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Journal of Clinical Microbiology, June 2007, p. 2034-2036, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02538-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Metallo-ß-Lactamase or Extended-Spectrum ß-Lactamase: a Wolf in Sheep's Clothing{triangledown}

Björn A. Espedido, Lee C. Thomas, and Jonathan R. Iredell*

Centre for Infectious Diseases and Microbiology, University of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia

Received 19 December 2006/ Returned for modification 20 February 2007/ Accepted 23 March 2007


   ABSTRACT
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Diagnostic algorithms in commonly used automated bacterial identification systems fail to reliably identify a metallo-ß-lactamase in the Enterobacteriaceae. Misidentification as an extended-spectrum ß-lactamase may result in inappropriate dismissal of drugs such as aztreonam in favor of carbapenems, which may in turn select for a highly carabapenem resistant phenotype.


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In Australia, several members of the Enterobacteriaceae with decreased susceptibility to carbapenems have recently emerged, associated with a carbapenem-hydrolyzing metallo-ß-lactamase (MBL) encoded by blaIMP-4 (7). Where this MBL is present (3), an elevated MIC of carbapenems can be modified by specific inhibitors in vitro (2, 4). Similarly, specific inhibition of MBL-mediated resistance to extended-spectrum cephalosporins requires that no other cause of resistance coexist in the test strain. Recognition is therefore complicated by highly efficient transmission of the MBL to host strains in which the carbapenem-resistant phenotypes differ (3) and in which a variety of other antibiotic resistance mechanisms may occur, including extended-spectrum ß-lactamases (ESBLs) (8).

In this study, we report our experience with two widely used automated systems, the Vitek (bioMérieux Vitek Systems Inc., Hazelwood, MO) and the Phoenix Microbiology System (Becton, Dickinson & Co., Franklin Lakes, NJ), for identification of selected blaIMP-4-positive wild-type (wt) strains (Table 1) and their transconjugants into UB5201Rf (a rifampin-resistant attenuated laboratory strain of Escherichia coli). Plasmid pJIBE404 is isogenic with pJIBE401 except that the first two cassettes in the array (blaIMP-4-qacG2) have spontaneously excised. Plasmid pJIBE402 contains the blaIMP-4-qacG2-aacA4-catB3 gene cassette array cloned into pGEM-T Easy (Promega Corp., Madison, WI) and is subject to the SP6 promoter in the lacZ orientation. Antibiotic susceptibility profiles were generated using the Phoenix NMIC/ID-101 panel (Becton, Dickinson & Co.) and using Vitek 1 GNS-424, Vitek 2 AST-N044, and Vitek 2 AST-N019 gram-negative cards (bioMérieux Vitek Systems Inc.) as indicated. Different carbapenems were present in the Phoenix panels (imipenem [IPM]) and Vitek 2 cards (IPM for AST-N019 and meropenem [MEM] for AST-N044) examined here, but the result of one can generally be used to predict the other for the Enterobacteriaceae. Phoenix and Vitek 2 "Advanced Expert System" (AES) data (AST-N044 cards) were obtained in mid-2006, and Vitek 2 AES data (AST-N019 cards) were obtained in December 2004. Vitek 1 data were obtained in December 2003. All systems had current software updates. Double-disk synergy tests (DDST) (6) were performed to detect the presence of an ESBL. ESBL and carbapenem Etests (AB Biodisk, Solna, Sweden) were used according to the manufacturer's instructions.


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  		TABLE 1. Isolates used in this study

 
Initial data obtained from the Vitek 2 (AST-N019) AES reported "susceptibility results inconsistent with organism identification" and corrected the IPM MIC from >16 to ≤2 µg ml–1 but cautiously advised a final result of "resistant" and a warning, "unusual resistance observed," for Kp1239::pJIBE401 (wt plasmid with blaIMP-4) (Tables 2 and 3). The same strain carrying pJIBE404 (wt plasmid without blaIMP-4) was reported accurately without any special remark. The same advice and warnings accompanied the AST-N019 profiles of pJIBE401 and pJIBE402 (cloned blaIMP-4) in E. coli UB5201Rf. The IPM MICs for UB5201Rf::pJIBE401 and UB5201Rf::pJIBE402 were corrected from 8 ("intermediate") to ≤0.5 ("intermediate") µg ml–1 and from 4 ("susceptible") to ≤0.5 ("susceptible") µg ml–1, respectively. "ESBL plus impermeability" was also advised for UB5201Rf::pJIBE402 along with a comment that the phenotype match was "very typical," albeit rare (data not shown). Interestingly, the simpler algorithms in Vitek 1 reported both Kp1239 and UB5201Rf with pJIBE401 as "ESBL negative," correctly identifying high-level resistance to both IPM and MEM in the former and MICs of <4 and <2 µg ml–1, respectively, for the latter.


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  		TABLE 2. MEM susceptibility interpretation by the bioMérieux VITEK 2

 

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  		TABLE 3. IPM susceptibility interpretation by the BD Phoenix

 
The Vitek 2 (AST-N044) AES warned of a possible carbapenemase in strains carrying a wt plasmid but not in E. coli UB5201Rf carrying the cloned array (pJIBE402) (Tables 2 and 3). The BDXpert system reported an increased IPM MIC but did not warn of a carbapenemase in any of the isolates tested. No ESBL activity was detected in any isolates by phenotypic testing (Etest and DDST) (data not shown), but E. coli (n = 4) and Klebsiella pneumoniae (n = 15) isolates repeatedly triggered BDXpert system rules 1505 and 1529 (Table 3).

Finding IMP-4 in an ESBL-positive organism is not unexpected (8). However, while all other IMP-4-positive strains were ESBL negative (by Etest and DDST) and were paradoxically reported as ESBL positive by the AES (AST-N044) and BDXpert, neither of the two enterobacters tested were reported ESBL positive by the AES or BDXpert despite the presence of blaSHV-12 (an ESBL gene) in Enterobacter cloacae El3518 (S. Partridge, personal communication). The Phoenix reported a probable ESBL (BDXpert rule 106) for E. coli UB5201Rf::pJIBE402, which contains blaTEM in the cloning vector, but no ESBL gene. pJIBE401, but not pJIBE404, triggered ESBL reports by both the AES and BDXpert for both E. coli (UB5201Rf) and K. pneumoniae (wt) backgrounds. Thus, while AmpC and IMP-4 may mask a true ESBL phenotype, the MBL goes unrecognized and triggers an incorrect diagnosis of an ESBL in both of the automated systems currently in widespread use. This is particularly counterproductive because the usual prescriber response to ESBL is a carbapenem, which is likely to select specifically for high-level pan-ß-lactam resistance in the presence of IMP-4 (3). In addition, IMP-4 and related enzymes are active against all ß-lactams except aztreonam, and the additional correction of aztreonam to "resistant" may therefore prevent prescribers from considering one of the few remaining antibiotics with activity against the MBL (1, 11).

It is useful to highlight possible misidentifications when resistance phenotypes are inconsistent, but inappropriate bacterial identification changes were also proposed by the AES: reassignment as Enterobacter aerogenes was recommended for carbapenem-resistant wt E. cloacae and K. pneumoniae, and reassignment as Citrobacter spp. was recommended for E. coli (UB5201Rf::pJIBE401and wt Ec9381).

Rather than relying on growth inhibition in the presence of clavulanic acid, the AES determines ESBL status by comparing the ß-lactam susceptibility patterns of individual isolates to an extensive database of MIC distributions of various ß-lactams (5, 9, 10). The BDXpert did not alter suggested identifications but was less able than the AES to suggest a carbapenemase. We found that susceptibility results correlated reasonably well between the VITEK 2 and Phoenix systems overall, despite minor differences in the antibiotic MIC end points (data not shown).

While carbapenem MICs were mostly within the Clinical and Laboratory Standards Institute (formerly NCCLS) breakpoints (≤4 and ≥16 µg ml–1 for MEM and IPM, respectively), the combination of inappropriate recommendations for bacterial identification changes and inaccurate identification of ESBL/MBL activity in these strains makes the automated systems in their present configurations somewhat problematic. The software used to manage these automated identifications and interpretations is subject to frequent updating, but the present algorithms may result in inappropriate therapeutic responses. It seems likely that the accumulation of various antibiotic resistance mechanisms, many of which move freely between species, will increasingly challenge the reliability of systems based purely on phenotypes.


   ACKNOWLEDGMENTS
 
B.A.E. is supported by an Australian Postgraduate Award and a Westmead Millennium Institute scholarship. This work was supported by NSW Health (CIDM-PH) and the National Health and Medical Research Council of Australia.

We are very grateful to Helen Ziochos for technical assistance and to T. Olma, M. Leroi, and T. Gottlieb for the clinical isolates used in this study. We thank the other members of the CIDM Research team for support and useful discussions, especially S. Partridge and Z. Zong.


   FOOTNOTES
 
* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology, Level 3 ICPMR Building, Westmead Hospital, Westmead, NSW 2145, Australia. Phone: 612-9845-6255. Fax: 612-9891-5317. E-mail: joni{at}icpmr.wsahs.nsw.gov.au Back

{triangledown} Published ahead of print on 4 April 2007. Back


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Journal of Clinical Microbiology, June 2007, p. 2034-2036, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02538-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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