JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2009, p. 294���299 Vol. 47, No. 2 0095-1137/09/$08.00 0 doi:10.1128/JCM.01797-08 Copyright �� 2009, American Society for Microbiology. All Rights Reserved. Identification of Plasmid-Mediated AmpC -Lactamases in Escherichia coli, Klebsiella spp., and Proteus Species Can Potentially Improve Reporting of Cephalosporin Susceptibility Testing Results Fred C. Tenover,1 Shannon L. Emery,2 Carol A. Spiegel,3 Patricia A. Bradford,4 Samantha Eells,2��� Andrea Endimiani,5 Robert A. Bonomo,5 and John E. McGowan, Jr.2* Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Atlanta, Georgia 303331 Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, Georgia 303222 Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 537923 Wyeth Research, Pearl River, New York 109654 and Louis Stokes Department of Veterans Affairs Medical Center, Cleveland Ohio 441065 Received 17 September 2008/Returned for modification 29 October 2008/Accepted 18 November 2008 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 -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 -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 -lactamases and ESBLs. Thus, additional data are needed before formal recommendations can be made on changing the reporting of ASC test results. -Lactamase-mediated resistance to penicillins and cepha- losporins is a significant problem among gram-negative bacte- ria worldwide (2, 4, 17). -Lactamases can be divided into four major classes (A, B, C, and D) based on substrate profiles and amino acid sequences (3, 5). Class A -lactamases include enzymes that are active against penicillins and cephalosporins. Mutations in key positions in class A -lactamase genes can produce extended-spectrum -lactamases (ESBLs) that medi- ate resistance to extended-spectrum cephalosporins (ESCs), such as cefotaxime (CTX), ceftriaxone, and ceftazidime (CAZ), and, in some cases, the advanced-spectrum cephalo- sporins (ASCs) cefepime and cefpirome (2, 13, 14). Class C -lactamases, such as the AmpC enzymes, also mediate resis- tance to ESCs but often remain susceptible to the ASCs, which could be important information for clinicians considering ther- apy for gram-negative infections. Studies by Coudron (9), Jacoby et al. (12), and Yagi et al. (33) have validated the use of boronic acid derivatives to detect AmpC -lactamases in gram-negative bacilli. Coudron���s study (9) included a recommendation that for isolates positive by the boronic acid test all cephalosporin test results be reported as resistant, similar to the Clinical and Laboratory Standards In- stitute (CLSI) ESBL reporting algorithm. It is not clear if Coudron���s recommendation included changing the results with regard to ASCs. The other two studies did not comment on the issue of how to interpret cephalosporin results. CLSI has yet to establish a testing and reporting algorithm specifically for or- ganisms containing AmpC -lactamases. CLSI���s algorithm to detect and confirm the presence of ESBLs is limited to isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, and Proteus mirabilis (6���8). Using the algorithm is critical for accurate test reporting since ESBL- producing organisms often test susceptible to one or more ESCs even though these drugs are not clinically active against infections caused by ESBL-producing organisms (20, 21). Thus, confirmation of the presence of an ESBL in an isolate of E. coli, P. mirabilis, or a Klebsiella species indicates that all penicillin and cephalosporin results should be reported as in- dicating resistance. However, resistance to ESCs in E. coli, Enterobacter species, and several other species of Enterobacte- riaceae can also be the result of mutations that upregulate production of chromosomal AmpC -lactamases or can be the result of acquisition of plasmid-encoded AmpC enzymes among organisms that do not contain a chromosomal AmpC -lactamase gene (24, 32). Pai and colleagues (19) have indi- cated that organisms that produce AmpC -lactamases, similar to those that produce ESBLs, may test as susceptible to ESCs, * Corresponding author. Mailing address: Department of Epidemi- ology, Rollins School of Public Health, Emory University, 1518 Clifton Rd., Atlanta, GA 30322. Phone and fax: (404) 727-9365. E-mail: jmcgowa@sph.emory.edu. ��� Present address: Los Angeles BioMedical Research Institute, Tor- rance, CA 90502. Published ahead of print on 26 November 2008. 294

thus producing misleading clinical information. This suggests that the identification of AmpC -lactamases among E. coli, P. mirabilis, or a Klebsiella species could also increase the accu- racy of antimicrobial susceptibility test reports for ESCs if the results were used to modify the interpretations of cephalospo- rin results. As noted above, in contrast to bacterial isolates containing ESBLs, isolates containing AmpC -lactamases typically re- main susceptible to ASCs (13, 14, 26, 27, 31). Although the occurrence is rare, AmpC -lactamases, such as CMY-19, show modest cefepime hydrolyzing activity (31) even though the organisms carrying this -lactamase remain fully suscepti- ble to both cefepime and cefpirome in vitro. Thus, there may be clinical relevance to distinguishing between bacterial iso- lates containing ESBLs, for which ASC results should be re- ported as resistant, and those containing AmpC -lactamases, for which ESC results would be reported as resistant but the ASC results would be reported as they test. The major goals of this study were to determine (i) whether testing 3-aminophenylboronic acid hemisulfate (APBA) in conjunction with CAZ and cefotetan (CTT) disks could im- prove the reporting of results for ESCs and ASCs by indicating which strains contained AmpC -lactamases, (ii) whether us- ing the combination of resistance to amoxicillin-clavulanate and cefoxitin as a surrogate had equivalent sensitivity and specificity to the APBA assay for identifying AmpC -lacta- mase-producing organisms, (iii) whether organisms containing both ESBLs and AmpC -lactamases would be consistently detected with both clavulanic acid (CLAV) and APBA, and (iv) whether a disk diffusion test using APBA dissolved in water gave comparable results to dissolving the free acid com- pound in dimethyl sulfoxide (DMSO). With regard to the last goal, one of the drawbacks to using boronic acid is the require- ment to dissolve the free acid in DMSO, a toxic chemical not often used in microbiology laboratories. Thus, we hypothesized that a boronic acid compound that did not have to be dissolved in DMSO could improve the general acceptance of the test in clinical laboratories. MATERIALS AND METHODS Bacterial strains. A total of 80 clinical isolates of E. coli, Klebsiella species, and Proteus species collected during a 5-year period from 15 hospitals in the United States that participate in Project ICARE (Intensive Care Antimicrobial Resis- tance Epidemiology) (11) were tested (Table 1). Sixty-one isolates were previ- ously characterized for ESBL production (18, 28, 30). All isolates were resistant to at least one ESC (i.e., CTX, CAZ, ceftriaxone, or ceftizoxime). The study was conducted in three phases. In phase I, all 80 isolates were tested. In phase II, a subset of 24 isolates from phase I (21 E. coli, 1 K. oxytoca, and 2 K. pneumoniae isolates), which had been collected prospectively at the University of Wisconsin because they were nonsusceptible to both cefoxitin (FOX) and amoxicillin- clavulanate, were further examined (Table 2). In phase III, an additional 11 isolates (8 E. coli and 2 K. pneumoniae isolates and 1 P. mirabilis isolate) containing multiple -lactamases from the Wyeth Research collection were tested (Table 3). Isolates from phase I were identified using Vitek 2 (bioMe��rieux, Durham, NC) or standard biochemical methods (10). Isolates from phase II were identified using Vitek, conventional biochemical tests, and phenotypic tests, and isolates in phase III were identified using API-20E strips (bioMe ��rieux). All isolates were stored at 70��C prior to testing. Each isolate was subcultured on Trypticase soy agar supplemented with 5% sheep blood (BD BioSciences, Sparks, MD) twice before testing. Control strains were kindly provided by the following individuals: E. coli TG1 containing the -lactamase FOX-1 was from George Jacoby (Lahey Clinic, Boston, MA) E. coli isolates containing the DHA-1 and ACC-1 -lactamases were obtained from Nancy Hanson (Creighton University School of Medicine, Omaha, NB) K. pneumoniae containing the CMY-8 was from Jing-Jou Yan (National Cheng Kung University Medical Col- lege, Taiwan) and Salmonella enterica AM03430 containing a CMY-2 -lacta- mase was obtained from Jean Whichard (Centers for Disease Control and Prevention, Atlanta, GA). Disk diffusion testing. BBL Sensi-Disc (6 mm) disks containing 30 g of either CTT, 30 g of CAZ, or 30 g of CAZ plus 10 g of CLAV were purchased from BD BioSciences (Sparks, MD). APBA hemisulfate (120 mg) was dissolved in 6 ml of distilled H2O, and 100 mg of APBA was dissolved in DMSO 30 l of either solution was added to CAZ and CTT disks. Disks were dried for 30 min prior to use or stored at 4��C for up to 1 month without loss of activity. Organisms were suspended in Mueller-Hinton broth to the turbidity of a 0.5 McFarland standard and inoculated onto 150-mm Mueller-Hinton agar plates (BD Biosciences) as for disk diffusion (7). Disks containing 30 g of CTT, 30 g of CAZ, 30 g of CTT plus 600 g of APBA, 30 g of CAZ plus 600 g of APBA, and 30 g of CAZ plus 10 g of CLAV were placed on the plate. (All isolates had previously been TABLE 1. Results of APBA, CLAV, and AmpC PCR tests for all study isolates Organism No. of isolates testedg No. of isolates positive by APBA test by substrate (family)a No. of isolates positive with CLAV and APBA (family) No. of isolates positive with CLAV onlyb No. of isolates negative with both ABPA and CLAV CAZ only CTT only CAZ and CTT Escherichia coli 53 7 (2 CIT, 5 NEGc) 1 (1 NEGd) 17 (4 CIT, 1 DHA, 1 FOX, 5 MOX, 6 NEGe) 2 (1 EBC, 1 NEGf) 11 15 Klebsiella oxytoca 7 1 (CIT) 6 0 Klebsiella pneumoniae 14 3 (2 FOX, 1 MOX) 10 1 Proteus mirabilis 1 1 Proteus penneri 1 1 Proteus vulgaris 4 4 Total 80 7 1 21 2 27 22 a Multiplex PCR was performed as described by Perez-Perez and Hanson (23) only on isolates that were positive in the APBA disk diffusion test. NEG, negative PCR assay. PCR results represent families of AmpC genes, not individual determinants (See Materials and Methods). b Organisms were tested previously with CAZ and CTT with and without CLAV per CLSI criteria (7). Results of ESBL production were consistent with previous results. c pI values of -lactamases determined by aIEF of five PCR negative isolates: (i) 8.5 (ii) 8.9, 8.1, 7.7, 7.0, 6.5 (iii) 9.0 (iv) 9.0 (v) 9.0, 8.8, 5.4. d pI values of -lactamases determined by aIEF of PCR negative isolate, 6.4. e aIEF of six PCR negative isolates: (i) 8.4, 5.4 (ii) 8.4, 5.4 (iii) 8.9 (iv) 8.4, 5.3 (v) 9.0, 8.4, 8.0, 7.6, 7.0, 6.4 (vi) 9.2, 8.0, 6.8, 5.4. f aIEF of PCR negative isolate, 7.3. g All ESBL screen test positive. VOL. 47, 2009 BORONIC ACID TESTING FOR AmpC -LACTAMASES 295