1 Institut für Infektionsmedizin, Zentrum für Klinisch-Theoretische Medizin, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany; 2 Chair of Medical Microbiology and Infectious Diseases, Swansea Clinical School, University of Wales Swansea SA2 8PP, UK
Received 6 July 2004; returned 14 August 2004; revised 6 September 2004; accepted 8 September 2004
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Abstract |
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Methods: Organisms tested included 57 extended-spectrum ß-lactamase (ESBL) strains comprising Enterobacter aerogenes (3), Enterobacter cloacae (10), Escherichia coli (11), Klebsiella pneumoniae (26), Klebsiella oxytoca (3) and Proteus mirabilis (4). Also included were 30 strains resistant to oxyimino cephalosporins but lacking ESBLs, which were characterized with other resistance mechanisms, such as inherent clavulanate susceptibility in Acinetobacter spp. (4), hyperproduction of AmpC enzyme in Citrobacter freundii (2), E. aerogenes (3), E. cloacae (3), E. coli (4), Hafnia alvei (1) and Morganella morganii (1), production of plasmid-mediated AmpC ß-lactamase in K. pneumoniae (3) and E. coli (3) or hyperproduction of K1 enzyme in K. oxytoca (6).
Results: The MicroScan MIC-based clavulanate synergy correctly classified 50 of 57 ESBL strains as ESBL-positive and 23 of 30 non-ESBL strains as ESBL-negative (yielding a sensitivity of 88% and a specificity of 76.7%, respectively). False negatives among ESBL producers were highest with Enterobacter spp. due to masking interactions between ESBL and AmpC ß-lactamases. False-positive classifications occurred in two Acinetobacter spp., one E. coli producing plasmid-mediated AmpC ß-lactamase and two K. oxytoca hyperproducing their chromosomal K1 ß-lactamase.
Conclusion: The MicroScan clavulanate synergy test proved to be a valuable tool for ESBL confirmation. However, this test has limitations in detecting ESBLs in Enterobacter spp. and in discriminating ESBL-related resistance from the K1 enzyme and from inherent clavulanate susceptibility in Acinetobacter spp.
Keywords: ESBLs , ESBL detection , ESBL discrimination , MIC-based clavulanate synergy test
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Introduction |
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Unfortunately, current diagnostic means failed to keep pace with changing conditions. The NCCLS has issued guidelines for phenotypic confirmation of suspected ESBL strains among E. coli, Klebsiella oxytoca and Klebsiella pneumoniae, but there are currently no interpretative criteria available for other genera, or the methods to be applied for ESBL detection against the background of coexisting resistance mechanisms.16 The NCCLS recommendation still relies on the MIC difference test, in which a ß-lactamase inhibitor is used to protect the activity of an indicator drug against an ESBL-producing strain. This test is considered positive when the suspected organism exhibits a lowering of 3 two-fold dilution steps in its MICs of ceftazidime or cefotaxime in the presence of a fixed concentration (4 mg/L) of clavulanic acid, versus its MIC when tested alone.16 For daily routine, this protocol has been translated into several customized microtitre formats, the MicroScan ESBL plus panel (Dade Behring) being a newer one among them. Its advantage is that it can be processed in a fully automated fashion, by using the Walkaway 96 SI instrument (Dade Behring, Schwalbach, Germany).
In this study, we aimed to determine whether the MicroScan ESBL plus panel would be able to discriminate between ESBLs, AmpC ß-lactamases, high levels of K1 ß-lactamase and inherent clavulanate susceptibility in isolates of Acinetobacter spp., Enterobacter aerogenes, Enterobacter cloacae, E. coli, Hafnia alvei, K. oxytoca, K. pneumoniae and Morganella morganii. Some of these strains had been used in previous studies of commercially available Etest strips and microdilution panels.17,18 Molecular procedures including PCR and nucleotide sequencing were used as the reference method.
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Materials and methods |
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The bacterial strains selected for this study included 87 oxyimino-cephalosporin-resistant isolates of Gram-negative bacteria, the majority of which have been characterized previously.17,18 The remaining strains were recent isolates gathered from patients attending the university hospital Hamburg-Eppendorf, Germany, affiliated with our institution. Species identification was performed by routine laboratory methods, such as the API32E (bioMérieux, Marcy l'Étoile, France).
PCR of ß-lactamase-encoding genes
Reference confirmation for ESBL production was by molecular characterization, i.e. by PCR analysis for ß-lactamase genes of the families CTX-M, SHV, TEM, OXA, VEB and PER and where applicable by nucleotide sequencing. The primers used to amplify the targeted genes were: TEM-F, 5'-TCCGCTCATGAGACAATAACC-3' and TEM-R, 5'-TTGGTCTGACAGTTACCAATGC-3'; SHV-F, 5'-TTATCTCCCTGTTAGCCACC-3' and SHV-R, 5'-GATTTGCTGATTTCGCTCGG-3';19 CTX-M-F, 5'-TCTTCCAGAATAAGGAATCCC-3' and CTX-M-R, 5'-CCGTTTCCGCTATTACAAAC-3'; OXA-1F, 5'-ACACAATACATATCAACTTCGC-3' and OXA-1R, 5'-AGTGTGTTTAGAATGGTGATC-3';20 OXA-2F, 5'-TTCAAGCCAAAGGCACGATAG-3' and OXA-2R, 5'-TCCGAGTTGACTGCCGGGTTG-3';20 OXA-10F, 5'-CGTGCTTTGTAAAAGTAGCAG-3' and OXA-10R, 5'-CATGATTTTGGTGGGAATGG-3';20 VEB-1F, 5'-CGACTTCCATTTCCCGATGC-3' and VEB-1R, 5'-GGACTCTGCAACAAATACGC-3';21 PER-F, 5'-ATGAATGTCATTATAAAAGC-3' and PER-R, 5'-AATTTGGGCTTAGGGCAGAA-3'.21 The nucleotide sequences were determined by bidirectional sequencing of PCR products, carried out by the BigDye dideoxy chain termination method on an ABI Prism 310 DNA sequencer (Perkin-Elmer Corp., Foster City, CA, USA). The nucleotide sequences were analysed using commercial software (Vector NTI suite; InforMax Inc.) against the GenBank database.
ESBL-positive strains
Sensitivity was calculated with 57 ESBL-positive strains, which included 40 bacteria (E. coli, K. oxytoca and K. pneumoniae) targeted by the NCCLS MIC difference test and 17 non-target bacteria, such as E. aerogenes, E. cloacae and Proteus mirabilis. In detail, the clinical isolates consisted of E. aerogenes (n=3), E. cloacae (n=10), E. coli (n=11), K. oxytoca (n=3), K. pneumoniae (n=26) and P. mirabilis (n=4). The E. aerogenes strains analysed harboured CTX-M-1 (n=2) and SHV-5 (n=1) ESBLs. The isolates of E. cloacae included SHV-12 (n=3) and TEM-type (ABL: A184V) (n=7) ESBLs. The E. coli strains included SHV-2 (n=2), SHV-5 (n=1), SHV-12 (n=3), TEM-26 (n=1), TEM-52 (n=1), TEM-111 (n=1), CTX-M-1 (n=1) and CTX-M-23 (n=1).22 The K. oxytoca isolates harboured CTX-M-1 (n=2) and SHV-12 (n=1). The K. pneumoniae strains harboured SHV-2 (n=5), SHV-2a (n=1), SHV-5 (n=4), SHV-12 (n=11), SHV-18 (n=1), SHV-19 (n=1), LEN-type (ABL: N53S, A201P, P218A) (n=1), TEM-47 (n=1) and TEM-110 (n=1). The P. mirabilis strains included CTX-M-1 (n=1), CTX-M-15 (n=1), CTX-M-22 (n=1) and TEM-92 (n=1).
ESBL-negative strains
Specificity was calculated with 30 clinical strains resistant to oxyimino cephalosporins that failed to produce any ESBLs according to the PCR-based technique. Rather, in these strains, the likely type of ß-lactamase present was inferred on the basis of the overall susceptibility profile.23,24 The following patterns were used for phenotypic detection of (i) hyperproduction of AmpC chromosomal ß-lactamase (AmpC high): resistant to ureidopenicillins and to cephalosporins in the oxyimino group (cefotaxime, cefotaxime/clavulanate, ceftazidime, ceftazidime/clavulanate and cefpodoxime) and the 7--methoxy group (cefoxitin and cefotetan).23 (ii) Within the AmpC high group, there were E. coli and K. pneumoniae strains that produced a plasmid-mediated AmpC ß-lactamase, as detected by a multiplex PCR assay for detection of plasmid-mediated ampC ß-lactamase genes (plasmid AmpC).8,25 (iii) Hyperproduction of K. oxytoca chromosomal K1 ß-lactamase (K1 high): resistant to ureidopenicillins, resistant to cefuroxime and aztreonam, and resistant or intermediate to cefotaxime and ceftriaxone, but susceptible to ceftazidime.23
By these criteria, the non-ESBL strains consisted of four strains of Acinetobacter spp. (interpretative reading not applicable); two strains of Citrobacter freundii (likely mechanism of resistance: AmpC high); three strains of E. aerogenes (AmpC high); three strains of E. cloacae (AmpC high); three strains of E. coli (plasmid AmpC, multiplex PCR: CIT-type); four strains of E. coli (AmpC high), one strain of H. alvei (AmpC high); six strains of K. oxytoca isolates (K1 high); three strains of K. pneumoniae (plasmid AmpC, multiplex PCR: CIT-type); one strain of M. morganii (AmpC high).
Panel processing and interpretation
Antibiotic susceptibilities were determined by overnight microdilution, with commercial dehydrated panels provided by Dade Behring. The ESBL plus panels were prepared and inoculated according to the manufacturer's recommended procedures. The inoculated panels were then placed into the Walkaway 96 SI instrument (Dade Behring) for incubation, final reading and interpretation of the results, which was conducted according to NCCLS criteria.16 The following antimicrobial agents (concentration ranges in mg/L) were used in the MicroScan ESBL plus panel: aztreonam, 0.516; cefepime, 132; cefotetan, 132; cefpodoxime, 0.564; cefotaxime, 0.5128; cefotaxime/clavulanate, 0.125/416/4; cefoxitin, 232; ceftazidime, 0.5128; ceftazidime/clavulanate, 0.125/416/4; ceftriaxone, 164; imipenem, 0.516; meropenem, 0.516; piperacillin, 1664.
Data analysis
For isolates for which ceftazidime or cefotaxime MICs were >128 mg/L, the MIC was taken as 256 mg/L and the reduction in the ceftazidime (cefotaxime) MIC was calculated from that value. Strains for which the results between the methods were discordant were tested again, and only concordant and reproducible data were analysed.
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Results |
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If a lowering of 3 two-fold dilutions in the MIC of either ceftazidime or cefotaxime was taken as the criterion for ESBL confirmation, the MIC difference test was able to detect 50 ESBL strains out of the total of 57 identified by molecular characterization (yielding 88% sensitivity). False negatives among ESBL producers were highest in tests with Enterobacter spp. (Table 1 and Figure 1). Addition of clavulanic acid to ceftazidime (or cefotaxime) failed to lower MICs at least eight-fold in tests with nine (or seven) out of 13 ESBL-producing Enterobacter strains. This poor performance was partly due to the ability of clavulanate to induce the chromosomal AmpC ß-lactamase, which often resulted in MICs of the combinations higher than that of the drug alone (Table 1 and Figure 1).
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Among the 30 isolates resistant to oxyimino cephalosporins but lacking ESBLs, we observed apparently positive results in MIC difference testing (three- to eight-fold increase in the MIC) with cefotaxime and ceftazidime in seven and five cases, respectively (Table 1 and Figure 2). Since the NCCLS requires only one of the ESBL tests to be positive for an organism to confirm ESBL production, this resulted in an overall specificity of only 76.7% (23/30 strains). Misidentification occurred in Acinetobacter spp. (two strains), E. coli producing plasmid-mediated AmpC ß-lactamase (three strains) and K. oxytoca hyperproducing their chromosomal K1 ß-lactamase (two strains). An effect of clavulanic acid on the activities of both cefotaxime and ceftazidime was noted in Acinetobacter spp. and E. coli producing plasmid-mediated AmpC enzymes, whereas in K. oxytoca hyperproducing K1 enzyme only cefotaxime was affected (Table 1 and Figure 2).
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Discussion |
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One issue is that the presence of ESBLs can be masked by the expression of AmpC ß-lactamases, which can be generated by chromosomal (e.g. in most Enterobacter spp., Serratia spp., C. freundii, M. morganii, Providentia spp. and Pseudomonas aeruginosa) or plasmid genes (mostly in E. coli and Klebsiella spp.). Plasmid-mediated AmpC ß-lactamases are thought to have originated from the chromosomes of the former Enterobacteriaceae species.27 Regardless of background, AmpC enzyme can interfere with clavulanate synergy tests.28,29 With the chromosomal type of enzyme, clavulanic acid may act as an inducer of high-level production, and may then attack the indicator cephalosporin, thus masking any synergy arising from a coexisting ESBL.28,29 Accordingly, in the present study, clavulanate-based synergy testing failed to detect reliably ESBL production in tests with 54%69% ESBL-producing Enterobacter strains. The overall ability of clavulanic acid to lower the MICs of either cefotaxime or ceftazidime, due to these constraints, was diminished to 50/57 strains (88% sensitivity) and 46/57 strains (81% sensitivity), respectively.
Even though they are not inducible, plasmid-encoded AmpC ß-lactamases typically are expressed at medium to high levels. Like their counterpart on the chromosome, plasmid-encoded AmpC ß-lactamases provide a broader spectrum of resistance than ESBL and are not blocked by commercially available inhibitors.8,27 Thus, high-level expression of a plasmid-mediated AmpC enzyme may also prevent recognition of an ESBL. In our study, two E. coli strains produced both a CTX-M ß-lactamase and a CIT-type plasmid-mediated AmpC ß-lactamase. In preliminary studies with these strains, dominant AmpC production covered and masked underlying ESBL production. Thus, these strains were initially considered only in the AmpC group until retesting was performed and the ESBL content was detected.
Approaches to overcome these difficulties include the use of tazobactam or sulbactam, which are much less likely to induce AmpC ß-lactamases and are therefore preferable inhibitors for ESBL detection tests with these organisms, or testing cefepime as an ESBL detection agent.28 Cefepime is a more reliable detection agent for ESBLs in the presence of an AmpC ß-lactamase, as this drug is stable to AmpC ß-lactamases, but labile to ESBLs.28 In a previous study employing the Etest ESBL, cefepime with and without clavulanic acid had promising utility in identifying ESBLs in isolates that also contained an AmpC ß-lactamase.18
Another issue of vital importance is which indicator cephalosporin should be tested. Synergy testing may fail to identify CTX-M positive isolates as ESBL producers if ceftazidime is used as the sole detection agent (as seen in the subset analysis of the CTX-M producers tested in this study). Relying on cefotaxime alone, on the other hand, is unreliable in distinguishing ESBL producers from hyperproducers of the K1 enzyme in K. oxytoca. As shown previously with the ESBL Etest and (as here) with a microdilution technique, many of the cefotaxime synergy-positive K. oxytoca isolates are hyperproducers of their chromosomal K1 ß-lactamase and are lacking an ESBL.17,18,30
Which bacteria should be excluded from routine ESBL testing has not received much attention. In the present study, we have tested six Acinetobacter strains. We observed ostensibly positive results with cefotaxime and ceftazidime in two out of six strains, although PCR analysis showed that these strains were lacking an ESBL. These results confirmed the suspicion that Acinetobacters often give apparently positive results, due to their susceptibility of the inhibitors themselves, and thus should not be tested for ESBL production by inhibitor-based methods.3133 A similar difficulty exists with Stenotrophomonas, where clavulanate synergy tests can produce false-positive results via inhibition of the L2 chromosomal ß-lactamase.34 Given that ESBLs are spreading and are reaching non-fermenters as well, principally Acinetobacter, these particularities could become clinically important in the future.3537
Overall, this study demonstrated that the MicroScan MIC difference test, using both ceftazidime and cefotaxime, is a sensitive tool in its ability to confirm the ESBL status of a given Gram-negative isolate. However, as with other techniques, its constraints must be recognized. First, and most importantly, clavulante synergy can give false-negative results in an AmpC environment, whether chromosomal or plasmid-encoded. Secondly, false-positive ESBL results can occur, due to the presence of other clavulanate-responsive ß-lactamases, mainly the K1 ß-lactamase of K. oxytoca, or due to clavulanate susceptibility, which is inherent to the species, as for Acinetobacter spp. As ESBL phenotypes become more and more complex, emphasis on an upgrade of the NCCLS recommendations seems to be warranted.
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Acknowledgements |
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This study was supported by Dade Behring.
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Footnotes |
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