ICESag08: A new composite integrative and conjugative element from Streptococcus agalactiae mediating resistance to chloramphenicol and macrolides

• Identifies a novel 133-kb composite ICE (ICE Sag 08) in Streptococcus agalactiae carrying multidrug resistance to chloramphenicol and macrolides. • Demonstrates horizontal transferability of ICE Sag 08 via conjugation, albeit at low frequency (1.05 × 10 −9 ). • Confers stable resistance to chloramphenicol and macrolides but significant fitness cost in transconjugants carrying ICE Sag 08 with recipient. • Reveals the potential for ICEs or elements from different streptococci to assemble into composite elements through specific integration and/or recombination Streptococcus agalactiae poses increasing clinical challenges owing to emerging antibiotic resistance, particularly to chloramphenicol, mediated by the conserved catQ - mef (I) (IQ module). To date, IQ module-carrying elements have been described: ICE Spn 529IQ (ICE Sa 2603 family) from Streptococcus pneumoniae , ICE Spy 029IQ and ICE Spy 005IQ (Tn 5253 family) from Streptococcus pyogenes , and composite ICE Sag 236 and ICE Sag 403 from Streptococcus agalactiae . This study characterized a novel composite integrative and conjugative element (ICE) carrying an IQ module by bioinformatics analysis of sequenced chloramphenicol-resistant S. agalactiae. Transferability was confirmed by conjugation assays. Fitness costs were assessed through growth and competition assays, and stability was evaluated in serial passage experiments. Comparative analysis with known ICEs from GenBank revealed that ICE Sag 08 is a 133-kb composite ICE that integrates four distinct elements: ICE Spn 529IQ, ICE San 95_ hsdM , ICE Spy 009, and IME Sp 2907. The catQ – mef (I) locus is located within the ICE Spn 529IQ, whereas erm (TR) is carried by IME Sp 2907, which is embedded within ICE San 95_ hsdM . Conjugation experiments and sequencing confirmed that ICE Sag 08 is transferable to recipient strains at frequency of 1.05 × 10⁻⁹. Transconjugants exhibited impaired growth and reduced competitive ability compared with those of the recipient strain. Stability assays performed after successive passages showed that ICE Sag 08 remained stable and conferred resistance. These results highlight the role of composite ICEs in the horizontal transfer of multidrug resistance, including to chloramphenicol ( catQ ) and macrolides [ mef (I), erm (TR)], and reveal an intricate interplay between different ICEs. These insights advance our understanding of how resistance disseminates and evolves and support the development of effective strategies to limit the spread of antimicrobial resistance.