Diversity of Plasmids Encoding Virulence and Resistance Functions in Salmonella enterica subsp. enterica Serovar Typhimurium Monophasic Variant 4,[5],12:i:- Strains Circulating in Europe

Patricia García,Katie L Hopkins,Janine Beutlich,Vanesa García,Reiner Helmuth,M Carmen Mendoza,Beatriz Guerra,John Threlfall,Dik Mevius,M Rosario Rodicio,Dipshikha Chakravortty

doi:10.1371/journal.pone.0089635

Abstract

Plasmids encoding resistance and virulence properties in multidrug resistant (MDR) Salmonella enterica (S.) serovar Typhimurium monophasic variant 4,[5],12:i:- isolates recovered from pigs and humans (2006-2008) in Europe were characterised. The isolates were selected based on the detection by PCR-amplification of S. Typhimurium virulence plasmid pSLT genes and were analysed by multi-locus sequence typing (MLST). The resistance genes present in the isolates and the association of these genes with integrons, transposons and insertion sequences were characterised by PCR-sequencing, and their plasmid location was determined by alkaline lysis and by S1-nuclease pulsed-field gel electrophoresis (PFGE) Southern-blot hybridisation. Plasmids were further analysed by replicon typing, plasmid MLST and conjugation experiments. The 10 S. 4,[5],12,i:- selected isolates belonged to ST19. Each isolate carried a large plasmid in which MDR with pSLT-associated virulence genes were located. After analysis, eight different plasmids of three incompatibility groups (IncA/C, IncR and IncF) were detected. Two IncA/C plasmids represented novel variants within the plasmid family of the S. 4,[5],12:i:- Spanish clone, and carried an empty class 1 integron with a conventional qacEΔ1-sul1 3′ conserved segment or an In-sul3 type III with estX-psp-aadA2-cmlA1-aadA1-qacH variable region linked to tnpA440-sul3, part of Tn2, Tn21 and Tn1721 transposons, and ISCR2. Four newly described IncR plasmids contained the resistance genes within In-sul3 type I (dfrA12-orfF-aadA2-cmlA1-aadA1-qacH/tnpA440-sul3) and part of Tn10 [tet(B)]. Two pSLT-derivatives with FIIs-ST1+FIB-ST17 replicons carried cmlA1-[aadA1-aadA2]-sul3-dfrA12 and bla TEM-1 genes linked to an In-sul3 type I integron and to Tn2, respectively. In conclusion, three emerging European clones of S. 4,[5],12:i:- harboured MDR plasmids encoding additional virulence functions that could contribute significantly to their evolutionary success.

Highlights

The emergence of multiple clones of Salmonella enterica (S.) serovar Typhimurium-like strains lacking expression of the second flagellar antigen (S. 4,[5],12:i:- throughout the manuscript) has been reported worldwide [1]
The two major clones circulating in Europe show multidrug resistance (MDR) to four or more unrelated classes of antimicrobials, making continued surveillance of their emergence and spread important
In order to investigate the presence and diversity of plasmids encoding resistance and virulence properties, which could give a selective advantage to emerging clones of S. 4,[5],12:i:- in Europe, isolates collected from different sources and countries within the European Union Network of Excellence (NoE) Med-Vet-Net were analysed

Summary

Introduction

The emergence of multiple clones of Salmonella enterica (S.) serovar Typhimurium-like strains lacking expression of the second flagellar antigen (S. 4,[5],12:i:- throughout the manuscript) has been reported worldwide [1]. The two major clones circulating in Europe (labelled as Spanish and European clones) show multidrug resistance (MDR) to four or more unrelated classes of antimicrobials, making continued surveillance of their emergence and spread important. Isolates belonging to the European clone exhibit resistance to ampicillin, streptomycin, sulphonamides and tetracycline (tetraR-type AMP-STR-SUL-TET). The isolates belonging to the Spanish clone show a MDR phenotype with additional resistance to chloramphenicol, gentamicin and trimethoprim (AMP-CHLGEN-STR/SPE-SUL-TET-TMP or variants therein) mediated by large non-conjugative plasmids (IncA/C or IncA/C+IncN plasmids of the pUO-STmRV1-like group) carrying the spv locus of the Salmonella virulence plasmids [1,5,6]. In order to investigate the presence and diversity of plasmids encoding resistance and virulence properties, which could give a selective advantage to emerging clones of S. In order to investigate the presence and diversity of plasmids encoding resistance and virulence properties, which could give a selective advantage to emerging clones of S. 4,[5],12:i:- in Europe, isolates collected from different sources and countries within the European Union Network of Excellence (NoE) Med-Vet-Net were analysed

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