ABSTRACTThe rapid increase of phenicol-oxazolidinone (PhO) resistance inStreptococcus suisdue to transferable resistance geneoptrAis a matter of concern. However, genetic mechanisms for the dissemination of theoptrAgene remain to be discovered. Here, we selected 33optrA-positiveS. suisisolates for whole-genome sequencing and analysis. The IS1216Eelement was present in 85% of theoptrA-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 IS1216EinoptrAspreading. ThreeoptrA-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 alternativeSSU1943attachment site along with the primarySSU1797attachment site (type 1) or into the singleSSU1797attachment site (type 2). In addition, conjugative transfer of anoptrA-carrying plasmid and prophage in streptococci was validated for the first time. Considering the abundance of MGEs inS. suisand 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-resistantS. suis.IMPORTANCEAntimicrobial resistance to phenicols and oxazolidinones by the dissemination of theoptrAgene leads to treatment failure in both veterinary and human medicine. However, information about the profile of these MGEs (mobilome) that carryoptrAand their transferability in streptococci was limited, especially for the zoonotic pathogenS. suis. This study showed that theoptrA-carrying mobilome inS. suisincludes integrative and conjugative elements (ICEs), plasmids, prophages, and antibiotic resistance-associated genomic islands. IS1216E-mediated formation ofoptrA-carrying translocatable units played important roles inoptrAspreading between types of MGEs, and conjugative transfer of variousoptrA-carrying MGEs (ICEs, plasmids, and prophages) further facilitated the transfer ofoptrAacross strains, highlighting a nonignorable risk to public health ofoptrAdissemination to other streptococci and even to bacteria of other genera.