Host Preference of Beneficial Commensals in a Microbially-Diverse Environment

Abstract

Gut bacteria are often described by the neutral term commensals. However, the more we learn about their interactions with hosts, the more apparent it becomes that gut commensals often contribute positively to host physiology and fitness. Whether hosts can prefer beneficial bacteria, and how they do so, is not clear. This is of particular interest in the case of the bacterivore C. elegans, which depends on bacteria as food source, but also as gut colonizers that contribute to its physiology, from development to immunity. It is further unclear to what extent worms living in their microbially-diverse habitats can sense and distinguish between beneficial bacteria, food, and pathogens. Focusing on Enterobacteriaceae and members of closely related families, we isolated gut bacteria from worms raised in compost microcosms, as well as bacteria from the respective environments and evaluated their contributions to host development. Most isolates, from worms or from the surrounding environment, promoted faster development compared to the non-colonizing E. coli food strain. Pantoea strains further showed differential contributions of gut isolates versus an environmental isolate. Characterizing bacterial ability to hinder pathogenic colonization with Pseudomonas aeruginosa, supported the trend of Pantoea gut commensals being beneficial, in contrast to the environmental strain. Interestingly, worms were attracted to the beneficial Pantoea strains, preferring them over non-beneficial bacteria, including the environmental Pantoea strain. While our understanding of the mechanisms underlying these host-microbe interactions are still rudimentary, the results suggest that hosts can sense and prefer beneficial commensals.

Keywords: C. elegans; Enterobacteriaceae; Pantoea; commensal; microbiota; preference.

Figure 1

Effects of environmental and worm gut isolates on C. elegans development. (A) Bacterial isolates distribution. Relative abundance of genera recovered from soil microcosms and from worm guts, identified based on full length 16S Sanger sequencing and colored as in panel B x-axis (see also Supplementary Table 1 ). (B) Percentage of L4 larvae in worm populations raised from eggs for 40 hours at 25°C on environmental or gut bacterial isolates, as designated, grouped at the genus level; N=25/strain/experiment, dots represent averages (± SE) of 2 independent experiments. E. coli strain OP50 and Enterobacter hormaechei strain CEent1, in the gray and the hatched box, respectively, represent values from 41 experiments and provide reference for comparisons. Dotted lines represent the mean value for development on OP50, mean + SD and mean + 2 SD’s. Boxes extend from first to third quartiles, with black lines representing the median. *p < 0.05; **p < 0.001, in comparison to E. coli (Student’s t-test, corrected for multiple testing using Bonferroni). Black dots at x-axis labels indicate non-Enterobacteriales isolates.

Figure 2

Gut isolates but not an environmental Pantoea strain provide protection from pathogenic colonization by Pseudomonas aeruginosa PA14. (A) Worm colonization by PA14-GFP following 43-45 hours of exposure. (B) Quantification of PA14-GFP fluorescent signal in images as in A; N=36-75 worms from two or three independent experiments. (C) Worm colonization by individual Pantoea strains. CFU counts were estimated in worms raised for 72 hours starting at the L1 stage. Shown are measurements performed in triplicate (N=10 worms each). B and (C) * and **, indicate p < 0.05 and p < 0.001, respectively (Student’s t-test). (D) Gut colonization in individual worms raised on BIGb0393 and shifted for 44 hours to P. aeruginosa PA14-GFP. No correlation is observed between Pantoea colonization (left y-axis, based on CFU counts), and PA14-GFP colonization [right y-axis, fluorescent signal, arbitrary units (AU)].

Figure 3

C. elegans shows preference towards beneficial Pantoea. (A) Multi-strain plates. Shown are percentages of young adult worms localized to spots of the designated strains after a three-hour incubation at 25°C. Shown are averages ± SEM for 10 plates (N=36-164 worms each). *p < 0.01 (Student’s t-test). (B) Pairwise preference comparisons. Shown are choice index values of young adult worms exposed to designated strains for 3 hours. Each dot represents an independent pairwise comparison between the designated Pantoea strains (N=21-123 worms per comparison), with boxes defining the 25 to 75 percentile values and lines representing the median value.