Various Mobile Genetic Elements Involved in the Dissemination of the Phenicol-Oxazolidinone Resistance Gene optrA in the Zoonotic Pathogen Streptococcus suis: a Nonignorable Risk to Public Health

ABSTRACT The rapid increase of phenicol-oxazolidinone (PhO) resistance in Streptococcus suis due to transferable resistance gene optrA is a matter of concern. However, genetic mechanisms for the dissemination of the optrA gene remain to be discovered. Here, we selected 33 optrA-positive S. suis isolates for whole-genome sequencing and analysis. The IS1216E element was present in 85% of the optrA-carrying contigs despite genetic variation observed in the flanking region. IS1216E-optrA-carrying segments could be inserted into larger mobile genetic elements (MGEs), including integrative and conjugative elements, plasmids, prophages, and antibiotic resistance-associated genomic islands. IS1216E-mediated circularization occurred to form the IS1216E-optrA-carrying translocatable units, suggesting a crucial role of IS1216E in optrA spreading. Three optrA-carrying MGEs (ICESsuAKJ47_SSU1797, plasmid pSH0918, and prophage ΦSsuFJSM5_rum) were successfully transferred via conjugation at different transfer frequencies. Interestingly, two types of transconjugants were observed due to the multilocus integration of ICESsuAKJ47 into an alternative SSU1943 attachment site along with the primary SSU1797 attachment site (type 1) or into the single SSU1797 attachment site (type 2). In addition, conjugative transfer of an optrA-carrying plasmid and prophage in streptococci was validated for the first time. Considering the abundance of MGEs in S. suis and the mobility of IS1216E-optrA-carrying translocatable units, attention should be paid to the potential risks to public health from the emergence and spread of PhO-resistant S. suis. IMPORTANCE Antimicrobial resistance to phenicols and oxazolidinones by the dissemination of the optrA gene leads to treatment failure in both veterinary and human medicine. However, information about the profile of these MGEs (mobilome) that carry optrA and their transferability in streptococci was limited, especially for the zoonotic pathogen S. suis. This study showed that the optrA-carrying mobilome in S. suis includes integrative and conjugative elements (ICEs), plasmids, prophages, and antibiotic resistance-associated genomic islands. IS1216E-mediated formation of optrA-carrying translocatable units played important roles in optrA spreading between types of MGEs, and conjugative transfer of various optrA-carrying MGEs (ICEs, plasmids, and prophages) further facilitated the transfer of optrA across strains, highlighting a nonignorable risk to public health of optrA dissemination to other streptococci and even to bacteria of other genera.

S treptococcus suis is one of the major pathogens of swine, which has been increasingly recognized as an emerging zoonotic agent. Outbreaks of S. suis infections in China and Southeast Asia have caused serious threats to public health (1)(2)(3). S. suis is also considered to be an antimicrobial resistance reservoir contributing to the spread of antibiotic resistance genes to major streptococcal pathogens (4,5). High rates of resistance to commonly used antimicrobials including tetracyclines and macrolides-lincomycins-streptogramin B (MLS B ) have been reported worldwide in S. suis in the past decades (4,5). More recently, a rapid increase of S. suis resistant to phenicols and oxazolidinones (PhO) has been documented (6,7).
Oxazolidinones, including linezolid, tedizolid, and contezolid, represent the lastresort antimicrobial agents against infections caused by multidrug-resistant Gram-positive pathogens (8,9). However, several studies had confirmed that the exclusive use of florfenicol in veterinary medicine could coselect PhO resistance (10,11). The PhO resistance in bacteria was originally associated with mutations in the domain V region of 23S rRNA and in genes coding for ribosomal proteins L3, L4, and L22 (12). This situation had changed in 2000, when the first transferable oxazolidinone resistance gene, cfr, was identified and reported in a Staphylococcus sciuri isolate (13). Successively, cfr variants, optrA, and poxtA have recently been reported in a variety of Gram-positive and -negative bacteria (14,15). To date, only cfr and optrA have been detected in S. suis, with the latter predominantly being isolated (16,17). For instance, a screening of resistance mechanisms in PhO-resistant S. suis showed that the genes optrA and cfr were present in 100% and 2.4% of the isolates, respectively (6).
The optrA gene encodes an ABC-F superfamily protein which confers PhO resistance by targeting the large subunit of the ribosome. Since the first report of optrA in enterococci from humans and food-producing animals (11), the genetic basis responsible for its dissemination has been extensively studied in enterococci, involving different mobile genetic elements (MGEs), particularly the plasmids and transposons (15,(18)(19)(20). The optrA gene was frequently flanked by a variety of insertion sequences (ISs), including IS1216E (21), ISEfa15 (22), ISChh1-like (23) and ISVlu1 (24), which might have facilitated the dissemination of the optrA gene across strains, species, or even genus boundaries. However, the transmission of antibiotic resistance genes in streptococci is primarily mediated by integrative and conjugative elements (ICEs) and the recently emerged prophages (5,25,26). ICEs are self-transmissible MGEs that primarily reside in the host cell's chromosome and yet have the ability to be transferred between cells by conjugation (27). ICEs encode the conjugation machinery not only for their self-transfer but also to mobilize other MGEs, including integrative and mobilizable elements and mobilizable genomic islands (28,29). Until now, information about the profile of these MGEs (mobilome) that carry optrA and their transferability in streptococci was limited, especially for the zoonotic pathogen S. suis (7,26,(30)(31)(32).
In this study, we investigated the optrA-carrying mobilome in S. suis strains isolated from our previous PhO resistance surveillance project (6, 7) and characterized various optrA-carrying MGEs, including ICEs, plasmids, prophages, and antibiotic resistanceassociated genomic islands. We further evaluated the role of the IS1216E element in dissemination of optrA and the transferability of optrA-carrying MGEs. Our work demonstrated critical roles of IS1216E in mediating movement of optrA between types of MGEs. The conjugative transfer of various optrA-carrying MGEs (ICEs, plasmids, and prophages) further facilitated the transfer of optrA in S. suis.

RESULTS AND DISCUSSION
Analysis of optrA-carrying mobilome in S. suis. We previously observed a rapid increase of optrA-mediated PhO resistance among S. suis isolates (6,7). To further characterize the genetic basis for this rapid dissemination of optrA among S. suis isolates, a total of 33 optrA-positive S. suis isolates were subjected to whole-genome sequencing (WGS) (see Table S1 in the supplemental material). WGS analysis identified the presence of optrA-carrying contigs which ranged from a 2,459-bp single optrA-carrying contig to a 12,571-bp segment comprising the IS1216E-4hp-DaraC-optrA-hp-erm(A)-like-met-2hp-IS1216E structure (Fig. 1A). Most optrA-carrying contigs had an araC-optrA organization (n = 16) or an optrA-erm(A)-like structure (n = 11) (Fig. 1A). Despite genetic variation observed in the flanking region of the optrA gene, insertion sequence IS1216E or truncated IS1216E (DIS1216E) was present upstream or downstream of optrA in 26 of the 33 isolates ( Fig. 1A and Table S1), suggesting a critical role of IS1216E in the spreading of optrA among S. suis isolates.
Due to the limitation of sequencing depth, flanking sequences are missing in many optrA-carrying contigs. Nevertheless, two copies of IS1216E were found in the upstream and downstream regions of optrA in the same orientation in sequenced contigs (Fig. 1A), including the smallest IS1216E-optrA-hp-IS1216E (4,608 bp), the IS1216E-araC-optrA-hp-DIS1216E (6,892 bp), the IS1216E-DaraC-optrA-hp-DIS1216E (4,934 bp), and the largest IS1216E-4hp-DaraC-optrA-hp-erm(A)-like-met-2hp-IS1216E segments, apart from the previously identified IS1216E-araC-optrA-hp-cat pC194 -IS1216E (6,446 bp) in strain YSJ17 (7). In addition, the optrA-carrying segments could be inserted into larger MGEs, including the IS1216E-araC-optrA-hp-IS1216E integrated into ICEs, prophages, and antibiotic resistanceassociated genomic islands from multiple strains, the IS1216E-optrA-hp-IS1216E integrated into plasmid pSH0918 from the early isolate SH0918 in 2009, and the IS1216E-4hp-DaraC-optrA-hp-erm(A)-like-met-2hp-IS1216E integrated into ARGI1 from strain SFJ44 (Fig. 1A). The integration of optrA-carrying segments into different MGEs provide the possibility for their intra-and interspecific transfer by hijacking MGE-encoded transfer machinery. IS1216E elements mediate the generation of optrA-carrying TUs. Two identical or closely related copies of the IS elements flanking optrA might be capable of circularization, thus generating a circular form containing one intact IS element and the sequence between the two copies of IS elements, known as translocatable units (TUs) (33), which can integrate into an ICE or a plasmid or t different chromosomal sites, thereby fostering the dissemination of optrA (Fig. 1A). To verify if this was the case in S. suis, the presence of optrA-carrying TUs mediated by IS1216E was examined by PCR ( Fig. 1A). As expected, all the IS1216E-optrA-carrying transposons in the strains SH0918, AKJ47, FJSM5, and SFJ44 could be circularized to generate the IS1216E-optrA-carrying TUs (Fig. 1B). Interestingly, due to the downstream IS1216E being interrupted by ISSsu6, we observed two types of TUs in strain AKJ47, IS1216E-araC-optrA-hp (4,788 bp) and araC-optrA-hp-DIS1216E-ISSsu6-DIS1216E (6,211 bp), respectively, indicating that the IS1216E element from either upstream or downstream of optrA could drive the formation of TUs. These results further suggested that IS1216E-mediated circularization of IS1216E-optrA-carrying transposons from different MGEs might have played an important role in facilitating its dissemination between different MGEs, bacterial strains, and even species (15,21).
Conjugative transfer of optrA between S. suis strains. To test transferability of the optrA gene located on different MGEs, conjugation experiments were performed using the above-mentioned four strains with different optrA-carrying MGEs as donors. Transfer was observed in strains AKJ47, SH0918, and FJSM5 but not in SFJ44 ( Table 1). The three corresponding transconjugants cAKJ47, cSH0918, and cFJSM5 exhibited elevated MICs to linezolid (4 to 8 mg/L) and florfenicol (32 to 64 mg/L) ( Table 1). In addition, transconjugant cFJSM5 also exhibited resistance to erythromycin (MIC, 32 mg/L) and clindamycin (MIC, 64 mg/L) ( Table 1), suggesting the cotransfer of the upstream erm(B) with the IS1216E-DaraC-optrA-hp-DIS1216E transposon. The donor strains and their corresponding transconjugants cAKJ47, cSH0918, and cFJSM5 were further completely genomically sequenced for the characterization of the MGEs responsible for optrA transfer, confirming that interstrain transfer of optrA was mediated by the self-transferable MGEs (ICESsuAKJ47_SSU1797, plasmid pSH0918, and prophage USsuFJSM5_rum; see below) rather than the IS1216E-optrA-carrying TUs, under laboratory experimental conditions. Differences in transfer frequencies were observed between ICEs, plasmids, and prophages. The ICESsuAKJ47_SSU1797 exhibited much higher transfer frequency than pSH0918 (;100-fold) and USsuFJSM5_rum (;500-fold) ( Table 1). One possible explanation is that the ICESsuAKJ47_SSU1797 encoded the intact conjugation machinery for its self-transfer at a much higher transfer frequency, while the plasmid pSH0918 and prophage USsuFJSM5_rum lacked conjugation machinery. However, a coresident ICE was present on both host chromosomes (data not shown), suggesting that the successful transfer of pSH0918 and USsuFJSM5_rum might be mobilized by ICE at low frequency. It is reasonable that ICEs were distributed more frequently than plasmids and prophages in streptococci (5,25,26).
Transfer and multilocus integration of a novel optrA-carrying ICE, ICESsuAKJ47_ SSU1797. In strain AKJ47, the optrA-carrying ICESsuAKJ47_SSU1797 was 68,648 bp in length and comprised 89 predicted open reading frames (ORFs) with an imperfect direct repeat of 2 or 4 nucleotides (nt) (59-GC-39/59-TCCC-39) in its flanking region, which was integrated into the SSU1797 gene, encoding a MutT/Nudix family protein, MutX ( Fig. 2A). The ICESsuAKJ47_SSU1797 backbones (except the integration module) exhibited 94.1 to 99.3% identity to ICESsuYZDH1 at the SSU0877 gene (encoding another MutT/Nudix family hydrolase) in S. suis strain YZDH1, the founder of a The transfer frequency of ICESsuAKJ47_SSU1797 to the recipient strain S. suis P1/7RF was (5.99 6 1.53) Â 10 26 , a rate similar to that of ICESsuYZDH1_SSU0877 and higher than that of the ICESa2603 family of ICEs (34,35). DNA hybridization of AKJ47 conjugation pairs with a specific probe for optrA suggested that the transferred ICESsuAKJ47_SSU1797 was located on an ;160-kb band and an ;590-kb band, respectively (Fig. 2B), indicating that the acquisition of ICE occurred by stable integration into additional attachment sites (secondary integration site) except for the primary SSU1797 site. Complete genome analysis of transconjugant cAKJ47 revealed the presence of an additional copy of ICESsuAKJ47 by integration into the SSU1943 gene (encoding another MutT/Nudix family hydrolase), a previously nonreported integration site. Sequence analysis revealed the presence of imperfect direct repeats flanking the ICE at the SSU1943 site (59-AC-39/59-TCCC-39). One hundred positive clones in transconjugants from conjugation experiments were then randomly selected and screened for the multilocus integration of ICESsuAKJ47. Subsequently, two types of transconjugants were identified, one with ICESsuAKJ47 integrated at both SSU1797 and SSU1943 sites (type 1), and the other integrated at only the SSU1797 site (type 2), accounting for 82.0% and 18.0% of the transconjugants, respectively. To test the ability of ICESsuAKJ47 to integrate into and excise from the primary site (SSU1797) and the secondary site (SSU1943), an inverse PCR amplification followed by Sanger sequencing analysis was performed (Fig. 2C). PCR amplification confirmed the integration and excision/circularization of ICESsuAKJ47 in both sites in transconjugant cAKJ47, but for donor strain AKJ47, only one copy of ICESsuAKJ47 was observed at the primary SSU1797 site (Fig. 2D). We did not observe the integration of ICESsuAKJ47_SSU1797 into SSU0468, SSU0877, and SSU1262 sites in donors and transconjugants (data not shown). Accumulation of IS1216E-optrA-carrying TUs into the novel ICESsuYZDH1 family of ICEs with its multilocus integration into different attachment sites of the host bacteria could have benefited from its dissemination, posing a great challenge to the control of optrA spreading.
Characterization and transfer of a novel optrA-carrying plasmid, pSH0918. The optrA-carrying plasmid pSH0918 in strain SH0918 was 26,155 bp in length and comprised 28 predicted ORFs (Fig. 3A), with an average lower GC content (33.2%) than the chromosome (41.1%). The repR gene of plasmid pSH0918 had 95.6% identity to the replicon of the broad-host-range Inc18 family plasmids, which have been associated with the dissemination of a variety of resistance genes, including the MLS B resistance gene erm(B), the tetracycline resistance gene tet(S), the vancomycin resistance gene vanA, and, more recently, the oxazolidinone resistance genes optrA and poxtA among Gram-positive coccus isolates (36,37). Sequence analysis showed that the nonconjugative plasmid pSH0918 shared conserved core structure with plasmid pHN105 (38). The insertion of the 4.7-kb transposon IS1216E-optrA-hp-IS1216E into the core promoterbinding protein (CPBP) family intramembrane metalloprotease gene at the 59 start position might have resulted in the acquisition of optrA by plasmid pSH0918 (Fig. 3B). This was supported by the observation of integration and excision of IS1216E-optrA-hp-IS1216E from the plasmid (Fig. 1B).
Previous reports have shown that the optrA gene can be carried by plasmids of enterococci and staphylococci, while for S. suis, a single report of plasmid-borne optrA was recently described (30). However, the optrA-carrying locus on the plasmid of HNAY3 had a single ISEnfa1-like element upstream of optrA, and this plasmid was Transfer of optrA-Carrying MGEs in S. suis Microbiology Spectrum transferred into Staphylococcus aureus and S. suis by electrotransformation rather than conjugation (30), which prompted us to continue exploring the role of IS elements in transmission of optrA into plasmids and the transferability of optrA-carrying plasmids between strains. For the first time, we demonstrated the conjugative transfer of the optrA-carrying plasmid pSH0918 to the recipient strain S. suis P1/7RF at a frequency of (5.70 6 1.06) Â 10 28 . S1-pulsed-field gel electrophoresis (PFGE) and DNA hybridization revealed that optrA was transferred along with the plasmid pSH0918 (Fig. 3C).
Identification and transfer of a novel optrA-carrying prophage, USsuFJSM5_rum. The optrA gene in strain FJSM5 was located on a prophage at the rum site (Fig. 4A). The USsuFJSM5_rum was 57,765 bp in length and comprised 64 predicted ORFs. USsuFJSM5_rum shared conservative syntenic core structure with Streptococcus pyogenes prophage Um46.1 (39), which contains a conserved region with six modules flanked by two variable regions (VRs) (Fig. 4A). The optrA gene and the MLS B resistance gene erm(B) were located on VR1, while the aminoglycoside resistance genes aadE and ant(99)-I accumulated on VR2. Interestingly, Um46.1 and related prophages have been documented in many streptococcal species, which carried determinants of resistance to MLS B , mef(A), erm(B), lnu(B), and lnu(C); tetracyclines, tet(O), tet(O/W/32/O), and tet (W); aminoglycosides, ant(6)-Ib, aphA3, sat4, and aadE; and PhO, cat PC194 and optrA (40), highlighting that antibiotic resistance genes can also be transferred by prophages. Currently, two optrA-carrying prophages have been reported, including USC181, which also carried mef(A), aacA-aphD, and cat, and USsuYSJ17-3, which also contained erm (B), aphA3, aac(69)-aph(299), and cat (7,17,26); however, transfer of these prophages was not successful (7,26). Transfer of USsuFJSM5_rum using a standard transduction protocol was not achievable even after more than three attempts. We noted in vitro evidence of transfer of prophage Um46.1. However, the transfer experiment was performed with a virtual conjugation protocol (41), suggesting the transfer of prophage was assisted by conjugative elements via conjugation. To test this possibility, conjugative transfer of USsuFJSM5_rum was performed. FJSM5 transferred the optrA gene to the recipient strain S. suis P1/7RF by conjugation at a frequency of (1.01 6 2.71) Â 10 28 . SmaI-PFGE and DNA hybridization revealed that optrA was transferred along with the self-transfer prophage USsuFJSM5_ rum (Fig. 4B). Complete genome analysis of transconjugant cFJSM5 showed that the resistance genes aadE and ant(99)-I were not transferred to the recipient strain with the prophage USsuFJSM5_rum. Further analysis showed that the USsuFJSM5_rum region was bounded by a single attL and two attR sites (attR1 and attR2); this suggests that two circular episomes may be formed, either a small prophage, USsuFJSM5_rum1 (attLÂattR1), or a To verify if this was the case, two circular forms of prophage were tested by inverse PCR and sequence analysis (Fig. 4C). As expected, two types of prophages could be circularized to form the episome by using P2/P3 (or P2/P5) primer pairs and the empty site attB by using P1/P4 (or P1/P6) primer pairs (Fig. 4D). Analysis of the att sequences allowed us to identify an imperfect 8-bp nucleotide match (attL/attP, 59-CACGTCGA-39; attR1/attB1, 59-CACATAGA-39; attR2/attB2, 59-CACATAGA-39), which is inconsistent with the 2-bp GA sequence in Um46.1 of a previous analysis (39). In addition, in the conjugation pairs of FJSM5 and P1/7RF, only the transfer of the small prophage USsuFJSM5_rum1 to P1/7RF was observed, which caused the recipient to acquire only the resistance genes optrA and erm(B).
In conclusion, different optrA-carrying MGEs, including plasmids, ICEs, and prophages, and antibiotic resistance-associated genomic islands are associated with the rapid increase of PhO resistance in S. suis. The high prevalence of IS1216E flanking the optrA gene and IS1216E-mediated formation of different IS1216E-optrA-carrying TUs might have played a vital role in the dissemination of optrA between MGEs. Moreover, the horizontal transfer of optrA-carrying ICEs, plasmids, and even prophages might explain the higher prevalence of optrA and diversity of MGEs in S. suis than in other streptococci. Considering the abundance of MGEs in S. suis and the mobility of optrA-carrying TUs, attention should be paid to the spread of optrA-mediated PhO resistance in S. suis, highlighting the risks of horizontal gene transfer (HGT) of optrA-carrying MGEs to other streptococci and even to bacteria of other genera.

MATERIALS AND METHODS
Bacterial strains and antimicrobial susceptibility testing. S. suis strains were grown in Todd-Hewitt broth (THB) or on Todd-Hewitt agar (THA) plates supplemented with 5% (vol/vol) calf serum and incubated at 37°C. Antimicrobial susceptibility testing was performed using the broth microdilution method according to CLSI guideline M100-ED29 (42). The antimicrobial agents used in this study are listed in Table 1.
PCR amplification analysis. IS1216E-mediated circularization of the optrA-carrying TUs was detected by an inverse PCR in selected strains with different genetic organizations, including within the SH0918 plasmid pSH0918, the AKJ47 ICESsuAKJ47_SSU1797, the FJSM5 prophage USsuFJSM5_rum, and the SFJ44 ARGI1, followed by sequencing analysis. Integrase-mediated excision and integration of ICESsuAKJ47_ SSU1797 and prophage USsuFJSM5_rum were performed with inverse PCR to detect the extrachromosomal circular form and integrated form of MGEs in both donors and their corresponding transconjugants. The specific primers are listed in Table S2 in the supplemental material.
Transfer experiments. The transferability of different optrA-carrying MGEs was determined by filter mating assays as described previously (35). Strains SH0918 (with optrA-carrying plasmid pSH0918), AKJ47 (with optrA-carrying ICESsuAKJ47_SSU1797), FJSM5 (with optrA-carrying prophage USsuFJSM5_rum), and SFJ44 (with optrA-carrying ARGI1) were selected as donors, and S. suis P1/7RF served as a recipient. The transfer frequency was calculated as the mean number of transconjugants per donor in each assay of three duplicates.
PFGE and DNA hybridization. The genomic location of optrA and the sizes of optrA-carrying MGEs in both the donors and related transconjugants were examined by S1-PFGE (for plasmid-borne optrA) or SmaI-PFGE (for chromosome-borne optrA) as previously described (37,45), followed by Southern blotting and DNA hybridization using a digoxigenin (DIG)-labeled probe specifically targeting the optrA gene with primers listed in Table S2.