2.6.1. Susceptibility testing of biofilms: Minimum biofilm eradication concentration (MBEC)

The Calgary Biofilm Device (CBD, MBEC Assay®, Innovotech Inc Edmonton, Canada) allows for the in vitro growth of biofilms [26,27]. A custom-made microbroth dilution plate (CML2FNUN; Sensititre™, Thermo Scientific™, MA, USA) was produced with increasing concentrations of eight common antimicrobials in PJI treatment (Fig. 3). The concentrations ranged from MIC determining levels to above breakpoint values (0.25–1216 ​μg/mL).

Antimicrobial agents and concentrations (μg/mL) included in the custom-made antimicrobial susceptibility plate used for the determination of MIC (minimum inhibitory concentration) and MBEC (minimum biofilm eradication concentration). First four empty wells had no antimicrobial agent. The following antimicrobial agents were included: ciprofloxacin (CIP), clindamycin (CLI), oxacillin + 2% NaCl (OXA), fusidic acid (FA), linezolid (LZD), rifampicin (RIF), trimethoprim/sulfamethoxazole (SXT) and vancomycin (VAN). One well was used as a positive control (+Ctrl) and another as a negative control (-Ctrl).

The CBD and the Sensititre™ antimicrobial plates were combined to determine the MBEC of the strains. The procedure for MBEC determination was done in accordance to Zaborowska et al., summarized in Fig. 2B [21]. In brief, 150 ​μL of an inoculum (10⁷ ​CFU/mL) of each strain in Mueller-Hinton Broth 2 (Sigma Aldrich, MO, USA) was added to a CBD and incubated at 37 ​°C and 125 ​rpm for 24 ​h, for the formation of biofilms on the pegs of the CBD lid. Four pegs were removed with sterile pliers and placed in saline, vortexed for 1 ​min, sonicated for 1 ​min (40 ​Hz) and the detached cell solution was 10-fold serially diluted and cultured on blood agar plates o.n. at 37 ​°C, to quantitate the number of viable bacteria in the biofilms (CFU/peg). To determine the MBEC, biofilms grown on peg lids of the CBD were rinsed in saline and placed in the antimicrobial plate and incubated at 37 ​°C for approx. 20 h. Each peg lid was rinsed twice, placed in a neutralizing recovery plate, sonicated for 1 ​min to detach the treated biofilm into each well, and incubated o.n. at 37 ​°C. MBEC was determined by ocular inspection using the Sensititre™ Manual Viewbox. In cases of growth in the highest concentration, the calculations were based on the next doubling concentration.