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Citation: Egorova, Svetlana, Timinouni, Mohammed, Demartin, Marie, Granier, Sophie, Whichard,
Jean, Sangal, Vartul, Fabre, Laëtitia, Delauné, Aurélia, Pardos, Maria, Millemann, Yves, Espié,
Emmanuelle, Achtman, Mark, Grimont, Patrick and Weill, François-Xavier (2008) Ceftriaxone-resistant
salmonella enterica serotype Newport, France. Emerging Infectious Diseases, 14 (6). pp. 954-7. ISSN
1080-6059
Published by: Centres for Disease Control and Prevention
URL: http://dx.doi.org/10.3201/eid1406.071168 <http://dx.doi.org/10.3201/eid1406.071168>
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DISPATCHES
Ce ft ria xone Re sist a nt
Salmonella enterica
Se rot ype N e w por t ,
Fra nc e
Svetlana Egorova,*1 Mohammed Timinouni,*2
Marie Demartin,* Sophie A. Granier,†
Jean M. Whichard,‡ Vartul Sangal,§
Laëtitia Fabre,* Aurélia Delauné,* Maria Pardos,*
Yves Millemann,¶ Emmanuelle Espié,#
Mark Achtman,§** Patrick A.D. Grimont,*
and François-Xavier Weill*
The multidrug-resistant (MDR) Salmonella enterica serotype Newport strain that produces CMY-2 β-lactamase
(Newport MDR-AmpC) was the source of sporadic cases and
outbreaks in humans in France during 2000–2005. Because
this strain was not detected in food animals, it was most likely
introduced into France through imported food products.
T
hird-generation cephalosporins are drugs of choice for
treatment of persons with nontyphoidal Salmonella infections that require chemotherapy or when fluoroquinolones are contraindicated. A new public health concern is
the emergence of third-generation cephalosporin–resistant
Salmonella isolates (1). Multidrug-resistant (MDR) Salmonella enterica serotype Newport isolates that produce
CMY-2, a β-lactamase that inactivates third-generation
cephalosporins, were first reported in the United States in
1998 (2). These isolates, known as Newport MDR-AmpC,
have quickly spread through the United States in cattle and
humans (3–5). It has been hypothesized that use of ceftiofur, a third-generation cephalosporin licensed in the United
States for use in cattle, could have selected for Newport
MDR-AmpC (2–4,7). Several observations and case-control studies suggested beef and milk from dairy cattle were
substantial sources of Newport MDR-AmpC infection in
humans (6–8).
These isolates seem to be extremely rare in Europe.
Two surveys performed in England and Wales (278,308 human Salmonella isolates tested, 1992–2003) and Spain (959
human Salmonella isolates, 1999–2000) did not detect New-
*Institut Pasteur, Paris, France; †Agence Française de Sécurité
Sanitaire des Aliments, Maisons-Alfort, France; ‡Centers for Disease Control and Prevention, Atlanta, Georgia, USA; §Max-PlanckInstitute für Infektionsbiologie, Berlin, Germany; ¶Ecole Nationale
Vétérinaire d’Alfort, Maisons-Alfort; #Institut de Veille Sanitaire,
Saint-Maurice, France; and **University College, Cork, Ireland
954
port MDR-AmpC (9,10). In St. Petersburg, Russia, only 1
Newport MDR-AmpC isolate was reported among 1,078
Salmonella isolates during 2002–2005 (11). In France, a
small outbreak (14 cases) of Newport MDR-AmpC was detected in 2003 and linked to consumption of imported horse
meat (12). We undertook the present study to acquire more
knowledge on circulation of Newport MDR-AmpC in humans, animals, and animal-derived food in France.
The Study
From 2000 through 2005, the French National Reference Centre for Salmonella at the Institut Pasteur in Paris
reported 829 Newport isolates among 69,759 Salmonella
clinical isolates. During this period and depending on the
year, serotype Newport ranked between 6th and 10th in
prevalence among human serotyped isolates. From 2000
through 2005, the Agence Française de Sécurité Sanitaire des Aliments reported 2,160 Newport isolates among
101,791 Salmonella isolates collected from animals and
food products.
Antimicrobial drug susceptibility testing was performed on 585 human Newport isolates and 342 nonhuman Newport isolates by disk diffusion with 32 antimicrobial drugs (additional information available from fxweill@
pasteur.fr). Data for Newport human isolates are shown in
the Table. Of 585 isolates tested, 46 (7.9%) were resistant
to third-generation cepalosporins. The geographic origin
of the isolates was mainly the Paris metropolitan area and
northern France (online Appendix Table, available from
www.cdc.gov/EID/content/14/6/954-appT.htm). There was
a high prevalence of third-generation cephalosporin–resistant isolates during 2000 (15%) and 2003 (17.5% caused
by a small outbreak). No third-generation cephalosporin
resistance was detected in any of the nonhuman Newport
isolates tested.
Experiments were performed on the 46 third-generation cephalosporin–resistant Newport isolates (additional
information available from fxweill@pasteur.fr). All but 1
of the Newport isolates were resistant to cefoxitin (online
Appendix Table). These isolates showed 4 resistance phenotypes; most (41, 89.1%) were resistant to streptomycin,
sulfonamides, chloramphenicol, and tetracycline. PCR and
sequencing showed that the 45 isolates resistant to cefoxitin
were positive for the blaCMY-2 gene, and cefoxitin-susceptible
isolates contained the extended-spectrum β-lactamase gene
blaCTX-M-1. Ceftriaxone MICs of Newport MDR-AmpC isolates ranged from 32 mg/L to >256 mg/L, and ceftazidime
MICs ranged from 64 mg/L to >256 mg/L. No blaTEM genes
were detected. Three isolates with additional resistance to
aminoglycosides contained a class 1 integron with the 1-kb
gene cassette aadA24 (known to encode resistance to strep1
2
Current affiliation: Institut Pasteur, St. Petersburg, Russia.
Current affiliation: Institut Pasteur, Casablanca, Morocco.
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 6, June 2008
Ceftriaxone-Resistant S. enterica Newport, France
Table. Resistance to specific antimicrobial drugs in Salmonella enterica serotype Newport from humans in France, 2000–2005*
% Resistant isolates
2000 (n = 100)
2001 (n = 124)
2002 (n = 66)
2003 (n = 126)
2004 (n = 91)
2005 (n = 78)
Drug
(N = 109)
(N = 134)
(N = 71)
(N = 138)
(N = 94)
(N = 80)
Amoxicillin
27
9.7
1.5
19.8
8.8
3.8
Ceftriaxone/ceftazidime
15
4
1.5
17.5
2.2
0
Gentamicin
4
1.6
0
1.6
2.2
0
Nalidixic acid
23
7.3
4.5
1.6
4.4
2.6
Ciprofloxacin
0
0
0
0
0
0
Sulfonamides
29
10.5
4.5
19.8
8.8
0
Trimethoprim
10
4
3
1.6
4.4
0
Chloramphenicol
25
9.7
1.5
15.9
8.8
0
Tetracycline
27
11.3
3
19
9.9
3.8
*n, no. of isolates studied; N, no. of isolates received at the French National Reference Centre for Salmonella (1 per patient).
tomycin and spectinomycin) (11). The chloramphenicol/
florfenicol resistance gene floR was detected in all but 1
CMY-2–producing Newport isolate.
Clonal relatedness of Newport isolates was assessed by
multilocus sequence typing (MLST) and PulseNet standard
method pulsed-field gel electrophoresis (PFGE) (Figure 1).
All 16 Newport MDR-AmpC isolates tested had a common sequence type (ST), ST45. XbaI-PFGE identified 10
distinct profiles (similarity 76.7%) among all 45 Newport
MDR-AmpC isolates. Single enzyme matches were found
for 3 of the profiles (15 isolates) in the US PulseNet national
database (www.cdc.gov/pulsenet; online Appendix Table;
Figure 2). Two PFGE types (New6 and New8) were divided into 2–4 subtypes because of additional band(s) <100
kb. Isolates from the 2003 outbreak showed 4 similar but
distinct PFGE profiles that differed by 1–2 bands, migrated
between 60 and 100 kb, and were attributed to plasmid(s)
(additional information available from fxweill@pasteur.
fr). If only cases with indistinguishable PFGE profiles had
been tested, potentially related cases would not have been
linked to this outbreak. Therefore, during an outbreak investigation of Newport MDR-AmpC, analysis of plasmid
content (either by alkaline lysis or S1 nuclease, depending
on size of additional bands) might complete XbaI-PFGE
profiles for isolates whose profiles differ by 1 or 2 additional bands of low molecular mass.
Alkaline lysis extraction showed that all but 1 of the
Newport MDR-AmpC isolates harbored a plasmid >125 kb
that hybridized with a blaCMY-2 probe; the remaining isolate harbored a plasmid of 100 kb (online Appendix Table).
Analysis with S1 nuclease showed that these plasmids were
100 kb–370 kb. Up to 3 additional plasmids (3.5 kb–100
kb) that did not have blaCMY-2 were detected in most isolates (online Appendix Table). Cephalosporin resistance
was transferred by electroporation of plasmid DNA to
Eschericha coli DH10B for all 38 CMY-2–positive isolates tested. When present in the donor strain, resistance
to sulfonamides, chloramphenicol, and tetracycline was
also transferred. Restriction analysis of plasmids isolated
from transformants showed 6 similar restriction profiles
for Newport isolates (R1–R6) (Figure 2, online Appendix
Table). R1 was predominant (found in 26 isolates among
35 tested, 74.3%). Newport plasmids R1–R6 and Agona
plasmid R8 were shown by PCR to contain variant A/C2
replicons (13), whereas Typhimurium plasmid R7 contained the I1 replicon.
PstI-digested plasmids analyzed by Southern hybridization with a blaCMY-2 probe (Figure 2) showed 4 hybridization profiles among Newport isolates. Profile H1 corresponded to plasmid type C described by Carattoli et al.
(14). Profiles H2, H3, and H4 differed from H1 by 1 additional band (>10 kb for H2, 3.2 kb for H3, and >18 kb for
H4), which indicated that the blaCMY-2 gene was partially or
totally duplicated.
Conclusions
Newport MDR-AmpC isolates have been the source
of sporadic cases and small outbreaks in humans in France
during 2000–2005. All isolates had the same MLST type,
ST45, and highly similar XbaI-PFGE profiles. Their plas-
Figure 1. Representative XbaI pulsed-field gel electrophoresis
(PFGE) profiles of third-generation cephalosporin–resistant
Salmonella Newport isolates studied. A dendrogram was generated
with Bionumerics software (Applied Maths, Sint-Martens-Latem,
Belgium). The PFGE profile (and if there were indistinguishable
isolates in the PulseNet USA database [www.cdc.gov/pulsenet],
the corresponding Centers for Disease Control and Prevention
PulseNet profile), the number of isolates, and the β-lactamase
genes are indicated.
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 6, June 2008
955
DISPATCHES
mids carrying blaCMY-2 were homogeneous (same incompatibility group A/C2, a main restriction type R1, and a main
hybridization type H1). These results support clonal expansion of 1 Newport strain (or a limited number of genetically related Newport strains) able to acquire and maintain
a large incA/C2 MDR plasmid.
The source of the French isolates remains unknown.
However, this strain was not found in French food animals
or domestically produced food products (additional information available from fxweill@pasteur.fr). One outbreak
during the study period was linked to imported horse meat.
Further investigation identified the source as a wholesaler
who imported meat from Belgium, the United Kingdom,
Hungary, Canada, Brazil, Argentina, Uruguay, and Australia (12). In contrast to Europe, Newport MDR-AmpC has
been frequently seen in the United States during the past decade. Furthermore, several characteristics were shared between US and French Newport MDR-AmpC isolates: ST45
(15), PFGE profiles New5, New6a, and New6b (displayed
by 15 isolates among the 45 studied), and blaCMY-2 plasmid
hybridization type H1 (14). We can reasonably hypothesize
that during 2000–2005 some isolates likely entered France
from North America through imported food. Alternatively,
they could have come to France and North America from
some other country.
Acknowledgments
We thank all corresponding laboratories of the French National Reference Centre for Salmonella and the Agence Française
de Sécurité Sanitaire des Aliments networks for participation in
the study, and Peter Gerner-Smidt for comparing PFGE profiles
with those of the PulseNet USA database.
S.E. and M.T. were supported by a grant from the Réseau
International des Instituts Pasteur. M.A. was supported by grant
05/FE1/B882 from the Science Foundation of Ireland.
Dr Egorova is a microbiologist in the laboratory of intestinal infections at the Institut Pasteur in St. Petersburg, Russia.
Her primary research interests are molecular epidemiology and
antimicrobial resistance surveillance of S. enterica.
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Figure 2. Representative PstI restriction profiles (A) and blaCMY-2
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Address for correspondence: François-Xavier Weill, Centre National
de Référence des Salmonella, Laboratoire des Bactéries Pathogènes
Entériques, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX
15, France; email: fxweill@pasteur.fr
All material published in Emerging Infectious Diseases is in the
public domain and may be used and reprinted without special
permission; proper citation, however, is required.
Se a rc h
pa st I ssue s
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