Anaerocult

The strains were grown on YEL agar at 30 °C for 7 days under near-anaerobic atmosphere (Anaerocult, Merck) Single colonies were picked and suspended in 0.1 M PIPES buffer, pH 6.8. An aliquot of 3 μl was added to Pioloform-coated 200-mesh copper grids previously glow discharged (Emitech K100X, Emitech Ltd., UK) to ensure even adhesion of the bacterial cells to the grids. After 1 min incubation, the excess suspension fluid was removed by soaking with a filter paper. The grids were negatively stained with 1% neutral uranyl acetate for 15 s and air-dried. Images were acquired at 120 kV with a Jeol JEM-1400 microscope (Jeol Ltd., Tokyo, Japan) using an Orius SC 1000B CCD-camera (Gatan Inc., USA).

Sterile micro compresses of 10 mm diameter were then soaked with 30 μL of each obtained compound. Therefore, the amount of Doxy contained in the tested nanofibre mass was 5.04 ± 0.01 µg, reconstituted in the form of 30 μL of sterile saline solution. Fifteen minutes after the Petri dishes were flooded with the inoculum, three different micro compresses were radially placed on the medium. The dishes were anaerobically incubated using a GENbag-GENbox type system (bioMérieux S.A., Marcy l’Etoile, France), at a controlled temperature of 37 °C. The anaerobiotic state was obtained using an anaerobiosis atmosphere generation system (Anaerocult™, Merck KGaA, Darmstadt, Germany, and further validated using dedicated colour-changing strips (Anaerotest™, Merck KGaA, Darmstadt, Germany)).
The results were recorded after seven days. The diameters of the resulting inhibition zones for both the controls and test samples were measured using an electronic calliper gauge, and the values were expressed in millimetres with two decimals. All tests were run in triplicate, and an average value of the inhibition zones was calculated. Data were statistically analysed, and the graphs were generated using the GraphPad Prism Software (GraphPad Software, Inc., San Diego, CA, USA).

C. baratii C3 was routinely cultured in Reinforced Clostridium Medium (RCM, Becton-Dickinson, Franklin Lakes, NJ, USA) supplemented with 0.5 g/L of L-cysteine (RCM-cys; Loba Chemie, India). All incubations were performed at 37 °C for 24–48 h in an anaerobic jar (Anaerocult, Merck, Darmstadt, Germany) with anaerobic packs (Gaspak EM, Becton-Dickinson, Franklin Lakes, NJ, USA). Two overnight cultures of C. baratii were made on Blood Agar and Starch Agar plates (Winkler, Santiago, Chile). The bacteria were also inoculated in API-20A strips (Biomerieux, Craponne, France) following the manufacturer’s instructions. Finally, antibiotic resistance patterns were evaluated using antibiotic discs in RCM agar plates incubated for 48 h (Lilfilchem, Roseto degli Abruzzi TE, Italy) for 48 h. Cefazolin (30 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), erythromycin (15 µg), penicillin G (10 µg), piperacill/tazobactam (110 µg), trimethoprim-sulfamethoxazole (25 µg), and vancomycin (30 µg) were used.

Bacteriodes coprophilus DSM 18228^T, B. coprosuis DSM 18011^T, B. eggerthii DSM 20697^T, B. finegoldi DSM 17565^T, B. fragilis DSM 2151^T, B. gallinarum DSM 18171^T, B. intestinalis DSM 17393^T, B. ovatus DSM 1896^T, B. pyogenes DSM 20611^T, B. salanitronis DSM 18170^T, B. suis DSM 20612^T, B. thetaiotaomicron DSM 2079^T, B. uniformis DSM 6597^T, B. vulgatus DSM 1447^T, and Parabacteroides distasonis DSM 20701^T were used as reference strains to establish the optimal DGGE gradient and conditions.
Type strains were grown using BPRM (Bacteroides Phage Recovery Media) broth (Tartera et al. 1992) and BPRM agar (Pronadisa, Laboratorios Conda, Madrid, Spain) without antibiotics at 37°C in anaerobic conditions (Anaerocult^®; Merck Millipore, Darmstadt, Germany).

Clinical materials were cultured on yeast cysteine blood agar (in-house abbreviation HCB) and incubated for 2–10 days (according to sample type) under anaerobic conditions in a jar or in plastic bags using either the Genbox ANAER (bioMérieux, Marcy-l’Étoile, France) or the Anaerocult (Merck, Darmstadt, Germany) system. Identification of anaerobic bacteria was carried out using a MALDI-TOF mass spectrometer (Bruker Daltonics, Leipzig, Germany).

The haemolytic capability of the strains was assessed by growth on tryptic-soy agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 5% sheep blood (Labema, Helsinki, Finland). A fresh colony from a YEL agar plate was streaked on blood agar and incubated for one week at 30 °C under an anaerobic atmosphere (Anaerocult, Merck, Darmstadt, Germany) after which the plates were examined for haemolysis. The presence of clearing around the bacterial growth was interpreted as β-haemolysis. For detection of pigmentation, fresh colonies were streaked on YEL agar plates and incubated as above followed by visual inspection. Staphylococcus aureus 8325-4 [41 (link)] was used as a positive control of haemolytic activity. The assay was repeated at least three times for each tested strain.