Caso bouillon

E. faecalis 12030 is a clinical strain originally isolated in Cleveland, OH [16] (link). It is a strong biofilm producer and is opsonized by antibodies against lipoteichoic acid (LTA) [16] (link). Two non-polar deletion mutants, E. faecalis 12030ΔbgsB and E. faecalis 12030ΔbgsA together with their reconstituted strains E. faecalis 12030ΔbgsB rec and E. faecalis 12030ΔbgsA rec, have been constructed and characterized previously [13] (link), [14] (link). All bacterial strains were grown at 37°C without agitation in Caso Bouillon (Carl Roth). All reagents used in this study were obtained from Sigma Aldrich if not stated otherwise.

Bovine pericardia from two to eight years old cattle (n = 5) were kindly provided by a local slaughterhouse (Vorwerk Podemus, Dresden, Germany). Pericardia were dissected and washed in PBS to remove the connective tissue. The SULEEI treatment [12 (link),13 (link)] was performed at the Fraunhofer Institute for Organic Electronics, Electron Beam, and Plasma Technology FEP (Dresden, Germany). Briefly, the bovine pericardia were at first decellularized according to Roosens et al. [32 (link)], secondly they were incubated in a 2% dextran solution (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) containing 0.1% riboflavin (Serva Electrophoresis GmbH, Heidelberg, Germany) prior to UV irradiation to crosslink collagen fibers, and lastly the pericardia were sterilized by low-energy electron irradiation (Table 1). As a control material, conventionally GA-fixed bovine pericardia (in 0.625% GA in 4.863 g/l HEPES supplemented with 2.65 g/L MgCl₂*6 H₂O, 4.71 g/L NaCl, pH 7.4 for 3 h at room temperature) were prepared. Pericardial thickness was measured using a thickness gauge Model FD50 (Käfer Messuhrenfabrik GmbH and Co. KG, Villingen-Schwenningen, Germany).
Verification of the sample sterility performed as described before (incubation in a CASO bouillon (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) at 30 °C for 14 days) was the basis for the sample implementation in this study [12 (link)].

Cells were grown in 24-well plates to a density of 1*10⁵ cells/well for 16 hours. After 16 hours, cells were counted using trypsin treatment and trypan blue. Prior to infection of cells, bacteria were grown to mid-log phase at 37°C without agitation in Caso Bouillon (Carl Roth), washed, and resuspended in DMEM supplemented with 5% FBS. The number of bacteria in the inoculum was first estimated from previously derived growth curve determinations and confirmed by serial dilutions and viable counts for each experiment. T24 cells were incubated with bacteria at 37°C for 2 hours at a multiplicity of infection of 100∶1. After infection of the monolayers, T24 cells were washed five times with phosphate saline buffer (PBS, Biochrom AG) and lysed with DMEM F-12 Ham containing 5% FBS and 0.25% Triton-X100 buffer for 15 min. Bacteria attached and internalized by the T24 cells were quantified by cultivation of serial dilutions of the cell-culture lysates. Pilot experiments confirmed the proportion of non-adherent bacteria to be <1% of the total colony-forming units (CFUs) in the lysates. Invasion experiments were performed as described elsewhere, using the gentamycin protection assay [17] (link).