ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2005, p. 1319–1322 Vol. 49, No.

4
0066-4804/05/$08.00⫹0 doi:10.1128/AAC.49.4.1319–1322.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

blaCTX-M Genes in Clinical Salmonella Isolates Recovered from

Humans in England and Wales from
1992 to 2003
M. Batchelor,1 K. Hopkins,2 E. J. Threlfall,2 F. A. Clifton-Hadley,1 A. D. Stallwood,1
R. H. Davies,1 and E. Liebana1*
Department of Food and Environmental Safety, Veterinary Laboratories Agency, Addlestone, Surrey,1 and Laboratory
for Enteric Pathogens, Health Protection Agency, London,2 United Kingdom
Received 11 August 2004/Returned for modification 4 October 2004/Accepted 22 November 2004

Cefotaximases (CTX-M) are a rapidly growing class A ␤-lactamase family that has been found among a wide
range of clinical bacteria. One hundred and six isolates were selected from 278,308 Salmonella isolates based
on resistance to ampicillin and cephalosporins and subjected to further characterization. Fourteen isolates
were blaCTX-M PCR positive, and cefotaxime MICs for these isolates were >16 mg/liter. Furthermore, sequence
analysis revealed the presence of type CTX-M9, -15, or -17 to -18. All 14 isolates presented different PFGE
restriction profiles, although six Salmonella enterica serotype Virchow isolates formed a tight cluster. The
blaCTX-M genetic determinants were present in transferable plasmids of ⬃63, 105, and >148 kb. Plasmid
restriction analysis showed that both horizontal transfer of similar plasmids among different clones and
transfer of genes between different plasmids were likely mechanisms involved in the spread of blaCTX-M genes.
We have found that CTX-M enzymes have emerged in community-acquired infections both linked to foreign
travel and domestically acquired. This is the first report of a CTX-M enzyme in Salmonella in the United
Kingdom. Also, it represents the first report of a blaCTX-M gene in Salmonella enterica serotype Stanley and a
blaCTX-M-15 gene in Salmonella enterica serotypes Anatum, Enteritidis, and Typhimurium.

Antimicrobials are essential for the treatment of invasive
infections caused by Salmonella spp. However, it is well known
that these organisms have been successful at acquiring resis-
tance to a broad range of commonly used antimicrobials (5, 6).
Extended-spectrum cephalosporins and fluoroquinolones are
often used in the treatment of invasive cases of salmonellosis.
However, the utilization of these drugs in treating complicated
Salmonella infections is becoming seriously compromised by
the increasing development of fluoroquinolone and extended-
spectrum cephalosporin resistance. A recent example of this is
treatment failure due to in vivo acquisition of a plasmid con-
taining the blaCTX-M-3 gene by Salmonella enterica serotype
Anatum (12).
Cefotaximases (CTX-M) are class A ␤-lactamases that in
general present higher levels of hydrolytic activity against ce-
fotaxime than against ceftazidime. Ceftazidime MICs for or-
ganisms producing these enzymes are sometimes in the sus-
ceptible range. Many laboratories use ceftazidime resistance
alone as an indicator of extended-spectrum ␤-lactamase pro-
duction. For this reason, CTX-M-producing isolates may be
missed by routine susceptibility testing performed by clinical
microbiology laboratories. CTX-M enzymes comprise a rapidly
growing family distributed both over wide geographic areas
and among a wide range of bacteria of clinical significance.
There are five major CTX-M groups (groups 1, 2, 8, 9, and 25)
(3).
In order to assess the current presence of organisms and
* Corresponding author. Mailing address: Department of Food and
Environmental Safety, Veterinary Laboratories Agency, Addlestone,
Surrey KT153NB, United Kingdom. Phone: 44-1932359582. Fax: 44-
1932357595. E-mail: E.Liebana@VLA.DEFRA.gsi.gov.uk.
genes of concern in England and Wales, the present work
aimed to screen a large collection of Salmonella isolates of
human origin with for the presence of blaCTX-M genes. Subse-
quently, the genes and gene-bearing plasmids of the positive
strains were characterized in detail.
MATERIALS AND METHODS
Isolates and determination of ␤-lactam resistance phenotype. Isolates from
the Salmonella strain collection at the Laboratory for Enteric Pathogens, Health
Protection Agency, Colindale, United Kingdom, from the period 1992 to 2003
(total ⫽ 278,308; 1992, n ⫽ 31,339; 1993, n ⫽ 29,276; 1994, n ⫽ 29,514; 1995, n
⫽ 28,588; 1996, n ⫽ 28,210; 1997, n ⫽ 31,480; 1998, n ⫽ 23,161; 1999, n ⫽ 16,957;
2000, n ⫽ 14,465; 2001, n ⫽ 16,038; 2002, n ⫽ 14,427; 2003, n ⫽ 14,853) were
scrutinized for phenotypes consistent with possible extended-spectrum ␤-lacta-
mase production. These represent all human clinical and food Salmonella iso-
lates submitted to the reference laboratory for England and Wales. Resistance to
antimicrobials was determined using a breakpoint method in isosensitest agar
(13). The breakpoints (in milligrams per liter) for resistance to the ␤-lactams
were as follows: ampicillin (Amp) (high-level resistance), ⬎128; cephalexin
(Lex), ⬎16; cephradine (Rad), ⬎16; cefuroxime (Cxm), ⬎16; ceftriaxone (Cro),
⬎1; and cefotaxime (Ctx), ⬎1. The breakpoints for other antibiotics were as
described previously (13). The full MICs of cefotaxime and ceftazidime were also
determined for the blaCTX-M-positive isolates following previously described
methods (7).
Genetic characterization of blaCTX-M genes. Confirmation of the presence of a
blaCTX-M ␤-lactamase gene was done by PCR with primers CTX-M universal F
(5⬘-CGA TGT GCA GTA CCA GTA A-3⬘) and R (5⬘-TTA GTG ACC AGA
ATC AGC GG-3⬘), designed from the alignment of blaCTX-M sequences repre-
sentative of each group. A 35-cycle program with annealing at 60°C was used,
resulting in a 585-bp amplicon. Identification of the blaCTX-M gene group was
carried out initially by sequencing this short fragment. Specific primers were then
designed for the CTX-M-1 (F, 5⬘-ATG GTT AAA AAA TCA CTG CG-3⬘, and
R, 5⬘-TTA CAA ACC GTC GGT GAC-3⬘) and CTX-M-9 (F, 5⬘-ATG GTG
ACA AAG AGA GTG CAA C-3⬘, and R, 5⬘-TTA CAG CCC TTC GGC GAT
G-3⬘) groups in order to amplify the complete genes. A 30-cycle program with
annealing at 50 or 60°C was used, resulting in 876-bp amplicons. These were
sequenced on both strands by automated PCR cycle sequencing using the Ap-

1319
1320 BATCHELOR ET AL. ANTIMICROB. AGENTS CHEMOTHER.

Chloramphenicol (Chl), gentamicin (Gen), neomycin (Ne), kanamycin (Kan), streptomycin (Str), sulfonamide (Su), spectinomycin (Spt), tetracycline (Tet), trimethoprim (Tmp), furazolidone (Fu), nalidixic acid (Nal),
plied Biosystems Prism 3100-Avant Genetic Analyzer. PCR for detection of In60

Transformation

Transformation
Transferable
sequences was performed with primers IATGR plus 341STOP and O3000SR plus

Conjugation
Conjugation
Conjugation

Conjugation

Conjugation

Conjugation

Conjugation
Conjugation

Conjugation
ISTOPR specific for the novel class 1 integron In60 as described previously (10).
Molecular typing. Preparation of DNA for pulsed-field gel electrophoresis
(PFGE) was as described by the Centers for Disease Control and Prevention (4).

No

No

No
Assessment of transferability of resistance. Conjugations were performed with
a rifampin-resistant recipient, Escherichia coli K-12 20R764, using in-broth meth-
ods (7). Conjugation mixtures were plated on CHROMagar ECC (M-Tech

Foreign travel
Diagnostics) containing rifampin (100 mg/liter) and cefotaxime (1 mg/liter) and

association

Thailand
Pakistan

Minorca
then incubated for 24 and 48 h at 37°C. When transfer was not achieved by

Spain
Spain
Spain
conjugation, transformation experiments were conducted as follows. Plasmid

No
No

No
No

No
No
No
No
DNA was prepared with a QIAGEN high-speed MIDI kit. ElectroMAX DH10B
cells (Invitrogen) were transformed with 60 ng of DNA using a Bio-Rad Gene-
Pulser II electroporator and standard conditions (2 kV; 200 ⍀; 25 ␮F). Trans-
formants were selected on nutrient agar containing 1 mg of cefotaxime/liter after

CTX-M genes
16 h of incubation at 37°C.

17 or 18

17 or 18
TABLE 1. Characterization of blaCTX-M-containing Salmonella isolates from humans in England and Wales
No. of
Plasmid profiling and restriction fragment length polymorphism of plasmid

15

9
9
9
9
9
9

9
15
15
15
9
DNA. Plasmid profiling was carried out according to methods described previ-
ously (7). Transferable plasmids were purified from recipient cells. Purified
plasmid DNA (2 ␮g) was digested with HpaI (Promega), and the resulting
products were separated by electrophoresis on 0.8% agarose gels at 45 V for 20 h
at 20°C.

Cazb MIC

0.75
0.75
(mg/liter)

ⱖ 256
3
1
1
1
1

3
1
ⱖ 256
ⱖ 256
ⱖ 256
2

ciprofloxacin (Cip). All isolates were sensitive to imipenem and cefoxitin when tested by a disk diffusion method according to NCCLS guidelines.
RESULTS
Isolate selection process. The criterion for selection of iso-

(mg/liter)
Ctx MIC
lates was high-level resistance to ampicillin plus at least one of

ⱖ 128
ⱖ 128
32
16
16
32
16
32
ⱖ 128
32
ⱖ 128
ⱖ 128
ⱖ 128
32
the cephalosporins in our panel (Lex, Rad, Cxm, Cro, or Ctx).
One hundred six isolates were selected from the total of
278,308 isolates analyzed based on the criterion and were sub-
sequently subjected to further characterization.
AmpLexRadCxmCroCtxChlGenNeKanStrSuSptTetNalCip
Resistance characterization. The 106 isolates selected were

AmpLexRadCxmCroCtxKanStrSuSptTetTmpFuNalCip
screened by the blaCTX-M universal PCR, and 14 of them were

AmpLexRadCxmCroCtxStrSuSptTetTmpFuNalCip
AmpLexRadCxmCroCtxNeKanStrSuSptTetTmpFu
AmpLexRadCxmCroCtxNeKanStrSuSptTetTmpFu
AmpLexRadCxmCroCtxNeKanStrSuSptTetTmpFu
AmpLexRadCxmCroCtxNeKanStrSuSptTetTmpFu

AmpLexRadCxmCroCtxNeKanStrSuSptTetTmpFu
proven to be positive. Within this group, Salmonella enterica
serotype Virchow isolates were the least resistant (cefotaxime

AmpLexRadCxmCroCtxSuSptTmpFuNalCip
MICs, 16 to 32 mg/liter); the cefotaxime MICs for the remain-
ing isolates were ⱖ128 mg/liter. The patterns of resistance to

AmpLexRadCxmCroCtxGenKanNalCip
Resistance patterna

the different antimicrobials are given in Table 1. Sequencing,

followed by BLAST searches, confirmed the identities of the
different genes, and this information is summarized in Table 1.
The In60 PCRs suggested that serotype Virchow isolates C5,
AmpLexRadCxmCroCtx

AmpLexRadCxmCroCtx

AmpLexRadCxmCroCtx
AmpLexRadCxmCroCtx
C7, C9, and C16 could carry a structure similar to the In60
integron described by Sabate et al. in 2002 (10), characterized
by insertion of IS3000 and the presence of orf513 and orf1005
and a second copy of the 3⬘-end conserved region. However,
full sequencing of these elements is required to fully demon-
strate the identities of these integrons. Serotype Virchow iso-
lates C4, C6, C8, and C11 were positive only by PCR with
primers 341STOP and IATGR, with the expected product of 907
bp, and were negative with the primers ISTOPR and O3000SR.
This suggests the presence of a transposase-like region (orf513)
Sept 01

Sept 97
Sept 97

Sept 99
Sept 01

Sept 03
isolated

Nov 02

Nov 01
Dec 97

Oct 01
Date

Jul 98

Jul 02
Jul 03
Jul 03

normally present in complex sul1-type integrons, but the struc-

ture is different from that of In60 and will be the subject of
future work.
All the isolates investigated presented different PFGE re-
Serotype or phagetype

Typhimurium UNT
Typhimurium UNT

striction profiles, although isolates C4 to C9 formed a cluster

Enteritidis 6a

with 72% similarity. Figure 1 shows the XbaI PFGE finger-

Enteritidis 1
Virchow 19
Virchow 19
Virchow 19
Virchow 19
Virchow 19
Virchow 37

Virchow 31

Virchow 19

prints for the 14 isolates.

Anatum

Caz, ceftazidime.
Stanley

Transferability of resistance. Transconjugants were ob-

tained for nine of the strains. E. coli transformants were ob-
tained for two of the isolates for which transfer by conjugation
was not successful (Table 1).
Isolate

Isolates C1, C10, C12, C13, and C15 transferred a plasmid of

C10
C11
C12
C13
C15
C16
C1
C3
C4
C5
C6
C7
C8
C9

⬃105 kb. Isolates C3, C11, and C16 transferred a plasmid of

VOL. 49, 2005 blaCTX-M IN SALMONELLA 1321

FIG. 1. Image generated by Bionumerics software showing the XbaI PFGE restriction profiles for 14 Salmonella CTX-M-positive isolates. The
sizes (in kilobases) of the fragments generated were calculated by comparison to a serovar Braenderup PulseNet universal control strain (H9812).

⬃63 kb. Isolates C4, C6, and C8 transferred a large low-copy
plasmid of ⬎148 kb. PCR analysis of the transconjugants and
transformants showed that the blaCTXM genetic determinants
were present in the specific plasmids acquired by the recipient
strains.
Restriction analysis of the CTX-M-containing plasmids was
carried out on plasmids extracted from transconjugants or
transformants and showed that two strains (C1 and C13) had
the same plasmid backbone (Fig. 2). These two isolates were of
different serotypes (Anatum and Typhimurium) isolated in
different years (2001 and 2003). The serotype Anatum isolate
was associated with a visit to Pakistan, while the serotype
Typhimurium isolate was acquired domestically. Also, strains
C12 and C13, both of serotype Typhimurium but isolated in
different years (2002 and 2003), showed very closely related
restriction fragment length polymorphism (RFLP) plasmid
patterns differing in only one band. Conversely, plasmids found
in C3 (serotype Stanley), C10 (serotype Enteritidis), C11 (se-
rotype Virchow), C15 (serotype Enteritidis), and C16 (sero-
type Virchow) presented distinct RFLP patterns (Fig. 2). De-
spite several attempts to extract plasmids for RFLP analysis
from isolates C4, C6, and C8, we consistently failed to obtain
sufficient plasmid for further experiments. The fact that these
plasmids were of the same approximate size and carried
blaCTX-M9 downstream of orf513 suggests that the same plas-
mid may be present in the three isolates.
DISCUSSION
The main enzymes found in our clinical Salmonella isolates
belonged to the groups M-1 and M-9. This is in agreement
with enzymes found in other countries in Europe (14). The
first report of CTX-M enzyme in the United Kingdom was
in a clinical isolate of Klebsiella oxytoca with blaCTX-M-9 (1).
blaCTX-M-9 was the most prevalent gene, found in 8 of the 14
isolates investigated in our study, and it was found exclusively
in serotype Virchow. Also belonging to CTX-M group 9, we
found blaCTX-M17-18 in serotype Enteritidis and serotype Stan-
ley. The second group of genes in our study was blaCTX-M-15,
found in three serotypes: serotype Anatum, serotype Typhi-
murium, and serotype Enteritidis. A report from the United
Kingdom of a CTX-M-15 enzyme in E. coli was published in
2001 (8), although it has never been found in Salmonella.
We have noticed that blaCTX-M determinants are often

FIG. 2. Image generated by Bionumerics software showing the HpaI restriction profile for CTX-M-containing plasmids.
1322 BATCHELOR ET AL.
linked to infections in travelers returning to the United King-
dom. The serotype Stanley-infected individual reported trav-
eling to Thailand, and the serotype Anatum-infected individual
reported traveling to Pakistan immediately before contracting
the infection. In addition, there seems to be an association
between traveling to Spain and infection with serotype Vir-
chow harboring blaCTX-M-9. Four of the eight serotype Vir-
chow clinical cases included in our study involved individuals
who reported traveling to Spain. The spread of a specific se-
rotype Virchow clone with blaCTX-M-9 genes in different loca-
tions in Spain during 1997 and 1998 has been reported recently
(11). All of the serotype Virchow isolates characterized in our
study presented different PFGE profiles, suggesting that the
spread of CTX-M-mediated resistance may be due to horizon-
tal transfer rather than to the spread of a single clone. Al-
though different, some of the serotype Virchow PFGE finger-
prints (C4, C6, and C8) were highly related (similarity, 90%),
which may also indicate clonal spread of this resistance. Our
data show that the United Kingdom serotype Virchow strains
were isolated from 1997 to 1999 and in 2001 and 2003. This
demonstrates that development of CTX-M-mediated resis-
tance within this serotype in England and Wales is not a phe-
nomenon isolated in time.
The plasmid analysis showed that horizontal transfer of plas-
mids is a likely mechanism involved in the epidemiology of
CTX-M-mediated resistance. The same backbone plasmids
seem to be circulating among different clones and serotypes of
Salmonella, and in these cases, we have shown that the plas-
mids are transferable by conjugation. A study from Poland has
also shown that the same plasmids can be found in different
species of Enterobacteriaceae (2). However, it has also been
demonstrated that genes that are geographically and tempo-
rally clustered can exist on different plasmids (9, 15). Our
plasmid RFLP data also suggest that transfer of the genes
between different plasmids is also a mechanism that contrib-
utes to dissemination of CTX-M resistance. In our study, we
found serotype Virchow clones with blaCTX-M-9 associated with
a possible In60 class 1 integron in two plasmids of different
sizes and RFLP patterns and blaCTXM-17-18 in two plasmids of
different sizes and RFLP patterns.
In summary, we have found that CTX-M enzymes have
emerged in community-acquired infections both linked to for-
eign travel and domestically acquired (in different regions:
Oxfordshire, London, Swansea, Northamptonshire, Lancash-
ire, Leicestershire, and Lincolnshire). This represents the first
report of a CTX-M enzyme in Salmonella in the United King-
dom. Also, it represents the first published report of a
ANTIMICROB. AGENTS CHEMOTHER.
blaCTX-M gene in serotype Stanley and of a blaCTX-M-15 gene in
serotypes Anatum, Enteritidis, and Typhimurium.
ACKNOWLEDGMENT
This work was funded by the Department for Environment, Food
and Rural Affairs, United Kingdom (project VM02136).
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