Computational protocol: Molecular typing and characterization of nasal carriage and community-onset infection methicillin-susceptible Staphylococcus aureus isolates in two Taiwan medical centers

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Protocol publication

[…] We used PCR to molecularly characterize isolates by spa type, multi-locus sequence typing (MLST), agr specificity group, and virulence genes. S. aureus DNA was extracted using a DNeasy Tissue Kit (Qiagen Inc., Valencia, USA), following the manufacturer’s instructions with a slight modification. An additional step was added just prior to proteinase K digestion: incubation with 5 μL of 5 mg/mL lysostaphin at 37°C for 30 min to 1 h until the cell suspension became clear.spa typing and the Based Upon Repeat Pattern (BURP) algorithm were performed using Ridom StaphType software (version 1.5; Ridom GmbH, Würzburg, Germany). Two clustering parameters were applied to BURP clusters, spa-clonal complexes (CCs), using dialog: “exclude spa types that are shorter than 5 repeats” and “spa types are clustered if cost is less or equal 4” [,]. The cost “6” parameter was also used as discussed by Strommenger et al. []. The cost matrix for distances of strains between spa types was also produced using the StaphType software, and the phylogenetic tree was conducted using MEGA version 5 by the neighbor-joining method []. The spa repeat region was amplified and sequenced using primers spa-1113f and spa-1514r, according to the manufacturer's instructions.MLST was performed as described previously on selected isolates of major PFGE clusters and all infection isolates []. Sequence type (ST) was assigned based on sequence allelic profiles using the MLST database website (http://www.mlst.net) [].agr specificity groups were determined by multiplex PCR as described by Lina et al. []. Non-typable strains were determined by agrD sequencing, which encodes the autoinducing peptide (AIP) precursor [].Virulence toxin genes for sea-e, seg-j, sem-o, tst, eta, etb, lukElukD, hla, hlb, hld, hlg, and hlg-2 were detected as described by Jarraud et al. []. Other bacterial adhesion genes, including fnbA, cna, sdrC, sdrD, sdrE, bbp, and icaA, were detected as described by Peacock et al. []. Sequences specific for Panton-Valentine leukocidin (PVL) genes (lukS-lukF) were detected as described previously []. […]

Pipeline specifications

Software tools StaphType, MEGA
Databases MLST.net
Application Phylogenetics
Organisms Staphylococcus aureus, Epipremnum aureum
Diseases Infection, Staphylococcal Infections
Chemicals Erythromycin, Methicillin