Computational protocol: Molecular characterization of Panton-Valentine leukocidin (PVL) toxin–encoding phages from South India

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Protocol publication

[…] A total of 19 nonduplicate MRSA isolates collected during 2015–2016 from patients with sepsis were included in this study. Antibiotic susceptibility testing was performed by the disk diffusion method for the following antibiotics: cefoxitin (30 μg), gentamicin (10 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), netilmicin (30 μg), rifampicin (5 μg), erythromycin (15 μg), clindamycin (2 μg), tetracycline (30 μg) and linezolid (30 μg). Inducible clindamycin resistance was detected by the D-zone test. Minimum inhibitory concentrations of vancomycin were determined by microbroth dilution method according to Clinical and Laboratory Standards Institute guidelines . SCCmec typing was performed as previously described .DNA isolation from pure cultures was performed using the QiAamp DNA mini Kit (Qiagen). The whole genome shotgun sequencing was performed for all the isolates using the Ion Torrent PGM system (Life Technologies) with 400 bp chemistry. The raw data generated were assembled de novo using AssemblerSPAdes v.5.0.0.0 embedded in Torrent suite server v.5.0.4. Genome sequence was annotated using PATRIC, the bacterial bioinformatics database and analysis resource (http://www.patricbrc.org) , and the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) (http://www.ncbi.nlm. nih.gov/genomes/static/Pipeline.html). Downstream analysis was performed using the Center for Genomic Epidemiology (CGE) server (http://www.cbs.dtu.dk/services) and PATRIC. Resistance gene profiles were analysed using ResFinder 2.1 from the CGE server (https://cge.cbs.dtu.dk//services/ResFinder/). ST was determined for all the isolates in the allele order of arcc, aroe, glpf, gmk, pta, tpi and yqil by comparing the sequences with the S. aureus database maintained at the MLST website (http://saureus.mlst.net/). PHASTER (PHAge Search Tool Enhanced Release) was used for annotation and identification of prophage sequences in the bacterial genome. […]

Pipeline specifications

Software tools PGAP, PHASTER, PHAST
Databases PATRIC MLST.net
Application Genome annotation