JAC Advance Access published online on January 25, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm535
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Evaluation of different laboratory tests for the detection of metallo-β-lactamase production in Enterobacteriaceae
4th Department of Internal Medicine, Molecular Biology Section, University of Athens Medical School, Athens, Greece
* Correspondence address. 4th Department of Internal Medicine, University General Hospital ‘Attikon’, Rimini 1, 124 62 Chaidari, Greece. Tel: +32-105831984; Fax: +32-105326426; E-mail: egalani{at}med.uoa.gr
Received 17 November 2007; returned 5 December 2007; revised 28 November 2007; accepted 11 December 2007
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Abstract |
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Objectives: Clinical isolates of Klebsiella pneumoniae (91), Escherichia coli (49), Enterobacter spp. (27), Proteus mirabilis (17), Citrobacter freundii (2), Providencia stuartii (3) and Serratia spp. (5), with various MICs of imipenem, were examined for production of metallo-β-lactamases (MBLs) with different phenotypic laboratory tests that have been previously published to detect MBLs in Pseudomonas aeruginosa and Acinetobacter spp.
Methods: A total of 194 (95 MBL-positive and 99 MBL-negative) clinical isolates with imipenem MICs 
0.25 to >256 mg/L were examined. All isolates were evaluated by the double-disc synergy test (DDST), the combination disc test (CDT), the MBL Etest and the modified Hodge test. The presence of blaVIM and blaIMP genes was evaluated by in situ hybridization with specific probes and was certified by PCR.
Results: In 30 blaVIM-positive isolates that exhibited MICs of imipenem 4 mg/L, MBL Etest could not be evaluated. CDT with ceftazidime and 1900 or 750 µg of EDTA, and DDST after applying an imipenem disc 10 mm apart from a disc containing 
1900 µg of EDTA, showed the highest sensitivity (97.9% to 100%) and specificity (87.9% to 96%) rates among the analysed procedures. CDT with imipenem and 1900 µg of EDTA exhibited a sensitivity of 94.7% and showed very good specificity (98%).
Conclusions: The CDT with imipenem/imipenem+0.5 M EDTA or ceftazidime/ceftazidime+0.2 M EDTA and the DDST with imipenem 10 mm apart from EDTA are the most effective methods for the detection of MBLs in Enterobacteriaceae.
Key Words: MBLs , carbapenemases , EDTA synergy test , Hodge test
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Introduction |
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The emergence of acquired metallo-β-lactamases (MBLs) in Gram-negative bacilli is becoming a therapeutic challenge, as these enzymes usually possess a broad hydrolysis profile that includes carbapenems and extended-spectrum β-lactams (ESBL) with the exception of monobactams. Because most of these carbapenemases confer only reduced susceptibility to carbapenems in Enterobacteriaceae, the presence of MBLs may remain underestimated. This, together with the mobile nature of the gene cassettes carrying the IPM- and VIM-type enzymes, may enhance their spread and compromise the future usefulness of carbapenems for the treatment of serious infections caused by Gram-negative bacilli, therefore requiring more attention than ever before. These facts highlight the challenge for clinical microbiologists and infectious disease specialists to accurately detect MBL-producing isolates, to implement prompt infection control, and to treat infections caused by MBL or MBL+ESBL producers appropriately.
There is an increasing number of studies reporting on the emergence of Enterobacteriaceae that produce MBLs, which invariably hydrolyse carbapenems.1–5 Outbreaks with VIM-type-producing isolates have been reported mostly with Pseudomonas aeruginosa, but also with Klebsiella pneumoniae in Greece.6
Microbiology laboratories must be prepared to screen for MBL-producing isolates by a low cost, convenient and sensitive procedure.
MBL activity is inhibited by chelating agents. Double-disc synergy tests (DDSTs) using a ceftazidime disc and a 2-mercaptopropionic acid (MPA) disc,7 or an imipenem disc and an EDTA disc,8 have been reported as simple methods to detect MBL-producing clinical isolates. However, occasional adjustment of the distance between the two discs is required to obtain optimal results.7,8 An imipenem disc with added EDTA (750 µg) was reported by Yong et al.9 to detect MBL-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. with high sensitivity. Chu et al.10 urged caution in the application of the imipenem/EDTA disc method and the double Etest, as although these techniques are adequate for preliminary screening, as they are not likely to give false-negative results, they should not be used as the sole indicator for the presence of MBLs because susceptibility to EDTA seems to be common among Gram-negative isolates and therefore the antimicrobial effect of imipenem/EDTA and EDTA alone on the test organism cannot be distinguished.10 A modified Hodge test (previously known as the cloverleaf test) was reported by Lee et al.,8 which can be used to screen carbapenemase-producing Gram-negative bacilli but cannot distinguish MBL-producing from non-MBL carbapenemase-producing Gram-negative bacilli.
Although different phenotypic methods have been described, the CLSI (formerly NCCLS) (along with other international committees) currently does not include standardized recommendations for MBL screening. Herein, we evaluate the laboratory tests that have been previously described to detect MBLs in P. aeruginosa and Acinetobacter spp. in clinical isolates of Enterobacteriaceae with various MICs of imipenem.
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Materials and methods |
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Bacterial strains
Clinical isolates of Enterobacteriaceae including K. pneumoniae (91), Escherichia coli (49), Enterobacter spp. (27), Proteus mirabilis (17), Citrobacter freundii (2), Providencia stuartii (3) and Serratia spp. (5), isolated from blood, central catheter tip, urine, bronchoalveolar lavage and sputum specimens from patients admitted to various wards in a Tertiary University Hospital in the Athens area, Greece, between August 2002 and June 2006 were included in the study.
Consecutive isolates and a collection of ceftazidime-non-susceptible isolates were subjected to screening. We tried to include 50% MBL-producing isolates. Repeated isolates from the same patient were excluded. All bacterial isolates were identified by conventional methods and by use of API ID32GN and API ID32E systems (bioMérieux, Marcy l'Étoile, France).
Antibiotic susceptibility testing was performed by the disc diffusion method using Mueller–Hinton agar (BBL, Becton Dickinson, Cockeysville, MA, USA) and interpreted according to the CLSI.11 The MIC of imipenem was determined by the Etest, according to the manufacturer's recommendations (AB Biodisk, Solna, Sweden). MICs falling between two values of the Etest were rounded up to the next 2-fold value for statistical analysis. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as controls. Organisms with MICs 4 mg/L were considered to be susceptible, whereas those with MICs 
16 mg/L were characterized as resistant.11 ESBL producers were identified by a modified CLSI confirmatory test using a disc of aztreonam (30 µg) and a disc of aztreonam/clavulanate (30+10 µg). ESBL production was inferred if the zone given by the aztreonam/clavulanate disc was 5 mm larger than the zone given by aztreonam alone. Aztreonam was selected because it is not affected by MBLs, and the aztreonam/clavulanate discs were prepared as reported by the CLSI.11
All strains were checked for production of MBL by the Etest, the EDTA DDST and the combination disc test (CDT), as described below.
MBL Etest was performed according to the recommendations of the manufacturer (AB Biodisk). A reduction in the MIC of imipenem of three or more 2-fold dilutions in the presence of EDTA was interpreted as a positive test indicative of MBL production.12
EDTA DDST was performed according to Arakawa et al.7 and Lee et al.,8 with slight modifications. Test strains were adjusted to a turbidity equivalent to that of a 0.5 McFarland standard and used to inoculate Mueller–Hinton agar plates. Depending on the test, a 10 µg imipenem disc or a 30 µg ceftazidime disc (Becton Dickinson) was placed on the plate, and a blank filter paper disc (6 mm in diameter, Whatman filter paper no. 2) was placed at a distance of 10, 15 and 20 mm (edge to edge). Ten microlitres of a 0.5 M EDTA solution (1900 µg of disodium salt, dihydrate) was added to the blank disc. After overnight incubation, the presence of any synergistic inhibition zone was interpreted as positive.
CDT was performed according to Yong et al.,9 with slight modifications. Two 10 µg imipenem discs and two 30 µg ceftazidime discs (Becton Dickinson) were placed on a plate inoculated with the test organism, and 10 µL of 0.5 or 0.2 M EDTA (750 µg of disodium salt, dehydrate) solution was added to one disc of each antibiotic. The inhibition zones of the imipenem and imipenem+EDTA and ceftazidime and ceftazidime+EDTA discs were compared after 18 h of incubation in air at 35°C. A zone diameter difference between the imipenem and imipenem+EDTA discs or the ceftazidime and ceftazidime+EDTA discs 7 mm was interpreted as a positive result for MBL production.9
Dot-blot hybridization of total DNA was carried out using digoxigenin (DIG)-labelled blaVIM-1- and blaIMP-1-containing PCR amplicons. Labelling and detection of the probes were carried out using a DIG DNA labelling and detection kit (Roche Diagnostics, Mannheim, Germany). The presence of a blaVIM type gene was certified by PCR with primers VIM-F (5'-AGTGGTGAGTATCCGACAG-3') and VIM-R (5'-ATGAAAGTGCGTGGAGAC-3'), corresponding to nucleotides 1339–1357 and 1599–1582, respectively, of the blaVIM-1 integron (GenBank accession no. Y18050 [GenBank] ). PCR-RFLP analysis was used to differentiate between the clusters of blaVIM-1 (blaVIM-1, blaVIM-4, blaVIM-5) and blaVIM-2 (blaVIM-2, blaVIM-3, blaVIM-6, blaVIM-8, blaVIM-9, blaVIM-10), as a SacI restriction site is present at position 515 in the genes of the blaVIM-1 cluster.13
All isolates were also tested for carbapenemase production with the Hodge test (previously known as the cloverleaf test) that was performed as modified by Lee et al.8 The indicator organism, E. coli ATCC 25922, at a turbidity equivalent to that of a 0.5 McFarland standard, was used to inoculate, with a swab, the surface of a Mueller–Hinton agar plate (Becton Dickinson), and the test strain was heavily streaked from the centre to the plate periphery. After brief drying, a 10 µg imipenem disc (Becton Dickinson) was placed at the centre, and the plate was incubated overnight at 35°C. The presence of a distorted inhibition zone was interpreted as a positive result for carbapenem hydrolysis screening.
The performance of the phenotypic MBL detection methods was evaluated using PCR as the gold standard.14 The sensitivity was based on the ratio a/(a+c), where a represented the number of strains that were correctly identified as MBL producers by the tested assay and c represented the number of true MBL producers incorrectly identified as non-producing strains. The specificity was based on the ratio d/(b+d), where d represented the true number of strains not producing MBLs correctly identified by the tested assay and b represented the number of strains that were incorrectly identified as MBL producers. The performance of the modified Hodge test was not evaluated, as this test is not specific for the detection of an MBL-producing isolate.
Isoelectric focusing was performed with sonic extracts on precast polyacrylamide gels with pH gradients of 3–10. TEM-1 (pI 5.4), TEM-3 (pI 6.3) and SHV-5 (pI 8.2) enzymes, produced by reference strains, were used as standards with known pIs for β-lactamase characterization.
SHV-type and CMY-type ESBL production was confirmed by PCR. PCR amplification was performed with primers 5'-ATG CGT TAT ATT CGC CTG TG-3' and 5'-GTT AGC GTT GCC AGT GCT CG-3', which amplified a 865 bp sequence of the blaSHV-like gene, and primers 5'-CTG CTG CTG ACA GCC TCT TT-3' and 5'-TTT TCA AGA ATG CGC CAG GC-3', which amplified a 1108 bp sequence of the blaCMY-2-like gene.
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Results |
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From the 194 clinical isolates that were studied, 95 isolates proved to harbour a blaVIM-type gene by PCR and hybridization experiments. Seventy-eight (82.1%) were isolated from patients in the intensive care unit. K. pneumoniae isolates constituted 76.8%, Enterobacter spp. 12.6%, and E. coli only 4.2% of the total isolates (Table 1). Among MBL-positive K. pneumoniae isolates, 79.5% were also ESBL producers as detected by the CLSI modified confirmatory test. Also, two E. coli and two Enterobacter spp. isolates were both MBL and ESBL producers (Table 1). Imipenem MICs varied from 0.5 to >32 mg/L in blaVIM-positive isolates and between 0.064 and 1 mg/L in MBL non-producers. An ESBL-producing Enterobacter spp. isolate with an imipenem MIC of 12 mg/L and a P. mirabilis isolate with an imipenem MIC of 4 mg/L, both negative in VIM and IMP PCR experiments but also negative for MBL production with all methods tested, were further examined for their β-lactamase content. The Enterobacter spp. isolate produced a β-lactamase with a pI of around 9.0 corresponding to AmpC and another with a pI of 7.6 corresponding to an SHV-like β-lactamase, whereas the P. mirabilis isolate exhibited one nitrocefin hydrolysing zone with a pI of around 9.0, which was confirmed to be a CMY-like β-lactamase by PCR.
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The labelled blaVIM-1 probe hybridized with 95 of the isolates tested. None of the isolates hybridized with the labelled blaIMP probe. The same 95 isolates yielded a 510 bp amplification product in PCR experiments, which suggested the presence of a blaVIM allele in these isolates (Table 1). PCR-RFLP analysis showed that all but one isolate harboured a blaVIM gene of the blaVIM-1 cluster.
The MBL Etest proved to be inappropriate as 31.6% of the PCR-positive isolates (VIM producers) exhibited MICs of 4 mg/L. The Etest strip contains imipenem concentrations 4 mg/L and imipenem+EDTA 1 mg/L (based on imipenem MIC) and cannot be used as a screening test in isolates exhibiting imipenem MICs 4 mg/L, unless modified to contain lower concentrations of imipenem.
Among the different combinations of carbapenemase inhibitor (at EDTA concentrations of 0.2 or 0.5 M) and imipenem or ceftazidime discs used in this study, the CDTs with imipenem/imipenem+0.5 M EDTA (1900 µg) and ceftazidime/ceftazidime+0.2 M EDTA (750 µg) gave the most indisputable results, with sensitivities of 94.7% and 97.9% and specificities of 98.0% and 96.0%, respectively (Table 2). For MBL-positive isolates, impregnation of imipenem discs with 750 or 1900 µg of EDTA increased inhibition zones from 0 to 29 mm (mean, 10.6 mm) and from 3 to 32 mm (mean, 13 mm), respectively, whereas impregnation of ceftazidime discs increased inhibition zones from 0 to 27 mm (mean, 14.7 mm) and 8 to 31 mm (mean, 18.4 mm), respectively. For MBL-negative isolates, inhibition zones of imipenem increased from 0 to 5 mm (mean, 0.6 mm) and from 0 to 8 mm (mean, 0.8 mm) and inhibition zones of ceftazidime increased from 0 to 16 mm (mean, 1.5 mm) and from 0 to 20 mm (mean, 2.5 mm), when impregnated with 750 or 1900 µg of EDTA, respectively. Ninety (94.7%) of the MBL-positive and 97 (98%) of the MBL-negative isolates were categorized according to the criterion of a 7 mm increase in the inhibition zone with the imipenem discs to which 1900 µg of EDTA was added. However, by the same criterion, when 750 µg of EDTA was added, all the MBL-negative isolates were well separated from MBL-positive isolates, but 20% of MBL-positive isolates showed false-negative results. Increased concentration of EDTA in CDTs resulted in false positives, in which the extended zone size difference between the imipenem or ceftazidime and imipenem+EDTA or ceftazidime +EDTA discs was due to susceptibility of the organism to EDTA rather than attenuation of any MBL through the EDTA chelation of zinc ions.
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In DDST, the 10 mm distance between the imipenem and EDTA discs exhibited excellent sensitivity (100%) and a specificity of 91.9% (Table 2). Increasing the distance of the discs to 20 mm resulted in the reduction of sensitivity to only 77.9% and enhancement of specificity to 96.0% (Table 2).
This was also confirmed with ceftazidime DDST, in which the sensitivity reduced from 77.9% to 20% and the specificity increased from 89.9% to 99.0%. Non-ESBL isolates exhibited better sensitivity rates than ESBL isolates (89.7 versus 72.7%), but poorer specificity rates (89.2 versus 93.8%) (Table 2).
The modified Hodge test is a method used for screening carbapenemase activity but not specifically MBL production, as other enzymes such as oxacillinases can be detected. Among 95 MBL-positive isolates, 4 (4.2%) were characterized as false negatives (Table 1) while results for 4 isolates were difficult to interpret. Eight isolates were positive in the modified Hodge test but negative in PCR and hybridization experiments, suggesting the presence of a carbapenemase other than an MBL enzyme. These isolates were further examined for their β-lactamase content. The four P. mirabilis isolates exhibited one nitrocefin hydrolysing zone with a pI of around 9.0, which was confirmed by PCR to be CMY-2-like, and another band with a pI of 5.6 or 5.4 corresponding to a TEM-like β-lactamase. The four Enterobacter isolates produced two β-lactamases; one with a pI of around 9.0, probably corresponding to AmpC, and the other with a pI of 7.1, which was proved by PCR to be a SHV-type β-lactamase.
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The implementation of a simple and accurate laboratory method to detect MBL production in Gram-negative bacilli is useful, particularly in countries where MBL strains are increasingly reported.1–6
PCR analysis is the gold standard method for the detection of MBL producers,14 but it is not suitable for daily testing in clinical laboratories due to the cost and inconvenience.
Arakawa et al.7 first described a simple disc diffusion test for the detection of IMP-1-type MBL-producing Gram-negative bacteria. They used a ceftazidime disc and two MBL inhibitors [EDTA and 2-mercaptopropionic acid (2-MPA)]. The specificity and sensitivity of this test were comparable with those of PCR analysis using blaIMP-specific primers and the authors concluded that this convenient test could be valuable for daily use in clinical laboratories. Later, Lee et al.8,15 reported that the Hodge test was reliable when zinc sulphate was added to imipenem discs, whereas imipenem/EDTA, ceftazidime/MPA and ceftazidime/sodium mercaptoacetic acid (SMA) DDSTs had both strengths and weaknesses for the detection of MBL-producing isolates of Pseudomonas spp. and Acinetobacter spp. Finally, they proposed the use of an imipenem disc and an EDTA (1900 µg) or an EDTA (750 µg)+SMA (2 mg) disc, which improved performance.15 Yong et al.9 reported a CDT that was simple to perform and highly sensitive in differentiating MBL-producing isolates. With Pseudomonas spp., all the MBL-positive isolates were well separated from MBL-negative isolates by the criterion of a 7 mm increase in the inhibition zone with the discs to which 750 µg of EDTA was added. The specificity was excellent for pseudomonads and good for acinetobacters as 4.3% MBL-positive and 9.1% MBL-negative acinetobacters showed false-negative and false-positive results, respectively. However, Chu et al.10 reported later that susceptibility to EDTA seems to be common among these species. Whether the CDT is able to differentiate the EDTA-induced membrane permeabilization effect and the zinc ion chelation MBL inhibition effect remains to be investigated.
In the present study, we tested 194 clinical isolates of Enterobacteriaceae with various MICs of imipenem (95 MBL producers), with the above-mentioned laboratory tests that have been previously described to detect MBLs in P. aeruginosa and Acinetobacter spp. We have also tried to alter the distance between the imipenem and EDTA discs to see whether the synergy can be more visible, and we used two different concentrations of EDTA in CDT (1900 or 750 µg) to separate MBL-positive from MBL-negative isolates.
Eight imipenem-susceptible isolates with positive Hodge test, but negative for all other methods tested, including the hybridization and PCR experiments, proved to produce chromosomal or plasmid AmpC-like together with a non-ESBL SHV- or TEM-like β-lactamase. Hodge-equivocal isolates must, therefore, be confirmed by another method, especially in P. mirabilis isolates where swarming is a factor that may complicate the reading and evaluation of the results.
CDT with ceftazidime and EDTA (1900 or 750 µg), and DDST with an imipenem disc 10 or 15 mm apart from a disc soaked with 10 µL of a 0.5 M EDTA solution (1900 µg), showed the highest sensitivity (97.9% to 100%) and specificity (87.9% to 96%) among the analysed procedures (Table 2). We also tried to alter the distance between the imipenem and EDTA discs to see whether the synergy could be more visible, but we found 10 mm to be optimal, as described by Arakawa et al.7 CDT with imipenem and 1900 µg of EDTA exhibited a sensitivity of 94.7% and showed a very good specificity of 98%. The lower amounts of EDTA employed in CDT resulted in higher specificity rates, but lower sensitivity rates. In some cases, however, the high amounts of the chelating agent employed resulted in inhibition zones of bacterial growth, giving rise to confusing results.
The DDST with the ceftazidime disc showed the lowest sensitivity (20% to 77.9% depending on the distance between the two discs). Nevertheless, DDST with imipenem and EDTA discs 10 mm apart provides a convenient assay for the initial screening of potential MBL producers in the clinical setting. With a sensitivity of 100% and a specificity of 91.9%, this method is a low-cost approach appropriate for routine use in clinical settings, but should not be used as the sole indicator for the presence of MBLs.
Although the laboratory tests were evaluated using VIM-producing clinical isolates only, the tests can also be used to screen for isolates producing MBLs other than VIM-type MBLs. Some reports describe the use of the tests for A. baumannii producing an IMP-type MBL, but also for P. aeruginosa, Enterobacter spp., K. pneumoniae, P. mirabilis, C. freundii and S. marcescens.7–9
On the basis of the findings of our study, we conclude that for the detection of MBLs in Enterobacteriaceae, the EDTA DST is better than CDT and MBL Etest. This method might be useful for routine use in microbiology laboratories to screen for the emergence of MBL producers for clinical and surveillance purposes.
It should be pointed out that CLSI documents do not yet propose a method for the detection of MBL production in Gram-negative isolates, and hence the methods evaluated here could be of use.
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No specific funding has been received.
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None to declare.
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Acknowledgements |
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We thank Zoi Chryssouli and Konstantina Orlandou for their technical work.
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