Determination of the minimum inhibitory concentration (MIC)
Initially, an overnight culture for every bacterial and fungal strain was prepared following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Each microorganism was plated onto Columbia blood agar plates (CBA) or yeast-cysteine blood agar plates (HCB). Facultative anaerobic bacteria and Candida albicans were cultivated on CBA agar plates at 37°C in a 5%-10% CO2 atmosphere for 24 hours. Meanwhile, the anaerobic bacteria were plated on HCB agar plates and incubated at 37°C for 48 hours within an anaerobic chamber (Anaerocult, Merck Chemicals GmbH, Darmstadt, Germany). A 0.5 A / 1 A McFarland standard suspension was generated in 0.9% saline (NaCl) for facultative anaerobic bacteria and Candida albicans , respectively. For the microdilution assay, all facultative anaerobic strains and Candida albicans were subsequently 1:10 diluted in BBL™ Mueller Hinton II Broth-Cation-Adjusted (MHB, BD, Heidelberg, Germany). The anaerobic bacteria were prepared in Wilkins–Chalgren broth (WCB) at a 0.5 A McFarland standard suspension. As stipulated by ISO 20776-1:2006, tests involving facultative anaerobic bacteria required a cell density of approximately 5 x 10^5colony forming units (CFU) per ml, while fungi tests utilized 5 x 10^4 CFU / ml, and tests involving obligate anaerobic bacteria used 5 x 10^6 CFU / ml. Subsequently, suitable volumes of the MHB / WCB microbial cultures were transferred into a 96-well microtiter plate using a multi-channel pipette. The prepared natural plant extracts were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at a concentration of 100 mg/ml. Concentration series of extract solutions in DMSO ranged from 10 mg/ml to 0.02 mg/ml, employing dilution levels from 10-fold to 5120-fold. Each well in the 96-well microtiter plate held a total volume of 100 µl. To rule out any potential antimicrobial effects of residual DMSO, a parallel dilution series of DMSO was investigated. Wells containing solely MHB / WCB, as well as a dilution series of 0.1% chlorhexidine (CHX), served as negative and positive controls for bacterial growth, respectively. Additionally, wells containing MHB / WCB and the added microbial strain were designated as growth controls. Contamination risks were minimized through the use of sterile MHB / WCB. Subsequently, E. coli , S. aureus , E. faecalis , andC. albicans were incubated at 37°C for 18 hours, while the three streptococci strains were incubated at 37°C under a 5%-10% CO2 atmosphere for 24 hours. Anaerobic bacteria were maintained at 37°C for 48 hours within an anaerobic jar (Anaerocult, Merck Chemicals GmbH, Darmstadt, Germany). All assays for each bacterial and fungal strain were carried out in duplicates, and the highest minimum inhibitory concentration (MIC) values were considered if MIC values exhibited minor discrepancies. If differences between two rows exceeded two dilution levels, the determination involving that specific extract was repeated. MIC was defined as the lowest concentration of each natural plant extract that visibly inhibited bacterial growth. The inhibitory impact of DMSO was factored in if bacterial growth was observed within the concurrently tested DMSO dilution series.