Computational protocol: Evolution and Diversity of the Antimicrobial Resistance Associated Mobilome in Streptococcus suis: A Probable Mobile Genetic Elements Reservoir for Other Streptococci

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Protocol publication

[…] The whole genomes of seven strains were sequenced and assembled on the Roche 454 GS Junior System at the Bioinformatics Center of Nanjing Agricultural University (Nanjing, China). These scaffolds of each genome were ordered according to the reference genome of the S. suis strain P1/7 (AM946016), using the Mauve v2.4.0 software (Darling et al., ). PCR assays were employed to close the gaps to obtain whole sequences of predicted MGEs. [...] MLST classification was derived directly from WGS data by the Web-based method (Larsen et al., ), and further confirmed with traditional PCR method (http://www.mlst.net/). Acquired antimicrobial resistance genes were identified using ResFinder 2.1 (Zankari et al., ).MGEs candidates were roughly located when genomes were compared with S. suis reference strain P1/7 by MAUVE 2.4.1 (Darling et al., ). Prophages were predicted with Phage_Finder (Fouts, ). ICEs were predicted with the present of type IV secretion systems (T4SSs) and integrases (Int). Boundaries and insertion sites of both prophages and ICEs were manually checked. ISs were identified with ISfinder (Siguier et al., ). Transposons were identified by comparing candidate regions containing associated transposase genes against the NCBI nucleotide database. Putative insertion sites and att sequences were manually identified. [...] For ICE identification, signature proteins of integrase, relaxase and VirB4 were searched by BLASTp comparison. Filters were defined by the coverage (>25% and cover function domain), E-value (1e-5) and length (>300 aa for Int, >150 aa for relaxases, and >320 aa for VirB4). If all three proteins were present, the elements were considered ICEs, elements that only harbored integrase and partially or no relaxase and VirB4 were considered defective ICEs (dICEs). Similar analysis of insertions was also done in 637 S. agalactiae, 5063 S. pneumoniae, and 268 S. pyogenes.Phylogenetic tree of integrases, relaxases and VirB4 proteins were further analyzed. Protein identity of less than 60% was considered as different clade. ICEs were classified by the presence of signature integrases, relaxase, VirB4, and AMR profiles.Two-hundred and ten ICESa2603 families of ICEs were retrieved from insertions at rplL and rum sites. BLAST atlas maps were built by comparing ICEs with reference sequences of 89 K (CP000407) using BLASTn with a >80% identity threshold within the CGView Comparison Tool (Grant et al., ). […]

Pipeline specifications

Software tools Mauve, Phage_Finder, BLASTP, BLASTN, CCT
Applications Genome annotation, Phylogenetics, WGS analysis, Nucleotide sequence alignment, Genome data visualization
Organisms Streptococcus suis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes