Proof of concept for detection of staphylococcal enterotoxins in platelet concentrates as a novel safety mitigation strategy.

BACKGROUND AND OBJECTIVES: Staphylococcus aureus is a predominant contaminant of platelet concentrates (PCs) that can evade detection during screening with culture methods. Importantly, S. aureus produces staphylococcal enterotoxins (SEs) during PC storage, which are linked to slow growth and enhanced biofilm formation. This study investigated timing of SE production during PC storage and feasibility of SE detection as a PC safety strategy. MATERIALS AND METHODS: Genomic and transcriptomic data of transfusion-relevant S. aureus PS/BAC/169/17/W, PS/BAC/317/16/W, CI/BAC/25/13/W and CBS2016-05 were used to determine the presence and differential expression of exotoxin genes in PCs. Trypticase soy broth (TSB) and PCs were inoculated with 1.0E+06 cfu/mL of S. aureus PS/BAC/169/17/W and CBS2016-05. Expression of SEs at different growth phases was confirmed with Western blotting. PCs were inoculated with 30 cfu/unit of the same strains, and SE detection during PC storage was optimized with a sandwich dot-ELISA assay. RESULTS: S. aureus genomes contain multiple exotoxin genes including those encoding for SEs. Transcriptome data revealed significant upregulation (0.5-6.7-fold, p < 0.05) of SE genes in PCs versus TSB. Western blots demonstrated SE production at all growth phases. Notably, dot-ELISA detected clinically relevant concentrations of SEs (~0.2 µg/mL) at 32 h of PC storage when S. aureus PS/BAC/169/17/W and CBS2016-05 counts were 1.8E+04 and 1.4E+04 cfu/mL, respectively. CONCLUSION: Genomic analyses revealed that staphylococcal exotoxins are widely distributed and highly conserved among transfusion-relevant S. aureus isolates. Furthermore, SEs are significantly upregulated in PCs and detected at 30 h of PC storage. Therefore, bacterial toxin detection could supplement mitigation strategies to enhance PC safety.

Staphylococcal Enterotoxins Enhance Biofilm Formation by Staphylococcus aureus in Platelet Concentrates.

Biofilm formation and slow growth by Staphylococcus aureus in platelet concentrates (PCs) cause missed detection of this bacterium during routine PC screening with automated culture systems. This heightens the chances of false-negative screening transfusions and pre-disposes transfusion patients to an elevated risk of sepsis due to secretion of staphylococcal enterotoxins (SEs) in PCs. A hybrid approach of comparative RNAseq analyses and CRISPR mutagenesis of SE genes was employed to investigate the effect of SEs in S. aureus growth and biofilm formation in PCs. RNAseq data showed no differential expression for key biofilm genes, whereas SE genes were upregulated (>0.5- to 3.6-fold change) in PCs compared to trypticase soy broth (TSB). Remarkably, growth and biofilm formation assays revealed increased growth for the S. aureus SE mutants, while their ability to form biofilms was significantly impaired (−6.8- to −2.4-fold change) in comparison to the wild type strain, in both PCs and TSB. Through the well-established superantigen mechanism of SEs, we propose three roles for SEs during biofilm development in PCs: (1) provide a scaffold for biofilm matrix, (2) mediate cell-to-cell aggregation, and (3) guarantee biofilm survival. Furthermore, SE contribution to both growth and biofilm development seems to be centrally regulated by agr via quorum sensing and by saeSR and sigB. This study reveals new roles for SEs, which enforce their relevance in ensuring PC safety for transfusion patients. It further deciphers the underlying reasons for failed S. aureus detection in PCs during screening with automated culture systems.

Complete Genome Sequence of Staphylococcus aureus CI/BAC/25/13/W, Isolated from Contaminated Platelet Concentrates in England.

We present the genome sequence of Staphylococcus aureus CI/BAC/25/13/W, which was isolated in 2013 as a contaminant of a platelet concentrate with abnormal clotting at the National Health Service Blood and Transplant. Assessment of the genome sequence showed the presence of one chromosome (2,719,347 bp) and one plasmid (1,533 bp).

Complete Genome Sequence of Staphylococcus aureus PS/BAC/169/17/W, Isolated from a Contaminated Platelet Concentrate in England.

We report the genome sequence of Staphylococcus aureus PS/BAC/169/17/W, which was isolated in 2017 from a contaminated platelet concentrate at the National Health Service Blood and Transplant. Assessment of the genome sequence of this strain showed the presence of a 2,753,746-bp chromosome and a plasmid of 2,762 bp.

Giant group I intron in a mitochondrial genome is removed by RNA back-splicing.

BACKGROUND: The mitochondrial genomes of mushroom corals (Corallimorpharia) are remarkable for harboring two complex group I introns; ND5-717 and COI-884. How these autocatalytic RNA elements interfere with mitochondrial RNA processing is currently not known. Here, we report experimental support for unconventional processing events of ND5-717 containing RNA. RESULTS: We obtained the complete mitochondrial genome sequences and corresponding mitochondrial transcriptomes of the two distantly related corallimorpharian species Ricordea yuma and Amplexidiscus fenestrafer. All mitochondrial genes were found to be expressed at the RNA-level. Both introns were perfectly removed by autocatalytic splicing, but COI-884 excision appeared more efficient than ND5-717. ND5-717 was organized into giant group I intron elements of 18.1 kb and 19.3 kb in A. fenestrafer and R. yuma, respectively. The intron harbored almost the entire mitochondrial genome embedded within the P8 peripheral segment. CONCLUSION: ND5-717 was removed by group I intron splicing from a small primary transcript that contained a permutated intron-exon arrangement. The splicing pathway involved a circular exon-containing RNA intermediate, which is a hallmark of RNA back-splicing. ND5-717 represents the first reported natural group I intron that becomes excised by back-splicing from a permuted precursor RNA. Back-splicing may explain why Corallimorpharia mitochondrial genomes tolerate giant group I introns.

Deep-water sea anemone with a two-chromosome mitochondrial genome.

Mitochondrial genome organization of sea anemones appears conserved among species and families, and is represented by a single circular DNA molecule of 17 to 21 kb. The mitochondrial gene content corresponds to the same 13 protein components of the oxidative phosphorylation (OxPhos) system as in vertebrates. Hallmarks, however, include a highly reduced tRNA gene repertoire and the presence of autocatalytic group I introns. Here we demonstrate that the mitochondrial genome of the deep-water sea anemone Protanthea simplex deviates significantly from that of other known sea anemones. The P. simplex mitochondrial genome contains a heavily scrambled order of genes that are coded on both DNA strands and organized along two circular mito-chromosomes, MCh-I and MCh-II. We found MCh-I to be representative of the prototypic sea anemone mitochondrial genome, encoding 12 OxPhos proteins, two ribosomal RNAs, two transfer RNAs, and a group I intron. In contrast, MCh-II was found to be a laterally transferred plasmid-like DNA carrying the conserved cytochrome oxidase II gene and a second allele of the small subunit ribosomal RNA gene.

Expression of homing endonuclease gene and insertion-like element in sea anemone mitochondrial genomes: Lesson learned from Anemonia viridis.

The mitochondrial genomes of sea anemones are dynamic in structure. Invasion by genetic elements, such as self-catalytic group I introns or insertion-like sequences, contribute to sea anemone mitochondrial genome expansion and complexity. By using next generation sequencing we investigated the complete mtDNAs and corresponding transcriptomes of the temperate sea anemone Anemonia viridis and its closer tropical relative Anemonia majano. Two versions of fused homing endonuclease gene (HEG) organization were observed among the Actiniidae sea anemones; in-frame gene fusion and pseudo-gene fusion. We provided support for the pseudo-gene fusion organization in Anemonia species, resulting in a repressed HEG from the COI-884 group I intron. orfA, a putative protein-coding gene with insertion-like features, was present in both Anemonia species. Interestingly, orfA and COI expression were significantly up-regulated upon long-term environmental stress corresponding to low seawater pH conditions. This study provides new insights to the dynamics of sea anemone mitochondrial genome structure and function.

Zoantharian mitochondrial genomes contain unique complex group I introns and highly conserved intergenic regions.

Complex group I introns represent hallmarks of hexacoral mitochondrial genomes (mtDNAs). These intron elements are expected to influence the gene organization and gene expression. We sequenced the mitochondrial genome and transcriptome of Zoanthus sansibariscus and Palythoa heliodiscus, two zoantharian species (colonial anemones) representing different families within the suborder Brachycnemina. The circular and approximately 21kb mtDNAs contained two group I introns, one in ND5 and another in COI. The ND5-717 intron harbored two conventional mitochondrial genes (ND1 and ND3) within its structure and revealed several conserved features compared to ND5-717 in sea anemones. The COI intron, however, was inserted at a unique location (after position 867), which was different from that in sea anemones (position 884) and stony corals (position 720). COI-867 contained a homing endonuclease gene (HEG) with remarkable features, including species-specific length variations and only one copy of the essential LAGLIDADG motif. Whereas transcriptome analysis indicated that all conventional mtDNA genes were expressed, HEG expression appeared significantly repressed. Finally, we identified absolutely conserved non-coding repeat motifs with antisense features and potential regulatory functions.