JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2011, p. 2798–2803 Vol. 49, No.

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0095-1137/11/$12.00 doi:10.1128/JCM.00404-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Practical Approach for Reliable Detection of AmpC

Beta-Lactamase-Producing Enterobacteriaceae䌤
Silke Polsfuss,† Guido V. Bloemberg,† Jacqueline Giger, Vera Meyer,
Erik C. Böttger, and Michael Hombach*
Institut für Medizinische Mikrobiologie, Universtät Zürich, 8006 Zürich, Switzerland
Received 25 February 2011/Returned for modification 5 April 2011/Accepted 18 May 2011

In this prospective study all Enterobacteriaceae isolates (n ⴝ 2,129) recovered in the clinical microbiology
laboratory during October 2009 to April 2010 were analyzed for AmpC production. Clinical and Laboratory
Standards Institute (CLSI) cefoxitin and cefotetan susceptibility breakpoints and CLSI critical ESBL diam-
eters were used to screen for potential AmpC producers. In total, 305 isolates (211 potential AmpC producers
and 94 AmpC screen-negative isolates as a control group) were further analyzed by multiplex PCR for the
detection of plasmid-encoded ampC beta-lactamase genes and by ampC promoter sequence analysis (consid-
ered as the gold standard). Cefoxitin and cefotetan were assessed as primary screening markers. The sensi-
tivities of cefoxitin and cefotetan for the detection of AmpC production were 97.4 and 52.6%, respectively, and
the specificities were 78.7 and 99.3%, respectively. As a phenotypic confirmation test, the Etest AmpC and the
cefoxitin-cloxacillin double-disk synergy method (CC-DDS) were compared. The sensitivities for the Etest
AmpC and the CC-DDS method were 77.4 and 97.2%, respectively, and the specificity was 100% for both
methods. The results of the Etest AmpC were inconclusive for 10 isolates. With the CC-DDS method 2
inconclusive results were observed. Based on this study, we propose a comprehensive diagnostic flow chart for
the detection of AmpC production consisting of a simple phenotypic screening and a single phenotypic
confirmation test with inconclusive results being resolved by molecular analysis. For the proposed flow chart
using (i) cefoxitin as a screening marker for AmpC production, (ii) the CC-DDS method as phenotypic
confirmation, and (iii) molecular methods in case of inconclusive results, the sensitivity and specificity for
AmpC detection would have been 97.4 and 100%, respectively, with respect to the studied isolates. The
phenotypic methods used in the AmpC algorithm are simple to perform and easy to implement in the
diagnostic laboratory.

In recent years, the prevalence of infections with multidrug- tams, and beta-lactam inhibitors. In contrast to expanded-spec-
resistant Enterobacteriaceae has steadily increased (18). Entero- trum beta-lactamases (ESBLs), AmpC beta-lactamases are
bacteriaceae producing AmpC beta-lactamases (AmpCs) have inhibited by boronic acid and cloxacillin (2, 9, 25). In E. coli,
become a major therapeutic challenge. The detection of regulation of chromosomal ampC expression differs signifi-
AmpC-producing Klebsiella spp., Escherichia coli, P. mirabilis, cantly from that of other Enterobacteriaceae. In E. coli ampC is
and Salmonella spp. is of significant clinical relevance since regulated by a weak promoter and a strong attenuator resulting
AmpC producers may appear susceptible to expanded-spec- in a constitutive low-level ampC expression (11). Diverse mu-
trum cephalosporins when initially tested (13, 27, 28). This may tations in the ampC promoter region leading to overexpression
lead to inappropriate antimicrobial regimens and therapeutic have been described (3, 4, 7, 11, 12, 24, 29). In addition to
failure (24). Thus, a simple and reliable detection procedure chromosomal ampC, Enterobacteriaceae can acquire plasmid-
for AmpC producers is needed. encoded ampC genes (9). In general, plasmid-encoded AmpC
Many Gram-negative bacteria harbor chromosomal ampC beta-lactamases are expressed constitutively and are readily
beta-lactamase genes, which are constitutively expressed at low detected by a multiplex PCR (17).
level. In general, the expression of chromosomally located Different phenotypic AmpC detection tests have been de-
ampC genes is inducible by beta-lactam antibiotics, such as scribed in the literature (9). A standardized diagnostic ap-
cefoxitin, cefotetan, and imipenem, and mediated by the reg- proach integrating screening and confirmation tests for the
ulator AmpR. Mutations in the repressor gene ampD are the detection of AmpC beta-lactamase-producing Enterobacteria-
most common cause of constitutive (hyper-)production of ceae has not been established to date. We sought here to
AmpC beta-lactamases (23). AmpC beta-lactamases degrade develop a comprehensive diagnostic flow chart integrating a
penicillins, expanded-spectrum cephalosporins (with the ex- simple phenotypic screening and confirmation for implemen-
ception of cefepime and cefpirome), cephamycins, monobac- tation in the routine diagnostic laboratory.

MATERIALS AND METHODS

* Corresponding author. Mailing address: Institut für Medizinische
Mikrobiologie, Universität Zürich, Gloriastr. 30/32, 8006 Zürich, Swit- Clinical isolates. In this prospective study all nonduplicate clinical Enterobac-
zerland. Phone: 0041 44 634 27 00. Fax: 0041 634 49 06. E-mail: teriaceae isolates (n ⫽ 2,129) from the diagnostic laboratory isolated over a
[email protected]. period of 7 months from October 2009 until April 2010 were screened for AmpC
† S.P. and G.V.B. contributed equally to this study. production (see Fig. 2). Only isolates that were considered clinically relevant
䌤
Published ahead of print on 1 June 2011. were included, i.e., isolates that were considered as normal flora or commensals

2798
VOL. 49, 2011 DETECTION OF AmpC BETA-LACTAMASES 2799

were disregarded. The isolates examined here included Escherichia coli, Kleb- TABLE 1. Species distribution and numbers of AmpC-producing
siella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Salmo- Enterobacteriaceae isolatesa
nella enterica, and Citrobacter koseri. With the exception of E. coli, we excluded
species with known chromosomal AmpC production, e.g., Enterobacter cloacae, No. (%) No. (%) of
Species
Enterobacter aerogenes, Citrobacter freundii, Serratia marcescens, Hafnia alvei, and of isolates ampC-positive isolatesb
Morganella morganii (9). Escherichia coli 1,435 (67.4) 33 (2.3)
Susceptibility testing. For susceptibility testing, the Kirby-Bauer disk diffusion Klebsiella pneumoniae 360 (16.9) 2 (0.6)
method was used. Antibiotic disks were purchased from Becton Dickinson Klebsiella oxytoca 99 (4.7) 0
(Franklin Lakes, NJ), and results were interpreted according to the Clinical and Salmonella enterica 4 (0.2) 2 (50.0)
Laboratory Standards Institute (CLSI) 2009 guidelines (5). For cefoxitin (30 Proteus vulgaris 26 (1.2) 0
␮g/disk) and cefotetan (30 ␮g/disk), screening cutoffs of ⱕ18 and ⱕ16 mm, Proteus mirabilis 131 (6.2) 1 (0.8)
respectively, were used (i.e., the CLSI susceptible breakpoints). In addition, the Citrobacter koseri 74 (3.5) 0
following ESBL CLSI screening cutoff values for expanded-spectrum cephalo-
sporins were used to select for potential AmpC-producing isolates as follows: Total 2,129 (100) 38
cefpodoxime (10 ␮g/disk), ⱕ17 mm; ceftazidime (30 ␮g/disk), ⱕ22 mm; cefo-
taxime (30 ␮g/disk), ⱕ27 mm; and ceftriaxone (30 ␮g/disk), ⱕ25 mm. Suscepti- a
Numbers are given for all Enterobacteriaceae species that were tested for
bility testing was performed on Mueller-Hinton agar (bioMérieux, Marcy antibiotic susceptibility.
b
l’Etoile, France) using McFarland 0.5 from overnight cultures, followed by in- A total of 19 E. coli strains harbored a plasmidic ampC, and 14 E. coli strains
cubation at 35°C for 16 to 18 h. contained mutations in the chromosomal ampC promoter which resulted in
overexpression. E. coli strains with both plasmidic ampC and chromosomal ampC
Phenotypic AmpC confirmation testing. The Etest AmpC (AB bioMérieux,
promoter mutations present were not detected.
Solna, Sweden) was performed according to the manufacturer’s instructions. The
test principle comprises a strip impregnated with a concentration gradient of
cefotetan on one half of the strip and cefotetan with cloxacillin on the other half
of the strip. MICs of cefotetan alone and cefotetan with cloxacillin were deter- Of the 2,129 isolates, 211 were categorized as potential
mined as recommended by the manufacturer. Ratios of cefotetan versus cefo-
tetan/cloxacillin of ⱖ8 were considered positive for AmpC beta-lactamase pro-
AmpC producers on the basis of (i) cefoxitin inhibition zone
duction. diameters of ⱕ18 mm, (ii) cefotetan inhibition zone diameters
The cefoxitin-cloxacillin double disc synergy test (CC-DDS) was performed as of ⱕ16 mm, and/or (iii) positive ESBL screening diameters
described previously (25). This test is based on the inhibitory effect of cloxacillin according to CLSI guidelines. To further assess the sensitivity
on AmpC. Disks containing either 30 ␮g of cefoxitin or 30 ␮g of cefoxitin plus
and specificity of the screening procedure, 94 of 1,922 isolates
200 ␮g of cloxacillin were manufactured for the present study (Liofilchem,
Roseto degli Abruzzi, Italy). The strains were inoculated on Mueller-Hinton agar with (i) cefoxitin inhibition zones of ⬎18 mm, (ii) cefotetan
using McFarland 0.5, followed and incubated at 35°C for 16 to 18 h. A difference inhibition zone diameters of ⬎16 mm, and (iii) negative ESBL
in the cefoxitin-cloxacillin inhibition zones minus the cefoxitin alone zones of ⱖ4 screening diameters according to the CLSI were included in
mm was considered indicative for AmpC production. the analysis. In all, 305 isolates (211 potential AmpC producers
ampC promoter sequencing (E. coli only). DNA was extracted from colonies
grown on sheep blood agar medium using the InstaGene Matrix (Bio-Rad,
and 94 determined to be negative by the AmpC screening
Switzerland) according to the manufacturer’s instructions. For the ampC pro- procedure) were characterized by phenotypic methods, multi-
moter mutation analysis, a 271-bp fragment was amplified by using the primers plex PCR and, in part, DNA sequence analysis (gold standard,
AB1(5⬘-GATCGTTCTGCCGCTGTG-3⬘) and ampC2 (5⬘-GGGCAGCAAATG see Fig. 2).
TGGAGCAA-3⬘) (4). PCR amplicons were purified with a QIAquick PCR
Of the 211 potential AmpC producers, 37 were confirmed as
purification kit (Qiagen, Hombrechtikon, Switzerland), followed by cycle se-
quencing using a BigDye reagent kit (Applied Biosystems, Switzerland). Se- AmpC-producing isolates by phenotypic and molecular meth-
quence analysis was performed on an ABI Prism 3100 DNA sequencer (Applied ods (see Fig. 2). Of 211 isolates with a CIT type plasmid-
Biosystems) according to standard protocols. Sequences were analyzed and ed- encoded AmpC beta-lactamase, 1 was detected by molecular
ited by using Lasergene 7 MegAlign software (DNASTAR, Inc.). The ampC methods exclusively. AmpC production in this isolate was not
promoter sequences were compared to the wild-type ampC sequence of E. coli
strain ATCC 25922.
detected by either the cefoxitin or the cefotetan disk diffusion
Detection of plasmid-mediated ampC beta-lactamase genes. For the detection test (which showed inhibition zone diameters of 21 or 27 mm,
of plasmid-mediated ampC beta-lactamase genes, a multiplex PCR was used respectively), nor was it detected by the cefoxitin-cloxacillin
(17), which detects the six plasmid-mediated ampC families. When necessary, double-disk synergy test. The cefoxitin and cefotetan MICs for
PCR amplicons were sequenced with the amplification primers according to the
this isolate were 4 and 0.75 mg/liter, respectively. Both values
protocol described above. Sequences were analyzed for homology by using the
National Center for Biotechnology Information GenBank database (http://www are in the susceptible range of the CLSI 2009 guidelines. The
.ncbi.nlm.nih.gov/). majority of plasmid-encoded AmpCs belonged to the CIT type
Interpretation. Molecular methods were considered the gold standard for (22 of 24 isolates), and two plasmid-encoded AmpCs were
calculation of the performance parameters. The results of the CC-DDS and/or identified as the DHA type.
Etest AmpC analyses were considered inconclusive if visible zones of inhibition
were lacking (i) with cefotetan or cefoxitin alone or (ii) with cefotetan-cloxacillin
The prevalence of AmpC production among all tested iso-
or cefoxitin-cloxacillin. lates was 1.8%. Most frequently, AmpC production was ob-
served in E. coli (33 of 38 potential AmpC-producing isolates);
19 of these isolates were plasmid encoded, and 14 were due to
RESULTS
mutations in the ampC promoter region (Table 1). The ma-
Analysis of Enterobacteriaceae isolates for AmpC production jority of the isolates with AmpC production were isolated from
in clinical isolates. A total of 2,129 nonduplicate clinical urine (52.6%), respiratory tract (18.4%), rectogenital (7.9%),
strains of the Enterobacteriaceae family isolated in the diagnos- and wound (7.9%) specimens. A total of 13.9% of the speci-
tic microbiological laboratory during a 7-month period were mens represent swabs without localization indicated that were
screened for AmpC production. Species with known chromo- originally sent to the laboratory for ESBL screening.
somally encoded AmpC beta-lactamases (9) were not included, Comparison of primary screening markers for AmpC pro-
except for E. coli. The majority of the isolates were identified duction. Cefoxitin and cefotetan were compared as primary
as E. coli (n ⫽ 1,435) and K. pneumoniae (n ⫽ 360) (Table 1). screening markers. For cefoxitin, an 18-mm inhibition zone
2800 POLSFUSS ET AL. J. CLIN. MICROBIOL.

TABLE 2. Performance of screening tests, confirmation tests, and the proposed AmpC detection algorithm
No. of isolates examineda %
Examination method
Total TP FP TN FN IR Sensitivity Specificity

Screening tests
Cefoxitin screening 305 37 57 210 1 0 97.4 78.7
Cefotetan screening 305 20 2 265 18 0 52.6 99.3

Confirmation tests
Etest AmpC 305 24 0 264 7 10 77.4 100.0
Cefoxitin ⫾ cloxacillin 305 35 0 267 1 2 97.2 100.0

AmpC algorithm 305 37 0 267 1 0 97.4 100.0

a
TP, true positive; TN, true negative; FP, false positive; FN, false negative; IR, inconclusive result. Results were rated inconclusive if the MICs exceeded the scale
of the Etest for cefotetan alone and/or cefotetan in combination with cloxacillin (Etest AmpC) or when no inhibition zone could be detected for both cefoxitin alone
or in combination with cloxacillin (CC-DDS). Inconclusive results were excluded from the calculation of performance parameters.

diameter was chosen as the cutoff, and 16-mm diameter was inconclusive results were not included in the calculation of
used for cefotetan (i.e., the CLSI 2009 susceptibility break- performance parameters. The sensitivities for Etest AmpC and
points). Performance parameters were calculated considering CC-DDS were 77.4 and 97.2%, respectively, and the specificity
molecular methods as the gold standard (multiplex PCR for was 100% when both methods were combined (Table 2).
the detection of plasmid-mediated ampC beta-lactamase genes Development of an algorithm for AmpC detection in Entero-
and E. coli chromosomal ampC promoter sequence analysis). bacteriaceae. Combining the most sensitive screening method
The sensitivities of cefoxitin and cefotetan for the detection of with the most accurate confirmation assay for AmpC produc-
AmpC production were 97.4 and 52.6%, respectively, and the tion, we developed a comprehensive diagnostic flow chart (Fig.
specificities were 78.7 and 99.3%, respectively. The absolute 3), which consists of (i) cefoxitin as a screening marker for
numbers of isolates and calculated performance parameters AmpC production and (ii) CC-DDS as phenotypic confirma-
are summarized in Table 2. tion, along with (iii) molecular methods in the case of incon-
Figure 1 shows the zone diameter distributions for cefoxitin clusive results. For AmpC detection in the isolates of the
and cefotetan in all isolates with a genotypically confirmed present study, this diagnostic approach would have displayed a
ampC. An AmpC screening cutoff for cefoxitin of ⱕ18 mm calculated sensitivity and specificity of 97.4 and 100%, respec-
(CLSI susceptibility breakpoint) missed only one isolate with a tively (Table 2), with molecular analysis for inconclusive results
genotypically detected CIT type AmpC that produced a diam- only necessary for two isolates (1% of all isolates positive in the
eter of 21 mm. Plasmid-encoded AmpCs clustered at a cefoxi- AmpC screening procedure).
tin inhibition zone of 6 mm (19 of 24 isolates), which corre-
sponds to the absence of a visible inhibition zone since the disc DISCUSSION
diameter itself is 6 mm. For isolates with plasmid-encoded
AmpCs, the largest cefoxitin inhibition zone observed was 13 Detection of AmpC production in pathogens might be impor-
mm, in an isolate that was identified as Salmonella enterica tant for ensuring effective antibiotic therapy (20) since the pres-
serovar Typhimurium with a CIT-type AmpC. In contrast, the ence of an AmpC beta-lactamase frequently seems to result in
majority of E. coli isolates with promoter mutations produced therapeutic failure when broad-spectrum cephalosporins are used
inhibition zone diameters of ⱖ13 mm (10 of 14 isolates). (14, 24). However, further studies are required to assess whether
Cefotetan, in general, showed higher variation in zone diam- AmpC production is an independent risk factor for clinical out-
eters than cefoxitin. An AmpC screening cutoff of ⱕ16 mm come. Several methods have been evaluated for phenotypic
(CLSI susceptibility breakpoint) missed all 14 E. coli isolates screening and confirmation of AmpC beta-lactamase production
with AmpC promoter mutations and 5 of 24 isolates with a (9, 25). However, a comprehensive diagnostic algorithm integrat-
plasmid-encoded AmpC. All isolates with promoter mutations ing both screening and confirmation has not been established. In
showed cefotetan zone diameters of ⱖ18 mm and the majority the present study we evaluated individual screening and confir-
of isolates with plasmid-encoded AmpCs showed cefotetan mation methods for AmpC production. Subsequently, we devel-
zone diameters of ⱕ17 mm (20 of 24 isolates). oped a diagnostic algorithm that (i) combines the most efficient
Comparison of confirmation assays for AmpC production. and accurate methods, (ii) is simple, and (iii) can be implemented
The Etest AmpC and the cefoxitin-cloxacillin CC-DDS in the diagnostic laboratory (Fig. 2).
method were compared as phenotypic confirmation tests. In 10 When cefoxitin and cefotetan (both cephamycins) were
of the 305 isolates, the results of the Etest AmpC analysis were compared as the primary screening marker, cefoxitin was
inconclusive since MICs exceeded the scale of the test for clearly superior to cefotetan regarding sensitivity (see Table 2).
cefotetan alone and/or cefotetan in combination with cloxacil- Our results for cefoxitin are in agreement with those of other
lin (Fig. 2). Therefore, the calculation of a ratio was not pos- authors (20, 25). However, the specificity in the present study
sible. With the CC-DDS, two inconclusive results were ob- was significantly lower, e.g., 78.7% versus the 95% reported by
served. In these two isolates no inhibition zone was present for Tan et al. (25). In contrast to MIC determination by automated
cefoxitin alone or in combination with cloxacillin. Isolates with systems, the determination of drug susceptibility by disc diffu-
VOL. 49, 2011 DETECTION OF AmpC BETA-LACTAMASES 2801

FIG. 1. Inhibition zone diameter distributions in AmpC beta-lactamase-producing isolates. The inhibition zone diameters of cefoxitin (A) and
cefotetan (B) are presented. The numbers of Enterobacteriaceae isolates with plasmidic ampC (u) and chromosomal ampC promoter mutations
(f, E. coli only) are given; the CLSI 2009 susceptibility breakpoints are indicated by black arrows.

sion may further enhance sensitivity since synergy and antag- type enzymes are characteristically inhibited by cefoxitin visible
onism phenomena are readily observed, e.g., when placing a as enhancement of the inhibition zones (synergy phenomena)
cefoxitin disc near a expanded-spectrum cephalosporin disc. of expanded-spectrum cephalosporins and cefoxitin. With this
For example, the presence of DHA type enzymes will lead to strategy, the detection of ACC-type AmpC enzymes is possi-
flattening of inhibition zones (antagonism phenomena) of ex- ble, although ACC enzymes appear to be cefoxitin susceptible
panded-spectrum cephalosporins toward inducers such as (1, 22). In contrast, cefoxitin screening by MIC alone would
cefoxitin, carbapenems, or clavulanic acid. Otherwise, ACC- miss ACC types. Other authors recommend additional screening
2802 POLSFUSS ET AL. J. CLIN. MICROBIOL.

FIG. 2. Study layout and numbers of isolates. CPD, cefpodoxime; CRO, ceftriaxone; CAZ, ceftazidime; CTX, cefotaxime; CC-DDS, double-
disk synergy test. #, AmpC missed by the algorithm.

criteria for ACC enzymes such as critical inhibition zone diame- Therefore, we chose cloxacillin as AmpC inhibitor in our al-
ters for amoxicillin-clavulanic acid or expanded-spectrum cepha- gorithm. Regarding sensitivity, the CC-DDS was clearly supe-
losporins (26). To date, the ACC types seem to be the only known rior to the Etest AmpC (97.2% versus 77.4%, respectively, see
enzymes that can be missed by cefoxitin screening. The isolation Table 2). This result may be explained by the use of cefotetan
numbers of ACC enzymes are still significantly lower than those in the Etest AmpC. Cefotetan has a lower sensitivity than
of CIT (CMY), FOX, and DHA types (10, 14, 19, 25, 26). No cefoxitin concerning the detection of AmpC production. This
ACC-type AmpC was detected in the present study. is also apparent when cefotetan disc diffusion was used as a
The AmpC flow chart (Fig. 2) can be combined with a flow screening test (see Table 2). Ten inconclusive results were
chart for ESBL detection (unpublished data). If cefoxitin is not obtained with the Etest AmpC, due to MICs exceeding the
routinely tested, an alternative branch may be chosen that Etest scale of cefotetan with or without cloxacillin (Table 2). In
substitutes the cefoxitin screening criteria by CLSI screening routine use, this may hamper the sensitivity and practicability
criteria for ESBL (Fig. 3). With a combined ESBL/AmpC of this method. In contrast, with CC-DDS only two inconclu-
screening strategy, ACC enzymes will readily be detected. sive results were obtained. For both isolates with an inconclu-
ACC confers high resistance to expanded-spectrum cephalo- sive result, no inhibition zone for cefoxitin was observed both
sporins, which serve as primary screening markers for ESBL with or without cloxacillin. Eventually, AmpC enzymes of the
detection (19, 21). Thus, corresponding isolates will be as- CIT type were found in both strains. The results for the CC-
signed to a combined ESBL/AmpC confirmation test via the DDS are in agreement with other studies that reported a high
CLSI screening criteria for ESBL (5, 6). sensitivity and specificity for this test (25).
The single false-negative result for the cefoxitin screening Combining the high sensitivity of cefoxitin screening with
test in the present study (Fig. 2) resulted from the presence of the high specificity of the cefoxitin-cloxacillin CC-DDS confir-
a CIT-type ampC detected by multiplex PCR. MICs of this mation test, we propose a flow chart for the phenotypic detec-
isolate for cefoxitin and cefotetan were well within the suscep- tion and characterization of AmpC beta-lactamases (Fig. 3). In
tible range, and both phenotypic confirmation tests were the case of (rarely occurring) inconclusive results, molecular
clearly negative (Etest AmpC ratio of 1.0; CC-DDS, no differ- methods are used for resolution. The proposed flow chart
ence). Sequence analysis of the CIT ampC gene did not reveal would have a calculated sensitivity and specificity of 97.4 and
any mutation affecting the structure and/or function of the 100%, respectively, with respect to the isolates in the present
enzyme. However, mutations in the regulatory regions may study. Phenotypic AmpC screening and confirmation tests are
result in very low expression or no expression of the structural inexpensive but nevertheless highly sensitive and specific.
gene (8). If the CIT type enzyme in this isolate were nonfunc- Therefore, it can be performed in all types of clinical labora-
tional, the sensitivity of the cefoxitin screening procedure tories, whereas the implementation of molecular methods is
would be close to 100%. often complex, requires specially trained personnel, and is as-
We compared the performance of the Etest AmpC and the sociated with higher costs.
cefoxitin-cloxacillin CC-DDS as a phenotypic confirmation test In conclusion, the proposed flow chart for detection of
(25). Boronic acid can be used as alternative to cloxacillin as AmpC is simple to use and easy to implement in a diagnostic
AmpC inhibitor, since it was found to be almost as sensitive laboratory. If molecular methods are not available, the few
and specific as cloxacillin by Tan et al. (25). However, boronic inconclusive isolates can be submitted to a reference labora-
acid may produce false-positive results in isolates carrying class tory for further investigations. In parallel, we have developed a
A carbapenemases, whereas cloxacillin does not (15, 16). flow chart for ESBL detection (unpublished), which in combi-
VOL. 49, 2011 DETECTION OF AmpC BETA-LACTAMASES 2803

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ACKNOWLEDGMENTS not responsive to extended-spectrum-cephalosporin treatment. Antimicrob.
Agents Chemother. 47:2138–2144.
We thank R. Zbinden for critical discussions and Claudia Merkofer 25. Tan, T. Y., L. S. Ng, J. He, T. H. Koh, and L. Y. Hsu. 2009. Evaluation of
for expert technical assistance. screening methods to detect plasmid-mediated AmpC in Escherichia coli,
Klebsiella pneumoniae, and Proteus mirabilis. Antimicrob. Agents Che-
This study was supported in part by the University of Zurich.
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26. Tenover, F. C., et al. 2009. Identification of plasmid-mediated AmpC beta-
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