The inhibitory effect of Bifidobacterium longum and Bifidobacterium infantis isolated from a Lac-B preparation on the growth of Escherichia coli O157: H7 strains, NK2, No.61468, and No.33009, and the production of verotoxins by these strains in vitro was investigated. In the control experiment, all three strains of E. coli O157: H7 grew well in GAM broth under aerobic incubation (35°C, 18 hr), and strains NK2 and No.61468 produced high levels of VT2 (400 ng/ml each) and of VT1 (25 and 3.13 ng/ml, respectively); however, strain No.33009 produced only VT2 (25 ng /ml), but no VT1 at any detectable level. The growth of these strains and their production of verotoxins in vitro were strongly inhibited by their incubation (35°C, 18 hr) in culture broths of B. longum or B. infantis, or in their supernatants obtained by centrifugation. When the strains of bifidobacteria were cultured in GAM broth by the conventional method, the pH of the culture broth shifted from the neutral region to the acidic region (about pH 5.0). The growth and verotoxin production of E. coli O157: H7 NK2 in GAM broth acidified by three types of low-molecular-weight fatty acids and hydrochloric acid were tested. It was found that the growth and verotoxin production of the test strain were inhibited to different degrees by the acids used; they were only slightly inhibited by lactic acid and hydrochloric acid, but strongly inhibited by acetic acid and butyric acid. The in vitro activity of bifidobacteria, the main components in a Lac-B preparation, corresponded to their excellent efficacy in intestinal infection with E. coli O157: H7 previously observed in germ-free mice. These results suggest that the oral administration of bifidobacteria could prevent the development of intestinal infections by enterohemorrhagic E. coli, including E. coli O157: H7.
A modification of bifidus-blood agar (A-W) containing peptones combination “A” (peptone from casein trypsin digested, peptone from meat pepsin digested, peptone from meat trypsin digested) and water-soluble aniline blue “W” was evaluated for its use in enumeration of Bifidobacterium species in faeces. This medium was originally described by Reuter in 1963. Colonies of bifidobacteria appeared copper coloured and had an elevated morphology on bifidus-blood agar (A-W). This distinct appearance allowed an easy and quick differentiation of bifidobacteria in faecal samples.Using bifidus-blood agar (A-W), bifidobacteria were isolated from 24 mouse faecal samples (n = 26), 16 human faecalsamples (n =16), and two rat faecal samples (n = 3). Ten known strains of Bifidobacterium and 50 strains of Bifidobacterium species isolated from faecal samples and commercial products all grew as elevated, copper colonies on bifidus-blood agar (A-W). Colonies of lactobacilli were distinctly different in colour and morphology, as were all other faecal bacteria in the samples tested. When recovery of bifidobacteria on bifidus-blood agar (A-W) was compared with that on three non-selective and two selective media, the recovery on bifidus-blood agar (A-W) was equivalent to that of two of the non-selective media. However, A-W agar provided a significantly higher recovery than the third non-selective medium and both selective media tested. The method of preparation and the choice of ingredients used were important.
A total of 17 Bifidobacterium pseudocatenulatum strains isolated from infant faeces and 1 Bifidobacterium pseudocatenulatum type strain were compared for their clonal relatedness. Clonal relatedness was compared by randomly amplified polymorphic DNA analysis (RAPD) using three selected primers (GEN 1-60-03, GEN 1-60-06 and GEN 1-60-07). RAPD patterns with DNA fragments ranging from approximately 0.3 to 10.0 kb in size were observed. Examination of the DNA fingerprints using cluster analysis showed a significant genetic diversity among the strains tested.