Rose bengal uptake by E. faecalis and F. nucleatum and light-mediated antibacterial activity measured by flow cytometry

Abstract

Rose bengal uptake by E. faecalis and F. nucleatum and light-mediated antibacterial activity measured by flow cytometry Antibacterial photodynamic therapy (aPDT) using rose bengal (RB) and blue-light kills bacteria through the production of reactive oxygen derivates. However, the interaction mechanism of RB with bacterial cells remains unclear. This study investigated the uptake efficiency and the antibacterial activity of blue light-activated RB against Enterococcus faecalis and Fusobacterium nucleatum. Spectrophotometry and epifluorescence microscopy were used to evaluate binding of RB to bacteria. The antibacterial activity of RB after various irradiation times was assessed by flow cytometry in combination with cell sorting. Uptake of RB increased in a concentration dependent manner in both strains although E. faecalis displayed higher uptake values. RB appeared to bind specific sites located at the cellular poles of E. faecalis and at regular intervals along F. nucleatum. Blue-light irradiation of samples incubated with RB significantly reduced bacterial viability. After incubation with 10 μM RB and 240 s irradiation, only 0.01% (± 0.01%) of E. faecalis cells and 0.03% (±0.03%) of F. nucleatum survived after treatment. This study indicated that RB can bind to E. faecalis and F. nucleatum in a sufficient amount to elicit effective aPDT. Epifluorescence microscopy showed a yet-unreported property of RB binding to bacterial membranes. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to form colonies on agars after cell sorting.

Photodynamic inactivation of foodborne bacteria by eosin Y

“…aureus was treated with curcumin and illuminated for 60 min, 28Á31% of dead cells were obtained, while the control containing only the bacteria had 1Á27% of dead cells (Jiang et al 2014). Manoil et al (2016) reported 2% of dead cells in the control containing only Ent. faecalis and 3Á5% in F. nucleatum, but after treatment with rose bengal and irradiation for 240 s 98Á8% of dead cells were obtained for Ent.…”

“…Manoil et al . () reported 2% of dead cells in the control containing only Ent. faecalis and 3·5% in F. nucleatum, but after treatment with rose bengal and irradiation for 240 s 98·8% of dead cells were obtained for Ent.…”

“…), and flow cytometry technique allows the detection of different microbial subpopulations based on membrane integrity through the retention or exclusion of dyes (Manoil et al . ).…”

“…faecalis and Fusobacterium nucleatum by rose bengal and blue light (Manoil et al . ). Both studies demonstrated increased numbers of PI marked cells (dead cells) when bacteria were treated with the photosensitizer and light, indicating membrane damage.…”

“…Bacteria exposed to photodynamic inactivation lose membrane integrity resulting in cell death (Jiang et al 2014), and flow cytometry technique allows the detection of different microbial subpopulations based on membrane integrity through the retention or exclusion of dyes (Manoil et al 2016).…”