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).     

In vitro studies on distribution of indigenous lactobacilli of the gastrointestinal tract of rats

To learn the biochemical mechanisms controlling the distribution of indigenous lactobacilli in the gastrointestinal tracts of rats, the effect of pH and stomach and cecal contents on lactobacillus distribution was investigated in vitro with a mixed culture of three lactobacillus strains isolated from the rat intestine. The pH of the growth medium affected the growth of lactobacilli strongly, irrespective of the lumenal contents. Lactobacillus fermentum outnumbered L. acidophilus and L. murini at low pH (PH 4.5; average pH of stomach contents of conventional rats) but at near neutral pH (pH 6.5; average pH of cecal contents of conventional rats), the growth of L. murini was predominant with all strains. More lactic acid was formed by lactobacilli in medium consisting of stomach contents than in cecal contents medium. L. murini grew in the nondialyzable fraction of the stomach contents and L. fermentum grew in the dialyzable fraction, but L. acidophilus did not grow in either fraction. L. murini grew in the nondialyzable fraction treated with hyaluronidase. In contrast, the nondialyzable fraction treated with pronase or chondroitinase did not allow L. murini to grow at all.     

Relationships between the probiotic and host indigenous lactobacilli under the conditions of mixed cultivation in vitro

Relationships between 5 Lactobacillus manufacturing strains, 458 cultures of indigenous lactobacilli isolated from the human digestive and vaginal tracts and 98 isolates from the feces of white rats and mice were under study. The study demonstrated that under the conditions of mixed in vitro cultivation of paired cultures, probiotic strains inhibited more than 60% of the indigenous lactobacilli isolates. L. acidophilus strain K3 III 24 had the widest spectrum of antagonistic activity. Antagonistic relationships between indigenous lactobacilli depended on the origin, individual features and the anatomical sites of the culture isolation. Based on these results it has to be suggested that probiotic lactobacilli are capable of inducing disbalance in the host indigenous lactoflora. While choosing probiotics the character of relationships between probiotic microorganisms and the indigenous lactobacilli of the future recipient is recommended to be preliminarily tested in vitro.     

Reverse effect of gram-positive bacteria vs. gram-negative bacteria on adjuvant-induced arthritis in germfree rats

Germfree (GF) F344 rats developed severe adjuvant-induced arthritis with a 100% incidence after a single intradermal injection of heat-killed Mycobacterium bovis (BCG). Specific pathogene-free (SPF) rats developed less severe arthritis with a lower incidence. The rats colonized with Escherichia coli or Bacteroides developed mild disease comparable to that in SPF rats. The rats colonized with Bifidobacterium, Propionibacterium acnes, Lactobacillus casei, L. fermentum, L. murini, and L. acidophilus developed more severe disease than that in GF rats. Furthermore, the rats colonized with a mixture of E. coli and the above lactobacilli developed very mild disease similar to that in SPF rats. These results suggest that gram-negative bacteria, such as E. coli and Bacteroides, may suppress the disease, possibly through their lipopolysaccharides, and may be responsible for the lower susceptibility of SPF rats; gram-positive bacteria, such as Bifidobacterium, P. acnes, and lactobacilli, may enhance the disease, possibly through their peptidoglycans; and E. coli may play a dominant role in modulating the development of adjuvant-induced arthritis.     

Decrease in ovalbumin specific IgE of mice serum after oral uptake of lactic acid bacteria

Different kinds of lactobacilli and Bifidobacteria fermented milk were fed to ovalbumin-specific IgE-elevated mice for 3 days, and after the final administration, changes in the ovalbumin-specific IgE values for each sample were compared to the value for non-fermented milk. Seven of the Lactobacillus-fermented milks caused a significant decrease in the serum ovalbumin-specific IgE levels. Above all, Lactobacillus acidophilus L92, Lactobacillus acidophilus CP1613, and Lactobacillus fermentum CP34 fermented milk had the most significant effects of decreasing the serum ovalbumin-specific IgE levels compared to a control group. The L. acidophilus L92 and L. fermentum CP34 cells also showed significant ovalbumin-specific IgE lowering activities. From these results, an active component seems to exist in the cells of L. acidophilus L92 and L. fermentum CP34 strains. Recovery of the radiolabeled L. acidophilus L92 and L. fermentum CP34 cells from the small intestine and the large intestine of the mouse 13 h after oral administration were higher than the recovery of any other strain.     

Lactobacilli and azoreductase activity in the murine cecum.

Azoreductase activity in the ceca of mice lacking lactobacilli as members of the normal microflora (reconstituted-lactobacillus-free [RLF] mice) was compared with that of RLF mice whose gastrointestinal tracts were colonized by strains of Lactobacillus delbrueckii and Lactobacillus fermentum. Azoreductase activity was 31% lower in the ceca of mice colonized by lactobacilli.     

Lactobacilli and azoreductase activity in the murine cecum.

Azoreductase activity in the ceca of mice lacking lactobacilli as members of the normal microflora (reconstituted-lactobacillus-free [RLF] mice) was compared with that of RLF mice whose gastrointestinal tracts were colonized by strains of Lactobacillus delbrueckii and Lactobacillus fermentum. Azoreductase activity was 31% lower in the ceca of mice colonized by lactobacilli.     

Species composition of intestinal lactoflora of healthy rhesus monkeys

The results of the study of the species composition of the fecal microflora of 15 healthy rhesus macaques, from whom 204 strains of lactobacilli have been isolated and identified, are presented. The intestinal microflora has been shown to include different species, subspecies and biovars of homo- and heterofermentative lactobacilli and to possess definite individual properties. L. acidophilus, mannose-negative biovar I, and L. fermentum, mannose-negative biovars I and II, occur most frequently in feces. Certain differences in the species composition of lactoflora in monkeys and humans have been revealed: in the former, L. fermentum, mannitose-positive biovar III, and L. casei occur considerably more seldom. The inclusion of L. acidophilus strain I L. fermentum strains I and II isolated from rhesus macaques into eubiotics for these monkeys has been suggested.     

The antibacterial resistance of the strains making up the components of probiotic preparations

The spectrum of the antibiotic resistance of 21 strains of normal microflora, mainly forming constituents of widely used probiotics to 25 antibacterial preparations. Lactobacillus spp. were prevalent in the strains under test. The spectrum of the antibiotic resistance of lactobacilli varied, the gradation of resistance being more pronounced with respect to strains and not species. Both highly sensitive and highly resistant Lactobacillus strains were found: L. acidophilus (a component of biopreparation "Linex"), L. plantarum 8RA3, L. fermentum 90T4C (probiotic "Lactobacterin"), L. fermentum BL96. Bacteria used as the components of combined preparation "Linex" exhibited the highest resistance to a number of modern antibiotics. Strains of bifidobacteria were found to be highly sensitive to antibiotics.     

Some probiotic properties of chicken lactobacilli

The beneficial effect of lactobacilli has been attributed to their ability to colonize human and animal gastrointestinal tracts. In this work, adhesion assays with three lactobacillus strains and intestinal fragments obtained from chickens were assessed. Lactobacillus animalis and L. fermentum were able to adhere to three kinds of epithelial cells (crop, small and large intestines) with predominance to small intestine. Among the strains considered, L. fermentum subsp. cellobiosus showed the lowest and L. animalis the highest adhesion ability. Scanning electron microphotographs showing L. animalis and L. fermentum adhering to intestinal cells were obtained. The characterization of L. animalis adhesion indicated that lectin-like structure of this strain has glucose/mannose as specific sugars of binding. However, a calcium requirement was not observed. The adhesion of L. fermentum was reduced by addition of sialic acid or mannose (P < 0.01). These carbohydrates can be involved in the interaction between adhesin and epithelial surface. In this case, the dependence on bivalent cations was demonstrated. Lactobacillus fermentum was effective in reducing the attachment of Salmonella pullorum by 77%, while L. animalis was able to inhibit (90%, 88%, and 78%) the adhesion of S. pullorum, S. enteritidis, and S. gallinarum to host-specific epithelial fragments respectively. Our results from this in vitro model suggest that these lactobacilli are able to block the binding sites for Salmonella adhesion.     

Lactobacilli isolated from the stomach of conventional mice.

Twenty strains of lactobacilli isolated from the stomach of conventional mice were tested for their ability to ferment or hydrolyze substrates that may be present in the stomach habitat. The lactobacilli could be placed in four groups (A to D) depending on their ability to ferment N-acetylglucosamine, dextrin, cellobiose, gum arabic, and xylan. The majority of the isolates belonged to groups A and D. Group A strains did not resemble previously described Lactobacillus species, but group D strains were identified as L. leichmannii. A representative group A isolate colonized the surface of the nonsecretory epithelium of the stomach of gnotobiotic mice; a group D isolate did not.     

Metabolism and some characteristics of lactobacilli isolated from the rumen of young calves

Fourteen strains of lactobacilli isolated from the rumen of young calves were studied to determine their biochemical characteristics, growth parameters, metabolism on lactose and sensitivity to 28 antimicrobial agents. Thirteen homofermentative strains belonged to Lactobacillus acidophilus and one heterofermentative strain resembled Lact. fermentum. The relevance of rumen lactobacilli to the nutrition of calves is discussed.     

Metabolism and some characteristics of lactobacilli isolated from the rumen of young calves

Fourteen strains of lactobacilli isolated from the rumen of young calves were studied to determine their biochemical characteristics, growth parameters, metabolism on lactose and sensitivity to 28 antimicrobial agents. Thirteen homofermentative strains belonged to Lactobacillus acidophilus and one heterofermentative strain resembled Lact. fermentum. The relevance of rumen lactobacilli to the nutrition of calves is discussed.     

Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus

A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.     

Effect of adherent Lactobacillus spp. on in vitro adherence of salmonellae to the intestinal epithelial cells of chicken

Single strains of Lactobacillus acidophilus and Lact. fermentum, isolated from chicken intestine, were used to study in vitro interactions with Salmonella enteritidis, Salm. pullorum or Salm. typhimurium in an ileal epithelial cell (IEC) radioactive assay. Exclusion, competition and displacement phenomena were investigated by respectively incubating (a) lactobacilli and IEC together, prior to addition of salmonellae, (b) lactobacilli, IEC and salmonellae together, and (c) salmonellae and IEC, followed by the lactobacilli. Lactobacilli were selected for study because of their strong ability to adhere to IEC and poor aggregation with salmonellae. The results demonstrated that Lact. acidophilus significantly reduced ( P < 0.05) the attachment of Salm. pullorum to IEC in the tests for exclusion and competition, but not in the displacement tests. Lactobacillus fermentum was found to have some ability to reduce the attachment of Salm. typhimurium to IEC under the conditions of exclusion ( P < 0.08), competition ( P < 0.09), but not displacement. However, both Lact. acidophilus and Lact. fermentum were unable to reduce the adherence of Salm. enteritidis to IEC under any of the conditions.     

Lactobacillus fermentum CRL 722 is able to deliver active alpha-galactosidase activity in the small intestine of rats

alpha-galactooligosaccharides (alpha-GOS) found in legumes such as soybeans can cause gastrointestinal disorders since mammals lack alpha-galactosidase (alpha-Gal) in the small intestine which is necessary for their hydrolysis. Lactobacillus fermentum CRL 722 is a lactic acid bacterium (LAB) capable of degrading alpha-GOS due to its elevated alpha-Gal activity. When conventional rats were fed live L. fermentum CRL 722 or cell-free extracts of this strain, a short-lived alpha-Gal activity was detected in the upper gastrointestinal tract. The safety of this LAB was also assessed. L. fermentum CRL 722 could thus be used as a vehicle to safely confer alpha-Gal in the small intestine of monogastric animal.     

Lactic bacteria in the digestive tract of poultry

Lactic bacteria predominate in the microflora of the digestive tract of chicken and turkey. They are represented mainly by Lactobacillus acidophilus, L. salivarius, L. fermentum and L. buchneri. Streptococcus faecium is always isolated. L. ruminis, L. vitulinus, L. delbrueckii, L. coryniformis and L. viridescens were found in this ecological niche for the first time. S. faecium and S. faecalis prevail in the digestive tract of geese and ducks, while lactobacilli are detected in a lesser amount and are represented mainly by L. plantarum. L. salivarius cells isolated from the digestive tract of poultry are highly polymorphous. Most of the lactic acid bacteria found in the digestive tract of poultry can grow at 45-50 degrees C whatever is the species they belong to.     

Comparison of faecal Lactobacillus populations in experimental animals from different breeding facilities and possible consequences for probiotic studies

AIMS: The effect of probiotic lactobacilli is likely dependent on the indigenous Lactobacillus strains in the intestinal tract. Since a substantial number of probiotic studies is performed in rodents, we compared the Lactobacillus strains of different rat and mouse populations in three animal facilities. METHODS AND RESULTS: SDS-PAGE and 16S rDNA analysis of cultured faecal lactobacilli revealed that different Lactobacillus strains were detected in genetically similar Wistar rats bred at different locations. Further, within the same animal facility host genetics did not affect the types of the predominant lactobacilli strains. CONCLUSIONS: Our results show that the environmental background of laboratory animals rather than host genetics determines the indigenous Lactobacillus strains that are found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings underline the importance of microflora analysis in probiotic studies.     

Variable biotherapeutic effects of Lactobacillus acidophilus isolates on orogastric and systemic candidiasis in immunodeficient mice

Two commercially available isolates of Lactobacillus acidophilus (NCFM and LA-1) were compared for their capacities to protect immunodeficient bg/bg-nu/un and bg/bg-nu/+ mice from candidiasis. L. acidophilus NCFM prolonged survival of adult and neonatal bg/bg-nu/nu mice, inhibited disseminated candidiasis in both mouse strains, suppressed weight loss associated with Candida albicans infection in bg/bg-nu/nu females, but did not decrease the severity or the incidente of orogastric candidiasis in gnotobiotic mice. L. acidophilus LA-1 suppressed numbers of C. albicans in the alimentary tracts of bg/bg-nu/+ mice and reduced the severity of mucosal candidiasis in bg/bg-nu/nuand bg/bg-nu/+ mice; however, L. acidophilus LA-1 did not improve the survival of bg/bg-nu/nu mice after oral challenge (colonization) with C. albicansand it was associated with lethality in gnotobiotic adult female bg/bg-nu/nu mice. These results demonstrate that the two isolates of L. acidophilus differed in their capacity to protect immunodeficient mice from candidiasis.     

The adherence of lactic acid bacteria to the columnar epithelial cells of pigs and calves

The adhesion of various lactobacilli and streptococci to columnar epithelial cells of pigs and calves were studied, by in vitro methods. The porcine strains isolated most frequently were Lactobacillus delbrueckii, Lact. acidophilus and Lact. fermentum. Thirteen of the 22 lactobacilli were adhesive. All the streptococci isolated belonged to Lancefield's D-group; none of them adhered to pig epithelial cells. The adhesive strains (9 of 22) of calves were identified as Lact. fermentum. Adherence was variable even between strains of the same species. Isolates from plant material, cultured milk and cheese did not adhere to the columnar epithelial cells in vitro. The adhesive porcine strains tolerated low pH and bile acids, which is important for their survival under conditions in the stomach and intestine.