Macrophage adaptation leads to parallel evolution of genetically diverse Escherichia coli small‐colony variants with increased fitness in vivo and antibiotic collateral sensitivity

Abstract

Small‐colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole‐genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP) and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron‐transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and showed increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host‐microbe interactions.

Adaptation Through Lifestyle Switching Sculpts the Fitness Landscape of Evolving Populations: Implications for the Selection of Drug-Resistant Bacteria at Low Drug Pressures

“…SCVs of bacteria such as S. aureus (von Eiff 2008), E. coli (Negishi et al 2018) and Pseudomonas aeruginosa (Malone 2015) have also been isolated from patient samples, and, hence, represent a clinically relevant phenotype of bacterial pathogens. SCV formation has typically been associated with mutations that lower metabolic activity in bacteria conferring resistance to antibiotics as a consequence (Pränting and Andersson 2011;Proctor et al 2014;Ramiro et al 2016;Santos and Hirshfield 2016). In this study, however, two features of the SCVs isolated from alternate-low lineages set them apart from earlier reports of SCVs.…”

“…E. coli SCVs are reported to have low growth rates, which allow them to resist killing by antibiotics (Ramiro et al 2016;Santos and Hirshfield 2016). In this case, however, there was no appreciable change in the growth rates of the SCVs relative to the wild type (Figure 3b and Figure S4).…”

“…Indeed, SCVs isolated by us grew to higher density than wild type, which indicates that respiratory pathways, which are normally implicated in SCV formation (Pränting and Andersson 2011;Proctor et al 2014;Ramiro et al 2016;Santos and Hirshfield 2016), were unaffected. Second, genome sequencing did not reveal any of the commonly encountered mutations in respiratory enzymes found in SCVs documented thus far (Ramiro et al 2016;Santos and Hirshfield 2016). Thus, our study reiterates the relatively less-appreciated impact of fimbriation on SCV formation in E. coli.…”

“…Further, the small-colony phenotype was enriched over passages (Figure 2, b and c) and undetectable in control lineages ( Figure S2). Formation of SCVs is triggered in bacteria by environmental stresses, including antibiotics (Wei et al 2011;Frenzel et al 2015;Ramiro et al 2016;Tuchscherr et al 2016). Hence, we hypothesized that SCV formation in the alternate-low lineages may allow adaptation to lowdrug pressure without acquisition of costly drug-resistance Figure 3 Small colony phenotype is associated with an increase in competitive fitness without conferring rifampicin resistance.…”

From the Past to the Future: Considering the Value and Limits of Evolutionary Prediction

“…The overall change in mean fitness also depends on genetic variation in sensitivity to such environmental change (i.e., the interaction between genotype and temporally varying environment), which signifies temporal change in genetic selection, whether this change is erratic or due to more regularly fluctuating selection. Moreover, even in experimentally controlled laboratory environments, rates of change in mean fitness can vary substantially among replicate microbial populations subject to the same selection regime (e.g., Travisano et al 1995;Ramiro et al 2016), although it is important to note that these studies typically span 1,000 generations or many more. The match between realized and predicted change in mean fitness for wild populations-and explanations for deviations between them-are empirical questions whose answers can come only from direct experimental study.…”