EDTA and Taurolidine Affect Pseudomonas aeruginosa Virulence In Vitro-Impairment of Secretory Profile and Biofilm Production onto Peritoneal Dialysis Catheters

Abstract

Peritoneal catheter-associated biofilm infection is reported to be the main cause of refractory peritonitis in peritoneal dialysis patients. The application of antimicrobial lock therapy, based on results on central venous catheters, may be a promising option for treatment of biofilm-harboring peritoneal catheters. This study investigated the effects of two lock solutions, EDTA and taurolidine, on an in vitro model of Pseudomonas aeruginosa biofilm-related peritoneal catheter infection. Silicone peritoneal catheters were incubated for 24 h with a bioluminescent strain of P. aeruginosa. Then, serial dilutions of taurolidine and/or EDTA were applied (for 24 h) once or twice onto the contaminated catheters, and P. aeruginosa viability/persistence were evaluated in real time up to 120 h using a Fluoroskan reader. On selected supernatants, high-performance liquid chromatography mass spectrometry (HPLC-MS) analysis was performed to measure the production of autoinducers (AI), phenazines, and pyocyianines. Taurolidine alone or in combination with EDTA caused a significant decrease of bacterial load and biofilm persistence on the contaminated catheters. The treatment did not lead to the sterilization of the devices, yet it resulted in a substantial destructuration of the catheter-associated P. aeruginosa biofilm. HPLC-MS analysis showed that the treatment of biofilm-harboring catheters with taurolidine and EDTA also affected the secretory activity of the pathogen. EDTA and taurolidine affect P. aeruginosa biofilm produced on peritoneal catheters and profoundly compromise the microbial secretory profile. Future studies are needed to establish whether such lock solutions can be used to render peritoneal catheter-related infections more susceptible to antibiotic treatment. IMPORTANCE An in vitro model allows studies on the mechanisms by which the lock solutions exert their antimicrobial effects on catheter-associated biofilm, thus providing a better understanding of the management of devise-associated infections.

Keywords: Pseudomonas aeruginosa; biofilm; lock solutions; peritoneal dialysis catheters.

FIG 1

EDTA and/or taurolidine effects on a PDC-associated biofilm: microbial growth during treatment and biofilm production at time 48 h. PDC pieces (0.5 cm) were contaminated with BLI-Pseudomonas (10⁵/ml) for 24 h. Then, the PDC-associated biofilm (24 h-old) was exposed to EDTA and/or taurolidine at the indicated doses from 24 h to 48 h, at 37°C. During such incubation time, the BLI signal was recorded (A) and, at the end of the treatment, the PDC were washed again and the persistent 48-h-old biofilm was assessed (B). The values were expressed as the mean ± SEM of the RLU of 8 replicates obtained in two independent experiments. Statistical analysis was performed according to Student’s t test using GraphPad Prism 8. **, P < 0.005; ***, P < 0.001.

FIG 2

Microbial regrowth after the 1st treatment and biofilm persistence at 72 h. At time 48 h, namely, at the end of the 1st treatment, the PDC were suspended in fresh medium, incubated at 37°C from 48 h to 72 h and kinetically checked for microbial regrowth by RLU measurement (A). At time 72 h, the PDC were washed again, and the RLU was measured to quantify the persistent 72-h-old biofilm (B). The values were expressed as the mean ± SEM of the RLU of 8 replicates obtained in two independent experiments. Statistical analysis was performed according to Student’s t test using GraphPad Prism 8. *, P < 0.05; **, P < 0.005.

FIG 3

Effects of the 2nd treatment with EDTA and/or taurolidine: microbial growth (72 to 96 h) in the presence of the lock solutions, biofilm production (96 h), and regrowth after removal of EDTA and/or taurolidine (96 to 120 h). At time 72 h, the PDC pieces were subjected to a 2nd treatment with ETDA and/or taurolidine, at the indicated doses, at 37°C. During such incubation time (72 to 96 h), the BLI signal was kinetically recorded (A); at time 96 h, the PDC were washed, and the 96-h-old biofilm was assessed (B). After that, the PDC suspended in fresh medium were incubated from 96 to 120 h to assessed the regrowth (C). The values were expressed as the mean ± SEM of the RLU of 8 replicates obtained in two independent experiments. Statistical analysis was performed according to Student’s t test using GraphPad Prism 8. *, P < 0.05; **, P < 0.005; ***, P < 0.001.

FIG 4

Flow chart of the protocol for assessing taurolidine and/or EDTA effects on PDC-associated biofilm. PDC pieces (0.5 cm; pretreated overnight with FBS) were exposed to BLI-Pseudomonas (10⁵/ml) for 1.5 h, washed and incubated in fresh medium (0 to 24 h) to allow biofilm formation. After PDC washing, the 1st treatment with the lock solutions was performed (24 to 48 h). During that time, microbial growth was kinetically measured and biofilm was assessed after PDC washing (48 h). The PDC were further incubated in fresh medium to allow microbial regrowth (48 to 72 h). Then, the 2nd treatment with the lock solutions was performed (72 to 96 h). During that time, microbial growth was kinetically measured, and biofilm was assessed after PDC washing (96 h). Finally, the PDC were further incubated in fresh medium to allow microbial regrowth (96 to 120 h). Additional details are given in Materials and Methods.