Computational protocol: Genotypic and phenotypic analysis of clinical isolates of Staphylococcus aureus revealed production patterns and hemolytic potentials unlinked to gene profiles and source

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Protocol publication

[…] Bacterial strains were obtained from the general hospital in Vienna and the general hospital in Linz, Austria. As clinical isolates were anonymous and data of the patients were not accessible, the study was exempt from ethical approval, which was testified by the Ethics Committee of the Immunology Outpatient Clinic (www.itk.at/news) after detailed evaluation of the study protocol. Isolates were identified as S. aureus by standard laboratory protocols, grown on tryptic soy broth agar plates. Isolates were not subcultured and thereafter stored at −80 °C for further exploration. Bacteremia isolates were designated with the letter B. All strains were identified as MSSA, and genotyped for twenty-four superantigen (−like) genes, four cytolysin genes, the agr groups I-IV, and the spa gene. All primer pairs are listed in Additional file : Table S1. None of the primer pairs reacted with our negative control strain Staphylococcus epidermidis ATCC 49461. Genomic DNA templates were purified from overnight cultures according to manufacturer’s protocols using the Wizard Purification Kit (Promega). For cell wall disruption cells were treated with lysostaphin and lysozyme (Sigma). DNA was amplified in a T3 thermocycler (Biometra) by 28 cycles of 95 °C (denaturation) for 30 s, specific annealing temperature for 45 s, and 68 °C (elongation) for 60 s, using the Platinum Taq PCRx DNA polymerase (Invitrogen). The reaction was initiated by 10 min incubation at 95 °C, and terminated by 10 min incubation at 68 °C. Primer sequences and PCR annealing temperatures are shown in Additional file : Table S1. Absence of the gene selv was verified through analysis of the genes sei and selm. Distribution statistics were done using the Pearson’s chi-squared test (χ2).Isolated genomic DNA of all strains was sequenced using the Illumina MiSeq sequencer, the Nextera XT library kit, and the MiSeq reagent kit as instructed by the manufacturer (Illumina). Upon checking the average size of amplicons using the BioAnalyzer (Agilent), and measuring the concentration using the QuBit system (Life technologies), four genomic libraries were combined for sequencing. Reads were mapped to S. aureus gene sequences extracted from GenBank (accession numbers: sea NC_003923.1, seb NC_002951.2, sec KF386012.1, sed AF053140.1, see M21319.1, tst AB678405.1, seg NC_009782.1, seh NC_002953.3, sei NC_009782.1, selj AF053140.1, selk NC_007793.1, sell NC_009782.1, selm NC_002745.2, seln EF531605.1, selo CP002388.1, selp NC_002745.2, selq NC_017347.1, selr AB330135.1, sels AB330135.1, selt AB330135.1, selu AY205307.1, selu2 EF030428, selv EF030427, selw CP000046, selx CP007447.1, hla BX571857.1, hlg1 S65052.1, hlg2 S65052.1, agr type I AF210055.1, agr type II AF001782.1, agr type III AF001783, agr type IV AF288215.1) using the alignment program CLC (CLCbio, Qiagen). All isolates were sequenced with a minimum coverage of 20-fold. In order to analyse repeat patterns in spa, de novo assembly of reads was done using CLC []. Blastn of contigs against spa (NC002952.2) was performed, and identified gene variants were assigned using the SPATYPEMAPPER software (download at http://www.clondiag.com/fileadmin/Media/Downloads/SPATypeMapper_0_6.zip). All unknown repeat patterns were re-sequenced (Sanger).Multilocus sequence typing (MLST) was performed as described in []. The seven included genes are arcC, aroE, glpF, gmk, pta, tpi, and ypiL. Loci information and primer sequences are available on the MLST website, which was also used to analyse all alleles (http://www.mlst.net). New spa type and new MLST type were uploaded to the spa website (http://www.spaserver.ridom.de) and MLST website. Newly identified selx and hla mutations have been deposited at GenBank (accession numbers KT943499 and KU236387). […]

Pipeline specifications

Software tools CLC Assembly Cell, BLASTN
Application Immune system analysis
Organisms Oryctolagus cuniculus, Staphylococcus aureus, Epipremnum aureum, Homo sapiens