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A Simplified
method of Three
Dimensional
Technique For The
Detection Of AmpC
Beta-Lactamases

2013
ARCHIVES OF CLINICAL MICROBIOLOGY

Vol. 4 No. 1:3
doi: 10.3823/268

R.K. Manojkumar Singh1, Nishith Kumar Pal2, Mandira
Banerjee3, Soma Sarkar4, Puranjoy Saha4,Manideepa
SenGupta4
1 Department of
Microbiology,Jawaharlal
Nehru Institute of Medical
Sciences, Imphal;
2 Institute of Postgraduate
Medical Education &
Research, Kolkata.

3 Nilratan Sarkar Medical
college, Kolkata.
4 Medical College & Hospital,
Kolkata.

Correspondence:

rkmksingh@gmail.com

Dr. R.K.Manojkumar Singh.
Department of Microbiology,
Jawaharlal Nehru Institute of
Medical Sciences, Porompat,
Imphal(East)-795005, India.

Abstract
Background: AmpC beta-lactamases are cephalosporinases that confer resistance to a wide variety of beta-lactam antibiotics. Detecting AmpC-mediated resistance in gram negative organisms is a challenge for laboratories due to misleading
results in phenotypic tests. There is always a search for newer and reliable methods
which will be more user-friendly to detect these enzymes in routine diagnostic
laboratories.

Objective: To design a simplified three-dimensional test for detecting the occurrence of AmpC β-lactamases.

Material and Methods: A total of 201 consecutive, non-duplicate, gram-negative clinical isolates of E.coli (n=65), Klebsiella spp.(n=70), Proteus spp.(n=4), citrobacter spp. (n=9), Enterobacter spp.( n=8), P.aeruginosa (n=27) and Acinetobacter
spp.(n=18), obtained over a period of six months (July to December, 2011) from
patients with nosocomial infections were screened for AmpC producers by Kirby
Bauer disk diffusion method using cefoxitin (30μg). Isolates with zone diameter less
than 18 mm were tested for inducible AmpC beta-lactamases by disk antagonism
test, and plasmid mediated AmpC activity by indigenously developed simplified
three dimensional and AmpC disk tests.

Results: Amongst the 201 gram-negative clinical isolates tested, the simplified
three dimensional test identified 60(29.8%) and AmpC disk test yielded 51(25.3%)
as plasmid AmpC producers from 69 screening positive isolates. Production of inducible AmpC beta-lactamase was shown in 3(1.5%) isolates by disk antagonism
test. Co-productions of extended spectrum beta-lactamases and metallo-betalactamases with AmpC beta-lactamases were observed in 3.9% and 4.9% isolates
respectively.

Conclusion: This simplified three dimensional technique is simple, reproducible,
convenient, reliable and accurate means of detection of AmpC beta-lactamases. It
can serve as a potentially useful diagnostic tool in those health care settings where
polymerase chain reaction and inhibitor based methods for the detection of AmpC
enzymes are costly and not readily available.

This article is available from:
www.acmicrob.com

Keywords: AmpC, three-dimensional technique.

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Introduction
AmpC β-lactamases are cephalosporinases that are poorly
inhibited by β-lactamase inhibitors such as clavulanic acid
[1]. They can be differentiated from extended spectrum
β-lactamases (ESBLs) by their ability to hydrolyse cephamycins as well as other extended-spectrum cephalosporins [2].
Over the last few years, numerous outbreaks of infections
with organisms producing AmpC β-lactamase, have been
observed worldwide.
Coudron et al used the standard disk diffusion breakpoint
for cefoxitin (zone diameter <18 mm) to screen isolates and
used a three-dimensional extract test as a confirmatory test
for isolates that harbour AmpC β-lactamases [3]. The current
Clinical and Laboratory Standards institute (CLSI) guidelines
do not describe any method for detection of isolates producing AmpC β-lactamases [4]. Various phenotypic techniques to
identify AmpC β-lactamase producing isolates are available
but are still evolving and are not yet optimized for the clinical
laboratory, which probably now underestimates this resistance mechanism. Several researchers have tried the three dimensional test with different modifications but it is laborious,
time consuming and needs experience. Despite of the fact
that multiplex PCR is the gold standard and highly accurate
for testing, it is beyond the routine microbiology laboratories.
The present study was conducted in a tertiary care hospital of
eastern India with an attempt to develop a simplified threedimensional test and evaluate the effectiveness to be applied
as a phenotypic method for detection of AmpC-harbouring
gram negative organisms.

Material and Methods
A total of 201 consecutive, non-duplicate gram-negative
clinical isolates over a period of six months (July to December, 2011) were collected from clinical specimens such as
urine, wound, blood, tracheal aspirates, endotracheal tube,
endotracheal aspirates or central venous catheter of hospitalised patients suspected with nosocomial infections. The
study included only the patients of all age groups and both
sexes developing infections after 48 hours of hospital stay
and those with infections at the time of admission or within
48 hours of hospitalization were excluded.
The isolates were identified on the basis of conventional
microbiological procedures [5]. Antibiotic susceptibility was
determined by using the Kirby Bauer’s disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [4] using the Mueller Hinton agar

2

Vol. 4 No. 1:3
doi: 10.3823/268

(Hi-Media, Mumbai,India) and commercially available 6mm
antimicrobial disks of cefoxitin (30µg), cefotaxime (30µg),
ceftriaxone (30µg), ceftazidime (30µg), cefpodoxime (30µg),
aztreonam (30µg) imipenem (10μg), amoxicillin/clavulanate
(20/10μg), amikacin (30μg), piperacillin (100μg), piperacillintazobactam(100/10μg), ciproploxacin (5μg), and cotrimoxazole (1.25/23.75μg) (Hi-Media, Mumbai, India).
Screening for AmpC β-lactamase production was performed
by Cefoxitin disk test [3, 6]. Isolates showing inhibition zone
diameter less than 18mm (screening positive) were further
subjected to disk antagonism test for inducible AmpC enzyme, and simplified three dimensional and AmpC disk tests
for the detection of plasmid AmpC β-lactamases.

Detection of AmpC β-lactamases
1.  Inducible AmpC β-lactamases [6]
The disk antagonism test was used for detection of inducible
AmpC β-lactamase in all the screening positive AmpC isolates.
A 0.5 McFarland of test isolate was swabbed on Mueller-Hinton agar plate and cefotaxime (30μg) or ceftazidime(30μg) or
ceftriaxone(30μg) and cefoxitin (30μg) disks were placed 20
mm apart from centre to centre. Isolates showing blunting of
the cefotaxime or ceftazidime or ceftriaxone zone of inhibition adjacent to the cefoxitin disk were considered inducible
AmpC β-lactamase (Figure 1.).

2.  Plasmid mediated AmpC β-lactamases
I.  Simplified three dimensional tests
Briefly, fresh overnight growth from Mueller–Hinton agar was
transferred to a pre-weighed sterile micro-centrifuge tube
of 2ml capacity. The tube was weighed again to check the
weight of the bacterial mass in order to obtain 10 mg of bacterial wet weight for each isolate. The growth was suspended
in 1.5 ml of peptone water and incubated at 35oC for 4
hours. This was subsequently concentrated by centrifugation
at 3000 rpm for 15 minutes and the supernatant was used
as a crude enzyme extract for the test. Lawn culture of E.
coli ATCC 25922 was prepared on MHA plates and cefoxitin
(30 µg) disk were placed on the plates. With a sterile surgical scapel blade (No. 15), linear slits (3 cm) beginning 3 mm
from the edge of the cefoxitin disk were cut in an outward
radial direction. At the other end of the slit a small triangular
well was made using the same scapel blade after flaming
and the enzyme extract was loaded for a total of 30 µl by a
micropipette using sterile tips. The plates were kept upright
for 5 minutes until the liquid dried and incubated at 35°C
for 18-24hours.

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Isolates showing clear distortion of the zone of inhibition of
cefoxitin were considered as strong AmpC producers, minimal distortion as intermediate and no distortion as negative
strains (Figure 2, 3, 4, 5).
II. AmpC disk test [7]
Sterile disks (6 mm) were moistened with sterile saline (20
µl) and inoculated with several colonies of test organism was
placed beside a cefoxitin disk (almost touching) on the MHA
plate lawned with a culture of E. coli ATCC 25922. The plates
were incubated overnight at 35°C. A positive test appeared
as a flattening or indentation of the cefoxitin inhibition zone
in the vicinity of the test disk [8]. A negative test had an
undistorted zone (Figure 6, 7).

Vol. 4 No. 1:3
doi: 10.3823/268

organisms were inoculated on Mueller Hinton agar plates as
recommended by the CLSI. Two 10 μg imipenem disks were
placed on the plate, and 10 μL of EDTA solution was added
to one of them to obtain the desired concentration (750 μg).
The inhibition zones of the imipenem and imipenem-EDTA
disks were compared after 16 to 18 hours of incubation at
35°C. An increase of ≥ 7 mm in zone inhibition diameter
around the imipenem and EDTA disk in comparison to the
imipenem disk alone was interpreted as a positive result for
MBL production.
Quality control: Quality control was achieved using K.
pneumonia (ATCC 700603), E. coli (ATCC 25922) and known
AmpC positive E.coli.
Statistical Analysis: Data obtained from this study were analysed using descriptive statistics like percentage and proportion. Sensitivity value was calculated for comparing the results
of three dimensional test performed by various researchers.

Detection of other β-lactamases in presence of
AmpC β-lactamases:
1. ESBLs: The original double disk synergy test was modified
for detecting ESBLs in AmpC-producing isolates by placing
a disk of piperacillin-tazobactam (100/10 μg) at a distance
of 20 mm from cefepime (30 μg) disc [9]. Briefly, a disk of
amoxycillin/clavulanate (20/10μg) was placed in the centre
of Mueller Hinton agar; then 30 μg disks of cefpodoxime,
ceftazidime, cefotaxime and cefepime were kept at a distance
of 20 mm from the amoxycillin/clavulanate disk (centre to
centre), and a disk of piperacillin-tazobactam was placed at
a distance of 20 mm from the cefepime disk. The organisms
were considered to be producing ESBL when the zone of
inhibition around cefepime or any of the extended-spectrum
cephalosporin disks showed a clear-cut increase towards the
piperacillin- tazobactam, or amoxicillin-clavulanate disks [9].
2. MBLs: The combined disk test or the disk enhancement
test was performed as described by Yong et al [10]. Test

Results
The isolated gram-negative organisms were E.coli (n = 65),
Klebsiella spp.(n = 70),Proteus spp.(n=4),Citrobacter spp.
(n = 9), Enterobacter spp.(n = 8), P.aeruginosa (n = 27) and
Acinetobacter spp.(n = 18) . The potential AmpC β-lactamase
producers detected by the screening test were 69.

Inducible AmpC production
Among the 69 screening positive isolates, one each of Proteus
spp, Pseudomonas aeruginosa and Acinetobacter spp (1.5%)
revealed the presence of inducible AmpC β-lactamases by
disk antagonism test. (Table 1).

Table 1. Detection of AmpC β-lactamases (Inducible & Plasmid).

3-DT(%)

AmpC disk test (%)

23(35.4)

Inducible AmpC
Disk antagonism
test (%)
0

21(32.3)

19(29.2)

22(31.4)

0

19(27.1)

16(22.9)

Microorganisms
( No. of isolates)

Screening positive
AmpC (%)

E.coli (65)
Klebsiella spp. (70)

Plasmid mediated AmpC

Proteus spp. (4)

1(25)

1(25)

0

0

Enterobacter spp. (8)

2(25)

0

2(25)

2(25)

Citrobacter spp. (9)

3(33.3)

0

3(33.3)

2(22.2)

Pseudomonas aeruginosa (27)

8(29.6)

1(3.7)

7(25.9)

5(18.5)

Acinetobacter spp. (18)

10(55.6)

1(5.6)

8(44.4)

7(38.9)

Total (202)

69(34.2)

3(1.5)

60(29.7)

51(25.3)

AmpC: AmpC beta-lactamases, 3-DT: Three dimensional tests.

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Plasmid-mediated AmpC production

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Klebsiella spp, Citrobacter spp and P.aeruginosa respectively.
Indentation indicating strong AmpC producer was observed
in 40(19.8%) isolates and flattening (weak AmpC) was seen
in 11(5.4) isolates (Table 1, 2).

Plasmid-mediated AmpC β-lactamase production was detected in 60(29.8%) isolates by the simplified three-dimensional test with the highest occurrence in Acinetobacter spp
(46.4% ) followed by Citrobacter spp (33.3%), E.coli (32.2%),
Klebsiella spp (27%), P.aeruginosa (25.9%) and Enterobacter
spp (25). A clear distortion of the zone of inhibition of Cefoxitin was observed in 52(25.8%), minimal distortion in 8(3.9%)
and no distortion in 9(4.4%) isolates (Table 1, 2).

ESBL and MBL production in presence of AmpC
β-lactamases:
The co-existence of ESBL and AmpC was detected by modified double disk synergy test in 8(3.9%) isolates whereas coproduction of MBL and AmpC was shown by 10(4.9%) isolates. All the MBL producers were found to show resistance
to imipenem (Table 3).

AmpC disk test was able to show 51(25.3%) isolates as AmpC
producers of which 38.9%, 29.2%, 25%, 22.9%, 22.2%
and 18.5% are Acinetobacter spp, E.coli, Enterobacter spp,

Table 2. Detection of AmpC β-lactamases by Simplified three dimensional & AmpC disk tests.
Plasmid mediated AmpC β-lactamases

Microorganisms
( No. of isolates)

Screening
positive
AmpC
isolates
(%)

Simplified three dimensional test (%)

AmpC disk test(%)

Distortion
(%)

Minimal
distortion
(%)

No.
distortion
(%)

Indentation
(%)

Flattening
(%)

No.
Distortion
(%)

E.coli (65)

23(35.4)

19(29.2)

2(3)

2(3)

15(23)

4(6.2)

4(6.2)

Klebsiella spp. (70)

22(31.4)

18(25.7)

1(1.4)

3(4.3)

13(18.6)

3(4.3)

6(8.6)

Proteus spp. (4)

1(25)

0

0

1(25)

0

0

1(25)

Enterobacter spp. (8)

2(25)

2(25)

0

0

2(25)

0

0

Citrobacter spp. (9)

3(33.3)

2(22.2)

1(11)

0

1(11)

1(11)

1(11)

Pseudomonas aeruginosa (27)

8(29.6)

5(18.5)

2(7.4)

1(3.7)

4(14.8)

1(3.7)

3(11)

Acinetobacter spp. (18)

10(55.6)

6(33.3)

2(11)

2(11)

5(27.8)

2(11)

Total (202)

69(34.2)

52(25.7)

8(3.9)

9(4.4)

40(19.8)

11(5.4)

3(16.7)
18(8.9)

Table 3. Detection of ESBLs,MBLs & pure AmpC in screening positive AmpC isolates.
Microorganisms
(No. of isolates)

Screening positive
AmpC (%)

AmpC+
ESBLs(%)

AmpC+
MBLs(%)

Pure
AmpC(%)

E.coli (65)

23(35.4)

4(6.2)

2(3)

15(23)

Klebsiella spp. (70)

22(31.4)

3(4.3)

1(1.4)

15(21.4)

Proteus spp. (4)

1(25)

0

0

1(25)

Enterobacter spp. (8)

2(25)

0

0

2(25)

Citrobacter spp. (9)

3(33.3)

0

0

3(33.3)

Pseudomonas aeruginosa (27)

8(29.6)

1(3.7)

3(11)

4(14.8)

Acinetobacter spp. (18)

10(55.6)

0

4(22.2)

5(27.8)

Total (202)

69(34.2)

8(3.9)

10(4.9)

45(22.3)

AmpC: AmpC beta-lactamases, ESBLs: Extended spectrum beta-lactamases, MBLs: Metallo-beta-lactamases.

4

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Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Vol. 4 No. 1:3
doi: 10.3823/268

Figure 1. Positive disk antagonism test. (Proteus spp.)
[cefotaxime(R) showing blunting & cefoxitin (L)].
Figure 2. Simplified three dimensional showing distortion in two
isolates of E.coli.
Figure 3. Simplified three dimensional showing distortion in two
isolates of Klebsiella spp.
Figure 4. Simplified three dimensional showing minimal
distortion (E. coli).
Figure 5. Negative three dimensional tests.
Figure 6. AmpC disk test showing indentation (R) & flattening(L).
Figure 7. AmpC disk test showing indentation (R) & negative(L).

Figure 7.

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Discussion
The three-dimensional test was designed to detect both
AmpC and ESBL production. In the “indirect” form used first
for AmpC detection by Thomson KS et al, a conventional disk
diffusion susceptibility assay is carried out with a susceptible
strain, such as E. coli ATCC 25922, as the lawn and a suspension of the test organism, which is added to a circular slit in
the agar 3 mm from a disk containing cefoxitin or some other
agent. Distortion of the zone of inhibition indicates a positive
test, as cefoxitin is hydrolyzed by the presence of an AmpC
enzyme. This technique has its own limitations, despite being
increasingly sensitive [11]. In subsequent modification by Coudron et al, a radial slit was employed, and rather than using
intact cells, the test organisms were concentrated by centrifugation, and the pellet was freeze-thawed five to seven times
to release β-lactamase [3]. The method could overcome the
need for rotator, but filling these slits without spillage was not
solved. Manchanda V et al further modified the procedure by
creating a well at the outer edge of the slit and the enzyme
extract was put into the well [2]. The method overcomes all
the problems of three-dimensional test but it is technically
demanding and needs experience.
A simplified method of three dimension test was developed
with few modifications from the previous one. We prepared
the enzyme extract by centrifugation after allowing incubating the organisms for 4 hours and it was not freeze thawed. A
small triangular well was made at the end of the slit through
which the enzyme extract was loaded using micropipette
with sterile tips so that any spillage while loading an enzyme
extract was far from the area where distortion of zone had
to be observed and filling the slits through these wells was
easy and rapid. This technique had advantages in terms of
less expertise and minimal equipment required, but great care
had to be taken while filling the slits and incubating the plates
to prevent spillover of the suspension.
In our study, simplified three dimensional test detected 60
AmpC producers from 69 screening positive isolates showing a sensitivity of 86.9%. This suggests that the technique
can be used for routine detection of the AmpC enzyme in a
clinical laboratory. Manchanda V et al, Coudron et al, Singhal
S et al, Bosak S et al, Mohamudha PR et al and Rudresh SM
et al found 75.6%, 35.5%, 36%, 100%, 93.6% and 70%
sensitivity rate for modified three dimensional test respectively [2-3, 8, 12-14].
It was found that the sensitivity of the three dimensional
test was more (86.9%) when compared to Amp C disk test
(73.9%). However, Amp C disk test was relatively easier to
perform when compared to three dimensional test. We employed disk antagonism test to identify inducible expression

6

Vol. 4 No. 1:3
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of chromosomal AmpC β-lactamases where three isolates
were positive ( one each of Proteus spp, Pseudomonas aeruginosa and Acinetobacter spp), thus revealing the presence
of only plasmid mediated resistance in E.coli and Klebsiella
spp, and this finding is well correlated with previous Indian
studies [6,15] .
A limitation of methods used to detect the AmpC enzyme
is that an increasing number of clinical isolates have multiple
beta-lactamases, which in turn can make inhibition patterns
complex and difficult to interpret [16]. Our study demonstrated the co-existence phenotype of both ESBL and AmpC
in 6.2%, 4.3% and 3.7% isolates of E. coli, Klebsiella spp.
and Pseudomonas aeruginosa respectively. This could be due
to the fact that plasmid-mediated AmpC enzymes have been
shown to disseminate among Enterobacteriaceae , sometimes in combination with ESBLs. In our study, cefepime was
the best and reliable cephalosporin in detecting ESBL in presence of AmpC producer as high level AmpC production has
minimal effect on the activity of cefepime, and tazobactam
turned out to be the best β-lactamase inhibitor in detecting
ESBL production followed by clavulanic acid [17]. It has been
reported that clavulanic acid may induce expression of high
level AmpC production in organisms producing both ESBL
and AmpC together, and may antagonize rather than protect the antibacterial activity of the partner β-lactam, thereby
making any synergy arising from inhibition of an ESBL, and
much better inhibition is achieved with the sulphones, such
as tazobactam and sulbactam which are preferable inhibitors
for ESBL detection tests in AmpC producers [16, 18-19].
Our study also demonstrated the presence of MBL (4.9%)
among AmpC producing isolates and imipenem was found to
be the most effective drug, showing maximum susceptibility
of 93.2% which is in agreement with earlier studies [6, 20].
Isoelectric focusing and genotypic characterization based on
multiplex PCR which are considered gold standard could not
carried out due to lack of infrastructure in our institution.

Conclusion
This three-dimensional test was easier to perform and interpret. It was found as sensitive and specific as other modifications and the additional cost is minimal. Clinical laboratories
which lack the infrastructure of advanced molecular methods
may use this technique to confirm the presence of AmpCmediated resistance.

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