A New Composite Integrative and Conjugative Element Mediates Multiple Drug Resistance from Streptococcus anginosus to Streptococcus agalactiae

Abstract

Introduction: Integrative and conjugative elements (ICEs) are a category of horizontal mobile genetic elements (MGEs) that play important roles in mediating the spread of antimicrobial resistance in Streptococci.

Methods: In this study, a novel ICE, namely ICESan26_rplL, was identified from commensal Streptococcus anginosus through routine silico analyses. The genetic characterization of ICESan26_rplL was explored based on a comparison with known ICEs. The cyclization and cross-species transferability (from Streptococcus anginosus to Streptococcus agalactiae) of ICESan26_rplL were explored using inverse PCR and conjugation transfer experiments.

Results: ICESan26_rplL is 61.618 kb in length at downstream of rplL and carries multiple antibiotic resistance genes, including erm(B) [for erythromycin, clindamycin, and streptococcin B (MLS_B)], tetM-tetL (tetracycline), lnu and lsa(E) (clindamycin), and ant(6)-I (aminoglycosides). The comparative analysis results showed that ICESan26_rplL was a composite ICE with a mosaic structure composed of modules in the ICESa2603 and TnGBS families. The inverse PCR results demonstrated that ICESan26_rplL could be excised from the chromosome to form a circular intermediate. The conjugation transfer experiment and sequencing results confirmed the cross-species transfer of ICESan26_rplL to S. agalactiae recipients at a relatively low frequency (2.13 × 10^-8). Moreover, core functional modules were retrieved from GenBank to search for any ICEs related to ICESan26_rplL. Eventually, 53 putative ICEs were identified, including three composite ICEs with high similarity to ICESan26_rplL, three ICEs in the TnGBS family, and 47 ICEs in the ICESa2603 family.

Discussion: In this study, a novel mosaic ICE was reported for the first time. In addition, its transfer to the major pathogen Streptococcus agalactiae was characterized and proved. These findings suggest that this ICE is expected to become a vehicle for the dissemination of multiple antimicrobial resistance through the wider pathogen Streptococci.

Keywords: ICEs; Streptococcus agalactiae; Streptococcus anginosus; antibiotic resistance genes; composite; conjugation transfer.

Figure 1

Comparison analysis of ICESan26_rplL with known ICEs. (a) comparison of ICESan26_rplL with ICESa2603, ICESsuYS388, ICESpy009, ICESp1116 and TnGBS2.6. (b) comparison of resistance genes cluster of ICESan26_rplL with plasmid pNUITMV6_2. Integrase genes are showed by blue arrows, relaxase by purple arrows, T4SSs genes by yellow arrows, antibiotics resistance genes by red arrows, putative ISs sequence by green arrows and others by white arrows. Areas shaded French grey represent regions of >60% identity.

Figure 2

Diagram and PCR detection of integration and excision/circularization of ICESan26_rplL. Black rectangles represent TnGBS-like ICE within ICESan26_rplL, the white combinate black rectangles represent composite ICESan26_rplL, Primers used are indicated by black arrows. The bottom panel shows the PCR results and PCR marker is Vazyme DL2000 DNA.

Figure 3

(a) Schematic of ICE conjugation transfer from donor to recipient and obtained transconjugant in cell perspective. (b) Alignment of genome drafts of recipient S. agalactiae SagR331 and transconjugant SagR331_TC. The different regions between transconjugant and recipient represents transferred ICESan26_rplL.