ABSTRACT
Virulence plasmids and antibiotic resistance plasmids are usually maintained separately in Salmonella spp.; however, we report an instance of a hybrid plasmid (pN13-01125) in Salmonella enterica serovar Dublin. Review of the complete sequence of the 172,265-bp plasmid suggests that pN13-01125 is comprised of the previously described pSDVr and pSH696_135 plasmids and that the mechanism of hybridization likely involves IS6 (IS26) insertion sequence elements. The plasmid has a low conjugation frequency, confers resistance to six classes of antimicrobials, and contains a complete spv virulence operon.
TEXT
Salmonella enterica serovar Dublin (S. Dublin) is a bovine-adapted pathogen that can cause systemic and enteric infections in livestock (1, 2). Exposure to contaminated milk, meat, and infected animals can cause rare cases of human infections and invasive bacteremia (3, 4). Two related ∼80-kbp virulence plasmids are found in S. Dublin and are collectively referred to as pSDV, which encodes factors important for virulence (5, 6), and S. Dublin isolates are increasingly found to maintain plasmids that confer antimicrobial resistance (AMR) (7). We report a novel S. Dublin plasmid isolated from a bovine fecal sample that is a hybrid of known AMR and virulence plasmids.
In 2010, surveillance of clinical bovine samples by the Canadian Integrated Program for Antimicrobial Resistance Surveillance identified an S. Dublin stool isolate (N13-01125) from the western region of Canada with multiple-drug resistance (MDR) to six classes of antibiotics. Whole-genome sequencing of isolate N13-01125 DNA was carried out on the long-read-length Pacific Biosciences RSII platform using a single cell; 38.8-K reads yielded 519 Mb of sequence with an N50 read length of 19.6 kb. Genome assembly was carried out with SMRT analysis software using the hierarchical genome assembly protocol (HGAP) 3.0, and the draft assembly was polished using the RS_Resequencing.1 protocol (8). Plasmid pN13-01125 was resolved to 215-fold coverage and circularized using the Circlator v1.1.1 software (9) to a final length of 172,265 bp. BLAST analysis showed that pN13-01125 had high sequence identity to the S. Dublin virulence plasmid pSDVr (also called pOU1115; accession no. DQ115388) (10), which was likely formed as a cointegrate of pOU1114 and pOU1113 and notably replaced the tra operon for the pil operon. Additionally, pN13-01125 exhibited high sequence identity to the AMR plasmid pSH696_135 (accession no. JN983048) (11). By submission to the Center for Genomic Epidemiology typing web tool (www.genomicepidemiology.org), plasmid incompatibility matches were found to both IncX1 and IncA/C2, which is consistent with the parental plasmids.
Mapping of the contribution of the parental plasmids to the pN13-01125 sequence was carried out using Double Act v2 (http://www.hpa-bioinfotools.org.uk/pise/double_actv2.html) (12). Plasmids pSH696_135 and pSDVr contribute three alternating blocks of sequence each to pN13-01125 (Fig. 1). By querying the ISFinder web tool (http://www-is.biotoul.fr), 11 IS6 family elements were identified (IS26-1 through IS26-10 and IS15DI). An IS26 element unique to pN13-01125 (IS26-1, IS26-2, IS26-3, IS26-4, IS26-5, IS26-8, IS26-9, IS26-10) was found at the junction between each parental sequence. Additionally, IS26-6 is found at a parental junction but was present within pSH696_135.
Sequence features of the hybrid plasmid pN13-01125 (GenBank accession no. KX815983). Protein-coding sequences are represented by boxes along the plasmid backbone and are colored by function (see legend at top right). Outer and inner boxes are drawn 5′→3′ in the clockwise and anticlockwise directions, respectively. Backbone shading indicates the origin of each plasmid sequence, either pSDVr (magenta) (accession no. DQ115388) or pSH696_135 (blue) (accession no. JN983048). Dashed origin shading indicates sequence inversion with respect to parental plasmid. Complete insertion sequence elements are labeled, and their boundaries are indicated with square brackets. Pairs of 8-bp target site duplications are shown (innermost and outermost boxes); each pair has the same color, and a line indicates their position relative to their respective IS6 element. Gene functions are derived from manual BLAST searches, comparison with the annotated sequences of the parental plasmids, and the study review by Harmer and Hall (24).
The sequence of pN13-01125 between IS26-10 and IS26-2 (inclusive of IS26-1) is identical in structure to that of the parental plasmids, while the remainder of the plasmid contains many inversions. The sequence inversions were likely driven by intramolecular IS6 replication in a manner described by He and coworkers (13), where IS6 family replication leaves 8-bp inverted-target site duplications (TSDs). Mapping of the 8-bp regions surrounding each IS6 element revealed pairs of TSDs in the region where there were also many sequence inversions, suggesting that sequence inversion is driven by IS replication (Fig. 1). The rearrangements of plasmid structure is reminiscent of the structure of the AMR-virulence plasmid hybrid pSUO-SEVR1 recently found in S. enterica serovar Enteritidis (14).
MICs were determined by broth microdilution with the Sensititre system (Trek Diagnostic Systems Ltd., Westlake, OH, USA) using established breakpoints from the Clinical and Laboratory Standards Institute for the original isolate N13-01125 and Escherichia coli TOP10 cells transformed with pN13-01125 (15) (Table 1). The patterns of resistance between the N13-01125 isolate and the E. coli isolate carrying pN13-01125 were identical, suggesting that the plasmid was the sole determinant of antibiotic resistance. Streptomycin susceptibility was equivocal because of the intrinsic resistance of E. coli TOP10 cells to this antibiotic. Elevated MICs to streptomycin, β-lactams, gentamicin, chloramphenicol, sulfisoxazole, and tetracycline were attributed to the presence of plasmid-borne strAB, blaCMY-2/blaTEM-1, aadB, floR/cmlA, sul2, and a class A tetA, respectively. N13-01125 was susceptible to the combination of trimethoprim-sulfamethoxazole, meropenem, azithromycin, and both ciprofloxacin and nalidixic acid; no plasmid-borne resistance determinant for these antimicrobials was found. Resistance to xenobiotics and disinfectants may be preserved, as an intact mer operon similar to that found in pDU1358, qacED1, and sugE was maintained in the hybrid plasmid.
MICs of S. Dublin N13-01125 isolate and E. coli TOP10 cell harboring pN13-01125
We found pSH696_135 to be the best single model for the AMR parental origin of pN13-01125; however, two regions of this plasmid differ from pN13-01125. First, an IS1294b at 6.3 kb in pSH696_135 is not present in pN13-01125. As with pN13-01125, this element is not present in some IncA/C plasmids and has been found to promote the mobilization of the nearby blaCMY-2 (16, 17). Second, blaCMY-2 is a part of an ISEcp1-blaCMY-2-blc-sugE transposition unit previously described in some IncA/C plasmids; this region interrupts a tra operon and is responsible for a low observed conjugation frequency in plasmids in which it is found (18). In pSH696_135 and other plasmids, this region is duplicated and divergently arranged, whereas it is found in a single copy in pN13-01125. To test whether the pN13-01125 plasmid is mobile, we carried out conjugation experiments with an isolate of S. Dublin harboring pN13-01125 as the potential donor and either a ciprofloxacin-resistant S. Dublin or a sodium azide-resistant E. coli (J53) isolate as the potential recipient using a previously described protocol (10). Although we were able to produce transconjugants, the frequency was low at ∼10−8, which is consistent with the conjugation frequency of the virulence plasmid pSDVr and the observation that Salmonella virulence plasmids are normally vertically inherited (19).
A single continuous section, from 9.3 to 32.4 kbp, of the virulence plasmid pSDVr was not maintained in pN13-01125. Notably, the two-component systems ccdAB and vagCD involved in plasmid maintenance and the replication genes repAC from pSDVr were not present in pN13-01125. Despite the loss of these genes, pN13-01125 was stably maintained after three rounds of subculture in its native background and in E. coli TOP10 cells. Plasmid replication and stability were likely inherited from the other parental plasmid; with the exception of ∼12 kb of DNA, the entire pSH696_135 plasmid was present in the hybrid.
The spv virulence operon was present in pN13-01125, which was essential and sufficient to restore virulence in various plasmid-cured S. enterica serovars, including S. Dublin (20, 21). The hybrid plasmid also inherited the macrophage-inducible carbonic anhydrase mig-5 and the quorum-sensing factor srgB. The fimbriae biosynthetic cluster (faeAIH and fedH) was not present in the hybrid plasmid, but the impact of the loss of this biosynthetic cluster on S. Dublin virulence is unknown.
Previously, AMR-virulence hybrid plasmids were reported in S. enterica serovars Typhimurium, Cholerasuis, and Enteritidis (14, 22, 23); however, to our knowledge, this report is the first characterization of a hybrid plasmid in S. Dublin. Although pN13-01125 has not maintained fimbriae biosynthesis via the fae cluster, it has maintained the critical spv operon; furthermore, it contains 11 antibiotic and xenobiotic resistance modules, representing the greatest number found in a Salmonella AMR-virulence hybrid plasmid to date. Plasmid hybridization and gene loss may represent a focusing of advantageous genetic content, although the effects on pathogenesis and fitness are unknown. The emergence of a novel virulence-AMR plasmid in S. Dublin and the potential transmission to other Salmonella serovars are of serious concern.
Accession number(s).The sequence of plasmid pN13-01125 was deposited in GenBank under the accession number KX815983.
ACKNOWLEDGMENT
We acknowledge David Boyd and Amrita Bharat for helpful suggestions on the figure and the manuscript.
FOOTNOTES
- Received 9 December 2016.
- Returned for modification 8 January 2017.
- Accepted 28 February 2017.
- Accepted manuscript posted online 20 March 2017.
- © Crown copyright 2017.
The government of Australia, Canada, or the UK (“the Crown”) owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.
