Research Study Abstract
Probiotic Clinical Trial
Persistence of Lactobacilli acidophilus DDS-1 in the
human gastrointestinal tract
Bob Hutkins and Jens Walter
University of Nebraska Department of Food Science
Project Summary:
Numerous studies have shown that many of the Lactobacillus strains used as probiotics are generally non-persistent in the human gastrointestinal tract. Nonetheless, it does appear that survival of these organisms through the stomach and small intestine is essential if that strain is to provide health benefits to the host. That is, for a probiotic to contribute intestinal health benefits, that organism must, at minimum, be able to reach the colon. Whether that organism can also persist after administration is stopped, however, depends largely entirely on the colonization factors express by that organism.
The inability to colonize the intestinal tract after probiotic administration is likely due to several factors. The general view is that probiotic strains are not normal inhabitants and simply lack the ability to compete well in the gut environment. In contrast, strains derived from humans (on a persistent basis) could, in theory, be better suited for colonizing other human hosts. However, this has never been tested, and it may well be that probiotic strains persist the same as human strains. To test this hypothesis, we propose to conduct a study to compare the persistence of Lactobacillus ruminis strain L5, which was detected in two human subjects in high numbers over the duration of 15 months, with a well-studied probiotic strain, Lactobacillus acidophilus DDS-1. This study will provide important knowledge about the ecology and persistence of intestinal and probiotic lactobacilli and the modulation of the gut microbiota through probiotics. In addition, it will reveal to what extent a well-characterized probiotic strain (DDS-1) itself persists in the GI tract and also how it influences other intestinal organisms.
Background
Most of the Lactobacillus strains used as probiotics are generally unable to persist in the human gastrointestinal tract (GIT). The main reasons for this observation are due to (1) competitive exclusion or colonization resistance conferred by the resident gut bacteria, and (2) host factors conferred by individuals. Moreover, ecological studies on the dynamics, over time, of resident Lactobacillus populations have shown that even these Lactobacillus strains are only present in low numbers and, like probiotic strains, are only temporarily detectable. These findings indicate that most Lactobacillus species are what ecologists refer to as “allochthonous” or “outsiders” to the human gut and are inherently not good competitors in the GIT. In contrast, a few Lactobacillus species, including Lactobacillus ruminis L5, have been shown to be more stable in human subjects. Therefore, it is possible that L. ruminis is autochthonous or a normal resident of the human GIT. The question remains, however, if an autochthonous strain would be a better “universial colonizer” (i.e., in other humans as well).
Hypotheses and Objectives:
We hypothesize that an autochthonous Lactobacillus strain can be established for significantly longer times than probiotic strains, and that its administration may have a greater impact on other members of the gut microbiota. To test this hypothesis, we propose to conduct a study to compare the persistence of Lactobacillus ruminis L5with a commercially available probiotic strain (Lactobacillus acidophilus DDS-1)in 6 human subjects. The specific aims of this study are:
- Conduct a study where L. acidophilus and L. ruminis is administered to 6 human subjects in two individual time periods.
- Compare the persistence of the probiotic strains using a combination of molecular methods.
- Compare the impact of probiotic administration on the composition of the fecal microbiota.
This study will provide important knowledge about how the gut microbiota can be modulated through probiotics, and will show if ecological criteria should be applied in the selection of probiotic strains if longer persistence is desired.
Research plan
Bacterial strains: The probiotic strain used in this study will be Lactobacillus acidophilus DDS-1 from Nebraska Cultures. Lactobacillus ruminis strain L5 will be provided by Gerald Tannock (University of Otago, New Zealand). The latter will prepared under the same conditions as L. acidophilus by a contract culture facility (Jeneil Biotech, Saukville, Wisconsin). Cell concentrations of more than 5 x 109/g are expected. Viability of the probiotic powders will be regularly tested.
Subjects, probiotic administration, and sampling: Six healthy subjects will be recruited. The study will take place over five periods (I, II, III, IV, and V). Period 1 (4 weeks) will serve as a control period. In Period II, volunteers will consume a daily dose of 1010 CFU for 7 days of Lactobacillus acidophilus DDS-1 followed by an 8-week wash out period (Period III). Then, volunteers will consume a daily dose of 1010 CFU of Lactobacillus ruminis (Period IV) followed again by a 12-week wash out period (Period V). Two fecal samples will be collected from each subject at week one and week four of the control period. After that, fecal samples will be collected at day 0 (which is day 7 of the feeding period II), 7, 14, 28, 42, 56 of the washout period III and at day 0 (which is day 7 of the feeding period IV), 7, 14, 28, 42, 56, 80 of the washout period V.
Examination of fecal samples to analyze the gut microbiota: Fecal samples will be serially diluted and plated on Rogosa SL agar (selective for lactobacilli), MacConkey agar (enterobacteria), and bile esculin azide agar (enterococci). Numbers of enterobacteria and enterococci will be determined after 24 hour aerobic incubation at 37°C. Numbers of lactobacilli will be determined after 48 hours anaerobic incubation at 37°˚C. To analyze the total Lactobacillus population, 10 colonies will be picked at random from a dilution agar plate containing 30-300 colonies. The isolates will be differentiated by a Amplified Fragment Length Polymorphism (AFLP) technique that we have developed for the differentiation of Lactobacillus and Streptococcus strains. This technique will allow the probiotic strain to be identified and to determine how long it persists in the human subjects.
The impact of probiotic administration on the resident Lactobacillus population will be determined by a method called DGGE. The effect of probiotic administration on other members of the gut microbiota will be performed by fluorescent in situ hybridization (FISH)/flow cytometry.
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