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. 2021 May 18;16(5):e0251784.
doi: 10.1371/journal.pone.0251784. eCollection 2021.

Physical properties of lactic acid bacteria influence the level of protection against influenza infection in mice

Takumi Watanabe  1   2 , Kyoko Hayashi  2 , Isao Takahashi  3 , Makoto Ohwaki  4 , Tatsuhiko Kan  1 , Toshio Kawahara  5
Affiliations

Physical properties of lactic acid bacteria influence the level of protection against influenza infection in mice

Takumi Watanabe et al. PLoS One. .

Abstract

We evaluated whether the water dispersibility of lactic acid bacteria (Enterococcus faecalis KH2) affects their efficacy. When cultured lactic acid bacteria are washed, heat-killed, and powdered, adhesion occurs between results in aggregation (non-treated lactic acid bacteria, n-LAB). However, dispersed lactic acid bacteria (d-LAB) with a lower number of aggregates can be prepared by treating them with a high-pressure homogenizer and adding an excipient during powdering. Mice were administered n-LAB or d-LAB Peyer's patches in the small intestine were observed. Following n-LAB administration, a high amount of aggregated bacteria drifting in the intestinal mucosa was observed; meanwhile, d-LAB reached the Peyer's patches and was absorbed into them. Evaluation in a mouse influenza virus infection model showed that d-LAB was more effective than n-LAB in the influenza yield of bronchoalveolar lavage fluids on day 3 post-infection and neutralizing antibody titers of sera and influenza virus-specific immunoglobulin A in the feces on day 14 post-infection. Therefore, the physical properties of lactic acid bacteria affect their efficacy; controlling their water dispersibility can improve their effectiveness.

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Conflict of interest statement

Takumi Watanabe and Tatsuhiko Kan are employed by Bio-Lab Co., Ltd. Isao Takahashi is employed by ICAM Co., Ltd. None of the authors had a personal or financial conflict of interest. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. In addition, all authors involved with the present manuscript declare that they have no competing interests of any kind, including financial and non-financial competing interests.

Figures

Fig 1
Fig 1. Experimental procedure of influenza virus infection.
Mice in the control, n-LAB, d-LAB, and OSL groups were administered distilled water, n-LAB (5 mg/day), d-LAB (5 mg/day, two doses daily) and OSL (0.2 mg/day, two doses daily), respectively, during the study period (days −7 to 14). Mice were intranasally infected with IFV on day 0. On day 3 after IFV infection, 5 mice from each group were euthanized to quantify virus load in the lungs and BALF. Further, 5 mice were euthanized for measuring neutralizing antibody and IgA levels on day 14. BALF, bronchoalveolar lavage fluid; d-LAB, dispersed lactic acid bacteria; IFV, influenza A virus; n-LAB, non-treated lactic acid bacteria; OSL, oseltamivir phosphate.
Fig 2
Fig 2. Measurement of n-LAB and d-LAB using a laser diffraction particle size analyzer (SALD-2300).
n-LAB (a) and d-LAB (b) were suspended in distilled water and the relative particle mass (frequency and integration) was measured using a laser diffraction particle size analyzer. d-LAB, dispersed lactic acid bacteria; n-LAB, non-treated lactic acid bacteria; n = 3.
Fig 3
Fig 3. Microscopic images of the Peyer’s patch in mice after LAB and d-LAB administration and Peyer’s patches images after d-LAB administration with cLSM.
n-LAB (a) or d-LAB (b) stained with Cy3 and Peyer’s patches were imaged using a fluorescence microscope. The uptake of d-LAB by Peyer’s patches after d-LAB administration was imaged using cLSM [(c); image from the lumen and (d); cross-sectional image]; actin was stained with phalloidin and nuclei were stained with DAPI. The white triangular arrow shows the bacteria. cLSM, confocal laser scanning microscopy; d-LAB, dispersed lactic acid bacteria; n-LAB, non-treated lactic acid bacteria.
Fig 4
Fig 4. Effect of n-LAB and d-LAB administration on IL-12 production in mouse splenocytes.
n-LAB and d-LAB were co-cultured with mouse splenocytes for 24 h. IL-12 concentration in the culture supernatant was measured using enzyme-linked immunosorbent assay. Control, culture medium only; d-LAB, dispersed lactic acid bacteria; n-LAB, non-treated lactic acid bacteria. Each value is presented as the mean ± SD; n = 6; **p < 0.01 vs. n-LAB.
Fig 5
Fig 5. Body weight change in mice infected with IFV.
IFV-infected mice were orally administered distilled water (control, filled circle), 0.2 mg/day of oseltamivir (OSL, white circle), 5 mg/day of non-treated lactic acid bacteria (n-LAB, filled square), and 5 mg/day of dispersed lactic acid bacteria (d-LAB, white square) from 7 day pre-infection to 14 day post-infection. Body weights are relative to those on the day of viral infection (day 0), which was set as 100%. Each value is presented as the mean ± SD; n = 5; IFV, influenza A virus.
Fig 6
Fig 6. Effect of LAB or d-LAB administration on viral load in mice.
Virus yield in BALF (a) and lung samples (b) were measured using a plaque assay on day 3 post-infection. Each value is presented as the mean ± SD; n = 5; **p < 0.01; *p < 0.05. BALF, bronchoalveolar lavage fluid; d-LAB, dispersed lactic acid bacteria; n-LAB, non-treated lactic acid bacteria; OSL, oseltamivir; PFU, plaque-forming unit.
Fig 7
Fig 7. Effects of n-LAB or d-LAB administration on the neutralizing antibody titer and IFV-specific IgA production in mice.
The titer of the virus-neutralizing antibody is presented as the reciprocal of the dilution of BALF samples (a) and sera (b) that reduced the plaque number to a level below 50% of that observed in the virus control. The IFV-specific IgA levels in BALF (c) and fecal samples (d) were determined using enzyme-linked immunosorbent assay. Each value is presented as the mean ± SD; n = 5; **p < 0.01; *p < 0.05. BALF, bronchoalveolar lavage fluid; IFV, influenza A virus; d-LAB, dispersed lactic acid bacteria; n-LAB, non-treated lactic acid bacteria; OSL, oseltamivir.
Fig 8
Fig 8. Image of n-LAB or d-LAB in contact with the small intestinal Peyer’s patches.
n-LAB could not reach the Peyer’s patches because of large particle size, which is blocked by mucus (a). d-LAB were small particle size and can pass via mucus to reach the Peyer’s patches (b). d-LAB, dispersed lactic acid bacteria; n-LAB, non-treated lactic acid bacteria.
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Grants and funding

The research received support from Bio-Lab Co., Ltd. and ICAM Co., Ltd. The funder provided support in the form of salaries for authors (Takumi Watanabe, Tatsuhiko Kan, and Isao Takahashi) but had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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