Permanent Colonization by Lactobacillus casei Is Hindered by the Low Rate of Cell Division in Mouse Gut

Abstract

Long residence times of probiotics in the intestinal tract would prolong their potential beneficial health effects and assist colonization. This study investigated the colonization potential of Lactobacillus casei Shirota in mouse intestine by using 5 (and 6)-carboxyfluorescein diacetate, succinimidyl ester (cFDA-SE) for assessment of doubling times in different parts of the intestine. The amounts of intestinal water overlying the surfaces of the duodenum, jejunum, ileum, and colon in BALB/c mice were 34.4 ؎ 2.9, 58.8 ؎ 6.8, 21.6 ؎ 2.2, and 8.0 ؎ 1.0 mg, respectively. Based on the residual concentrations of cFDA-SE-labeled lactobacilli on intestinal mucosal surfaces, the average half times for the wash-out of lactobacilli fed were estimated at 3.98, 1.55, 1.34, and 2.48 days in the duodenum, jejunum, ileum, and colon, respectively. The average doubling times of the lactobacilli, estimated from the residual fluorescent levels of surface-adhered cells, were 4.10, 4.78, 4.56, and 5.59 days in the duodenum, jejunum, ileum, and colon, respectively. It is estimated that the lactobacilli would have to achieve an average doubling time of 1.03 to 2.04 days to colonize the various sections of the mouse intestinal tract more permanently.Probiotic intestinal bacteria beneficially influence the health of the host by modulating the metabolic activities, immunity, and microbiota in the host's intestine (7,12). Lactobacilli have been used as antigen and cytokine delivery vehicles for oral immunization and disease treatment (11,16). Probiotic bacteria are selected for their beneficial health properties as well as their ability to tolerate intestinal conditions and achieve high growth rates in culture (13). However, no probiotic lactobacilli used in clinical trials and commercial production have been demonstrated to persist in fecal samples for more than a few weeks after their administration has been stopped (4,5,14,15). Such an effect is termed colonization resistance. The ability of exogenously administered probiotics to adhere to the mucosal cells and multiply in the intestinal tract has been questioned (2). There are recent reports on the recovery of consumed lactobacilli from human colonic biopsies after discontinuation of probiotic administration (1, 3, 18), thus providing direct evidence that probiotic lactobacilli are able to temporary colonize colonic mucosae. Prolonged adhesion and colonization of probiotic bacteria on intestinal mucosal surfaces could favor probiotic effects. The aim of this study was to understand the growth and colonization of lactobacilli in the intestinal tract, using the mouse as the model system. MATERIALS AND METHODSPreparation of fluorogenic dye. Five (and 6)-carboxyfluorescein diacetate, succinimidyl ester (cFDA-SE), is a nonfluorescent membrane-permeative ester which nonspecific prokaryotic and eukaryotic intracellular esterases convert to a fluorescent derivative that in turn is then covalently linked to intracellular proteins via the probe's succinimidyl group (19). cFDA-SE (2 mg) (Molecul...

Impact of probiotic supplements on microbiome diversity following antibiotic treatment of mice

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“…The membrane was incubated overnight at 4°C in phosphate-buffered saline (PBS) containing 10% (vol/vol) nonfat dry milk and 0.1% Tween 20 and then for 1 h at room temperature with a 1/2,000 dilution of a rabbit antiserum directed against the D region of FnBPA. The membrane was rinsed and incubated in PBS containing nonfat dry milk (5% vol/vol), 0.1% Tween 20, and horseradish peroxidase-conjugated secondary antibody (14). Bacteria were washed in PBS and used to perform invasiveness assays with Caco-2 cells grown as described above on glass coverslips pretreated with 10 g/ml poly-D-lysine (Sigma-Aldrich).…”

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