JAC Advance Access originally published online on February 22, 2007
Journal of Antimicrobial Chemotherapy 2007 59(4):812-813; doi:10.1093/jac/dkm001
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Correspondence |
Prospective evaluation of imipenem/EDTA combined disc and Etest for detection of metallo-ß-lactamase-producing Pseudomonas aeruginosa
Microbiology Department, Erasme Hospital-ULB, 808 route de Lennik, 1070 Brussels, Belgium
* Corresponding author. Tel: +3225554518; Fax: +3225553110; E-mail: lberges{at}ulb.ac.be
Keywords: carbapenems , resistance , P. aeruginosa , MBLs , ß-lactamases
Nosocomial infection involving multiresistant Pseudomonas aeruginosa is a growing problem worldwide. Rapid detection of metallo-ß-lactamases (MBLs) is crucial for patient management and appropriate infection control procedures. We sought to compare the accuracy of MBL enzyme detection in carbapenem-resistant P. aeruginosa isolated at Erasme Hospital by using two MBL screening tests: the imipenem/EDTA combined disc test (MBL-CD) and the imipenem/EDTA Etest (MBL-Etest), both of which are based on the ability of EDTA to sequester zinc ions and to inactivate the metalloenzymes.
From May 2004 to February 2005, the MBL-CD (Rosco®, Denmark) method was included in the routine antimicrobial susceptibility testing of P. aeruginosa by disc diffusion method. Antibiogram was performed and interpreted according to CLSI criteria.1 We prospectively tested all consecutive P. aeruginosa isolates during the study period, including non-duplicate clinical isolates and ceftazidime-resistant isolates from surveillance cultures in ICU patients. All meropenem and imipenem co-resistant clinical isolates and carbapenem-susceptible P. aeruginosa isolates showing a positive MBL-CD test were analysed. In MBL-CD, imipenem and combined imipenem/EDTA (750 µg) discs (Rosco® Neosensitabs) were placed on the agar plates. After overnight incubation at 35°C, inhibition zones of the imipenem with and without EDTA were compared. The test is considered positive if a 
6 mm increase in the zone diameter for imipenem/EDTA is observed. The MBL-Etest strip (AB Biodisk, Solna, Sweden) contains a double-sided dilution range of imipenem (4–256 mg/L) and imipenem (1–64 mg/L) combined with EDTA (320 mg/L); inoculation and incubation were performed according to the manufacturer's instructions. The test was considered positive if a reduction of imipenem MIC by three or more 2-fold dilutions was observed in the presence of EDTA. Two consensus PCR assays for detection of all blaVIM- and blaIMP-encoding genes were performed [ref. 2 and L. Poirel (Hôpital Kremlin Bicêtre, Paris, France), personal communication]. Carbapenem hydrolysis was performed by UV spectrophotometry on isolates with positive phenotypic MBL detection tests and negative PCR for IMP/VIM genes.
Of the 587 P. aeruginosa isolates analysed during the period, 42 (7.2%) were co-resistant to imipenem and meropenem. The MBL-CD screening was positive for 26 of these carbapenem-resistant strains. The 16 MBL-CD-negative P. aeruginosa isolates were negative by both Etest and PCR analysis. Among the 26 MBL-CD-positive P. aeruginosa, 23 isolates were MBL-Etest positive and 3 were negative by Etest and PCR. Of these 23 isolates, 16 carried the VIM gene, 3 the IMP gene and 4 showed negative VIM/IMP-consensus PCR. Of the four MBL-Etest-positive P. aeruginosa isolates that were PCR negative, only one showed carbapenemase activity by UV spectrophotometry, suggesting the presence of an MBL from another enzyme family. The three others were interestingly recovered from mucoid isolates of cystic fibrosis patients.
Ten carbapenem-susceptible P. aeruginosa isolates with positive MBL-CD showed negative MBL-Etests and VIM/IMP-consensus PCR .
It is known that EDTA may increase bacterial cell-wall permeability and that zinc (chelated by EDTA) accelerates imipenem decomposition and decreases OprD expression of P. aeruginosa.3–5 These non-specific effects cause MBL-CD false-positive results.
The ability of MBL-CD and MBL-Etest to predict MBL production in carbapenem co-resistant strains showed a sensitivity of 100% [95% confidence interval (95% CI): 80–100] for both tests; the specificity was 72.7% (95% CI: 49.6–88.4) and 86.4% (95% CI: 64–96.4), respectively. In this sample, the positive predictive value for those tests was 76.9% (95% CI: 55.9–90.2) and 86.9% (95% CI: 65.3–96.6) and the negative predictive value was 100% (95% CI: 75.9–100) and 100% (95% CI: 79.1–100), respectively. Excluding cystic fibrosis patients' isolates (n = 3), specificity was, respectively, 84.2% (95% CI: 59.5–95.8) and 100% (95% CI: 79.1–100) for the CD and Etests; the positive predictive value was 86.9% (95% CI: 63.3–96.6) and 100% (95% CI: 80–100) and the negative predictive value was 100% (95% CI: 75.9–100) and 100% (95% CI: 79.1–100), respectively.
MBL-mediated carbapenem resistance is expressed at high level. Therefore contact growth (no inhibition zone) around imipenem/meropenem discs has been suggested as a clue to MBL production. We therefore studied the inhibition zone diameter around imipenem and meropenem discs (12 mm diameter) for the 42 carbapenem-resistant P. aeruginosa. Of the 20 confirmed MBL-producing isolates (16 VIM, 3 IMP and 1 non-VIM/non-IMP with carbapenemase activity detected by UV spectrophotometry), 17 had a meropenem and imipenem inhibition zone diameter of <13 mm (contact growth) and 3 had a zone between 13 and 15 mm.
Of the 22 MBL-negative isolates, 16 showed a >15 mm imipenem/meropenem inhibition zone and 6 were 
15 mm. On the basis of these observations, we propose an MBL detection algorithm (Figure 1) for carbapenem-resistant P. aeruginosa, excluding mucoid isolates from cystic fibrosis patients, with selective use of secondary confirmation tests.
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To validate this algorithm, we analysed 568 isolates of P. aeruginosa non-susceptible by disc diffusion to carbapenems, which were prospectively collected from March 2005 to October 2006 in our laboratory. After excluding isolates from cystic fibrosis patients, 105 of these isolates showed a positive MBL-CD test. These 105 isolates were all tested by MBL-Etest and PCR. Of these, 77 were MBL-Etest positive, 74 carried the VIM gene and 2 harboured the IMP gene. In this prospective sample, the algorithm for MBL detection had a sensitivity of 100% (95% CI: 94–100), a specificity of 96.6% (95% CI: 80.4–99.8), a positive predictive value of 98.7% (95% CI: 92–99.9) and a negative predictive value of 100% (95% CI: 85–100%). One isolate had a ‘contact growth’ with no inhibition zone, positive MBL-Etest but negative VIM/IMP-consensus PCR. In addition, two P. aeruginosa strains carrying other carbapenem resistance mechanisms with derepressed AmpC, loss of OprD porin and/or MexXY overexpressed efflux pump systems were tested and were classified as non-carbapenemase-producing strains by the algorithm.6
In summary, we found that the MBL-CD test was a rapid and sensitive method for MBL production screening in P. aeruginosa but lacks specificity. Contact growth or carbapenem inhibition diameter of <15 mm performed equally well. For mucoid strains from cystic fibrosis patients, false-positive results occurred with phenotypic MBL screening methods, and molecular confirmation was needed. When these strains are excluded, MBL-Etest appeared to be a specific tool to confirm MBL production in high-level carbapenem-resistant strains. This algorithm can be implemented to guide the clinical and epidemiological patient management in centres lacking molecular biology tools until confirmation by a reference centre. Further studies are needed to confirm the accuracy of this simple detection strategy in other clinical settings and with strains harbouring other MBL enzymes.
None to declare.
Acknowledgements
A part of this work was presented at the Sixteenth European Congress of Clinical Microbiology and Infectious Diseases, Nice, 2006. We wish to thank Laurent Poirel for providing control strains (VIM-2-producing P. aeruginosa COL-1 and IMP-4-producing Escherichia coli EC158) and primers used in this study, and Giuseppe Cornaglia and Annarita Mazzariol for carbapenem hydrolysis detection by UV spectrophotometry.
References
1 Clinical and Laboratory Standards Institute. (2005) Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15(CLSI, Wayne, PA, USA).
2
Pitout JD, Gregson DB, Poirel L, et al. (2005) Detection of Pseudomonas aeruginosa producing metallo-ß-lactamases in a large centralized laboratory. J Clin Microbiol 43:3129–35.
3 Livermore DM and Brown DFJ. Detection of ß-Lactamase-Mediated Resistance http://www.bsac.org.uk/_db/_documents/Chapter_6.pdf (18 October 2006, date last accessed).
4
Baxter IA and Lambert PA. (1997) The effect of zinc on imipenem. J Antimicrob Chemother 39:838–9.
5
Conejo MC, Garcia I, Martinez-Martinez L, et al. (2003) Zinc eluted from siliconized latex urinary catheters decreases OprD expression, causing carbapenem resistance in Pseudomonas aeruginosa.. Antimicrob Agents Chemother 47:2313–5.
6
Deplano A, Denis O, Poirel L, et al. (2005) Molecular characterization of an epidemic clone of panantibiotic-resistant Pseudomonas aeruginosa.. J Clin Microbiol 43:1198–204.
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