Recurrent Reverse Evolution Maintains Polymorphism after Strong Bottlenecks in Commensal Gut Bacteria.

The evolution of new strains within the gut ecosystem is poorly understood. We used a natural but controlled system to follow the emergence of intraspecies diversity of commensal Escherichia coli, during three rounds of adaptation to the mouse gut (∼1,300 generations). We previously showed that, in the first round, a strongly beneficial phenotype (loss-of-function for galactitol consumption; gat-negative) spread to >90% frequency in all colonized mice. Here, we show that this loss-of-function is repeatedly reversed when a gat-negative clone colonizes new mice. The regain of function occurs via compensatory mutation and reversion, the latter leaving no trace of past adaptation. We further show that loss-of-function adaptive mutants reevolve, after colonization with an evolved gat-positive clone. Thus, even under strong bottlenecks a regime of strong-mutation-strong-selection dominates adaptation. Coupling experiments and modeling, we establish that reverse evolution recurrently generates two coexisting phenotypes within the microbiota that can or not consume galactitol (gat-positive and gat-negative, respectively). Although the abundance of the dominant strain, the gat-negative, depends on the microbiota composition, gat-positive abundance is independent of the microbiota composition and can be precisely manipulated by supplementing the diet with galactitol. These results show that a specific diet is able to change the abundance of specific strains. Importantly, we find polymorphism for these phenotypes in indigenous Enterobacteria of mice and man. Our results demonstrate that natural selection can greatly overwhelm genetic drift at structuring the strain diversity of gut commensals and that competition for limiting resources may be a key mechanism for maintaining polymorphism in the gut.

A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut.

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10-5. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.

Enhanced Survival of Rifampin- and Streptomycin-Resistant Escherichia coli Inside Macrophages.

The evolution of multiple-antibiotic-resistant bacteria is an increasing global problem. Even though mutations causing resistance usually incur a fitness cost in the absence of antibiotics, the magnitude of such costs varies across environments and genomic backgrounds. We studied how the combination of mutations that confer resistance to rifampin (Rif(r)) and streptomycin (Str(r)) affects the fitness of Escherichia coli when it interacts with cells from the immune system, i.e., macrophages (Mϕs). We found that 13 Rif(r) Str(r) doubly resistant genotypes, of the 16 tested, show a survival advantage inside Mϕs, indicating that double resistance can be highly beneficial in this environment. Our results suggest that there are multiple paths to acquire multiple-drug resistance in this context, i.e., if a clone carrying Rif(r) allele H526 or S531 acquires a second mutation conferring Str(r), the resulting double mutant has a high probability of showing increased survival inside Mϕs. On the other hand, we found two cases of sign epistasis between mutations, leading to a significant decrease in bacterial survival. Remarkably, infection of Mϕs with one of these combinations, K88R+H526Y, resulted in an altered pattern of gene expression in the infected Mϕs. This indicates that the fitness effects of resistance may depend on the pattern of gene expression of infected host cells. Notwithstanding the benefits of resistance found inside Mϕs, the Rif(r) Str(r) mutants have massive fitness costs when the bacteria divide outside Mϕs, indicating that the maintenance of double resistance may depend on the time spent within and outside phagocytic cells.