Among the 1233 Salmonella enterica isolates obtained in two Spanish hospitals, five isolates (0.4%) (serovars: Virchow, four; Livingstone, one) had the phenotype of an extended-spectrum -lactamase (ESBL) producer. The genetic characterization of the ESBL of S. enterica Livingstone revealed a bla SHV-2 gene. The bla CTX-M-10 gene in a phage-related genetic environment was found in one S. enterica Virchow isolate, and the bla CTX-M-9 gene within the In60 integron was found in the three remaining Virchow isolates. These three isolates presented indistinguishable or closely related pulsed-field gel electrophoresis patterns among themselves and also as compared with the two other bla CTX-M-9-containing isolates previously obtained from animals. ESBL production is an emerging mechanism of resistance in S. enterica in the two studied hospitals. © 2009 The Authors Journal compilation © 2009 European Society of Clinical Microbiology and Infectious Diseases

García-Campello, M.

Lantero, M.

Moreno, M.Á.

Riaño, I.

Sáenz, Y.

Torres, C.

Vinué, L.

Zarazaga, M.

Álvarez, P.

AbstractAmong the 1233 Salmonella enterica isolates obtained in two Spanish hospitals, five isolates (0.4%) (serovars: Virchow, four; Livingstone, one) had the phenotype of an extended-spectrum β-lactamase (ESBL) producer. The genetic characterization of the ESBL of S. enterica Livingstone revealed a blaSHV–2 gene. The blaCTX-M-10 gene in a phage-related genetic environment was found in one S. enterica Virchow isolate, and the ЫACTX-M-9 gene within the In60 integron was found in the three remaining Virchow isolates. These three isolates presented indistinguishable or closely related pulsed-field gel electrophoresis patterns among themselves and also as compared with the two other blaCTX-M-9-containing isolates previously obtained from animals. ESBL production is an emerging mechanism of resistance in S. enterica in the two studied hospitals

Elsevier - Publisher Connector 

5. Wey SB, Mori M, Pfaller MA, Woolson RF, Wenzel RP. Hospital-
acquired candidemia: the attributable mortality and excess length of
stay. Arch Intern Med 1988; 148: 2642–2645.
6. Falagas ME, Apostolou KE, Pappas VD. Attributable mortality of can-
didemia: a systematic review of matched cohort and case–control
studies. Eur J Clin Microbiol Infect Dis 2006; 25: 419–425.
7. Ascioglu S, Rex JH, de Pauw B et al. Deﬁning opportunistic invasive
fungal infections in immunocompromised patients with cancer and
hematopoietic stem cell transplants: an international consensus. Clin
Infect Dis 2002; 34: 7–14.
8. Vardakas KZ, Samonis G, Michalopoulos A, Soteriades ES, Falagas
ME. Antifungal prophylaxis with azoles in high-risk, surgical intensive
care unit patients: a meta-analysis of randomized, placebo-controlled
trials. Crit Care Med 2006; 34: 1216–1224.
9. Reagan DR, Pfaller MA, Hollis RJ, Wenzel RP. Characterization of the
sequence of colonization and nosocomial candidemia using DNA ﬁn-
gerprinting and a DNA probe. J Clin Microbiol 1990; 28: 2733–2738.
10. Swoboda SM, Merz WG, Lipsetta PA. Candidemia: the impact of anti-
fungal prophylaxis in a surgical intensive care unit. Surg Infect (Larch-
mt) 2003; 4: 345–354.
11. Blumberg HM, Jarvis WR, Soucie JM et al. Risk factors for candidal
bloodstream infections in surgical intensive care unit patients: the
NEMIS prospective multicenter study. The National Epidemiology of
Mycosis Survey. Clin Infect Dis 2001; 33: 177–186.
12. Yamamura DL, Rotstein C, Nicolle LE, Ioannou S. Candidemia at
selected Canadian sites: results from the Fungal Disease Registry,
1992–1994. Fungal Disease Registry of the Canadian Infectious Dis-
ease Society. CMAJ 1999; 160: 493–499.
13. Cheng MF, Yang YL, Yao TJ et al. Risk factors for fatal candidemia
caused by Candida albicans and non-albicans Candida species. BMC
Infect Dis 2005; 5: 22.
14. McKinnon PS, Goff DA, Kern JW et al. Temporal assessment of Can-
dida risk factors in the surgical intensive care unit. Arch Surg 2001;
136: 1401–1408.
Occurrence of extended-spectrum
b-lactamase-producing Salmonella enterica
in northern Spain with evidence of CTX-M-9
clonal spread among animals and humans
I. Rian˜o1, M. Garcı´a-Campello2, Y. Sa´enz1, P. A´lvarez2,
L. Vinue´1, M. Lantero3, M. A´. Moreno4, M. Zarazaga1 and
C. Torres1
1) A´rea de Bioquı´mica y Biologı´a Molecular, Universidad de La Rioja,
Logron˜o, 2) Servicio de Microbiologı´a, Complejo Hospitalario de Ponteve-
dra, Galicia, 3) Servicio de Microbiologı´a, Hospital Universitario Central de
Asturias, Oviedo and 4) Departamento de Sanidad Animal, Facultad de
Veterinaria, Universidad Complutense de Madrid, Madrid, Spain
Abstract
Among the 1233 Salmonella enterica isolates obtained in two
Spanish hospitals, ﬁve isolates (0.4%) (serovars: Virchow, four;
Livingstone, one) had the phenotype of an extended-spectrum
b-lactamase (ESBL) producer. The genetic characterization of
the ESBL of S. enterica Livingstone revealed a blaSHV-2 gene. The
blaCTX-M-10 gene in a phage-related genetic environment was
found in one S. enterica Virchow isolate, and the blaCTX-M-9 gene
within the In60 integron was found in the three remaining
Virchow isolates. These three isolates presented indistinguish-
able or closely related pulsed-ﬁeld gel electrophoresis patterns
among themselves and also as compared with the two other
blaCTX-M-9-containing isolates previously obtained from animals.
ESBL production is an emerging mechanism of resistance in
S. enterica in the two studied hospitals.
Keywords: CTX-M-9, CTX-M-10, Salmonella enterica, SHV-2
Original Submission: 14 February 2008; Revised Submis-
sion: 9 July 2008; Accepted: 15 July 2008
Editor: L. Peixe
Clin Microbiol Infect 2009; 15: 292–295
10.1111/j.1469-0691.2008.02673.x
Corresponding author and reprint requests: C. Torres, A´rea
de Bioquı´mica y Biologı´a Molecular, Departamento de Agricultura y
Alimentacio´n, Universidad de La Rioja, Madre de Dios 51, 26006
Logron˜o, Spain
E-mail: carmen.torres@unirioja.es
Salmonella enterica is a zoonotic bacterium, transmitted
through the food chain, which causes gastroenteritis and
other types of infection that require, in some cases, the use
of antimicrobial agents. S. enterica isolates harbouring
extended-spectrum-b-lactamases (ESBLs) have emerged
worldwide during the last years, and ESBLs of the CTX-M
type are increasingly reported in Salmonella, as well as in
other members of the Enterobacteriaceae [1,2]. The occur-
rence of different genes encoding CTX-M enzymes within
sul1-type integron structures that facilitate their dissemina-
tion has been previously reported [3,4], although other
genetic environments have also been described for blaCTX-M
genes [5,6]. The objective of this study was to detect and
characterize the ESBLs in S. enterica isolates recovered in
two Spanish hospitals, and also to determine the genetic
environment of ESBL genes and its possible location within
integron structures.
During the period 2000–2004, a total of 1038 clinical
isolates of S. enterica were recovered from unrelated
292 Clinical Microbiology and Infection, Volume 15 Number 3, March 2009 CMI
ª2009 The Authors
Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 286–295
patients in the Complejo Hospitalario of Pontevedra
(a 624-bed institution located in Pontevedra, Galicia, in the
northwest of Spain) (2000–2002, 471 isolates; 2003, 310
isolates; and 2004, 257 isolates). For four of these isolates,
all belonging to serovar Virchow, the MIC of cefotaxime
and/or ceftazidime was ‡2 mg/L and the ESBL screening
test was positive; they were included in this study for
b-lactamase characterization. These isolates were obtained
from ambulatory patients with acute gastroenteritis: one
during the period 2000–2002, one in 2003, and two
in 2004, representing 0.2%, 0.3% and 0.8%, respectively,
of the total isolates in each of the studied periods in this
hospital.
In addition, 195 isolates of S. enterica were recovered dur-
ing 2003 in the Hospital Central of Asturias (a 1357-bed
institution of Oviedo, Asturias, in the north of Spain). For
one isolate (0.5%) (serovar Livingstone), which was recov-
ered from a hospitalized patient with acute gastroenteritis,
the MIC of cefotaxime and/or ceftazidime was ‡2 mg/L and
the ESBL screening test was positive; it was also included in
this study for b-lactamase characterization.
Antimicrobial susceptibility to amoxycillin, amoxycillin–
clavulanic acid, cefotaxime, ceftazidime, cefepime, aztreonam,
imipenem, nalidixic acid, ciproﬂoxacin, gentamicin, amikacin,
tobramycin, streptomycin, fosfomycin, tetracycline, chloram-
phenicol, sulphonamide and trimethoprim–sulphamethoxaz-
ole was determined using disk diffusion and broth
microdilution methods [7]. Escherichia coli ATCC 25922 was
used as a quality control strain. The double-disk synergy test
using amoxycillin–clavulanic acid and cefotaxime or
ceftazidime disks was performed with all isolates to detect
ESBL production [8].
The presence of genes encoding TEM-, SHV-, OXA-,
CTX-M- and CMY-type b-lactamases was studied using spe-
ciﬁc PCRs [9]. All obtained amplicons were sequenced on
both strands, and the sequences were compared with those
included in the GenBank database and at the website http://
www.lahey.org/Studies/, in order to ascribe the speciﬁc type
of b-lactamase gene. The presence of the sul1, sul2 and sul3
genes, associated with sulphonamide resistance, and the
tet(A) and tet(B) genes, associated with tetracycline resis-
tance, were also determined by PCR in the ﬁve isolates [10].
Positive and negative controls from the University of La Rioja
collection were used in all PCRs.
The presence of class 1 integrons as well as the character-
ization of the gene cassettes included in their variable
regions was studied by PCR and sequencing [10]. The PCR
‘primer-walking’ strategy, with a wide variety of primers
based on known sequences [6, 9], was used to amplify over-
lapping fragments, in order to determine the genetic environ-
ment of the blaCTX-M genes.
The ﬁve clinical S. enterica isolates included in this study
represented 0.4% of the total S. enterica isolates obtained in
both hospitals during the studied period. Table 1 shows the
characteristics of the ﬁve ESBL-positive isolates. The genes
encoding both CTX-M-9 and TEM-1b b-lactamases were
detected in three S. enterica serovar Virchow isolates, those
encoding CTX-M-10 and TEM-1b in another serovar
Virchow isolate, and that encoding SHV-2 in the serovar
Livingstone isolate.
The CTX-M-10 b-lactamase was initially described in an
E. coli strain isolated in 1997 in a hospital of Madrid, Spain
[6], and it was later reported in different species of Enterob-
acter and Klebsiella [1,11,12]. This enzyme was found in an
E. coli isolate recovered in 2000 in the north of Spain, and
recently also in an S. enterica serovar Virchow isolate from
the same region [13,14]; no transconjugants of the described
S. enterica strain were obtained after conjugation experi-
ments, and the same result was found (data not shown) with
the S. enterica isolate C683 (Table 1).
The genetic environment of the blaCTX-M-10 gene in the
S. enterica isolate C683, obtained by the PCR primer-walking
TABLE 1. Phenotypes, resistance genes and integron elements detected in the ﬁve extended-spectrum b-lactamase-producing
Salmonella enterica isolates
S. enterica name/
serovar
MIC (mg/L)
Resistance
phenotype
(excluding b-lactams)
Antibiotic resistance genes detected Class integron
AMX AMC CTX CAZ IPM FEP ATM bla Othersa intI1
Gene cassettes
inside VR
C516/Virchow >16 8/4 >128 1 <0.5 >32 4 STR–TET–SUL–SXT–NAL CTX-M-9, TEM-1b tet(A), sul1, sul2 – dfrA16, aadA2
C650/Virchow >16 16/8 >128 1 <0.5 8 2 STR–SUL–SXT–NAL CTX-M-9, TEM-1b sul1, sul2 – dfrA16, aadA2
C651/Virchow >16 8/4 >128 2 <0.5 8 8 STR–TET–SUL–SXT–NAL CTX-M-9, TEM-1b tet(A), sul1, sul2 – dfrA16, aadA2
C683/Virchow >16 <4/2 128 1 <0.5 4 4 SUL–NAL CTX-M-10, TEM-1b – – –
C493/Livingstone >16 <4/2 64 8 <0.5 8 ND STR–TET–SUL SHV-2 tet(A), sul1 – aadA1
AMX, amoxycillin; AMC, co-amoxiclav; CTX, cefotaxime; CAZ, ceftazidime; IPM, imipenem; FEP, cefepime; ATM, aztreonam; STR, streptomycin; TET, tetracycline; SUL, sul-
phonamide; SXT, trimethoprim–sulphamethoxazole; NAL, nalidixic acid; VR, variable region; ND, not determined.
aAntimicrobial resistance genes detected outside of the variable regions of class 1 integrons.
CMI Research Notes 293
ª2009 The Authors
Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 286–295
strategy [6], is shown in Fig. 1. This structure is similar to
that previously reported by Oliver et al. [6] in a blaCTX-M-10-
containing E. coli strain (GenBank accession number
AY598759). This result reveals a linkage of this bla gene to a
phage-related element, in contrast to the truncated ISEcp1
element upstream from the blaCTX-M-10 gene that has been
previously detected in the ﬁrst blaCTX-M-10-containing E. coli
isolate in France [5]. This work provides another example of
the inﬂuence of the genomic environment on local dissemi-
nation of resistance genes, even among different bacterial
genera [6].
The CTX-M-9 group (mainly CTX-M-9 and CTX-M-14
enzymes) is highly represented in Spain and, in the case of
CTX-M-9, is usually linked to class 1 integrons associated
with an ISCR1 element [2,4,15]. For that reason, a wide
variety of primers based on the In60 integron structure
(GenBank accession number AF174129) were used [9,10]
(Fig. 1) in order to explore the genetic environment of the
blaCTX-M-9 gene in the three S. enterica serovar Virchow iso-
lates. Curiously, the intI1 gene was not found by PCR in
any of these three CTX-M-9-producing isolates, even using
other reported primers (data not shown). The genetic envi-
ronment found in the present three clinical blaCTX-M-9-con-
taining isolates was identical to the surrounding region
previously found in S. enterica serovar Virchow isolates of
animal origin [9].
The clonal relationship among the blaCTX-M-9-containing
isolates was studied by pulsed-ﬁeld gel electrophoresis,
following the PulseNet Europe One-day protocol with XbaI
as the restriction enzyme (http://www.pulsenet-europe.org/
docs.htm), and the obtained patterns were found to be indis-
tinguishable from those found in two blaCTX-M-9-containing
S. enterica serovar Virchow isolates previously obtained from
animal samples [9].
All ﬁve isolates of human or animal origin showed indistin-
guishable or closely related patterns, indicating the clonal
relationship among them. This observation is of interest and
suggests a relationship among animal and human blaCTX-M-9-
containing S. enterica isolates that are emerging in both popu-
lations [16]. In addition, it is important to underline that the
observed close clonal relationship of blaCTX-M-9-containing
Salmonella isolates is at variance with the high diversity
among blaCTX-M-9-containing E. coli isolates previously
reported [17–19].
All three blaCTX-M-9-containing isolates showed resistance
to streptomycin, sulphonamide, trimethoprim–sulphamethox-
azole and nalidixic acid, and two of them were also resistant
to tetracycline. In addition to the resistance genes included
in the In60 integron, these isolates also harboured the sul2
gene, in addition to the tet(A) gene in the two tetracycline-
resistant isolates.
The blaSHV-2-containing S. enterica serovar Livingstone iso-
late harboured a class 1 integron that included the aadA1
gene cassette, but the ESBL gene was not found to be associ-
ated with this class 1 integron.
S. enterica isolates producing ESBLs, mainly of the CTX-M
group, seem to be an emerging problem in human medicine.
The detection of closely related or indistinguishable pulsed-
ﬁeld gel electrophoresis patterns among CTX-M-9-producing
S. enterica isolates of both human and animal origin could
indicate the transfer of ESBL-producing S. enterica among
animals and humans. The detection of the blaCTX-M-10 gene
in S. enterica isolates is interesting; this is the second time
that it has been reported in the literature, and its genetic
environment was linked to a phage-related element previ-
ously detected among Enterobacteriaceae. More studies
should be performed in the future to track the evolution of
ESBLs in S. enterica isolates from different environments.
DNA
invertaseorf2 orf3 blactx-m-10
int11 sullaadA2 qacEΔ1dfrA16
orf4 IS4321
transposaseorf8orf7
bla
ctx-m-9orf513
(a)
(b)
Phage related region
5,CS 3,CS
orf3-like orf1005
FIG. 1. Genetic structures detected surrounding: (a) the blaCTX-M-10 gene in Salmonella enterica serovar Virchow isolate C683; and (b) the
blaCTX-M-9 gene in the three remaining S. enterica serovar Virchow isolates (C516, C650 and C651). The discontinued arrow indicates that the
intI1 gene was not detected; open box, the region that shows high similarity to orf3 of Kluyvera ascorbata; d, 59-be; diagonally striped box,
the recombination site attI; c, indicates the speciﬁc positions of the primers used for PCR primer-walking strategy.
294 Clinical Microbiology and Infection, Volume 15 Number 3, March 2009 CMI
ª2009 The Authors
Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 286–295
Transparency Declaration
This work was supported in part by Project SAF2006-14207-
C02. The authors have no conﬂict of interest to declare.
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CMI Research Notes 295
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Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 286–295


Occurrence of extended-spectrum β-lactamase-producing Salmonella enterica in northern Spain with evidence of CTX-M-9 clonal spread among animals and humans 

Repositorio de Universidad de La Rioja

5. Wey SB, Mori M, Pfaller MA, Woolson RF, Wenzel RP. Hospital-acquired candidemia: the attributable mortality and excess length ofstay. Arch Intern Med 1988; 148: 2642–2645.6. Falagas ME, Apostolou KE, Pappas VD. Attributable mortality of can-didemia: a systematic review of matched cohort and case–controlstudies. Eur J Clin Microbiol Infect Dis 2006; 25: 419–425.7. Ascioglu S, Rex JH, de Pauw B et al. Defining opportunistic invasivefungal infections in immunocompromised patients with cancer andhematopoietic stem cell transplants: an international consensus. ClinInfect Dis 2002; 34: 7–14.8. Vardakas KZ, Samonis G, Michalopoulos A, Soteriades ES, FalagasME. Antifungal prophylaxis with azoles in high-risk, surgical intensivecare unit patients: a meta-analysis of randomized, placebo-controlledtrials. Crit Care Med 2006; 34: 1216–1224.9. Reagan DR, Pfaller MA, Hollis RJ, Wenzel RP. Characterization of thesequence of colonization and nosocomial candidemia using DNA fin-gerprinting and a DNA probe. J Clin Microbiol 1990; 28: 2733–2738.10. Swoboda SM, Merz WG, Lipsetta PA. Candidemia: the impact of anti-fungal prophylaxis in a surgical intensive care unit. Surg Infect (Larch-mt) 2003; 4: 345–354.11. Blumberg HM, Jarvis WR, Soucie JM et al. Risk factors for candidalbloodstream infections in surgical intensive care unit patients: theNEMIS prospective multicenter study. The National Epidemiology ofMycosis Survey. Clin Infect Dis 2001; 33: 177–186.12. Yamamura DL, Rotstein C, Nicolle LE, Ioannou S. Candidemia atselected Canadian sites: results from the Fungal Disease Registry,1992–1994. Fungal Disease Registry of the Canadian Infectious Dis-ease Society. CMAJ 1999; 160: 493–499.13. Cheng MF, Yang YL, Yao TJ et al. Risk factors for fatal candidemiacaused by Candida albicans and non-albicans Candida species. BMCInfect Dis 2005; 5: 22.14. McKinnon PS, Goff DA, Kern JW et al. Temporal assessment of Can-dida risk factors in the surgical intensive care unit. Arch Surg 2001;136: 1401–1408.Occurrence of extended-spectrumb-lactamase-producing Salmonella entericain northern Spain with evidence of CTX-M-9clonal spread among animals and humansI. Riaño1, M. Garcı́a-Campello2, Y. Sáenz1, P. Álvarez2,L. Vinué1, M. Lantero3, M. Á. Moreno4, M. Zarazaga1 andC. Torres11) Área de Bioquı́mica y Biologı́a Molecular, Universidad de La Rioja,Logroño, 2) Servicio de Microbiologı́a, Complejo Hospitalario de Ponteve-dra, Galicia, 3) Servicio de Microbiologı́a, Hospital Universitario Central deAsturias, Oviedo and 4) Departamento de Sanidad Animal, Facultad deVeterinaria, Universidad Complutense de Madrid, Madrid, SpainAbstractAmong the 1233 Salmonella enterica isolates obtained in twoSpanish hospitals, five isolates (0.4%) (serovars: Virchow, four;Livingstone, one) had the phenotype of an extended-spectrumb-lactamase (ESBL) producer. The genetic characterization ofthe ESBL of S. enterica Livingstone revealed a blaSHV-2 gene. TheblaCTX-M-10 gene in a phage-related genetic environment wasfound in one S. enterica Virchow isolate, and the blaCTX-M-9 genewithin the In60 integron was found in the three remainingVirchow isolates. These three isolates presented indistinguish-able or closely related pulsed-field gel electrophoresis patternsamong themselves and also as compared with the two otherblaCTX-M-9-containing isolates previously obtained from animals.ESBL production is an emerging mechanism of resistance inS. enterica in the two studied hospitals.Keywords: CTX-M-9, CTX-M-10, Salmonella enterica, SHV-2Original Submission: 14 February 2008; Revised Submis-sion: 9 July 2008; Accepted: 15 July 2008Editor: L. PeixeClin Microbiol Infect 2009; 15: 292–29510.1111/j.1469-0691.2008.02673.xCorresponding author and reprint requests: C. Torres, Áreade Bioquı́mica y Biologı́a Molecular, Departamento de Agricultura yAlimentación, Universidad de La Rioja, Madre de Dios 51, 26006Logroño, SpainE-mail: carmen.torres@unirioja.esSalmonella enterica is a zoonotic bacterium, transmittedthrough the food chain, which causes gastroenteritis andother types of infection that require, in some cases, the useof antimicrobial agents. S. enterica isolates harbouringextended-spectrum-b-lactamases (ESBLs) have emergedworldwide during the last years, and ESBLs of the CTX-Mtype are increasingly reported in Salmonella, as well as inother members of the Enterobacteriaceae [1,2]. The occur-rence of different genes encoding CTX-M enzymes withinsul1-type integron structures that facilitate their dissemina-tion has been previously reported [3,4], although othergenetic environments have also been described for blaCTX-Mgenes [5,6]. The objective of this study was to detect andcharacterize the ESBLs in S. enterica isolates recovered intwo Spanish hospitals, and also to determine the geneticenvironment of ESBL genes and its possible location withinintegron structures.During the period 2000–2004, a total of 1038 clinicalisolates of S. enterica were recovered from unrelated292 Clinical Microbiology and Infection, Volume 15 Number 3, March 2009 CMIª2009 The AuthorsJournal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 286–295patients in the Complejo Hospitalario of Pontevedra(a 624-bed institution located in Pontevedra, Galicia, in thenorthwest of Spain) (2000–2002, 471 isolates; 2003, 310isolates; and 2004, 257 isolates). For four of these isolates,all belonging to serovar Virchow, the MIC of cefotaximeand/or ceftazidime was ‡2 mg/L and the ESBL screeningtest was positive; they were included in this study forb-lactamase characterization. These isolates were obtainedfrom ambulatory patients with acute gastroenteritis: oneduring the period 2000–2002, one in 2003, and twoin 2004, representing 0.2%, 0.3% and 0.8%, respectively,of the total isolates in each of the studied periods in thishospital.In addition, 195 isolates of S. enterica were recovered dur-ing 2003 in the Hospital Central of Asturias (a 1357-bedinstitution of Oviedo, Asturias, in the north of Spain). Forone isolate (0.5%) (serovar Livingstone), which was recov-ered from a hospitalized patient with acute gastroenteritis,the MIC of cefotaxime and/or ceftazidime was ‡2 mg/L andthe ESBL screening test was positive; it was also included inthis study for b-lactamase characterization.Antimicrobial susceptibility to amoxycillin, amoxycillin–clavulanic acid, cefotaxime, ceftazidime, cefepime, aztreonam,imipenem, nalidixic acid, ciprofloxacin, gentamicin, amikacin,tobramycin, streptomycin, fosfomycin, tetracycline, chloram-phenicol, sulphonamide and trimethoprim–sulphamethoxaz-ole was determined using disk diffusion and brothmicrodilution methods [7]. Escherichia coli ATCC 25922 wasused as a quality control strain. The double-disk synergy testusing amoxycillin–clavulanic acid and cefotaxime orceftazidime disks was performed with all isolates to detectESBL production [8].The presence of genes encoding TEM-, SHV-, OXA-,CTX-M- and CMY-type b-lactamases was studied using spe-cific PCRs [9]. All obtained amplicons were sequenced onboth strands, and the sequences were compared with thoseincluded in the GenBank database and at the website http://www.lahey.org/Studies/, in order to ascribe the specific typeof b-lactamase gene. The presence of the sul1, sul2 and sul3genes, associated with sulphonamide resistance, and thetet(A) and tet(B) genes, associated with tetracycline resis-tance, were also determined by PCR in the five isolates [10].Positive and negative controls from the University of La Riojacollection were used in all PCRs.The presence of class 1 integrons as well as the character-ization of the gene cassettes included in their variableregions was studied by PCR and sequencing [10]. The PCR‘primer-walking’ strategy, with a wide variety of primersbased on known sequences [6, 9], was used to amplify over-lapping fragments, in order to determine the genetic environ-ment of the blaCTX-M genes.The five clinical S. enterica isolates included in this studyrepresented 0.4% of the total S. enterica isolates obtained inboth hospitals during the studied period. Table 1 shows thecharacteristics of the five ESBL-positive isolates. The genesencoding both CTX-M-9 and TEM-1b b-lactamases weredetected in three S. enterica serovar Virchow isolates, thoseencoding CTX-M-10 and TEM-1b in another serovarVirchow isolate, and that encoding SHV-2 in the serovarLivingstone isolate.The CTX-M-10 b-lactamase was initially described in anE. coli strain isolated in 1997 in a hospital of Madrid, Spain[6], and it was later reported in different species of Enterob-acter and Klebsiella [1,11,12]. This enzyme was found in anE. coli isolate recovered in 2000 in the north of Spain, andrecently also in an S. enterica serovar Virchow isolate fromthe same region [13,14]; no transconjugants of the describedS. enterica strain were obtained after conjugation experi-ments, and the same result was found (data not shown) withthe S. enterica isolate C683 (Table 1).The genetic environment of the blaCTX-M-10 gene in theS. enterica isolate C683, obtained by the PCR primer-walkingTABLE 1. Phenotypes, resistance genes and integron elements detected in the five extended-spectrum b-lactamase-producingSalmonella enterica isolatesS. enterica name/serovarMIC (mg/L)Resistancephenotype(excluding b-lactams)Antibiotic resistance genes detected Class integronAMX AMC CTX CAZ IPM FEP ATM bla Othersa intI1Gene cassettesinside VRC516/Virchow >16 8/4 >128 1 <0.5 >32 4 STR–TET–SUL–SXT–NAL CTX-M-9, TEM-1b tet(A), sul1, sul2 – dfrA16, aadA2C650/Virchow >16 16/8 >128 1 <0.5 8 2 STR–SUL–SXT–NAL CTX-M-9, TEM-1b sul1, sul2 – dfrA16, aadA2C651/Virchow >16 8/4 >128 2 <0.5 8 8 STR–TET–SUL–SXT–NAL CTX-M-9, TEM-1b tet(A), sul1, sul2 – dfrA16, aadA2C683/Virchow >16 <4/2 128 1 <0.5 4 4 SUL–NAL CTX-M-10, TEM-1b – – –C493/Livingstone >16 <4/2 64 8 <0.5 8 ND STR–TET–SUL SHV-2 tet(A), sul1 – aadA1AMX, amoxycillin; AMC, co-amoxiclav; CTX, cefotaxime; CAZ, ceftazidime; IPM, imipenem; FEP, cefepime; ATM, aztreonam; STR, streptomycin; TET, tetracycline; SUL, sul-phonamide; SXT, trimethoprim–sulphamethoxazole; NAL, nalidixic acid; VR, variable region; ND, not determined.aAntimicrobial resistance genes detected outside of the variable regions of class 1 integrons.CMI Research Notes 293ª2009 The AuthorsJournal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 286–295strategy [6], is shown in Fig. 1. This structure is similar tothat previously reported by Oliver et al. [6] in a blaCTX-M-10-containing E. coli strain (GenBank accession numberAY598759). This result reveals a linkage of this bla gene to aphage-related element, in contrast to the truncated ISEcp1element upstream from the blaCTX-M-10 gene that has beenpreviously detected in the first blaCTX-M-10-containing E. coliisolate in France [5]. This work provides another example ofthe influence of the genomic environment on local dissemi-nation of resistance genes, even among different bacterialgenera [6].The CTX-M-9 group (mainly CTX-M-9 and CTX-M-14enzymes) is highly represented in Spain and, in the case ofCTX-M-9, is usually linked to class 1 integrons associatedwith an ISCR1 element [2,4,15]. For that reason, a widevariety of primers based on the In60 integron structure(GenBank accession number AF174129) were used [9,10](Fig. 1) in order to explore the genetic environment of theblaCTX-M-9 gene in the three S. enterica serovar Virchow iso-lates. Curiously, the intI1 gene was not found by PCR inany of these three CTX-M-9-producing isolates, even usingother reported primers (data not shown). The genetic envi-ronment found in the present three clinical blaCTX-M-9-con-taining isolates was identical to the surrounding regionpreviously found in S. enterica serovar Virchow isolates ofanimal origin [9].The clonal relationship among the blaCTX-M-9-containingisolates was studied by pulsed-field gel electrophoresis,following the PulseNet Europe One-day protocol with XbaIas the restriction enzyme (http://www.pulsenet-europe.org/docs.htm), and the obtained patterns were found to be indis-tinguishable from those found in two blaCTX-M-9-containingS. enterica serovar Virchow isolates previously obtained fromanimal samples [9].All five isolates of human or animal origin showed indistin-guishable or closely related patterns, indicating the clonalrelationship among them. This observation is of interest andsuggests a relationship among animal and human blaCTX-M-9-containing S. enterica isolates that are emerging in both popu-lations [16]. In addition, it is important to underline that theobserved close clonal relationship of blaCTX-M-9-containingSalmonella isolates is at variance with the high diversityamong blaCTX-M-9-containing E. coli isolates previouslyreported [17–19].All three blaCTX-M-9-containing isolates showed resistanceto streptomycin, sulphonamide, trimethoprim–sulphamethox-azole and nalidixic acid, and two of them were also resistantto tetracycline. In addition to the resistance genes includedin the In60 integron, these isolates also harboured the sul2gene, in addition to the tet(A) gene in the two tetracycline-resistant isolates.The blaSHV-2-containing S. enterica serovar Livingstone iso-late harboured a class 1 integron that included the aadA1gene cassette, but the ESBL gene was not found to be associ-ated with this class 1 integron.S. enterica isolates producing ESBLs, mainly of the CTX-Mgroup, seem to be an emerging problem in human medicine.The detection of closely related or indistinguishable pulsed-field gel electrophoresis patterns among CTX-M-9-producingS. enterica isolates of both human and animal origin couldindicate the transfer of ESBL-producing S. enterica amonganimals and humans. The detection of the blaCTX-M-10 genein S. enterica isolates is interesting; this is the second timethat it has been reported in the literature, and its geneticenvironment was linked to a phage-related element previ-ously detected among Enterobacteriaceae. More studiesshould be performed in the future to track the evolution ofESBLs in S. enterica isolates from different environments.DNAinvertaseorf2 orf3 blactx-m-10int11 sullaadA2 qacEΔ1dfrA16orf4 IS4321transposaseorf8orf7blactx-m-9orf513(a)(b)Phage related region5,CS 3,CSorf3-like orf1005FIG. 1. Genetic structures detected surrounding: (a) the blaCTX-M-10 gene in Salmonella enterica serovar Virchow isolate C683; and (b) theblaCTX-M-9 gene in the three remaining S. enterica serovar Virchow isolates (C516, C650 and C651). The discontinued arrow indicates that theintI1 gene was not detected; open box, the region that shows high similarity to orf3 of Kluyvera ascorbata; d, 59-be; diagonally striped box,the recombination site attI; c, indicates the specific positions of the primers used for PCR primer-walking strategy.294 Clinical Microbiology and Infection, Volume 15 Number 3, March 2009 CMIª2009 The AuthorsJournal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 286–295Transparency DeclarationThis work was supported in part by Project SAF2006-14207-C02. The authors have no conflict of interest to declare.References1. Livermore DM, Cantón R, Gniadkowski M et al. CTX-M: changing theface of ESBLs in Europe. J Antimicrob Chemother 2007; 59: 165–174.2. Rossolini GM, D’Andrea MM, Mugnaioli C. The spread of CTX-M-typeextended-spectrum b-lactamases. Clin Microbiol Infect 2008; 14: 33–41.3. Sabaté M, Navarro F, Miró E et al. 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Briñas L, Lantero M, de Diego I et al. Mechanisms of resistance toexpanded-spectrum cephalosporins in Escherichia coli isolates recoveredin a Spanish hospital. J Antimicrob Chemother 2005; 56: 1107–1110.19. Garcı́a A, Navarro F, Miró E et al. Characterization of the highly vari-able region surrounding the blaCTX-M-9 gene in non-related Escherichiacoli from Barcelona. J Antimicrob Chemother 2005; 56: 819–826.CMI Research Notes 295ª2009 The AuthorsJournal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 286–295

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