M I N I R E V I E W

Anepidemicof plasmids?Disseminationof extended-spectrumcephalosporinases amongSalmonella and otherEnterobacteriaceaeLin-Hui Su1,2, Chishih Chu3, Axel Cloeckaert4 & Cheng-Hsun Chiu2,5

1Department of Clinical Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 2Chang Gung University College of Medicine, Taoyuan, Taiwan;3Department of Microbiology and Immunology, National Chiayi University, Chiayi, Taiwan; 4INRA, UR1282, Infectiologie Animale et Sante Publique,

Nouzilly, France; and 5Department of Pediatrics, Chang Gung Children’s Hospital, Taoyuan, Taiwan

Correspondence: Cheng-Hsun Chiu,

Department of Pediatrics, Chang Gung

Children’s Hospital, 5 Fu-Hsin Street,

Kweishan, Taoyuan 333, Taiwan. Tel.: 1886 3

328 1200, ext.: 8896; fax: 1886 3 328 8957;

e-mail: chchiu@adm.cgmh.org.tw

Received 6 August 2007; revised 29 October

2007; accepted 30 October 2007.

First published online 19 December 2007.

DOI:10.1111/j.1574-695X.2007.00360.x

Editor: Willem van Leeuwen

Keywords

plasmid; antimicrobial resistance; extended-

spectrum b-lactamase; AmpC; Salmonella ;

Enterobacteriaceae .

Abstract

CTX-M- and AmpC-type b-lactamases comprise the two most rapidly growing

populations among the extended-spectrum cephalosporinases. The evolution and

dissemination of resistance genes encoding these enzymes occur mostly through

the transmission of plasmids. The high prevalence of clinical isolates of Enter-

obacteriaceae producing the plasmid-mediated extended-spectrum cephalospor-

inases resembles an epidemic of plasmids, and has generated serious therapeutic

problems. This review describes the emergence and worldwide spread of various

classes of plasmid-mediated extended-spectrum cephalosporinases in Salmonella

and other Enterobacteriaceae, the transfer mechanism of the plasmids, detection

methods, and therapeutic choices.

Introduction

Many Gram-negative bacteria are able to express chromo-

some-mediated b-lactamases, which represent a major

resistance mechanism to b-lactam antibiotics. The first

plasmid-mediated b-lactamase to be discovered, TEM-1,

was described in 1965 (Datta & Kontomichalou, 1965). The

subsequently discovered TEM-2 and TEM-13 were derived

from TEM-1 with a similar hydrolytic spectrum (Jacoby &

Medeiros, 1991). SHV-1 is another widespread plasmid-

mediated b-lactamase; its hydrolytic spectrum of activity is

similar to that of TEM-1, but it achieves better activity

against ampicillin (Bush et al., 1995). SHV-1-related se-

quences can be found on the chromosome of most strains

of Klebsiella pneumoniae (Haeggman et al., 1997). This may

explain the common ampicillin resistance expressed by this

organism, and also suggests that the K. pneumoniae species-

specific chromosomal b-lactamase is the ancestor of plas-

mid-mediated SHVs. In some way, possibly as a result of

environmental selective pressure, the gene was transferred

into a plasmid where it acquired the potential for

horizontal dissemination to other bacterial species. These

early plasmid-mediated b-lactamases are broad-spectrum

b-lactamases capable of hydrolyzing both penicillins and

narrow-spectrum cephalosporins (Bush et al., 1995). How-

ever, the capture of the gene by the plasmid has facilitated

the widespread distribution of such resistance determinants

among many bacterial species, leading to subsequent global

dissemination.

The development of the third-generation, or extended-

spectrum, cephalosporins in the early 1980s was a milestone

in the history of the battle against the antimicrobial resis-

tance of pathogenic bacteria, because these antimicrobial

agents initially were resistant to all known b-lactamases,

which are frequently produced by many Gram-negative

bacteria. However, many hospital epidemics since then have

been described to be related to the spread of multidrug-

resistant bacteria under the selective pressure of extensive

use of extended-spectrum cephalosporins (Bradford, 2001;

Paterson & Bonomo, 2005). The major mechanism for the

FEMS Immunol Med Microbiol 52 (2008) 155–168 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

spread of such resistance among members of the family

Enterobacteriaceae is also associated with the transfer of

resistance plasmids. This review aims to provide an overview

of the emergence and wide dissemination of several major

classes of plasmid-mediated extended-spectrum cephalos-

porinases in Enterobacteriaceae, the mechanisms by which

the resistance plasmids disseminate, and the clinical impact

of the production of these enzymes on laboratory detection

and therapy.

Emergence of extended-spectrumcephalosporinases

The first plasmid-encoded b-lactamase to be defined that is

capable of hydrolyzing the extended-spectrum cephalospor-

ins was SHV-2 (Kliebe et al., 1985). Sequence analysis

indicated that SHV-2 differed from SHV-1 by only one

mutational change, resulting in the replacement of glycine

by serine at codon 238 as well as in a substantial expansion

of its hydrolytic spectrum. Since then, other extended-

spectrum cephalosporin-hydrolyzing b-lactamases have

been discovered and increasingly described in several species

of Enterobacteriaceae (Bradford, 2001; Paterson & Bonomo,

2005). These new b-lactamases have collectively been

termed extended-spectrum b-lactamases (ESBLs).

Historically, however, the first appearance of ESBLs can

be traced back to as early as 1982, when a strain of Klebsiella

oxytoca demonstrating ceftazidime resistance was recovered

in England (Du Bois et al., 1995). The b-lactamase respon-

sible, reported in 1995 as TEM-12, was carried on a plasmid.

The subsequent dissemination of such plasmid-mediated

resistance to extended-spectrum cephalosporins was so fast

that it soon affected several bacterial species. To date, such

reports have been described in many countries, and several

continents have been affected. Plasmid-mediated resistance

is one of the most important problems of antimicrobial

resistance that has been encountered in the past two

decades.

Following the introduction of more b-lactam antibiotics

with greater b-lactamase stability, including the fourth-

generation cephalosporins, carbapenems and monobactams

in the late 1980s, some Gram-negative bacteria, notably

Citrobacter freundii, Enterobacter cloacae, Escherichia coli,

Morganella morganii, Providencia rettgeri, and Serratia mar-

cescens, evolved by mutation to overexpress their chromo-

somal AmpC-type b-lactamases, thus providing resistance

to extended-spectrum cephalosporins and monobactams

(Sanders, 1987; Caroff et al., 2000; Philippon et al., 2002;

Walther-Rasmussen & Høiby, 2002). Worst of all, such

resistance phenotypes soon appeared in other species,

including K. pneumoniae, Proteus, Salmonella and Shigella,

that lack an intrinsic AmpC-type enzyme; the spread of this

resistance was also mediated by plasmids (Chiu et al., 2004;

Huang et al., 2005; Su et al., 2005, 2006; Su & Chiu, 2007;

Wu et al., 2007).

AmpC-type b-lactamases, when encoded by genes origin-

ally located on the chromosome, are inducible via a regula-

tion system, which primarily involves AmpR (Philippon

et al., 2002). Until recently, plasmid-encoded ampC genes

were thought to be noninducible owing to the lack of a

functional ampR gene or the absence of an AmpR binding

site (Philippon et al., 2002). However, plasmid-encoded

blaDHA genes originating from M. morganii are nearly

always mobilized together with ampR (Verdet et al., 2006).

Similar situations are found in other inducible plasmid-

mediated non-DHA ampC genes, including ACT-1 (Reisbig

& Hanson, 2002), CFE-1 (Nakano et al., 2004), and CMY-13

(Miriagou et al., 2004). The mechanism that determines

whether the ampC alone or ampC together with ampR are to

be mobilized from a chromosome onto a plasmid needs

further study.

Classification of extended-spectrumcephalosporinases

Plasmid-mediated extended-spectrum cephalosporinases

can be grossly divided into two groups, ESBLs and AmpC-

type b-lactamases, according to the source of origin,

spectrum of hydrolysis, and susceptibility to b-lactamase

inhibitors. There are currently more than 300 ESBLs that

have been described. To maintain the most up-to-date

information and standardize the nomenclature for this

rapidly growing population, a website (http://www.lahey.

org/studies/webt.htm) hosted by George Jacoby and Karen

Bush has been established and made accessible to the public.

According to the latest revision of the website as of 28

November 2007, the two largest ESBL families are TEM-type

derivatives, which have reached TEM-161, and SHV-type

derivatives, which have reached SHV-105. All of them are

the progeny of classical b-lactamases, TEM-1, TEM-2, or

SHV-1, which are not ESBLs. The early predominance of

TEM- and SHV-type variants among ESBLs appears to

reflect the widespread distribution of their plasmid-bound

ancestor enzymes (TEM-1 and SHV-1) in the 1970s.

In addition to SHV- and TEM-type enzymes, other types

of plasmid-mediated ESBLs have increased dramatically in

recent years. Among them, CTX-M-type b-lactamases re-

present the most rapidly growing group of ESBLs (Bonnet,

2004; Walther-Rasmussen & Høiby, 2004). The first CTX-

M-like ESBL to be discovered, designated FEC-1, was from

a cefotaxime-resistant isolate of E. coli in Japan in 1986

(Matsumoto et al., 1988). In 1989 in Germany, Bauernfeind

et al. (1990) reported the discovery of a non-TEM, non-SHV

ESBL, designated CTX-M-1, from a clinical cefotaxime-

resistant E. coli isolate. Although the appearance of CTX-M

enzymes was only a few years later than that of TEM- or

FEMS Immunol Med Microbiol 52 (2008) 155–168c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

156 L.-H. Su et al.

SHV-type ESBLs, the global expansion of CTX-M-produ-

cing strains was not found until 1995 (Bonnet, 2004;

Walther-Rasmussen & Høiby, 2004). The CTX-M-type

derivatives have now reached CTX-M-69, and only CTX-

M-1 and CTX-M-2 were identified before 1995 (Bauern-

feind et al., 1990, 1992). Owing to the high homology in the

sequences of these b-lactamase genes and the surrounding

genetic environment, the chromosomal cephalosporinases

of Kluyvera spp. are considered to be the progenitors of the

plasmid-encoded CTX-M-type ESBLs (Decousser et al.,

2001; Humeniuk et al., 2002; Poirel et al., 2002).

Other minor ESBL families have also been reported,

including the PER, VEB, TLA, GES, and IBC types that

preferentially hydrolyze ceftazidime (Vahaboglu et al., 1995;

Bauernfeind et al., 1996; Poirel et al., 1999, 2000; Silva et al.,

2000; Girlich et al., 2001; Vourli et al., 2003), and the SFO,

BES, and FEC types that preferentially hydrolyze cefotaxime

(Matsumoto et al., 1988; Matsumoto & Inoue, 1999; Bonnet

et al., 2000). These ESBLs are probably plasmid-encoded

and have been found in a wide range of geographic

locations. Recent reports, however, have indicated that genes

encoding PER-1 can be found inserted into the chromo-

some of isolates in some members of the family Enterobac-

teriaceae (Perilli et al., 2007). VEB, TLA, GES, and IBC genes

also can be found located in integrons (Poirel et al., 1999,

2000; Girlich et al., 2001; Vourli et al., 2003; Szczepanowski

et al., 2004; Bae et al., 2007).

Plasmid-mediated AmpC b-lactamases represent another

large group of extended-spectrum cephalosporinases that

are of great clinical concern. The nomenclature of plasmid-

mediated AmpC b-lactamases is much more complicated

than that of ESBLs (Philippon et al., 2002). On the basis of

amino acid similarities as well as of their putative progenitor

chromosomal enzymes, these plasmid-mediated AmpC b-

lactamases can be classified into distinct genetic clusters

(Perez-Perez & Hanson, 2002; Philippon et al., 2002;

Walther-Rasmussen & Høiby, 2002): the Aeromonas caviae

group with FOX-type enzymes; the Aeromonas spp. group

with MOX types and certain CMY types (CMY-1, -8 to -11,

etc.); the C. freundii group with BIL-, CFE-, LAT-, and some

CMY-type enzymes (CMY-2 to -7, etc.); the E. cloacae group

with ACT and MIR types; the Hafnia alvei group with ACC

types; and the M. morganii group with DHA types. Plasmids

encoding these AmpC b-lactamases were all derived from

the chromosomally encoded AmpC b-lactamases of the

individual representative bacterial species.

Global dissemination

The early appearance of ESBLs in Europe may reflect the fact

that clinical use of extended-spectrum cephalosporins

started in this region. The phenomenon soon proliferated

to become a global epidemic, as it did not take long for

countries in other parts of the world to report the emergence

of various ESBL-producing bacteria. However, the preva-

lence of ESBL production varies significantly among bacter-

ial species and ESBL types as well as among countries and

institutions.

The SENTRY Antimicrobial Surveillance Program has

been established for several years to monitor the prominent

pathogens and antimicrobial resistance patterns of nosoco-

mial and community-acquired infections via a broad net-

work of sentinel hospitals distributed by geographic location.

The most recent reports from the SENTRYdata are summar-

ized in Table 1 and may represent the current global status of

ESBL production among the Enterobacteriaceae (Winokur

et al., 2001; Bell et al., 2003; Nijssen et al., 2004; Hirakata

et al., 2005; Deshpande et al., 2006). In general, the highest

rate of ESBL production was found in K. pneumoniae,

particularly among isolates recovered from eastern and

southern Europe, Latin America, and some countries in the

Asia-Pacific region (Winokur et al., 2001; Nijssen et al., 2004;

Hirakata et al., 2005). Escherichia coli isolates from these

areas also expressed a relatively higher rate of ESBL produc-

tion. Similar findings have been reported from another

large series of antimicrobial susceptibility surveillance

programs – the Meropenem Yearly Susceptibility Test Infor-

mation Collection (MYSTIC) study (Turner, 2004).

Large-scale surveillance, specifically on the prevalence

of plasmid-mediated AmpC b-lactamases among Gram-

negative bacteria, has not been well described until recently.

From a batch of 752 isolates collected over a wide geographic

area in the United States, plasmids encoding AmpC-type

b-lactamases were found in K. pneumoniae (8.5%),

K. oxytoca (6.9%), and E. coli (4%), and 20 of the 70

collection sites in 10 of the 25 states were involved (Alvarez

et al., 2004). AmpC-mediated resistance was found in only

nine clinical isolates of Salmonella from a large collection of

278 308 Salmonella isolates recovered from humans during

the period 1992�2003 in England and Wales (Batchelor

et al., 2005).

Transmission from animal to humans

Antimicrobial resistance occurs in several ways, respectively

or concurrently. Pre-existing susceptible bacteria could

become resistant through horizontal gene transfer origi-

nating from other resistant bacteria. Resistant bacteria could

be selected under antimicrobial pressure from a heteroge-

nous population already existing in the host. A susceptible

host could acquire resistant bacteria from exogenous sources

during antimicrobial therapy. For extended-spectrum

cephalosporinases, the characteristic of being plasmid-

mediated provides an efficient mechanism for the transmis-

sion of resistance. Actually, many epidemiological reports

have demonstrated that common conjugative resistance

FEMS Immunol Med Microbiol 52 (2008) 155–168 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

157Epidemic of plasmids

plasmids as well as the same resistant clones can be found

simultaneously from food animals and humans, suggesting

that transmission of extended-spectrum cephalosporin

resistance between animals and humans does occur (Weill

et al., 2004; Yan et al., 2004; Bertrand et al., 2006).

The transmission can also cross bacterial species borders,

and, moreover, bacteria can spread these resistance determi-

nants across animal species and their environment and

further affect humans through various cryptic routes yet to

be elucidated. Clinical studies have indicated that in vivo

transmission of ceftriaxone resistance among members of

the family Enterobacteriaceae could occur during antimicro-

bial therapy in patients (Su et al., 2003). Using a turkey

model, a recent report demonstrated that a blaCMY-2-carry-

ing conjugative plasmid could be transferred from donor

E. coli to Salmonella enterica serotype Newport and then

further to another serotype of E. coli in the gut (Poppe et al.,

2005). Furthermore, during an outbreak investigation, the

same clonal group of multidrug-resistant E. coli was recov-

ered from clinical samples of hospitalized dogs with extra-

intestinal infections and from rectal swabs of staff working

in a veterinary hospital (Sidjabat et al., 2006). A large field

study demonstrated that the patterns of antimicrobial

resistance were similar among isolates from faecal samples

of farm animals, regardless of the animal species, and farm

environment samples, which were considered to be related

to surface-water contamination with antimicrobial resistant

bacteria (Sayah et al., 2005). The resistant determinants may

subsequently enter human bodies through water consump-

tion and affect the therapeutic value of the associated

antimicrobial agents when they are applied.

Many human antibiotics or their analogues have been

used for therapeutic purposes in veterinary medicine and

as growth promoters in animal feed (Shryock, 2000).

Table 1. Percentage of organisms expressing an ESBL phenotype in the SENTRYAntimicrobial Surveillance Program for various regions and countries in

the past decade

Year(s)

of study

Region

Country

No. of isolates tested and rate (%) of isolates with an ESBL phenotype

References

Total

Escherichia

coli

Klebsiella

pneumoniae

Klebsiella

oxytoca

Proteus

mirabilis

Enterobacter

cloacae�

No. % No. % No. % No. % No. % No. %

1997–1998 Europe 4707 4.9 3325 1.3 767 18.1 215 12.6 400 5.3 Nijssen et al. (2004)

France 1020 1.1 776 0 117 4.3 36 8.3 91 3.3

Spain 799 1.6 581 0.3 104 5.8 38 10.5 76 1.3

Germany 466 1.1 341 0 70 2.9 31 9.7 24 0

Greece 357 12.6 220 2.0 94 38 9 22.2 34 5.9

Italy 320 11.9 219 7.0 40 30.0 16 12.5 45 28.9

Switzerland 303 0.7 228 0.9 47 0 17 0 11 0

Turkey 294 23.8 172 8.0 103 48.5 14 42.9 5 0

The Netherlands 279 2.5 173 0 48 8.3 24 12.5 34 0

Portugal 230 10 145 5.0 69 23.2 0 0 16 0

Poland 217 4.6 159 2.0 33 15.2 7 14.3 18 5.6

Austria 137 0.7 105 0 16 0 8 12.5 8 0

Belgium 117 1.7 84 0 12 0 12 16.7 9 0

1998� 2002 Asia-Pacific 3655 5.9 1738 17.3 250 6.8 338 3.3 587 16 Bell et al. (2003),

Australia 1311 0.5 328 3.7 130 0.8 151 0 178 4 Hirakata et al. (2005)

China 163 24.5 75 30.7 10 30.0 9 0 38 37

Hong Kong 608 14.3 224 11.6 10 0 37 8.1 36 6

Japan 337 2.4 210 10.0 51 5.9 27 3.7 101 3

Philippines 338 5.0 319 21.9 13 38.5 33 0 94 35

Singapore 318 11.3 225 35.6 3 33.3 39 17.9 27 44

South Africa 261 1.5 135 28.1 16 0 27 0 54 20

Taiwan 319 5.6 222 13.5 17 23.5 15 0 59 19

1997� 1999 Western Pacific 1104 7.9 560 24.6 111 1.8 Winokur et al. (2001)

Europe 3822 5.3 946 22.6 442 11.1

Latin America 2026 8.5 897 45.4 196 22.4

United States 4966 3.3 2017 7.6 589 4.9

Canada 1203 4.2 368 4.9 97 3.1

2004 North America 1429 4.5 Deshpande et al.

(2006)

�Data for E. cloacae isolates are from Bell et al. (2003).

FEMS Immunol Med Microbiol 52 (2008) 155–168c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

158 L.-H. Su et al.

Increasing numbers of reports have indicated that this

practice may contribute greatly to the increase of antimicro-

bial resistance in human pathogens through the food chain

and that it should therefore be prohibited (Barton, 1998;

Witte, 1998).

Mechanisms of plasmid transfer

A number of reports have indicated that genes encoding

SHV (Haeggman et al., 1997), CTX-M (Decousser et al.,

2001; Humeniuk et al., 2002; Poirel et al., 2002), and AmpC

b-lactamases (Walther-Rasmussen & Høiby, 2002) were

derived from chromosomal b-lactamase genes of certain

bacterial species. The subsequent horizontal transfer of

plasmids encoding these genes that occurred not just

between organisms of the same species but also, in some

cases, between quite distantly related species is the major

cause of the rapid dissemination of resistance to b-lactam

antibiotics. Mechanisms for the transfer of such resistance

genes can be assigned to three categories: (1) conjugation of

self-transmissible plasmids, genomic islands or elements

(Pembroke & Murphy, 2000); (2) mobilization of a coexist-

ing but physically independent plasmid consisting of an oriT

(origin of transfer) sequence for DNA processing by a

coresident self-transmissible plasmid; and (3) transfer of a

cointegrate formed by the fusion of a conjugative element

to another plasmid or to the bacterial chromosome.

In general, classification of plasmids is based on their

replication mechanisms in each incompatibility group (Sha-

piro, 1977; Lawley et al., 2004), and a PCR-based typing

method will be discussed in the next section. To accomplish

conjugation, conjugative plasmids posses some transfer-

related functions, including an exclusion system located on

the bacterial surface to avoid the redundant transfer between

organisms with the same or related plasmids (Harrison

et al., 1992), and randomly dispersed conjugative pili on

the bacterial surface to provide one organism with multiple

junctions with the neighboring organisms (Lawley et al.,

2002). During mating, bacteria form aggregates, and the

genetic elements then unwind, nick, and transport to the

recipient organism (Wilkens & Lanka, 1993). However,

some plasmids may not be able to conjugate, but they may

be mobilized. Mobilizable plasmids consist of their own nic

or oriT sites and proteins for recognition and cleavage, but

the direction of the complex to the transferosome is

determined by other conjugative elements.

The majority of plasmid-encoded ESBL and AmpC genes

are located on conjugative plasmids (Walther-Rasmussen &

Høiby, 2002, 2004; Rupp & Fey, 2003; Liu et al., 2007;

Wu et al., 2007). Only a few of these genes are found on

nonself-transmissible plasmids, but such plasmids may be

mobilizable (Winokur et al., 2000; Chiu et al., 2004; Liebana

et al., 2004). The resistance genes are usually associated with

insertion sequences (IS) and transposons. The class 1

integron-associated ORF, orf513, has been shown to be

involved in the mobilization of several classes of resistance

genes, including plasmid-encoded ampC and blaCTX-M

genes (Arduino et al., 2002, 2003; Weill et al., 2004; Verdet

et al., 2006; Wachino et al., 2006). Among class 1 integrons, a

group of similar genetic elements, termed common regions

(CRs), are usually found beyond but close to their 30

conserved sequences. CRs demonstrate IS91-like features,

which mobilize adjacent DNA through an atypical transpo-

sition mechanism termed rolling circle replication (Toleman

et al., 2006a). They were thus considered as transferable

elements and renamed ‘insertion sequence CRs’ (ISCRs)

(Toleman et al., 2006a, b). The element orf513 was incorpo-

rated in the first CR element to be discovered, which was

reported in the early 1990s as a DNA sequence of 2154 bp

embedded in the complex class 1 integrons In6 and In7

(Stokes et al., 1993). The orf513-associated CR was thus

called ISCR1 and has since been linked to various resistance

genes (Toleman et al., 2006a, b). One interesting finding is

that ISCR1 has thus far not been associated with the CMY-2

subgroup genes, which are invariably linked to ISEcp1

(Toleman et al., 2006b).

ISEcp1 is also frequently associated with resistant isolates

that express plasmid-mediated CTX-M- or AmpC-type b-

lactamases (Chiu et al., 2004; Giles et al., 2004; Lartigue

et al., 2004; Eckert et al., 2006; Su et al., 2006; Liu et al., 2007;

Wu et al., 2007). Plasmid-encoded CTX-M-type ESBLs have

been shown to originate from chromosomal cephalospor-

inases of Kluyvera spp. (Decousser et al., 2001; Humeniuk

et al., 2002; Poirel et al., 2002), and ISEcp1 appears to play a

key role in mediating such an evolutional change (Bonnet,

2004). Although the mechanism associated with ISEcp1

transposition is not fully understood, a closely related

insertion sequence, ISEcp1B, has been shown to recognize a

variety of similar nucleotide sequences as the right inverted

repeat during a mobilization process and to lead to the

insertion of ISEcp1B at various sites (Poirel et al., 2005). It

is possible that ISEcp1 functions through a similar mechan-

ism and results in the formation of a diverse genetic

environment surrounding various plasmid-borne CTX-M-

type b-lactamase genes (Lartigue et al., 2004; Eckert et al.,

2006; Liu et al., 2007). In addition, typical � 35 and � 10

promoter sequences have been found to be located within

the ISEcp1B upstream of a blaCTX-M-19 gene in a K. pneumo-

niae clinical isolate (Poirel et al., 2003) and within another

ISEcp1-like element in front of a blaCMY-7 gene in a

S. enterica serotype Typhimurium strain (Hossain et al.,

2004). Thus, ISEcp1 or its derivatives may be responsible not

only for the mobilization of plasmid-mediated CTX-M- and

AmpC-type b-lactamase genes but also for their expression,

a phenomenon that explains the frequent association of

ISEcp1 with the two types of resistance genes.

FEMS Immunol Med Microbiol 52 (2008) 155–168 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

159Epidemic of plasmids

In contrast to the diverse genetic environment found in

CTX-M-type genes, however, a number of reports from

different geographic areas all indicated that a conserved

DNA fragment consisting of a specific ISEcp1-blaCMY-2-blc-

sugE structure is distributed among various Salmonella

serotypes and several members of the family Enterobacter-

iaceae (Chiu et al., 2004; Giles et al., 2004; Su et al., 2006;

Su & Chiu, 2007; Wu et al., 2007). A hypothesis was thus

generated that the blaCMY-2 gene may have been dissemi-

nated among various genomic backgrounds through the

transfer of plasmids containing such a conserved DNA

fragment, rather than the resistance gene itself having been

moved to separate plasmids or chromosomes (Giles et al.,

2004; Su et al., 2006). Furthermore, the conserved genetic

structure surrounding the blaCMY-2 gene has been described

in a blaCMY-5-carrying plasmid from K. oxytoca, although

the flanking regions beyond the structure were not explored

(Wu et al., 1999; Giles et al., 2004). Based on the similarity

between this conserved structure and the genetic organiza-

tions flanking the chromosomal ampC gene of C. freundii, it

was postulated that plasmid-mediated CMY-type b-lacta-

mase genes may originate from the chromosomal ampC

gene of C. freundii (Wu et al., 1999). Although the role of

these cotransferred genes in cephalosporin resistance re-

mains unclear, the possible functions of blc (an outer

membrane lipoprotein) and sugE (a member of the small

multidrug resistance gene family encoding multidrug efflux

proteins) indicate that they may be associated with multi-

drug resistance (Chung & Saier, 2002; Campanacci et al.,

2006). Furthermore, the majority of this conserved genetic

structure was found inserted into the same region of a finQ

gene (Chiu et al., 2004; Su et al., 2006), which is a fertility

inhibition gene of the F plasmid (Ham & Skurray, 1989). It

is possible that disruption of the finQ gene may break its

fertility inhibition function and presumptively could restore

the conjugation ability of the plasmid itself. Whether or not

it is through such an efficient mechanism that the blaCMY-2

gene is able to propagate and become relatively more

prevalent than other ampC genes is a question that requires

further study.

Table 2 lists the four plasmids, encoding either blaCTX-M

or blaCMY-2, the complete nucleotide sequences of which

have been fully analysed. These b-lactamase genes are all

linked to the ISEcp1, and the plasmids invariably belong to

the IncI1 or IncI1-like incompatibility group (Table 2).

Generally, the conjugation systems of the plasmids coevolve

with their replicons, as conjugative plasmids of a given

incompatibility group usually share similar, if not identical,

transfer systems. This phenomenon suggests that the plas-

mids encoding extended-spectrum cephalosporinases may

have evolved to be well adapted to the intracellular environ-

ment, along with a property of easy dissemination among

various species of Enterobacteriaceae.

Recent reports also indicate that a variety of widely

disseminated conjugative plasmids are associated with the

spread of CTX-M- and Amp-C-type enzymes. Some of the

plasmids belong to the so-called broad-host-range plasmid

groups, such as IncN (Carattoli et al., 2006; Hopkins et al.,

2006; Novais et al., 2007), IncP1-a (Novais et al., 2006),

IncL/M (Novais et al., 2007), and IncA/C2 (Carattoli et al.,

2006; Novais et al., 2007), and thus their further spread to

other members of Enterobacteriaceae and other bacterial

species is a cause for concern.

Replicon typing of plasmids that carry genesconferring resistance to extended-spectrumcephalosporins

Plasmids with the same replication machinery cannot be

propagated together, and therefore can be classified in

incompatibility groups (Datta & Hughes, 1983; Couturier

et al., 1988). Methods for determining incompatibility

groups described in earlier reports involved conjugation/

transformation and hybridization experiments (Datta &

Hedges, 1971; Couturier et al., 1988) and were quite

laborious and time-consuming. Recently a PCR-based re-

plicon typing method was developed that allowed the rapid

identification of major plasmid incompatibility groups

among Enterobacteriaceae, i.e. FIA, FIB, FIC, HI1, HI2, I1-

Ig, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA (Carattoli et al.,

2005). This PCR-based method has been used to monitor

the dissemination and evolution of resistance plasmids, in

particular those conferring resistance to extended-spectrum

cephalosporins. Thus, in E. coli and Salmonella, CMY-2-type

Table 2. Genetic features of four fully sequenced plasmids encoding blaCTX-Mor blaCMY-2

Species Gene

Plasmid

size (bp) Incompatibility group

b-Lactamase

linked genes

Accession

number Reference

Citrobacter freundii blaCTX-M-3 89 468 IncI1-like plasmid ISEcp1 NC_004464

AF550415

Boyd et al. (2004)

Escherichia coli blaCTX-M-15 92 353 IncFII (R100) plasmid ISEcp1 NC_005327

Salmonella enterica

serotype Choleraesuis

blaCMY-2 138 742 IncI1 (R64)-like plasmid ISEcp1-blaCMY-2-blc-sugE AY509004 Chiu et al. (2005)

Salmonella enterica blaCMY-2 99 331 IncI1 (ColIB-P9)-like plasmid ISEcp1-blaCMY-2-blc-sugE DQ017661

FEMS Immunol Med Microbiol 52 (2008) 155–168c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

160 L.-H. Su et al.

A and CMY-2-type B plasmids have been shown to belong to

the A/C2 and I1 incompatibility groups, respectively (Car-

attoli et al., 2006; Hopkins et al., 2006). These plasmids have

spread in the United States and in Europe. Depending on the

blaCTX-M gene, different CTX-M plasmids have recently

been identified and found to be of the I1, FII, HI2, K, and

N incompatibility groups (Hopkins et al., 2006). These

results demonstrate the association of certain b-lactamase

genes with specific plasmid backbones. Very recently, the

dissemination and persistence of blaCTX-M-9 were shown to

be linked to CR1-containing class 1 integrons linked

to defective transposon derivatives from Tn402 located in

old antibiotic resistance plasmids of the HI2, P1-a, and FI

incompatibility groups (Novais et al., 2006). It was suggested

that the presence of this ESBL gene on broad-host-range

IncP1-a plasmids might contribute to its dissemination to

hosts that are not members of the family Enterobacteriaceae.

Laboratory detection

The rapid and accurate identification of organisms that

express resistance to extended-spectrum cephalosporins

among clinical isolates is a prerequisite for an effective

antimicrobial therapy and the prevention of their further

spread. The problem associated with the detection of such

organisms is that they may appear susceptible to cephalos-

porins in in vitro phenotypic screening of antimicrobial

susceptibility using conventional breakpoints (Paterson &

Bonomo, 2005). Reports have documented that failure rates

are unacceptably high when cephalosporins are used in the

treatment of serious infections caused by organisms able to

produce ESBLs or AmpC-type enzymes (Paterson et al.,

2001; Pai et al., 2004), even with the evidence of in vitro

susceptibility. One explanation for such clinical failure is

that the extended-spectrum cephalosporinases produced

by the organisms are able to hydrolyze the agents in vivo.

Another important factor is the inoculum effect for ESBL-

producers, because the MICs of cephalosporins rise as the

inoculum of ESBL-producing organisms increases (Thom-

son & Moland, 2001). Thus, for infection sites where higher

concentrations of bacterial organisms are present (e.g. those

involved in intra-abdominal abscesses and pneumonia), or

that are difficult to reach with certain antimicrobial agents

(e.g. those involved in endocarditis, meningitis, and osteo-

myelitis), clinical failure of cephalosporin therapy would be

expected even if the serum levels of antibiotics far exceeded

the MIC of the antibiotic when tested in vitro at the

conventional inoculum of 105 CFU mL�1.

In view of these problems, the Clinical and Laboratory

Standards Institute (CLSI, formerly the National Committee

for Clinical Laboratory Standards) has recommended that

ESBL production should be screened for E. coli, K. pneumo-

niae, K. oxytoca, and Proteus mirabilis (CLSI, 2006). A

standard disk diffusion method, or alternatively a dilution

method, has been proposed by the CLSI for screening ESBL-

producing bacteria. Suspected ESBL-producing strains are

selected and subjected to further phenotypic confirmatory

tests (CLSI, 2006).

According to the CLSI guideline, isolates with a positive

phenotypic confirmatory test should be reported as resistant

to all penicillins, cephalosporins (including cefepime, but

excluding cephamycins), and monobactams, no matter what

the original susceptibility of the particular agent is (CLSI,

2006). However, false identification of ESBLs has been

reported for isolates expressing only non-ESBL b-lactamases

as a result of the concurrent outer membrane protein

deficiency or mutations in the promoter sequence (Rice

et al., 2000; Wu et al., 2001). Some of these studies, although

not all, indicated that successful treatment can be achieved

with the use of third-generation cephalosporins (Wu et al.,

2001). On the other hand, false-negative detection may also

occur if the test isolate produces both ESBLs and AmpC-

type b-lactamases, probably because the latter enzymes are

resistant to the inhibition by clavulanate and hence mask the

synergistic effect of cephalosporins and clavulanate against

ESBLs (Steward et al., 2001).

Currently there are no CLSI-recommended tests for detect-

ing AmpC-type b-lactamases. However, several phenotypic

methods for the screening of AmpC-type b-lactamases have

been reported, including the three-dimensional test (Man-

chanda & Singh, 2003), the cefoxitin-agar method (Nasim

et al., 2004), the Hodge test (Yong et al., 2002), and the

double-disk test with various inhibitors or inducers to AmpC

enzymes (Coudron, 2005; Dunne & Hardin, 2005). A recently

reported new method, the AmpC disk test, using filter-paper

disks impregnated with EDTA to detect the presence of

plasmid-mediated AmpC-type b-lactamases, appears to re-

present a sensitive, specific, and convenient method for the

detection of plasmid-mediated AmpC-type b-lactamases in

organisms lacking a chromosomally mediated AmpC enzyme

(Black et al., 2005). A multiplex PCR method for the detection

of plasmid-meditated AmpC b-lactamases has also been

devised (Perez-Perez & Hanson, 2002).

Molecular methods for the identification of ESBL genes,

such as blaSHVs and blaTEMs, usually involve amplification

and sequencing of the target genes. For the much more

diverse blaCTX-Ms, various primer pairs are required to

amplify the target genes from various groups of CTX-M

genes. The subsequent sequencing analysis then leads to the

identification of the corresponding CTX-M genes. As clin-

ical isolates are frequently associated with multiple resis-

tance genes, the PCR/sequencing strategy described here

appears simple but time-consuming. A novel system, which

comprises a dual strategy that first uses multiplex PCR to

detect blaSHVs, blaCTX-M-3-like and blaCTX-M-14-like genes, and

then a modified SHV melting-curve mutation detection

FEMS Immunol Med Microbiol 52 (2008) 155–168 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

161Epidemic of plasmids

method to rapidly differentiate six blaSHV genes (blaSHV-1,

blaSHV-2, blaSHV-2a, blaSHV-5, blaSHV-11, and blaSHV-12), has

been developed (Chia et al., 2005). The system was designed

to detect simultaneously the predominant ESBL genes in

the local area where the study was conducted. For other

areas, where different ESBLs are prevalent, the system can be

easily modified to suit the individual situations.

Antimicrobial therapy

Considering the high prevalance of extended-spectrum

cephalosporinase-producing strains among Enterobacteria-

ceae, selection of empirical antimicrobial therapy for infec-

tions caused by these bacteria should take into account the

possibility that the isolate is an ESBL-producer. The judg-

ment could be based on local susceptibility patterns of the

individual bacteria species. Risk factors for infection with

an ESBL-producing strain, including known colonization

with an ESBL-producing organism, prolonged hospital stay,

accommodation in an intensive care unit or other hospital

area where ESBL-producers are known to be endemic, the

use of invasive medical devices (e.g. urinary catheters,

endotracheal tubes, central venous lines, etc.) for a pro-

longed period, and the use of various antibiotic classes,

including quinolones, trimethoprim-sulfamethoxazole,

aminoglycosides, metronidazole, and third-generation

cephalosporins (Graffunder et al., 2005; Kanafani et al.,

2005; Paterson & Bonomo, 2005), should also be considered.

Critically ill patients with nosocomial K. pneumoniae or

E. coli infections should probably be treated with antibiotics

active against ESBL-producers until the absence of an ESBL

is definitively established.

As mentioned earlier, the use of cephalosporins to treat

serious infections caused by ESBL-producers is associated

with high rates of treatment failure, even in cases that

the in vitro susceptibility has been clearly demonstrated

(Paterson et al., 2001). Regarding cephamycins (for example

cefoxitin, cefotetan, and flomoxef), although the agents are

not liable to hydrolysis by ESBLs, clinical experience with

their use in the treatment of ESBL-producing enterobacter-

ial infections is limited (Paterson et al., 2001). A recent

report indicated that flomoxef might have potential ther-

apeutic effects in treating infections caused by ESBL-produ-

cing organisms (Lee et al., 2006). However, similar to the

results described in early reports regarding the emerging

resistance during therapy with cefoxitin (Pangon et al.,

1989) or cefotetan (Bradford et al., 1997), collateral

damage of prolonged flomoxef therapy could occur and

lead to full resistance to flomoxef and ertapenem and

reduced susceptibility to imipenem (Lee et al., 2007a).

The resistance was attributable to the acquisition of a

plasmid-mediated blaDHA-1 gene and the depletion of

OmpK36 production in an original OmpK35-deficient,

ESBL-producing K. pneumoniae isolate after flomoxef treat-

ment (Lee et al., 2007a).

Carbapenems are the treatment of choice for infections

caused by AmpC- and ESBL-producing Enterobacteriaceae

(Paterson, 2000; Paterson & Bonomo, 2005). Recently, a

large prospective, multi-country study of K. pneumoniae

bacteremia demonstrated that mortality in patients with

infection caused by ESBL-producing K. pneumoniae was

significantly lower when a carbapenem was used compared

with other drug classes (Paterson et al., 2004). The greatest

published experience has been with imipenem, but MICs for

meropenem are only slightly lower than that for imipenem

(Paterson et al., 2000). These two antibiotics may be

interchangeable in the treatment of ESBL-producing entero-

bacterial infections. There is no published clinical experi-

ence with ertapenem. However, if an isolate is susceptible

in vitro to ertapenem, it appears reasonable to use ertape-

nem for the treatment of the infection.

Carbapenem resistance in Gram-negative bacteria could,

however, occur through the production of carbapenem-

hydrolyzing b-lactamases (Walsh et al., 2005), including

serine carbapenemases (class A) (Lomaestro et al., 2006),

metallo-b-lactamases (class B) (Yan et al., 2001), and

oxacillinases (class D) (Poirel et al., 2004). Expression of

plasmid-mediated class C b-lactamases (Bradford et al.,

1997; Cao et al., 2000; Bidet et al., 2005; Kaczmarek et al.,

2006; Lee et al., 2007a, b) or ESBLs (Crowley et al., 2002;

Elliott et al., 2006; Mena et al., 2006; Kim et al., 2007)

paralleling membrane impermeability has also been shown

to confer carbapenem resistance.

Caution must be exercised in the use of cefepime, a

fourth-generation cephalosporin, in infections caused by

AmpC-producing organisms. Novel AmpC-type b-lacta-

mases conferring resistance to this antibiotic have been

increasingly reported (Wachino et al., 2006). Treatment

failure resulting from the emergence of resistance to

cefepime itself or from the acquisition of other ESBL genes

by the isolate, possibly during therapy, has also been

reported (Song et al., 2005). In addition, cefepime is less

reliable for therapy in cases of high-inoculum infections

(Kang et al., 2004).

Fluoroquinolones should be regarded as the second-line

therapy for patients with K. pneumoniae bacteremia that

may result from an ESBL-producer (Paterson, 2000). It

should be noted that the probability of fluoroquinolone

resistance is greater in isolates that are ESBL-producing than

in those that are not (Paterson et al., 2000). Combinations of

b-lactam/b-lactamase inhibitors may appear active in vitro

against ESBL-producing K. pneumoniae but are subject to

rising MICs as the inoculum of infecting organisms rises

(Thomson & Moland, 2001), leading to clinical failures

(Pillay et al., 1998). On the other hand, if other classes

of antibiotics are to be selected as alternative therapeutic

FEMS Immunol Med Microbiol 52 (2008) 155–168c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

162 L.-H. Su et al.

agents, antimicrobial susceptibility testing for these agents

should be determined before use.

Conclusions

The high prevalence of clinical isolates producing plasmid-

mediated extended-spectrum cephalosporinases is an epi-

demic of plasmids, which has generated serious therapeutic

problems. The treatment of infections in hospitalized

patients caused by these resistant bacteria is and will remain

an important medical problem. It is likely that resistance

among various Enterobacteriaceae to extended-spectrum

cephalosporins will continue to increase owing to the wide

dissemination of the resistance plasmids. This situation

gives strong testimony to the resilience of microorganisms

and their ability to adapt to their environment, such that

the rate of increase of resistance will largely depend on the

antimicrobial regimens in use. The ability to treat infections

caused by these resistant organisms successfully demands a

multifaceted approach combining continued research into

and development of novel classes of antimicrobial agents,

more prudent use of existing agents, and an emphasis on

more effective infection control measures.

Acknowledgements

The work performed in Chang Gung Memorial Hospital

and Chang Gung Children’s Hospital on antimicrobial

resistance in Enterobacteriaceae is in part supported by

grants NSC95-2314-B-182A-025 from the National Science

Council, Executive Yuan, Taiwan, and CMRPG350021 from

Chang Gung Memorial Hospital, Taiwan.

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