amp c

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Evaluation of four phenotypic methods to detect plasmid-mediated AmpC β-
lactamases in clinical isolates
Article in European Journal of Clinical Microbiology & Infectious Diseases · January 2012
DOI: 10.1007/s10096-011-1537-y · Source: PubMed
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2. ARTICLE
Evaluation of four phenotypic methods to detect
plasmid-mediated AmpC β-lactamases in clinical isolates
M. J. Gude & C. Seral & Y. Sáenz &
M. González-Domínguez & C. Torres & F. J. Castillo
Received: 17 October 2011 /Accepted: 21 December 2011 /Published online: 26 January 2012
# Springer-Verlag 2012
Abstract Four phenotypic methods (three dimensional test,
AmpC test, cloxacillin synergy test and cefotetan/cefotetan-
cloxacillin E-test) to detect plasmid-mediated AmpC β-
lactamases (pAmpC) were compared in 125 clinical Enter-
obacteriaceae isolates with AmpC profile: 74 E. coli
(blaCMY-2: 70; blaDHA-1: 4), five K. pneumoniae (blaCMY-2:
2; blaDHA-1: 3), six P. mirabilis (blaCMY-2: 6) and 40 nega-
tive isolates for pAmpC β-lactamases. All evaluated meth-
ods showed a good sensitivity (>95%) but low values of
specificity (<60%) in E. coli, explained by an increase of
AmpC expression caused by chromosomal ampC promoter/
attenuator mutations (−42, −18, −1, +58, predominantly).
The cefotetan/cefotetan-cloxacillin or cloxacillin synergy
test may be advocated as phenotypic screening test, and
the AmpC test as confirmatory test for detection of pAmpC
in isolates that lack or minimally express chromosomally
encoded AmpC β-lactamases. In the case of E. coli, the
phenotypic evaluated tests were not able to differentiate
between chromosomal ampC overexpression or acquisition
of plasmid-encoded ampC genes.
Introduction
Plasmid-mediated AmpC β-lactamases (pAmpC) belong to
Ambler class C and have been reported worldwide in Gram-
negative bacteria since their first description in 1989 [1].
pAmpC are clinically important because they confer trans-
ferable resistance to all β-lactams (including cephamycins)
except fourth-generation cephalosporins and carbapenems.
Treatment options are severely limited because pAmpC are
often associated with other multiple resistance genes, such
as those of resistance to aminoglycosides, chloramphenicol,
quinolones, sulfonamides, tetracycline and trimethoprim as
well as other β-lactamase genes [2–5]. The pAmpC genes
are derived from the chromosomal ampC genes that have
been mobilized. Some Enterobacteriaceae, such as Citro-
bacter freundii, Enterobacter cloacae, Hafnia alvei, Morga-
nella morganii and Aeromonas spp., carry an inducible
chromosomal ampC gene. However, the chromosomal
ampC gene in Escherichia coli is expressed constitutively
due to the absence of the regulator ampR gene and the
presence of a weak promoter and a transcriptional attenuator
[2, 4]. E. coli strains containing the wild-type gene produce
a low basal level of the enzyme and are susceptible to
cephalosporins. However, mutations in the promoter/atten-
uator region can result in ampC constitutive overexpression
and cephalosporin resistance [6–10].
The “gold standard” for pAmpC detection requires
molecular methods that are not suitable for routine use
in clinical microbiology laboratories [11]. Phenotypic
methods proposed to date for detection of AmpC can be
divided in those that detect AmpC activities in enzyme
M. J. Gude :M. González-Domínguez
Departamento de Microbiología,
Hospital Clínico Universitario Lozano Blesa,
Zaragoza, Spain
C. Seral (*) :F. J. Castillo
Departamento de Microbiología, Hospital Clínico Universitario
Lozano Blesa and Universidad de Zaragoza,
Zaragoza, Spain
e-mail: cseral@unizar.es
Y. Sáenz
Área de Microbiología Molecular,
Centro de Investigación Biomédica de La Rioja (CIBIR),
Logroño, Spain
C. Torres
Área de Bioquímica y Biología Molecular and Área de
Microbiología Molecular, Universidad de La Rioja and Centro de
Investigación Biomédica de La Rioja (CIBIR),
Logroño, Spain
Eur J Clin Microbiol Infect Dis (2012) 31:2037–2043
DOI 10.1007/s10096-011-1537-y

3. extracts and those that evaluate the effects induced by
AmpC inhibitors such as cloxacillin, boronic acid and
monobactam derivates [12]. Reference guidelines for
performing in vitro susceptibility testing still do not in-
dicate either phenotypic screening or confirmatory tests
that should be used for isolates suspicious of harbouring
pAmpC. Several studies have validated the use of boron-
ic acid as inhibitor of AmpC [13–16], but it is not
specific for AmpC because boronic acid is known to
inhibit Klebsiella pneumoniae carbapenemase (KPC)-type
β-lactamases [14, 17–19].
Purpose
Our study was designed to assess four phenotypic methods
for pAmpC detection in a clinical laboratory, using two
cloxacillin tests and two previously described methods com-
pared with a genetic analysis in E. coli, K. pneumoniae and
Proteus mirabilis isolates.
Material and methods
A total of 21,376 Enterobacteriaceae were isolated in the
Microbiology Laboratory of a 803-bed University Teaching
Tertiary Hospital in the North of Spain, covering an area of
286,774 inhabitants over 30 months (June 2008 to Decem-
ber 2010). The identification and the susceptibility to 22
antibiotics were performed by microdilution using the
WIDER I System (Francisco Soria-Melguizo, Madrid,
Spain). Susceptibility results were interpreted according to
CLSI guidelines [20].
The screening criteria for isolate inclusion in this study
for further analysis was cefoxitin resistance (MIC>16 mg/l),
with (i) reduced susceptibility to amoxicillin-clavulanic acid
(MIC>8/4 mg/l), and/or (ii) reduced susceptibility to cefo-
taxime (MIC >1 mg/l) or ceftazidime (MIC >4 mg/l) in
Enterobacteriaceae isolates that lack inducible chromosom-
al AmpC β-lactamases. A total of 125 clinical isolates (only
one isolate per patient was considered) that fulfilled these
criteria (E. coli 105 out of 11,164 isolates, K. pneumoniae
13/1,765 and P. mirabilis 7/1,598) were tested for the pres-
ence of pAmpC by four phenotypic methods, all of them
previously described by different authors [2, 21–23], and a
genotypic one (used as a gold standard method) [11]. The
four phenotypic methods used in this study include the
three-dimensional test, the AmpC test, the cloxacillin syn-
ergy test (Cloxa test) and the cefotetan/cefotetan-cloxacillin
test (Fig. 1).
Three-dimensional test (3D) This method is a modified
Hodge test described by Coudron et al. [23]. A lawn of E.
coli ATCC 25922 was inoculated on a Mueller-Hinton agar
plate. After the agar surface dried, a 30-μg cefoxitin disk
was placed at the center and 20 μl of a 0.5 McFarland
suspension of the test isolate was dispensed into a radial slit
performed in the plate. After overnight incubation at 37°C,
Fig. 1 Methods evaluated for
detection of pAmpC enzymes.
a Three dimensional (3D) test.
b AmpC test. c Cloxacillin
synergy test. d cefotetan/
cefotetan-cloxacillin test
2038 Eur J Clin Microbiol Infect Dis (2012) 31:2037–2043

4. enhanced growth of the surface organism in the inhibition
zone along with the test strain was interpreted as evidence
for the presence of AmpC β-lactamase (see Fig. 1a).
AmpC test The AmpC disks were prepared as previously
described, applying 20 μl of a 1:1 mixture of saline and
100x Tris-EDTA solution to sterile filter paper disks. The
surface of a Mueller-Hinton agar plate was inoculated with a
lawn of E. coli ATCC 25922. Prior to use, AmpC disks were
rehydrated with 20 μl of saline and several colonies of the test
organism were applied to a disk and placed face down on the
agar almost touching a 30 μg cefoxitin disk. After overnight
incubation at 37°C, plates were examined for either an inden-
tation or a flattening of the zone of inhibition, which was
interpreted as a positive result for AmpC [22]. See Fig. 1b.
Cloxacillin synergy test (Cloxa test) As previously de-
scribed, this test is based on the use of cloxacillin as inhib-
itor of AmpC enzymes. Each isolate was inoculated on a
Mueller-Hinton agar according to the CLSI disk-diffusion
method. A disk of cloxacillin (500 μg) was placed between
ceftazidime (30 μg) and cefotaxime (30 μg). After incuba-
tion, an organism that demonstrated an enhancement in the
inhibition zone around the antibiotic disks was considered to
be an AmpC producer [21] (see Fig. 1c.)
Cefotetan/cefotetan-cloxacillin test (CN/CNI) This test is
based on diffusion test by Epsilon-test (E-test®, bioMérieux,
Marcy-l'Etoile, France) using cefotetan and cefotetan with
cloxacillin. Each isolate was inoculated on a Mueller-Hinton
agar according to the CLSI disk-diffusion method [20].
After incubation overnight at 37°C, a reduction of cefotetan
MIC of at least three dilutions in the presence of cloxacillin
was interpreted as a positive test [2] (see Fig. 1d).
All phenotypic methods were also tested for the presence
of scattered colonies located near the edge of the inhibition
halo of the antibiotics disks [21].
The genetic analysis used to detect the presence of six
families of plasmid-mediated AmpC β-lactamases genes
was a multiplex PCR assay described by Pérez-Pérez et al.
[11]. This multiplex PCR included the genes within the
following families: ACC, CIT (including LAT-1 to LAT-4,
CMY-2 to CMY-7, and BIL-1), DHA (DHA-1 and DHA-2),
EBC (including MIR-1 and ACT-1), FOX (including FOX-
1 to FOX-5b), and MOX (including MOX-1, MOX-2,
CMY-1, and CMY-8 to CMY-11). Five reference isolates
harbouring pAmpC previously characterised, two E. coli
(ACC-1 and CMY-2), one K. pneumoniae (FOX-5), one A.
hydrophila (MOX-3), one K. oxytoca (DHA-1) [21, 24, 25]
and six strains harboring other β-lactamases were included
to investigate the specificity of tested phenotypic methods.
The DHA-1 producing isolates were tested for induction
using cefoxitin and imipenem as inductors.
Negative E. coli isolates for pAmpC genes were further
selected for molecular analysis based on ampC promoter/atten-
uator sequencing to assess the chromosomal AmpC overpro-
duction. For the chromosomal ampC promoter/attenuator
mutation analysis, a 271-bp fragment was amplified and se-
quenced using primers AB1 (5′-GATCGTTCTGCCGCTG
TG-3′) and AmpC2 (5′-GGGCAGCAAATGTGGAGCAA-
3′) containing the fragment of the chromosomal ampC gene
promoter enclosing the −35 box, the −10 box and the attenuator
of E. coli [26]. The sequences were compared with the same
region of the E. coli K12 ampC gene, constitutively expressed
at a low level. The E. coli K12 ampC gene is the reference used
by several authors to assess the overexpression due to promot-
er/attenuator mutations [7, 8, 27].
Statistical analysis
Validity tests including sensitivity, specificity, positive pre-
dictive value and negative predictive value were calculated.
Sensitivity was defined as the percentage of PCR positive
isolates determined to be susceptible to the phenotypic
testing and specificity was defined as the percentage of
PCR negative isolates determined to be non-susceptible by
phenotypic testing. False positive for pAmpC was defined
as isolates with AmpC profile but negative for plasmid-
mediated AmpC β-lactamases genes.
Results
Among 21,376 Enterobacteriaceae collected prospectively in
our laboratory from clinical samples, 125 isolates (0.86%)
fulfilled the screening criteria. Multiplex PCR amplification
and sequencing analysis showed that 85 isolates (0.58%)
harbored a pAmpC gene including 74 E. coli (blaCMY-2: 70;
blaDHA-1: 4), five K. pneumoniae (blaCMY-2: 2; blaDHA-1: 3)
and six P. mirabilis (blaCMY-2: 6). No enzymes belonging to
the ACC, FOX, MOX or EBC families were detected among
these isolates. The results of the four phenotypic methods for
detection of pAmpC are shown in Table 1. The validity of the
results for all tested methods in E. coli is shown in Table 2. All
tests displayed similar sensitivities, above 95%, being slightly
higher in the cefotetan/cefotetan-cloxacillin test. In terms of
specificity, we observed differences among the four tests; the
AmpC test was found as the best confirmatory one in E. coli
showing a specificity of 58%, with 13 false positives and three
false negatives. No test showed specificity values over 60%.
In the case of K. pneumoniae and P. mirabilis, the findings
varied widely in all tests, ranging in sensitivity from 60% to
100% and specificity from 62.5% to 87.5% for K. pneumo-
niae, and in sensitivity from 83.3% to 100% and 100% of
specificity for P. mirabilis.
Eur J Clin Microbiol Infect Dis (2012) 31:2037–2043 2039

5. pAmpC E. coli producers exhibited the presence of scat-
tered colonies located near the edge of the inhibition halo of
cefoxitin, cefotaxime, ceftazidime and/or aztreonam. DHA-
1 producing E. coli isolates were positive for induction test
with cefoxitin and/or imipenem.
Several mutations in the chromosomal ampC promoter/
attenuator region of E. coli were identified in isolates with
negative pAmpC genes (Table 3). These detected mutations
included changes that created an alternate displaced promot-
er (−42), mutations in the wild-type promoter/attenuator
(−32, +22, +24, +26, +27, +32, +37), mutations in the
spacer length between the −35 and −10 box (−28, −18)
and single-base-pair deletion/insertion in the attenuator re-
gion (+21, +34). Mutations at positions −1, +37, +49, and
+58 were detected outside of the promoter/attenuator region.
Discussion
pAmpC have become a serious concern and have been
reported worldwide [28–31]. Their detection in a clinical lab-
oratory is very important for infection control purposes and for
ensuring effective therapeutic options. The recognition of
pAmpC producers can be difficult in the routine laboratory
practice, although resistance to cefoxitin can help to identify
most of them. Unfortunately, cefoxitin resistance is not only
due to AmpC production, but may also be due to decreased
permeability of the outer membrane [32] and rarely, by other
enzymes. In addition, various mutations in the ampC promoter/
attenuator region have been identified in cephamycin-resistant
E. coli isolates, which result in overproduction of naturally
chromosomal AmpC β-lactamase [7, 8, 33]. Several pheno-
typic methods have been described and used in laboratories for
detection of pAmpC in clinical isolates but few studies have
compared the existing methods [12, 33–35]. Most of these
Table 1 Correlation between the results of the four phenotypic methods and multiplex ampC PCR to detect plasmid-mediated AmpC
Variable pAmpC (n) Positive phenotypic tests (n1/n)
3D test AmpC test Cloxa test CN/CNI test
Positive multiplex ampC PCR (n085)
E. coli (n074) CMY-2 (70) 67/70 68/70 68/70 70/70
DHA-1 (4) 4/4 3/4 3/4 3/4
K. pneumoniae (n05) CMY-2 (2) 1/2 1/2 2/2 2/2
DHA-1 (3) 3/3 2/3 2/3 3/3
P. mirabilis (n06) CMY-2 (6) 6/6 6/6 6/6 5/6
Negative multiplex ampC PCR (n040)
E. coli (n031) – 17/31 13/31 22/31 20/31
K. pneumoniae (n08) – 1/8 1/8 3/8 1/8
P. mirabilis (n01) – 0/1 0/1 0/1 0/1
Reference isolates
E. coli (n02) ACC-1 1/1 1/1 1/1 1/1
CMY-2 1/1 1/1 1/1 1/1
K. pneumoniae (n01) FOX-5 1/1 1/1 1/1 1/1
K.oxytoca (n01) DHA-1 1/1 1/1 1/1 1/1
A.hydrophila (n01) MOX-3 1/1 1/1 1/1 1/1
ESBL
E. coli (n05) 0/5 0/5 0/5 0/5
K. pneumoniae (n01) 0/1 0/1 0/1 0/1
n number of isolates, n1 positive test, 3D three-dimensional test, Cloxa test cloxacillin synergy test, CN/CNI test cefotetan/cefotetan-cloxacillin E-test
Table 2 Validity test to detect AmpC in E. coli (multiplex-PCR as
gold standard)
Methods Sensitivity (%) Specificity (%) PPV (%) NPV(%)
3D test 96 45 81 82.35
AmpC test 96 58 84.5 85.7
Cloxacillin test 96 29 76.8 75
CN/CNI test 98.6 35.4 78.9 91.66
PPV positive predictive value, NPV negative predictive value
2040 Eur J Clin Microbiol Infect Dis (2012) 31:2037–2043

6. studies have assessed the usefulness of boronic acid as AmpC
inhibitor but false positives have been described in KPC-
production strains [14, 17–19]. There is a need, therefore, for
alternative methods that can be integrated into diagnostic lab-
oratories. For this reason, we chose the cloxacillin synergy test
to be evaluated against classical tests. In this study, all methods
evaluated showed a good sensitivity. Our data showed that,
overall, the best results as screening methods were obtained by
using the cefotetan/cefotetan-cloxacillin test. In terms of spec-
ificity, the best values were found with the AmpC test. Cefo-
tetan/cefotetan-cloxacillin and cloxacillin synergy tests are the
easiest to perform because they are commercially available.
The AmpC and 3D tests are less expensive but have the
disadvantage of requiring E. coli ATCC 25922 and of taking
more time to complete. In addition, the 3D test has a drawback
that interpretation of the results is difficult and subjective,
requiring some experience.
We found similar sensitivities than previously described
studies [12, 33–35], but lower values of specificities in E. coli.
The low specificity led us to examine the chromosomal ampC
promoter/attenuator mutations to determine the ampC
overexpression. Most of the mutations found in our study had
been previously described [8–10, 26, 33]. The mutations at
positions −42 or −32, which are linked to AmpC overexpres-
sion, were found in all but six of the 31 pAmpC-negative E. coli
isolates. For that reason, the false positives found in the
phenotypic methods might be associated with chromosomal
AmpC hyperproduction.
Our study had some limitations. The phenotypic methods
were only assessed in E. coli with DHA-1 and CMY-2
enzymes because they were the only two enzymes found
during the 30 months surveyed in our clinical strains (no
pAmpC from other families were detected). Considering that
the criteria for isolate inclusion was cefoxitin resistance, some
pAmpC isolates (such as ACC-producing ones) could be
overlooked or not detected. It should be noted that, due to
the small number of isolates harboring pAmpC in K. pneumo-
niae and P. mirabilis, it is unclear whether the high sensitivity
and specificity observed in these isolates for all methods were
due to its detection ability or to the low number of isolates.
The capability to detect AmpC enzymes is important to
improve the therapeutic management of infections and
Table 3 Mutation analysis of the chromosomal ampC promoter/attenuator region of 31 cefoxitin-resistant pAmpC-negative producing E. coli and
results of the four phenotypic methods
Nº strain Phenotypic Tests Position of mutations in ampC promoter/attenuator region
3D AmpC CLOXA CN/CNI
C68, C70, C94, C115, C130 + + + + −42; −18; −1; +58
C140, C142 + + + + −42; −18; −1
C42 + + + + −42; −18; −1; +24; +37; +58
C29 + + + + −42; −18; −1; +24; +37; +49; +58
C117 + + + + −32; +21:INS; +58
C10 + + + − −32; +22; +26; +27; +32
C26 + + − + −32; +22; +26; +27; +32
C89 + − + + −42; −18; −1; +58
C5 + − + + −42; −18; −1; +34:DEL; +58
C7 + − + − −42; −18; −1; +58
C3 + − + − −32; −18; −1
C92, C120, C32 − − + + −42; −18; −1; +58
C63 − + − + −42; −18; −1; +58
C64, C138 − − − + −42; −18; −1; +58
C149 − − − + −42; −18; −1
C78 − − + − −42; −18; −1
C45, C50 − − + − −18; −1; +58
C73 − − + − −28
C112 + − − − −32, −28
C87 − − − − −28
C31 − − − − −28; −1
C123 − − − − −18; −1; +58
Mutations at positions −1, +49, +58 are outside of promoter/attenuator region. INS and DEL indicate insertion and deletion of nucleotides,
respectively
Eur J Clin Microbiol Infect Dis (2012) 31:2037–2043 2041

7. provide useful epidemiological data. Our results showed that
all evaluated methods had high sensitivity so they could be
used as a screening test according to the facilities of each
clinical laboratory. None of the tests was able to discriminate
between plasmidic and/or hyperproduction of the chromo-
somal enzyme in E. coli isolates. Mirelis et al. [21] have
described a simple method based on the presence of scattered
colonies located near the edge of the inhibition halo of cefox-
itin, cefotaxime, ceftazidime and aztreonam (only present in
pAmpC) to distinguish between pAmpC or hyperproduction
chromosomal enzyme in E. coli, as we could appreciate in all
positive pAmpC isolates.
Our conclusion is that cefotetan/cefotetan-cloxacillin or
cloxacillin synergy tests may be advocated as phenotypic
screening tests in a routine clinical laboratory. In pAmpC
producing E. coli isolates, the complementary test described
by Mirelis et al. [21] might be recommended to differentiate
overexpression of chromosomal AmpC enzyme or acquisi-
tion of plasmid-encoded ampC genes. In DHA-1 producing
E. coli isolates, an induction test with cefoxitin and/or
imipenem could help to differentiate acquisition of the
plasmid-encoded ampC gene, due to their inducible
expression.
Acknowledgments This work was supported by Departamento de
Ciencia, Tecnología and Universidad del Gobierno de Aragón, Spain
(Project DGA/Grupos consolidados, B24-211130). We are grateful to
Mirelis B., Miró E., and Navarro F. for providing reference strains.
MJG received a grant from the S.E.I.M.C (Sociedad Española de
Enfermedades Infecciosas y Microbiología Clínica).
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