Optimization of Streptococcus agalactiae Biofilm Culture in a Continuous Flow System for Photoinactivation Studies

Abstract

Streptococcus agalactiae is a relevant cause of neonatal mortality. It can be transferred to infants via the vaginal tract and cause meningitis, pneumonia, arthritis, or sepsis, among other diseases. The cause of therapy ineffectiveness and infection recurrence is the growth of bacteria as biofilms. To date, several research teams have attempted to find a suitable medium for the cultivation of S. agalactiae biofilms. Among others, simulated vaginal fluid has been used; however, biofilm production in this medium has been found to be lower than that in tryptic soy broth. We have previously shown that S. agalactiae can be successfully eradicated by photoinactivation in planktonic culture, but there have been no studies on biofilms. The aim of this study was to optimize S. agalactiae biofilm culture conditions to be used in photoinactivation studies. We compared biofilm production by four strains representing the most common serotypes in four different broth media with crystal violet staining. Then, we evaluated stationary biofilm culture in microtiter plates and biofilm growth in a CDC Biofilm Reactor® (BioSurface Technologies, Bozeman, MT, USA) under continuous flow conditions. Subsequently, we applied Rose Bengal-mediated photoinactivation to both biofilm models. We have shown that photoinactivation is efficient in biofilm eradication and is not cyto/phototoxic to human keratinocytes. We found conditions allowing for stable and repetitive S. agalactiae biofilm growth in continuous flow conditions, which can be successfully utilized in photoinactivation assays and potentially in all other antibacterial studies.

Antimicrobial Photodynamic Inactivation: An Alternative for Group B Streptococcus Vaginal Colonization in a Murine Experimental Model

“…The main goal was to obtain a lethal dose of aPDI, i.e., viability reduction by >3 log 10 CFU/mL. These parameters were chosen according to the previous experiments [ 21 ] using the ATCC strain. Figure 1 A presents viability reduction in S. agalactiae caused by aPDI with different RB concentrations for the ATTC strain and six clinical isolates representing six different serotypes.…”

“…The current study describes aPDI employing a harmless photoactive compound, i.e., rose bengal (RB), which was used in our previous mechanistic studies concerning aPDI treatment [ 21 , 42 , 43 ]. RB is a xanthene dye, which has been widely applied as a photoactive compound in photodynamic treatment [ 15 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ].…”

“…Photodynamic inactivation protocol was previously described [ 21 ]. In short, overnight cultures of S. agalactiae or Lactobacilli were adjusted to 2.4 or 2.0 McFarland units, respectively, which corresponds to a cell density of approx.…”

“…Biofilm culture and photodynamic inactivation of biofilm were previously described [ 21 ]. In short, biofilms were cultured on 96-well flat-bottom microtiter plates.…”

Post grafted gallic acid to chitosan-Ag hybrid nanoparticles via free radical-induced grafting reactions

“…The antibiofilm activity was evaluated as reported previously [35]. S. aureus and E. coli biofilms were cultured in Tryptic-Soy Broth with 1 % glucose or 8-times diluted Luria Broth respectively.…”