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Many clinically relevant species of Gram-negative bacilli are often resistant to β-lactam antibiotics, including extended-spectrum cephalosporins, but rarely to carbapenems [1]. Carbapenem resistance was mainly due to decreased outer-membrane permeability [2]. However, IMP-1 metallo-β-lactamase-producing Pseudomonas aeruginosa emerged in Japan [3], and then the resistance spread to other species. Recently, IMP-2-producing Acinetobacter baumannii[4] and VIM-1- and VIM-2-producing strains of P. aeruginosa have been reported in Europe [5–7]. The metallo-β-lactamase-producing strains of P. aeruginosa which we reported in 1998 [8] were subsequently identified as VIM-2 β-lactamase producers. As metallo-β-lactamase genes reside in mobile integrons, rapid detection of metallo-β-lactamase-producing bacteria is necessary not only to augment our understanding of the resistance, but also to control the spread of the resistance.

Hodge et al [9] developed a test to detect penicillinase-producing Neisseria gonorrhoeae and other species of bacteria. A double disk synergy test using β-lactam and β-lactamase-inhibitor disks is a convenient method of detecting extended-spectrum β-lactamase-producing Gram-negative bacilli [10]. EDTA inhibition of β-lactamase activity is used to differentiate a metallo-β-lactamase from other β-lactamases [11]. We found that the tests of modified Hodge and EDTA-disk synergy were simple to use to screen metallo-β-lactamase-producing strains [12]. The aim of this study was to evaluate the usefulness of the modified Hodge and EDTA-disk synergy tests for the screening of metallo-β-lactamase-producing strains from a large number of imipenem-resistant clinical isolates of Pseudomonas spp. and Acinetobacter spp.

Pseudomonas spp. and Acinetobacter spp. were isolated in 1995–99 from urine, sputum and other clinical specimens and identified by conventional methods and by the ID 32 GN system (bioMerieux Vitek, Marcy-l'Etoile, France). Isolates resistant to imipenem by the disk diffusion test [13] were kept at −76 °C in 20% skimmed milk until used in this study. P. aeruginosa strains producing IMP-1, VIM-1 and VIM-2 metallo-β-lactamases, and a Serratia marcescens strain producing Sme-1 serine β-lactamase, were used as control strains.

The test of Hodge et al [9] was modified by substituting Escherichia coli ATCC 25922 for penicillin-susceptible Staphylococcus aureus ATCC 25923, and 10-µg imipenem disk for a 10-U penicillin disk. The surface of a Mueller–Hinton agar plate was inoculated evenly using a cotton swab with an overnight culture suspension of E. coli, which was adjusted to one-tenth turbidity of the McFarland no. 0.5 tube. After brief drying, an imipenem disk was placed at the center of the plate, and imipenem-resistant test strains from the overnight culture plates were streaked heavily from the edge of the disk to the periphery of the plate. The presence of a distorted inhibition zone after overnight incubation was interpreted as modified Hodge test positive.

For the EDTA-disk synergy test, an overnight culture of the test strain was suspended to the turbidity of a McFarland no. 0.5 tube and used to swab inoculate a Mueller–Hinton agar plate. After drying, a 10-µg imipenem disk (BBL, Cockeysville, MD) and a blank filter paper disk were placed 10 mm apart from edge to edge, and 10 µL of 0.5 M EDTA solution was then applied to the blank disk, which resulted in approximately 1.5 mg/disk. After overnight incubation, the presence of an enlarged zone of inhibition was interpreted as EDTA-synergy test positive.

Tests used to determine the characteristics of the EDTA-disk synergy-positive strains were agar dilution susceptibility [14] to imipenem (Merck Sharp & Dohme, West Point, PA, USA), and imipenem-hydrolyzing activity [15] of the crude cell sonicate with or without EDTA treatment at 30 °C by using a UV spectrophotometer (Shimadzu Corp., Tokyo, Japan). Isoelectric focusing of the β-lactamase [16] was performed using a polyacrylamide gel (pH 3–10) and a ThermoFlow ETC Unit (Novex Experimental Technology, San Diego, CA, USA), and visualization of the bands with a 1 mg/mL solution of nitrocefin (Unipath Ltd, Basingstoke, UK). The blaVIM-2 gene was detected by PCR using the following primers: forward 5′-ATT GGT CTA TTT GAC CGC GTC-3′ (nucleotides 1342–1362), and reverse 5′-TGC TAC TCA ACG ACT GAG CG-3′ (nucleotides 2102–2121) [7]. Template DNA was extracted by boiling for 5 min. The PCR conditions were one cycle of predenaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s and extension at 72 °C for 1 min, and one cycle of final extension at 72 °C for 7 min.

Among a total of 530 imipenem-resistant isolates screened by the modified Hodge test, 43 were positive and 31 were equivocal (Table 1, Figure 1). Six of nine imipenem-resistant P. putida isolates showed positive results at 35 °C, but the remaining three equivocal isolates became positive at 30 °C. Among the 28 isolates of Acinetobacter spp. tested, nine were positive and four were equivocal. The distortion patterns shown by all of the isolates were similar to those of the control strains producing IMP-1, VIM-1, VIM-2 and Sme-1.

Table 1.  Screening of metallo-β-lactamase-producing species of Pseudomonas and Acinetobacter by the modified Hodge test and the EDTA-disk synergy test
Modified Hodge test (no. tested)No. (%) of isolates with EDTA-disk synergyaNo. (%) of isolates with imipenem hydrolysisa
PositiveNegativePositiveNegative
  • a

    Sensitivities and specificities for the modified Hodge test were 100% and 88%, respectively, and for the EDTA-disk synergy test both 100%, as compared to imipenem hydrolysis. Only 54 of 456 Hodge and EDTA-disk synergy-negative isolates and nine Hodge-equivocal, but EDTA-disk synergy-negative isolates were tested for imipenem hydrolysis.

  • b

    Seven isolates were A. baumannii and two were Acinetobacter spp.

  • c

    Equivocal when incubated at 35 °C, but positive at 30 °C.

Positive (43)
 P. aeruginosa (28)28 (100)0 (0)28 (100)0 (0)
 P. putida (6)6 (100)0 (0)6 (100)0 (0)
 Acinetobacter spp. (9)b9 (100)0 (0)9 (100)0 (0)
Equivocal (31)
 P. aeruginosa (24)15 (62.5)9 (37.5)15 (62.5)9 (37.5)
 P. putida (3)c3 (100)0 (0)3 (100)0 (0)
 A. baumannii (4)4 (100)0 (0)4 (100)0 (0)
Negative (456)
 P. aeruginosa (441)0 (0)441 (100)0 (0)39 (100)
 Acinetobacter spp. (15)0 (0)15 (100)0 (0)15 (100)
Total (530)654656563
image

Figure 1. Modified Hodge test. A Mueller–Hinton agar plate was inoculated with E. coli ATCC 25922. An imipenem disk was put in place and imipenem-resistant test isolates were streaked from the edge of the disk to the periphery of the plate and incubated overnight. (A) and (C) are imipenem-hydrolyzing strains which distorted the inhibition zone. (B) and (D) are imipenem-non-hydrolyzing strains with no effect on the zone.

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All of the 43 modified Hodge test-positive isolates showed an EDTA-disk synergy-positive pattern (Figure 2A, C) similar to those shown by IMP-1- VIM-1- and VIM-2-producing control strains. Twenty-two of 31 equivocal strains were also synergy positive. The inhibition zones which were measured from the edge of the disk were 0–3 mm with imipenem disks alone, while those between EDTA and imipenem disks were 6–14 mm. The inhibition zones were enlarged at least 6 mm by the presence of the EDTA disk.

image

Figure 2. EDTA-disk synergy test. A Mueller–Hinton agar plate was inoculated with an imipenem-resistant test isolate, and a 10-µg imipenem disk (left) and a 5 mM EDTA disk (right) were put in place and incubated overnight. (A) A synergistic inhibition zone is shown between an imipenem disk (left) and an EDTA disk (right) by a VIM-2 metallo-β-lactamase-producing strain. (B) Only a partially inhibited zone around an EDTA disk is shown by a metallo-β-lactamase-non-producing strain. (C) A metallo-β-lactamase-producing strain showing a small enhanced inhibition zone when the disks were placed 15 mm apart.

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All of the 65 EDTA-disk synergy-positive strains hydrolyzed imipenem. Fifty-four of the 456 Hodge- and EDTA-disk synergy-negative isolates and nine Hodge-equivocal but EDTA-disk synergy-negative isolates were spectrophotometrically negative for imipenem hydrolysis (Table 1). Sensitivities and specificities were for the modified Hodge test 100% and 88%, respectively, and for the EDTA-disk synergy test both 100% as compared to imipenem hydrolysis.

The hydrolysis of imipenem by all of the EDTA-disk synergy-positive strains was inhibited by 10 mM EDTA, but not by 50 µM clavulanic acid. The MIC range of imipenem for the isolates was 8–256 mg/L. Relative rates of hydrolysis of imipenem by representative isolates were 24–47% when compared to that of ampicillin. The blaVIM-2 gene was detected from all of the EDTA-synergy-positive strains by PCR. The pI of VIM-2 β-lactamase was approximately 5.3.

In the original test of Hodge et al [9], a penicillin G disk and penicillin G-susceptible Staphylococcus aureus strain were used to differentiate penicillinase-producing gonococci. In this study, it was found that background-lawn formation by Staphylococcus aureus was inhibited by some test strains of P. aeruginosa and that the inhibition zones with imipenem disks were too large. Our modified Hodge test, using a commercially available 10-µg imipenem disk and an indicator E. coli strain at one-tenth the turbidity of a McFarland 0.5 tube, detected not only IMP-1, VIM-1 and VIM-2, but also an Sme-1 producer. The test was simple to use for screening carbapenem-hydrolyzing strains out of a large number of imipenem-resistant isolates. The distortion of the inhibition zone was less distinct compared to that shown by gonococci [9], but in most cases it was not difficult to suspect carbapenem hydrolysis (Figure 1). Three of nine P. putida isolates which were modified Hodge test equivocal at 35 °C became positive at 30 °C, possibly because of the lower optimal growth temperature of the organism.

Our preliminary results [12] showed that a double disk synergy test using a 10-µg imipenem disk was easier to read when the EDTA disk content was 1.5 mg than when it was 3 or 4.5 mg. An approximately 1.5-mg EDTA disk can be prepared by adding 10 µL of a readily available 0.5 M EDTA solution (pH 8.0) to a blank filter paper disk. The test results were similar with both dried and wet disks. Occasional imipenem-hydrolyzing strains did not show enhanced inhibition zones or showed only small zones when the two disks were placed 15 mm apart from edge to edge (Figure 2C). In general, the results were more distinct when the distance between the two disks was 10 mm.

It was recently reported by Arakawa et al that a disk containing 2-mercaptopropionic acid gave the clearest synergy in screening IMP-1-producing strains [17]. However, 0.5 M EDTA solution (pH 8.0) is readily available in most laboratories. Arakawa et al used ceftazidime disks instead of imipenem disks to increase the sensitivity of the test, as some strains were resistant to low levels of imipenem only. However, ceftazidime should be less specific for the detection of metallo-β-lactamase producers. McLaughllin and Laconis [18] used a disk containing 20 µL of a 5 mM EDTA solution to screen for the presence of metallo-β-lactamase in exponentially growing cell sonicates. However, it takes time to prepare cell sonicates.

Although the usefulness of our modified Hodge and EDTA-disk synergy tests was evaluated using VIM-2-producing clinical isolates only, the results with control strains producing IMP-1, VIM-1, VIM-2 and Sme-1 β-lactamase suggested that the tests could also be used to screen for strains producing carbapenemases other than VIM-2. A limitation of this study is that although many VIM-2 metallo-β-lactamase-producing isolates were tested, PFGE patterns suggest that some of them belong to the same clone.

In conclusion, the tests of modified Hodge and EDTA-disk synergy are simple to perform and should be suitable to screen metallo-β-lactamase-producing clinical isolates of P. aeruginosa, P. putida and Acinetobacter spp., to characterize the β-lactamases and to control spread of the resistance.

Acknowledgments

  1. Top of page
  2. Acknowledgments
  3. References

We thank Dr David M. Livermore, Central Public Health Laboratory, London, UK, for providing IMP-1 and VIM-2 β-lactamase-producing P. aeruginosa and Sme-1-producing Serratia marcescens, and Professor Gian M. Rossolini, Universita di Siena, Siena, Italy for VIM-1-producing P. aeruginosa. We thank Young Hee Seo for her technical assistance.

References

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