AmpC b-lactamases and bacterial resistance:

an updated mini review

Mohammad Shahida, Farrukh Sobiaa, Anuradha Singha, Haris M. Khana,
Peter M. Hawkeyb,c, Anwar Huqd and Nancy Khardorie

Reviews in Medical Microbiology 2009, 20:41–55

Keywords: AmpC b-lactamases, antibiotic resistance, mini review

Introduction resistance in Klebsiella pneumoniae isolates from patients

The accumulation of bacterial antibiotic resistance is a
dramatic demonstration of Darwin’s dictum of the
survival of the fittest, with serious practical consequence
for treatment failure. The patterns and mechanisms of
resistance undergo continuous evolution and other
resistances are proliferating rapidly, notably those to
cephalosporins and quinolones among Gram-negative
bacteria and carbapenem resistance is emerging too,
especially in Acinetobacter spp. For a resistance to succeed,
it needs to have a mechanism that imposes little fitness
burden, along with a biologically fit host strain or strains.
The principal bacterial defence mechanism is the
production of b-lactamases; enzymes which produce a
biologically inactive product by hydrolyzing the b-lactam
ring [1]. b-Lactamases can also be described as bacterial
enzymes that inactivate b-lactam antibiotics by hydroly-
sis, which results in ineffective compounds. To date, at
least 400 different types of b-lactamases have been
described (www.lahey.org). Some enteric pathogens may
also be cephamycin-resistant by virtue of porin mutation.
Plasmid-mediated AmpC (pMAmpC) enzymes may
confer broad resistance to all b-lactams other than
carbapenems and hence pose a major therapeutic
challenge [2]. Carbapenems are active against strains that
harbour these enzymes, but several researchers have
described the emergence of resistance due to a dual
mechanism. Bradford et al. [3] described imipenem
in New York and these organisms produced multiple
b-lactamases, including an AmpC-type enzyme with pI
of 9.0. AmpC b-lactamases are produced by numerous
pathogens [4] and are emerging as an increasing cause of
resistance in Enterobacteria [5]. Interestingly, increasing
frequency of co-existence of blaampC with blaCTX-M has
also been reported recently [6]. Acquisition of AmpC-
type genes by plasmids in Escherichia coli and K. pneumoniae
has been known since the 1980s [7]. Plasmidic ampC
genes are derived from the chromosomal ampC genes of
several (not all) members of the family Enterobacter-
iaceae, including Enterobacter cloacae, Citrobacter freundii,
Morganella morganii, and Hafnia alvei [4].
To meet the challenge of resistance due to production of b-
lactamases, b-lactams with greater b-lactamase stability,
including cephalosporins, carbapenems, and monobac-
tams, were introduced in the 1980s. Resistance appeared
initially in organisms such as E. cloacae, C. freundii, Serratia
marcescens and Pseudomonas aeruginosa that could, by
mutation, overproduce their chromosomal AmpC (also
termed class C or group 1) b-lactamase, thus providing
resistance to both oxyimino- and 7-a-methoxy-cepha-
losporins and monobactams [8]. Later, resistance appeared
in bacterial species that lack an inducible AmpC enzyme,
such as K. pneumoniae, E. coli, Salmonella spp. and Proteus
mirabilis, and this resistance was found to be mediated
by plasmids encoding extended-spectrum b-lactamases

a
Section of Antimicrobial Resistance Researches and Molecular Biology, Department of Microbiology, Jawaharlal Nehru Medical
College & Hospital, Aligarh Muslim University, Aligarh, Uttar Pradesh, India, bAntimicrobial Agents Research Group, Institute of
Biomedical Research, University of Birmingham, Edgbaston, cWest Midlands Health Protection Agency, Heart of England NHS
Foundation Trust, Bordesley Green East, Birmingham, UK, dMaryland Pathogen Research Institute, Bioscience Research Building,
University of Maryland, College Park, Maryland, and eDivision of Infectious Diseases, Department of Internal Medicine, Southern
Illinois University School of Medicine, Springfield, Illinois, USA.
Correspondence to Dr Mohammad Shahid, Assistant Professor & Consultant Microbiologist, Department of Microbiology,
Jawaharlal Nehru Medical College & Hospital, Aligarh Muslim University, Aligarh 202 002, Uttar Pradesh, India.
Tel: +91 571 2720382; fax: +91 571 2721776/2720382; e-mail: shahidsahar@yahoo.co.in; drmohdshahid123@yahoo.com
Received: 25 June 2009; accepted: 8 August 2009.

DOI:10.1097/MRM.0b013e328331ad83

ISSN 0954-139X Q 2009 Wolters Kluwer Health I Lippincott Williams & Wilkins 41
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
42 Review in Medical Microbiology 2009, Vol 20 No 3
(ESBLs), which are enzymes that arose by mutations in
TEM (named after patient Temoneira from Athens,
Greece) or sulfhydryl variable (SHV) b-lactamases of
more limited hydrolytic capacity [9–11]. Such resistance
included oxyimino-cephalosporins and monobactams
but not 7-a-methoxy-cephalosporins and was blocked
by clavulanate, sulbactam or tazobactam, which are
inhibitors that are generally ineffective against class C
enzymes [8,12]. With continuing use of 7-a-methoxy-
cephalosporins (cefoxitin and cefotetan) and the clinical
introduction of b-lactamase inhibitor combinations,
plasmids encoding class C b-lactamases appeared [13].
Like their counterpart on the chromosome, such
enzymes provided a broader spectrum of resistance than
ESBLs and were not blocked by commercially available
inhibitors. Furthermore, in a strain with decreased outer
membrane permeability, such enzymes can provide
resistance to carbapenems as well, as has been observed
with clinical isolates of K. pneumoniae during an out-
break in New York [3] and in individual isolates of
E. coli in the United Kingdom [14] and K. pneumoniae in
Sweden [15].
History and chronology of AmpC
b-lactamases
b-Lactamases, demonstrated or presumed to be chro-
mosomally mediated, have been described in Acinetobacter
spp., Aeromonas spp., Chromobacterium violaceum, C.
freundii, Enterobacter spp., E. coli, H. alvei, Lysobacter
lactamgenus, M. morganii, Ochrobactrum anthropi, Proteus
rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Psy-
chrobacter immobilis, Rhodobacter sphaeroides, S. marcescens,
and Yersinia enterocolitica. In Shigella flexneri and Shigella
dysenteriae, ampC is included in a large deletion [16]. An
ampC locus appears on the genetic map of Salmonella
[17], but the evidence for its existence was indirect and its
presence has not been confirmed in the sequenced
genomes of Salmonella enterica serotypes Typhimurium or
Paratyphi [18], so that Salmonella is considered to be
AmpC
[13]. A chromosomal ampC gene is also lacking
in Klebsiella spp. and P. mirabilis [18]. Bobrowski et al. [19]
described a plasmid-mediated b-lactamase indistinguish-
able from the AmpC enzyme of E. coli in a strain of P.
mirabilis and Levesque et al. [20] reported a plasmid-
mediated cephalosporinase in Achromobacter spp. In 1983,
Knothe et al. [21] reported the transfer of cefoxitin
resistance from S. marcescens to Proteus or Salmonella spp.,
but resistance segregated on transfer to E. coli and no
biochemical or molecular studies were done [21]. In
1989, Bauernfeind et al. [22] described a K. pneumoniae
isolate from South Korea that could transfer resistance to
cefoxitin and cefotetan as well as to penicillins, oxyimino-
cephalosporins and monobactams to E. coli. The enzyme,
termed CMY-1 for its cephamycinase activity, had an
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
isoelectric point (pI) of 8.0 and was more sensitive to
inhibition by sulbactam than by clavulanate or tazobac-
tam, suggesting that it might be a class C enzyme.
However, the first proof that a class C b-lactamase had
been captured on a plasmid was provided by Papanicolaou
et al. [23], who described transmissible resistance to
a-methoxy-b-lactam and oxyimino-b-lactam mediated
by an enzyme (MIR-1) with the biochemical properties
of a class 1 b-lactamase and showed that part of the MIR-1
gene was 90% identical to the ampC gene of E. cloacae.
Subsequently, plasmid-mediated class C b-lactamases
have been discovered worldwide; an updated information
regarding the detection of AmpC types from various
Gram-negative bacterial species, their accession numbers
and country of origin is shown in Table 1. These
b-lactamases have been named with inconsistency typical
of b-lactamase nomenclature according to the resistance
produced to cephamycins (CMY), cefoxitin (FOX) and
moxalactam (MOX) or latamoxef (LAT), to the type of
b-lactamase, such as AmpC type (ACT) or Ambler class C
(ACC), and to the site of discovery, such as the Miriam
Hospital in Providence, Rhode Island, USA (MIR-1) or
Dhahran Hospital in Saudi Arabia (DHA). BIL-1 was
characterized and even named after the patient (Bilal)
who provided the original sample [24] (for detailed
descriptions readers are encouraged to read [25]).
Bou et al. [26] analyzed the nucleic acid sequence and
reported a new ampC b-lactamase gene that was closely
related to those encoding FOX-1, FOX-2, and FOX-3
b-lactamases, but its product had four novel amino acid
mutations at position 11 (M!T), 43 (A!E), 233
(V!A), and 280 (Y!H). They designated this product as
FOX-4. In 2003, Nakano et al. [27] described a novel
plasmid-encoded ampC-type b-lactamase, CFE-1, with
an ampR gene derived from C. freundii. DNA sequence
analysis also identified a gene upstream of ampC whose
sequence was 99% identical to ampR gene from C. freundii
GC3. In addition, a fumarate operon (frd ABCD) and an
outer membrane lipoprotein (blc) surrounding the ampR-
ampC genes in C. freundii were identified and insertion
sequence (IS26) elements were observed on both sides of
sequence identified (forming an IS26 composite trans-
poson) [27]. They concluded that these results confirmed
the evidence of translocation of a b-lactamase-associated
gene region from chromosome to plasmid. Doi et al. [28]
characterized a novel plasmid-mediated cephalosporinase
CMY-9 and revealed its genetic environment in an
E. coli clinical isolate. After determining the amino acid
sequence, it was found that CMY-9 had a single amino
acid substitution (E 85 D), the residue reported to be part
of the recognition site for R1 side chain of b-lactams,
compared with amino acid sequence of CMY-8 and also
had 78% identity with the amino acid sequence of Cep H
(chromosomal cephalosporinase of Aeromonas hydrophi-
lia). In the same year, Hujer et al. [29] identified a new
allelic variant of Acinetobacter baumanii cephalosporinase,
ADC-7 b-lactamase. The amino acid composition of this
 AmpC b-lactamases – an update Shahid et al. 43

Table 1. Various chromosomal and plasmid-mediated AmpC b-lactamases reported in various Gram-negative organisms, their year and country of origin,
and accession numbers analyzed in the present review.

Organisms/ Accession Year reported Country of origin Chromosomal/

AmpC variants numbers (in chronological order) (based on GenBank data) plasmid-mediated

Escherichia coli
BIL-1 X74512 1993 United Kingdom (Pakistan) Plasmid
FOX-2 Y10282 1996 Germany (Guatemala) Plasmid
LAT-3 Y15411 1997 Greece Plasmid
LAT-4 Y15412 1997 Greece Plasmid
CMY-3 Y16783 1998 Plasmid
CMY-4 AJ007826 1998 United Kingdom Plasmid
CMY-7 AJ011291 1998 India Plasmid
CMY-6 AJ011293 1998 India Plasmid
FOX-4 AJ277535 2000 Spain Plasmid
CMY-9 AB061794 2001 Japan Plasmid
CMY-11 AF381626 2001 Korea Plasmid
CMY-13 AY339625 2003 Greece Plasmid
CMY-18 AY743434 2004 Korea Plasmid
CMY-21 DQ139328 2005 United Kingdom Plasmid
CMY-22 DQ256079 2005 China Plasmid
CMY-23 DQ438952 2006 United Kingdom Plasmid
CMY-24 EF415650 2007 Singapore Plasmid
CMY-28 EF561644 2007 Ireland Plasmid
CMY-29 EF685371 2007 New Zealand Plasmid
CMY-30 EF536872 2007 New Zealand Plasmid
CMY-32 EU496815 2008 United States Plasmid
CMY-33 EU496816 2008 United States Plasmid
DHA-1 2008 India Plasmid
Klebsiella pneumoniae
MOX-1 D13304 1992 Japan Plasmid
MIR-1 M37839 1993 United States Plasmid
FOX-1 X77455 1994 Argentina Plasmid
LAT-1 X78117 1994 Greece Plasmid
CMY-2 X91840 1995 Greece Plasmid
CMY-1 X92508 1995 South Korea Plasmid
ACT-1 U58495 1996 United States Plasmid
LAT-2 S83226 1996 Greece Plasmid
CMY-8 AF167990 1999 Taiwan Plasmid
MOX-2 AJ276453 2000 France (Greece) Plasmid
DHA-2 AF259520 2000 France Plasmid
FOX-6 AY034848 2001 United States Plasmid
CMY-10 AF381618 2001 Korea Plasmid
DHA-3 AY494945 2003 Taiwan Plasmid
FOX-7 AJ703795 2004 Italy Plasmid
CMY-19 AB194410 2004 Japan Plasmid
FOX-5 AY007369 2005 United States Plasmid
CMY-31 EF622224 2007 Switzerland Plasmid
Klebsiella oxytoca
FOX-3 Y11068 1997 Italy Plasmid
CMY-5 Y17716 1998 Sweden Plasmid
CMY-26 AB300358 2007 Japan Plasmid
Enterobacter cloacae
MHN1 X08082 1988 Chromosomal
GC1 D44479 1994 Japan Chromosomal
K992004.1 AF411144 2001 Korea Chromosomal
K995120.1 AF411145 2001 Korea Chromosomal
K99230 AF411146 2001 Korea Chromosomal
K9911729 AF411147 2001 Korea Chromosomal
K9973 AF411148 2001 Korea Chromosomal
K9914325 AF411149 2001 Korea Chromosomal
Salmonella enteridis
DHA-1 AJ237702 1999 France Plasmid
Proteus mirabilis
CMY-12 Y16785 1998 France Plasmid
CMY-14 AJ555825 2003 Poland Plasmid
CMY-16 AJ781421 2004 Italy Plasmid
Aeromonas sobria
AER14 ASU10251 1994 United States Chromosomal
CEPS X80277 1994 United Kingdom Chromosomal
Serratia marcescens
SRT-1 AB008454 1997 Japan Chromosomal
SST-1 AB008455 1997 Japan Chromosomal
Hafnia alvei
ACC-1 AJ270941 2000 France (Saudi Arabia) Plasmid
ACC-2 AF180952 1999 France Plasmid
ACC-3 AF180958 1999 France Plasmid
ACC-4 EF504260 2007 Greece Plasmid

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
44 Review in Medical Microbiology 2009, Vol 20 No 3
enzyme did not match with known class C b-lactamases
and hence they proposed a uniform designation for this
family of cephalosporinases, ADC (Acinetobacter-derived
cephalosporinase) and identified enzyme as ADC-7 [29].
In 2006, Wachino et al. [30] suggested that the genetic
environment of bla CMY-19 was identical to that of bla
CMY-9 and a single amino acid substitution, I 292 S,
adjacent to the H-10 helix region was observed between
CMY-9 and CMY-19. This substitution was suggested to
be responsible for the expansion of hydrolyzing activity
against several broad-spectrum cephalosporins. The
chronology of discovery of AmpC b-lactamases is
provided in Table 1.
Origin and evolution of AmpC
b-lactamases
By observing phylogeny, it was determined that the ampC
gene has been mobilized at least six times [31]. The LAT
alleles and most of CMYalleles were descended from a C.
freundii ampC gene. This group includes CMY-3b, which
was found in the chromosome of P. mirabilis [32] and must
have entered that chromosome by horizontal transfer
from C. freundii. ACT-1 and MIR-1 descended from an
ampC gene of E. cloacae, whereas, DHA-1 and DHA-2
were descended from M. morganii ampC [33,34]. An M.
morganii chromosomal ampC allele that was identical to
plasmid-borne DHA-1 allele had been reported [34],
suggesting that the ampC gene from M. morganii has been
mobilized twice. ACC-1 was descended from H. alvei
ampC. The FOX alleles were descended from an
Aeromonas sobria ampC gene. CMY-1, CMY-8, and
CMY-9 and the MOX alleles were also mobilized from
the Aeromonadaceae group. The phylogeny showed that
although the ampC gene had been mobilized at least six
times, with the exception of Morganella, it had only been
mobilized once from any given species [31]. Hence, it was
concluded that when the antibiotic resistance gene from
any given species was mobilized, it is less likely that the
gene will be successfully mobilized again from that species
than from a different species. Once a gene has been
mobilized and is free to move horizontally, it will spread
through closely related members of that species and
perhaps other species [31].
Epidemiology of plasmid-mediated AmpC
enzymes
The ampC genes are widely distributed among the
Enterobacteriaceae [35]. In typical E. coli and Shigella
species, ampC is expressed at such low levels that deletion
of ampC does not affect the sensitivity to b-lactams, but in
C. freundii and E. cloacae, ampC is inducible both by
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
penicillins, such as ampicillin, and by cephalosporins [35].
Because of their location on the host chromosome, ampC
genes were not initially subjected to the rapid dissemina-
tion allowed by the horizontal transmission of plasmid-
borne genes. A survey of 20 US hospitals showed that
plasmid-borne ampC genes were more prevalent than
TEM-type ESBL genes, although they were less common
than SHV-type ESBLs (G.A. Jacoby, P. Han, M. Alvarez,
and F. Tenover, Program Abstract of the 35th Interscience
Conference on Antimicrobial Agents and Chemotherapy,
abstract C49, 1995).
pMAmpC b-lactamases have been discovered most
frequently in isolates of K. pneumoniae and also in
naturally ocurring AmpC
species like Klebsiella oxytoca,
Salmonella spp., and P. mirabilis. E. coli can also increase
production of its normally weakly expressed chromoso-
mal AmpC promoter or attenuator with consequent
enhanced gene expression [36,37]. Plasmid-determined
cephalosporinase have been found in Africa (Algeria,
Tunisia), Asia (India, Japan, Pakistan, South Korea),
Europe (France, Germany, Greece, Italy, Sweden, United
Kingdom), the Middle East (Saudi Arabia), North
America (United States), and South and Central
Americas (Argentina, Guatemala).
Several geographic clusters have been described [38].
These include a North American cluster (of MIR-1 and
ACT-1), a Central and South American cluster (of FOX-
1 and FOX-2), and an Asian cluster (of CMY-2, CMY-2b,
LAT-1, and LAT-2). Just as with strains producing ESBLs,
travel and transfer of patients has allowed importation of
BIL-1 (CMY-2) enzymes from the Indian subcontinent
to London [39], several CMY types (CMY-2, CMY-6,
and CMY-7) from Punjab (India) to London [15], CMY-
2 from Algeria to France [40], CMY-4 from India to
Sweden, FOX-2 from Guatemala to Germany, ACC-1
from Tunisia to France [41,42], and MOX-2 from Greece
to France (S. Boyer-Mariotte, L. Raskine, B. Hanau, A.
Philippon, M.M. Sanson-LePors, and G. Arlet, Program
Abstract of the 38th Interscience Conference on
Antimicrobial Agents and Chemotherapy, abstract C7,
1998).
As described earlier, CMY-type enzymes are prevalent
and widely distributed [43], whereas DHA-type enzymes
have been isolated less often. CMY-2 is the most prevalent
pMAmpC as well as the most widely distributed
geographically. It has been reported in Algeria, France,
Germany, Greece, India, Pakistan, Taiwan, Turkey,
United Kingdom, and the United States [4,44]. First
DHA-type enzymes were isolated in Saudi Arabia in
1992; then DHA-2-producing strains of K. pneumoniae
were isolated in France [45]. At the end of the 1990s,
DHA-1 was detected in two K. pneumoniae isolates from
California and Florida [7,46], whereas in Taiwan K.
pneumoniae isolates producing DHA-1 were reported
between 1999 and 2001 [47]. DHA-1 was also reported in

Seoul, South Korea. Recently blaDHA-1 has been found in
S. enterica serovar Montevideo in Korea [48] and S. enterica
serotype Seftenberg in United Kingdom [49] and very
recently from an E. coli isolate in India (GenBank
Accession No. EU589460).
Many strains with plasmid-determined AmpC enzyme
also produce TEM-1, TEM-2, SHV-5 [39,50], or even
CTX-M-15 [5].
Relationship of chromosomal and plasmid-
encoded AmpC enzymes
A dendrogram of chromosomal and plasmid-encoded
AmpC enzymes demonstrated the diversity of chromo-
somal AmpC genes and the close relationship of some
plasmid-mediated enzymes to chromosomal enzymes of
particular organisms. The plasmid-mediated enzymes
could be divided into five or six clusters: the C. freundii
group with LAT types and certain CMY types, the
Enterobacter group with MIR-1 and ACT-1, the M.
morganii group with DHA-1 and DHA-2, the H. alvei
group represented by ACC-1, and the Aeromonas group
with MOX-type, FOX-type, and other CMY-type
enzymes [25]. The relationship between plasmid-
encoded enzymes and certain chromosomal b-lactamases
was found to be very close: there was 100% amino acid
homology within the M. morganii and H. alvei groups and
more than 94% homology within the C. freundii group.
ACT-1 and MIR-1 shared 91.4% amino acid identity
with each other but only 85–87% identity with most E.
cloacae AmpC enzymes. However, the enzyme from E.
cloacae strain GN7471 [51] had 91.1% identity to ACT-1
and MIR-1, and an environmental strain of Enterobacter
was 98% identical, so that origin from some Enterobacter
species was likely suggested (M. Rottman and G. Arlet,
personal communication). FOX enzymes have been
reported to have 95% or more sequence identity within
the group, and CMY-1, CMY-8, and CMY-9 are more
than 97% identical, but either group has only about 74%
identity with the available A. sobria AmpC sequences,
which, in turn, differ from each other by more than 25%
[25]. The AmpC sequence of P. aeruginosa is even more
distant, so the origin of these enzymes remains uncertain
[25].
According to PCR and sequencing results, the b-
lactamases of E. cloacae K992004.1 and E. cloacae
K995120.1 were identical to the chromosomal AmpC
b-lactamases of E. cloacae MHN1 [52] and those of E.
cloacae K99230, Enterobacter aerogenes K9911729, E. cloacae
K9973, and E. cloacae K9914325 were similar to that of E.
cloacae MHN1 (98.5, 98.2, and 98.2% identity of amino
acid sequence). E. aerogenes K9911729 was found to be
closely related to CMY-1 and showed 99.7% identity of
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
AmpC b-lactamases – an update Shahid et al. 45
amino acid sequence [53]. Figure 1 shows the clonal
relationship of various plasmid and chromosomal AmpCs
whose sequences were available in GenBank at the time of
writing this review.
Genetic environment of bla AmpC genes
Some ampC genes are located within or near mobilizing
elements, such as transposons or integrons [54]. Integrons
have been implicated in the horizontal transfer of class C
b-lactamases, but not as gene cassettes, as has been seen
for the Ambler class A, B, and D b-lactamases [55]. Many
resistance genes are located on gene cassettes with a
downstream 59-base element that acts as a specific
recombination site for incorporation into integrons [54].
Analysis of published sequences indicates that AmpC
genes found on plasmids are not linked to 59-base
elements. In fact, several ampC genes, such as blaDHA-1,
blaFOX-4, blaCMY-1, blaCMY-8, and blaMOX-1, have been
described in a particular class 1 integron, originally
identified in In6 and In7, which contain two copies of
the 30 conserved segment (30 -CS1 and 30 -CS2) surround-
ing a common region and an antibiotic resistance gene
[25,56–58]. The common regions are always located
downstream of truncated 30 CS of integrons and at the
same site approximately 0.2 kb downstream of the 30 end
of Orf 513. 2.1 kb common regions represent one end of
an undefined transposon, which comprises these unique
sulI-type integrons with resistance gene cassettes. More-
over, a variety of genes conferring resistance to antibiotics
such as broadspectrum b-lactams, chloramphenicol, and
trimethoprim are located immediately downstream of
this putative transposon. Other antibiotic resistant genes
have now been reported adjacent to the common region
of similar integrons, such as qnr in In 37, blaCTX-M-9 in In
60, dfrA10 in In 34, dfrA10 in variant Salmonella genomic
island (SGI1) from S. enterica serovar Agona, blaCMY-9 in
pCMXR1 from E. coli and catA2 in pAr-32 from
Aeromonas salmonicida [59]. It is, therefore, speculated that
these Gram-negative bacteria have accumulated an array
of antibiotic resistance genes through transposition of a
putative transposon, which carries integrons with gene
cassettes conferring resistance to aminoglycosides, into
the vicinity of other classes of resistance genes, effectively
acquiring phenotypes of multidrug resistance. DHA-1
structural and regulatory genes are present in an integron
that includes a site-specific integrase, two copies of
qacED1 SulI, an aad A2 gene for aminoglycoside
resistance with its downstream 59-base element, and orf
341, a postulated recombinase [59]. ampC and ampR
genes from the chromosome of M. morganii [34,60], thus,
appears to have inserted into a complex sul I-type
integron (readers are encouraged to see [59] for a detailed
description of genetic organization of ampC and ampR
genes originating from M. morganii). Nucleotide
sequences of In6 and In7 have now been updated to
46 Review in Medical Microbiology 2009, Vol 20 No 3

Fig. 1. Neighbour joining tree with branch length self-drawn using the Clustal X software based on the sequences of various
plasmid and chromosomal blaampCs available in GenBank.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

contain orf 513, not orf 341 (GenBank Accession
Nos. L06418 and L06822, respectively). Partridge and
Hall [58] showed that CR1 containing open reading
frame (ORF) orf513, CR2 (containing orfA), and CR3
(containing orf2) form a family of genetic elements
involved in the mobilization of resistance genes in Gram-
negative bacteria. It has been proposed that orf513
catalyzes the incorporation of a resistance gene at the 30
end of the common region.
Characterization of genetic environment of E. coli
revealed the presence of plasmid located class 1 integron,
In53. The integrase gene of In53 was found to be
interrupted by an IS26 insertion sequence, which was also
present in 30 CS. Thus, In53 is a truncated integron
located on a composite transposon, named Tn2000
bounded by two IS26 elements in opposite direction [61].
Genes for the AmpC enzymes have been located on
plasmids of sizes varying from 7 to 180 kb [14,62]. A few
of the plasmids have not been self-transmissible but are
transferable by transformation [14,23,63] or mobilization
[50,54,64,65]. Plasmids encoding AmpC enzymes often
carry multiple other resistances, including resistance to
aminoglycosides, chloramphenicol, sulphonamide, tetra-
cycline, trimethoprim, or mercuric ion [3,14,23,66,67].
A plasmid encoding a FOX-type enzyme even carried a
gene for fluoroquinolone resistance [68]. Clinical isolates
often produce other b-lactamases in addition to an AmpC
enzyme. The bla genes may be on different plasmids, but
often they coexist on the same plasmid. For example, the
gene for ACT-1 was found in clinical isolates along with a
pI 5.6 b-lactamase consistent with TEM-10 or TEM-26
and a pI 7.6 enzyme consistent with SHV-1, and on
cloning the ACT-1 gene was found in a 15-kb cluster
with genes for a b-lactamase of pI 5.4, presumably TEM-
1, and pI 7.0, possibly another SHV-type enzyme [3].
Furthermore, an ACT-1 probe hybridized to chromo-
somal DNA of these clinical strains, implying mobility of
the gene by carriage on a transposon. Indirect evidence
suggests that CMY-3 and CMY-4 could be transposon-
mediated as well: CMY-3 because its gene is located on
the chromosome of a species (P. mirabilis) lacking a native
ampC gene [32], and CMY-4 because in E. coli clinical
isolates from London a CMY-4 probe hybridized to both
7 and 45-kb plasmids, a dual location that could be
explained by transposability [14]. The MIR-1 gene is
located near a sequence closely related to an insertion
sequence transposase, but direct attempts to demonstrate
transposability of MIR-1 or BIL-1 (CMY-2) have not
been successful [69,70].
Most plasmid-encoded AmpC b-lactamases, like CMY-
2, CMY-4, and LAT-1, lack the ampR gene. Citrobacter
spp. and Enterobacter spp. possess ampR and ampC genes,
the fumarate operon frdABCD immediately downstream
of the ampR gene, and also outer membrane lipoprotein
blc immediately downstream of the ampC gene [71]. In
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
AmpC b-lactamases – an update Shahid et al. 47
contrast, M. morganii possesses the ampR and ampC genes
but not the fumarate operon [34]; and hybF is substituted
for the fumarate operon upstream from the ampC gene.
Enzymatic properties
pMAmpCs have pIs between 6.4 and 9.4. In strains
containing several b-lactamases, AmpC enzymes can be
identified after isoelectric focusing by differential
inhibition of nitrocefin reactivity with 5 mg of cefox-
itin/ml [23]. Few plasmids were found to carry both
ampR and ampC and were inducible (DHA-1 and DHA-
2), but most pMAmpCs genes expressed constitutively
(MIR-1, which expressed constitutively even in the
presence of complete system for induction) [70]. The
apparent molecular size of mature pMAmpCs vary from
38 to 42 kDa. The amino acid sequence of enzymes
revealed an active site serine in motif Ser-X-X-Lys (X is
any amino acid) at residues 64–67 of the mature protein.
A Lys-Ser/Thr-Gly motif has been found at residues
315–317 and forms the tertiary structure of the active
site. A tyrosine residue at position 150 forms part of class
C typical motif Tyr-X-Asn and is important for catalysis
of b-lactam hydrolysis [72]. The plasmid-mediated
enzymes can be divided into following clusters: C.
freundii group (group with LAT and certain CMY types),
Enterobacter group (group with MIR-1 and ACT-1), M.
morganii group (group with DHA-1 and DHA-2), and H.
alvei group are represented by ACC-1, whereas Aeromonas
group is represented by MOX, FOX, and other CMY
types of enzymes. Very recently, a detailed review
covering their physical and enzymatic properties (includ-
ing enzyme kinetics), structure and essential sites,
regulation, their pumps and porins, has been published
[73]; and, therefore, these parameters have not been
described in detail in the present article.
Susceptibility to different antimicrobials
and mechanisms of resistance
Strains with plasmid-mediated AmpC enzymes were
consistently resistant to the aminopenicillins (ampicillin
or amoxicillin) and carboxypenicillins (carbenicillin or
ticarcillin) and ureidopenicillins (piperacillin), whereas
among penicillins, strains were found to be susceptible only
to amidinocillin or temocillin. AmpC enzymes provided
resistance to cephalosporins in the oxyimino group
(ceftazidime, cefotaxime, ceftriaxone, and cefuroxime)
and 7-a-methoxy group (cefoxitin, cefotetan, and
moxalactam). The enzymes were also active against the
monobactam, aztreonam. Alteration in antibiotic access to
the enzyme can markedly change the susceptibility profile.
The Gram-negative bacteria have outer membrane
48 Review in Medical Microbiology 2009, Vol 20 No 3
proteins (OMPs) and one family of OMPs is a porin, which
forms water-filled channels that permit diffusion of small
hydrophilic solutes. OmpK 36 porins in K. pneumoniae
were found to augment resistance provided by ESBLs and
pMAmpC to include resistance to oxyimino-b-lactams
and carbapenems [74]. Hyperproduction of chromosomal
AmpC together with porin loss in E. coli [75] or porin
deficiency alone in K. pneumoniae can give rise to
cephamycin and oxyimino-b-lactam resistance. According
to Pai et al. [76], pMAmpCs are associated with potentially
fatal errors of false susceptibility in routine susceptibility
tests.
Mechanism of inducibility of AmpC gene
AmpC is inducible through a system involving ampD,
ampG, ampR, and intermediates in peptidoglycan
recycling. ampD encodes a cytosolic N-acetyl-anhydro-
muramyl-L-alanine amidase that hydrolyze 1,6-anhydro-
muropeptides, whereas ampG encodes a trans-membrane
permease and ampR a transcriptional regulator of LysR
family [27,77,78].
During normal bacterial growth, in the absence of b-
lactam, anhydromuropeptides released from bacterial
peptidoglycan in the periplasm are imported into the
cytoplasm by AmpG and hydrolyzed by amidase AmpD,
and the resulting components are then used for synthesis
of murein precursor. One of the precursors, UDP-Mur-
Nac pentapeptide (uridine-pyrophosphoryl-N-acetyl
muramyl- L -alanyl- D -glutamyl-meso-diaminopimelic
acid-D-alanyl-D-alanine) binds to AmpR, causing AmpR
to assume configuration that does not activate ampC
promoter, thereby resulting in low level of ampC
expression. In contrast, in the presence of b-lactam,
anhydromuropeptides accumulate in the cytoplasm and
thus displace UDP-Mur-Nac pentapeptide from its
AmpR-binding site. AmpR thereby assumes an active
configuration and enhances ampC expression. In the
absence of b-lactamase inducer, ampR represses the
synthesis of b-lactamase 2.5-fold, whereas expression
induced 10–200-fold in the presence of b-lactamase
inducer [79]. Mutations in the specific site of ampR work
as an activator of ampC and results in hyperproduction of
AmpC [80,81]. Deletion mutation of the ampR gene
results in a slightly higher level of basal expression of the
C. freundii b-lactamase, but enzyme synthesis can no
longer be induced. Knockout mutations in the ampD
gene result in constitutive hyperproduction of the b-
lactamase, even in the absence of a b-lactam inducer [82].
Until recently, plasmid-encoded ampC genes were
considered noninducible because they lacked the
regulator gene ampR [25]. However, this generalization
is no longer valid, as three inducible plasmid-encoded
AmpC b-lactamases, DHA-1, DHA-2, and ACT-1, have
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
been described and all of them carry the ampR and ampC
genes [33,45,83] The ampC gene of E. coli is normally
expressed at a low level, regulated by a growth rate
dependent attenuation mechanism but not by induction,
because ampR is missing. Owing to mutations, the
expression of a chromosomal gene encoding AmpC
b-lactamase can be increased during therapy with third-
generation cephalosporins, but as to what extent fourth-
generation cephalosporins are affected by this mechanism
is not fully understood. Genetically, promoter changes
[36,37], insertion of IS2 [84], an optimized distance [85]
in the pribnow box (-35 and -10), and the presence of
more than one copy of ampC have been described as
crucial factors for ampC hyperproduction. The presence
of mutations in the promoter region is the mechanism
most frequently reported to be responsible for the
hyperproduction of b-lactamases.
Detection of AmpC producers
The current CLSI documents (formerly NCCLS) do
not indicate the screening and confirmatory tests that
should be used for the detection of AmpC b-lactamases.
However, methods of detecting ESBL-producing bacteria
have been evolving for more than a decade, beginning
with the description of the disk approximation test by
Brun-Buisson et al. [86] and subsequently described by
Jarlier et al. [87]. The three-dimensional test, originally
described by [88] is a more sensitive procedure for the
detection of ESBLs. However, this technique had its own
limitations. The test was modified with the use of a slit
technique with bacterial suspensions and enzyme extracts,
but filling these slits was a fastidious procedure [89–91].
Subsequently, a simple and user-friendly procedure was
developed using punched wells in place of slits [92].
An AmpC disk test was developed by Black et al. [93]
that was found to be a simple, convenient, and accurate
means of detection of pMAmpCs in organisms lacking
chromosomal AmpC b-lactamase. The test accurately
distinguished between cefoxitin insusceptibility caused
by AmpC production and nonb-lactamase mechanism
like reduced outer membrane permeability (porin
mutation), but this test was not able to discriminate
between positive results due to upregulated chromo-
some-mediated AmpC and those due to pMAmpCs.
Later, in the same year, Black et al. [94] described the
diagnostic utility of AmpC b-lactamase inhibitors, LN-
2-128, RO 48–1220 (Fig. 2a), Syn 2190 (Fig. 2b) in
combination with cefotetan or cefoxitin in a disk test for
detection of clinical isolates of Klebsiella spp. producing
pMAmpCs. LN-1220 is a C-3 substituted cephalo-
sporin-derived inhibitor with a broad spectrum of
inhibition and inhibits both class A (TEM and SHV) and
class C (Amp C type) b-lactamases, whereas RO 48-1220
is a 2b alkenyl penicillanic acid sulfone inhibitor also with

Fig. 2. AmpC b-lactamase inhibitors RO 48-1220 (a) and Syn
2190 (b).
a broad spectrum of inhibition and Syn 2190 is a
monobactam derivative containing 1,5-dihydroxy-4-
pyridone as a C-3 side chain and is a potent inhibitor of
class C b-lactamases. The combination of Syn 2190 and
cefotetan was found to have sensitivity of 91% and
specificity and reproducibility of 100% respectively.
Suspected AmpC producers can be studied further for
cephamycin hydrolysis with the three-dimensional
extract test. Cloxacillin can be used to block AmpC
activity selectively after isoelectric focusing. Lack of
inhibition of activity against oxyimino-b-lactams or
cephamycins by clavulanate is indirect evidence for
presence of AmpC enzyme, but some AmpCs were
found unusually susceptible to inhibition by tazobactam
[95].
Partially derepressed strains could be mistaken for AmpC
producers if they are tested in the absence of inducer
cefoxitin. Jacoby et al. [96] have proposed a disc-based
method that could be used to detect various b-lactamases
in E. coli and K. pneumoniae. Recently, a test for AmpC-
type b-lactamases that involves augmentation of the
inhibition zone around ceftazidime and cefotaxime discs
by boronic acid has been proposed by Yagi et al. [97].
Testing is complicated by the fact that the pattern of
resistance may be altered by porin loss [98]. Researchers
have used commercially available boronic acid com-
pounds, APB (3-aminophenyl boronic acid), NPB (3-
nitrophenyl boronic acid), and TPB (2-thiophene
boronic acid) and found APB as most practical candidate
among specific inhibitors of class C b-lactamase because
NPB and TPB were found to have antibacterial activity
by themselves at concentrations of about 300 mg/ml,
leading to misinterpretation of the change in diameter of
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
AmpC b-lactamases – an update Shahid et al. 49
the growth inhibitory zone [99]. Enlargement of zone of
inhibition by more than 5 mm was adopted as positive
result with APB-inhibited ceftazidime and cefotaxime
resistance in AmpC-producing strains. The exceptions
found were ACT-1-producing and MOX-2-producing
strains that failed with ceftazidime plus APB and DHA-1
and FOX-1 producing strains that failed with cefotaxime
plus APB. A positive response to APB indicates pro-
duction of an AmpC b-lactamase, but not necessarily
a plasmid-mediated enzyme, as in E. coli strains, this
phenotype may result also from an overexpression of
chromosomal ampC gene. In 2006, Ruppe et al. [100]
proposed a simple cefoxitin-based double-disc synergy test
(DDST) for specific detection of ACC-1 in members of
Enterobacteriaceae, including natural AmpC producers, in
association with the cloxacillin-based DDST as a first-line
AmpC-type b-lactamase screening test. Application of
combined multiple double-disc tests integrated in routine
antibiogram was considered as reliable and easy approach
by Apfalter et al. [101] to identify the prevalence of
ESBLs in AmpC producers. Some bacterial species may
produce an inducible AmpC b-lactamase, which can be
overlooked by routine susceptibility tests. Cantarelli et al.
[102] described a new test based on the strong inducible
effect of imipenem on AmpC and consequent antagonism
with ceftazidime. Ceftazidime–imipenem antagonism test
(CIAT) can be used to confirm the presence of known,
as well as new, inducible enzymes, among enterobacterial
strains.
Genetic testing can be done by using multiplex PCR (for
AmpC) as described by Perez-Perez and Hanson [103].
One can identify family-specific AmpC b-lactamase genes
through this method, in which six sets of ampC-specific
primers resulting in amplicons that range from 190 to
520 bp that can be distinguished by gel electrophoresis.
This PCR-based assay differentiated multiple genes within
one reaction. WAVE DNA fragment analysis was
performed in order to shorten the time required for
analysis and it increases sensitivity of multiple-gene assay
[103]. WAVE technology is a high-pressure liquid
chromatography-based nucleic acid analysis and peaks
are observed where each peak had a retention time
equivalent to retention time observed in the single template
amplification, and hence amplified PCR products can be
compared with chromatogram obtained in WAVE analysis.
To determine the genetic environment of bla genes,
standard PCR amplification and sequencing analyses were
performed with sets of primers that were designed on the
basis of nucleotide sequence deposited in EMBL/
GenBank/DDBJ databases.
In 2007, Zhu et al. [104] developed Multiplex Asymmetric
PCR (MAPCR)-based Oligonucleotide Microarray for
detection of 10 known ESBLs and pMAmpC b-lactamase
genes in Gram-negative bacteria. MAPCR is based on a
two-round reaction to promote the accumulation of single
stranded amplicon amenable for microarray hybridization
50 Review in Medical Microbiology 2009, Vol 20 No 3
by employing multiple universal unrelated sequence-
tagged primers and elevating annealing temperature at
second round of amplification.
SDS-PAGE whole-cell protein profile analysis was used
for the first time by Costas et al. [105] for typing clinical
isolates of Klebsiella aerogenes. Manchanda et al. [106] used
this technique as an effective adjunct to other methods of
typing of isolates of AmpC-producing K. pneumoniae.
This study also demonstrated the usefulness of SDS-
PAGE and ribotyping as epidemiological typing tech-
niques, the latter being more discriminatory.
Readers are also encouraged to read a recently published
article [73] for details of AmpC detection in clinical
laboratories.
Replicon typing
Understanding the molecular epidemiology of resistance
plasmids has proven to be a complex task due to diversity
and promiscuity of these elements. The relatedness of
these plasmids can be demonstrated either by restriction
fragment pattern analysis or by classification into
incompatibility groups (Inc groups) and replicon (rep)
typing [107]. A PCR-based replicon typing (PBRT)
method has been recently developed and can be easily
applied to a larger number of strains [108]. Replicon
typing of plasmids carrying blaCMY b-lactamase genes
indicates a predominance of T1 and A/C replicons
among blaCMY-carrying plasmids [109]. Inc T1 plasmids
carrying blaCMY genes show some difference with respect
to the additional plasmid-located resistance (mainly
sulfonamide or trimethoprim resistance) or to a capability
of self-transferability. Furthermore, as the blaCMY-2,
blaCMY-4, and blaCMY-21 gene differ from each other by
only one or a few nucleotide substitutions, they could,
therefore, simply be variants of one another and this
evolution could have occurred within the same Inc I1 or
Inc A/C plasmid scaffold by accumulation of single-
point mutations.
The detection of same resistance gene (blaCMY-2) on an
Inc A/C plasmid carrying the same repA gene variant
strongly indicates that the blaCMY- A/C plasmid could
be a successful and widely distributed plasmid circulat-
ing on different continents. Results obtained by PBRT
indicates the high prevalence of some replicons, such as
rep I1 and rep A/C, in association with relevant
extended-spectrum cephalosporin-resistant genes, such
as the blaCMY-2 gene, suggesting a large diffusion of
particular plasmid prevailing in bacterial populations of
the United Kingdom, but also identified in bacterial
populations from other continents. These epidemic
plasmids seem to prevail in different environments and
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
might spread across different bacterial species within the
human host [109].
The association of replicons with specific plasmid-borne
resistance genes opens the possibility of easily detecting
and tracing the diffusion of successful plasmids as well as
detecting the mobilization capability of a resistance gene
among different plasmids.
Regions responsible for the extended
substrate spectrum in class C b-lactamase
Wachino et al. [30] designated the amino acid sequence
from 279 to 287 as H-10 helix, which may be based on
crystal structure of AmpC K-12, but Lee et al. [110] showed
that sequence from 289 to 294 was H-10 (a10) helix, based
on superimposed crystal structure among CMY-10, P99,
and GC1 b-lactamases. There are different positions of
H-10 helix that are related to the extended substrate
spectrum in class C b-lactamase, hence it has been
proposed that the exact region responsible for the extended
substrate spectrum is the R2 loop (residues 289–307)
[111]. The following reasons support the proposal whereby
the exact region is R2 loop in R2 active site:
(1) The R2 loop includes all regions responsible for the
extended substrate spectrum in all reported class C
extended spectrum b-lactamases except HKY 28 [112].
These regions are six amino acid deletion (residues 289–
294) of CHE [113], single amino acid substitution (L
296 H) of AmpC KL, four amino acid deletions
(residues 293–296) of HD [114], three amino acid
deletions (residues 303–305) of CMY-10 [111], single
amino acid substitution (I 292 S) of CMY-19 [29], and
the L 293 P substitution of Ear 2 [115].
(2) Mutations in the R2 loop can change the architecture of
the active site in class C ESBLs. Owing to the deletion in
CMY-10, the R2 loop in R2 active site results in
shortened path of the connection R2 loop between a10
and b11, which induces a nearly 2.5 Å shift of a9 and
a10 relative to adjacent helix a11 in CMY-10 and
opening the gap between a9, a10, and a11 [111];
hence, bulky R2 side chain of extended spectrum
cephalosporins could fit snugly into significantly
widened R2 active site [116].
Contribution of ampC promoter mutations
to cefoxitin resistance in Escherichia coli
The AmpC resistance phenotype in E. coli can be a result
of overexpression of the chromosomal ampC gene,
acquisition of plasmidic ampC, alterations in the
permeability of the cell to cefoxitin or a combination
of these factors. Analysis of cefoxitin-resistant strains has

revealed that ampC promoter and attenuator regions often
carry mutations leading to overproduction of enzymes
[37,117–120]. These AmpC overproducing strains are
not only resistant to ampicillin but also usually have
reduced susceptibilities to expanded-spectrum cephalo-
sporins. Genetically the most common mutations are
those that create a strong promoter that more closely
resembles the E. coli consensus promoter [121] and
mutations that destabilize the attenuator. The lack of
clonal strain dissemination, the high numbers of
mutations in the promoter and attenuater regions, and
the small numbers of acquired AmpC resistance genes
suggest that antimicrobial selective pressure may be
playing a significant role in the potential emergence of
cefoxitin resistance during treatment of a patient.
Analysis of promoter regions in E. coli showed the
presence of two conserved regions, the -35 box and -10
box, also called the pribnow box [121]. For most
promoters, the degree of homology to the -35 consensus
sequence, TTGACA, and the -10 consensus sequence,
TATAAT, is directly related to promoter strength.
Furthermore, the spacing between these two regions
plays a role in promoter strength, with the optimal
distance being 17 bp. Various studies have reported
that variations in the promoter and attenuator regions
of ampC are a mechanism of hyperproduction of
AmpC protein that results in cefoxitin resistance
[37,117,119,120,122]. Changes at position -42 (C!T)
in the ampC promoter were shown to increase
transcription by 43-fold [37] or 18-fold, depending on
the type of transcriptional reporter system used. The
C!T transition at position -42 results in modification of
the transcriptional initiation site of up to 5 bp upstream
from original site [36]. Almost all of promoters harboring
this change also had a corresponding T!A transversion at
position -18, and this change resulted in a new -42 box,
resulting in the formation of a strong promoter as has been
described in cefoxitin-resistant E. coli strains [36,37,122].
Mutations at position -42 and -18 or at position -18 only
were the most common promoter-region variations,
suggesting that these changes may be favoured in situ over
other promoter-region mutation that may affect ampC
expression [123]. Changes in the -35 and -10 boxes or in
the spacer region that result in ampC promoter sequences
more closely resemble the E. coli consensus promoter
sequence [117,119,120,122]. A T!A transversion at
position -32 resulted in the formation of a -35 consensus
box and increased the level of ampC expression [117].
C!T transition at the -11 position resulted in six-fold
increase in promoter strength. A doublet of T!A inserted
between nucleotides -14 and -13 creates a novel 18 bp
spacer region [123].
Jurian et al. [124] first described a transcriptional
attenuator region in the E. coli ampC promoter region.
It was hypothesized that mutations in this region con-
tribute to AmpC overproduction by causing destabil-
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
AmpC b-lactamases – an update Shahid et al. 51
ization of the hairpin structure, resulting in increased
transcription. Insertion of IS elements in the promoter
region causes up-regulation of gene expression by
producing an alternate promoter [84]. In E. coli, insertion
of IS911 in the promoter spacer region resulted in the
possible generation of a stronger -35 sequence and 17 bp
spacer region, thereby creating a hybrid promoter
sequence that more closely resembled the E. coli
consensus promoter sequence. Insertion of IS10 element
between attenuator and the start codon presumably
allowed ampC to be transcribed and also nullified any
effect from attenuator region.
Conclusion
Resistant bacteria are emerging worldwide as a threat to
favorable treatment outcome of common infections in
hospital and community. b-Lactamase production by
Gram-negative (especially members of Enterobacteria-
ceae) and some Gram-positive bacteria is perhaps the
most important resistant mechanism; however, other
mechanisms including porin loss, AmpC b-lactamase
production, or carbapenemase production are also
increasing markedly. AmpC-type resistance in the
hospital setting is not primarily due to the dissemination
of clonal strains or the spread of resistant plasmids but is
due to the emergence of resistant strains in patients,
possibly through selective pressure from antibiotic
prescribing. Therefore, in our opinion, more focused
studies on AmpC b-lactamases and an urgent need by
CLSI to frame guidelines for AmpC detection are
required in the near future.
Acknowledgement
M. Shahid is grateful to Department of Science &
Technology, Ministry of Science & Technology, Govern-
ment of India for the award of ‘Young Scientist Project
Award’ (FT/SR-L-111/2006)
References
1. Sykes RB, Bush K. Physiology, biochemistry and inactivation
of b-lactamases. In: Morin RB, Gorman M, editors. The
chemistry and biology of beta-lactam antibiotics. New York:
Academic Press; 1982. pp. 155–208.
2. Shahid M, Sobia F, Singh A, Malik A, Khan HM, Jonas D,
Hawkey PM. Beta-lactams and beta-lactamase-inhibitors in
current or potential clinical practice: a comprehensive up-
date. Crit Rev Microbiol 2009; 35:81–108.
3. Bradford PA, Urban C, Mariano N, Projan SJ, Rahal JJ, Bush K.
Imipenem resistance in Klebsiella pneumoniae is associated
with the combination of ACT-1, a plasmid-mediated AmpC
b-lactamase, and the loss of an outer membrane protein.
Antimicrob Agents Chemother 1997; 41:563–569.
52 Review in Medical Microbiology 2009, Vol 20 No 3
4. Bauernfeind A, Chong Y, Lee K. Plasmid-encoded AmpC
b-lactamases: how far have we gone 10 years after the
discovery. Yonsei Med J 1998; 39:520–525.
5. Ensor VM, Shahid M, Evans JT, Hawkey PM. Occurrence,
prevalence and genetic environment of CTX-M b-lactamases
in Enterobacteriaceae from Indian Hospitals. J Antimicrob
Chemother 2006; 58:1260–1263.
6. Shahid M, Ensor VM, Hawkey PM. Emergence and dissemina-
tion of Enterobacteriaceae with plasmid-mediated CMY-6 and
CTX-M-15 beta-lactamases in community in North-India.
World J Microbiol Biotechnol 2009; 25:1439–1446.
7. Alvarez M, Tran JH, Chow N, Jacoby GA. Epidemiology of
conjugative plasmid-mediated AmpC b-lactamases in the
United States. Antimicrob Agents Chemother 2004; 48:533–
537.
8. Sanders CC. b-Lactamases of gram-negative bacteria: new
challenges for new drugs. Clin Infect Dis 1992; 14:1089–1099.
9. Philippon A, Labia R, Jacoby G. Extended-spectrum b-lacta-
mases. Antimicrob Agents Chemother 1989; 33:1131–1136.
10. Jacoby GA, Medeiros AA. More extended-spectrum b-lacta-
mases. Antimicrob Agents Chemother 1991; 35:1697–1704.
11. Jacoby GA. Genetics of extended-spectrum b-lactamases. Eur
J Clin Microbiol Infect Dis 1994; 13:2–11.
12. Livermore DM. b-Lactamases in laboratory and clinical
resistance. Clin Microbiol Rev 1995; 8:557–584.
13. Medeiros A. Evolution and dissemination of b-lactamases
accelerated by generations of b-lactam antibiotics. Clin Infect
Dis 1997; 24:S19–S45.
14. Stapleton PD, Shanon KP, French GL. Carbapenem resistance
in Escherichia coli associated with plasmid-determined CMY-
4 b-lactamase production and loss of an outer membrane
protein. Antimicrob Agents Chemother 1999; 43:1206–1210.
15. Cao VT, Arlet G, Ericsson BM, Tammelin A, Courvalin P,
Lambert T. Emergence of imipenem resistance in Klebsiella
pneumoniae owing to combination of plasmid-mediated
CMY-4 and permeability alteration. J Antimicrob Chemother
2000; 46:895–900.
16. Maurelli AT, Fernández RE, Bloch CA, Rode CK, Fasano A.
Black holes and bacterial pathogenicity: a large genomic
deletion that enhances the virulence of Shigella spp. and
enteroinvasive Escherichia coli. Proc Natl Acad Sci U S A
1998; 95:3943–3948.
17. Sanderson KE, Hessel A, Rudd KE. Genetic map of Salmonella
typhimurium, edition VIII. Microbiol Rev 1995; 59:241–303.
18. Morosini MI, Ayala JA, Baquero F, Martı́nez JL, Blázquez J.
Biological cost of AmpC production for Salmonella enterica
serotype Typhimurium. Antimicrob Agents Chemother 2000;
44:3137–3143.
19. Bobrowski MM, Matthew M, Barth PT, Datta N, Grinter NJ,
Jacob AE, et al. Plasmid-determined b-lactamase indistin-
guishable from the chromosomal b-lactamase of Escherichia
coli. J Bacteriol 1976; 125:149–157.
20. Levesque R, Roy PH, Letarte R, Pechère JC. A plasmid-
mediated cephalosporinase from Achromobacter species.
J Infect Dis 1982; 145:753–761.
21. Knothe H, Shah P, Krcmery V, Antal M, Mitsuhashi S. Trans-
ferable resistance to cefotaxime, cefoxitin, cefamandole and
cefuroxime in clinical isolates of Klebsiella pneumoniae and
Serratia marcescens. Infection 1983; 11:315–317.
22. Bauernfeind A, Chong Y, Schweighart S. Extended broad
spectrum b-lactamase in Klebsiella pneumoniae including
resistance to cephamycins. Infection 1989; 17:316–321.
23. Papanicolaou GA, Medeiros AA, Jacoby GA. Novel plasmid-
mediated b-lactamase (MIR-1) conferring resistance to
oxyimino- and a-methoxy b-lactams in clinical isolates of
Klebsiella pneumoniae. Antimicrob Agents Chemother 1990;
34:2200–2209.
24. Payne DJ, Woodford N, Amyes SGB. Characterization of the
plasmid mediated b-lactamase BIL-1. J Antimicrob Chemother
1992; 30:119–127.
25. Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC
type b-lactamases. Antimicrob Agents Chemother 2002;
46:1–11.
26. Bou G, Oliver A, Ojeda M, Monzon C, Beltran J-M. Molecular
characterization of FOX-4, a new AmpC-type plasmid-
mediated b-lactamase from an Escherichia coli strain iso-
lated in Spain. Antimicrob Agents Chemother 2000; 44:2549–
2553.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
27. Nakano R, Okamoto R, Nakano Y, Kaneko K, Okitsu N,
Hosaka Y, Inoue M. CFE-1, a novel plasmid encoded AmpC
b-lactamase with an ampR gene originating from Citrobacter
freundii. Antimicrob Agents Chemother 2004; 48:1151–
1158.
28. Doi Y, Shibata N, Shibayama K, Kamachi K, Kurokawa H,
Yokoyama K, et al. Characterization of a novel plasmid-
mediated cephalosporinase (CMY-9) and its genetic environ-
ment in an E. coli clinical isolate. Antimicrob Agents
Chemother 2002; 46:2427–2434.
29. Hujer KM, Hamza NS, Hujer AM, Perez F, Helfand MS, Bethel
CR, et al. Identification of a new allelic variant of the Acine-
tobacter baumanii cephalosporinase, ADC-7 b-lactamase:
defining a unique family of class C enzymes. Antimicrob
Agents Chemother 2005; 49:2941–2948.
30. Wachino J, Kurokawa H, Suzuki S, Yamane K, Shibata N,
Kimura K, et al. Horizontal transfer of blaCMY- bearing plas-
mids among clinical Escherichia coli and Klebsiella pneumo-
niae isolates and emergence of cefepime-hydrolyzing CMY-
19. Antimicrob Agents Chemother 2006; 50:534–541.
31. Barlow M, Hall BG. Origin and evolution of the AmpC
b-lactamases of Citrobacter freundii. Antimicrob Agents
Chemother 2002; 46:1190–1198.
32. Bret L, Chanal-Claris C, Sirot D, Chaibi EB, Labia R, Sirot J.
Chromosomally encoded AmpC-type beta-lactamase in
a clinical isolate of Proteus mirabilis. Antimicrob Agents
Chemother 1998; 42:1110–1114.
33. Barnaud G, Arlet G, Verdet C, Gaillot O, Lagrane PH, Phillipon
A. Salmonella enteritidis: AmpC plasmid mediated inducible
b-lactamase (DHA-1) with an ampR gene from Morgenella
morganii. Antimicrob Agents Chemother 1998; 42:2352–
2358.
34. Poirel L, Guibert M, Girlich D, Nass T, Nordmann P. Cloning,
sequence analysis, expression, and distribution of ampC-
ampR from Morganella morganii clinical isolates. Antimicrob
Agents Chemother 1999; 43:769–776.
35. Lindberg F, Normark S. Contribution of chromosomal beta-
lactamases to beta-lactam resistance in enterobacteria. Rev
Infect Dis 1986; 8 (Suppl 3):S292–S304.
36. Nelson EC, Elisha BG. Molecular basis of AmpC hyperproduc-
tion in clinical isolates of Escherichia coli. Antimicrob Agents
Chemother 1999; 43:957–959.
37. Caroff N, Espaze E, Gautreau D, Richet H, Reynaud A. Analysis
of the effects of -42 and -32 ampC promoter mutations in
clinical isolates of Escherichia coli hyperproducing AmpC.
J Antimicrob Chemother 2000; 45:783–788.
38. Odeh R, Kelkar S, Hujer AM, Bonomo RA, Schreckenberger
PC, Quinn P. Broad resistance due to plasmid mediated AmpC
b-lactamases in clinical isolates of Escherichia coli. Clin Infect
Dis 2002; 35:140–145.
39. M’Zali FH, Heritage J, Gascoyne-Binzi DM, Denton M, Todd
NJ, Hawkey PM. Transcontinental importation into the UK of
Escherichia coli expressing a plasmid-mediated AmpC-type b-
lactamase exposed during an outbreak of SHV-5 extended-
spectrum b-lactamase in a Leeds hospital. J Antimicrob
Chemother 1997; 40:823–831.
40. Koeck JL, Arlet G, Philippon A, Basmaciogullari S, Thein HV,
Buisson Y, Cavello JD. A plasmid-mediated CMY-2 b-lacta-
mase from an Algerian clinical isolate of Salmonella seften-
berg. FEMS Microbiol Lett 1997; 152:255–260.
41. Girlich D, Karim A, Spicq C, Nordmann P. Plasmid-mediated
cephalosporinase ACC-1 in clinical isolates of Proteus mir-
abilis and Escherichia coli. Eur J Clin Microbiol Infect Dis 2000;
19:893–895.
42. Nadjar D, Rouveau M, Verdet C, Donay J, Herrmann J,
Lagrange PH, et al. Outbreak of Klebsiella pneumoniae
prodicing transferable AmpC-type b-lactamase (ACC-1)
originating from Hafnia alvei. FEMS Microbiol Lett 2000;
187:35–40.
43. Yan JJ, Ko W-C, Wu HM, Tsai SH, Chuang CL, Wu JJ. Complex-
ity of Klebsiella pneumoniae isolates resistant to both cepha-
mycins and extended-spectrum cephalosporins at a teaching
hospital in Taiwan. J Clin Microbiol 2004; 42:5337–5340.
44. Huang F, Chiu C, Wang M, Wu C, Hsieh K, Chiou CC.
Outbreak of dysentery associated with Shigella sonnei: first
report of plasmid-mediated CMY-2-type AmpC b-lactamase
resistance in Shigella sonnei. J Clin Microbiol 2005; 43:2608–
2612.

45. Fortineau N, Poirel L, Nordmann P. Plasmid-mediated and
inducible cephalosporinase DHA-2 from Klebsiella pneumo-
niae. J Antimicrob Chemother 2001; 47:207–210.
46. Moland ES, Black JA, Ourada J, Reisbig MD, Hanson ND,
Thomson KS. Occurrence of newer b-lactamases in Klebsiella
pneumoniae isolates from 24 US hospitals. Antimicrob Agents
Chemother 2002; 46:3837–3842.
47. Yan JJ, Ko W-C, Jung Y-C, Chuang C-L, Wu JJ. Emergence of
Klebsiella pneumoniae isolates producing inducible DHA-1
b-lactamase in a university hospital in Taiwan. J Clin Microbiol
2002; 40:3121–3126.
48. Kim JY, Park YJ, Lee SO, Song W, Jeong SH, Yoo YA, Lee KY.
Case report: bacteremia due to Salmonella enterica serotype
Montevideo producing plasmid-mediated AmpC beta-lacta-
mase (DHA-1). Ann Clin Lab Sci 2004; 34:214–217.
49. Liebana E, Batchelor M, Clifton-Hadley FA, Davies RH,
Hopkins KL, Threlfall EJ. First report of Salmonella isolates
with the DHA-1 AmpC b-lactamase in the United Kingdom.
Antimicrob Agents Chemother 2004; 43:4492.
50. Gazouli M, Tzouvelekis LS, Prinarakis E, Miriagou V, Tzelepi E.
Trasferable cefoxitin resistance in enterobacteria from Greek
hospitals and characterization of a plasmid-mediated group 1
b-lactamase (LAT-2). Antimicrob Agents Chemother 1996;
40:1736–1740.
51. Kuga A, Okamoto R, Inoue M. ampR gene mutations that
greatly increase class C b-lactamase activity in Enterobacter
cloacae. Antimicrob Agents Chemother 2000; 44:561–567.
52. Bush K, Jacoby GA, Medieros AA. A functional classification
scheme for beta-lactamases and its correlation with molecu-
lar structure. Antimicrob Agents Chemother 1995; 39:1211–
1233.
53. Lee SH, Kim JY, Shin SH, An YJ, Choi YW, Jung YC, et al.
Dissemination of SHV-12 and characterization of new AmpC-
type beta-lactamase genes among clinical isolates of Enter-
obacter species in Korea. J Clin Microbiol 2003; 41:2477–
2482.
54. Verdet C, Arlet G, Barnaud G, Lagrange PH, Phillipon A.
A novel integron in Salmonella enterica serovar Enteridis,
carrying the bla(DHA-1) gene and its regulator gene ampR,
originated from Morganella morganii. Antimicrob Agents
Chemother 2000; 44:222–225.
55. Hall RM, Collis CM. Antibiotic resistance in Gram-negative
bacteria: the role of gene cassettes and integrons. Drug Resist
Updates 1998; 1:109–119.
56. Stokes HW, Tomaras C, Parsons Y, Hall RM. The partial 3’-
conserved segment duplications in the integrons In6 from pSa
and In7 from pDGO100 have a common origin. Plasmid 1993;
30:2233–2239.
57. Valentine CR, Heinrich MJ, Chissoe SL, Roe BA. DNA
sequence of direct repeats of the SulI gene of plasmid
pSa. Plasmid 1994; 32:222–227.
58. Partridge SR, Hall RM. In34, a complex In5 family class 1
integron containing orf513 and dfrA10. Antimicrob Agents
Chemother 2003; 47:342–349.
59. Verdet C, Benzerara Y, Gautier V, Adam O, Ould-Hocine Z,
Arlet G. Emergence of DHA-1 producing Klebsiella spp. in the
Parisian region: genetic organization of the ampC and ampR
genes originating from Morganella morganii. Antimicrob
Agents Chemother 2006; 50:607–617.
60. Barnaud G, Arlet G, Danglot C, Phillipon A. Cloning and
sequencing of the gene encoding the AmpC b-lactamase
of Morganella morganii. FEMS Microbiol Lett 1997; 148:
15–20.
61. Naas T, Mikami Y, Imami T, Poirel L, Nordman P. Char-
acterization of In53, a class1 plasmid- and composite
transposon-located integron of Escherichia coli which
carries an unusual array of gene cassettes. J Bactriol 2001;
183:235–249.
62. Horii T, Arakawa Y, Ohta M, Sugiyama T, Wacharotayankun
R, Ito H, Kato N. Characterization of plasmid-borne and
costitutively expressed blaMOX-1 gene encoding AmpC type
b-lactamase. Gene 1994; 139:93–98.
63. Wu SW, Dornbusch K, Kronvall G, Norgren M. Characteriza-
tion and nucleotide sequence of a Klebsiella oxytoca cryptic
plasmid encoding a CMY-type b-lactamase: confirmation that
the plasmid-mediated cephamycinase originated from the
Citrobacter freundii AmpC b-lactamase. Antimicrob Agents
Chemother 1999; 43:1350–1357.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
AmpC b-lactamases – an update Shahid et al. 53
64. Tzouvelekis LS, Tzelepi E, Mentis AF, Tsakris A. Identifica-
tion of a novel plasmid-mediated b-lactamase with chro-
mosomal cephalosporinase characteristics from Klebsiella
pneumoniae. J Antimicrob Chemother 1993; 31:645–654.
65. Gazouli M, Tzouvelekis LS, Vatopoulos AC, Tzelepi E. Trans-
ferable class C b-lactamase in Escherichia coli isolated in
Greek hospitals and characterization of two enzyme variants
(LAT-3 and LAT-4) closely related to Citrobacter freundii
AmpC b-lactamase. J Antimicrob Chemother 1998; 42:419–
425.
66. Bauernfeind A, Stemplinger I, Jungwirth R, Wilhelm R,
Chong Y. Comparative characterization of the cephamyci-
nase blaCMY-1 gene and its relationship with other b-lacta-
mase genes. Antimicrob Agents Chemother 1996; 40:1926–
1930.
67. Bauernfeind A, Wanger S, Jungwirth R, Schneider I, Meyer D.
A novel class C b-lactamase (FOX-2) in Escherichia coli
conferring resistance to cephamycins. Antimicrob Agents
Chemother 1997; 41:2041–2046.
68. Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone re-
sistance from a transferable plasmid. Lancet 1998; 351:797–
799.
69. Fosberry AP, Payne DJ, Lawlor EJ, Hodgson JE. Cloning and
sequence analysis of blaBIL-1, a plasmid-mediated class C
b-lactamase in Escherichia coli BS. Antimicrob Agents
Chemother 1994; 38:1182–1185.
70. Jacoby GA, Tran J. Sequence of the MIR-1 b-lactamases.
Antimicrob Agents Chemother 1999; 43:1759–1760.
71. Bishop RE, Penfold SS, Frost LS, Holtje JV, Weiner JH. Sta-
tionary phase expression of a novel Escherichia coli outer
membrane lipoprotein and its relationship with mammalian
apolipoprotien D. Implications for the origin of lipocalins.
J Biol Chem 1995; 270:23097–23103.
72. Oefner C, D’Arcy A, Daly JJ, Gubernator K, Charnas RL,
Heinze I, et al. Refined crystal structure of b-lactamase from
Citrobacter freundii indicates a mechanism for b-lactam
hydrolysis. Nature 1990; 343:284–288.
73. Jacoby GA. AmpC b-lactamases. Clin Microbiol Rev 2009;
22:161–182.
74. Rice LB. Successful intervention for Gram-negative resistance
to extended-spectrum beta-lactamase antibiotics. Pharma-
cotherapy 1999; 19:120S–128S.
75. Martinez-Martinez L, Conejo MC, Pascual A, Harnandez-
Alles S, Ballesta S, Ramirez De Arellano-Ramos E, et al.
Activities of imipenem and cephalosporins against
clonally related strains of Escherichia coli hyperproducing
chromosomal b-lactamase and showing altered porin
profiles. Antimicrob Agents Chemother 2000; 44:2534–
2536.
76. Pai H, Kang CI, Byeon JH, Lee KD, Park WB, Kim HB, et al.
Epidemiology and clinical features of bloodstream infections
caused by AmpC-type beta-lactamase-producing Klebsiella
pneumoniae. Antimicrob Agents Chemother 2004; 48:3720–
3728.
77. Jacobs C, Frere J-M, Normark S. Cytosolic intermediates for
cell wall biosynthesis and degradation control inducible b-
lactam resistance in Gram-negative bacteria. Cell 1997;
88:823–832.
78. Normark S. b-Lactamase induction in Gram-negative bacteria
is intimately linked to peptidoglycan recycling. Microb Drug
Resist 1995; 1:111–114.
79. Lindberg F, Westman L, Normark S. Regulatory components in
Citrobacter freundii ampC b-lactamase induction. Proc Natl
Acad Sci U S A 1985; 82:4620–4624.
80. Bartowsky E, Normark S. Purification and mutant analysis
of Citobacter freundii AmpR, the regulator of chromo-
somal AmpC b-lactamases. Mol Microbiol 1991; 5:1715–
1725.
81. Bartowsky E, Normark S. Interaction of wild type and mutant
ampR of Citrobacter freundii with target DNA. Mol Microbiol
1993; 10:555–565.
82. Bennett PM, Chopra I. Molecular basis of b-lactamase induc-
tion in bacteria. Antimicrob Agents Chemother 1993; 37:153–
158.
83. Reisbig MD, Hanson ND. The ACT-1 plasmid-encoded AmpC
beta lactamase is inducible: detection in a complex beta-
lactamase background. J Antimicrob Chemother 2002; 49:
557–560.
54 Review in Medical Microbiology 2009, Vol 20 No 3
84. Jaurin B, Normark S. Insertion of IS2 creates a novel ampC
promoter in Escherichia coli. Cell 1983; 32:809–816.
85. Aoyama T, Takanami M, Ohtsuka E, Taniyama Y, Marumoto R,
Sato H, Ikehara M. Essential structure of E. coli promoter:
effect of spacer length between the two consensus sequence
on promoter function. Nucleic Acids Res 1983; 11:5855–
5864.
86. Brun-Buisson C, Legrand P, Philippon A, Montravers F, An-
squer M, Duval J. Transferable enzymatic resistance to third-
generation cephalosporins during nosocomial outbreak of
multiresistant Klebsiella pneumoniae. Lancet 1987; 2:302–
306.
87. Jarlier V, Nicolas MH, Fournier G, Philippon A. Extended
broad-spectrum b-lactamases conferring transferable resis-
tance to newer b-lactam agents in Enterobacteriaceae: hos-
pital prevalence and susceptibility patterns. Rev Infect Dis
1988; 10:867–878.
88. Thomson KS, Mejglo ZA, Pearce GN, Regan TJ. 3-Dimensional
susceptibility testing of beta-lactam antibiotics. J Antimicrob
Chemother 1984; 13:45–54.
89. Vercauteren E, Descheemaeker P, Ieven M, Sanders CC,
Goossens H. Comparison of screening methods for detection
of extended-spectrum beta-lactamases and their prevalence
among blood isolates of Escherichia coli and Klebsiella spp. in
a Belgian teaching hospital. J Clin Microbiol 1997; 35:2191–
2197.
90. Coudron PE, Moland ES, Thomson KS. Occurrence and
detection of AmpC b-lactamases among Escherichia coli,
Klebsiella pneumoniae and Proteus mirabilis isolates at a
Veterans Medical Centre. J Clin Microbiol 2000; 38:1791–
1796.
91. Manchanda V, Singh NP. Occurrence and detection of AmpC
b-lactamases among gram-negative clinical isolates using a
modified three-dimensional test at Guru Tegh Bahadur Hos-
pital, Delhi, India. J Antimicrob Chemother 2003; 51:415–
418.
92. Shahid M, Malik A, Agrawal M, Singhal S. Phenotypic detec-
tion of the extended-spectrum and AmpC b-lactamases by a
new spot inoculation method and modified three-dimen-
sional extract test: comparison with conventional three-
dimensional extract test. J Antimicrob Chemother 2004;
54:684–687.
93. Black JA, Moland ES, Thomson KS. AmpC disk test for detec-
tion of plasmid-mediated AmpC b-lactamases in Enterobac-
teriaceae lacking chromosomal AmpC b-lactamases. J Clin
Microbiol 2005; 43:3110–3113.
94. Black JA, Thomson KS, Buynak JD, Pitout JDD. Evaluation of
b-lactamase inhibitors in disk tests for detection of plasmid-
mediated AmpC b-lactamases in well characterized clinical
strains of Klebsiella spp. J Clin Microbiol 2005; 43:4168–
4171.
95. Babiani GS, Danel F, Munro SD, Micklesen PA, Livermore
DM. Unusual tazobactam-sensitive AmpC b-lactamase from
two Escherichia coli isolates. J Antimicrob Chemother 1998;
41:115–118.
96. Jacoby GA, Walsh KE, Walker VJ. Identification of extended-
spectrum, AmpC, and carbapenem-hydrolyzing b-lactamases
in Escherichia coli and Klebsiella pneumoniae by disk tests.
J Clin Microbiol 2006; 44:1971–1976.
97. Yagi T, Wachino J, Kurokawa H, Suzuki S, Yamane K, Doi Y,
et al. Practical methods using boronic acid compounds for
identification of class C b-lactamase-producing Klebsiella
pneumoniae and Escherichia coli. J Clin Microbiol 2005;
43:2551–2558.
98. Hernandez-Alles S, Conejo MC, Pascual A, Tomas JM, Benedi
VJ, Martinez-Martinez L. Relationship between outer mem-
brane alterations and susceptibility to antimicrobial agents
in isogenic strains of Klebsiella pneumoniae. J Antimicrob
Chemother 2000; 46:273–277.
99. Song W, Bae IK, Lee YN, Lee CH, Lee SH, Jeong SH. Detection
of extended spectrum b-lactamases by using boronic acid
as an AmpC b-lactamase inhibitor in clinical isolates of
Klebsiella spp. and Escherichia coli. J Clin Microbiol 2007;
45:1180–1184.
100. Ruppe E, Bidet P, Verdet C, Arlet G, Bingen E. First detection of
an AmpC b-lactamase in Citrobacter freundii by a new,
simple double-disc synergy test. J Clin Microbiol 2006;
44:4204–4207.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
101. Apfalter P, Assadian O, Daxbock F, Hirschl AM, Rotter ML,
Makristathis A. Extended double disc synergy testing reveals a
low prevalence of extended-spectrum b-lactamases in Enter-
obacter spp. in Vienna, Austria. J Antimicrob Chemother
2007; 59:854–859.
102. Cantarelli VV, Inamine E, Brodt TCZ, Secchi C, Cavalcante BC,
Periera FS. Utility of ceftazidime–imipenem antagonism test
(CIAT) to detect and confirm the presence of inducible AmpC
b-lactamases among Enterobacteriaceae. Braz J Infect Dis
2007; 11:237–239.
103. Perez-Perez FJ, Hanson ND. Detection of plasmid-
mediated AmpC b-lactamase genes in clinical isolates by
using multiplex PCR. J Clin Microbiol 2002; 40:2153–
2162.
104. Zhu L, Zhang Z, Liang D, Jiang D, Wang C, Du N, et al.
Multiplex asymmetric PCR-based oligonucleotide microarray
for detection of drug resistance genes containing single muta-
tions in Enterobacteriaceae. Antimicrob Agents Chemother
2007; 51:3707–3713.
105. Costas M, Holmes B, Sloss LL. Comparison of SDS–PAGE
protein patterns with other typing methods for investigating
the epidemiology of Klebsiella aerogenes. Epidemiol Infect
1990; 104:455–465.
106. Manchanda V, Singh NP, Shamweel A, Eideh HK, Thukral KK.
Molecular epidemiology of clinical isolates of AmpC pro-
ducing Klebsiella pneumoniae. Ind J Med Microbiol 2005;
24:177–181.
107. Couturier M, Bex F, Bergquist PL, Mass WK. Identification and
classification of bacterial plasmids. Microbiol Rev 1988;
52:375–395.
108. Carattoli A, Bertani A, Villa L, Falbo V, Hopkins KL, Threlfall
EJ. Identification of plasmids by PCR-based replicon typing.
J Microbiol Methods 2005; 63:219–228.
109. Hopkins KL, Liebana E, Villa L, Batchelor M, Threlfall EJ,
Carattoli A. Replicon typing of plasmids carrying CTX-M or
CMY b-lactamases circulating among Salmonella and Escher-
ichia coli isolates. Antimicrob Agents Chemother 2006;
50:3203–3206.
110. Lee SH, Lee JH, Heo MJ, Bae IK, Jeong SH, Cha SS. Exact
location of the region responsible for the extended substrate
spectrum in class C b-lactamases. Antimicrob Agents
Chemother 2007; 51:3778–3779.
111. Kim JY, Jung HI, An YJ, Lee JH, Kim SJ, Jeong SH, et al.
Structural basis for the extended substrate spectrum of
CMY-10, a plasmid-encoded class C b-lactamase. Mol Micro-
biol 2006; 60:907–916.
112. Doi Y, Wachino J, Ishiguro M, Kurokawa H, Yamane K,
Shibata N, et al. Inhibitor-sensitive AmpC b-lactamase variant
produced by an Escherichia coli clinical isolate resistant
to oxyiminocephalosporins and cephamycins. Antimicrob
Agents Chemother 2004; 48:2652–2658.
113. Barnaud G, Labia R, Raskine L, Sanson-Le Pors MJ, Philip-
pon A, Arlet G. Extension of resistance to cefepime
and cefpirome associated to a six amino acid deletion in
the H-10 helix of the cephalosporinase of an Enterobacter
cloacae clinical isolate. FEMS Microbiol Lett 2001; 195:
185–190.
114. Mammeri H, Poirel L, Bemer P, Drugeon H, Nordman P.
Resistance of cefepime and cefpirome due to a four amino
acid deletion in the chromosome-encoded AmpC b-lacta-
mase of a Serratia marcescens clinical isolate. Antimicrob
Agents Chemother 2004; 48:716–720.
115. Barnaud G, Benzerara Y, Gravisse J, Raskine L, Sanson-Le Pors
MJ, Labia G, Arlet G. Selection during cefepime treatment of a
new cephalosporinase variant with extended-spectrum resis-
tance to cefepime in an E. aerogenes clinical isolate. Anti-
microb Agents Chemother 2004; 48:1040–1042.
116. Lee SH, Jeong SH, Cha SS. Minimising antibiotic resistance.
Lancet Infect Dis 2005; 5:668–670.
117. Caroff N, Espaze E, Berard I, Richet H, Reynaud A. Muta-
tions in the ampC promoter of Escherichia coli isolates
resistant to oxyiminocephalosporins without extended-
spectrum b-lactamase production. FEMS Microbiol Lett
1999; 173:459–465.
118. Brinas L, Zarazaga M, Saenz Y, Ruiz-Laerra F, Torres C. b-
Lactamases in ampicillin-resiostant Escherichia coli isolates
from foods, humans, and healthy animals. Antimicrob Agents
Chemother 2002; 46:3156–3163.

119. Corvec S, Caroff N, Espaze E, Marraillac J, Reynaud A. 11
mutation in the ampC promoter increasing resistance to b-
lactams in a clinical Escherichia coli strain. Antimicrob Agents
Chemother 2002; 46:3265–3267.
120. Siu LK, Lu P-L, Chen J-Y, Lin FM, Chang S-C. High level of
expression AmpC b-lactamase due to insertion of nucleotides
between -10 and -35 promoter sequences in Escherichia coli
clinical isolates: cases not responsive to extended-spectrum
cephalosporin treatment. Antimicrob Agents Chemother 2003;
47:2138–2144.
121. Hawley DK, McClure WR. Compilation and analysis of
Escherichia coli promoter DNA sequences. Nucleic Acids
Res 1983; 11:2237–2255.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
AmpC b-lactamases – an update Shahid et al. 55
122. Forward KR, Wiley BM, Low DE, McGeer A, Kapala MA,
Kapala MM, Burrows LL. Molecular mechanisms of cefoxitin
resistance in Escherichia coli from Toronto area hospitals.
Diagn Microbiol Infect Dis 2001; 41:57–63.
123. Mulvey MR, Bryce E, Boyd DA, Agostini MO, Land AM, Simor
AE, Paton S, the Canadian Hospital Epidemiology-committae
and Canadian nosocomial Infection Surveillance Program,
Health Canada. Molecular characterization of cefoxitin-
resistant Escherichia coli from Canadian hospitals. Antimicrob
Agents Chemother 2005; 49:358–365.
124. Jurian B, Grundstorm T, Edlund T, Normark S. The E. coli
b-lactamase attenuator mediates growth rate-dependent
regulation. Nature 1981; 290:221–225.