Indian J Med Res 129, June 2009, pp 707-712

Simple screening tests for detection of carbapenemases in clinical

isolates of nonfermentative Gram-negative bacteria

M.J.C. Noyal, G.A. Menezes, B.N. Harish, S. Sujatha & S.C. Parija

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research

Puducherry, India

Received April 10, 2008

Background & objectives: The production of carbapenemases is an important mechanism responsible

for the carbapenem resistance. A simple and inexpensive testing method for screening of carbapenemase
producers is essential. A prospective study was undertaken to detect metallo--lactamases (MBLs) and
AmpC -lactamases in nonfermentative Gram negative bacteria and to evaluate the various methods for
detection of carbapenemases and MBLs.
Methods: A total of 100 Acinetobacter spp. (78 A. baumannii and 22 A. lwoffii) and 140 Pseudomonas spp.
(103 P. aeruginosa and 37 other Pseudomonas spp.) were screened for meropenem resistance by Kirby-
Bauer disc diffusion method. Modified Hodge test, EDTA disk synergy (EDS) test and AmpC disk test
were used for the detection of carbapenemases, MBLs and AmpC -lactamases, respectively.
Results: Forty six (59.0%) A. baumannii, 7 (31.8%) A. lwoffii, 32 (31.1%) P. aeruginosa and 7 (18.9%)
Pseudomonas spp. were resistant to meropenem. Among the 32 meropenem resistant P. aeruginosa,
15 (46.9%) were AmpC -lactamase producers, 16 (50.0%) MBL producers by EDS test, but only 9
(28.1%) found positive for carbapenemases by modified Hodge test. Among the 46 meropenem resistant
A. baumannii, 31 (67.4%) were AmpC -lactamase producers, 3 (6.5%) MBL producers, but only 1
(14.3%) was positive for carbapenemases by modified Hodge test. One P. aeruginosa was positive for
carbapenemase by modified Hodge test, but was negative for MBL and AmpC -lactamase.
Interpretation & conclusions: MBL production is an important mechanism of carbapenem resistance
among Pseudomonas species but not among Acinetobacter species. EDS is more sensitive for detection
of MBLs than modified Hodge test. Both EDTA-meropenem and EDTA-ceftazidime combination must
be used to detect all the MBL producers. Carbapenemases other than MBL may also be responsible
for carbapenem resistance. AmpC -lactamase is also a contributory factor for carbapenem resistance
among the isolates in the hospital.

Key words AmpC - lactamases - AmpC disk test - EDTA disk synergy test - metallo--lactamases - modified Hodge test -
nonfermentative bacteria

One of the most important mechanisms coding for -lactamase enzymes mutate continuously
of microbial resistance to -lactam antibiotics in response to the heavy pressure of antibiotic use
(penicillins, cephalosporins, monobactams, and leading to development of newer -lactamases with a
carbapenems) is hydrolysis by -lactamases. Genes broad spectrum of activity. Among the -lactamases,
707
708 INDIAN J MED RES, JUNE 2009
the carbapenemases especially transferrable metallo-
-lactamases (MBLs) are the most feared because of
their ability to hydrolyze virtually all drugs in that
class, including the carbapenems. There are several
mechanisms for carbapenem resistance such as the
lack of drug penetration due to mutation in porins,
loss of certain outer membrane proteins and efflux
mechanisms1.
In addition to their resistance to all -lactams,
the MBL producing strains are frequently resistant
to aminoglycosides and fluoroquinolones2. However,
these usually remain susceptible to polymyxins2.
Unlike carbapenem resistance due to several other
mechanisms, the resistance due to MBL and other
carbapenemase production has a potential for rapid
dissemination, as it is often plasmid mediated2.
Consequently, the rapid detection of carbapenemase
production is necessary to initiate effective infection
control measures to prevent their dissemination.
Modified Hodge test is a phenotypic method
for detection of carbapenemases3. Various methods
like, EDTA disk synergy (EDS) test3, MBL E-test1,
EDTA-based microbiological assay4 are used for
detection of MBLs. But EDS test is a relatively
simple and sensitive method for MBL detection.
There are no established phenotypic methods for
detection of specific serine carbapenemases. AmpC
disk test5 and three-dimensional extract method6 are
the commonly used tests for detection of AmpC -
lactamases.
Although different phenotypic methods have
been described, for a long time there was no
standard guideline for screening of carbapenemases.
Recently, CLSI has recommended modified Hodge
test for detection of carbapenemases activity in
Enterobacteriaceae, but not in non-fermenters7.
Detection of genes coding for carbapenem resistance
by PCR, usually give reliable and satisfactory results8,
but this method is of limited practical use for daily
application in clinical laboratories because of the cost.
Thus, a simple and inexpensive testing method for
screening of carbapenemase producers is necessary.
Therefore this study was undertaken to detect
MBLs and AmpC -lactamases in clinical isolates
of nonfermentative Gram-negative bacteria. Clinical
usefulness of the various methods for detection of
carbapenemases and MBLs as a screening test was
also evaluated.
Material & Methods
A prospective study was conducted over a period
of nine months (June 2007 to February 2008) at the
Department of Microbiology, Jawaharlal Institute
of Postgraduate Medical Education and Research
(JIPMER), Puducherry. A total of 100 Acinetobacter
species and 140 Pseudomonas species were included
in this study. These organisms were isolated from
specimens like sputum, tracheal aspirate, pus, urine,
blood, pleural fluid and ascitic fluid of patients admitted
to different wards, which were sent to the microbiology
laboratory for routine culture identification and
sensitivity testing.
The Acinetobacter species and Pseudomonas
species were identified based on standard
bacteriological techniques9,10. All these isolates were
screened for meropenem resistance by Kirby-Bauer
disk diffusion method according to CLSI guidelines11.
Modified Hodge test: The meropenem resistant strains
were subjected to modified Hodge test for detection
of carbapenemases3. An overnight culture suspension
of Escherichia coli ATCC 25922 adjusted to 0.5
McFarland standard12 was inoculated using a sterile
cotton swab on the surface of a Mueller-Hinton agar
(MHA) (HI-MEDIA, Mumbai, India). After drying,
10 g meropenem disk (HI-MEDIA, Mumbai, India)
was placed at the center of the plate and the test
strain was streaked from the edge of the disk to the
periphery of the plate in four different directions. The
plate was incubated overnight at 37oC. The presence
of a cloverleaf shaped zone of inhibition due to
carbapenemase production by the test strain was
considered as positive (Fig. 1).
EDTA disk synergy (EDS) test: EDTA disk synergy
(EDS) test was done with simultaneous testing of two
different -lactams (meropenem and ceftazidime), for
Fig. 1. Modified Hodge test. Positive strain shows a cloverleaf
shaped zone of inhibition due to carbapenemase production, while
the negative strain shows an undistorted zone of inhibition.
 NOYAL et al: DETECTION OF CARBAPENEMASES IN GNBS
Fig. 2. EDTA disc synergy test. Positive strain shows a synergistic
zone of inhibition between ceftazidime or meropenem disc and
EDTA disc, while the negative strain shows no synergistic zone of
inhibition.
Fig. 3. AmpC disc test. Indentation of the cefoxitin zone of
inhibition is seen in the vicinity of the disk with positive strain,
while there is an undistorted zone of inhibition near the negative
strain.
detection of metallo--lactamases in the meropenem
resistant isolates3.
A 0.5 M EDTA solution was prepared by dissolving
186.1 g of disodium EDTA. 2H2O (REACHEM,
Chennai, India) in 1,000 ml of distilled water. The
pH was adjusted to 8.0 by using NaOH (HI-MEDIA,
Mumbai, India) and was sterilized by autoclaving13.
An overnight liquid culture of the test isolate was
adjusted to a turbidity of 0.5 McFarland standard12
and spread on the surface of a MHA plate. A 10
g meropenem disk or 30 g ceftazidime disk (HI-
MEDIA, Mumbai, India) was placed on the agar.
A blank disk (6 mm in diameter, Whatmann filter
paper no. 1) was kept on the inner surface of the
lid of the MHA plate and 10 l of 0.5 M EDTA is
added to it. This EDTA disk was then transferred to
the surface of the agar and was kept 10 mm edge-to-
edge apart from the meropenem or ceftazidime disk.
After incubating overnight at 37oC, the presence
of an expanded growth inhibition zone between
the two disks was interpreted as positive for MBL
production (Fig. 2).
709
Amp C ddisk test: AmpC disk test was also done for the
meropenem resistant strains for detection of AmpC
-lactamases5. On a MHA plate, lawn culture of E.
coli ATCC 25922 was made from an overnight culture
suspension adjusted to 0.5 McFarland standard12. A 30
g cefoxitin disk was kept on the surface of the agar. A
blank disk (6 mm in diameter, Whatmann filter paper no.
1) was moistened with sterile saline and inoculated with
a few colonies of the test strain. The inoculated disk was
then placed beside the cefoxitin disk almost touching it.
The plate was incubated overnight at 37oC. A flattening
or indentation of the cefoxitin inhibition zone in the
vicinity of the disk with test strain was interpreted as
positive for the production of AmpC -lactamase. An
undistorted zone was considered as negative (Fig. 3).
Results
Of the 100 clinical isolates of Acinetobacter
species, 78 were A. baumannii, while 22 were A.
lwoffii. Among the 140 Pseudomonas isolates screened,
103 were P. aeruginosa, while the remaining 37 were
other Pseudomonas spp.
Forty six (59.0%) A. baumannii, 7 (31.8%) A. lwoffii,
32 (31.1%) P. aeruginosa and 7 (18.9%) Pseudomonas
spp. were found resistant to meropenem.
Among the 32 meropenem resistant P. aeruginosa,
15 (46.9%) were AmpC -lactamase producers,
16 (50.0%) were MBL producers by EDTA disk
synergy test, but only 9 (28.1%) were positive for
carbapenemases by modified Hodge test (Table).
Two isolates were positive for both MBL and AmpC
-lactamase. One was positive for carbapenemase by
modified Hodge test, but was negative for MBL and
AmpC -lactamase by EDTA disk synergy test and
AmpC disk test respectively. Of the 16 MBL producers,
8 were detected by simultaneously testing with both
meropenem and ceftazidime in EDS, 6 were detected
only using EDTA-ceftazidime combination and 2 were
positive by EDTA-meropenem combination alone.
Of the 7 meropenem resistant Pseudomonas spp., 4
(57.1%) were AmpC -lactamase producers, 2 (28.6%)
were MBL producers by EDTA disk synergy test, but
only 1 (14.3%) was positive for carbapenemases by
modified Hodge test (Table). Of the 2 MBL producers,
one was detected by simultaneously testing with
both meropenem and ceftazidime in EDS, while the
other was detected only using EDTA-ceftazidime
combination.
710 INDIAN J MED RES, JUNE 2009
Table. Results of modified Hodge test, EDTA disc synergy test &
AmpC disc test
Bacteria No. of No. of positives (%)
meropenem
resistant Modified EDTA disc AmpC
Hodge synergy disc
isolates
test test test
P. aeruginosa 32 9 (28.1) 16 (50.0) 15 (46.9)
Pseudomonas spp. 7 1 (14.3) 2 (28.6) 4 (57.1)
A. baumannii 46 1 (2.2) 3 (6.5) 31 (67.4)
A. lwoffii 7 0 (0) 0 (0) 4 (57.1)
Among the 46 meropenem resistant A. baumannii,
31 (67.4%) were AmpC -lactamase producers, 3
(6.5%) were MBL producers, but only 1 (14.3%) was
positive for carbapenemases by modified Hodge test
(Table). Among the 3 MBL producers, one was detected
by simultaneously testing with both meropenem and
ceftazidime in EDS and 2 were detected only using
EDTA-ceftazidime combination.
Of the 7 meropenem resistant A. lwoffii, 4 (57.1%)
were AmpC -lactamase producers, but none were
positive for MBL and other carbapenemases (Table).
Discussion
Carbapenems have a broad spectrum of antibacterial
activity, and these are resistant to hydrolysis by most -
lactamases including extended spectrum -lactamases
(ESBL) and AmpC -lactamases14. These are often
used as a last resort in infections due to multidrug-
resistant Gram-negative bacilli. However there is an
alarming increase in reports of carbapenem resistance
in Acinetobacter species and P. aeruginosa15. The first
metallo--lactamase-producing P. aeruginosa strain
was isolated in Japan in 198816. For many years, these
MBL producing isolates were restricted to Japan, but
now it has disseminated worldwide17-19. In India, MBL
producing P. aeruginosa was first reported in 200220.
In our study we report a high prevalence of
meropenem resistance among A. baumannii. In a
prospective study with 150 Acinetobacter species
conducted at Bangalore, India, 14.0 per cent resistance
to carbapenems was reported21. Surveillance in
Brooklyn, New York, revealed that approximately
2 of every 3 isolates of Acinetobacter species were
resistant to carbapenem22. In an Australian study with
ninety A. baumannii isolates recovered from blood,
64 per cent resistance to meropenem was noted23.
Though our result was contradictory to the relatively
lesser resistance rates among Acinetobacter species
documented in India, it was similar to the New York
and Australian studies, which also have reported
high prevalence of carbapenem resistance among
Acinetobacter species. High antibiotic pressure due
to indiscriminate use of carbapenems in this part
of the world could have resulted in the increase in
carbapenem resistant Acinetobacter spp.
Among the P. aeruginosa 31.1 per cent meropenem
resistance was documented in this study. A 5 year
longitudinal study involving many centers from Latin
America has reported that, P. aeruginosa resistance to
carbapenems has risen to 40.0 per cent 24. In a study
done at a tertiary care hospital in Puducherry (India),
with 266 isolates of P. aeruginosa, 10.9 per cent
resistance to carbapenems was reported25. Though the
carbapenems resistance among P. aeruginosa in our
study was lesser than the Latin American surveillance
study24, it was higher than the resistance documented
in 2006 from this part of the world25. These findings
clearly show a rising trend in the carbapenem resistance
among the nonfermenters.
We also found that 50 per cent of the carbapenem
resistance among P. aeruginosa was attributable to the
production of MBLs. This was surprisingly higher
than the documented report in Korea, where 11.4 per
cent of imipenem-resistant P. aeruginosa isolates
produced MBLs26. But another Indian study has
reported MBL production among 75 per cent of the
imipenem resistant Pseudomonas species27. Among the
meropenem resistant A. baumannii, 6.5 per cent were
MBL producers, which was relatively less compared to
14.2 per cent imipenem-resistance attributable to the
production of MBLs reported in Korea26. In an Indian
study on meropenem resistant Acinetobacter species
none of the isolates produced MBLs21. Consequently
in the Indian scenario production of MBL may not
play a major role in carbapenem resistance among
Acinetobacter species.
EDTA disk synergy test detected MBL
production in additional 9 Pseudomonas species and
2 Acinetobacter species, which were missed by the
modified Hodge test. Based on these findings, EDTA
disk synergy test seems to be a better method for MBL
detection then modified Hodge test. Though the reason
for the difference in the performance of these two
tests is not clear, similar results have been observed
in other studies3,27. In the EDS test, EDTA-ceftazidime
combination detected additional MBL producers
which were not identified by EDTA-meropenem
 NOYAL et al: DETECTION OF CARBAPENEMASES IN GNBS
combination. The reason for the increased sensitivity
of EDTA-ceftazidime combination is the ability of
ceftazidime to produce a marked inhibitory effect
with EDTA. Therefore, ceftazidime appears to be the
better substrate for EDS. Similar results were observed
in a study done at the National Institute of Infectious
Diseases, Tokyo, Japan28.
Two MBL producing P. aeruginosa were detected
only by EDTA-meropenem combination, but not
by EDTA-ceftazidime combination. A synergistic
zone of inhibition which must have been normally
present between EDTA and ceftazidime due to MBL
production was not observed with these two isolates.
These two MBL producers were also simultaneously
producing AmpC -lactamase. Hence it could be
proposed that the synergistic zone of inhibition was
masked by the resistance to ceftazidime conferred
by the AmpC -lactamase, which is independent of
zinc ions for its action. Based on this study it is clear
that both EDTA-meropenem and EDTA-ceftazidime
combination must be used simultaneously to detect all
the MBL producers, which may otherwise be missed
by using either of this combination alone.
One of the meropenem resistant P. aeruginosa was
positive for carbapenemase by modified Hodge test, but
was negative for MBL and AmpC -lactamase. This
may be because of the production of carbapenemase
other than MBL, which is not dependent on zinc
ion for its action. Accordingly, apart from MBL,
other classes of carbapenemases (class A or D) can
also be responsible for meropenem resistance in P.
aeruginosa29.
AmpC -lactamase was produced by 46.9 and
67.4 per cent of the meropenem resistant P. aeruginosa
and A. baumannii respectively. Therefore, AmpC -
lactamase could be an important contributory factor
for meropenem resistance among the isolates in our
hospital similar to other studies22,30,31. This seems to be
more likely with A. baumannii, most of which in our
study, despite being meropenem resistant, did not show
production of MBL or other carbapenemases.
The detection of MBL and other carbapenemases
is of utmost importance in deciding the most
appropriate therapeutic regimen for treatment of
the carbapenem resistant nonfermenters. In several
studies, intravenous colistin combined with rifampin
and imipenem was recommended for the treatment
of carbapenem-resistant isolates lacking MBLs,
whereas the combination of colistin and rifampicin
711
(with or without tigecycline) was recommended for
treatment of MBL producing carbapenem-resistant
isolates30,31. Moreover, studies have shown that efflux
pumps mainly affect meropenem, while specific -
lactamases (carbapenemases) hydrolyze imipenem
more efficiently30. So, detection of carbapenemases is
necessary for administration of appropriate therapy.
The drawbacks in our study are the relatively
small sample size and failure to evaluate the clinical
usefulness of detection of carbapenemases. Our study
was restricted mainly to detection of carbapenemases
and the comparison of the efficacy of different
techniques for detection of carbapenemases. Further
studies are needed to evaluate the clinical usefulness of
detection of carbapenemases.
In conclusion, MBL production is an important
mechanism of carbapenem resistance among
Pseudomonas species, while it may not play a major
role in carbapenem resistance among Acinetobacter
species. Modified Hodge test may not be a useful
screening test for carbapenemases as many MBL
producing isolates were not detected by this test. EDS
is a more sensitive method for detection of MBLs.
Ceftazidime appears to be the better substrate for EDS
compared to meropenem, but both meropenem and
ceftazidime must be used simultaneously to detect all
the MBL producers. Carbapenemases other than MBL
may also be responsible for carbapenem resistance
in these nonfermenters. AmpC -lactamase is also a
contributory factor for carbapenem resistance among
the isolates in the hospital.
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