Characterization of the growth dynamics and biofilm formation of Staphylococcus epidermidis strains isolated from contaminated platelet units

Abstract

Bacterial contamination of platelet concentrates (PCs) poses the highest transfusion-associated infectious risk, with Staphylococcus epidermidis being a predominant contaminant. Herein, the growth dynamics of 20 S. epidermidis strains in PCs and regular media were characterized. Strains were categorized as fast (short lag phase) or slow (long lag phase) growers in PCs. All strains were evaluated for the presence of the biofilm-associated icaAD genes by PCR, their capability to produce extracellular polysaccharide (slime) on Congo red agar plates and their ability to form surface-attached aggregates (biofilms) in glucose-supplemented trypticase soy broth (TSBg) using a crystal violet staining assay. A subset of four strains (two slow growers and two fast growers) was further examined for the ability for biofilm formation in PCs. Two of these strains carried the icAD genes, formed slime and produced biofilms in TSBg and PCs, while the other two strains, which did not carry icaAD, did not produce slime or form biofilms in TSBg. Although the two ica-negative slime-negative strains did not form biofilms in media, they displayed a biofilm-positive phenotype in PCs. Although all four strains formed biofilms in PCs, the two slow growers formed significantly more biofilms than the fast growers. Furthermore, growth experiments of the two ica-positive strains in plasma-conditioned platelet bags containing TSBg revealed that a slow grower isolate was more likely to escape culture-based screening than a fast grower strain. Therefore, this study provides novel evidence that links S. epidermidis biofilm formation with slow growth in PCs and suggests that slow-growing biofilm-positive S. epidermidis would be more likely to be missed with automate culture.

Antimicrobial peptides: an effective approach to prevent bacterial biofilm formation in platelet concentrates

“…In this study, we demonstrate that a combination of synthetic AMPs is effective in inhibiting biofilm formation by three biofilm‐forming strains of S. epidermidis in PCs. Previously, we have shown that the PC storage environment promotes biofilm formation by S. epidermidis isolates traditionally considered to be biofilm‐negative . Importantly, biofilms formed in PCs have increased pathogenicity as demonstrated using nematode‐killing assays .…”

“…It is also acknowledged that clinical studies in humans are needed before the use of AMPs have a practical application in transfusion settings. It is nevertheless important to highlight that bacterial detection during PC screening would be enhanced by using AMPs since we have demonstrated that biofilm formation during PC storage results in missed detection with culture systems . We have also shown that biofilms formed during PC storage have enhanced pathogenicity and could pose a higher safety risk to transfusion patients, which would be averted by inhibiting biofilm formation with AMPs.…”

“…Importantly, biofilms formed in PCs have increased pathogenicity as demonstrated using nematode‐killing assays . Biofilm formation by S. epidermidis has also been linked to missed detection during PC screening .…”

“…Wells of six‐well polystyrene tissue culture plates (Corning, Inc.) were seeded with 3 mL of this suspension and incubated at 37°C with no agitation for 24 hours to obtain mature biofilms. For biofilm formation in PCs, a similar approach was used but instead of preparing the inocula in TSBg, the suspensions were prepared in PCs, which were then dispensed into six‐well plates and incubated under PLT storage conditions, at 22 ± 2°C on a platform PLT agitator for 5 days to produce mature biofilms . After incubation, each well was gently washed three times with 3 mL of PBS to remove free‐floating cells and bacterial aggregates (biofilms) attached to the plastic of the culture plates were stained for 30 minutes with 3 mL of 0.3% crystal violet (Becton, Dickinson, and Company).…”

“…100 rpm) for 15 minutes to elute the bound crystal violet. This elution was done once; subsequently, six 0.2‐mL aliquots taken from the 3‐mL eluted solution were transferred to six wells of a 96‐well plate, and the absorbance of each sample seeded in the well was read at a wavelength 492 nm in a microplate reader (Expert Plus, ASYS Hitech GmbH) . Absorbance of the bacterial cultures was obtained by averaging the values obtained for the six aliquots.…”