A Small Multihost Plasmid Carrying erm(T) Identified in Enterococcus faecalis

The aim of this study was to determine the mobile genetic elements involved in the horizontal transfer of erm(T) in Enterococcus faecalis, and its transmission ability in heterologous hosts. A total of 159 erythromycin-resistant enterococci isolates were screened for the presence of macrolide resistance genes by PCR. Whole genome sequencing for erm(T)-carrying E. faecalis E165 was performed. The transmission ability in heterologous hosts was explored by conjugation, transformation, and fitness cost. The erm(T) gene was detected only in an E. faecalis isolate E165 (1/159), which was located on a 4,244-bp small plasmid, designed pE165. Using E. faecalis OG1RF as the recipient strain, pE165 is transferable. Natural transformation experiments using Streptococcus suis P1/7 and Streptococcus mutans UA159 as the recipients indicated it is transmissible, which was also observed by electrotransformation using Staphylococcus aureus RN4220 as a recipient. The erm(T)-carrying pE165 can replicate in the heterologous host including E. faecalis OG1RF, S. suis P1/7, S. mutans UA159, and S. aureus RN4220 and conferred resistance to erythromycin and clindamycin to all hosts. Although there is no disadvantage of pE165 in the recipient strains in growth curve experiments, all the pE165-carrying recipients had a fitness cost compared to the corresponding original recipients in growth competition experiments. In brief, an erm(T)-carrying plasmid was for the first time described in E. faecalis and as transmissible to heterologous hosts.


INTRODUCTION
Macrolides are a class of important natural or semisynthetic antibiotics that bind to the 50S ribosomal subunit and inhibit protein synthesis (1,2). They have antimicrobial activity against Gram-positive and selected Gram-negative organisms (3,4). The frequent use of macrolides in medical clinics and animal husbandry is accompanied by increased macrolide resistance, which may result in a failure of the treatment (5).
There are three ways to acquire macrolide resistance: modification of the target site, efflux pump, and drug inactivation (2,5). Modification of the target site is mediated by 23S rRNA methylation enzymes, encoded by erm genes that confer resistance to macrolides; in the case of constitutive expression, they can also confer resistance to lincosamides and streptogramin B (6)(7)(8)(9)(10)(11). Among the different erm genes currently known to occur in the different species of bacteria, erm(A) and erm(B) are most frequent (12,13), mainly carried by a plasmid, transposons, translocatable units (TUs), and integrative and conjugative elements (ICEs) (13). Ribosomal protection gene msr codes for ABC-F protein confers macrolide and streptogramin B resistance. The mef gene codes for an efflux pump confers macrolide resistance only. Drug inactivation enzymes including phosphotransferases and esterases, which are encoded by mph genes and ere genes, respectively, confer macrolide resistance (13,14).
Since erm(T) had been detected in Lactobacillus, it has also been described in isolates of the bacterial species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus gallolyticus, Staphylococcus aureus, Erysipelothrix rhusiopathiae, Haemophilus parasuis, and Enterococcus faecium and Streptococcus suis (15)(16)(17)(18)(19)(20)(21)(22), which revealed its widespread presence. However, whether it is transmissible and whether the erm(T)-carrying mobile genetic elements can replicate in a heterologous host had not been fully explored. So, in this study, the presence of erm(T) in enterococci was investigated and the associated mobile genetic elements involved in the horizontal transfer of erm(T) were explored. In addition, the transmission ability and maintenance of erm(T)-carrying plasmid in the heterologous host was elucidated using conjugation, transformation, and fitness cost experiments.

Bacterial Strains and Antimicrobial Susceptibility Testing
During the routine survey for the presence of erm(T) in enterocci of swine origin, a total of 159 non-duplicate enterococci isolates with erythromycin MICs of no <8 mg/L were collected in July and September 2018 from anal swabs of healthy pigs at two farms in Henan Province, China.
Antimicrobial susceptibility testing (AST) was carried out by broth microdilution according to recommendations given in document M100 (Twenty-Eighth Edition) issued by CLSI (21). S. aureus ATCC 29213 served as the quality control strain.

PCR Analysis
The aforementioned erythromycin-resistant enterococci strains were detected for the presence of macrolide resistance gene erm(T) and other resistance genes by PCR using the primers listed in Table 1 (15,(23)(24)(25)(26). PCR products for erm(T) in E. faecalis E165 and its transconjugants and transformants were subjected to Sanger sequencing.

Whole Genome Sequencing (WGS) and Analysis
Whole genome DNA of E. faecalis E165 was sequenced by the PacBio RS and Illumina MiSeq platforms (Shanghai Personal Biotechnology Co., Ltd, China). The PacBio sequence reads were assembled with HGAP4 and CANU (Version 1.6), and corrected by Illumina MiSeq with pilon (Version 1.22). The prediction of ORFs and their annotation was performed using Glimmer 3.0.

Intraspecies Transformation and Interspecies Transformation
To investigate whether the erm(T)-carrying plasmid could be transferred into bacteria of the same and other species, plasmid DNA extracted from E. faecalis E165 was obtained by using Qiagen Mini-prep kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
To determine the intraspecies transmissibility of the erm(T)-carrying plasmid pE165, conjugation experiments were performed using E. faecalis E165 as the donor and E. faecalis OG1RF as the recipient as previously described (27). Transconjugants were screened on brain heart infusion (BHI, Oxoid, British) agar supplemented with 32 mg/L rifampicin, 32 mg/L fucidin acid, and 8 mg/L erythromycin. The corresponding transconjugant was designated E. faecalis OG1RF-Tc.
Natural transformation experiment using the recipient S. suis P1/7 was performed as described to investigate the interspecies transmissibility of erm(T)-carrying plasmid pE165 (28). The peptide (GNWGTWVEE) was dissolved in Milli-Q water at a final concentration of 5 mM and was used as a pheromone for the transformation. The erythromycin-susceptible recipient strain S. suis P1/7 was grown overnight in THY broth (3 g Todd-Hewitt Broth and 2 g yeast for 100 ml, Oxoid, British) at 37 • C under 5% CO 2 . The overnight culture was diluted 1:40 into pre-warmed THY broth, and grown at 37 • C under 5% CO 2 without shaking. Plasmid DNA (1.2 µg) and stock peptide (5 µl) were added when the recipient culture reached an OD 600 between 0.035 and 0.058, and then incubated for 2h at 37 • C under 5% CO 2 . The samples were diluted and plated on THA (Todd-Hewitt Broth with 1.3% agar powder, Oxoid, British) containing 8 mg/L erythromycin. The corresponding transformant was designated S. suis P1/7-Tm.
Natural transformation experiment with the recipient S. mutans UA159 was performed following procedures as previously described (29). For the screening of the transformants, THA was supplemented with 8 mg/L erythromycin. The corresponding transformant was designated S. mutans UA159-Tm.
The electrotransformation experiment with recipient strain S. aureus RN4220 was performed as described in a previous study (30). The corresponding transformant was designated S. aureus RN4220-Tm.

Identification and Characterization of erm(T)-Carrying Plasmid in E. faecalis
In addition, erm(A) gene carried by Tn6674 was located on the chromosome. Three copies of erm(B) gene were located on a 65,052 bp conjugative plasmid (designated pE165-2) and the conjugative region from pE165-2 exhibits 99% DNA identity to pL15 described in an E. faecalis isolated from swine in Brazil (CP042214). pE165-2 also includes tet(M) and tet(L) conferring resistance to tetracyclines, dfrG conferring resistance to trimethoprim, aacA-aphD conferring resistance to aminoglycosides, and cat encoding chloramphenicol acetyltransferase. A 2,836 bp small plasmid that did not carry any resistance gene was also detected in E. faecalis E165 (designated pE165-3).
Previous studies identified a complete translational attenuator immediately upstream of the erm(T) gene which consisted of two pairs of inverted-repeat sequences of 12 bp each and a reading frame for a regulatory peptide of 19 aa (15,17,33). Inducible erm gene expression often required an intact translational attenuator, while deletions or duplications that appeared in the regulatory region would cause constitutive erm gene expression (34). A comparison of the erm(T) regulatory region of pE165 with that of plasmids pRW35 (EU192194) revealed that the erm(T) regulatory region of pE165 had four bp point mutations and one bp deletion in the regulatory peptide ORF. This single nucleotide deletion resulted in a frame shift mutation, which  extended the reading frame for the regulatory peptide from 19 aa to 22 aa (Figure 2). In addition, erm(T) regulatory region of pE165 was compared to those of pSC262 and pUR2940 with mutations in previous reports (22,35), and the results are shown in Supplementary Figures S1, S2.  Table 2. Compared to E. faecalis OG1RF, OG1RF-Tc displayed a higher erythromycin MIC (>512 mg/L) and higher clindamycin MIC (256 mg/L). Interspecies transmissibility of pE165 was investigated by natural transformation using S. suis P1/7 and S. mutans UA159 as the recipients and electrotransformation using S. aureus RN4220 as the recipient. Transformants P1/7-Tm, UA159-Tm, and RN4220-Tm were successfully obtained, and the transfer frequencies were 0.63 × 10 2 µg −1 ,2.1×10 2 µg −1 and 4.9×10 4 µg −1 . The transformants were confirmed by AST and sequencing of 16S rRNA. PCR assay also revealed that only erm(T)  could be detected in these transformants. MICs of E. faecalis E165, S. suis P1/7, S. mutans UA159, S. aureus RN4220, and their transformants are shown in Table 2. S. suis transformant P1/7-Tm displayed a higher erythromycin MIC (>512 mg/L) and a higher clindamycin MIC (32 mg/L) compared with S. suis P1/7. S. mutans transformant UA159-Tm displayed higher erythromycin MIC (>512 mg/L) and clindamycin MIC (256 mg/L) compared with S. mutans UA159. S. aureus transformant RN4220-Tm displayed higher erythromycin MIC (>512 mg/L) and higher clindamycin MIC (128 mg/L) compared with S. aureus RN4220.

The erm(T)-Carrying Plasmid Is Transmissible
The physical map of pE165 and restriction enzyme-digested plasmid profiles of the plasmids from E. faecalis E165, transconjugant E. faecalis OG1RF-Tc, transformants S. suis P1/7-Tm, S. mutans UA159-Tm, and S. aureus RN4220-Tm are shown in Figure 3. The results indicated that only pE165 can transfer into the recipient strains and pE165 can replicate in heterogenous hosts. The result of the southern bolt revealed that erm(T) gene was located on pE165 in heterogenous hosts. The results of AST indicated that pE165 can constitutively express erythromycinand clindamycin-resistance phenotype in heterogenous hosts.

Fitness Cost
The growth curve of E. faecalis E165, S. suis P1/7, S. mutans UA159, S. aureus RN4220, and their transconjugants and transformants in the absence of erythromycin are shown in Figure 4. The results showed that no significant fitness burden for pE165-carrying transconjugants and transformants was observed compared with the recipient strains in the absence of selective pressure.
Competition experiments can offer a more discriminative and precise measurement of fitness, and the competitive disadvantage of the fitness burden caused by pE165 can be reflected during all the phases of the growth cycle and in successive cycles. During the competition experiment between E. faecalis OG1RF and OG1RF-Tc, from day 1 on, a successive decrease in the proportion of E. faecalis OG1RF-Tc was observed, and all the colonies tested were pE165 free on day 14 ( Figure 5A). From day 1 on, a fast and constant decrease in the proportion of S. suis P1/7-Tm was observed and all the strains were tested pE165 free on day 6 ( Figure 5B). In the process of competition experiment between S. mutans UA159 and UA159-Tm, an obvious decrease in the proportion of UA159-Tm was observed. On day 9, all the colonies tested were pE165 free ( Figure 5B). For the result of the competition experiment between S. aureus RN4220 and RN4220-Tm, it had an obvious decrease from day 3 on, and RN4220-Tm could not be detected on day 15 ( Figure 5A). The above results suggested that all the pE165-carrying transconjugants and transformants had a fitness cost compared to the recipient strains without pE165, but the fitness cost among the different transconjugants and transformants differed.
In this study, erm(T)-positive plasmid pE165 was identified in a E. faecalis strain E165. Whole-genomic sequencing for E165 was performed. The genetic environment of erm(T) in this study was then analyzed by comparing it with similar erm(T) genetic environments published previously (19,20,33,35,39,40). The homology analysis of the rep and mob genes located on pE165 suggested that this plasmid had the potential ability to transfer into enterococci and other species. Transformation experiments confirmed that pE165 was successfully transferred into Enterococcus, Streptococcus, and Staphylococcus. Elevated MICs of erythromycin and clindamycin were conferred by erm(T) in the recipient strains. According to recommendations given in CLSI, inducible expression of erm(T) cannot produce clindamycin resistance unless it is induced by erythromycin (41). In this study, the inducible clindamycin resistance tests indicated that the transconjugant E. faecalis OG1RF-Tc, transformants P1/7-Tm, UA159-Tm, and RN4220-Tm were resistant to both erythromycin and clindamycin ( Table 2), which revealed the expression of erm(T) in these strains was constitutive. This is also in agreement with the observation that deletions or duplications appeared in the regulatory region of erm(T) in these strains.
It can be found that the proportion of pE165-carrying strains constantly decreased until undetectable in the competition experiments between transformants and original recipients performed by successive culturing in the absence of antibiotic pressure. Although plasmids can mediate the horizontal transmission of resistance genes between bacteria and facilitate their adaptation to the pressure of antibiotics, they also entail a metabolic burden that reduces the competitiveness of the plasmid-carrying clone in the absence of selection (42). Acquisition and maintenance of a plasmid are directly associated with fitness effects on the recipient strain. The constitutive expression of erm(T) will produce a burden (fitness cost) in the recipient strain, and the low prevalence of erm(T) gene in many genera of bacteria may be explained in this way.

CONCLUSIONS
The erm(T) gene was first reported in an E. faecalis strain. A 4244 bp erm(T)-positive plasmid pE165 was characterized. The transmissibility of pE165 was investigated between intraand inter-species. The presence of pE165 greatly elevated the MICs of erythromycin and clindamycin which indicated the expression of erm(T) in the recipient strains was constitutive. Although the fitness cost showed us this plasmid reduced the competitiveness of the host strain, the potential possibility of dissemination of erm(T) among species of bacteria should not be ignored.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below https://www.ncbi.nlm. nih.gov/, CP089585.

AUTHOR CONTRIBUTIONS
X-DD and DL designed the research and supervised the study. X-YL, RY, CX, and YS performed the experiments and analyzed the data. X-YL, RY, and X-DD wrote the manuscript. All authors revised the manuscript and approved the final version for submission.

FUNDING
This work was supported by grants from the Program for Innovative Research Team (in Science and Technology) at the University of Henan Province (No. 18IRTSTHN020).