JAC Advance Access originally published online on January 28, 2008
Journal of Antimicrobial Chemotherapy 2008 61(4):827-830; doi:10.1093/jac/dkn016
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Original research |
Evaluation of phenotypic tests for the detection of metallo-β-lactamase-producing Pseudomonas aeruginosa in a low prevalence country
1 Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway 2 Karolinska-Institutet-MTC, Karolinska University Hospital, Solna, Department of Clinical Microbiology, Stockholm, Sweden 3 Division of Infection Control, Norwegian Institute of Public Health, Oslo, Norway 4 Department of Microbiology and Virology, University of Tromsø, Tromsø, Norway
* Corresponding author. Tel: +47-77627043/+47-97653716; Fax: +47-77627015; E-mail: orjan.samuelsen{at}unn.no
Received 12 November 2007; returned 6 December 2007; revised 3 January 2008; accepted 4 January 2008
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Abstract |
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Objectives: To evaluate four phenotypic tests for the detection of metallo-β-lactamase (MBL) production in Pseudomonas aeruginosa in a low MBL prevalence setting.
Methods: Sixty clinical isolates of P. aeruginosa resistant to imipenem and/or meropenem and seven MBL-positive control strains were examined by: (i) MBL Etest; (ii) combined imipenem discs supplemented with EDTA (IPM-EDTA); (iii) β-lactam discs on dipicolinic acid plates (DF-DIPI); and (iv) the Cica-beta test. Spectrophotometric analysis of crude cell extracts for imipenem hydrolysis along with consensus PCRs for blaVIM and blaIMP was used as reference methods.
Results: Two clinical isolates (3%) were MBL-positive. The MBL Etest and IPM-EDTA test scored positive for all MBL-positive isolates, but showed specificities of 86% and 91%, and positive predictive values (PPVs) of only 20% and 29%, respectively. Adding resistance to ceftazidime (MIC >8 mg/L) as a criterion for MBL testing would reduce the number of isolates to be screened by 50% and increase the PPVs of the MBL Etest and IMP-EDTA test to 29% and 40%, respectively. The Cica-beta test correctly identified all MBL-negative isolates, but misidentified one MBL-positive clinical isolate as an extended-spectrum β-lactamase (ESBL)-producer and one as inconclusive (producing multiple β-lactamases). No reliable breakpoints could be defined for the DF-DIPI test due to overlapping inhibition zone diameters for MBL-positive and -negative isolates.
Conclusions: None of the phenotypic tests were optimal due to low sensitivity or specificity, resulting in low PPVs. Including ceftazidime resistance to the MBL-screening criteria would significantly improve the performance of the MBL Etest and IPM-EDTA disc test.
Keywords: metal-chelators , inhibitors , MBL Etest , combined disc , Cica-beta , dipicolinic acid
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Introduction |
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The prevalence of Pseudomonas aeruginosa producing metallo-β-lactamases (MBLs) is increasing worldwide, and transferable multidrug resistance associated with IMP and VIM MBLs is emerging in other clinically relevant species such as Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii.1 Thus, rapid and accurate detection of MBLs is necessary to implement adequate infection control measures to prevent nosocomial spread.
This study was designed to evaluate the screening criteria and four phenotypic tests, as second-line tests for the detection of MBL production in a Norwegian collection of carbapenem (imipenem and/or meropenem)-resistant clinical isolates of P. aeruginosa with a presumed low prevalence of MBLs.
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Materials and methods |
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Bacterial isolates
Imipenem- and/or meropenem-resistant clinical isolates of P. aeruginosa (n = 138) were submitted to the Reference Centre for Detection of Antimicrobial Resistance from various Norwegian clinical microbiology laboratories between 2004 and 2006 for the analysis of MBL production. All isolates were examined consecutively with an extended β-lactam susceptibility profile using Etest (AB Biodisk, Solna, Sweden), MBL Etest (AB Biodisk) and consensus PCRs for blaVIM and blaIMP in parallel with MBL-positive and -negative control strains. The EUCAST clinical MIC breakpoints were used for interpretation. All isolates were identified as P. aeruginosa using VITEK 2 (bioMérieux, Marcy l’Étoile, France). Sixty isolates were selected to evaluate four phenotypic MBL detection methods and screening criteria for MBL testing. Thirty-three isolates expressed a positive MBL Etest when examined upon submission to the Reference Centre. In addition, 27 MBL Etest-negative isolates were selected randomly from the collection to cover strains from all laboratories and throughout the whole collection period. Spectrophotometric analysis of crude cell extracts and subsequent inhibition by EDTA and PCR analysis for blaVIM and blaIMP were used as reference methods for the detection of MBL-positive isolates. Seven P. aeruginosa strains with known MBL production [VIM-1, VIM-2, VIM-7, IMP-16, GIM-1 (n = 2) and SPM-1] were used for quality control. P. aeruginosa ATCC 27853 (LGC Promochem, Boras, Sweden) was included as a negative quality control.
Performance and interpretation of the MBL Etest (AB Biodisk) and Cica-beta test (MAST Diagnostic, UK) were according to manufacturers’ instructions. A 0.5 McFarland inoculum on Mueller–Hinton (MH) agar (Becton Dickinson, Cockeysville, MD, USA) was used for the disc diffusion methods. Dipicolinic acid (200 mg/L) (Sigma-Aldrich, Oslo, Norway) was added to the MH agar plates for the disc diffusion test with dipicolinic acid (DF-DIPI).2 The combined disc test with imipenem plus EDTA (IPM-EDTA; Calbiochem, EMD Biosciences, La Jolla, CA, USA) was performed using 10 µg imipenem discs (Oxoid Ltd, Basingstoke, UK) imipenem discs supplemented with EDTA (930, 744, 518 and 292 µg/disc) in parallel with blank paper discs (Oxoid) supplemented with EDTA alone. Discs containing 10 µg of imipenem, 10 µg of ceftazidime and 30 µg of aztreonam were used for the DF-DIPI test.
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Results and discussion |
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The overall results from the different tests are presented in Table 1. Spectrophotometric analysis of crude cell extracts showed that two (3%) of the clinical isolates were MBL producers (data not shown). PCR and DNA sequence analysis showed that one isolate harboured blaVIM-2, whereas the other isolate harboured blaVIM-4. None of the other isolates showed any carbapenemase activity in the hydrolysis assay.
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MBL Etest
The MBL Etest has been evaluated in several studies and found to be a sensitive method for detection of MBL production in P. aeruginosa.3–5 However, some reports have shown that the specificity of the test is low, thus overestimating the number of MBL-positive isolates.6,7 Thirty-three of the clinical isolates had a positive MBL Etest when examined consecutively upon arrival in our laboratory. However, only 10 isolates (including both MBL-positive isolates) revealed a reproducible positive MBL Etest when retested as part of this study. The 27 isolates with an initial negative MBL Etest showed reproducible negative MBL Etest results upon retesting. These results indicate a previous problem with the reproducibility of borderline positive MBL Etest results. In summary, the MBL Etest correctly identified both MBL-positive clinical isolates and the seven MBL-positive control strains (Table 1). However, an additional eight MBL-negative isolates were also falsely identified as MBL-positive. The overall performance of the MBL Etest revealed a sensitivity, specificity and positive predictive value (PPV) of 100%, 86% and 20%, respectively.
Combined disc tests using various amounts of EDTA/disc have previously been evaluated for detection of MBLs.5,8 Initial studies examining 30 of the clinical isolates (including the two MBL-positive isolates) plus the MBL-positive control strains showed that discs containing 930, 744, 518 or 292 µg of EDTA/disc did not perform well in separating the MBL-positive and -negative isolates (Figure 1). In contrast to Pitout et al.,5 many isolates in our strain collection gave large inhibition zones around blank discs containing 930 and 744 µg of EDTA/disc (data not shown) suggesting that the optimal amount of EDTA may depend on the strain collection studied. The best separation between MBL-positive and -negative isolates was obtained using 518 µg of EDTA/disc and a breakpoint of 
8 mm (Figure 1c). However, these criteria gave five false-positive isolates, resulting in a sensitivity, specificity and PPV of 100%, 91% and 29%, respectively. All the MBL-positive control strains were correctly identified with the 518 µg of EDTA disc. A breakpoint of 7 mm as used previously would have further increased the number of false positive results.5,8 Interestingly, the five false-positive isolates in the IMP-EDTA assay were also among the eight false positives in the MBL Etest.
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DF-DIPI
Dipicolinic acid has been described as an effective inhibitor of MBLs and has no hydrolytic effect on imipenem as observed with mercaptopropionic acid.2 Kimura et al.2 showed a clear reduction of MICs in the presence of dipicolinic acid using a microbroth dilution assay. However, our observations showed that no reliable breakpoints could be defined for the DF-DIPI test due to significant overlapping inhibition zone diameters for MBL-positive and -negative isolates (data not shown).
The Cica-beta test utilizes the chromogenic cephalosporin HMRZ-86 along with inhibitors of MBLs, extended-spectrum β-lactamases (ESBLs) and AmpC enzymes.9 The test correctly identified 53 MBL-negative isolates as non-MBL producers as they could not hydrolyse HMRZ-86. Five of the MBL-negative isolates were able to hydrolyse HMRZ-86, but were not inhibited on the MBL test strip. The clinical isolate producing VIM-2 was misidentified as an ESBL, while the isolate producing VIM-4 was inconclusive (multiple β-lactamases). Thus, the sensitivity and specificity of the Cica-beta test were 0% and 100%, respectively, in our collection of Norwegian clinical strains. MBL production was correctly identified in six of seven MBL-positive control strains by the Cica-beta test. The VIM-7-producing control strain was only inhibited in the C test strip, indicating the presence of a derepressed AmpC. The zero sensitivity of the Cica-beta test in our collection of clinical strains is obviously biased due to the low number of MBL-positive isolates. The control isolates producing VIM-1 and VIM-2 were correctly identified. In contrast, the clinical isolates producing VIM-2 and VIM-4 were misidentified or gave inconclusive test results, respectively. Our observations are in accordance with the recently reported Cica-beta-based misidentification of 4 out of 10 VIM-producing clinical isolates of P. aeruginosa.9 These results indicate that VIM-producing isolates, which are considered the most prevalent MBL type in Europe,1 may go undetected if the Cica-beta test were to be used as the only second-line screening test for MBL production. The sensitivity of the Cica-beta would have increased to 67% if the MBL control strains were included in the overall results. This is in line with the recently reported sensitivity of 75% in a larger collection of MBL-positive P. aeruginosa strains.9
Various criteria for screening for MBL production in P. aeruginosa have been suggested. Although the number of MBL-positive clinical isolates in our strain collection was low, the susceptibility profile of the isolates indicates that imipenem and/or meropenem resistance alone as criteria for MBL screening is suboptimal. Including resistance to ceftazidime (MIC > 8 mg/L) along with resistance to imipenem and/or meropenem (MIC > 8 mg/L) as additional criteria would reduce the number of isolates to be screened by 30 (50%). Moreover, the number of false-positive results in the MBL Etest and IPM-EDTA test would also be reduced, thereby increasing the PPVs to 29% and 40%, respectively. These observations are in accordance with the recent report by Giske et al.,10 showing that decreased permeability and increased efflux are the most prevalent carbapenem resistance mechanisms in Norwegian and Swedish clinical isolates of P. aeruginosa. These observations support the use of ceftazidime resistance (MIC > 8 mg/L) along with carbapenem resistance as criteria for MBL screening.
The low PPVs of the MBL Etest and IPM-EDTA test make them suboptimal for the detection of MBLs in P. aeruginosa in a low MBL prevalence setting. Further refinement of the dipicolinic acid inhibition assay and the Cica-beta test is required before they can be recommended for the phenotypic detection of MBL in P. aeruginosa.
Careful evaluation of the susceptibility profile before testing and inclusion of ceftazidime resistance as an additional criterion along with imipenem and meropenem resistance for MBL screening would significantly reduce the number of isolates to be tested as well as the number of false-positive results in the MBL Etest and IPM-EDTA test.
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Funding |
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This study was funded through internal funding from the University Hospital of North Norway. Ø. S. is funded by a grant from the Northern Norway Regional Health Authority Medical Research Programme.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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This study was presented in part at the Seventeenth ECCMID, Munich, Germany, 2007. We are grateful to the clinical microbiology laboratories in Norway for sending clinical isolates to the Reference Centre and T. R. Walsh for the MBL-positive control strains. We also thank Bjørg Haldorsen for skilful technical assistance.
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References |
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1 Walsh TR, Toleman MA, Poirel L, et al. Metallo-β-lactamases: the quiet before the storm? Clin Microbiol Rev (2005) 18:306–25.
2 Kimura S, Ishii Y, Yamaguchi K. Evaluation of dipicolinic acid for detection of IMP- or VIM-type metallo-β-lactamase-producing Pseudomonas aeruginosa clinical isolates. Diagn Microbiol Infect Dis (2005) 53:241–4.[CrossRef][Web of Science][Medline]
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Walsh TR, Bolmstrom A, Qwarnstrom A, et al. Evaluation of a new Etest for detecting metallo-β-lactamases in routine clinical testing. J Clin Microbiol (2002) 40:2755–9.
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Lee K, Yong D, Yum JH, et al. Evaluation of Etest MBL for detection of blaIMP-1 and blaVIM-2 allele-positive clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol (2005) 43:942–4.
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Pitout JD, Gregson DB, Poirel L, et al. Detection of Pseudomonas aeruginosa producing metallo-β-lactamases in a large centralized laboratory. J Clin Microbiol (2005) 43:3129–35.
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Berges L, Rodriguez-Villalobos H, Deplano A, et al. Prospective evaluation of imipenem/EDTA combined disc and Etest for detection of metallo-β-lactamase-producing Pseudomonas aeruginosa. J Antimicrob Chemother (2007) 59:812–3.
7 Chu YW, Cheung TK, Ngan JY, et al. EDTA susceptibility leading to false detection of metallo-β-lactamase in Pseudomonas aeruginosa by Etest and an imipenem-EDTA disk method. Int J Antimicrob Agents (2005) 26:340–1.[CrossRef][Web of Science][Medline]
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Yong D, Lee K, Yum JH, et al. Imipenem-EDTA disk method for differentiation of metallo-β-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol (2002) 40:3798–801.
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Livermore DM, Warner M, Mushtaq S. Evaluation of the chromogenic Cica-β-test for detecting extended-spectrum, AmpC and metallo-β-lactamases. J Antimicrob Chemother (2007) 60:1375–9.
10 Giske CG, Buarø L, Sundsfjord A, et al. Alterations of porin, pumps and penicillin-binding proteins in carbapenem resistant clinical isolates of Pseudomonas aeruginosa. Microb Drug Res (2008) 14. in press.
11
Ilstrup DM. Statistical methods in microbiology. Clin Microbiol Rev (1990) 3:219–26.
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