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

## TL;DR

This study shows how Tn1806 integrates into another ICE in Streptococcus agalactiae, contributing to antibiotic resistance spread.

## Contribution

First experimental evidence of Tn1806 nesting within an ICESa2603-family ICE in S. agalactiae.

## Key findings

- Tn1806-like ICEs were found in 24 out of 161 S. agalactiae isolates.
- Three Tn1806-like ICEs were nested within ICESpy009, an ICESa2603-family ICE.
- Composite ICEs were transferable at low frequencies, with some nested ICEs excising and transferring independently.

## 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 Tn1806 into ICESa2603-family ICEs in Streptococcus agalactiae by conjugation experiments. PCR screening of 161 S. agalactiae clinical 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 the snf2 (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 Tn1806 nesting within another ICE as a mechanism for resistance accumulation and mobile element evolution in S. agalactiae. The spread of such composite ICEs may confer multiple forms of resistance to new hosts, challenging infection treatment and raising public health concerns.