Api 20a

Two test systems, API® 20A (Biomerieux) and RapID^TM ANA II (Remel/Thermo Fisher), were used according to the instructions of the manufacturers. In brief, Finegoldia strains were grown on blood agar plates for 4 days under anaerobic conditions; cells were harvested and resuspended in the test system’s recommended inoculation fluids in the desired densities. For the RapID^TM ANA II kit the bacterial suspension had a visual turbidity equal to a no. 4 McFarland turbidity standard. After inoculation, the RapID^TM ANA II panel was incubated at 37 °C for 5 h. The inoculated API 20 A kit panel was incubated for 24 h. Additional substances were added after inoculation, and results were interpreted as described in the instructions of the manufacturers.

All fecal specimens were inoculated on selective cycloserine-cefoxitin-fructose agar plates (CCFA, Oxoid, United Kingdom) with 5% egg yolk after ethanol shock treatment and incubated in an anaerobic jar (Mart, NL) at 37°C for 48 h. After being vacuumed, an anaerobic atmosphere of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide were injected. C. difficile colonies were identified on the basis of their typical morphology on agar plates and by Gram stain as well as the characteristic odor. Suspected colonies were further confirmed by API 20A (BioMerieux, France) for their biochemical characteristics and amplification of the GDH gene (Bauer et al., 2011 (link)), and the 16S rRNA gene (Lin et al., 2011 (link)).
DNA extraction was performed on all C. difficile isolates using a commercial DNA extraction kit (Tiangen, Beijing) based on the manufacturers’ instruction. The DNA samples were stored at -20°C for further use.

Strain KD22^T cell morphology was assessed by transmission electron microscopic as previously reported (Routy et al. 2022 (link)). Gram stain was assessed using the standard protocol. The bacterium motility was investigated using a Leica DM 1000 photonic microscope (Leica Microsystems) at 100 X magnification. To test sporulation, a thermal shock at 80 °C for 20 min of strain KD22^T was performed. The growth temperature range was determined by culturing strain KD22^T on Columbia agar and incubated for 2 days at various temperatures (room, 28, 37, 42, and 56 °C) under different atmospheres (anaerobic, microaerophilic, and aerobic conditions). The pH range growth was also tested at pH 5, 6, 6.5, 7, 7.5, and 8.5. Tolerance of NaCl was determined for concentrations ranked between 0 and 100 g/l. Catalase and oxidase productions were also detected (BioMérieux). Enzymatic and biochemical properties of strain KD22^T were determined in duplicate using the API^® 20A, API^® ZYM, and Rapid ID 32A identification systems (BioMérieux). Short-chain fatty acids were analyzed using both a gas chromatograph (Hewlett Packard) and Microbial Identification System software.

Biochemical tests were performed using API ZYM, API 20A, and API 50CH strips (bioMérieux) according to the manufacturer's instructions. The strips were incubated for 4, 24, and 48 hr respectively.
Cellular fatty acid methyl ester (FAME) analysis was performed using Gas Chromatography/Mass Spectrometry (GC/MS). Strain Marseille‐P2341^T was grown on Columbia agar enriched with 5% sheep's blood (bioMérieux). Two samples were then prepared with approximately 50 mg of bacterial biomass per tube harvested from several culture plates. Fatty acid methyl esters were prepared as described by Sasser (Sasser, 2006). GC/MS analyses were carried out as previously described (Dione et al., 2016). In brief, fatty acid methyl esters were separated using an Elite 5‐MS column and monitored by mass spectrometry (Clarus 500—SQ 8 S, Perkin Elmer, Courtaboeuf, France). A spectral database search was performed using MS Search 2.0 operated with the Standard Reference Database 1A (NIST, Gaithersburg, USA) and the FAMEs mass spectral database (Wiley, Chichester, UK).
Antibiotic susceptibility was tested using the disc diffusion method (Le Page et al., 2015). The results were read using Scan 1200 (Interscience, Saint‐Nom‐la‐Bretèche, France).

A total of 35 clinical isolates were used. Included were Porphyromonas saccharolytica (n=9), Porphyromonas gingivalis (n=13), Prevotella intermedia (n=9), Prevotella melaninogenica (n=4). All isolates belonged to a collection of the microbiology laboratory at UHS. For quality control, two reference strains, namely Porphyromonas gingivalis (ATCC 33277) and Bacteroides fragilis (ATCC 25285), were included in the study obtained from reference center (MicroBioLogics, USA) and identified by morphological, cultural, and biochemical profile (API -20A, bio Merieux, France). These reference strains were included in every cycle of culture and susceptibility testing to monitor the consistency of the procedure.