Reversion to sensitivity explains limited transmission of resistance in a hospital pathogen

Reviewer #1 (Public review):

Summary:

Tracy and colleagues study the loss of daptomycin resistance in Enterococcus faecium isolates from bloodstream infections using in vitro evolution experiments in the absence of antibiotics. They test the hypothesis that antibiotic resistance arising de novo during treatment will carry a higher fitness cost and will revert more readily than resistance isolates which have been transmitted and have therefore already survived in the absence of antibiotic selection pressure.

Strengths:

This is an important question as a fitness cost to resistance is typically found in lab evolution experiments and assumed in modelling studies, but often not identified in clinical isolates. Here the authors find examples of clinical isolates which do and don't revert to sensitivity in in vitro evolution in the absence of antibiotics. Sequencing of the lab evolved isolates revealed that reversal of resistance was often due to mutations in the same gene that evolved in vivo, which is nice evidence that these resistance mutations did confer a fitness cost.

Weaknesses:

Although this is an interesting study on an important topic, currently the results are overinterpreted do not justify the title of the paper 'Reversion to sensitivity explains limited transmission of resistance in a hospital pathogen' for several reasons. Firstly, the patient group, e.g. 'putatively transmitted' isolates vs 'de novo' isolates was not a significant predictor of change in MIC. Instead the change in MIC in the absence of antibiotics was significantly associated with the starting MIC of the isolate in the evolution experiments, but this would be expected since isolates with a higher MIC have more potential to decrease in MIC in the evolution experiments. The abstract and some conclusion do not match the results in some instances, for example the abstract states 'resistance that arose de novo within patients was higher level but exhibited greater declines in resistance in vitro'. In the discussion: they state "these findings support our hypothesis that transmitted resistance strains are less likely to revert". However, on page 14 the initial MICs between DNR and PTR were not significantly different and patient group was not a significant predictor of change in MIC. Sequencing of the lab evolved isolates revealed that reversal of resistance was often due to mutations in the same gene that evolved in vivo. However, there were also some example of mutations in the same genes within the PTR isolates, so it remains unclear if there is a significant difference in behaviour between the DNR and PTR isolates in terms of reversion mutations. Significance testing, controlling for the starting MIC, would help confirm this.

Secondly, the 'putatively transmitted isolates', i.e. isolates that were resistant in the first positive blood culture, do not necessarily represent resistant isolates that have been transmitted between hosts. E. faecium is primarily a commensal of the intestinal tract, but which can cause opportunistic extra-intestinal infections. These bacteremia cases were most likely caused by within-host translocation of a strain already colonizing the intestine to the bloodstream - indeed, it has been shown that antibiotics can lead to Enterococcus overgrowth in the intestine and subsequent bloodstream invasion (DOI: 10.1172/JCI43918). The 'putatively transmitted isolates' may have initially colonised the intestine via between host transmission in an already resistant state, as assumed by the authors, but they may also have evolved resistance de novo within the host's intestine prior to causing bloodstream infections. Since they do not have data on past daptomycin exposure in these individuals it cannot be assumed that these isolates were transmitted with high resistance between hosts. An alternative explanation for any differences between the 'de novo' and 'putatively transmitted' could be the environment where resistance evolved, e.g. the intestine with strong competition from other strains and species, or within the otherwise sterile bloodstream environment. The authors hypothesise that "newly resistant population must continue to transmit between hosts in antibiotic free conditions to ensure its survival" and that "transmission acts as a filter to select for resistance with a lower cost or lower chance of reversion". Rather than transmission per se, it is equally plausible that survival of the newly resistant population within the primary niche, the intestinal microbiota, is the crucial to filter for resistance with a lower cost.

Reviewer #2 (Public review):

Summary:

In this study, the authors combine the study of clinical samples of antibiotic resistant bacteria with experimental evolution and evolutionary genomics to address important questions about the propensity for reversion in two different schema: de novo resistance arising within a patient, and transmitted resistance. The authors' use of a combination of methods help to answer the question outlined in their hypothesis, that de novo resistance mechanisms appear to revert to sensitive phenotypes more readily in a drug-free environment.

Strengths:

This study is exceptionally well-written and organized. The authors state their hypothesis clearly, and follow it up with an impressive effort that is truly translational-they make direct use of clinical samples of bacteria, and combine that with approaches in experimental evolution and evolutionary genomics. The conclusions follow naturally from the results, and there are no irresponsible leaps made.

Weaknesses:

I will divide my criticism into two areas, conceptual (most of my critique), with a very small methodological question.

(1) In the end, the authors offer findings that appear to be correct, and (again) are reported very clearly. However, this study is very surface-level in its theoretical underpinnings and construction, which is puzzling, because the field of antibiotic resistance and adaptation more broadly, is full of relevant studies and explanatory tools. Below I'll identify several areas where this manifests.

For one, the authors do not engage with a large recent literature on reversion, reversal, and compensation. It provides much more conceptual grounding for what the authors observe, much of it compatible with the findings from this study:

To offer two quick examples:
- Avrani S, Katz S, Hershberg R. Adaptations accumulated under prolonged resource exhaustion are highly transient. MSphere. 2020 Aug 26;5(4):10-128.
- Pennings, P.S., Ogbunugafor, C.B. and Hershberg, R., 2022. Reversion is most likely under high mutation supply when compensatory mutations do not fully restore fitness costs. G3, 12(9), p.jkac190.

Examinations of the studies on adaptation and reversion offer a richer mechanistic take on what was observed. But this literature alone is less of a problem than the general offering of different takes for the results. One can turn to a different literature - from ecology - to find a different explanation that is compatible with the findings.

De novo evolution involves the strong selection and rapid fixation of populations that are evolving largely to a relatively simple ecological milieu: their only primary function is to promote replication and survival of populations experiencing the negative fitness effects of drug pressure. Alternatively, transmitted resistant populations must deal with a multitude of selective pressures, working dynamically across time and space. In such a scenario, one would expect populations to locate places on the fitness landscape that are commensurate with survival in both drug-poor and drug-rich environments, as this is the ecological reality of the transmitted resistant bacteria. I could envision selection for "generalism" in this setting, corresponding to populations that have fixed mutations that promote resistance, but also those that ensure replication in drug-free environments. This regime might even reflect selection for "generalism" or "increased niche breadth." That is, transmitted resistance may have adopted a "jack of all trades, master of none" phenotype. The de novo resistance strains, alternatively, are selected for "generalism."

See the following for examples (there are many):

- Kassen R. The experimental evolution of specialists, generalists, and the maintenance of diversity. Journal of evolutionary biology. 2002 Mar 1;15(2):173-90.
- Bell TH, Bell T. Many roads to bacterial generalism. FEMS microbiology ecology. 2021 Jan;97(1):fiaa240.

Note that this classically ecological explanation is only one of several other literatures that offer models for the findings in this study.

To the authors' credit, their study was about the very real-world problem of antibiotic resistance, using a system that is far less tractable than the model systems research that has generated a lot of data and theory. And sure: the study is valuable because it communicates an interesting finding using a combination of methods (impressively). But in some ways, the study almost reads as a descriptive exercise: it offers a good question (does de novo or transmitted resistance revert more readily), and tells you what they found (de novo does). However the explanatory mechanisms do not advance our understanding much. Reporting the presence of unstable and disruptive mutations in the de novo populations is hardly an explanation. That is, alternatively, data in support of a proper explanation. There is nothing magical about de novo evolution that should be selected for disruptive mutations.

The reasons for the different sorts of mutation could have to do with the population genetic particulars of the de novo regime: large populations, strong selective pressure, relatively static fitness landscape. In such a setting, selection marches a population greedily up a peak. Alternatively, a transmitted population arises from a lineage that has observed a multitude of ecologies, across different fitness landscapes and has fixed mutations that confer survival across all of them.

There's a literature that speaks to this:
- Miller CM, Draghi JA. Range expansion can promote the evolution of plastic generalism in coarse-grained landscapes. Evolution Letters. 2024 Apr 1;8(2):322-30.
- Bono LM, Draghi JA, Turner PE. Evolvability costs of niche expansion. Trends in genetics. 2020 Jan 1;36(1):14-23.

The findings are simple enough (a testament to the strong study design and execution) that supporting population genetic simulations, or analytical descriptions (maybe not relevant) could offer insight as to what really happened here.

(2) I recognize the challenge of working with clinical samples. It is very difficult to understand everything about them. But even having considered that, I might be missing something.

My main question here involves the origin of the putatively transmitted strains. The authors state that " Isolates were also obtained from six patients with a putatively transmitted resistant bacteria (hereafter PT), where a daptomycin-resistant, E. faecium bacteremia was identified on their first culture."

This seems like an awfully dubious way to identify transmitted resistance. I suppose I understand the logic (de novo evolution requires the observer to have seen the evolution happen in real-time). But this definition leaves the study wide open for an "apples to oranges" comparison that might render the other aspects questionable.

The de novo strains are being compared to transmitted strains that may have been part of lineages that had passed between many, many patients. If this were true, then we should expect the genomic architecture of the transmitted strains to be far different. The transmitted strains might have undergone more selection in different regimes and genetic drift. Drift might have fixed mutations in transmission bottlenecks, altering the genetic architecture. In such a scenario, one might expect these populations to have a more difficult time unwinding their resistance phenotype.

In the end, I applaud the authors on a well-done and well-written study.