Establishment of oral bacterial communities in germ-free mice and the influence of recipient age

Abstract

The acquisition of the oral microbiome is a complex process. We examined how the timing of microbial exposure alters bacterial colonization of the tooth surface. Germ-free mice were conventionalized by exposure to specific pathogen-free (SPF) mice to acquire a commensal microbiome over three distinct 4-week periods, 0-4 weeks of age (Conv0-4w), 4-8 weeks (Conv4-8w), or 8-12 weeks (Conv8-12w). Bacterial DNA was extracted from the tooth surface and analyzed by 16S rDNA sequencing. Total bacteria and inflammatory cytokine expression in gingiva were determined by quantitative real-time polymerase chain reaction. After co-housing with SPF mice, Conv0-4w and Conv4-8w mice had low bacterial diversity, whereas Conv8-12w mice had high bacterial diversity that was similar to that of SPF donor mice, as determined by both operational taxonomic units and the Shannon Index. Cluster analysis with unweighted Unifrac distance also supported these trends. This was surprising as the amount of maturation time, 4 weeks, was equal in all conventionalized mice and tooth eruption was largely completed by 4 weeks. This suggests that host factors that occur after tooth eruption have a significant effect on the microbial tooth colonization.

Keywords: 16S rDNA; age; gnotobiotic mice; inflammatory response; oral microbiota; sequencing.

FIGURE 1

Age influences the acquisition of the oral microbiota as measured by alpha-diversity. A & B. Alpha diversity was assessed by the number of OTUs. C & D. Alpha diversity was assessed by the Shannon Index. (*P<.05) between the indicated groups

FIGURE 2

Recipient mouse age influences oral microbial beta-diversity during conventionalization of germ-free mice. Results of Principal Component Analysis with unweighted UniFrac distance. (A) Comparison between Conv0-4w mice and specific pathogen-free (SPF) mice (P<.05). (B) Comparison between Conv4-8w mice and SPF mice (P<.05), (C) Comparison between Conv8-12w mice and SPF mice (P>.05). (D) Comparison between Conv0-4w mice and Conv4-8w mice (P<.05). (E) Comparison between Conv0-4w mice and Conv8-12w mice (P<.05). (F) Comparison between Conv4-8w mice and Conv8-12w mice (P<.05)

FIGURE 3

Recipient mouse age influences oral microbial composition during conventionalization of germ-free mice. (A) Area charts show the relative abundance of the predominant bacteria in each group. (B) The relative abundance ratio of the top five bacteria observed in different groups. (C-G) Relative abundance of Enterobacteriaceae, Enterococcus, Staphylococcus, Streptococcus and Lactobacillus, respectively. *Significant difference (P<.05) between the indicated groups, ± indicates the difference between the indicated groups is P=0.06.

FIGURE 3

Recipient mouse age influences oral microbial composition during conventionalization of germ-free mice. (A) Area charts show the relative abundance of the predominant bacteria in each group. (B) The relative abundance ratio of the top five bacteria observed in different groups. (C-G) Relative abundance of Enterobacteriaceae, Enterococcus, Staphylococcus, Streptococcus and Lactobacillus, respectively. *Significant difference (P<.05) between the indicated groups, ± indicates the difference between the indicated groups is P=0.06.

FIGURE 4

Conventionalization of neonatal germ-free mice induces elevated inflammatory cytokine expression. (A) The number of total oral bacteria on the tooth surface was determined using quantitative real-time polymerase chain reaction of the 16S rRNA gene. (B) Interleukin-1β (IL-1β) expression in the gingival tissue around molar teeth determined by quantitative real-time PCR. (C) IL-12β expression in the gingival tissue around molar teeth determined by quantitative real-time PCR. *Significant difference (P<.05) between the indicated groups