Abstract

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 Tn1806into ICESa2603-family ICEs inStreptococcus agalactiaeby conjugation experiments. PCR screening of 161S. agalactiaeclinical isolates identified potential Tn1806-like ICE carriers; whole-genome sequencing was performed to further characterize the macrolide-resistance isolates from this group. PCR detection resulted in 24 carrying Tn1806-like ICEs being found, five of which were macrolide-resistant. Genomic analysis for these five revealed distinct Tn1806-like ICEs (ICESag16, ICESag57, ICESag139, ICESag167, and ICESag220), three of which were found nested within another ICE (ICESpy009, an ICESa2603-family ICE). Conjugation experiments confirmed ICESag167 could integrate into thesnf2(methyltransferase containing a SNF2 helicase domain) of ICESpy009 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 ICESag167 were excised and transferred independently. This work provides first experimental evidence supporting Tn1806nesting within another ICE as a mechanism for resistance accumulation and mobile element evolution inS. agalactiae. The spread of such composite ICEs may confer multiple forms of resistance to new hosts, challenging infection treatment and raising public health concerns.