In all experiments, taurolidine 2% solution (Geistlich Pharma AG, Wolhusen, Switzerland) was used and diluted until to the necessary dilution. As positive control minocycline (Sigma-Aldrich, Buchs, Switzerland) and as negative control dH2O were used.
Several oral bacterial strains (mainly P. gingivalis and A. actinomycetemcomitans) were included in the experiments (Table 1). The clinical isolates (4 P. gingivalis, 3 A. actinomycetemcomitans, 2 T. forsythia, 2 Fusobacterium nucleatum, 4 oral streptococci) were obtained from subgingival biofilm samples. Culturing subgingival biofilm, and isolation of respective bacterial strains was approved by the Ethical Committee of the Canton Bern (KEK 096/15). Without removing of supragingival biofilm, sterile paper point (ISO 50) were inserted into the periodontal pocket for 30 s. Then the pooled paper points were placed into 1 ml of reduced transport fluid. After culturing, colonies typical for the respective species were isolated and subcultivated. Identity was confirmed by nucleic acid-based methods.
Bacterial strains were kept frozen at -80°C. About one week before experiments, they were subcultured and passaged 2 -3 times on tryptic-soy-agar plates with 5% of sheep blood (Oxoid, Basingstoke, GB).
Monocytic cells of human origin (MONO-MAC-6; DSMZ no. ACC 124) were maintained in RPMI 1640 medium containing 10% fetale bovine serum (FBS). For experiments cells were used in M199 media without FBS (Invitrogen; Carlsbad, CA, USA).
TEM and SEM photographs after exposure to 1% taurolidine
Three bacterial strains (P. gingivalis ATCC 33277, T. forsythia B13237 and P. micra ATCC 33270) were included. They were suspended to a density of about McFarland 4 in nutrient broth (Wilkins-Chalgren broth (Oxoid) supplemented with 5 mg/l β-NAD and 10 mg/ NAM (both Sigma-Aldrich, Buchs, Switzerland)) without and with 1% of taurolidine. After an incubation for 2 h in an anaerobic atmosphere at 37°C, these bacteria were prepared for (TEM) and scanning electron microscopy (SEM).
The samples were centrifuged and washed twice with 0.9% w/v NaCl and finally suspended to a 20-fold concentration of bacteria. For SEM, this suspension was transferred to slides and thereafter fixed with 2% glutaraldehyde solution in 0.1 M cacodylate buffer (pH 7.4) for 15 min. Then samples were washed 3-fold with 0.1 M cacodylate buffer and dehydrated in ascending ethanol concentrations (30, 50, 70, 90 and 100%) for 10 minutes each. Subsequently, the samples were critical-point dried using liquid CO2 and sputter coated with platinum (thickness approx. 1 nm) using a SCD005 sputter coater (BAL-TEC, Liechtenstein) to avoid surface charging. Finally, the specimens were investigated with a field emission (FE) scanning electron microscope LEO-1530 (Carl Zeiss NTS GmbH, Oberkochen, Germany).
For TEM, bacteria suspensions were fixed with 0.5% formaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 30 min. After washing 3-fold with 0.1 M cacodylate buffer and post-fixating with osmiumtetroxide for 1 h, dehydration in ascending ethanol series with post-staining in uranylacetate was performed. Afterwards the samples were embedded in epoxy resin (Araldite) and sectioned using a Leica Ultracut E (Leica, Wetzlar, Germany). Ultrathin sections (60 nm thickness) were mounted on filmed Cu grids, post-stained with lead citrate, and studied in a transmission electron microscope (EM 900, Zeiss, Oberkochen, Germany) at 80 kV and magnifications of 3,000x to 20,000x.
SEM and TEM photographs were taken at the Center for Electron Microscopy of the Jena University Hospital, Germany.
Interaction with bacterial virulence factors
To determine a potential effect of lipopolysaccharide (LPS), 10 pg and 100 pg LPS originated from Escherichia coli (Sigma-Aldrich) and 10 ng LPS from P. gingivalis (InvivoGen, Toulouse, France) were used. Taurolidine in the final concentrations of 0.01%, 0.25% and 1% and as control 16 µg/ml minocycline were added to the LPS for 2 h at 37°C. Thereafter, endotoxin activity was measured by Limulus amebocyte lysate assay (LAL) QCL-1000TM test (Lonza, Basel, Switzerland) according to the manufacturer’s recommendation. Also the two bacterial strains P. gingivalis ATCC 33277 and A. actinomycetemcomitans Y4 (about 108/ml) were exposed to same concentrations of antimicrobials for 2 h before measuring endotoxin activity.
Leukotoxin was purified as described by Kachlany et al. , added by a final centrifugation by using 10 kDa centrifugal filter to remove proteins of lower weights. Leukotoxin in a concentration of 4 µg/ml and two A. actinomycetemcomitans strains (108/ml in M199 media) were exposed to three concentrations of taurolidine (0.01%, 0.5%, 1.5%) and to 16 mg/ml minocycline for 2 h before monocytic cells were added. Vitality of MONO-MAC-6 cells was determined after 1 h and 20 h of incubation at 37°C with 5% of CO2 by using MTT assay according to Mosmann .
Experiments on LPS and leukotoxin activity were made in independent triplicates. One-way ANOVA with post-hoc Bonferroni compared different antimicrobials with controls each. Further, t-test was used to find differences in viability of MONO-MAC-6 cells after exposing A. actinomycetemcomitans strains and leukotoxin with non-stimulate cells (only controls). A difference with a p-value of <0.05 was considered as being statistically significant each.
A direct inhibitory effect on gingipain activity was assessed by adding three concentrations of taurolidine (0.01%, 0.25%, 1%) and of minocycline (0.5 µg/ml, 16 µg/ml, 128 µg/ml) for 2 h to 10 nM activated RgpB (being an arginine-specific gingipain; the RgpB was kindly provided by Jan Potempa, Jagiellonian University Krakow, Poland). Thereafter, P. gingivalis strains (108/ml Wilkins-Chalgren broth) were exposed to the same concentrations of the antimicrobials for 2 h before measuring arginine specific amidolytic activity in the suspensions by adding the chromogenic N-α-benzoyl-DL-arginine-p-nitroanilide (BApNA) substrate (Sigma-Aldrich), USA) with a final concentration of 2 mM in the assay buffer (0.2M Tris-HCl, 0,1M NaCl, 5 mM CaCl2, pH 7.6, supplemented with 10 nM cysteine hydrochloride solution in a ratio 1 : 1. The absorbance was read at 405 nm (37oC) at 30 s intervals for 1 h by using a spectrophotometer (BioTek EL808, BioTek, Luzern, Switzerland).
Potential development of resistance
Initially all clinical isolates were included in this experiment. However, the A. actinomycetemcomitans Be12206 strain did not survive the passages. So results from 14 strains were available. The method was adapted to the procedures made before [28, 29]. For that strains were transferred on Wilkins Chalgren agar plates (Oxoid) containing 1/4 - 1/8 of the MICs of taurolidine or minocycline up to 50 passages (about one passage each five days). Before and after each 10 passages MICs were determined again by agar dilution technique. If there was an increase or decrease of MICs after passaging, the concentration of the antimicrobial was adapted. Further strains with altered MICs were passaged 3 times on media free of antimicrobials to see the stability of the altered MIC. Strains showing changed MIC before and after 50 passages with subinhibitory concentrations of taurolidine were prepared for TEM and SEM as described above.
Strains with increasing MICs were screened for potential efflux resistance mechanisms. The efflux inhibitors 50 µg/ml NMP, 10 µg/ml CCCP, 100 µg/ml verapamil and 20 µg/ml reserpine (all inhibitors Sigma-Aldrich) were added to the nutrient media before adding antimicrobials and determining MIC again after incubation.