# Characterization of a novel composite ICE in Streptococcus agalactiae conferring resistance to macrolides [erm(TR)] and cadmium (cadA)

## TL;DR

A new composite genetic element in Streptococcus agalactiae was found to carry genes for resistance to antibiotics and cadmium, aiding in the spread of these resistance traits.

## Contribution

The discovery of a novel composite ICE, ICESag39, with a modular structure that promotes resistance gene dissemination.

## Key findings

- ICESag39 has a nested structure with an internal Tn1806-like ICE carrying resistance genes erm(TR) and cadA.
- ICESag39 transfers at a frequency of 8.2 × 10−9 and co-transfers both resistance determinants.
- Database screening revealed 199 similar ICEs, 62.8% of which co-carried erm(TR) and cadA.

## Abstract

Macrolide resistance genes (erm and mef families) and heavy metal resistance genes (cadA) are increasingly disseminated among streptococci via diverse mobile genetic elements.

Through whole-genome sequencing of 16 Streptococcus agalactiae isolates resistant to both erythromycin and clindamycin, we identified 19 integrative and conjugative elements (ICEs), a type of self-transfer genetic elements, conferring antibiotic resistance. Among these, a novel composite ICE designated ICESag39 was identified in S. agalactiae Sag39 through comparative analysis with the NCBI database.

ICESag39 measured 113,125 bp in length, and it featured a nested “Russian doll” structure comprising an ICESa2603 family backbone integrated with an internal Tn1806-like ICE. The embedded Tn1806-like ICE contained four variable regions (VR1-VR4) that serve as insertion hotspots; among these, VR3 and VR4 carry erm(TR) from ICESp2907 and the cadmium resistance gene cadA from an uncharacterized genetic element, respectively. Conjugation and excision assays confirmed that ICESag39 transfers at a frequency of 8.2 × 10−9 and co-transfers both resistance determinants. Under cadmium stress, transconjugants carrying ICESag39 displayed enhanced growth relative to the recipient. Although the internal Tn1806-like ICE was also capable of independent transfer, its efficiency was significantly lower (< 10−9), and its circular form is undetectable by PCR. Database screening identified 199 structurally similar ICEs (ICESag39-like ICE), 62.8% (125/199) of which co-carried erm(TR) and cadA, underscoring the prevalence of this ICE and its associated resistance traits.

This study characterizes a new composite ICE and elucidates a modular mechanism that facilitates the assembly and dissemination of resistance genes, thereby promoting bacterial genome diversification.