The spa-typing data from MRSA isolates were analyzed by the BURP (Based Upon Repeat Pattern) algorithm, using the StaphType software v. 2.2.1 (Ridom GmbH, Germany). Neighbor-Joining tree of the isolates was constructed using MEGA 6 (Molecular Evolutionary Genetics Analysis; Tamura et al., 2013 (link)).
Staphtype software v 2
Manufactured by Ridom
Sourced in Germany
Ridom StaphType software v.2.1.1 is a bioinformatics tool designed for the molecular typing of Staphylococcus aureus isolates. The software performs sequence-based analysis of the staphylococcal protein A (spa) gene to determine the corresponding spa type.
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Lab products found in correlation
5 protocols using staphtype software v 2
1
MRSA isolates spa-typing analysis
2
Comprehensive Molecular Profiling of Staphylococcus aureus
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All isolates were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion, staphylococcal protein A gene (spa) typing using StaphType software v. 2.2.1 (Ridom, Germany), multilocus sequence typing (MLST) using BioNumerics v. 6.6 (Applied Maths, Belgium) MLST online plugin, agr group determination [10] , SCCmec typing [11] , plasmid content analysis [12] , and prophage typing [13] . The genes for PVL [14] , leukocidin lukED [15] , staphylococcal enterotoxins [16] , enterotoxin gene cluster (EGC) [17] , exfoliative toxin genes [18] , immune evasion cluster (IEC) [19] , and arginine catabolic mobile element (ACME) [20] were screened using polymerase chain reaction (PCR) or multiplex PCR.
3
Standardized spa Typing and Clustering
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spa typing was performed according to Harmsen et al.9 (link) Nucleotide sequencing of the repeat-containing region of the spa gene was conducted on both DNA strands of the PCR product by GenoMed (St Louis, MO, USA), using BigDye Terminator Ready Reaction Cycle Sequencing Kit. The spa types were identified with Ridom StaphType software v.2.1.1 (Ridom GmbH, Münster, Germany),9 (link) and grouped using BURP, Ridom StaphType software. spa types were clustered if the cost between members of a given group was less than or equal to four. The BURP algorithm was used to assign spa types into spa clonal complexes (spa-CCs) with defined default parameters “exclude spa types shorter than five repeats” and “cluster spa types into the same group if cost distances are less than four”.10 (link) Since the results of spa typing and MLST are highly concordant,7 (link) the spa typing data could be easily mapped on the MLST types using SpaServer database (http://spaserver.ridom.de/ ).
4
Genetic Characterization of Staphylococcus aureus
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Initially, S. aureus isolates were screened by PCR for identification of CC398 [24 (link)] and in all accessory gene regulator (agr) allotypes were determined [25 (link)]. Amongst CC398 isolates discrimination between human and animal clade was performed [26 (link)].
spa typing was performed [27 (link)] and spa-types were determined by using the Ridom StaphType software v.2.1.1 (Ridom GmbH) [28 (link)]. The BURP algorithm was used to assign spa-types into spa-clonal complexes (spa-CCs) with defined default parameters "exclude spa-types shorter than 5 repeats" and "cluster spa-types into the same group if cost distances are less than 4" [29 (link)].
Multilocus Sequence Typing (MLST) was performed on selected isolates, 36 MRSA and 62 MSSA [30 (link)]. At least one isolate per each spa-type detected in each source of material was analyzed. The sequence type (ST) was also determined for three non-spa-typeable isolates. Allele numbers and STs were assigned through the S. aureus MLST database (http://saureus.mlst.net ).
Clonal complexes, CCs, were determined using the eBURSTv3 algorithm (http://saureus.mlst.net/eburst ) and restricted to STs that share five or more alleles of seven loci examined with founder or subfounders, within each predicted clonal group [31 (link)]. The analysis was performed on April 29th, 2016.
spa typing was performed [27 (link)] and spa-types were determined by using the Ridom StaphType software v.2.1.1 (Ridom GmbH) [28 (link)]. The BURP algorithm was used to assign spa-types into spa-clonal complexes (spa-CCs) with defined default parameters "exclude spa-types shorter than 5 repeats" and "cluster spa-types into the same group if cost distances are less than 4" [29 (link)].
Multilocus Sequence Typing (MLST) was performed on selected isolates, 36 MRSA and 62 MSSA [30 (link)]. At least one isolate per each spa-type detected in each source of material was analyzed. The sequence type (ST) was also determined for three non-spa-typeable isolates. Allele numbers and STs were assigned through the S. aureus MLST database (
Clonal complexes, CCs, were determined using the eBURSTv3 algorithm (
5
Staphylococcus aureus Typing via spa
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spa typing was performed according to Harmsen et al.22 (link) Nucleotide sequencing of the repeat-containing region of the spa gene was conducted on both DNA strands of the PCR product from Genomed (Warszawa, Poland), using BigDye Terminator Ready Reaction Cycle Sequencing kit (St Louis, MO, United States). spa-types were identified with Ridom Staph-Type software v.2.1.1 (Ridom GmbH, Münster, Germany).22 (link)
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