need to increase the levels of awareness of clinical laboratories about the detection of newer ␤-lactamases. Therefore, a study was conducted in 2000 to investigate the occurrence of these ␤-lactamases in Klebsiella pneumoniae isolates at 24 U.S. medical centers. To enhance the likelihood of detecting imported AmpC and carbapenem-hydrolyzing ␤-lactamases, participating laboratories were permitted to include archived strains (1996 to 2000) that were intermediate or resistant to either cefoxitin or imipenem. The ␤-lactamase production of 408 isolates positive by screening of 1,123 isolates was investigated by ESBL phenotypic confirmation tests; and for AmpC and carbapenem-hydrolyzing ␤-lactamases, three-dimensional tests, isoelectric focusing, ␤-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular tests were used. ESBL-producing isolates were detected at 18 of the 24 sites (75%), imported AmpC-producing isolates were detected at 10 sites (42%), inducible imported AmpC-producing isolates were detected at 3 sites (12.5%), and a molecular class A carbapenem-hydrolyzing enzyme was detected at 1 site (4%). No class B or D carbapenem-hydrolyzing enzymes were detected. ESBLs and imported AmpC ␤-lactamases were detected at a significant number of sites, indicating widespread penetration of these enzymes into U.S. medical institutions. Because these enzymes may significantly affect therapeutic outcomes, it is vital that clinical laboratories be aware of them and be able to detect their occurrence.

Resistance to the extended-spectrum cephalosporins can occur in Salmonella species via the production of extended-spectrum and AmpC ␤-lactamases. We describe human infections with Salmonella enterica serotype Newport phage type 14 strains resistant to ceftazidime (CAZ) and cefoxitin (FOX) related to the handling of pet treats containing dried beef. These strains were isolated from five patients in Calgary, Alberta, Canada, during 2002 and were compared to a strain cultured from a commercial pet treat present at the property of one of the patients. The strains were resistant to FOX, CAZ, cefpodoxime, ampicillin, and chloramphenicol; intermediate resistant to ceftriaxone and cefotaxime; and sensitive to the aminoglycosides, ciprofloxacin, cefepime, and imipenem. Isoelectric focusing, multiplex PCR, and sequencing of the amplicons showed that all strains produced the plasmid-encoded AmpC ␤-lactamase, CMY-2. Restriction analysis of plasmid DNA following transformation demonstrated that bla CMY-2 was encoded on an approximately 140-kb plasmid. Pulsed-field gel electrophoresis showed the human and pet treat Salmonella strains to be highly related. This study is the first to implicate the transfer of multidrug-resistant Salmonella species through the handling of commercial pet treats containing animal products. In addition to documenting the first cases of human infection caused by CMY-2-producing S. enterica serotype Newport strains in Canada, this study illustrates the necessity of rapid and accurate laboratory-based surveillance in the identification of novel types of antimicrobial resistance.

Detection of extended-spectrum ␤-lactamases (ESBLs) in AmpC-producing Enterobacteriaceae is problematic. A modification of the double-disk test (MDDT) has been developed for successful detection of ESBLs in gram-negative bacilli producing well-characterized ␤-lactamases as well as 212 clinical isolates of Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, and Citrobacter freundii. MDDT accurately differentiated between ESBL producers and derepressed chromosomal AmpC mutants. MDDT provides a cost-effective alternative approach for clinical microbiology laboratories for routine susceptibility testing with simultaneous detection of ESBLs in Enterobacteriaceae.Enterobacteriaceae producing both AmpC ␤-lactamases and extended-spectrum ␤-lactamases (ESBLs) have been increasingly reported worldwide (2-6, 12, 16, 18). Since AmpC-producing organisms can act as hidden reservoirs for ESBLs, it is important for clinical microbiology laboratories to be able to detect ESBL production in these organisms on a routine basis (9). The National Committee for Clinical Laboratory Standards (NCCLS) has published guidelines for the detection of ESBLs in clinical isolates of Escherichia coli and Klebsiella spp., but there are currently none available for other genera (10). Since high-level expression of AmpC ␤-lactamases may mask recognition of ESBLs (17), a unique modification of the double-disk test (MDDT) using a combination of cefepime (FEP) and piperacillin-tazobactam (TZP) was evaluated to detect ESBLs. This evaluation was carried out with well-characterized strains producing either AmpC ␤-lactamases and ESBLs or either ␤-lactamase alone. In addition, the MDDT determined the presence of an ESBL in an E. coli isolate that showed a negative result with the NCCLS disk confirmation test.The following strains producing known ␤-lactamases were used for this study (Table 1). A total of 212 clinical isolates were also evaluated by MDDT: 94 were E. cloacae, 32 were E. aerogenes, 25 were S. marcescens, and 61 were C. freundii. The isolates were nonrepetitive (one per patient) and were obtained from clinical specimens from Universitas and Pelonomi Hospitals, Bloemfontein, South Africa, over a 9-month period during 1998 and 1999.

High-level expression of AmpC beta-lactamases results in organisms resistant to multiple beta-lactam antibiotics. The mechanism of chromosomally mediated AmpC resistance has been elucidated, however the mechanism(s) driving plasmid-encoded AmpC resistance are unknown. Studies were designed to identify factors which influence expression of plasmid-encoded ampC genes and correlate these factors with resistance. As the model system, ampC genes of Enterobacter origin were used to determine how gene copy number, genetic background and genetic organization influenced resistance phenotypes. To this end, gene expression from the plasmid-encoded inducible blaACT-1 and non-inducible blaMIR-1 were compared with chromosomal ampC gene expression from both wild-type (WT) and derepressed Enterobacter cloacae isolates. RNA levels within the original clinical isolates were examined using primer extension analysis, whereas a new PCR strategy was developed to examine gene copy number. These data revealed that blaACT-1 and blaMIR-1 constitutive expression was 33- and 95-fold higher than WT expression, whereas copy numbers of the plasmid-encoded genes were 2 and 12, respectively. Differences in promoters and transcriptional starts for the respective plasmid-encoded genes were noted and contribute to increases observed in overall expression. Finally, beta-lactam MICs were increased two- to 16-fold when blaACT-1 was expressed in Escherichia coli AmpD- strains compared with E. coli AmpD+ strains. In conclusion, high-level expression of plasmid-encoded ampC genes requires interplay between multiple factors including genetic organization, promoter modifications, genetic background, and to some extent gene copy number. In addition, clinical laboratories need to be aware that genetic backgrounds of inducible plasmid-encoded genes can dramatically influence MICs for organisms not normally associated with derepressed phenotypes.

These findings indicate that the biological cost of high-level AmpC production can be compensated by plasmid-encoded factors and not by regulating ampC expression.