Growth and biofilm formation by Staphylococcus epidermidis and other relevant contaminant bacteria during storage of platelet concentrates.

摘要

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal adverse transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. Its ability to cause chronic nosocomial infections by forming surface-attached communities of organisms known as biofilms has imparted the status of major opportunistic pathogen to this otherwise innocuous, Gram-positive skin commensal. However, its virulence in the context of transfusable blood products has not been extensively studied. In particular, biofilm formation during platelet storage may be fundamental to missed bacterial detection by current screening methods. The preliminary objective of this thesis was to confirm that biofilm formation could be exhibited by S. epidermidis under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential. Confirmatory markers included (a) presence of the biofilm icaD gene (b) slime production on Congo red agar, (c) biofilm formation on polystyrene, and (d) polysaccharide intercellular adhesin detection via indirect immunoassay. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm phenotype.;The findings of this thesis research endorse novel proposals to minimize biofilm occurrence during platelet storage. Since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. As a final, proof-ofprinciple objective, methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed 'PEGylation'. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.;Validation of the threat of S. epidermidis biofilm formation during platelet storage signifies that any modifications to current platelet storage protocols should be rigorously evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example. Little is known about the effect of PAS on the growth dynamics of bacteria, and there have been no studies on the influence of bacteria on platelet quality when suspended in PAS. To this end, PCs suspended in either plasma or PAS were inoculated with S. epidermidis or the Gram-negative bacterium Serratia liquefaciens. Growth rates and biofilm formation were assayed for 5 days. Concurrently, platelet markers were measured by an assay panel and flow cytometry. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs. Reduced biofilm formation was exhibited by both species during storage in PAS-PCs, and is advantageous since it increases bacteria availability for sampling detection. Although S. liquefaciens altered several platelet quality markers by day 3-4 post-inoculation in both PAS-PCs and plasma-PCs, S. epidermidis contamination did not produce measurable platelet changes. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs, while changes of platelet quality can herald S. liquefaciens contamination in excess of 108 CFU/mL.