# Prevalence and Characterization of Extended-Spectrum Beta-lactamases-Producing Salmonella enterica Isolates in Saragossa, Spain (2001–2008)

María Pardos de La Gándara, Cristina Seral, Javier Castillo García, Carmen

Rubio Calvo, François-Xavier Weill

► To cite this version:

María Pardos de La Gándara, Cristina Seral, Javier Castillo García, Carmen Rubio Calvo, François-Xavier Weill. Prevalence and Characterization of Extended-Spectrum Beta-lactamases-Producing Salmonella enterica Isolates in Saragossa, Spain (2001–2008). Microbial Drug Resistance, 2011, 17 (2), pp.207-13. 10.1089/mdr.2010.0105. pasteur-01131166

HAL Id: pasteur-01131166

https://pasteur.hal.science/pasteur-01131166

Submitted on 12 Mar 2019

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Title: Prevalence and characterization of extended-spectrum-betalactamases-producing *Salmonella enterica* isolates in Saragossa, Spain (2001-2008)

Authors: María Pardos-de la Gándara, MD PhD¹, ², Cristina Seral-García, PharmD, PhD², Javier Castillo-García, MD, PhD², Carmen Rubio-Calvo, MD, PhD², François-Xavier Weill, MD, PhD¹.

¹ Institut Pasteur, Laboratoire des Bactéries Pathogènes Entériques, Centre National de Référence des *Salmonella*, Paris, France

² Departamento de Microbiología, Hospital Clínico Universitario “Lozano Blesa”, Departamento de Microbiología, Facultad de Medicina, Universidad de Zaragoza

Corresponding author: María Pardos de la Gándara: Centre Nacional de Référence des *Salmonella*, Laboratoire des Bactéries Pathogènes Entériques, Institut Pasteur, 28, rue du docteur Roux, 75724 Paris Cedex 15- France. Tel. + 33 (1) 45 68 83 39. Fax. + 33 (1) 45 68 88 37. E-mail: mariapardos@yahoo.es

## ABSTRACT

We analysed the prevalence of resistance to extended-spectrum cephalosporins (ESC) among clinical strains of *Salmonella enterica* collected by the Laboratory of Clinical Microbiology in the University Clinical Hospital Lozano Blesa in the region of Aragon (Spain), for which very few epidemiological information exists. A total of 2,092 strains of *S. enterica* were identified in stool samples from patients with gastroenteritis. Five isolates showed an ESBL phenotype: four isolates of *S. enterica* serotype Virchow harboured the extended-spectrum beta lactamase (ESBL) encoding *bla*<sub>CTX-M-9</sub> gene and an isolate of serotype Enteritidis carried a *bla*<sub>CTX-M-1</sub> gene that, to the best of our
knowledge, is being described here for the first time in this serotype of S. *enterica*. The
five ESC-resistant isolates were also resistant to spectinomycin, streptomycin,
kanamycin, sulfonamides, tetracycline, and trimethoprim as well as to nalidixic acid.
The ESBL isolate of serotype Enteritidis though remained susceptible to kanamycin and
nalidixic acid. A class 1 integron of 1.5 kb was detected for the four serotype Virchow
isolates with the gene cassette *dfrA16—aadA2*. The *bla*<sub>CTX-M-9</sub> gene was carried by a
~300 kb IncHI2 conjugative plasmid in the case of the S. *enterica* serotype Virchow
isolates. The *bla*<sub>CTX-M-1</sub> gene was carried by a ~100 kb IncI1-N conjugative plasmid for
the serotype Enteritidis ESC-resistant isolate. All the four ESC-resistant strains of S.
*enterica* serotype Virchow clustered together in a XbaI-pulsed-field gel electrophoresis,
which also revealed a strong similarity between them and some pulsotypes of S.
*enterica* serotype Virchow from France.
# 1 INTRODUCTION

Nontyphoidal salmonellosis is one of the most prevalent food borne bacterial infections in Europe and in North America. In the United States, the burden of this infection has been estimated annually to be 1.4 million of cases resulting in 400 deaths ³⁷. In Europe, 181,876 cases were reported in 2005, which meant a ratio of 39/100,000 inhabitants ¹¹. Most infections are self-limited and do not require antimicrobial treatment. However, severe, life-threatening bacteraemia sometimes occur, particularly in children and immunocompromised hosts and in these cases an antimicrobial therapy is recommended ³⁵. Appropriate drugs for *Salmonella* infections include ampicillin, extended-spectrum cephalosporins (ESC), fluoroquinolones, and trimethoprim-sulfamethoxazole. However, rising rates of resistance to ampicillin and trimethoprim-sulfamethoxazole have significantly reduced their efficacy and fluoroquinolones are not approved for the use in children. Consequently, ESC have become the current drugs of choice for the treatment for invasive infections in children ⁴. The number of cases of salmonellosis caused by isolates resistant to these ESC is in continuous increase since the very first case was detected in the early 1980s ¹. Nonetheless, they are still rare over the total of *Salmonella* foodborne infections, just reaching a 0.2 % in Europe in 2004 ²⁵. Resistance to these drugs is mainly mediated by the bacterial production of beta-lactamases that degrade ESC. Two main classes of plasmid beta-lactamases that inactivate ESC have been identified in *Salmonella*: the Ambler class A extended-spectrum beta-lactamases (ESBLs), the most prevalent class in this genus, and the Ambler class C cephamycinases. Most ESBLs belong to three families, TEM, SHV and CTX-M. Over the last decade, CTX-Ms have become the most prevalent family of ESBLs in the genus *Salmonella* in Europe ¹.
25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

In Spain, the first ESBL-producing *Salmonella enterica* isolate was described in 1996<sup>26</sup>. It was a *S. enterica* serotype Othmarschen producing TEM-27 causing a nosocomial outbreak in Madrid. Thereafter, *S. enterica* isolates producing ESBLs CTX-M-9, CTX-M-27, TEM-52 or cephramycinase CMY-2 were reported sporadically in humans or in animals<sup>4, 13, 34</sup>. A recent study on randomly selected strains from different hospitals in Spain identified 27 (0.26 %) human isolates of *S. enterica* producing ESBLs or cephramycinases between 2001 and 2005<sup>16</sup> and in a specific study about *S. enterica* serotype Virchow, 79 out of 504 (15%) isolates recovered from 14 of the 17 provinces in Spain were ESBL producers, 48 of them carrying a *bla*<sub>CTX-M-9</sub> gene<sup>18</sup>.

In 2000, in Aragón, a northwestern region of Spain (1,326,918 inhabitants), 129 foodborne outbreaks that affected 2,030 people (including 1,464 in Saragossa), resulting in 103 hospitalizations were reported. *S. enterica* was found to be the causative agent in 62% of the cases. In 2005, a foodborne outbreak was reported with 179 cases of salmonellosis from the whole region (109 occurring in Saragossa), 19 patients needed hospitalization. This outbreak was epidemiologically related to the consumption of locally farmed chicken (http://www.aragon.es). Very few studies have been published about the epidemiological situation of *S. enterica* or about the profile of antimicrobial resistances shown by them, both for the whole region of Aragon and for the Sanitary Area Saragossa 3. The last clinical study showed a continuous increase of isolations of *S. enterica*, from 1994 (118 isolates) to 2000 (287 isolates)<sup>29</sup>.

In this study, we assessed the frequency of different serotypes and the prevalence of ESC resistance among *S. enterica* isolates obtained in Saragossa during an eight-year period (2001-2008). The characterization of the beta-lactamase genes and their genetic support, and class 1 integron cassettes was done on ESC-resistant *S. enterica* isolates.
## 51 MATERIALS AND METHODS

### 52 *Strains*

S. *enterica* isolates were recovered from stool samples at the Laboratory of Clinical Microbiology of the University Hospital H.C.U. "Lozano Blesa" between 2001 and 2008. The Hospital "Lozano Blesa" is a 900-bed teaching hospital located in Saragossa, the capital city of Aragón. It is the reference hospital of the Sanitary Area Saragossa 3 (268,624 inhabitants in 2005). During the study period, the Laboratory of Clinical Microbiology received 59,977 stool samples, and a pathogen was found in roughly 10% of the samples. S. *enterica* was the main agent with 2,092 isolates (only one isolate per patient per month was considered).

S. *enterica* strains were identified using the WIDER system (Soria Melguizo, Madrid, Spain)³⁶ and serotyped on the basis of somatic O, and both phase 1 and phase 2 flagellar antigens by agglutination tests with antisera (Bio-Rad, Marnes la Coquette, France) as specified by the White-Kauffmann-Le Minor scheme ¹⁷.

### 66 *Antimicrobial susceptibility*

Antimicrobial susceptibility was first carried out for all the isolates using the WIDER system according to the recommendations of the Clinical and Laboratory Standards Institute guidelines ⁹. For ESC, the antibiotic concentration range was 0.12 to 8 µg/ml for cefotaxime and 0.5 to 16 µg/ml for ceftazidime. All strains of *Salmonella* showing a decreased susceptibility to one or both of these antibiotics, meaning a minimal inhibitory concentration (MIC) ≥ 1 µg/mL but remaining susceptible to cephamycins and to the association with clavulanic acid were selected for further analysis, according with the classical definition of ESBL ³¹.
75

ESBL phenotype was detected by the double-disk synergy method ²⁰ and
measuring the MIC for ceftriaxone (CRO) ceftazidime (CAZ) and cefotaxime (CTX)
with and without clavulanic acid, using the ESBL detection Etest strips (AB Biodisk,
Solna, Sweden).

76

77

78

79

Additional testing was carried out by the disk-diffusion method with 32
antimicrobial drugs (Bio-Rad), as previously described ³⁸.

80

81

82

PCR amplification of antimicrobial resistance genes and sequence analysis

83

Total DNA was extracted using the InstaGene matrix kit (Bio-Rad) according to
the manufacturer's recommendations. The resistance genes, *bla*<sub>TEM</sub>, *bla*<sub>SHV</sub>, *bla*<sub>OXA-1</sub>
group, *bla*<sub>CTX-M</sub>, and class 1 integron gene cassettes were amplified by PCR as
described previously <sup>38</sup>. Also *qnr*, *qepA* and *aac(6')-Ib* genes were investigated when
resistance to NAL was observed, as previously described <sup>30, 33</sup>.

84

85

86

87

88

Sequencing was performed at the “Plateforme de Génotypage des Pathogènes et Santé Publique, PF8” (Institut Pasteur). The nucleotide sequences and the deduced protein sequences were analyzed with EditSeq and Megalign software (Dnastar, Madison, WI). The BLASTN program of NCBI (National Center for Biotechnology Information, US National Library of Medicine, Bethesda, MD, U.S.A.) was used for database searches.

89

90

91

92

93

94

95

Resistance transfer determination

96

A resistance transfer experiment was carried out on solid media, using either
*Escherichia coli* K-12 BM14 resistant to sodium azide or *E. coli* C1a resistant to
nalidixic acid (NAL), as the recipient strain ¹². Transconjugants were selected on
Drigalski agar (Bio-Rad) supplemented with CRO (20 µg/mL) and sodium azide (500

97

98

99
100 µg/mL) or NAL (64 µg/mL). Three *E. coli* transconjugants were arbitrarily selected for each experiment.

102

103 Plasmid analysis

104 Plasmid DNA from parental and transconjugant strains was analyzed by
105 pulsed field gel electrophoresis (PFGE) after linearization with the S1 nuclease enzyme,
106 as described previously ¹². PCR-based replicon typing analysis was performed, as
107 described by Carattoli et al. ⁷. The 18 primer pairs targeting FIA, FIB, FIC, HI1, HI2,
108 I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F and FII replicons were used in separate
109 PCR reactions.

110

111 PFGE typing

112 The genetic diversity of the four *S. enterica* serotype Virchow isolates resistant
113 to ESC was assessed by PFGE of genomic DNA digested with *Xba*I (Roche,
114 Mannheim, Germany), as described previously ³⁸. The running conditions and the
115 molecular size marker were as described in the standardized PulseNet protocol
116 (http://www.cdc.gov/pulsenet/). BioNumerics 4.0 (Applied Maths, Saint-Martens-
117 Latem, Belgium) was used for image normalization and construction of similarity
118 matrices. Bands were assigned manually. Clustering was carried out by the unweighted
119 pair-group method with arithmetic averages (UPGMA) based on the Dice similarity
120 index, using a 1% optimization parameter and 1% band-position tolerance. The results
121 were compared to PFGE profiles of *S. enterica* from the French National Reference
122 Centre for *Salmonella* (FNRC-Salm) database.

123

124
125 **RESULTS**

Between 2001 and 2008, 2,092 *S. enterica* isolates were identified (one per patient within a period of 30 days), with a continuous tendency to decrease from 2002 (394 isolates) to 2008 (138 isolates). The distribution of serotypes is presented in **TABLE 1**. Enteritidis was the predominant serotype, accounting for 52% of all the isolates.

The results of susceptibility testing are shown in **TABLES 2 AND 3**. Of the 2,092 isolates, 387 (18.5 %) were susceptible to all antimicrobial agents tested. The most frequent types of resistance observed concerned ampicillin (increasing from 33.5 % in 2001 to 59.4 % in 2008), NAL (47.2 % in 2005 but decreasing since then to a 21.7 % in 2008) and trimethoprim/sulfamethoxazole (peaking at 15 % in 2004). Although up to 17 isolates had a MIC for ceftriaxone of ≥ 1 µg/mL, only five isolates (0.24 %) of serotypes Enteritidis (isolate 06-424914) and Virchow (isolates 02-236146, 02-214992, 03-1672608 and 04-1831083), showed an ESBL phenotype determined by double-disk diffusion test and ESBL-Etest® (TABLE 3). All five isolates were susceptible to cephamycins, association with clavulanic acid and carbapenems. The mechanisms of resistance to beta-lactam antibiotics of these five isolates were investigated and are shown below. The other 12 isolates with a MIC for ceftriaxone of ≥ 1 µg/mL were resistant to cefoxitin and gave a negative double disk synergy test (despite the use of several ESC disks placed at distances of both 15 and 20 mm from the clavulanic acid disk), therefore they were considered as cephamycinase-producing isolates. No further molecular characterization of these isolates has been possible, as they were accidentally discarded as no-ESBL-producers.

The serotype Enteritidis isolate 06-424914 was also resistant to aminoglycosides (streptomycin, spectinomycin), trimethoprim-sulfamethoxazole, and tetracycline (TABLE 3). The serotype Virchow isolates 02-236146, 02-214992, 03-1672608, 04-
1831083 had a similar susceptibility profile, except an additional resistance to kanamycin and NAL with a decreased susceptibility to ciprofloxacin (MIC of 0.25 to 0.5 mg/L).

PCR and sequence analysis detected in the five isolates both the penicillinase *bla*<sub>TEM-1</sub> gene and an ESBL *bla*<sub>CTX-M</sub> gene. Serotype Enteritidis isolate 06-424914 contained the *bla*<sub>CTX-M-1</sub> gene, whereas serotype Virchow isolates contained the *bla*<sub>CTX-M-9</sub> gene. The serotype Virchow isolates also harboured a 1.5 kb class 1 integron containing a *dfrA16-aadA2* gene cassette known to confer resistances to trimethoprim and streptomycin and spectinomycin respectively. No class 1 integrons were found for the Enteritidis isolate.

In serotype Enteritidis isolate 06-424914, the *bla*<sub>CTX-M-1</sub> gene was carried by a ≈100 kb conjugative IncI1-IncN multireplicon plasmid. Other resistance determinants, affecting streptomycin, spectinomycin, sulfamides, trimethoprim-sulfamethoxazole, and tetracycline were cotransfered to transconjugants with ceftriaxone resistance.

In serotype Virchow isolates, the *bla*<sub>CTX-M-9</sub> gene was carried by a ≈300 kb conjugative plasmid of replicon IncHI2. The same resistance determinants as listed for the serotype Enteritidis isolate 06-424914, were cotransfered to transconjugants with ceftriaxone resistance. The resistance to NAL was due to a chromosomal mutation on the QRDR region of the *gyrA* leading to a substitution of a serine in position 83 by a phenylalanine, as described previously²².

In this study, all the CTX-M-9-producing *S. enterica* serotype Virchow isolates clustered together independently to their geographic area of origin, might it be Spain or France, as it was shown in a database comparison between the isolates from Saragossa and some pulsotypes of the FNRC-Salm (FIGURE 1).

174
175 DISCUSSION

176 Since the first bla<sub>CTX-M</sub> genes were described in the early 1990s, *S. enterica* has
177 been one of the first species of *Enterobacteriaceae* to be identified harbouring this kind
178 of resistances ¹. CTX-M-9 was first reported in Spain in 1996, produced by an *E. coli*
179 human isolate and *S. enterica* serotype Virchow carrying a bla<sub>CTX-M-9</sub> appeared just a
180 few years later ³⁴. Retrospective studies in the United Kingdom have found strains that
181 were isolated in the 1990s, from patients with a history of travelling abroad ¹⁹. Strains
182 isolated from both humans and poultry were reported in France, suggesting an
183 interspecies transfer, which affected several serotypes (Virchow and Enteritidis among
184 them) and different ESBL enzymes (CTX-M-2, TEM-52, CTX-M-9) ³⁸. This is
185 supported as well by the latest studies on poultry in Spain ⁵.

186 The ESBL gene bla<sub>CTX-M-1</sub> was first reported in Germany in 1996, harboured by
187 an *E. coli* strain ² and the first case of *S. enterica* producing CTX-M-1 was a strain of
188 serotype Typhimurium isolated in France ²¹. Recent studies found *S. enterica* serotypes
189 Enteritidis, carrying the bla<sub>CTX-M-32</sub> gene, and Litchfield carrying the bla<sub>CTX-M-1</sub> gene in
190 Spain, in relation with conjugative plasmids of IncN and IncI1, respectively ¹⁶. The
191 plasmid carrying the bla<sub>CTX-M-1</sub> gene had also a similar size (110 kb) to the plasmid
192 found for the Enteritidis isolate of our study, and both share a similar multi-drug
193 resistance profile and the lack of a class 1 integron. *E. coli* strains carrying this ESBL
194 gene on a IncI1 plasmid have been reported Italy and France from both humans and
195 animals (poultry and dogs) ¹⁴, ¹⁵. Another study on *E. coli* strains recovered from human
196 samples in France showed that the bla<sub>CTX-M-1</sub> gene was carried by either IncI1 or IncN
197 plasmids ²⁴. Multireplicon plasmids do often occur ⁶, but an IncI1-N has not been
198 described yet. Previous findings in animals, mainly poultry, of both ESBL bla<sub>CTX-M-1</sub>
199

and *bla*<sub>CTX-M-9</sub> harboured in IncI or IncHI2 plasmids suggest that poultry might play an
important role as a reservoir for these bacteria<sup>16, 38</sup>.

200

201

The first ESBL S. *enterica* strains were detected in our laboratory in 2002 ⁸.

202

During this eight-year prospective study we have found a total of four isolates of serotype Virchow harbouring a *bla*<sub>CTX-M-9</sub> gene and one *S. enterica* serotype Enteritidis with the *bla*<sub>CTX-M-1</sub> gene, all of them during the period 2002-2006. ESBL strains were no longer recovered after 2006. Although the final rate of ESBL among the total figures was rather low (0.24 %), we call out the fact that those four isolates of serotype Virchow producing a CTX-M-9 occurred in 26.6 % of all isolates of that serotype during the time period of this study. The four strains of serotype Virchow appear to carry the same IncHI2 conjugative plasmid previously described in CTX-M-9 producing strains from Spain (in the region of La Rioja, adjacent to Aragon<sup>32</sup>, in Barcelona<sup>10</sup> and the city of Madrid<sup>28</sup>). These works describe plasmids of the same size and belonging to the same incompatibility group IncHI2, carrying the same gene cassette *dfrA16—aadA2* within the complex class 1 integron In60, altogether with the *bla*<sub>CTX-M-9</sub> gene<sup>3, 10, 27, 32</sup>. This has finally become one of the most predominant combinations of *S. enterica* serotype and ESBL in Spain and Portugal<sup>23</sup>.

203

204

205

206

207

208

209

210

211

212

213

214

215

216

Clonal transmission of multi-drug-resistance has been proven in isolates from
poultry and humans<sup>32, 38</sup>. The PFGE pulsotypes, with more than 96 % of similarity
among the four ESBL Virchow isolates and the phenotype, gene cassettes and plasmids
found suggest that they could be associated to the clonal spread of *S. enterica* serotype
Virchow PT19 previously described in Spain and France<sup>18, 38</sup>.

217

218

219

220
## ACKNOWLEDGEMENTS

We would like to acknowledge Dr. Joseph Dragavon for the careful reading of this manuscript.

María Pardos de la Gándara was the recipient of the following grants during this work:

1. Grant for Short Term Research in Clinical Centres Abroad by the S.E.I.M.C. (2007)

2. Grant "Project Europe XXI" of the CAI-Social Action and the Government of Aragón for Short Term Research Abroad (2008)

3. Postdoctoral / Post-Specialization Fellowship for Research in Universities or Centres Abroad from the "Alfonso Martín Escudero" Foundation, Spain (2009)

4. Student Travel Fellowship from the SEIMC (Spanish Society of Infectious Diseases and Clinical Microbiology) for the Meeting in Seville, Spain (2009)
## AUTHOR DISCLOSURE STATEMENT

The authors declare no commercial associations that might create a conflict of interest in connection with the submitted manuscript:

- María PARDOS DE LA GÁNDARA: has received research funding from the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (Beca Martín Luengo 2007), from the CAI-Gobierno de Aragón (Proyecto Europa XXI 2007-2008) and from the Fundación Alfonso Martín Escudero (Becas para estudios en el extranjero 2008-2009).

- Cristina SERAL GARCÍA: absence of any relationship or any degree of conflicting or dual interest, financial or of any other nature, that may affect professional judgment in relation to the submitted article.

- Francisco Javier CASTILLO GARCÍA: absence of any relationship or any degree of conflicting or dual interest, financial or of any other nature, that may affect professional judgment in relation to the submitted article.

- Carmen RUBIO CALVO: absence of any relationship or any degree of conflicting or dual interest, financial or of any other nature, that may affect professional judgment in relation to the submitted article.

- François Xavier WEILL: absence of any relationship or any degree of conflicting or dual interest, financial or of any other nature, that may affect professional judgment in relation to the submitted article.
REFERENCES

1. Arlet, G., T.J. Barrett, P. Butaye, A. Cloeckaert, M.R. Mulvey, and D.G. White. 2006. *Salmonella* resistant to extended-spectrum cephalosporins: prevalence and epidemiology. *Microbes Infect* **8**:1945-54.

2. Bauernfeind, A., I. Stemplinger, R. Jungwirth, S. Ernst, and J.M. Casellas. 1996. Sequences of beta-lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequences with those of other beta-lactamases. Antimicrob Agents Chemother **40**:509-13.

3. Bonnet, R. 2004. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother **48**:1-14.

4. Cantón, R., A. Novais, A. Valverde, E. Machado, L. Peixe, F. Baquero, and T.M. Coque. 2008. Prevalence and spread of extended-spectrum beta-lactamase-producing *Enterobacteriaceae* in Europe. Clin Microbiol Infect **14** **Suppl 1**:144-53.

5. Cápita, R., C. Alonso-Calleja, and M. Prieto. 2007. Prevalence of *Salmonella enterica* serovars and genovars from chicken carcasses in slaughterhouses in Spain. J Appl Microbiol **103**:1366-75.

6. Carattoli, A. 2009. Resistance plasmid families in *Enterobacteriaceae*. Antimicrob Agents Chemother **53**:2227-38.

7. Carattoli, A., A. Bertini, L. Villa, V. Falbo, K.L. Hopkins, and E.J. Threlfall. 2005. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods **63**:219-28.

8. Castillo García, F.J., C. Seral García, M. Pardos De la Gándara, M.I. Millán Lou, and C. Pitart Ferré. 2007. Prevalence of fecal carriage of ESBL-producing *Enterobacteriaceae* in hospitalized and ambulatory patients during two non-outbreak periods. Eur J Clin Microbiol Infect Dis **26**:77-8.

9. Clinical and Laboratory Standards Institute/NCCLS. Clinical and Laboratory Standards Institute/NCCLS. 2009. Performance Standards for Antimicrobial Susceptibility Testing: Nineteenth Informational Supplement. M100-S19. CLSI, Wayne, PA.

10. Diestra, K., C. Juan, T. Curiao, B. Moya, E. Miró, J. Oteo, T.M. Coque, M. Pérez-Vázquez, J. Campos, R. Cantón, A. Oliver, and F. Navarro. 2009. Characterization of plasmids encoding *bla*ESBL and surrounding genes in Spanish clinical isolates of *Escherichia coli* and *Klebsiella pneumoniae*. J Antimicrob Chemother **63**:60-6.
11. **European Centre for Disease Prevention and Control (ECDC).** Salmonellosis (non-typhi, non-paratyphi) - Epidemiological situation. 2008. European Centre for Disease Prevention and Control (ECDC). Available at [ecdc.europa.eu/en/publications](http://ecdc.europa.eu/en/publications). (Online.)

12. **Fabre, L., A. Delauné, E. Espié, K. Nygard, M. Pardos, L. Polomack, F. Guesnier, M. Galimand, J. Lassen, and F.X. Weill.** 2009. Chromosomal integration of the extended-spectrum beta-lactamase gene *blaCTX-M-15* in *Salmonella enterica* serotype Concord isolates from internationally adopted children. Antimicrob Agents Chemother **53:1808-16.**

13. **Fernández Vázquez, M., J.L. Muñoz Bellido, M.I. García García, and J.A. García-Rodríguez.** 2006. *Salmonella enterica* serovar Enteritidis producing a TEM-52 beta-lactamase: first report in Spain. Diagn Microbiol Infect Dis **55:245-6.**

14. **García-Fernández, A., G. Chiaretto, A. Bertini, L. Villa, D. Fortini, A. Ricci, and A. Carattoli.** 2008. Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum beta-lactamases in *Escherichia coli* and *Salmonella* of human and animal origin. J Antimicrob Chemother **61:1229-33.**

15. **Girlich, D., L. Poirel, A. Carattoli, I. Kempf, M.F. Lartigue, A. Bertini, and P. Nordmann.** 2007. Extended-spectrum beta-lactamase CTX-M-1 in *Escherichia coli* isolates from healthy poultry in France. Appl Environ Microbiol **73:4681-5.**

16. **González-Sánz, R., S. Herrera-León, M. de la Fuente, M. Arroyo, and M.A. Echeíta.** 2009. Emergence of extended-spectrum beta-lactamases and AmpC-type beta-lactamases in human *Salmonella* isolated in Spain from 2001 to 2005. J Antimicrob Chemother **64:1181-6.**

17. **Grimont, F., and F.X. Weill.** 2007. Antigenic Formulae of the *Salmonella* serovars. WHO-Institut Pasteur. Available at [www.pasteur.fr/sante/clre/cadrecnr/salmoms-index.html](http://www.pasteur.fr/sante/clre/cadrecnr/salmoms-index.html). (Online.)

18. **Herrera-León, S., R. González-Sanz, I. Rodríguez, M.R. Rodicio, and M.A. Echeíta.** 2010. Spread of a multiresistant CTX-M-9-producing *Salmonella enterica* serotype Virchow phage type 19 in Spain. Eur J Clin Microbiol Infect Dis **29:901-5.**

19. **Hopkins, K.L., A. Deheer-Graham, E.J. Threlfall, M.J. Batchelor, and E. Liébana.** 2006. Novel plasmid-mediated CTX-M-8 subgroup extended-spectrum beta-lactamase (CTX-M-40) isolated in the UK. Int J Antimicrob Agents **27:572-5.**

20. **Jarlier, V., M.H. Nicolas, G. Fournier, and A. Philippon.** 1988. Extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-
lactam agents in *Enterobacteriaceae*: hospital prevalence and susceptibility patterns. *Rev Infect Dis* **10**:867-78.

21. **Lartigue, M.F., L. Poirel, J.W. Decousser, and P. Nordmann.** 2005. Multidrug-resistant *Shigella sonnei* and *Salmonella enterica* serotype Typhimurium isolates producing CTX-M beta-lactamases as causes of community-acquired infection in France. *Clin Infect Dis* **40**:1069-70.

22. **Le, T.A., L. Fabre, P. Roumagnac, P.A. Grimont, M.R. Scavizzi, and F.X. Weill.** 2007. Clonal expansion and microevolution of quinolone-resistant *Salmonella enterica* serotype typhi in Vietnam from 1996 to 2004. *J Clin Microbiol* **45**:3485-92.

23. **Livermore, D.M., R. Cantón, M. Gniadkowski, P. Nordmann, G.M. Rossolini, G. Arlet, J. Ayala, T.M. Coque, I. Kern-Zdanowicz, F. Luzzaro, L. Poirel, and N. Woodford.** 2007. CTX-M: changing the face of ESBLs in Europe. *J Antimicrob Chemother* **59**:165-74.

24. **Marcadé, G., C. Deschamps, A. Boyd, V. Gautier, B. Picard, C. Branger, E. Denamur, and G. Arlet.** 2009. Replicon typing of plasmids in *Escherichia coli* producing extended-spectrum beta-lactamases. *J Antimicrob Chemother* **63**:67-71.

25. **Meakins, S., I.S. Fisher, C. Berghold, P. Gerner-Smidt, H. Tschape, M. Cormican, I. Luzzi, F. Schneider, W. Wannett, J. Coia, A. Echeíta, and E.J. Threlfall.** 2008. Antimicrobial drug resistance in human nontyphoidal *Salmonella* isolates in Europe 2000-2004: a report from the Enter-net International Surveillance Network. *Microb Drug Resist* **14**:31-5.

26. **Morosini, M.I., J. Blázquez, M.C. Negri, R. Cantón, E. Loza, and F. Baquero.** 1996. Characterization of a nosocomial outbreak involving an epidemic plasmid encoding for TEM-27 in *Salmonella enterica* subspecies *enterica* serotype Othmarschen. *J Infect Dis* **174**:1015-20.

27. **Novais, A., R. Canton, R. Moreira, L. Peixe, F. Baquero, and T.M. Coque.** 2007. Emergence and dissemination of Enterobacteriaceae isolates producing CTX-M-1-like enzymes in Spain are associated with IncFII (CTX-M-15) and broad-host-range (CTX-M-1, -3, and -32) plasmids. *Antimicrob Agents Chemother* **51**:796-9.

28. **Novais, A., R. Cantón, A. Valverde, E. Machado, J.C. Galán, L. Peixe, A. Carattoli, F. Baquero, and T.M. Coque.** 2006. Dissemination and persistence of *bla*CTX-M-9 are linked to class 1 integrons containing CR1 associated with defective transposon derivatives from Tn402 located in early antibiotic resistance plasmids of IncHI2, IncP1-alpha, and IncFI groups. *Antimicrob Agents Chemother* **50**:2741-50.
29. **Olivera, S., F.J. Castillo, M.T. Llorente, A. Clavel, M. Varea, C. Seral, and M.C. Rubio.** 2002. [Antimicrobial resistance of clinical strains of *Salmonella enterica* isolated in Zaragoza]. *Rev Esp Quimioter* **15**:152-7.

30. **Perichon, B., P. Courvalin, and M. Galimand.** 2007. Transferable resistance to aminoglycosides by methylation of G1405 in 16S rRNA and to hydrophilic fluoroquinolones by QepA-mediated efflux in *Escherichia coli*. *Antimicrob Agents Chemother* **51**:2464-9.

31. **Philippon, A., R. Labia, and G. Jacoby.** 1989. Extended-spectrum beta-lactamases. *Antimicrob Agents Chemother* **33**:1131-6.

32. **Riaño, I., M. García-Campello, Y. Sáenz, P. Álvarez, L. Vinué, M. Lantero, M.A. Moreno, M. Zarazaga, and C. Torres.** 2009. Occurrence of extended-spectrum beta-lactamase-producing *Salmonella enterica* in northern Spain with evidence of CTX-M-9 clonal spread among animals and humans. *Clin Microbiol Infect* **15**:292-5.

33. **Richter, S.N., I. Frasson, C. Bergo, R. Manganelli, A. Cavallaro, and G. Palu.** 2010. Characterisation of qnr plasmid-mediated quinolone resistance in *Enterobacteriaceae* from Italy: association of the *qnrB19* allele with the integron element *ISCR1* in *Escherichia coli*. *Int J Antimicrob Agents* **35**:578-83.

34. **Simarro, E., F. Navarro, J. Ruiz, E. Miró, J. Gómez, and B. Mirelis.** 2000. *Salmonella enterica* serovar virchow with CTX-M-like beta-lactamase in Spain. *J Clin Microbiol* **38**:4676-8.

35. **Threlfall, E.J.** 2002. Antimicrobial drug resistance in *Salmonella*: problems and perspectives in food- and water-borne infections. *FEMS Microbiol Rev* **26**:141-8.

36. **Valverde, A., T.M. Coque, L. García-San Miguel, F. Baquero, and R. Cantón.** 2008. Complex molecular epidemiology of extended-spectrum beta-lactamases in *Klebsiella pneumoniae*: a long-term perspective from a single institution in Madrid. *J Antimicrob Chemother* **61**:64-72.

37. **Voetsch, A.C., T.J. Van Gilder, F.J. Angulo, M.M. Farley, S. Shallow, R. Marcus, P.R. Cieslak, V.C. Deneen, and R.V. Tauxe.** 2004. FoodNet estimate of the burden of illness caused by nontyphoidal *Salmonella* infections in the United States. *Clin Infect Dis* **38 Suppl 3**:S127-34.

38. **Weill, F.X., R. Lailler, K. Praud, A. Kerouanton, L. Fabre, A. Brisabois, P.A. Grimont, and A. Cloeckaert.** 2004. Emergence of extended-spectrum-beta-lactamase (CTX-M-9)-producing multiresistant strains of *Salmonella enterica* serotype Virchow in poultry and humans in France. *J Clin Microbiol* **42**:5767-73.
TABLES AND FIGURE LEGENDS

<figure><img src="image_2.png" /><figcaption>FIGURE 1. CLONAL RELATEDNESS OF XbaI-PGFE PROFILES OBTAINED FROM S. ENTERICA SEROTYPE VIRCHOW ISOLATES FROM SARAGOSSA AND FRANCE</figcaption></figure>
FIGURE 1. The two first numbers indicate the year of isolation of the strain. The strains from Saragossa are outlined in bold and with asterisks (*). The remaining strains belong to the database of the French National Reference Centre for *Salmonella*. Am: amoxicillin, Cro: ceftriaxone, Caz: ceftazidime, S: streptomycin, Sp: spectinomycin, K: kanamycin, G: gentamicin, Su: sulfonamide, Te: tetracycline, Tm: trimethoprim, Nal: nalidixic acid.
<table><caption>TABLE 1. DISTRIBUTION OF SALMONELLA ENTERICA SEROTYPES IN THE CLINICAL MICROBIOLOGY
LABORATORY OF H.C.U. LOZANO BLESA IN ARAGÓN, SPAIN, DURING THE PERIOD 2001-2008</caption><thead><tr><th>Salmonella serotypes</th><th>2001<br>(n=304)</th><th>2002<br>(n=394)</th><th>2003<br>(n=334)</th><th>2004<br>(n=346)</th><th>2005<br>(n=197)</th><th>2006<br>(n=207)</th><th>2007<br>(n=172)</th><th>2008<br>(n=138)</th><th>total<br>(n=2092)</th></tr></thead><tbody><tr><td>O:9 (formerly D1 group)</td><td>183</td><td>233</td><td>219</td><td>197</td><td>97</td><td>83</td><td>59</td><td>32</td><td>1103</td></tr><tr><td><strong>Enteritidis</strong></td><td><strong>181</strong></td><td><strong>233</strong></td><td><strong>216</strong></td><td><strong>195</strong></td><td><strong>97</strong></td><td><strong>81</strong></td><td><strong>57</strong></td><td><strong>28</strong></td><td><strong>1088</strong></td></tr><tr><td>Others</td><td>2</td><td>0</td><td>3</td><td>2</td><td>0</td><td>2</td><td>2</td><td>4</td><td>15</td></tr><tr><td>O:4 (formerly B group)</td><td>72</td><td>115</td><td>67</td><td>89</td><td>61</td><td>72</td><td>65</td><td>72</td><td>613</td></tr><tr><td><strong>Typhimurium</strong></td><td><strong>68</strong></td><td><strong>107</strong></td><td><strong>66</strong></td><td><strong>89</strong></td><td><strong>58</strong></td><td><strong>68</strong></td><td><strong>59</strong></td><td><strong>67</strong></td><td><strong>582</strong></td></tr><tr><td>Others</td><td>4</td><td>8</td><td>1</td><td>0</td><td>3</td><td>4</td><td>6</td><td>5</td><td>31</td></tr><tr><td>O:6,7 (formerly C1 group)</td><td>13</td><td>22</td><td>21</td><td>14</td><td>18</td><td>11</td><td>9</td><td>9</td><td>117</td></tr><tr><td><strong>Virchow</strong></td><td><strong>0</strong></td><td><strong>3</strong></td><td><strong>3</strong></td><td><strong>2</strong></td><td><strong>3</strong></td><td><strong>1</strong></td><td><strong>1</strong></td><td><strong>2</strong></td><td><strong>15</strong></td></tr><tr><td>Others</td><td>13</td><td>19</td><td>18</td><td>12</td><td>15</td><td>10</td><td>8</td><td>7</td><td>102</td></tr><tr><td>O:6,8 (formerly C2 group)</td><td>21</td><td>17</td><td>5</td><td>8</td><td>17</td><td>13</td><td>4</td><td>11</td><td>96</td></tr><tr><td>Other groups</td><td>15</td><td>7</td><td>22</td><td>38</td><td>2</td><td>23</td><td>33</td><td>12</td><td>152</td></tr><tr><td>Non typable</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>5</td><td>2</td><td>2</td><td>11</td></tr></tbody></table>
<table><caption>TABLE 2. PERCENTAGE OF RESISTANCE TO SPECIFIC ANTIBIOTICS IN S. ENTERICA IN ARAGÓN, SPAIN FROM 2001 TO 2008</caption><thead><tr><th rowspan="2">Antibiotics</th><th colspan="8">% of isolates resistant</th><th rowspan="2">total<br>(n=2092)</th></tr><tr><th>2001<br>(n=304)</th><th>2002<br>(n=394)</th><th>2003<br>(n=334)</th><th>2004<br>(n=346)</th><th>2005<br>(n=197)</th><th>2006<br>(n=207)</th><th>2007<br>(n=172)</th><th>2008<br>(n=138)</th></tr></thead><tbody><tr><td>Ampicillin</td><td>33.5</td><td>38.8</td><td>24.0</td><td>35.2</td><td>43.8</td><td>36.2</td><td>34.8</td><td>59.4</td><td>36.3</td></tr><tr><td>Cefotaxime</td><td>0</td><td>0.2</td><td>0.9</td><td>0.3</td><td>1</td><td>1.9</td><td>1.1</td><td>2.9</td><td>0.8</td></tr><tr><td>Gentamicin</td><td>4.3</td><td>4</td><td>1.2</td><td>1.7</td><td>1</td><td>1.9</td><td>1.7</td><td>10.8</td><td>3</td></tr><tr><td>Nalidixic acid</td><td>28.6</td><td>27</td><td>31</td><td>31</td><td>47.2</td><td>33.3</td><td>30.8</td><td>21.7</td><td>31</td></tr><tr><td>Ciprofloxacin</td><td>0.3</td><td>0.7</td><td>0.2</td><td>0.3</td><td>0</td><td>0.9</td><td>0</td><td>0.7</td><td>0.4</td></tr><tr><td>Co-trimoxazole</td><td>7.8</td><td>12.6</td><td>7.8</td><td>15</td><td>12</td><td>10.6</td><td>12.7</td><td>5</td><td>10.8</td></tr></tbody></table>
<table><caption>TABLE 3. CHARACTERISTICS OF THE S. ENTERICA STRAINS AND THE TRANSCONJUGANTS IN THIS STUDY</caption><thead><tr><th>strain</th><th>serotype</th><th>year</th><th>coresistance phenotype</th><th>ESBL</th><th>CTX</th><th>CTL</th><th>CAZ</th><th>CZL</th><th>CPM</th><th>CPL</th><th>CRO</th><th>CIP</th><th>Inc</th><th>plasmid size</th></tr></thead><tbody><tr><td>02-236146</td><td>Virchow</td><td>2002</td><td>SSpKSuTeTmNal</td><td>CTX-M-9</td><td>&gt;16</td><td>0.047</td><td>&lt;0.5</td><td>0.19</td><td>1</td><td>0.064</td><td>15</td><td>28</td><td>HI2</td><td>290 kb</td></tr><tr><td>236146-TC2</td><td></td><td></td><td>SSpKSuTeTm</td><td>CTX-M-9</td><td>2</td><td>0.032</td><td>&lt;0.5</td><td>0.094</td><td>0.5</td><td>0.064</td><td>17</td><td>35</td><td>HI2</td><td>290 kb</td></tr><tr><td>02-214992</td><td>Virchow</td><td>2002</td><td>SSpKSuTeTmNal</td><td>CTX-M-9</td><td>&gt;16</td><td>0.064</td><td>&lt;0.5</td><td>0.25</td><td>2</td><td>0.064</td><td>16</td><td>26</td><td>HI2</td><td>320 kb</td></tr><tr><td>214992-TC1</td><td></td><td></td><td>SSpKSuTeTm</td><td>CTX-M-9</td><td>2</td><td>0.032</td><td>&lt;0.5</td><td>0.125</td><td>0.38</td><td>&lt;0.064</td><td>20</td><td>35</td><td>HI2</td><td>320 kb</td></tr><tr><td>03-1672608</td><td>Virchow</td><td>2003</td><td>SSpKSuTeTmNal</td><td>CTX-M-9</td><td>16</td><td>0.047</td><td>0.75</td><td>0.19</td><td>1</td><td>&lt;0.064</td><td>16</td><td>26</td><td>HI2</td><td>320 kb</td></tr><tr><td>1672608-TC</td><td></td><td></td><td>SSpKSuTeTm</td><td>CTX-M-9</td><td>2</td><td>0.032</td><td>&lt;0.5</td><td>0.125</td><td>0.38</td><td>&lt;0.064</td><td>16</td><td>35</td><td>HI2</td><td>320 kb</td></tr><tr><td>04-1831083</td><td>Virchow</td><td>2004</td><td>SSpKSuTeTmNal</td><td>CTX-M-9</td><td>16</td><td>0.047</td><td>0.75</td><td>0.125</td><td>1</td><td>0.064</td><td>16</td><td>26</td><td>HI2</td><td>320 kb</td></tr><tr><td>1831083-TC</td><td></td><td></td><td>SSpKSuTeTm</td><td>CTX-M-9</td><td>12</td><td>0.032</td><td>0.5</td><td>0.125</td><td>0.5</td><td>&lt;0.064</td><td>16</td><td>35</td><td>HI2</td><td>320 kb</td></tr><tr><td>06-424914</td><td>Enteritidis</td><td>2006</td><td>SSpSuTeTm</td><td>CTX-M-1</td><td>&gt;16</td><td>0.032</td><td>1.5</td><td>0.19</td><td>3</td><td>&lt;0.064</td><td>15</td><td>35</td><td>I1-N</td><td>100 kb</td></tr><tr><td>424914-TC1</td><td></td><td></td><td>SSpSuTeTm</td><td>CTX-M-1</td><td>&gt;16</td><td>0.023</td><td>1</td><td>0.094</td><td>2</td><td>&lt;0.064</td><td>15</td><td>35</td><td>I1-N</td><td>145 kb</td></tr></tbody></table>

TC: E. coli transconjugant; S: streptomycin; Sp: spectinomycin; K: kanamycin; Su: sulfonamides; Te: tetracyclin; Tm: trimethoprim; Nal: nalidixic acid; CTL: cefotaxime; CIP: ciprofloxacin; Inc: plasmid incompatibility group. All measurements are E-test MICs (µg/ml) except for CRO and CIP when figures represent diameters on disk diffusion test (mm).