Flucytosine → 5-Fluorouracil

Bifidobacterium longum/pAV001-HU-eCD obtained in Reference Example 1 was inoculated into 5 mL of MRS medium containing the antibiotic spectinomycin and 50 μg/mL of 5-FC, followed by anaerobic culture at 37° C. for 72 hours. Then, 1 mL of the culture medium was similarly inoculated into 9 mL of MRS medium containing the antibiotic spectinomycin and 50 μg/mL of 5-FC, and cultured for 24 hours under the same culture conditions. This inoculation step was repeated three rounds to produce the 5-FU-resistant Bifidobacterium longum/pAV001-HU-eCD. Next, the 5-FU-resistant growth of the produced 5-FU-resistant Bifidobacterium longum/pAV001-HU-eCD was confirmed by inoculating into MRS medium containing 20 μg/mL of 5-FU and the antibiotic spectinomycin and culturing for 24 hours under the same culture conditions as above. Then, the bacteria were suspended with glycerol and stored at −80° C. as glycerol stocks. The stored bacteria sample and wild-type Bifidobacterium longum were inoculated respectively into MRS media containing 250 μg/mL of 5-FU and the antibiotic spectinomycin and growths thereof were compared. The growth of bacteria from the stored sample was observed on the following day, indicating the maintenance of 5-FU resistance, while the wild type did not grow. Viable cell counts of the post-culture bacterial culture were determined by plate count method. The viable cell count was 2 to 3×109 CFU/mL for the bacterial culture of the stored sample, while that of the wild type was below the limit of detection (below 103 CFU/mL).

1. customBifidobacterium longum/pAV001-HU-eCD obtained in Reference Example 1
2. waitcultured for 24 hours under the same culture conditions

ORD-Eintrag

ord-0abf3ebb3adc4609a0644b8cc619f3c6

Quelldatensatz

uspto-grants-2014_05

DOI

10.6084/m9.figshare.5104873.v1

Patentnummer

US08734779B2