A Possible Role of Insertion Sequence IS1216V in Dissemination of Multidrug-Resistant Elements MESPM1 and MES6272-2 between Enterococcus and ST59 Staphylococcus aureus

Sequence type 59 (ST59) is the dominant type of community-associated methicillin-resistant Staphylococcus aureus (MRSA) in Taiwan. Previously, we reported that ST59 MRSA harbors enterococcal IS1216V-mediated multidrug-resistant composite transposons MESPM1 or MES6272-2. The MES were found to have a mosaic structure, largely originating in enterococci and partly native to S. aureus. The current study aimed to track the origin of the MES and how they disseminated from enterococci to ST59 S. aureus. A total of 270 enterococcal isolates were analyzed, showing that two ST64 Enterococcus faecalis isolated in 1992 and 11 clonal complex 17 Enterococcus faecium harbored MESPM1-like and MES6272-2-like structures, respectively. Sequence analysis revealed that ST64 E. faecalis strain N48 acquired the MESPM1-like structure on the plasmid pEflis48. The pEflis48 harbored the enterococci-originated region (erythromycin, kanamycin, and streptomycin resistances) and the S. aureus-originated region (chloramphenicol resistance) of MESPM1 but was separated by the replication region of the plasmid. Homologous recombination between the two direct repeats of IS1216V resulted in excision of the replication region of the plasmid to regenerate MESPM1. The p4780-1 and pV19 of E. faecium carried MES6272-2-like structures with IS1216V, albeit with multiple insertions by other insertion sequences. The findings show that IS1216V plays important roles in bidirectional gene transfer of multidrug resistance between enterococci and S. aureus.


PCR Mapping of MES Structures
The resistance determinants ermB, aph(3 )-IIIa, aadE, aacA-aphD, and cat were detected as previously described [4]. For ermB-, aph(3 )-IIIa-, and aadE-positive isolates, six primer sets (I-VI) were used to screen the MES structures. The entire region of MES PM1 -like structures in the two E. faecalis strains was further mapped using two PCR primer sets (A and B) with the methods we had previously described [5]. Information about the primers is indicated in Table 1, and the locations of the primers are indicated in Figure S1.

Antimicrobial Susceptibility Testing
Susceptibility testing of vancomycin, erythromycin, kanamycin, streptomycin, gentamicin, and chloramphenicol was performed by the agar dilution method according to the 2020 guidelines of the Clinical and Laboratory Standards Institute [18]. E. faecalis ATCC 29,212 was used as the reference strain. High-level resistance of streptomycin and gentamicin was defined as minimal inhibitory concentrations of >2000 and >500 mg/L, respectively.

Pulsed-Field Gel Electrophoresis (PFGE)
PFGE was performed as previously described [22]. In brief, agarose-embedded bacterial DNA was digested with SmaI (New England BioLabs, Ipswich, MA, USA) and then was separated using a CHEF-DRIII apparatus (Bio-Rad Laboratories, Hercules, CA, USA). PFGE was carried out at 200 V and 14 • C for 22.5 h, with the pulse times ranging from 5 to 35 s. The pulsotypes were analyzed by BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium). The dendrogram of pulsotype relationships was produced by the unweighted pair group method using arithmetic averages (UPGMA) based on Dice similarity indices.
2.6. S1 Nuclease-Digested PFGE S1 nuclease-digested PFGE analysis was performed as previously described to determine the sizes of the plasmids [23]. The agarose-embedded bacterial DNA was incubated at 37 • C for 45 min with 10 units of Aspergillus oryzae S1 nuclease (Invitrogen). The reaction was stopped by transferring the agarose-embedded bacterial DNA to ES buffer (0.5 mM EDTA, 1% (w/v) Sarkosyl, pH 9.0) at 4 • C for 10 min. The plugs were applied to wells of 1.2% (w/v) agarose gels (Bio-Rad) and run in a CHEF-DRIII apparatus (Bio-Rad) with a pulse angle of 120 • and pulse times of 45 s for 14 h and 25 s for 6 h at 200 V in 0.5× Tris-borate-EDTA (TBE). Each band was considered a unit length linear plasmid.

Southern Blot
The linear plasmids in the gel separated by S1-PFGE were depurinated, denatured, neutralized, and transferred to a Hybond-N+ nylon membrane (GE Healthcare, Chicago, IL, USA) by vacuum-blotting. Hybridization was achieved using the digoxigenin-labeled DNA probe specific for the aadE gene generated by PCR. The detection of hybridization was performed using an alkaline-phosphatase-conjugated anti-digoxigenin antibody (Roche Diagnostics GmbH, Mannheim, Germany) and the substrate CSPD (Roche Diagnostics GmbH) according to the manufacturer's instructions.

Sequencing and Analysis of the MES Structures and Their Adjacent Environments
The sequence of the MES 6272-2 -like structure in E. faecium 4780-1 was determined using the primer sets for PCR mapping described above, and extension of the IS12126V upstream sequence was achieved by inverse PCR. For E. faecalis strain N48 and E. faecium V19, Illumina MiSeq 300 bp paired-end sequencing was used to determine the MES PM1 -like and MES 6272-2 -like structures. Total Illumina sequencing output corresponded to approximately 146-and 301-folds of the N48 and V19 genome size, respectively. Contigs were obtained using de novo assembly with SPAdes v3.10.1 [24]. A total of 92 and 261 contigs were yielded for the N48 and V19 genomes, respectively. The gaps between the contigs were filled up by Sanger sequencing. The open reading frame (ORF) was determined using BLAST. The plasmid family was determined using PlasmidFinder 2.0 [25].

Filter Mating
Filter mating was carried out on BHI agar using E. faecalis N48 or E. faecium V19 as donors, and E. faecalis JH2-2 or S. aureus RN2677 as recipients. The donor-recipient mix cultures were placed on a filter with incubation at 37 • C for 24 h, followed by resuspension and shaking in BHI broth with incubation at 37 • C for 1 h. The cells were collected and plated on BHI agar containing 250 mg/L erythromycin, 100 mg/L rifampin, 100 mg/L streptomycin (if donor was E. faecalis N48), 100 mg/L gentamicin (if donor was E. faecium V19), or 25 mg/L fusidic acid (if recipient was E. faecalis JH2-2).

Nucleotide Sequence Accession Numbers
The nucleotide sequences (Supplementary Data) of pEflis48, p4780-1, and pV8919 characterized in this study have been deposited in GenBank under accession numbers MT877066-MT877068.

Localization of the MESPM1-or MES6272-2-Like Elements in Enterococcal Strains
To determine the location of the MES PM1 -or MES 6272-2 -like elements in enterococcal strains, E. faecalis strain N48 isolated in 1992, E. faecium 4780-1 isolated in 2003, and E. faecium V19 isolated in 2013 were chosen for S1 nuclease-digested PFGE analysis. The S1 nuclease-digested DNAs were separated by PFGE, followed by Southern blot hybridization with the aadE probe ( Figure 2). Southern blot hybridization after S1 nuclease-digested PFGE revealed that the MES-like elements of E. faecalis strain N48 and E. faecium 4780-1 were located on the plasmids with estimated sizes ranging from 48.5 to 97 kb, while the MES 6272-2 -like elements of E. faecium V19 were located on a relatively large plasmid with an estimated size ranging from 194 to 242.5 kb. The size of the MES PM1 -like-carrying plasmid of E. faecalis strain 46 and the MES 6272-2 -like-carrying plasmids of E. faecium strains V32, V35, V47, and V76 corresponded to E. faecalis N48 and E. faecium V19, respectively (data not shown).

Sequencing of the MESPM1-Like Elements and the Adjacent Genetic Environments in Enterococcal Strains
E. faecalis N48 was chosen for DNA sequencing to determine the MES-like structure and the surrounding genetic environment. An incomplete plasmid sequence of 50,564 bp in length was determined, and sequence analysis revealed that the MES PM1 -like structure was located on a mosaic plasmid, pEflis48 ( Figure 3a)

Localization of the MESPM1-or MES6272-2-Like Elements in Enterococcal Strains
To determine the location of the MESPM1-or MES6272-2-like elements in enterococcal strains, E. faecalis strain N48 isolated in 1992, E. faecium 4780-1 isolated in 2003, and E. faecium V19 isolated in 2013 were chosen for S1 nuclease-digested PFGE analysis. The S1 nuclease-digested DNAs were

Sequencing of the MESPM1-Like Elements and the Adjacent Genetic Environments in Enterococcal Strains
E. faecalis N48 was chosen for DNA sequencing to determine the MES-like structure and the surrounding genetic environment. An incomplete plasmid sequence of 50,564 bp in length was determined, and sequence analysis revealed that the MESPM1-like structure was located on a mosaic plasmid, pEflis48 (Figure 3a) Detailed sequence comparison of the MESPM1-like structure in E. faecalis N48 is shown in Figure  3b, and the results are highly identical to those for ST59/SCCmec V MRSA PM1, except (i) an additional 344 bp sequence near the IS1216V; (ii) an additional region corresponding to E. faecalis Figure 2. S1 nuclease PFGE analysis followed by Southern blot hybridization with the aadE probe of E. faecalis N48, E. faecium V19, and E. faecium 4780-1: (a) S1 nuclease-digested DNAs separated by PFGE; (b) Southern blot hybridization of the aadE probe of the S1 nuclease-digested DNAs.
Detailed sequence comparison of the MES PM1 -like structure in E. faecalis N48 is shown in Figure 3b, and the results are highly identical to those for ST59/SCCmec V MRSA PM1, except (i) an additional 344 bp sequence near the IS1216V 1 ; (ii) an additional region corresponding to E. faecalis plasmid pTEF3, including a rep gene belonging to the Inc18 family of broad-host-range conjugative plasmid, a truncated gene-encoding pheromone-binding protein, and a copy of IS1216V; and (iii) lack of a 298 bp noncoding sequence and an IS1216V on the right side of MES PM1 . Interestingly, the cat gene and its surrounding environment that originated in S. aureus were also found in pEflis48 and showed 99.9% sequence identity to MES PM1 . Moreover, PCR and DNA sequencing using the primer set A located in MES PM1 (one was located upstream of IS1216V 2 and the other was located downstream of IS1216V 3 , as indicated in Figure 3 and Figure S1) outside the pTEF3 region yielded a 1301 bp amplicon corresponding to a copy of IS1216V, indicating that homologous recombination occurred between the two direct repeats of IS1216V to excise the pTEF3 region. PCR of E. faecalis N46 generated the identical size to that of E. faecalis N48 ( Figure S1b).

Horizontal Transfer of MESPM1 or MES6272-2 Encoding Drug Resistance
Conjugative transfer of MES-like structures between enterococci and S. aureus was performed by filter mating. E. faecalis N48 or E. faecium V19 were used as donors, while E. faecalis JH2-2 or S. aureus RN2677 were used as recipients. The MES-related resistances were successfully transferred from E. faecalis N48 or E. faecium V19 to E. faecalis JH2-2, with transfer frequencies of 3.7 × 10 −2 and 2.5 × 10 −7 , Microorganisms 2020, 8, 1905 8 of 12 respectively. Resistances were transferred to transconjugants ( Table 3). All of the transconjugants tested acquired plasmids with sizes similar to their donors detected by S1 nuclease PFGE with Southern blot hybridization. However, no transconjugants were obtained in the cases of transfer from E. faecalis N48 or E. faecium V19 to S. aureus RN2677 (efficiency < 6.5 × 10 −10 ).

Discussion
Previously, we described MES PM1 and MES 6272-2 in ST59 MRSA, the novel multidrug-resistant composite transposons mediated by enterococcal IS1216V and mingled with S. aureusand enterococci-originated sequences [4,5]. This raised a question about how the mosaic composite transposons emerged and disseminated into ST59 MRSA, the dominant CA-MRSA in Taiwan identified in 1997 [26]. In the current study, we collected enterococcal clinical isolates from as early as 1991 and analyzed their resistance elements. As expected, MES PM1 -like and MES 6272-2 -like structures, both surrounded by IS1216V, were found in E. faecalis and E. faecium isolates, respectively. Furthermore, the MES PM1 -like structure of E. faecalis isolated in 1992 had already acquired the cat gene and its surrounding genetic environment that was native to S. aureus, suggesting that horizontal gene transfer from S. aureus to enterococci might have occurred before the emergence of multidrug-resistant ST59 MRSA.
The IS1216 isoform groups, including IS1216, IS1216E, and IS1216V, are one of the most multiply represented insertion sequences in enterococci and play important roles in the dissemination of resistance determinants [7,12]. They form composite transposons encoding antibiotic resistance in enterococci, such as Tn5385 (encoding gentamicin, streptomycin, penicillin, erythromycin, and tetracycline/minocycline resistances), Tn5482, and Tn5506 (both encoding vancomycin resistance) [27][28][29]. MES PM1 and MES 6272-2 are rare cases of composite transposons mediated by IS1216V found in S. aureus [4,5]. We previously reported that MES PM1 would be excised, resulting in a single IS1216V remaining in MES PM1 [4]. The same phenomenon has been demonstrated in pEf37BA of E. faecium with the pbp5 gene surrounded by two direct repeats of IS1216V [30]. In the current study, the pEflis48 of ST64 E. faecalis carried a MES PM1 -like structure, within which a region included the rep gene and a truncated prgZ gene encoding pheromone-binding proteins corresponding to that in pTEF3 of vancomycin-resistant E. faecalis (Figure 3). PCR with primer set A located upstream of IS1216V 2 and downstream of IS1216V 3 generated a 1031 bp amplicon, indicating that RecA-mediated homologous recombination would occur to delete the replication region of pTEF3 and leave a copy of IS1216V (Figure 3 and Figure S1). As a result, MES PM1 , a composite transposon mediated by IS1216V highly similar to that in ST59/SCCmec V MRSA with erythromycin, kanamycin, streptomycin, and chloramphenicol resistances, would be regenerated. This indicates the important role of IS1216V in the dissemination of multidrug resistance between enterococci and ST59 S. aureus, the most dominant CA-MRSA in Taiwan.
The cat genes found in staphylococci and enterococci are frequently located on small multicopy plasmids and are seldom associated with large multiresistant plasmids [31,32]. In pEflis48 of E. faecalis and MES PM1 of ST59/SCCmecV MRSA, the cat gene and its surrounding environments were both highly related to a small S. aureus plasmid SAP084A (>99.5% DNA sequence similarity, Figure 3), which indicates that this region originated in S. aureus. Furthermore, the S. aureus-originated cat region in pEflis48 and MES PM1 was flanked by two direct repeats of enterococcal IS1216V in which another copy embedded, resulting in disruption of the rep gene encoding replication initiator of the S. aureus plasmid. This implies that the region transferred from S. aureus to enterococci, mediated by enterococcal IS1216V. Therefore, we hypothesize that the S. aureus-originated cat region was initially acquired on a large multiresistant plasmid of enterococci by IS1216V from a small S. aureus plasmid, regenerated to MES PM1 by RecA-mediated homologous recombination via two direct repeats of IS1216V, and finally disseminated into ST59 S. aureus, the dominant CA-MRSA in Taiwan. This would be the first example of IS1216V-mediated bidirectional transfer of resistance in S. aureus and enterococci.
The MES PM1 -like structure of E. faecalis N48 was located on an Inc18 plasmid. The Inc18 plasmids usually possess the ability to confer antibiotic resistance and to transfer to a variety of Gram-positive cocci including enterococci and staphylococci [12,33]. Inc18 plasmids comprising insertion of Tn1546 to facilitate dissemination of vancomycin resistance from enterococci to MRSA have been reported [34]. In the current study, although pEflis48 displayed high transfer efficiency (3.7 × 10 −2 /donor) from donor E. faecalis N48 to recipient E. faecalis JH2-2, it failed to transfer to S. aureus RN2677. The reason is unknown and needs to be investigated in the future. A previous study reported that a pSK41-like plasmid is necessary to contribute to successful transfer of the Inc18-like vanA plasmid from E. faecalis to MRSA [35]. Further research should be carried out to illustrate the transfer mechanism of MES-like structures between enterococci and S. aureus.
ST64 E. faecalis belongs to CC8. Although CC8 was not included in the major global clusters associated with healthcare-associated infections such as CC2 [8,9], ST64 E. faecalis has been reported to be associated with multidrug resistance to erythromycin, kanamycin, streptomycin, gentamicin, and tetracycline [36,37]. In contrast to E. faecalis, all of the E. faecium isolates characterized in this study were CC17, the most prevalent cluster worldwide, notably in the nosocomial setting [8,38,39]. It has been proposed that progressive evolution to acquire resistance determinants in CC17 E. faecium is associated with its success to adapt to conditions of modern hospitals [38]. In the current study, the MES 6272-2 -like structures of the CC17 E. faecium strains isolated in 2003, 2013, and 2014 were inserted by several insertion sequences such as ISEfm1, ISEf1, and ISL3, and an additional cluster of resistance determinants lnu(B), lsa(E), spw, and aadE was acquired (Figure 4). The unpredictable genomic changes of CC17 E. faecium would hamper the tracking of the origin of MES-like structures.
In conclusion, the MES PM1 structure with the enterococci-originated region (erythromycin, kanamycin, and streptomycin resistances) and the S. aureus-originated region (chloramphenicol resistance) found in ST64 E. faecalis and ST59 S. aureus indicates that bidirectional gene transfer mediated by IS1216V can occur between enterococci and S. aureus. Our study is of great importance, as it is the first to demonstrate the role of IS1216V in interspecies transfer of multidrug resistance genes between enterococci and ST59 S. aureus.