Fermented Milk Containing Lacticaseibacillus rhamnosus SNU50430 Modulates Immune Responses and Gut Microbiota in Antibiotic-Treated Mice

Abstract

Antibiotics are used to control infectious diseases. However, adverse effects of antibiotics, such as devastation of the gut microbiota and enhancement of the inflammatory response, have been reported. Health benefits of fermented milk are established and can be enhanced by the addition of probiotic strains. In this study, we evaluated effects of fermented milk containing Lacticaseibacillus rhamnosus (L. rhamnosus) SNUG50430 in a mouse model with antibiotic treatment. Fermented milk containing 2 × 10⁵ colony-forming units of L. rhamnosus SNUG50430 was administered to six week-old female BALB/c mice for 1 week. Interleukin (IL)-10 levels in colon samples were significantly increased (P < 0.05) compared to water-treated mice, whereas interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) were decreased, of mice treated with fermented milk containing L. rhamnosus SNUG50430-antibiotics-treated (FM+LR+Abx-treated) mice. Phylum Firmicutes composition in the gut was restored and the relative abundances of several bacteria, including the genera Coprococcus and Lactobacillus, were increased in FM+LR+Abx-treated mice compared to PBS+Abx-treated mice. Interestingly, abundances of genus Coprococcus and Lactobacillus were positively correlated with IL-5 and IL-10 levels (P < 0.05) in colon samples and negative correlated with IFN-γ and TNF-α levels in serum samples (P < 0.001). Acetate and butyrate were increased in mice with fermented milk and fecal microbiota of FM+LR+Abx-treated mice were highly enriched with butyrate metabolism pathway compared to water-treated mice (P < 0.05). Thus, fermented milk containing L. rhamnosus SNUG50430 was shown to ameliorate adverse health effects caused by antibiotics through modulating immune responses and the gut microbiota.

Keywords: Antibiotic; Lacticaseibacillus rhamnosus; fermented milk; gut microbiota; immunomodulation; probiotic.

Fig. 1. The experimental scheme of this study.

A mixture of antibiotics, containing ampicillin, metronidazole, neomycin and vancomycin, was treated to 6 week-old female BALB/c mice via drinking water for 1 week. Then, 200 μl of fermented milk contained 2 × 10⁵ CFUs of L. rhamnosus SNUG50430 was administered to mice once daily by oral gavage for 1 week. Colon samples were homogenized and the supernatant was collected after centrifugation at 15,000 ×g for 10 min at 4°C. Cytokine levels in the supernatant were measured. The PBS-antibiotics (PBS+Abx)-treated, the fermented milk without L. rhamnosus SNUG50430-antibiotics (FM+Abx)–treated, and the fermented milk with L. rhamnosus SNUG50430-antibiotics (FM+LR+Abx) group, were designed as each experimental group. Water-treated group was used as a negative control.

Fig. 2. Effects of fermented milk containing L. rhamnosus SNUG50430 on cytokine levels in colon samples of antibiotic-treated mice.

(A) Interferon gamma (IFN-γ), (B) Interleukin (IL)-2, (C) IL-5, (D) IL-10, (E) Tumor necrosis factor alpha (TNF-α). Data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments. Asterisks indicate a statistically significant difference [*P < 0.05; **P < 0.01; Kruskal-Wallis one-way analysis of variance (ANOVA) with the Dunn’s post hoc test].

Fig. 3. Effects of fermented milk containing L. rhamnosus SNUG50430 on cytokine levels in serum samples of antibiotic-treated mice.

(A) IFN-γ, (B) TNF-α. Cytokine levels in the serum collected from mice were measured. Data are expressed as the mean ± SEM of three independent experiments. Asterisks indicate a statistically significant difference (**P < 0.01; Kruskal-Wallis one-way ANOVA with the Dunn’s post hoc test).

Fig. 4. Effects of fermented milk containing L. rhamnosus SNUG50430 on fecal microbiota in antibiotictreated mice.

(A) Observed species and (B) Shannon indices of each experimental group for Alpha-diversity, (C) Non-metric multi-dimensional scaling (NMDS) plot with Bray-Curtis distances for experimental groups, (D) Comparisons of microbial taxa of experimental group at phylum level. Data are expressed as the mean ± SEM of three independent experiments. Asterisks indicate a statistically significant difference [**P < 0.01; Kruskal-Wallis one-way ANOVA with the Dunn’s post hoc test].

Fig. 5. Relative abundances in microbial genera among experimental groups.

(A) Genus Coprococcus, (B) Genus Dehalobacterium, (C) Genus Dorea, (D) Genus Lactobacillus, (E) Genus Ruminococcus, (F) Genus Klebsiella, (G) Genus Proteus. Data are expressed as the mean ± SEM. Asterisks indicate a statistically significant difference (*P < 0.05; **P < 0.01; Kruskal-Wallis one-way ANOVA with the Dunn’s post hoc test).

Fig. 6. Spearman's correlations between relative abundances of microbial genera and cytokine levels in mice.

(A) Colon samples, (B) Serum samples. Colors indicate the degrees of correlation. Asterisks indicate statistical significance (*P < 0.05; ***P < 0.001).

Fig. 7. Alterations in short-chain fatty acid (SCFA) concentrations and butyrate metabolism according to the phylogenetic investigation of communities by reconstruction of unobserved state (PICRUSt) analysis in antibiotic-treated mice fed fermented milk containing L. rhamnosus SNUG50430.

(A) Acetate concentration, (B) Butyrate concentration, (C) PICRUSt analysis for butyrate metabolism. SCFAs in samples were measured using an Agilent 7890A gas chromatograph. Data are expressed as the mean ± SEM. Asterisks indicate a statistically significant difference (*P < 0.05; **P < 0.01; Kruskal-Wallis one-way ANOVA with the Dunn’s post hoc test for SCFA concentrations in experimental groups and Mann-Whitney U test for PICRUSt analysis).