Genome Sequencing of a Historic Staphylococcus aureus Collection Reveals New Enterotoxin Genes and Sheds Light on the Evolution and Genomic Organization of This Key Virulence Gene Family

Abstract

We take advantage of an historic collection of 133 Staphylococcus aureus strains accessioned between 1924 and 2016, whose genomes have been long-read sequenced as part of a major National Collection of Type Cultures (NCTC) initiative, to conduct a gene family-wide computational analysis of enterotoxin genes. We identify two novel Staphylococcal enterotoxin (pseudo)genes (sel29p and sel30), the former of which has not been observed in any contemporary strain to date. We provide further information on five additional enterotoxin genes or gene variants that have either recently entered the literature or for which the nomenclature or description is currently unclear (selz, sel26, sel27, sel28 and ses-2p). An examination of over 11,000 RefSeq genomes in search of wider support for these seven (pseudo)genes led to the identification of an additional three novel enterotoxin gene family members (sel31, sel32 and sel33) plus two new variants (seh-2p and ses-3p). We cast light on the genomic distribution of the enterotoxin genes, further defining their arrangement in gene clusters. Finally, we show that co-occurrence of enterotoxin genes is prevalent, with individual NCTC strains possessing as many as eighteen enterotoxin genes and pseudogenes, and that Clonal Complex membership rather than time of isolation is the key factor in determining enterotoxin load. Importance: Staphylococcus aureus strains pose a significant health risk to both human and animal populations. Key amongst this species’ virulence factors are the Staphylococcal enterotoxin gene family. Certain enterotoxin forms can induce a potentially life-threatening immune response, while others are implicated in less fatal though often severe conditions such as food poisoning. Genetic characterisation of Staphylococcal enterotoxin gene family members has steadily accumulated over recent decades, with over 20 genes now established in the literature. Despite the current wealth of knowledge on this important gene family, questions remain about the presence of additional enterotoxin genes and the genomic composition of family members. This study further expands knowledge of the Staphylococcal enterotoxins while shedding light on their evolution over the last century.

Homologous recombination between tandem paralogues drives evolution of a subset of type VII secretion system immunity genes in firmicute bacteria

“…The coordinates of the ess/T7 locus were identified in each strain using a custom bash script and the locus extracted using the coordinates for DNA immediately 5′ of essC and 3′ of focA (which defines the 3′ boundary of the T7 immunity island [ 10 ]; Supplementary Data 1). Next the clonal complex of each strain was annotated based on a core genome SNP tree constructed previously [ 35 ], because for the purpose of our analysis it was essential to analyse very closely related strains to ensure that any differences could not be due to divergent evolution. From the annotated tree we selected to analyse the ess/T7 locus from the 31 CC8 strains as there are a significant number of strains in this clonal complex (including NCTC8325) and all strains are extremely closely related to one another [ 35 ].…”

“…Next the clonal complex of each strain was annotated based on a core genome SNP tree constructed previously [ 35 ], because for the purpose of our analysis it was essential to analyse very closely related strains to ensure that any differences could not be due to divergent evolution. From the annotated tree we selected to analyse the ess/T7 locus from the 31 CC8 strains as there are a significant number of strains in this clonal complex (including NCTC8325) and all strains are extremely closely related to one another [ 35 ]. Using Easyfig to assess the genetic differences at this locus [ 32 ], we observed that the most common genetic composition was to encode 12 esaG paralogues, as seen for NCTC8325 and RN6390 (Fig.…”

Homologous recombination between tandem paralogues drives evolution of a subset of Type VII secretion system immunity genes in firmicute bacteria

“…The coordinates of the ess/T7 locus were identified in each strain using a custom bash script and the locus extracted using the coordinates for DNA immediately 5’ of essC and 3’ of focA (which defines the 3’ boundary of the T7 immunity island (10); Supplementary Data 1). Next the clonal complex of each strain was annotated based on a core genome SNP tree constructed previously (34), because for the purpose of our analysis it was essential to analyse very closely related strains to ensure that any differences could not be due to divergent evolution. From the annotated tree we selected to analyse the ess/T7 locus from the 31 CC8 strains as there are a significant number of strains in this clonal complex (including NCTC8325) and all strains are extremely closely related to one another (34).…”

“…Next the clonal complex of each strain was annotated based on a core genome SNP tree constructed previously (34), because for the purpose of our analysis it was essential to analyse very closely related strains to ensure that any differences could not be due to divergent evolution. From the annotated tree we selected to analyse the ess/T7 locus from the 31 CC8 strains as there are a significant number of strains in this clonal complex (including NCTC8325) and all strains are extremely closely related to one another (34). Using Easyfig to assess the genetic differences at this locus (35), we observed that the most common genetic composition was to encode 12 esaG paralogues, as seen for NCTC8325 and RN6390 (Fig S3).…”

Staphylococcus aureus in the Processing Environment of Cured Meat Products

“…The complete egc was sequenced with the designed primers. Four different egc variants were found (Figure 3): ST433 and ST30 belonged to egc3 [11] or OMIUNG according to other classification [20]. The isolate D41 (ST30) possessed the egc3 but also two amino acid substitutions in SEO (D49E) and SEG (V230F) regarding deposited sequences in Genbank.…”