Spaserver

The spa typing, based on amplification of the variable X region of protein A gene, was performed as described previously^{90 (link)}. The spa types were assigned using the Ridom StaphType software version 2.2.1 (https://www.ridom.de/ Ridom GmbH, Wurzburg, Germany) and the Ridom SpaServer database (SpaServer.ridom.de/">https://SpaServer.ridom.de/). The predicted MLST were assigned based on Ridom SpaServer. The based upon repeat pattern (BURP) algorithm was used to calculate spa clonal complexes (spa-CCs) with following parameters: (I) exclude spa types shorter than 5 repeats; (II) cost less or equal to 4; (III) cluster composed of 2 or more related spa types was regarded as CC; (IV) a spa type that was not grouped into a CC was considered as singleton.

Bacterial strains were cultured on blood agar at 35°C, after which extraction of DNA was performed by heat lysis. Briefly, a few colonies were suspended in molecular grade water and heated to 95°C for 15 minutes with shaking (300 rpm). After centrifugation at 14,500 rpm for 2 minutes, the supernatant was collected. Confirmation of MRSA was performed with a multiplex conventional PCR detecting the mecA gene, the S. aureus-specific spa gene, and the Panton-Valentine leukocidin (PVL) genes lukSF-PV, followed by gel electrophoresis [11 (link)]. For strains that were mecA negative, mecC PCR [5 (link)] was additionally performed.
All strains were spa-typed according to Harmsen et al. [11 (link)] with primers spa-1113f and spa-1514r [12 (link)] using the Ridom StaphType software and SpaServer [13 (link)]. The spa-types were assigned to known sequence types (ST) and/or clonal complexes (CC) based on Ridom Staphtyper [12 (link)] and pubMLST [14 (link), 15 (link)] databases. If a spa-type could not be assigned to a ST or CC, multi locus sequence typing (MLST) was performed, and CC was assigned using eBURST [16 (link)] software. A minimum spanning tree (MST) based on spa-repeats was constructed using Based Upon Repeat Pattern (BURP) clustering [17 (link)] with the Ridom SeqSphere + software [18 (link)]. To calculate genotypic diversity, Simpson’s diversity index [19 ] was used.

There is currently no established molecular marker for LA-S. aureus. LA-MDRSA has been consistently classified as: (i) MDRSA carriage or infection in humans arising from exposure to livestock, (ii) belonging to the clonal complex 398 (CC398) or CC9, and (iii) lacking the staphylococcal complement inhibitor gene scn (scn-). Here we used CC9 or CC398 within the IIa livestock clade of the CC398 phylogeny, and the absence of the scn gene (scn-) as indicators of LA-S. aureus. Putative clonal complex (CC) was previously assigned to each isolate based on spa type, using the Ridom StaphType software and the Ridom SpaServer (http://spa.ridom.de/index.shtml).

Staphylococcal protein A (spa) typing was performed by amplifying the spa gene as described above. PCR products were sequenced by Eton Biosciences, Inc. (Research Triangle Park, NC). spa types were assigned using the Ridom Staph Type standard protocol (http://www.ridom.com) and the Ridom SpaServer (http://spa.ridom.de/index.shtml). To assess nasal carriage prevalence of CC398 and CC9, spa types were assigned to these clonal complexes based on the scientific literature and analyses of S. aureus isolates collected from our prior livestock worker studies in North Carolina (Nadimpalli et al. 2015 (link); Rinsky et al. 2013 (link)). The spa types classified as CC398 and CC9 are listed in the Supplemental Material in “Assignment of spa types to clonal complexes (CCs).” For within household concordance, spa types were assigned to multi-locus sequence types (MLSTs) based on the scientific literature, analyses of S. aureus isolates collected from our prior livestock worker studies in North Carolina (Nadimpalli et al. 2015 (link); Rinsky et al. 2013 (link)), and queries of the Ridom SpaServer.

The polymorphic X region of the spa gene was amplified from all S. aureus isolates using the spa primers exhibited in Table 1. All sequencing reactions were performed at Bioneer (Korea) and then the data were analyzed using MEGA 4 software. Finally, spa types were assigned by Ridom SpaServer (SpaServer.ridom.de">http://SpaServer.ridom.de) [12 (link)].

The spa typing was performed for methicillin-resistant S. aureus strains as described previously [16 (link)]. The method is based on the sequence analysis of variable region of the protein A (spa) gene, resulting in spa types, assigned by the Ridom StaphType software version 2.2.1 (http://www.ridom.de/ Ridom GmbH, Wurzburg, Germany) and the Ridom SpaServer database (SpaServer.ridom.de/" xmlns:xlink="http://www.w3.org/1999/xlink">http://SpaServer.ridom.de/). The predicted MLST were assigned based on Ridom SpaServer.