Experimental bacterial adaptation to the zebrafish gut reveals a primary role for immigration

Abstract

All animals live in intimate association with microorganisms that profoundly influence their health and development, yet the traits that allow microorganisms to establish and maintain host associations are not well understood. To date, most investigations aimed at identifying traits required for host association have focused on intrahost niches. Consequently, little is known about the relative contribution of extrahost factors such as environmental growth and survival and immigration into hosts from the external environment, as promoters of host association. To address this, we developed a tractable experimental evolution system that investigates both intra- and extrahost factors contributing to bacterial adaptation to the vertebrate gut. We passaged replicate lines of a zebrafish bacterial isolate, Aeromonas veronii, through populations of germ-free larval zebrafish (Danio rerio), each time using gut-associated Aeromonas populations to inoculate the aquatic environment of the next zebrafish population. We observed rapid increased adaptation to the host in all replicate lines. The initial adaptations present in early-evolved isolates did not increase intrahost fitness but rather enhanced both immigration from the environment and interhost transmission. Only in later-evolved isolates did we find evidence for intrahost-specific adaptations, as demonstrated by comparing their competitive fitness in the host genotype to which they evolved to that in a different genotype. Our results show how selection for bacterial transmission between hosts and their environment can shape bacterial-host association. This work illuminates the nature of selective forces present in host–microbe systems and reveals specific mechanisms of increased host association. Furthermore, our findings demonstrate that the entire host–microbe–environment system must be considered when identifying microbial traits that contribute to host adaptation.

Phenotypic parallelism during experimental adaptation of a free-living bacterium to the zebrafish gut

“…Another option is that after bacterial cells migrate into the digestive tract, motility could help bacteria resist expulsion induced by intestinal contractions [39]. Ultimately, our results add to a growing body of evidence implicating motility as an important trait for host colonization [14, 17, 29, 59], and the phenotypic parallelism we observed suggests that traits associated with dispersal can play a critical role in the establishment of host-microbe symbioses [60].…”

“…Since Robinson and colleagues previously demonstrated that serially passaged Aeromonas populations, which were more motile than an unpassaged ancestor, were able to better colonize GF larval zebrafish [29], we hypothesized that MR-1 could similarly improve host colonization in our study via enhanced motility. A common assumption in microbiology is that there is a trade-off between adherence and motility [42–45], wherein cells that tend to be more adherent also tend to be less motile and vice versa.…”

“…Alternatively, once an evolved MR-1 cell encounters a host, faster swimming speeds could help strains traverse narrow junctions within the host to reach the gut more quickly than their ancestral competitor [14]. However, prior work showed no fitness advantage for a hypermotile Aeromonas strain gavaged into the oral cavity of larval zebrafish, suggesting this mode of fitness enhancement may be less likely [29]. Another option is that after bacterial cells migrate into the digestive tract, motility could help bacteria resist expulsion induced by intestinal contractions [39].…”

“…Our experimental evolution serial passage scheme was similar to the one outlined by Robinson et al 2018. 5 mL overnight tryptic soy broth (TSB) cultures of MR-1 isolates tagged with either green fluorescent protein (MR-1gfp) or dTomato fluorescent protein (MR-1dT) were diluted 1:100 in TSB and allowed to grow out to late log phase (4-5 hours).…”

“…Many insights have been gained from employing this type of approach in vitro , and experimental evolution practitioners have begun expanding into in vivo environments to investigate evolutionary dynamics within hosts [24–28]. A recent example of this was conducted by Robinson and colleagues who experimentally adapted an Aeromonas strain previously isolated from zebrafish ( Danio rerio ) to germ free (GF) larval zebrafish to explore how this bacterium might increase its association with its vertebrate host [29]. They found that evolved Aeromonas isolates achieved higher relative abundances in the digestive tracts of larvae during competition against their ancestral strain.…”