Masami Morotomi

Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: Selective isolation from faeces and identification using monoclonal antibodies

Lactobacillus casei strain Shirota (LCS) is a probiotic bacterium used in the production of fermented milk products and lactic acid bacteria preparations. To investigate the survival of LCS in the gastrointestinal tract, we have developed a selective medium and specific monoclonal antibodies to isolate and identify this strain. Selective LLV agar medium was prepared by modifying LBS medium, a selective medium for lactobacilli, through the replacement of glucose with lactitol as a carbon source and vancomycin as a selective antibiotic. Culture in LLV agar medium followed by ELISA using monoclonal antibodies specific for LCS was able to detect the organism in faeces. Using this method, we studied the faecal recovery of LCS in individuals who drank 125 ml of fermented milk which contained 10(10) live LCS for 3 days. The mean recovery was about 10(7) live bacteria per gram of faeces, indicating that LCS survived transit through the gastrointestinal tract after ingestion of the fermented milk.

Mutagenic activation of biliary metabolites of 1-nitropyrene by intestinal microflora

To investigate the modifying role of intestinal microflora in the metabolism of chemical carcinogens in vivo, we subjected bile from Fischer rats treated per os with chemical carcinogens and related compounds to a mutagenicity assay in the presence and absence of a cell-free extract from human feces. A mixture of the bile sample and potassium phosphate buffer was incubated in the presence or absence of human cell-free fecal extract and then further incubated with a bacterial suspension of Salmonella typhimurium tester strains TA98 or TA100. Bile from rats treated with 1-nitropyrene (1-NP) produced about 2700 and 400 revertants per plate in strain TA98 in the presence and absence of the fecal extract, respectively. There was a drug dose- and bile volume-related response. Treatment of 1-NP-bile with beta-glucuronidase, but not aryl sulfatase, enhanced its mutagenicity. Cell-free extracts of some strains of intestinal bacteria (Bacteroides fragilis ATCC 12044, B. vulgatus ATCC 8482, B. thetaiotaomicron ATCC 12290, Bacteroides sp. strain 524, Eubacterium eligens VPI C15-48, Peptostreptococcus sp. strain 204 and Escherichia coli A-5-18) also enhanced the mutagenicity of 1-NP-bile. These bacterial cell-free extracts hydrolyzed the synthetic beta-D-glucuronides of phenolphthalein and/or p-nitrophenol. These data indicate that the glucuronide(s) of 1-NP-metabolite(s) secreted into bile can be hydrolyzed in the intestine by bacterial beta-glucuronidases to potent mutagenic aglycone(s).

Distribution of Indigenous Lactobacilli in the Digestive Tract of Conventional and Gnotobiotic Rats

Distribution of indigenous lactobacilli in the gastrointestinal tracts of rats was investigated at the species level. The indigenous lactobacilli isolated from conventional rats were divided into three groups, Lactobacillus acidophilus and its related strains, L. fermentum, and L. murini. Localization of the Lactobacillus groups in the gastrointestinal tracts could be distinguished clearly based on arabinose and glucose fermentation reaction of isolates from each part of the gastrointestinal tract. Group I (L. acidophilus and the related strains) and Group II (L. fermentum) were the major populations of lactobacilli on the walls of the non‐glandular part and in the contents of the stomachs of both conventional and gnotobiotic rats. Group I predominated in all the parts of the digestive tract of conventional rats, whereas Group II was in the minority in the lower part of the gastrointestinal tracts of both groups of rats. Group III (L. murini) was the predominant population of Lactobacillus in the lower part of small intestine of conventional rats and in all parts of the gastrointestinal tracts of gnotobiotic rats except for the wall of the non‐glandular part of the stomach.

Intestinal Microflora and Bile Acids

In vitro cholic acid (CA) transformation by mixed fecal culture was investigated. Concentrations of glucose, peptone, and yeast extract in the medium and the initial pH of the medium markedly affected the CA transformation. Yeast extract enhanced the transformation, whereas high concentrations of glucose and peptone inhibited it. When the initial pH of the medium was below 6.5, CA was converted to 7‐keto‐deoxycholic acid (7KD),and formation of deoxycholic acid (DC) was not observed. In contrast, with an initial pH of 7.0, about 60% of the CA was converted to 7KD after 3 days of incubation, and then DC gradually formed after 4 days of incubation, following the disappearance of 7KD. The formation of DC in the cultured samples was paralleled in each case by disappearance of 7KD. In pure culture systems, Escherichia coli and some strains of Bacteroides formed 7KD from CA. No DC formation was observed in pure cultures of any of the strains examined.

Reduction of population levels of some indigenous bacteria by lactobacilli in the gastrointestinal tract of gnotobiotic rats.

Effects of each of three indigenous Lactobacillus groups on other bacterial populations were separately investigated in gnotobiotic rats. In the wall of the non‐glandular part of the stomach, contents of the stomach and contents of the upper part of the small intestine, some pre‐associated indigenous bacteria were reduced to conventional population levels by introducing three groups of lactobacilli: Group I (Lactobacillus acidophilus and related strains), Group II (L. fermentum) and the groups mixed. However, no obvious reduction in cell numbers of the pre‐associated bacteria occurred in the case of Group III (L. murini).