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. 2017 Mar 17:8:435.
doi: 10.3389/fmicb.2017.00435. eCollection 2017.

Safety Evaluation of a Novel Strain of Bacteroides fragilis

Ye Wang  1 , Huimin Deng  2 , Zhengchao Li  2 , Yafang Tan  3 , Yanping Han  3 , Xiaoyi Wang  3 , Zongmin Du  3 , Yangyang Liu  4 , Ruifu Yang  3 , Yang Bai  5 , Yujing Bi  3 , Fachao Zhi  5
Affiliations

Safety Evaluation of a Novel Strain of Bacteroides fragilis

Ye Wang et al. Front Microbiol. .

Abstract

Commensal non-toxigenic Bacteroides fragilis confers powerful health benefits to the host, and has recently been identified as a promising probiotic candidate. We previously isolated B. fragilis strain ZY-312 and identified it as a novel strain based on 16S rRNA sequencing and morphological analyses. We also determined that ZY-312 displayed desirable probiotic properties, including tolerance to simulated digestive fluid, adherence, and in vitro safety. In this study, we aim to investigate whether ZY-312 meets the safety criteria required for probiotic bacteria through comprehensive and systematic evaluation. Consequently, the fatty acid profile, metabolite production, and biochemical activity of strain ZY-312 were found to closely resemble descriptions of B. fragilis in Bergey's manual. Taxonomic identification of strain ZY-312 based on whole genome sequencing indicated that ZY-312 and ATCC 25285 showed 99.99% similarity. The 33 putative virulence-associated factors identified in ZY-312 mainly encoded structural proteins and proteins with physiological activity, while the lack of bft indicated that ZY-312 was non-toxigenic. In vivo safety was proven in both normal and immune-deficient mice. The 11 identified antibiotic resistance genes were located on the chromosome rather than on a plasmid, ruling out the risk of plasmid-mediated transfer of antibiotic resistance. In vitro, ZY-312 showed resistance to cefepime, kanamycin, and streptomycin. Finally, and notably, ZY-312 exhibited high genetic stability after 100 passages in vitro. This study supplements the foundation work on the safety evaluation of ZY-312, and contributes to the development of the first probiotic representative from the dominant Bacteroidetes phylum.

Keywords: Bacteroides fragilis; genetic stability; probiotic; safety evaluation; whole genome sequencing.

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Figures

FIGURE 1
FIGURE 1
ZY-312 is non-pathogenic in mice. (A) Changes in body weight (%) per day were observed for the experimental and control groups. (A-1) Specific pathogen-free (SPF) BALB/c normal mice (n = 8) were treated with 5 × 1011 colony forming units (cfu)/day ZY-312 for 5 days and observed for 18 days. A control group was treated with saline. (A-2) SPF normal mice (n = 16) were treated with culture supernatant (0.5 mL/day) for 5 days and observed for 17 days. Tryptic soy broth was used for the control group. (A-3) Nude mice (n = 5) were treated with ZY-312 at a concentration of 1 × 109 cfu/day for 3 days and observed for 7 days. The control group were treated with saline. (B) Light micrograph images of the stomach, colon, liver, and spleen from mice belonging to the high dosage group (upper) and control group (lower). No significant lesions were observed (mean ± SE; NS, not significant, t-test).
FIGURE 2
FIGURE 2
ZY-312 is genetically stable. (A) Gram-stained ZY-312, A100, and B100 cells following anaerobic culture on tryptic soy agar (5% sheep blood) for 48 h at 37°C. Observation were made using a light microscope (4000×) and a scanning electron microscope (30000×). (B) Growth curves of ZY-312, A100, and B100 cultured anaerobically for 24 h. (C) SPF BALB/c normal mice (n = 8) were treated with A100 or B100 at a concentration of 1 × 109 cfu/day for 3 days and observed for 7 days. A control group was treated with saline. (D) Average nucleotide identities were calculated between each generation.
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