Experimental Evaluation of Host Adaptation of Lactobacillus reuteri to Different Vertebrate Species

Abstract

The species Lactobacillus reuteri has diversified into host-specific lineages, implying a long-term association with different vertebrates. Strains from rodent lineages show specific adaptations to mice, but the processes underlying the evolution of L. reuteri in other hosts remain unknown. We administered three standardized inocula composed of strains from different host-confined lineages to mice, pigs, chickens, and humans. The ecological performance of each strain in the gastrointestinal tract of each host was determined by typing random colonies recovered from fecal samples collected over five consecutive days postadministration. Results revealed that rodent strains were predominant in mice, confirming previous findings of host adaptation. In chickens, poultry strains of the lineage VI (poultry VI) and human isolates from the same lineage (human VI) were recovered at the highest and second highest rates, respectively. Interestingly, human VI strains were virtually undetected in human feces. These findings, together with ancestral state reconstructions, indicate poultry VI and human VI strains share an evolutionary history with chickens. Genomic analysis revealed that poultry VI strains possess a large and variable accessory genome, whereas human VI strains display low genetic diversity and possess genes encoding antibiotic resistance and capsular polysaccharide synthesis, which might have allowed temporal colonization of humans. Experiments in pigs and humans did not provide evidence of host adaptation of L. reuteri to these hosts. Overall, our findings demonstrate host adaptation of L. reuteri to rodents and chickens, supporting a joint evolution of this bacterial species with several vertebrate hosts, although questions remain about its natural history in humans and pigs.IMPORTANCE Gut microbes are often hypothesized to have coevolved with their vertebrate hosts. However, the evidence is sparse and the evolutionary mechanisms have not been identified. We developed and applied an experimental approach to determine host adaptation of L. reuteri to different hosts. Our findings confirmed adaptation to rodents and provided evidence of adaptation to poultry, suggesting that L. reuteri evolved via natural selection in different hosts. By complementing phylogenetic analyses with experimental evidence, this study provides novel information about the mechanisms driving host-microbe coevolution with vertebrates and serve as a basis to inform the application of L. reuteri as a probiotic for different host species.

Keywords: Lactobacillus reuteri; host adaptation; probiotics; symbiosis.

FIG 1

Neighbor-joining phylogenetic tree of Lactobacillus reuteri based on the core genome alignment (900 genes) of 25 strains. Tips of the branches are color coded by lineage, and cohesive clades are labeled.

FIG 2

Graphic representation of the experimental design. Eighteen strains of different host origins and phylogenetic lineages were grouped into 3 different inocula containing 6 strains each. To facilitate differentiation, strains within the same inoculum were selected to carry distinct leuS alleles. Standardized inocula were prepared to contain equivalent cell numbers of each strain and administered to germfree mice (n = 5 per inoculum group), Lactobacillus-free chickens (n = 5 per inoculum group), germfree pigs (n = 3 per inoculum group), and humans with a low background of lactobacilli (n = 5 per inoculum group). Bacteria were cultured from the inocula and from fecal samples collected between days 1 and 5 after administration, and strain composition was determined by randomly typing colonies.

FIG 3

Cell numbers of L. reuteri in fecal samples (mice, pigs, and humans) or cloacal swabs (chickens), determined by quantitative culture. Data are presented as the log₁₀ CFU. Each data point represents a sample from individual animals or human volunteers, and horizontal bars represent means ± standard deviations.

FIG 4

Stacked bar plots show the relative abundance of L. reuteri strains in the inocula and feces of mice, pigs, and humans and cloacal swabs of chickens at baseline and during days 1 to 5 after oral administration of 3 different inocula (A, B, and C). An asterisk denotes when a strain from a host-specific lineage was significantly more abundant (P < 0.05) than all strains from other lineages in the native host. A white star denotes when the percentages of poultry VI and human VI strains were not significantly different (P < 0.05) in chickens. A triangle indicates when a strain became significantly enriched in a nonnative host. Adjacent bar graphs show the means and standard errors of the relative strain abundance of each strain from the colonies typed at day 5. Individual data points represent the percent colonies typed in each animal or human volunteer. Groups labeled with different letters are significantly different (P < 0.05). Statistical significance was determined by a one-way ANOVA (α = 0.05).

FIG 5

Inferred evolutionary history of L. reuteri-host associations. Ancestral states were inferred on the bacterial phylogeny, modified from a previous study (17). The tree is a maximum-likelihood reconstruction of a concatenated set of 7 single-copy genes from 116 strains. Colors represent host state on the tips of the tree and inferred states on ancestral nodes. Equivocal ancestral states are represented by mixed colors in the circle. Four host-switching events are highlighted as enlarged circles (labeled 1 to 4). The time scale is on a scale of thousands of years (kyrs), and estimates were obtained by using a Bayesian phylogenetic analyses.

FIG 6

Core- and strain-specific gene content of L. reuteri linage VI strains. The ovals represent the genomes of poultry VI (yellow) and human VI (orange) strains. The core gene set (genes unique to each strain) is indicated by the number in the center, and the value below it in parentheses is the number of unique lineage VI genes, compared with other L. reuteri strains (i.e., the value obtained after subtracting genes found in other L. reuteri strains).