Nisin, a Probiotic Bacteriocin, Modulates the Inflammatory and Microbiome Changes in Female Reproductive Organs Mediated by Polymicrobial Periodontal Infection

Abstract

Periodontitis-related oral microbial dysbiosis is thought to contribute to adverse pregnancy outcomes (APOs), infertility, and female reproductive inflammation. Since probiotics can modulate periodontitis and oral microbiome dysbiosis, this study examined the effects of a probiotic bacteriocin, nisin, in modulating the reproductive microbiome and inflammation triggered by periodontitis. A total of 24 eight-week-old BALB/cByJ female mice were randomly divided into four treatment groups (control, infection, nisin, and infection+nisin group), with 6 mice per group. A polymicrobial (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum) mouse model of periodontal disease was used to evaluate the effects of this disease on the female reproductive system, with a focus on the microbiome, local inflammation, and nisin's therapeutic potential in this context. Moreover, 16s RNA sequencing was used to evaluate the changes in the microbiome and RT-PCR was used to evaluate the changes in inflammatory cytokines. Periodontal pathogen DNA was detected in the reproductive organs, and in the heart and aorta at the end of the experimental period, and the DNA was especially elevated in the oral cavity in the infection group. Compared to the control groups, only P. gingivalis was significantly higher in the oral cavity and uterus of the infection groups, and T. forsythia and F. nucleatum were significantly higher in the oral cavity of the infection groups. The infection and nisin treatment group had significantly lower levels of P. gingivalis, T. forsythia, and F. nucleatum in the oral cavity compared with the infection group. Since periodontal pathogen DNA was also detected in the heart and aorta, this suggests potential circulatory system transmission. The polymicrobial infection generally decreased the microbiome diversity in the uterus, which was abrogated by nisin treatment. The polymicrobial infection groups, compared to the control groups, generally had lower Firmicutes and higher Bacteroidota in all the reproductive organs, with similar trends revealed in the heart. However, the nisin treatment group and the infection and nisin group, compared to the control or infection groups, generally had higher Proteobacteria and lower Firmicutes and Bacteroidota in the reproductive organs and the heart. Nisin treatment also altered the microbiome community structure in the reproductive tract to a new state that did not mirror the controls. Periodontal disease, compared to the controls, triggered an increase in inflammatory cytokines (IL-6, TNF-α) in the uterus and oral cavity, which was abrogated by nisin treatment. Polymicrobial periodontal disease alters the reproductive tract's microbial profile, microbiome, and inflammatory status. Nisin modulates the microbial profile and microbiome of the reproductive tract and mitigates the elevated uterine inflammatory cytokines triggered by periodontal disease.

Keywords: antimicrobial therapy; nisin; oral microbiome; periodontal disease; reproductive tract microbiome.

Figure 1

A diagram of the polymicrobial infection protocol. Following 1 week of acclimation and 1 week of antibiotic administration, the mice were infected with the polymicrobial infection from weeks (WKS) 2 to 10. Oral swab samples were collected at baseline after the antibiotic administration and at the end of the experimental period at 10 weeks. Tissues (uterus, ovary, vagina, heart, aorta) were also collected at 10 weeks.

Figure 2

Oral periodontal pathogens are present in the female upper reproductive organs, plus the heart and aorta, and nisin mitigates changes in the oral cavity. The graphs show the log copy number of the periodontal pathogens P. gingivalis (a), T. forsythia (b), T. denticola (c), and F. nucleatum (d) in the oral swabs (at baseline and infection after 8 weeks) and uterus, ovary, vagina, heart and aorta tissue. Data are represented as the mean ± SD. * indicates significant differences between marked groups (p ≤ 0.05).

Figure 3

Polymicrobial periodontal infection triggers an increase in TNF-α and IL-6 mRNA expression in the oral cavity and uterus; nisin mitigates these changes. The graphs show the mRNA cytokine expression levels, including TNF-α (a), IL-β (b), and IL-6 (c), in the oral swabs and uterus, ovary, vagina, heart and aorta. Data are represented as the mean ± SD. * indicates significant differences between the two marked groups (p ≤ 0.05).

Figure 4

Polymicrobial periodontal infection alters the microbiome alpha diversity of the female reproductive system and nisin mediates the changes. The graphs show the alpha diversity of the observed species (a) and the Simpson (b), Shannon (c), and Chao1 (d) indices for the uterus, ovary, vagina, and heart tissue. Data are represented as the median ± 95% CI. * indicates significant differences between the two marked groups (p ≤ 0.05).

Figure 5

Polymicrobial periodontal infection tends to alter the microbiome beta diversity of the female reproductive system and nisin mediates the changes. Principal coordinate analysis (PCoA) representing the beta diversity for the uterus (a), ovary (b), vagina (c), and heart (d).

Figure 6

Periodontal infection shifts the relative abundance and microbial composition in the uterus, ovary and vagina; nisin mitigates some changes and establishes a new state. Bar graphs show the relative abundance of each bacteria taxa at the phylum level (a) and genus level (b) in the oral cavity, uterus, ovary, vagina and heart. Data are represented as the mean. # indicates a significant difference between the control and infection groups (p ≤ 0.05). * indicates a significant difference between the infection and infection+nisin groups (p ≤ 0.05).

Figure 7

Periodontal infection significantly shifts the relative abundance and microbial phylum composition in the uterus, ovary and vagina; nisin mitigates some changes and establishes a new state. Bar graphs show the significant microbial differences between the groups at the phylum level in the uterus (a), ovary (b), vagina (c), and heart (d). Data are represented as the means in the bar graphs on the left, and as the 95% CIs in the scales on the right; p values are shown in each figure for each species.

Figure 8

Periodontal infection significantly shifts the relative abundance and microbial genus composition in the uterus, ovary and vagina; nisin mitigates some changes and establishes a new state. Bar graphs show the significant microbial differences between the groups at the phylum level in the uterus (a), ovary (b), and vagina (c) and heart (d). Data are represented as the means in the bar graphs on the left, and as the 95% CIs in the scales on the right; p values are shown in each figure for each species.