CONCISE COMMUNICATIONS
Modified Hodge and EDTA-disk synergy tests to screen metallo-ji-
lactamase-producing strains of Pseudomonas and Acinetobacter species
K. Lee, Y Chong', H. B. Shin, Y A. Kim, D. Yong and J. H. Yum
Department of Clinical Pathology and Research Institute of Bacterial Resistance, Yonsei University College of Medicine,
Box 8044, Seoul, Korea
*Tel: + 82 2361 5866 Fax: + 82 2313 0908 E-mail: whonetkor@yumc.yonsei.ac.kr
Accepted 30 November 2000
Many clinically relevant species of Gram-negative bacilli are
often resistant to p-lactam antibiotics, including extended-
spectrum cephalosporins, but rarely to carbapenems [1]. Car-
bapenem resistance was maiuly due to decreased outer-mem-
brane permeability [2]. However, IMP-l metallo-ji-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-l- and VIM-2-
producing strains of P aeruginosa have been reported in Europe
[5-7]. The metallo-p-lactamase-producing strains of P aerugi-
nosa which we reported in 1998 [8] were subsequently identi-
fied as VIM-2 p-lactamase producers. As metallo-ji-lactamase
genes reside in mobile integrons, rapid detection of metallo-ji-
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 p-lactam and p-lactamase-
inhibitor disks is a convenient method of detecting extended-
spectrum p-lactamase-producing Gram-negative bacilli [10].
EDTA inhibition of p-lactamase activity is used to differentiate
a metallo-ji-lactamase from other p-lactamases [11]. We found
that the tests of modified Hodge and EDTA-disk synergy were
simple to use to screen metallo-p-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-p-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 iden-
tified 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-I, VIM-l and VIM-2 metallo-ji-lactamases,
© 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases
c.P. o.
and a Serratia marcescens strain producing Sme-l serine 15-
lactamase, were used as control strains.
The test of Hodge et al [9] was modified by substituting
Escherichia coli ATCC 25922 for penicillin-susceptible Staphy-
lococcus aureus ATCC 25923, and 10-J-lg imipenem disk for a lO-
U penicillin disk. The surface of a Mueller-Hinton agar plate
was inoculated eveuly 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 1O-J-lg imipenem disk (BBL, Cockeysville, MD)
and a blank filter paper disk were placed 10 mm apart from edge
to edge, and 10 J-lL of0.5 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 eularged 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 p-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 ofthe bands with a 1 mg/mL solution ofnitrocefin
(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
 Concise Communications 89

Table 1 Screening of metallo-Il-Iactamase-producing species of Pseudomonas and Acinetobacter by the modified Hodge test and the EDTA-disk synergy test

No. (%) of isolates with EDTA-disk No. (%) of isolates with imipenem
svnerqv" hvdrolvsis"

Modified Hodge test

(no. tested) Positive Negative Positive Negative

Positive (43)
P aeruginosa (28) 28 (100) 0(0) 28 (100) 0(0)
P putida (6) 6 (100) 0(0) 6 (100) 0(0)
Acinetobacterspp. (9)b 9 (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)C 3 (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) 65 465 65 63

"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. "Seven isolates wereA. baumannii and two wereAcinetobacterspp. "Equivocal when incubated at 35 'C, but positive at
30 'C.

1342-1362), and reverse 5'-TGC TAC TCA ACG ACT- and specificities were for the modified Hodge test 100% and
GAG CG-3' (nucleotides 2102-2121) [7]. Template DNA 88%, respectively, and for the EDTA-disk synergy test both
was extracted by boiling for 5 min. The PCR conditions were 100% as compared to imipenem hydrolysis.
one cycle of predenaturation at 94°C for 5 min, 35 cycles of The hydrolysis of imipenem by all of the EDTA-disk
denaturation at 94°C for 30 s, annealing at 56°C for 30 sand synergy-positive strains was inhibited by 10 mM EDTA, but
extension at 72°C for 1 min, and one cycle offinal extension at
72 -c for 7 min.
Among a total of530 imipenem-resistant isolates screened by
the modified Hodge test, 43 were positive and 31 were equi-
vocal (Table 1, Figure 1). Six of nine imipenem-resistant P
putida isolates showed positive results at 35°C, but the remain-
ing three equivocal isolates became positive at 30 "C. Among
the 28 isolates ofAcinetobacter 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 Srne--L.
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 con-
trol 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.
All of the 65 EDTA-disk synergy-positive strains hydrolyzed 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-
imipenem. Fifty-four of the 456 Hodge- and EDTA-disk
resistant test isolates were streaked from the edge of the disk to the periph-
synergy-negative isolates and nine Hodge-equivocal but
ery of the plate and incubated overnight. (A) and (C) are imipenem-hydro-
EDTA-disk synergy-negative isolates were spectrophotometri- Iyzing strains which distorted the inhibition zone. (B) and (D) are imipenem-
cally negative for imipenem hydrolysis (Table 1). Sensitivities non-hydrolyzing strains with no effect on the zone.

© 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, GMI, 7, 88-102
90 Clinical Microbiology and Infection, Volume 7 Number 2, February 2001
Figure 2 EDTA-disk synergy test. A Mueller-Hinton agar plate was inocu-
lated with an imipenem-resistant test isolate, and a 10-l-'g imipenem disk
(left) and a 5 mM EDTA disk (right) were put in place and incubated over-
night. (A) A synergistic inhibition zone is shown between an imipenem disk
(left) and an EDTA disk (right) by a VIM-2 metallo-Il-Iactamase-producing
strain. (B) Only a partially inhibited zone around an EDTA disk is shown by
a metallo-Il-Iactamase-non-producing strain. (C) A metallo-Il-Iactamase-
producing strain showing a small enhanced inhibition zone when the disks
were placed 15 mm apart.
not by 50 J-lM clavulanic acid. The MI C range of imipenem for
the isolates was 8-256 mg/L. Relative rates of hydrolysis of
imipenem by representative isolates were 24-47% when com-
pared 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 If-Iactamase 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
© 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, GMI, 7, 88-102
aeruginosa and that the inhibition zones with imipenem
disks were too large. Our modified Hodge test, using a com-
merciallyavailable 1O-J-lg imipenem disk and an indicator E. coli
strain at one-tenth the turbidity of a McFarland 0.5 tube,
detected not only IMP-I, VIM-l and VIM-2, but also an
Sme-l 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 ofnine P putida isolates which were
modified Hodge test equivocal at 35°C became positive at
30 "C, possibly because of the lower optimal growth tempera-
ture of the organism.
Our preliminary results [12] showed that a double disk
synergy test using a 10-J-lg 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 J-lL 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-hydro-
lyzing strains did not show enhanced inhibition zones or
showed ouly small zones when the two disks were placed
15 mm apart from edge to edge (Figure 2e). 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 contain-
ing 2-mercaptopropionic acid gave the clearest synergy in
screening IMP-I-producing strains [17]. However, 0.5 M
EDTA solution (pH 8.0) is readily available in most labora-
tories. Arakawa et al used ceftazidime disks instead ofimipenem
disks to increase the sensitivity of the test, as some strains were
resistant to low levels of imipenem ouly. However, ceftazidime
should be less specific for the detection of metallo-If-Iactamase
producers. McLaughllin and Laconis [18] used a disk containing
20 J-lL of a 5 mM EDTA solution to screen for the presence of
metallo-If-Iactamase 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-I,
VIM-I, VIM-2 and Sme-l If-Iactamase suggested that the tests
could also be used to screen for strains producing carbapene-
mases other than VIM-2. A limitation of this study is that
although many VIM-2 metallo-If-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-If-Iactamase-producing clinical isolates of P aeruginosa,
P putida and Acinetobacter spp., to characterize the If-Iactamases
and to control spread of the resistance.

ACKNOWLEDGMENTS
We thank Dr David M. Livermore, Central Public Health
Laboratory, London, UK, for providing IMP-l and VIM-2
p-lactamase-producing P aeruginosa and Sme-l-producing Ser-
ratia marcescens, and Professor Gian M. Rossolini, Universita di
Siena, Siena, Italy for VIM-l-producing P aeruginosa. We thank
Young Hee Seo for her technical assistance.
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Center for Communicable Diseases, Institute of Public Health, Trubarjeva 2, 1000 Ljubljana, Slovenia
Fax: + 386 61 323940 E-mail: maja.socan@ivz-rs.si
Accepted 13 November 2000
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