Worldwide, nontyphoidal *Salmonella enterica* is a major cause of foodborne illness, and Enteritidis is one of the most commonly reported serovars ([www.who.int/salmsurv/links/GSSProgressReport2005.pdf](http://www.who.int/salmsurv/links/GSSProgressReport2005.pdf)). In industrialized countries, *S*. *enterica* serovar Enteritidis commonly causes self-limiting gastroenteritis, for which treatment with antimicrobial drugs is usually not needed. However, in developing countries, this serovar, together with serovar Typhimurium, frequently causes invasive infections and substantial illness and death among young children with underlying diseases and among adults with HIV infection ([@R1]). Although antimicrobial drug resistance is not as high in *S. enterica* serovar Enteritidis as in other zoonotic disease serovars, multidrug-resistance (resistance to [\>]{.ul}4 antimicrobial drugs) has been increasingly reported ([@R2]), threatening treatment success for patients with severe infections. In recent years, in association with multidrug resistance, another trend has arisen: the emergence of virulence-resistance (VR) plasmids; these are hybrid plasmids that harbor resistance (R) and virulence (V) determinants. The appearance of these plasmids is of concern because they could lead to the co-selection of virulence (in addition to resistance) through the use of antimicrobial drugs ([@R3]*,*[@R4]). One such plasmid, pUO-SeVR1, has been recently reported in a multidrug-resistant (MDR) clinical isolate of *S. enterica* serovar Enteritidis (CNM4839/03) from Spain ([@R5]). This mobilizable plasmid of ≈100 kb derives from pSEV, the serovar-specific V plasmid of *S. enterica* serovar Enteritidis, and carries most of its V determinants, including the *spvRABCD* locus (*Salmonella* plasmid virulence). This plasmid greatly increases the ability of salmonellae to proliferate intracellularly and has been associated with severe infections in humans ([@R6]). The plasmid also harbors several R genes---*bla*~TEM-1,~ *catA2, strA*-*strB, sul1, sul2, tet*(A)---and a class-1 integron with the 700-bp/*dfrA7* variable region, which confer resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim (R-type ACSSuTTm). To investigate their international spread, we studied the presence of *S. enterica* serovar Enteritidis isolates carrying pUO-SeVR1--like plasmids in the United Kingdom.

The Study
=========

We screened 31,615 *S. enterica* serovar Enteritidis isolates that had been collected from clinical specimens during 2005--2010 and deposited in the culture collection of the Health Protection Agency *Salmonella* Reference Unit. We screened the isolates for R-type ACSSuTTm. A total of 14 serovar Enteritidis isolates showing this resistance phenotype were detected and subsequently examined for the presence of integron-located *dfrA7*. Of the 14 isolates, 11 were positive and their plasmid content was analyzed by S1 pulsed-field gel electrophoresis (PFGE) ([@R2]) and by the Kado and Liu methods ([@R7]); we used serovar Enteritidis strains NRL-Salm-PT4 and CNM4839/03 as controls for pSEV-- and pUO-SeVR1--carrying isolates, respectively. The 11 isolates harbored 1 plasmid of variable size (60--95 kb); among these, 9 isolates hybridized with *dfrA7*-specific and *spvC*-specific probes (with plasmids of 85--95 kb) ([Figure](#F1){ref-type="fig"}). These 9 isolates contained a VR-hybrid plasmid similar to pUO-SeVR1 and were selected for further analyses ([Table 1](#T1){ref-type="table"}, [Table 2](#T2){ref-type="table"}). The remaining 2 isolates carried the normal pSEV plasmid (60 kb), in which *spvC* hybridized; *dfrA7* was chromosomally located.

![Genomic macrorestriction of *Salmonella* *enterica* serovar Enteritidis isolates: pulsed-field gel electrophoresis profiles for *Xba*I (left panel) and S1 (right panel). Lane M, *Xba*I-digested DNA of *S. enterica* serovar Braenderup H9812, used as size standard; lane 1, NRL-Salm-PT4; lane 2, CNM4839/03; lane 3, H051860415; lane 4, H070360201; lane 5, H070420137; lane 6, H073180204; lane 7, H091340084; lane 8, H091800482; lane 9, H095100307; lane 10, H100240198; lane 11, H101700366. The strain NRL-Salm-PT4 was used as control for the most commonly found *Xba*I-profile in *S*. *enterica* serovar Enteritidis.](12-0063-F){#F1}

###### Epidemiologic information for multidrug-resistant *Salmonella* *enterica* serovar Enteritidis isolates, 2005--2010, UK\*

  Isolate no.   Date of isolation   Source   Recent travel history   African patient name   Patient age, y
  ------------- ------------------- -------- ----------------------- ---------------------- ----------------
  CNM4839/03†   2003                Feces    Unknown                 No                     3
  H051860415    2005 Apr 19         Blood    Nigeria                 No                     38
  H070360201‡   2007 Jan 14         Blood    Unknown                 Yes                    35
  H070420137‡   2007 Jan 15         Feces    Unknown                 Yes                    35
  H073180204    2007 Jul 31         Blood    Unknown                 Yes                    34
  H091340084    2009 Mar 15         Feces    Uganda                  No                     59
  H091800482    2009 Apr 17         Blood    Unknown                 Yes                    30
  H095100307§   2009 Dec 7          Blood    Unknown                 Yes                    68
  H100240198§   2010 Jan 9          Blood    Unknown                 Yes                    68
  H101700366§   2010 Apr 22         Blood    Unknown                 Yes                    68

\*All isolates contained a virulence-resistance hybrid plasmid similar to pUO-SeVR1. †Control isolate from Spain. ‡Recovered from the same patient. §Recovered from the same patient.

###### Characteristics of *Salmonella* *enterica* serovar Enteritidis isolates harboring pUO-SeVR1-like plasmids, 2005--2010, UK\*

  Isolate no.   Phage type   Resistance phenotype/ genotype                                                              Class 1 integron†   pSEV genes‡                                                                            MLVA                  MLST     VR plasmid, kb
  ------------- ------------ ------------------------------------------------------------------------------------------- ------------------- -------------------------------------------------------------------------------------- --------------------- -------- ----------------
  CNM4839/03    PT 14b       AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/*dfrA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5,* *srgB, srgC, pefA, pefB, pefC, pefD*        2-12-9-4-4-3-NA-8-8   ST11     100
  H051860415    PT 42        AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/*dfrA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5, srgB, srgC, pefA, pefB, pefC, pefD*          2-13-9-4-4-3-NA-8-8   ST1479   95
  H070360201    PT 42        AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/*dfrA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5, ΔsrgA, srgB, srgC, pefA, pefB*               2-15-9-4-4-3-NA-8-8   ST1479   88
  H070420137    PT 42        AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/*dfrA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5, ΔsrgA, srgB, srgC, pefA, pefB*               2-15-9-4-4-3-NA-8-8   ND       88
  H073180204    PT 42        AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/*dfrA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5, srgB, srgC, pefA, pefB, pefC, pefD*          2-13-9-4-4-3-NA-8-8   ND       92
  H091340084    PT 42        AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/*dfrA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5, ΔsrgA, srgB, srgC, pefA, pefB, pefC, pefD*   2-9-9-4-4-3-NA-8-8    ST11     90
  H091800482    PT 42        AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/*dfrA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5, srgB, srgC, pefA, pefB, pefC, pefD*          2-13-9-4-4-3-NA-8-8   ND       92
  H095100307    PT 42        AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/*dfrA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5, ΔsrgA, srgB, srgC, pefA, pefB, pefC, pefD*   2-13-9-4-4-3-NA-8-8   ND       95
  H100240198    PT 42        AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/d*frA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5, ΔsrgA, srgB, srgC, pefA, pefB, pefC, pefD*   2-13-9-4-4-3-NA-8-8   ND       95
  H101700366    PT 42        AMP, CHL, STR, SUL, TET, TMP/*bla*~TEM-1,~*catA2, strA-strB, sul1, sul2, tet*(A), *dfrA7*   700 bp/*dfrA7*      *spvA, spvB, spvC, spvR, rsk, rck, mig-5, ΔsrgA, srgB, srgC, pefA, pefB, pefC, pefD*   2-13-9-4-4-3-NA-8-8   ND       95

\*pSEV, serovar-specific V plasmid of *S.enterica* serovar Enteritidis; MLVA, multilocus variable number tandem repeat analysis; MLST, multilocus sequence typing; VR, virulence-resistance; PT, phage type; NA, no amplification from this locus; AMP, ampicillin; CHL, chloramphenicol; STR streptomycin; SUL, sulfonamides; TET, tetracycline; TMP, trimethoprim; ST, sequence type; ND, not done. †Size of the variable region amplified with the 5′CS and 3′CS primers ([@R2]). ‡All plasmids were positive for IncFIIA, IncFIB and the *par* locus. Two new primer pairs were devised for detection of *srgA*: srgAB-Fw1/Rv1 (5′-CGCCTTCCGTGTATGTCC/GCGAGTCACTCACCGACAG-3′) and srgAB-Fw2/Rv2 (5′-GTTGCACAGGAGTGGGAGTC/GTCCGGGTTCCATGTCAG-3′). The forward primers anneal at different positions within *srgA*; the reverse primers anneal at different positions within *srgB*.

In the 9 isolates carrying VR-hybrid plasmids, the R-type ACSSuTTm was encoded by the R-genes *bla*~TEM-1,~ *catA2*, *strA-strB*, *sul1*, *sul2*, *tet*(A), and *dfrA7*, which were located on the pUO-SeVR1--like plasmids as determined by Southern blot hybridization. By PCR amplification, using previously described primers and conditions ([@R5]*,*[@R8]) ([Table 2](#T2){ref-type="table"}), and by Southern blot hybridization ([@R5]), we tested for the presence of IncFII and IncFIB replicons, *parAB* (partition), *spvRABCD*, *rck* (resistance to complement killing), *mig-5* (macrophage-induced gene), *pefABCDI* (Pef fimbriae operon), and *srgA* (SdiA-regulated gene; next to *orf7*), all carried by pSEV. The 9 plasmids were positive for the 2 replicons and for all genes screened except *pefC* and *pefD* (absent in H070360201 and H070420137), *pefI*-*orf7* (absent in all), and *srgA* (either absent \[H051860415, H073180204, and H091800482\] or truncated \[in the remaining isolates\]) ([Table 2](#T2){ref-type="table"}).

Isolate subtyping was conducted by phage typing, multilocus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), and *Xba*I-PFGE ([@R9]*,*[@R10]) (<http://mlst.ucc.ie/mlst/dbs/Senterica>; [www.pulsenetinternational.org](http://www.pulsenetinternational.org)). The 9 *S. enterica* serovar Enteritidis isolates belong to phage type (PT) 42, in contrast with CNM4839/03, which belongs to PT14b ([Table 2](#T2){ref-type="table"}). We identified 4 MLVA profiles, which were all single-locus variants of the highly variable locus SENTR5, indicating that the isolates are closely related ([Table 2](#T2){ref-type="table"}). The isolate from Spain and 3 of the 9 isolates from the United Kingdom, selected as representative of each MLVA profile, were also analyzed by MLST ([Table 2](#T2){ref-type="table"}). CNM4839/03 and H091340084 were assigned to sequence type (ST) 11, the most commonly found ST in serovar Enteritidis (<http://mlst.ucc.ie/mlst/>). The remaining 2 isolates could be ascribed to ST1479, the first examples of this single-locus variant of ST11 in the MLST database ([Table 2](#T2){ref-type="table"}). According to *Xba*I-PFGE, the control strain NRL-Salm-PT4 showed a clearly distinct profile in comparison with CNM4839/03 and the 9 isolates containing pUO-SeVR1--like plasmids, which generated 6 closely related patterns ([Figure](#F1){ref-type="fig"}). Most isolates differed by 1 band of variable size (85--95 kb), which corresponded to the *Xba*I-linearized VR-hybrid plasmids. As an exception, isolate H070420137 showed additional differences in chromosomal bands of ≈150 and ≈300 kb. Because isolates H070420137 and H070360201 came from the same patient ([Table 1](#T1){ref-type="table"}) and shared the same V and R genotypes and other typing markers, 1 isolate could have evolved from the other; however, co-infection of the patient with 2 closely related strains cannot be ruled out. In addition, considering the typing results of H095100307, H100240198, and H101700366, the identical size of their plasmids, and the fact that they were isolated from the same patient ([Table 1](#T1){ref-type="table"}), these 3 isolates can be considered the same strain.

All except 2 of the UK isolates carrying pUO-SeVR1--like plasmids were recovered from the blood of patients who had recently returned from an African country or who had an African name ([Table 1](#T1){ref-type="table"}). Supporting the possible African origin, similar *Xba*I-PFGE profiles and MDR phenotypes (ampicillin, trimethoprim, sulfonamides, and tetracycline) have been identified in clinical *S. enterica* serovar Enteritidis isolates involved in outbreaks and community infections in different African countries. These isolates caused bacteremia, meningitis, diarrhea ([@R11]*--*[@R13]), and high case-fatality rates; they affected mainly children, whereas most clinical isolates analyzed in our study were obtained from adults with bacteremia ([Table 1](#T1){ref-type="table"}). The detection of the 700-bp/*dfrA7* integron in *S. enterica* serovar Enteritidis isolates from Africa ([@R14]) also supports an African origin of the MDR serovar Enteritidis isolates harboring pUO-SeVR1--like plasmids. Of note, resistance derivatives of pSLT, the V-plasmid specific to *S*. Typhimurium, have been found in the epidemic ST313 clone of this serovar, which has been considered a major cause of invasive disease in sub-Saharan Africa ([@R15]).

Conclusions
===========

Closely related MDR *S. enterica* serovar Enteritidis isolates carrying pUO-SeVR1--like plasmids were recovered in the United Kingdom. Most were isolated from the blood of patients linked to Africa, and they showed common features with serovar Enteritidis isolates involved in outbreaks on that continent. The possibility that this potentially invasive clone of *S. enterica* serovar Enteritidis can be spread through human travel, together with the detection of VR-plasmids in the serovar most frequently associated with human infections, is of public health concern and requires surveillance.

*Suggested citation for this article*: Rodríguez I, Rodicio MR, Guerra B, Hopkins KL. Potential international spread of multidrug-resistant invasive *Salmonella enterica* serovar Enteritidis. Emerg Infect Dis \[serial on the Internet\]. 2012 Jul \[*date cited*\]. <http://dx.doi.org/10.3201/eid1807.120063>

We thank B. Malorny and E. Junker for help with MLVA typing; A. Schroeter, J. Ledwolorz, and staff of the Health Protection Agency *Salmonella* Reference Unit for phage typing; and I. Montero for help with MLST. We are grateful to R. Helmuth for his advice and critical reading of the manuscript.

This study was supported by the Health Protection Agency, the Federal Institute for Risk Assessment (BfR-46-001; 45-005), and the "Fondo de Investigación Sanitaria" of the "Instituto de Salud Carlos III" of Spain (project FIS PI11/00808, cofunded by the European Regional Development Fund of the European Union: a Way of Making Europe). I.R. received a postdoctoral grant from Fundación Ramón Areces, Madrid, Spain.

For strain requests, contact the Health Protection Agency Colindale, email: katie.hopkins\@hpa.org.uk.

Dr Rodríguez is a postdoctoral fellow at the Federal Institute for Risk Assessment in Berlin, Germany, in the Unit of Antimicrobial Resistance and Resistance Determinants of the Department of Biological Safety. Her research focuses on the genotypic characterization of antimicrobial drug resistance and the molecular epidemiology of gram-negative drug-resistant bacteria.
