Characterization of pTS14, an IncF2:A1:B1 Plasmid Carrying tet(M) in a Salmonella enterica Isolate

The objective of this study was to explore the genetic and biological features of the tet(M)-harboring plasmid pTS14 in Salmonella enterica strain S14 isolated from a chicken fecal sample. Plasmid pTS14 was identified by conjugation, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and plasmid sequencing. The biological characteristics of pTS14 were assessed via stability, growth kinetics, and starvation survival experiments. Strain S14, belonging to ST3007, harbored a 119-kb tet(M)-bearing IncF2:A1:B1 conjugative plasmid pTS14. The plasmid pTS14 contained a novel transposon Tn6709 with the genetic structure IS26-tnpA1-tnpA2-Δorf13-LP-tet(M)-tnpX-ΔtnpR-IS26, and the resistance genes tet(B), tet(D), strAB, sul2, and blaTEM–1b. In addition, pTS14 was found to be highly stable in the recipient strain E. coli J53. The transconjugant TS14 exhibited a higher survival ratio than E. coli J53 under permanent starvation-induced stress. The tet(M)-bearing IncF2 epidemic plasmid lineage may accelerate the dissemination of tet(M) and other genes by coselection, which could constitute a potentially serious threat to clinical treatment regimens.


INTRODUCTION
The tetracycline resistance gene tet(M) encodes a ribosomal protection protein that confers tetracycline resistance to a variety of bacterial species (Franke and Clewell, 1981;Roberts et al., 1986;Donhofer et al., 2012;Roberts and Schwarz, 2016), including 38 genera of Gram-positive bacteria and 39 genera of Gram-negative bacteria, with most associated with other tet genes 1 , likely through the association with integrative and conjugative transposons located on the chromosome or conjugative plasmids, which facilitate horizontal transfer (Bryan et al., 2004;Jones et al., 2006;Tuckman et al., 2007;de Vries et al., 2009;Hu et al., 2013). In Gram-negative bacteria, the tet(M) gene was first reported in Escherichia coli in 2006 (Jones et al., 2006) and later described in Salmonella enterica isolates from chicken and pig feces in China in 2017 (Ma et al., 2017). To date, several plasmids harboring the tet(M) gene have been reported in E. coli, which include the IncHI2-type plasmids p1106 (MG825373), pECAZ147_2 (CP018993), and pTW4-IncHI2 (MK293945), as well as the hybrid IncN1-IncHI2-type plasmid pHN6DS (MH459020) and IncX1-FI:A:B plasmid pYPE12 (CP041443). The incompatibility group IncF is a main vehicle for the dissemination of the rmtB and/or bla CTX−MS genes in Enterobacteriaceae (Deng et al., 2011;Hou et al., 2012). However, there are relatively few reports of tet(M)-harboring IncF plasmids from Salmonella. Here, we report the complete sequence of the tet(M)-harboring IncF2:A1:B1 plasmid pTS14 isolated from S. enterica. A novel transposon, Tn6709 harboring the tet(M) gene, as well as three other resistance modules, were located on the same plasmid, pTS14. Moreover, the biological characteristics of plasmid pTS14 in Salmonella were further investigated.

Bacterial Strains
During a survey of the tet(M) gene in Henan Province, China, conducted in December 2017, one tet(M)-positive Salmonella strain, named S14, was isolated from the feces of a chicken. Strain identification was confirmed by PCR analysis and 16S rRNA sequencing along with MALDI-TOF MS detection (AXIMA Performance; Shimadzu Corporation, Kyoto, Japan) as described previously (Weisburg et al., 1991;Pavlovic et al., 2013). Subsequently, strain S14 was serotyped according to the Kauffmann-White scheme with the use of commercial antiserum.

Susceptibility Testing and Detection of Tetracycline Resistance Genes
The susceptibility of Salmonella strain S14 to 13 antibiotics (Supplementary Table S1) was determined via the broth microdilution method and interpreted in accordance with the guidelines of the Clinical and Laboratory Standards Institute (2017). Minimum inhibitory concentrations were calculated on three independent occasions. E. coli ATCC 25922 was used as a quality control strain. The tet(A), tet(B), tet(C), and tet(D) genes were screened by PCR as described previously (Sun et al., 2018).

Multilocus Sequence Typing (MLST)
To investigate the genetic typing of the isolate, MLST of seven housekeeping genes (thrA, purE, sucA, hisD, aroC, hemD, and dnaN) was performed as described previously (Yap et al., 2016). The sequences were subsequently submitted to the MLST database 2 and assigned existing or novel allele type identification numbers. The corresponding sequence types were derived from the set of allelic profiles of each of the seven loci.

Conjugation Experiments
Mating experiments were conducted to evaluate the transferability of the tet(M) gene with the tet(M)-positive strain S14 as the donor and rifampicin-resistant Salmonella 2 http://mlst.warwick.ac.uk strain JS-500 and sodium azide-resistant E. coli strain J53 as the recipients. The transconjugants were selected on MacConkey agar plates supplemented with doxycycline (16 mg/L) and rifampicin (400 mg/L) for Salmonella JS-500 or doxycycline (16 mg/L) and sodium azide (200 mg/L) for E. coli J53. The conjugation frequency was calculated as the ratio of the number of transconjugants per recipient. All transconjugants were confirmed by pulsed-field gel electrophoresis (PFGE). The presence of the tet(M) and other tet genes in all transconjugants was confirmed by PCR analysis and sequencing.

S1-PFGE and Southern Hybridization
Prior to PFGE, DNA from the donor strain S14 and the corresponding E. coli J53 transconjugant (named TS14) were treated with S1 nuclease. The location of tet(M) gene was identified by Southern blot hybridization with the use of a probe for the tet(M) gene.

Plasmid Sequencing and Annotation
The plasmid from the transconjugant was extracted using the QIAGEN Plasmid Midi Kit (Qiagen, Hilden, Germany) and sequenced with Illumina Hiseq technology (Illumina, Inc., San Diego, CA, United States). Assembly of the generated sequences with Newbler software v2.6 (Roche Diagnostics Corporation, Indianapolis, IN, United States) generated nine contigs. Gaps between the contigs were closed by PCR and sequencing. The plasmid sequences were initially predicted and annotated using the Subsystem Technology (RAST v2.0) server (Aziz et al., 2008), and corrected manually using the BLASTn and BLASTp algorithms 3 . The plasmid replicon genotype was identified using the PlasmidFinder database 4 . Comparative analysis and generation of plasmid maps were performed using the Python application Easyfig and the BLAST Ring Image Generator (Alikhan et al., 2011;Sullivan et al., 2011).

Biological Characteristics of the tet(M)-Harboring Plasmid pTS14
To assess bacterial growth kinetics, the transconjugant TS14 and recipient E. coli J53 were incubated overnight at 37 • C in lysogeny broth (LB; 5 mL) and then diluted to an optical density at 600 nm (OD 600 ) of 0.004 in 20 mL of fresh LB with or without doxycycline (16 mg/L). Over a 15-h inoculation period, bacterial growth was measured and recorded every hour. To evaluate plasmid stability, transconjugant E. coli TS14 was maintained for 14 days in daily refreshed LB (100-fold dilution) without antibiotic selection, as previously described . Approximately 100 colonies were randomly chosen and replica plated onto LB agar plates with doxycycline. All colonies grown on doxycycline-supplemented agar were subjected to PCR analysis to confirm the presence of the tet(M) gene. M9-glycerin (0.2%) minimum medium was used for starvation survival experiments of E. coli J53, TS14, and a mixture of both. The percent survival was calculated as the ratio of the mean number of CFUs divided by the number of CFUs after overnight incubation FIGURE 1 | Whole-plasmid sequence of pTS14 and comparison of similar IncF2 plasmids. The 150326-bp pST90-1 was used as a reference plasmid at the highest coverage (79%). Key features of pST90-1 are highlighted in different colors. Replicon genes are in blue; transfer-associated genes in cyan; resistance genes in red; mobile elements in yellow; the loci of stability-associated genes (iutA-iucABCD and sitABCD) in maroon; and hypothetical proteins in black. The outer ring comprises the CoDing sequence of pST90-1. The plasmids in this study included pTS14 (IncF2:A1:B1, MN328348), pST90-1 (IncF2:A1:B58, CP050735), pYSP8-1-CTX-M-14 (IncF2:A1:B1, CP037912), pSI108-1 (IncF2:A1:B58, CP050770), pGDD25-16 (IncF2:A1:B1, MH316135), and pCA08 (IncF2:A16:B20, CP009233).

Nucleotide Sequence Accession Number
The complete sequence of plasmid pTS14 was submitted to the GenBank under the accession number MN328348.

RESULTS AND DISCUSSION
Characterization of Salmonella Strain S14 The tet(M)-positive strain S14 was serotyped as S. enterica, belonging to ST3007. The results of the conjugation assay indicated that the tet(M) gene can be successfully transferred to Salmonella JS-500 and E. coli J53 at frequencies of 1.638 × 10 −4 and 1.397 × 10 −4 , respectively, which were assigned as TS14-JS500 and TS14. Susceptibility testing showed that S14, TS14-JS500, and TS14 were all resistant to doxycycline, tetracycline, gentamicin, sulfamethoxazole-trimethoprim, and amoxicillin (Supplementary Table S1). The transconjugant TS14 was selected for further study. S1-PFGE and Southern hybridization indicated that the tet(M) gene was located on a plasmid of ∼119 kb in size, designated as pTS14 (Supplementary Figure S1). The results of the mating experiments demonstrated that plasmid pTS14 harboring tet(M) can be self-transmissible and spread between and across genera.

Characterization of Plasmid pTS14
Sequencing of the plasmid pTS14 from transconjugant TS14 revealed a 119,493-bp circular episome with a mean G + C content of 50.81% with 155 putative open reading frames (ORFs). The plasmid pTS14, belonging to IncF2:A1:B1, has a typical FII-type backbone and three conserved modules (Figure 1): (i) regions encoding the plasmid replication gene repA; (ii) regions responsible for plasmid transfer and two conserved pilusencoding loci (tra and trb); (iii) plasmid maintenance region, plasmid segregation system (including ParAB and PsiAB) and a multiple toxin-antitoxin (TA)-based addiction system (including PemI/PemK, CcdA/CcdB, and Hok); and two accessory regions (regions encoding antimicrobial resistance and virulence).
Comparative analysis revealed that the most noticeable difference among these IncF2 plasmids lay in the region encoding multidrug resistance (MDR) and virulence factors. The MDR region of pTS14 is composed of several segments, successively including a truncated transposon Tn10-like segment carrying the tet(B) and tet(D) genes, a Feo system for ferrous iron utilization, a tet(M)-containing segment, a resistance cluster of sul2-strA-strB, and an incomplete Tn3-harboring bla TEM−1b transposon (Figure 2). The truncated Tn10-like transposon, the first segment, has undergone deletion of IS10L and jemA, which are located upstream of the truncated jemB, and another IS10R that was divided into three segments, which were disrupted by the insertion of IS26 and IS1P with 8-bp direct repeats.
The tet(M)-bearing segment, an IS26-bracked composite transposon, was designated as Tn6709 using the Transposon Nomenclature Database 5 . Tn6709 consists of the insertion sequence IS26, an incomplete transposase tnpA from Tn5403, the incomplete transposase tnpA from the Tn3 family, truncated orf13, the tet(M) leader peptide (LP) gene, the tetracycline resistance gene tet(M), the tnpX and tnpR genes from the Tn1000 transposon, and the IS26 element (Figure 3). Comparative sequence analysis showed that fragment orf13-lptet(M) was relatively conserved with 100% sequence identity to the other six sequences displayed in Figure 3. Interestingly, the 3 -terminal end of the partial or complete sequence encoding a Tn3-family transposase was located upstream of orf13 in six similar sequences and Tn6709, indicating that the transfer of tet(M) was closely related to that of the Tn3 family. Besides, for Tn6709, only two sequences (p41-3 and pTW4) carried the IS26 element upstream of the tnpA or tnpA1 gene. Furthermore, the IS26 element was located downstream of the incomplete tnpR in plasmids pTW4, pYSP8-1-CTX-M-14, and pTS14. The IS26 element plays a key role in the reorganization of the MDR region of plasmids (Harmer et al., 2014). In this study, reverse PCR was performed to explore the existence of a circular intermediate of the IS26-bracked composite transposon Tn6709. However, no circular intermediate could be obtained from strain S14. In addition, sequence analysis revealed that there were no direct repeats flanking the IS26 element in transposon Tn6709, indicating that the IS26-bracked composite transposon Tn6709 acquired by pTS14 may have occurred by recombination rather than transposition.
The tet(M) gene was originally designated by Burdett et al. (1982), and was subsequently found to be frequently connected with transposons in Gram-positive bacteria, such as the Tn916 transposon of Streptococcus faecalis (Roberts and Kenny, 1987), the Tn5801-like transposons of Staphylococcus aureus and Enterococcus faecalis (de Vries et al., 2009), and the Tn5397-like transposons of Clostridioides difficile and Enterococcus faecium (Agerso et al., 2006). In Gram-negative bacteria, the tet(M) gene was first reported in E. coli in 2006 as being flanked by IS26 and ISVs1 (Jones et al., 2006), and was first described in S. enterica isolated from chicken and pig feces in China in 2017 (Ma et al., 2017). The tet(M) gene was found to be associated with the Tn6539 transposon (Sun et al., 2018). In the present study, the tet(M) gene was located on the novel composite transposon Tn6709 in IncF2:A1:B1, related to the epidemic plasmid lineage IncF2. Taken together, these results suggest that the tet(M) gene can transfer from Gram-positive bacteria to Gram-negative bacteria and disseminate between different plasmid groups.

The Biological Characteristics of the Plasmid pTS14
In the bacterial growth experiment, the growth rate of the transconjugant TS14 was similar to that of the recipient E. coli J53 in LB broth without doxycycline, while that with doxycycline was slightly lower than the recipient in LB broth without doxycycline ( Figure 4A). The plasmid pTS14 existed stably in transconjugant TS14 for at least 14 days of passage in an antibiotic-free environment. The survival rate of TS14 was highest, followed by the mixture of TS14 and E. coli J53, and lowest for E. coli J53, which demonstrates that the growth of the transconjugant TS14 harboring pTS14 was not lower than that of E. coli J53. After 15 days, the bacterial numbers of the three cultures all decreased to less than 10% ( Figure 4B). Overall, in this initial study, plasmid pTS14 carrying the Feo system did not affect bacterial growth, although there was no benefit to the transconjugant harboring pTS14. Further investigations are warranted to elucidate the role of the Feo acquisition system of plasmids in Salmonella.

CONCLUSION
In summary, the self-transmissible IncF2:A1:B1 plasmid pTS14 from Salmonella carried the tet(M), tet(B) and tet(D) genes, along with the sul2-strA-strB module and the incomplete bla TEM−1bbearing transposon Tn3. The tet(M) gene was located on the novel composite transposon Tn6709 and flanked by two IS26 elements oriented in the opposite direction. In addition, the plasmid pTS14 harbored a Feo system responsible for ferrous iron transportation.
The epidemic IncF2 plasmids have the potential to carry both virulence and antimicrobial resistance determinants, which might contribute to the further dissemination of the tet(M) gene via co-selection by other antimicrobials. Therefore, there is a need to monitor the dissemination of this MDR-and virulenceassociated plasmid among Enterobacteriaceae.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
GH and YP conceived and designed the experiments. YL, XL, and XC performed the experiments. MC, JL, and YL analyzed the data. SL, DH, and LY contributed to reagents, materials, and analysis tools. YL and GH wrote the manuscript. All authors contributed to the article and approved the submitted version.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.01523/full#supplementary-material FIGURE S1 | S1-PFGE (A) and Southern hybridization (B) of Salmonella strain S14 and the corresponding transconjugant TS14 with the tet(M) gene as a probe. Marker, Salmonella Braenderup H9812.
FIGURE S2 | Comparative analysis of pTS14 with other IncF-type plasmids. Homologous segments generated by a BLASTn comparison (≥97% identity) are shown as gray boxes. Genes are represented by thick arrows. The color code equates to that described in this figure legend.
TABLE S1 | The MICs of S14 and transconjugant in this study.