Site-Specific Nested Integration of Tn1806 into ICESa2603-Family Integrative and Conjugative Elements in Streptococcus agalactiae

Composite integrative and conjugative elements (ICEs) frequently mediate the co-transfer of multiple antibiotic resistance genes during horizontal gene transfer, but their formation mechanisms remain unclear. This study investigated the site-specific integration of Tn<i>1806</i> into ICE<i>Sa</i>2603-family ICEs in <i>Streptococcus agalactiae</i> by conjugation experiments. PCR screening of 161 <i>S. agalactiae</i> clinical isolates identified potential Tn<i>1806</i>-like ICE carriers; whole-genome sequencing was performed to further characterize the macrolide-resistance isolates from this group. PCR detection resulted in 24 carrying Tn<i>1806</i>-like ICEs being found, five of which were macrolide-resistant. Genomic analysis for these five revealed distinct Tn<i>1806</i>-like ICEs (ICE<i>Sag</i>16, ICE<i>Sag</i>57, ICE<i>Sag</i>139, ICE<i>Sag</i>167, and ICE<i>Sag</i>220), three of which were found nested within another ICE (ICE<i>Spy</i>009, an ICE<i>Sa</i>2603-family ICE). Conjugation experiments confirmed ICE<i>Sag</i>167 could integrate into the <i>snf2</i> (methyltransferase containing a SNF2 helicase domain) of ICE<i>Spy</i>009 in recipient cells, generating a composite ICE. Re-conjugation verified the transferability of composite ICE at low frequencies (8.63 × 10^-8), during which some nested ICE<i>Sag</i>167 were excised and transferred independently. This work provides first experimental evidence supporting Tn<i>1806</i> nesting within another ICE as a mechanism for resistance accumulation and mobile element evolution in <i>S. agalactiae</i>. The spread of such composite ICEs may confer multiple forms of resistance to new hosts, challenging infection treatment and raising public health concerns.