Clindamycin hydrochloride

The reagents used for the synthesis of hydroxyapatite, including phosphoric acid (HNO₃), calcium hydroxide (Ca(OH)₂) and ammonia water (NH₄OH, 25%), were purchased from Sigma-Aldrich (Darmstadt, Germany). The antibiotic used for the modification was clindamycin hydrochloride, purchased from Sigma-Aldrich (Darmstadt, Germany). The mobile phase used for HPLC was a combination of acetonitrile (CH₃CN) from Honeyweel (Seelze, Germany) and potassium dihydrogen phosphate (KH₂PO₄) from DOR-CHEM (Krakow, Poland). Phosphate-Buffered Saline Tablets were purchased from OXOID (Basingstoke, UK). A Hydrolab model HLP 5sp unit was used to obtain demineralized water for all solutions.

The minimum inhibitory concentration (MIC), which is a standard measure of the activity of antimicrobials, was determined according to the Clinical and Laboratory Standards Institute protocol (2018) Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria [44 ]. Bacterial suspensions of 10⁵ to 10⁶ CFU/mL in NYCIII+ 10% HS were treated with a 1:2 dilution series of either PM-477 (starting concentration 64 µg/mL) or the antibiotics metronidazole (Gatt-Koller), tinidazole (Sigma Aldrich), or clindamycin (Clindamycin hydrochloride, Sigma Aldrich), starting with a concentration of 128 µg/mL and twofold dilution down to 0.0625 µg/mL. Controls for growth in the absence of antimicrobials were also included. OD₆₂₀ was recorded by a microplate reader (Tecan, Grödig, Austria) after incubation at 37 °C for 48 h or, for some fast-growing strains, after incubation for 24 h, and compared to t = 0 values. Subsequently, to determine the minimum bactericidal concentration (MBC), 2 µL of the antimicrobial dilution series and bacteria suspension of 10⁵ to 10⁶ were spotted on NYC III + 10% HS agar plates and incubated anaerobically for 2–3 days. Colonies were counted and MBC_99.5 values were determined.

Antimicrobial susceptibility was determined according to the ISO 10932:2010 [31 ] broth microdilution procedure using eight antimicrobial agents (ampicillin sodium salt, chloramphenicol, clindamycin hydrochloride, erythromycin, gentamicin sulphate, streptomycin sulphate salt, tetracycline, vancomycin), all purchased from Sigma Aldrich (St. Louis, MO, USA). The Minimum Inhibitory Concentration (MIC), defined as the lowest concentration of antibiotic giving a complete inhibition of visible growth in comparison to an antibiotic-free control well, was determined by the microdilution method according to Russo et al. (2018) [32 (link)]. The experiments were conducted in triplicate.
The genomes of the BA15 and BA17 strains were analyzed for the presence of antibacterial resistant genes and other gene associations that can influence the safety profile of the strains. The analysis was performed using the Pathosystems Resource Integration Center (PATRIC) database [33 (link)].