Annals of Clinical & Laboratory Science 37:75-78 (2007)
© 2007 Association of Clinical Scientists
Evaluation of Phoenix Automated Microbiology System for Detecting Extended-Spectrum ß-Lactamases among Chromosomal AmpC-producing Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, and Serratia marcescens
Yeon-Joon Park,
Jin Kyung Yu,
Seungok Lee,
Jung-Jun Park and
Eun-Jee Oh
Department of Laboratory Medicine, College of Medicine, Catholic University of Korea, Seoul, Korea
Address correspondence to Yeon-Joon Park, M.D., Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Kangnam St. Mary’s Hospital, 505 Banpo-dong, Seocho-ku, Seoul 137-701, Korea; tel 82 2 590 1604; fax 82 2 592 4190; e-mail yjpk{at}catholic.ac.kr.
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Abstract
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We evaluated the BD Phoenix Extended-Spectrum ß-Lactamase (ESBL) detection test among chromosomal AmpC-producing Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, and Serratia marcescens. The study was conducted on 72 non-repetitive ESBL producers (33 E. cloacae, 13 E. aerogenes, 14 C. freundii, and 12 S. marcescens) and 77 ESBL non-producers (33 E. cloacae, 9 E. aerogenes, 6 C. freundii, and 29 S. marcescens). The organisms were selected as suspected ESBL-producers based on the double disk synergy test and confirmed by PCR amplification of blaTEM-1, blaSHV-1, blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9. The Phoenix ESBL test, using a 5-well confirmatory test and the BDXpert system, was evaluated. Of the 72 isolates identified as ESBL-producers based on the DDST, 46 isolates harbored CTX-M-type enzymes, 21 harbored TEM type enzymes, and 31 harbored SHV enzymes. The Phoenix system identified ESBL only in 15 isolates. Of the 77 ESBL non-producers, ths Phoenix system identified ESBL in 4 isolates, 3 of which were confirmed to be ESBL-producers. In this study, the Phoenix system was highly specific (76/77, 98.7%), and it identified 3 additional ESBL-producers that were not detected by DDST. However, the Phoenix system’s sensitivity was very low (15/72, 20.8%). Considering the increasing prevalence of ESBL production among AmpC-producers, the BD Phoenix system could not be considered a reliable stand-alone ESBL detection method for the strains tested in our study.
Keywords: Phoenix System, AmpC, extended-spectrum ß-lactamases, Gram-negative bacilli
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Introduction
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Extended-spectrum ß-lactamases (ESBLs) represent an ever-growing class of plasmid-mediated ß-lactamase found in Gram-negative bacilli. Although CLSI screening and confirmatory tests apply only to Escherichia coli, Klebsiella spp., and Proteus mirabilis, given the increasing prevalence of ESBLs among AmpC-producers [1,2], reliable automated methods for detection of ESBLs should be available.
ESBL detection in AmpC-producers is more difficult because AmpC type ß-lactamases can obscure the synergistic effects of clavulanate and cephalosporins against ESBLs. Therefore, use of the cefepime disk, which is less subject to hydrolysis by AmpC ß-lactamases than third-generation cephalosporins, increases the sensitivity of ESBL detection [3]. Becton-Dickinson Biosciences Corp. (Sparks, MD) has introduced a short-incubation system for bacterial identification and susceptibility testing, known as BD Phoenix. The Phoenix ESBL test uses growth response to cefpodoxime, ceftazidime, ceftriaxone, and cefotaxime, with or without clavulanic acid, to detect the production of ESBLs. The test algorithm has been delineated by Sanguinetti et al [4] and has been shown to detect ESBL production by AmpC-producers in addition to Klebsiellae spp, E. coli, and P. mirabilis.
In this study, we evaluated the BD Phoenix ESBL detection test among chromosomal AmpC-producers, and the results were compared with those obtained by molecular analysis.
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Materials and Methods
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Bacterial isolates and phenotypic characterization.
The study was conducted on 72 non-repetitive ESBL-producers (33 E. cloacae, 13 E. aerogenes, 14 C. freundii, and 12 S. marcescens) and 77 ESBL-non-producers (33 E. cloacae, 9 E. aerogenes, 6 C. freundii, and 29 S. marcescens). The organisms were selected as suspected ESBL-producers based on the double disk synergy test (DDST) [5]. Briefly, the surface of a Mueller-Hinton (MH) agar plate was inoculated evenly, using a cotton swab, with an overnight culture suspension of clinical isolate that was adjusted to the McFarland 0.5 standard. After inoculation, disks (BBL, Cockeysville, MD) containing 30 µg of ceftazidime (CAZ), cefotaxime (CTX), aztreonam (AZT), cefepime (FEP), and amoxicillin-clavulanic acid (AMC) (20/10 µg, BBL) were placed at distances of 20 mm (center to center). E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as negative and positive controls for ESBL production. In addition, for discrepant cases, DDST was repeated with the addition of 64 µg of boronic acid to an amoxicillin-clavulanic acid disk [6].
Antimicrobial susceptibility and ESBL test using the Phoenix system.
For the BD Phoenix system (Becton-Dickinson Diagnostic Systems, Sparks, MD), the combined ID and AST NMIC/ID 107 panel for gram-negative bacilli were used. The setup of panels was arranged according to the manufacturer’s instructions. Briefly, we used the PUD (version 4.11B) and the Phoenix ID broth was inoculated with bacterial colonies from MacConkey agar and adjusted to a 0.5 to 0.6 McFarland standard using the Crystal Spec Nephelometer (BD Diagnostic Systems). The Phoenix ESBL test used 5 wells containing the fixed concentrations of the following drugs or drug combinations: cefpodoxime, ceftazidime, ceftazidime plus clavulanate (CA), cefotaxime plus CA, and ceftriaxone plus CA. After inoculation with each of the isolates, the panel was placed in the instrument and continually monitored for growth. When the decision point was at the terminal node, the results were reported.
Molecular characterization of ESBL producers.
For 72 ESBL-producers based on DDST and isolates that were DDST-negative but Phoenix-ESBL-positive, a search for the blaTEM-1, blaSHV-1, blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9 was performed by PCR amplification with the following sets of primers: TEM-1F (5'-AGCCATACCAAACGACGAG-3') and TEM-1B (5'-ATTGTTGCCGGGAAGCTAGA-3') for blaTEM-1, SHV-1F (5'-TATCCCTGTTAGCCACCCTG-3') and SHV-1B (5'-CACTGCAGCAGCTGC(A/C)-TT-3') for blaSHV-1, CTX-1F (5'-GGTTTAAAAAATCACTGCGTC-3') and CTX-1B (5'-TTGGTGACGATTTTAGCC-GC-3') for blaCTX-M-1, CTX-2F (5'-ATGATGACTCAGAGCATTCG-3') and CTX-2B (5'-TGGGTTACGATTTTCGCCGC-3') for blaCTX-M-2, CTX-9F (5'-CGCTTTATG-CGCAGACGA-3') and CTX-9B (5'-GATTCTCGCCGCTGAAGC-3') for blaCTX-M-9. Strains encoding blaTEM, blaSHV, and blaCTX-M genes were used as positive controls for PCR amplification. To confirm that TEM and SHV type enzymes were not restricted-spectrum but extended-spectrum ß-lactamases, sequencing and Nhe I digestion experiments were performed [7].
Isoelectric focusing (IEF).
Isoelectric focusing was performed for isolates that were DDST-negative, but ESBL-positive by the Phoenix system. Briefly, crude ß-lactamase preparations, derived from the sonicated bacterial cultures, were assessed for the ß-lactamase pIs and the general inhibitor profile by IEF. The IEF was performed at room temperature on a Bio-Rad mini isoelectric focusing III (Bio-Rad Corp., Richmond, CA). The enzymes were visualized by staining the gel with a 0.5 mM nitrocefin solution (BBL, Cockeysville, MD). The isoelectric points of the enzymes from the Proteus isolates were estimated by comparison with TEM-1, TEM-10, SHV-1, SHV-5, and CMY-1.
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Results and Discussion
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Of the 72 isolates identified as ESBL-producers based on the DDST, 46 isolates harbored CTX-M-type enzymes, 21 isolates harbored TEM type enzymes, and 31 harbored SHV enzymes.
Phoenix identified ESBL only in 15 isolates (Table 1
). According to the species, 5 of 33 ESBL-positive E. cloacae, 6 of 13 E. aerogenes, 4 of 14 C. freundii, and none of 12 S. marcescens isolates were correctly identified. According to the type of ESBLs produced, 13.0% (6/46) of CTX-M-type producers, 28.6% (6/21) of TEM-producers, and 29.0% (9/31) of SHV-producers were correctly identified.
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Table 1. Distribution of ESBLs detected by BD Phoenix system in E. cloacae, E. aerogenes, C. freundii, and S. marcescens.
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Of the 77 ESBL-non-producers, Phoenix identified ESBL in 4 isolates. By the repeated DDST using boronic acid, 3 of them were ESBL-positive and they harbored blaCTX-M genes. The 1 remaining isolate was ESBL-PCR and DDST-negative. Isoelectric focusing of the isolate showed 1 band of pI 8.2, which was inhibited by cloxacillin, representing AmpC ß-lactamase.
In this study, the Phoenix system was highly specific (76/77, 98.7%), and it identified 3 additional ESBL-producers that were not detected by DDST. However, the Phoenix system’s sensitivity was very low (15/72, 20.8%). This result contrasts markedly with the report by Sanguinetti et al [4], in which the Phoenix system identified the ESBLs (SHV and CTX-M-types) in all the ESBL-producing E. cloacae, E. aerogenes, C. freundii, and S. marcescens. Although we cannot compare our findings with the study by Sanguinetti et al [4], because the antibiotic susceptibility of the ESBL-producing isolates was not indicated, the susceptibility to piperacillin/tazobactam in ESBL-producing isolates was very low (14/72, 19.4%) in the present study (Table 2), which suggests AmpC derepression in most of the ESBL producers [8]. This low sensitivity in detecting ESBL among AmpC-producing organisms may be due to the fact that the Phoenix system does not include cefepime, which is recommended for detecting ESBL among chromosomally-encoded AmpC-producers because these agents are less efficiently hydrolyzed by AmpC enzymes than other cephalosporins [5].
In a study that determined the utility of the Vitek 2 advanced expert system for ESBL detection in Enterobacter species, the sensitivity was higher (62.5%), but the Vitek 2 system erroneously reported cephalosporin susceptibility in nearly three-fourths of the discrepant cases [9]. In the present study, despite the very low susceptibility (1.4~4.2%) to ceftazidime, cefotaxime, and aztreonam in ESBL-producers, the susceptibility to cefepime was 33.3%, which should be regarded as resistant because of ESBL production. In conclusion, considering the increasing prevalence of ESBL production among AmpC-producers, there is a need for improvements in detecting ESBLs with the BD Phoenix system.
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Acknowledgements
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The authors thank the Becton-Dickinson Biosciences Corporation for placing the Phoenix instrument at our disposal and providing the consumables.
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References
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Abstract
Introduction
