Strain ZY-312 was isolated from the feces of a healthy infant, and was previously identified using 16S rRNA gene sequence analysis (Deng et al., 2016). B. fragilis strain ATCC 25285 (also known as NCTC 9343) was purchased from the American Type Culture Collection (ATCC). The culture conditions and materials were as described previously for strains ZY-312 and ATCC 25285 (Deng et al., 2016). For the microbial contamination experiment, E. coli CBSLAM00087, Staphylococcus aureus (S. aureus) CMCC(B) 26058, Salmonella enterica (S. enterica) serotype Paratyphi B CBSLAM00994, Pseudomonas aeruginosa (P. aeruginosa) CBSLAM00818, Candida albicans (C. albicans) CMCC(F)98001, and Clostridium sporogenes (C. sporogenes) CMCC(B) 64941 were obtained from the Academy of Military Medical Science, Beijing, China. All strains were verified by PCR amplification of 16S rRNA gene using the universal primers 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492 R (5′-GGTTACCTTGTTACGACTT-3′) (Lanes D–J). Amplified products were sequenced (Biomed).

For acute oral toxicity studies, 6- to 8-week-old female specific pathogen-free (SPF) BALB/c mice and nude mice were procured from the Animal Experiment Center of the Academy of Military Medical Sciences, Beijing, China. The animals had free access to tap water and standard rodent diet. All of the animal experiments were performed in accordance with the approval of the Animal Ethics Committee of the Beijing Institute of Microbiology and Epidemiology, Beijing, China.

Total DNA was extracted from all strains using a DNA extraction kit (Qiagen, USA) and the complete genome sequence of strain ZY-312 was determined using an Illumina Hiseq 2000 sequencing system (Xu et al., 2015). A genome sequencing library with an average insert size of 400 bp was generated, and raw short-read sequences were filtered using Seqprep¹ and Sickle² software. The genome was then assembled de novo using SOAP de novo software (Luo et al., 2012). Accuracy of the assembled genome sequence was evaluated by mapping all raw reads onto the scaffolds using SOAPaligner (Cui et al., 2013).

Gene prediction was carried out using Glimmer3.0 software (Xu et al., 2015). Putative antibiotic resistance genes and putative virulence factors were identified by BLAST analysis of the antibiotic resistance genes database (ARDB)³ (Liu and Pop, 2009) and the virulence factors database (VFDB)⁴ (Chen et al., 2005), respectively. A BLAST comparison was performed between the genome sequences of strain ZY-312 and B. fragilis strain NCTC 9343 (GenBank accession number NC_003228). A Perl script was written and the average nucleotide identity (ANI) was calculated using BLAST. A neighbor-joining phylogenetic tree was built using treebest based on the ZY-312 complete genome sequence, the complete shotgun sequences of other B. fragilis strains, and the complete genome sequences of B. fragilis strains NCTC 9343 (GenBank accession number NC_003228), YCH46 (GenBank accession number NC_006347), and B. fragilis 638R (GenBank accession number NC_016776).

ZY-312 was cultured on Eosin methylene blue agar for 24 h at 37°C (E. coli as positive control), SS agar for 24 h at 37°C (S. enterica serovar Paratyphi B as positive control), Columbia agar containing blood and gentamicin for 48 h at 37°C (C. sporogenes as positive control), mannitol sodium chloride agar for 24 h at 37°C (S. aureus as positive control), NAC agar for 24 h at 37°C (P. aeruginosa as positive control), Sabouraud dextrose agar for 72 h at 28°C (C. albicans as positive control), rose bengal agar for 96 h at 28°C (C. albicans as positive control), and agar agar for 48 h at 37°C (S. aureus as positive control) for excluding microbial contamination. The major fatty acids were analyzed using an HP6890 gas chromatograph (ver. A 5.01), as previously described (Tan et al., 2010). Supernatant from late-logarithmic phase ZY-312 and ATCC 25285 cultures was collected by centrifugation for 10 min at 3000 ×g, and then analyzed by gas chromatography-mass spectrometry. Carbon-source utilization analyses were carried out using a Biolog AN microPlate test panel (Biolog, USA). Bacterial cells were collected from logarithmic phase ZY-312 and ATCC 25285 cultures and resuspended at a concentration of 1.5 × 10⁸ colony forming units (cfu)/mL, and then inoculated into the test plate. After incubating anaerobically for 16–24 h, test results were read using a microplate reader (SpectraMax, M2), using deionized water as a negative control. To test catalase activity, 3–5 drops (about 100 μL) of 3% hydrogen peroxide (freshly prepared) were dropped onto the center of precipitated bacterial cells on microscope slides. To test gelatin liquefaction activity, bacteria were inoculated into gelatin medium, tryptone soy broth (TSB, OXIOD, UK) solidified with 15% gelatin, using a sterilized needle. Plates were incubated at 37°C for 48 h anaerobically, then placed at 4°C for 3–4 h. To determine hemolytic ability, bacteria were anaerobically cultured on tryptone soy agar (TSA, OXIOD, UK) supplemented with 5% (v/v) goat blood for 48 h at 37°C. To determine motility, bacteria were cultured on semi-solid medium (TSB solidified with 0.5% agar) at 37°C for 48 h anaerobically.

Antibiotic resistance testing was performed using the minimum inhibitory concentration (MIC) method as described previously (Fernandez et al., 2005). Test antibiotics were chosen based on the antibiotic resistance genes identified in the annotated genome of ZY-312, with the following antibiotics included in the analysis: cefoxitin, ceftriaxone, cefepime, trimethoprim, clarithromycin, chloromycetin, levofloxacin, streptomycin, kanamycin, tetracycline, vancomycin, and polymyxin B (NIFDC, China). Although bcrA was identified in the genome, bacitracin was not used because of high toxicity, while fosmidomycin (rosA) is still undergoing testing and was therefore also excluded. Quinupristin/dalfopristin, the targets of the vatB gene product are not used in China, and there is no corresponding antibiotic for ykkC. ZY-312 was cultivated anaerobically at 37°C for 48 h at a concentration of 10⁷ cfu/mL with antibiotics at different concentrations. MIC was determined by measuring optical density at 600 nm (OD₆₀₀) with a microplate reader (SpectraMax, M2).

To assess the acute toxicity of ZY-312, SPF BALB/c mice were randomly assigned into five groups (n = 5–8). Each group was administered with ZY-312 in a suspension containing 1 × 10⁹, 5 × 10¹⁰, or 5 × 10¹¹ cfu/day, 0.5 mL/day culture supernatant, or 0.5 mL/day saline via oral gavage for 5 days. General condition and body weight were observed daily for 17–18 days. At the end of the experimental period, blood samples were obtained for hematological and serum biochemistry analyses. Stomach, colon, liver, and spleen were collected, weighed, and prepared for histopathological examination. Nude mice were orally administered with ZY-312 suspension at a dosage of 1 × 10⁹ cfu/day for 3 days. General condition and bodyweight were observed for 7 days.

To explore whether genetic variation occurs in ZY-312 during in vitro passage, we continuously subcultured ZY-312 for 100 generations. Two parallel tubes were inoculated from an original culture of ZY-312 in TSB supplemented with 5% fetal bovine serum, and then incubated at 37°C in an anaerobic glove box (Bugbox, Ruskin) for 24 h. These initial cultures were designated A₀ and B₀. Subsequent passages were performed by inoculating 1% of each culture into fresh medium every 24 h, until A₁₀₀ and B₁₀₀ were obtained. Genetic variation was evaluated by complete genome sequencing of strains A₁₀, A₂₅, A₅₀, and A₁₀₀, and B₁₀, B₂₅, B₅₀, and B₁₀₀, and ANI was calculated for each generation. Morphological variations were observed by colony examination, Gram staining, and scanning electron microscopy (SEM) of A₁₀₀ and B₁₀₀, with wild-type ZY-312 used for comparison. Growth of ZY-312, A₁₀₀, and B₁₀₀ under anaerobic conditions was also examined over a 24-h period. Acute toxicity of A₁₀₀ and B₁₀₀ was detected in SPF mice at a dosage of 1 × 10⁹ cfu/day for 3 days, with general condition and bodyweight observed for 7 days.

All experiments were performed at least in triplicate using independent assays, and values were expressed as the mean ± standard error. An unpaired Student’s t-test was performed to determine statistically significant differences in the acute toxicity assays. A p-value of <0.05 was considered statistically significant.

Strain ZY-312 only grew under anaerobic conditions (Supplementary Figure S1), with no growth observed under aerobic conditions or in 5% CO₂, implying it was an obligate anaerobe. Microbial contamination was excluded by culturing ZY-312 in different culture media under different conditions (Supplementary Figure S2). ZY-312 formed circular, low convex, semi-opaque colonies following anaerobic cultivation on blood agar plates (Supplementary Figure S1). Cells were Gram-negative, and shown to be rod shaped with rounded ends by scanning electrochemical microscopy (Supplementary Figure S1). This morphology matched the descriptions of B. fragilis in Bergey’s manual (Krieg et al., 2001).

The major fatty acids of ZY-312 were C_15:0 anteiso, C_15:0 iso, C_16:0, and C_16:0 3-OH (Supplementary Figure S3 and Table 1A). The major products in the culture supernatant of ZY-312 were lactic acid, acetic acid, succinic acid, propionic acid, and phenylacetic acid (Table 1B). ZY-312 and ATCC 25285 differed in their ability to metabolize L-valine, salicin, L-alanine, L-alanyl-L-glutamine, and N-acetyl-D-glucosamine (Table 2A). Both strains were positive for catalase activity, weakly positive for gelatin liquefaction, and negative for hemolytic activity and motility (Table 2B). The physiological and biochemical characteristics of ZY-312 were in accordance with B. fragilis as described in Bergey’s manual (Krieg et al., 2001).

The phylogenetic trees generated from the whole genome sequence of ZY-312 and other B. fragilis strains are shown in Supplementary Figures S5, S6. ZY-312 and ATCC 25285 had an ANI of 99.99%. In total, 33 putative virulence factors (Table 3) and 11 antibiotic resistance genes (Table 4A) were annotated in the ZY-312 genome based on a minimum of 40% amino acid homology. The complete genome was 4,558,494 bp in length and contained on a single chromosome, with an average GC content of 43.08%, consistent with that of B. fragilis (41–44%) (Krieg et al., 2001). All drug-resistance genes were located on the chromosome rather than on plasmids.

Through BLAST analysis of the VFDB, a total of 33 virulence factor homologs were identified in the genome of ZY-312 (Table 3). Most of these putative virulence genes encoded proteins involved in cellular structure or physiological activities, and none had previously been reported as being related to the pathogenesis of B. fragilis. Notably, bft was not present in the genome of ZY-312, indicating that it is a non-toxigenic strain.

Antibiotic resistance tests were performed based on the antibiotic resistance genes identified in the genome of YZ-312 (Table 4A), and results are summarized in Table 4B. Based on guidelines for antibiotic resistance breakpoints (Russell and Sambrook, 2002; National Center for Clinical Laboratories, 2006), ZY-312 showed resistance to cefepime, kanamycin, and streptomycin, but was susceptible to ceftriaxone, trimethoprim, clarithromycin, chloramphenicol, tetracycline, and levofloxacin. Without available guidelines for B. fragilis, resistance of ZY-312 to vancomycin and polymyxin B could not be confirmed; however, we observed that 4–8 μg/mL vancomycin and >8 μg/mL polymyxin B were sufficient to inhibit ZY-312 growth in vitro.

To confirm the in vivo safety of ZY-312, acute toxicity experiments were performed in both normal SPF BALB/c mice and nude mice. Lacking a thymus and an immune response, nude mice are an ideal animal model for evaluating probiotic safety. No death was observed in the BALB/c mice during the toxicity experiments, no treatment-related toxicity was observed, and no significant difference in body weight was noted between low, medium (Supplementary Figure S4), and high (Figure 1A-1) dose treatment groups and the control group, respectively. Similarly, no difference was found between the culture supernatant-treated group and the control group (Figure 1A-2). In addition, there was no obvious histopathological damage in the stomach, colon, liver, or spleen of BALB/c mice from the high dosage group (Figure 1B). There were also no significant differences in blood routine index or hepatorenal function between the high dose and control groups (data not shown). For the immune-deficient mouse toxicity experiments, ZY-312 again had no deleterious effects on body weight (Figure 1A-3).

To explore whether ZY-312 undergoes major genomic rearrangements during passage, the stability of ZY-312 was examined after 100 generations in vitro. A₁₀₀ and B₁₀₀ were morphologically identical to the original ZY-312 strain (Figure 2A) based on visual inspection, optical microscopy, and SEM. The growth characteristics of A₁₀₀ and B₁₀₀ (Figure 2B) were not significantly different from those of ZY-312. Furthermore, following oral administration of A₁₀₀ or B₁₀₀ at a dose of 1 × 10⁹ cfu/day for 3 days, mice did not show any clinical symptoms or weight loss during the observation period (Figure 2C). The calculated ANI values from each generation were at least 99.98% (Figure 2D).