Dicloxacillin

The co-cultures were incubated for 168 h in conditions resembling those in the intestinal tract (anaerobic, 37 °C) in a Concept 400 Anaerobic Workstation (Baker Ruskinn, Sanford, ME, USA). The cultures were collected directly after the inoculation (0 h), then after 24, 48, 72, 96 and 168 h of incubation and diluted in physiological salt prior to selective plating (Koch’s plate method): Lactobacillus on Rogosa agar (Biomaxima, Lublin, Poland), Bifidobacterium on RCA agar (BTL, Lodz, Poland) with the addition of dicloxacillin (Sigma-Aldrich, Burlington, MA, USA), Escherichia coli on ENDO agar (Biomaxima, Poland), Enterococcus on bileaesculin agar (Biomaxima, Poland), Clostridium on DRCM agar (Biomaxima, Poland) and Bacteroides on Schaedler agar (Biomaxima, Poland) with Schaedler supplement (Biomaxima, Poland).
Afterwards, the plates were incubated for 48 h at 37 °C; Lactobacillus, Escherichia coli and Enterococcus under aerobic conditions and Bifidobacterium, Bacteroides and Clostridium under anaerobic conditions in a Concept 400 Anaerobic Workstation (Ruskinn Biotrace, Baker Ruskinn, Sanford, ME, USA). All cultures were done in duplicates. Simultaneously, the changes in pH were monitored using an Elmetron CP-401 pH-meter (Elmetron, Zabrze, Poland).

S. epidermidis ATCC 35984 (RP62A) was used in this study. For each experiment, isolated colonies were picked from tryptic soy agar (TSA, Liofilchem, Téramo, Italy) plates, inoculated into tryptic soy broth (TSB) (Liofilchem) and incubated overnight (~16 h) at 37 °C and 120 rpm (ES-20 Shaker-Incubator, BioSan, Riga, Latvia). Dicloxacillin, rifampicin, and teicoplanin were purchased from Sigma-Aldrich (St. Louis, MO, USA), tetracycline was purchased from GRiSP (Porto, Portugal), and vancomycin was purchased from PanReac Applichem (Darmstadt, Germany). Pooled human serum from clotted whole blood was purchased from Sigma-Aldrich, and pooled human plasma in lithium heparin was purchased from Tebu-Bio (Le Perray-en-Yvelines, France). TSB supplemented with lithium heparin (TSBH, BD Vacutainer, Franklin Lakes, NJ, USA) was used as a control in every experiment. Heat inactivation of serum/plasma was carried out at 56 °C for 30 min. Iron (III) chloride and human transferrin (partially iron-saturated) were purchased from Sigma-Aldrich.