Ceftriaxone-Resistant Salmonella enterica Serotype Newport, France

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.

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][2001][2002][2003][2004][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 fl uoroquinolones 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 fi rst 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)(4)(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)(3)(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)(7)(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-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 bla CMY-2 gene, and cefoxitin-susceptible isolates contained the extended-spectrum β-lactamase gene bla CTX-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 bla TEM 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 strep- tomycin and spectinomycin) (11). The chloramphenicol/ fl orfenicol resistance gene fl oR 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-fi eld gel electrophoresis (PFGE) (Figure 1). All 16 Newport MDR-AmpC isolates tested had a common sequence type (ST), ST45. XbaI-PFGE identifi ed 10 distinct profi les (similarity 76.7%) among all 45 Newport MDR-AmpC isolates. Single enzyme matches were found for 3 of the profi les (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 profi les 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 profi les 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 profi les for isolates whose profi les 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 bla CMY-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 bla CMY-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 profi les 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/C 2 replicons (13), whereas Typhimurium plasmid R7 contained the I1 replicon.

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 profi les. Their plas-Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 6, June 2008 955 mids carrying bla CMY-2 were homogeneous (same incompatibility group A/C 2 , 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/C 2 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 identifi ed 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 profi les New5, New6a, and New6b (displayed by 15 isolates among the 45 studied), and bla CMY-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. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 6, June 2008