1. Evolutionary Biology
Download icon

High potency of sequential therapy with only β-lactam antibiotics

  1. Aditi Batra
  2. Roderich Roemhild
  3. Emilie Rousseau
  4. Sören Franzenburg
  5. Stefan Niemann
  6. Hinrich Schulenburg  Is a corresponding author
  1. Department of Evolutionary Ecology and Genetics, University of Kiel, Germany
  2. Max Planck Institute for Evolutionary Biology, Germany
  3. Institute of Science and Technology, Austria
  4. Borstel Research Centre, National Reference Center for Mycobacteria, Germany
  5. Competence Centre for Genomic Analysis Kiel, University of Kiel, Germany
Research Article
  • Cited 1
  • Views 1,365
  • Annotations
Cite this article as: eLife 2021;10:e68876 doi: 10.7554/eLife.68876

Abstract

Evolutionary adaptation is a major source of antibiotic resistance in bacterial pathogens. Evolution-informed therapy aims to constrain resistance by accounting for bacterial evolvability. Sequential treatments with antibiotics that target different bacterial processes were previously shown to limit adaptation through genetic resistance trade-offs and negative hysteresis. Treatment with homogeneous sets of antibiotics is generally viewed to be disadvantageous as it should rapidly lead to cross-resistance. We here challenged this assumption by determining the evolutionary response of Pseudomonas aeruginosa to experimental sequential treatments involving both heterogenous and homogeneous antibiotic sets. To our surprise, we found that fast switching between only β-lactam antibiotics resulted in increased extinction of bacterial populations. We demonstrate that extinction is favored by low rates of spontaneous resistance emergence and low levels of spontaneous cross-resistance among the antibiotics in sequence. The uncovered principles may help to guide the optimized use of available antibiotics in highly potent, evolution-informed treatment designs.

eLife digest

Overuse of antibiotic drugs is leading to the appearance of antibiotic-resistant bacteria; this is, bacteria with mutations that allow them to survive treatment with specific antibiotics. This has made some bacterial infections difficult or impossible to treat. Learning more about how bacteria evolve resistance to antibiotics could help scientists find ways to prevent it and develop more effective treatments.

Changing antibiotics frequently may be one way to prevent bacteria from evolving resistance. That way if a bacterium acquires mutations that allow it to escape one antibiotic, another antibiotic will kill it, stopping it from dividing and preventing the appearance of descendants with resistance to several antibiotics. In order to use this approach, testing is needed to find the best sequences of antibiotics to apply and the optimal timings of treatment.

To find out more, Batra, Roemhild et al. grew bacteria in the laboratory and exposed them to different sequences of antibiotics, switching antibiotics at different time intervals. This showed that sequential treatments with different antibiotics can limit bacterial evolution, especially when antibiotics are switched quickly. Unexpectedly, one of the most effective sequences used very similar antibiotics. This was surprising because using similar antibiotics should lead to the evolution of cross-resistance, which is when a drug causes changes that make the bacterium less sensitive to other treatments. However, in the tested case, cross-resistance did not evolve when antibiotics were switched quickly, thereby ensuring efficiency of treatment.

Batra et al. show that alternating sequences of antibiotics may be an effective strategy to prevent drug resistance. Because the experiments were done in a laboratory setting it will be important to verify the results in studies in animals and humans before the approach can be used in medical or veterinary settings. If the results are confirmed, it could reduce the need to develop new antibiotics, which is expensive and time consuming.

Introduction

The efficacy of antibiotics for the treatment of infections is diminishing rapidly as bacteria evolve new mechanisms to resist antibiotics (Laxminarayan et al., 2013). Resistance evolution is frequently observed during antibiotic therapy and can happen within days (Bloemberg et al., 2015; Hjort et al., 2020; Tueffers et al., 2019). A failure to account for such rapid bacterial adaptation is likely a common reason for treatment failure (Woods and Read, 2015; Zhou et al., 2020). For this reason, the field of evolutionary medicine specifically accounts for bacterial evolvability and seeks treatment solutions that are hard to overcome by genetic adaptation (Andersson et al., 2020; Merker et al., 2020). While an evolution-proof antibiotic remains to be found, the mechanisms that restrict evolutionary escape are starting to be revealed (Bell and MacLean, 2018). Such evolutionary insight may guide the design of effective and sustainable antibiotic therapy.

An effective way of reducing the amount of evolutionary solutions is to administer several antibiotics either simultaneously (i.e., combination therapy) or sequentially (i.e., sequential therapy). Tailored combination treatments make use of physiological and evolutionary constraints (Baym et al., 2016). The emergence of resistance is delayed by combinations, when evolutionary escape requires multiple mutations and when drug interactions eliminate the intermediate genetic steps of single-drug resistance (Chait et al., 2007), antibiotic tolerance (Levin-Reisman et al., 2017), and heteroresistance (Band et al., 2019). However, when genetic resistance to the combination is easily accessible, for example, through gene amplification of efflux pumps, then combination therapy can accelerate resistance emergence (Pena-Miller et al., 2013). This undesired selective effect is potentially avoided by sequential drug application. Evolutionary escape from sequential treatments is constrained by negative hysteresis responses induced by specific antibiotics (Roemhild et al., 2018) and/or the emergence of genetic collateral sensitivity trade-offs (Barbosa et al., 2019; Yoshida et al., 2017). Negative hysteresis occurs when exposure to an antibiotic induces changes to bacterial physiology that transiently increase the killing efficacy of other antibiotics (Roemhild et al., 2018). Collateral sensitivity is a genetic side effect of evolved resistance that too increases the efficacy of other antibiotics (Szybalski and Bryson, 1952). Collateral sensitivity is prevalent among pathogens and occurs especially between antibiotics with distinct mechanism of action (i.e., heterogeneous sets of antibiotics), while cross-resistance often emerges towards antibiotics with similar mode of action (i.e., homogeneous sets of antibiotics) (Barbosa et al., 2017; Imamovic and Sommer, 2013; Lázár et al., 2013; Maltas and Wood, 2019). Thus, conventionally, multidrug treatments would avoid antibiotics from similar classes, with the rationale of limiting the overlap in the respective sets of resistance mutations, and thus the ensuing cross-resistance.

The particular efficacy of sequential therapy has been confirmed with the help of evolution experiments under controlled laboratory conditions. Different types of sequential treatments have been tested. Some regimens involved a single switch between antibiotics, while others included multiple switches at short time intervals. One of the main findings was that the efficacy of sequential treatments depended both on the included antibiotics and the particular treatment sequence (Fuentes-Hernandez et al., 2015; Maltas and Wood, 2019; Roemhild et al., 2015). While fast sequential treatments did not exclude the eventual emergence of multidrug resistance, many significantly delayed bacterial adaptation compared to monotherapy (Kim et al., 2014; Roemhild et al., 2015; Yoshida et al., 2017). A single antibiotic switch can also delay adaptation, dependent on the drug order, and it can additionally reverse previous resistance and resensitize bacterial populations to specific antibiotics (Barbosa et al., 2019; Hernando-Amado et al., 2020; Imamovic and Sommer, 2013; Yen and Papin, 2017). Moreover, our group previously demonstrated that fast sequential treatments with a heterogeneous set of three antibiotics – the fluoroquinolone ciprofloxacin (CIP), the β-lactam carbenicillin (CAR), and the aminoglycoside gentamicin (GEN) – delayed the emergence of multidrug resistance in the pathogen Pseudomonas aeruginosa (Roemhild et al., 2018). The observed inhibition of evolutionary escape was manifested by the occurrence of population extinction, although antibiotic concentrations were below the minimal inhibitory concentration (MIC). We further found that negative hysteresis at antibiotic switches reduced adaptation rates because it selected for distinct genetic changes. Several populations adapted to fast sequential treatment by independent mutations in the histidine kinase cpxS that only mildly increased resistance, thereby explaining the low rate of adaptation to the used antibiotics. Instead, the cpxS mutations suppressed negative hysteresis, demonstrating that adaptation was specific to the selective constraint imposed by the drug switches. Based on these findings, we assumed that the acting selective dynamics were ultimately a consequence of antibiotic heterogeneity. However, is this so? Do selective dynamics differ for a homogenous set of drugs?

The primary aim of our current study was to assess the efficacy of sequential treatments with either heterogeneous or homogeneous sets of three antibiotics. We focused on P. aeruginosa strain PA14 as a tractable pathogen model system, for which comprehensive experimental reference data is available on resistance evolution (e.g., Barbosa et al., 2019; Barbosa et al., 2018; Hernando-Amado et al., 2020; Roemhild et al., 2018; Sanz-García et al., 2018; Yen and Papin, 2017). We performed similar evolution experiments as before, with three new sets of bactericidal antibiotics, two of which included only β-lactams, and one the three previously considered modes of action (Figure 1—figure supplement 1A). The new heterogeneous drug set CIP, streptomycin (STR), and doripenem (DOR) involved drug synergy and was expected to contribute to collateral sensitivity (Barbosa et al., 2018; Barbosa et al., 2017). The drug sets comprising three β-lactams, however, had all properties that would typically be avoided for the design of multidrug treatments. The three β-lactams CAR, cefsulodin (CEF), and DOR have the same core structure and individually inhibit the DD-transpeptidase activity in cell-wall synthesis (Walsh, 2003). The collateral effects landscape between CAR-CEF-DOR was expected to be dominated by cross-resistance (Barbosa et al., 2017) and the three antibiotics showed neither synergy nor antagonism (Barbosa et al., 2018). Resistance to these antibiotics may potentially be achieved through single mutations. The situation is replicated by the set of ticarcillin (TIC), azlocillin (AZL), and ceftazidime (CEZ). In contrast to expectations, the triple β-lactam sequences showed high treatment potency. Therefore, the secondary aim of our study was to assess which characteristics constrained the ability of the bacteria to adapt to the β-lactam sequential treatments. We focused on one triple β-lactam set (CAR-CEF-DOR) and specifically tested the influence of antibiotic switching rate, switching regularity, negative hysteresis, the potential for spontaneous resistance evolution, and resulting cross-resistances on treatment efficacy.

Results

Triple β-lactam sequential treatments favor extinction of bacterial populations

We challenged a total of 756 replicate P. aeruginosa populations with sequential treatments across three fully independent evolution experiments, each focused on a different set of three antibiotics (Figure 1, Figure 1—figure supplement 1, Supplementary file 1A, Materials and methods). The antibiotic concentrations were calibrated to an inhibitory concentration of 75% (IC75), allowing bacteria to adapt to the imposed selection pressure. We used a serial dilution protocol for experimental evolution, with 2% culture transfer after 12 hr (one transfer) across a total of 96 transfers, equivalent to approximately 500 bacterial generations. Following the previous setup (Roemhild et al., 2018), we recorded the evolutionary dynamics in response to 16 different treatments, belonging to four main treatment types: monotherapy, fast-regular, slow-regular, and random sequential therapy (Figure 1).

Figure 1 with 1 supplement see all
Probability of evolutionary rescue depends on drug triplets and treatment type.

(A) The evaluated antibiotic combinations comprise different types of antibiotic targets. Fluoroquinolone antibiotics (FQ) target DNA gyrase, aminoglycosides (AG) inhibit translation, and β-lactams (BL) inhibit cell-wall synthesis. (B) The evaluated treatment protocols test the effects of switching rate and temporal regularity. (C) A fraction of lineages is eradicated by the sublethal dosage sequential treatments. Lineage extinction is high for combinations of cell-wall targeting β-lactams. (D) Variation in extinction for the β-lactam combinations by treatment type (n = 3–6 protocols per treatment type). (E) The distribution of evolutionary trajectories for Exp. 3 with CAR-DOR-CEF shows that the majority of extinction events occur within the first 12 serial transfers (n = 180 lineages). Growth of evolving lineages is quantified relative to untreated reference populations using the relative area under the growth curve (AUC). AZL: azlocillin; CAR: carbenicillin; CEF: cefsulodin; CEZ: ceftazidime; CIP: ciprofloxacin; DOR: doripenem; GEN: gentamicin; STR: streptomycin; TIC: ticarcillin. The following supplementary material is available for Figure 1: Figure 1—figure supplement 1, Figure 1—source data 1, Figure 1—figure supplement 1—source data 1, Supplementary file 1A.

Extinction of experimental populations differed considerably between the antibiotic sets. The two β-lactam sets produced a surprisingly high degree of extinction (CAR-CEF-DOR and TIC-AZL-CEZ; extinct fraction 27.2 and 13.3%, respectively, Figure 1C). The observed extinction frequency was comparable to that observed in the previous experiment with CAR-CIP-GEN (extinct fraction 15%, Figure 1C). CIP-DOR-STR caused no extinction, indicating that extinction was not explained by applying heterogeneous sets of antibiotics. Within the β-lactam sequential treatments, we observed that treatments that switched between antibiotics fast (every transfer) produced much higher extinction levels than those that switched slowly (every four transfers) or not at all (Figure 1D). Most of the extinction events happened early in the experiment (Figure 1E), indicating that the initial treatment steps are critical for adaptation of populations. We conclude that fast sequential β-lactam treatments showed a surprising ability to restrict bacterial adaptation. As this result was unexpected, we decided to research the mechanisms that constrain resistance emergence in β-lactam sequences. Given that the experiment involving CAR-CEF-DOR produced the highest fraction of extinct populations, we decided to focus further analyses on this set.

Resistance to doripenem was constrained in both monotherapy and switching treatments in the CAR-CEF-DOR triple β-lactam experiment

The CAR-CEF-DOR triple β-lactam experiment was characterized in detail for changes in growth, evolved resistance, and whole-genome sequences in order to assess the selection dynamics involved. We calculated the relative growth yield (see Materials and methods) at the end of each transfer and found growth dynamics to be divided into three phases: an early phase of rapid adaptation (transfers 1–12), followed by a phase of gradual growth yield convergence (transfers 13–48), and a final plateau phase (transfers 49–96) (Figure 2A; the growth phases are separated by vertical dotted lines). We compared the main treatment types using general linear models (GLM) for each phase separately (this fulfills the model assumption of response linearity). The early phase dynamics were characterized by significantly decelerated adaptation dynamics of the fast-regular group compared with monotherapy and slow-regular (GLM, post hoc test, p<0.037, Supplementary file 1B), but not random treatments. The slow-regular treatment did not differ significantly from monotherapy or random treatments (GLM, post hoc test, p=0.469, Supplementary file 1B). In the subsequent phase, growth yields of the groups converged to a plateau of roughly 90% relative yield, indicating similar final levels of adaptation (the growth yields of main treatment groups showed no statistical differences in phases 2 and 3, Supplementary file 1B). Alternating between the β-lactams fast and in a regular order therefore significantly constrained the growth of the bacterial populations. Intriguingly, in these fast sequential treatments, bacterial growth in the transfers with DOR was lower than in the transfers with the other two antibiotics (Figure 2—figure supplement 1), indicating an evolutionary constraint associated with the antibiotic DOR. We can rule out the alternative hypotheses that the reduced growth is explained by a stronger initial reduction in bacterial population size by DOR in comparison to the other two drugs or increased stochastic variation in dosage effects. All treatments were initiated using specifically standardized IC75 dosage (see Materials and methods) and at the IC75, DOR showed very little variation (Figure 1—figure supplement 1). We thus hypothesize that the observed evolutionary constraint may be due to lower rate of DOR resistance emergence.

Figure 2 with 5 supplements see all
Resistance to doripenem is constrained in the CAR-CEF-DOR triple β-lactam experiment.

(A) Rapid adaptive increase of biomass yields relative to the untreated reference populations (mean ± CI95; n = 3–6 protocols per treatment type and 12 biological replicates per sequence; extinct lineages excluded). Vertical dotted lines separate the three growth phases. Evolved changes in the susceptibility to the treatment antibiotics CAR, DOR, and CEF and the non-treatment antibiotics CIP and GEN after transfer 12 (B) or transfer 48 (C), evaluated with 20 isolates each for the 16 representative adapting populations at each time point. Mono 1 is monotherapy with CAR, mono 2 is monotherapy with DOR, and mono 3 is monotherapy with CEF. The evolution of resistance and hypersensitivity is indicated by red and blue colors, respectively, given for the considered isolates as horizontal lines (total of 640 isolates), sorted according to evolution treatment (main rows in the figures) and tested antibiotics (main columns; antibiotics given at the bottom). Pie charts on the right show phenotypic within-population diversity, where different colors indicate subpopulations inferred from hierarchical clustering of resistance phenotypes. CAR: carbenicillin; CEF: cefsulodin; CIP: ciprofloxacin; DOR: doripenem; GEN: gentamicin. The following supplementary material is available for Figure 2: Figure 2—figure supplement 1, Figure 2—figure supplement 2, Figure 2—figure supplement 3, Figure 2—figure supplement 4, Figure 2—figure supplement 5, Figure 2—source data 1, Figure 2—figure supplement 1—source data 1, Figure 2—figure supplement 2—source data 1, Figure 2—figure supplement 3—source data 1, Figure 2—figure supplement 5—source data 1, Supplementary file 1B.

To understand the dynamics of early adaptation in more detail, we measured the resistance profiles of 16 evolved populations after transfers 12 and 48 from the different antibiotic treatments (representing the end of phases 1 and 2, respectively; Figure 2B, C, Figure 2—figure supplements 25, Supplementary file 1B–F; see Materials and methods). We randomly sampled 20 bacterial colonies from each population and characterized their resistance profile by broth microdilution. Resistance was measured for the three antibiotics of the evolution experiment and two additional clinically relevant antibiotics from different classes, ciprofloxacin and gentamicin. The resistance profiles in the early and the mid phases were found to be distinctly different. Resistance to the used β-lactams increased across the two time points only in some treatments, but not all (Figure 2B, C, Figure 2—figure supplement 4, Figure 2—figure supplement 5, Supplementary file 1F), suggesting treatment-dependent evolutionary responses to the antibiotics. We assessed how the main treatment types varied in their β-lactam resistance using a GLM for each phase separately. Most treatment types varied significantly from each other in their multidrug β-lactam resistance in both phases (Supplementary file 1C, D). The multidrug resistance in the early phase was in most cases constrained by the susceptibility to DOR (e.g., in the switching and monotherapy treatments). We additionally observed collateral responses of the treatment to the two non-β-lactams, which increased over time. We further used hierarchical clustering of the resistance profiles to assess the presence of subpopulations, followed by calculation of Shannon diversity for each population at both transfers. We found population diversity to be significantly higher at transfer 48 as compared to transfer 12 (ANOVA, F = 6.2060, p=0.01893, Supplementary file 1E), indicating a diversification of the evolving lineages over time. Taken together, the population analysis of resistance profiles indicates that resistance evolution depends on the exact treatment protocol and that the dynamics of resistance emergence to DOR may be key for the observed deceleration of β-lactam adaptation in the fast-regular treatments.

To identify the genomic changes underlying the first steps of β-lactam adaptation, we sequenced 33 whole genomes of the evolved and characterized isolates from the monotherapy, fast-regular, and slow-regular treatment types. Specifically, we sequenced three isolates from each population representing the distinct phenotypic subpopulations, assessed above. We found that all isolates, except those that received DOR monotherapy, had mutations in known resistance genes by the end of the early phase (Table 1). This agreed with the inferred resistance profiles where isolates from the DOR monotherapy did not show a noticeable amount of resistance at that stage (Figure 2B). DOR resistance was, however, found at the end of the middle phase (Figure 2C), and this was mirrored in the genomics with a non-synonymous mutation in the gene ftsI. This gene codes for the penicillin binding protein 3 (PBP3) (Liao and Hancock, 1995), a common target of the three β-lactams (Davies et al., 2008; Fontana et al., 2000; Rodriguez‐Tebár et al., 1982; Rodríguez-Tebar et al., 1982; Zimmermann, 1980). ftsI was also found to be mutated in isolates from CAR monotherapy, although at a different site within the gene and associated with a different resistance profile than the DOR-associated ftsI variant (Figure 2B). Isolates from CEF monotherapy contained mutations in pepA. This gene is responsible for the production of a protein required for cytotoxicity and virulence in P. aeruginosa (Hauser et al., 1998). Although its role in antimicrobial resistance remains to be studied in detail, it was previously found to be mutated in P. aeruginosa strains resistant to certain β-lactams (Cabot et al., 2018; Sanz-García et al., 2018). The switching treatments selected for mutations in the above-listed and also in some additional genes. In particular, we identified mutations in nalD and phoQ, a negative regulator of the MexAB-OprM efflux pump and a two-component system, respectively. Mutations in these genes account for resistance to a variety of drugs in P. aeruginosa (Barbosa et al., 2021; Sobel et al., 2005). Further mutations were identified in some non-canonical β-lactam resistance genes such as rmcA, 23srRNA, 3-oxoacyl synthase, dnaX, and zipA (Table 1). Taken together, mutations in both canonical and non-canonical targets of β-lactam selection were identified in our experiment, and among these, DOR resistance mutations were found only later in the experiment, consistent with the obtained resistance profiles (Figure 2B, C).

Table 1
Evolved genetic changes inferred from whole-genome sequencing.
Treatment typeID*AA changeGene nameAnnotationFreq
Monotherapy1V471GftsIPeptidoglycan synthesis3/3
2§N242SftsIPeptidoglycan synthesis3/3
3T157PpepAVirulence3/3
Fast-rgular5V471GftsIPeptidoglycan synthesis3/3
6K26nalDEfflux3/3
S379ISRrmcABiofilm maintenance1/3
7R220CphoQTwo-component3/3
-PA14_5563123srRNA, translation1/3
Slow-rgular8V471GftsIPeptidoglycan synthesis3/3
9D357NpepAVirulence3/3
10T157PpepAVirulence3/3
E115VAAWIPKPA14_21540Lipid metabolism (3-exoacyl ACP synthase)1/3
Q117AEEQPA14_21540Lipid metabolism (3-exoacyl ACP synthase)1/3
R178CzipACell division2/3
P483PEPdnaXCell division1/3
  1. * Individual treatment of evolution experiment.

    Amino acid change.

  2. Occurrence frequency of the identified variant (before slash) out of the total number of isolates sequenced (behind slash).

    § Mutations listed are from isolates obtained from the populations frozen at transfer 48, no variants were found in the isolates from transfer 12.

Table 1—source data 1

Source data for the summary of the genome sequencing analysis shown in Table 1.

https://cdn.elifesciences.org/articles/68876/elife-68876-table1-data1-v2.xlsx

Based on our detailed characterization of the CAR-CEF-DOR triple β-lactam experiment, we conclude that DOR has a key role in restricting evolutionary rescue as evidenced by the delayed acquisition of genetic resistance to it.

Asymmetric bidirectional hysteresis was identified between doripenem and carbenicillin

As extinction was associated with antibiotic switches, we next focused on selective events that can occur at drug switches, such as hysteresis, an inducible physiological change. We characterized the complete hysteresis landscape between the three β-lactams: CAR, DOR, and CEF. We pretreated exponential phase cells with an antibiotic for only 15 min to ensure that cells are physiologically challenged but not subject to differential killing or replication. The pretreatment was followed by a change to fresh medium containing a second antibiotic as main treatment. We included controls of no pretreatment, or no main treatment (Figure 3A). We found that negative hysteresis existed for several switches between the β-lactams (Figure 3B, C, Figure 3—figure supplement 1, Figure 3—figure supplement 2). DOR and CAR displayed asymmetric bidirectional negative hysteresis with the switch from DOR to CAR, resulting in stronger negative hysteresis than the reverse. Negative hysteresis was also observed in the switch from CAR to CEF and CEF to CEF. To our surprise, only a single case of weak positive hysteresis was observed, although we generally anticipated it given that P. aeruginosa produces the AmpC β-lactamase (Livermore, 1995). We conclude that negative hysteresis is abundant between the studied β-lactams and is a potential predictor of treatment potency in the sequential β-lactam treatments.

Figure 3 with 2 supplements see all
Negative hysteresis is common among the tested β-lactam antibiotics.

(A) Hysteresis effects were measured using the previously established experimental approach (see Materials and methods). (B) Bacterial counts were plotted over time after the pretreatment to obtain time-kill curves (mean ± sem, n = 3). Level of hysteresis was quantified as the difference between the antibiotic switch and the only main curves. Negative values indicate negative hysteresis and positive values indicate positive hysteresis. (C) Heatmap of hysteresis levels between all nine combinations of the three β-lactams. DOR and CAR show asymmetric bidirectional negative hysteresis. Negative hysteresis is also observed in switches from CEF to CEF and CAR to CEF. Weak positive hysteresis is found for the switch from CEF to CAR. The following supplementary material is available for Figure 3: Figure 3—figure supplement 1, Figure 3—figure supplement 2, Figure 3—source data 1, Figure 3—figure supplement 1—source data 1. CAR: carbenicillin; CEF: cefsulodin; DOR: doripenem.

Probability of direct and indirect resistance was the least for doripenem

Since resistance to DOR was constrained in both the monotherapy and the switching treatments (Figure 2B), we hypothesized that DOR resistance was difficult to achieve compared to the other two β-lactams. Resistance against a given drug can arise because of spontaneous direct resistance and/or because of collateral resistance from the preceding antibiotics in the sequence. As a first step, we thus measured the spontaneous direct resistance rate with the classic fluctuation assay using identical inhibitory concentrations of the three antibiotics (Luria and Delbrück, 1943; Figure 4A, Supplementary file 1G). To determine the probability of indirect resistance in a second step, we isolated the obtained single-step mutants and quantified the fraction of cross-resistance towards the other two β-lactams with a patching assay (Figure 4A). We used a comparatively large number of spontaneous mutants for this analysis (n = 60 per antibiotic) to capture the stochastic nature of evolution and, in this context, the potential importance of collateral effects for bacterial adaptation, as previously emphasized (Nichol et al., 2019). We found that the spontaneous resistance rate was significantly lower for DOR than for CAR and CEF (likelihood ratio test, p<0.0001 and p<0.01, respectively; Supplementary file 1H, Figure 4B). Moreover, the resulting cross-resistance effects (Figure 4C) were particularly common towards CAR (93% of clones with spontaneous CEF resistance and 71% with DOR resistance) and CEF (73% of originally CAR-resistant clones and 67% DOR-resistant clones). By contrast, the smallest levels of cross-resistance were expressed towards DOR (36% of originally CAR-resistant clones and 50% CEF-resistant clones). The overall fraction of cross-resistant clones was significantly smaller towards DOR than either CEF or CAR (Fisher's exact test, p<0.0004; Supplementary file 1I). We conclude that of the three β-lactams DOR had the lowest probability for both direct and indirect resistance, thereby providing experimental support to the indication of constrained DOR resistance evolution obtained from the detailed phenotypic and genomic characterization of the evolved bacteria (Figure 2, Table 1).

Doripenem has the lowest rates of direct and indirect resistance.

(A) Schematic of the experimental protocol to determine spontaneous rates of resistance on each of the three β-lactams and the resulting collateral landscape. Briefly, an overnight culture was taken and split into 30 parallel cultures where bacteria were allowed to divide in the absence of an antibiotic and any other constraint. Spontaneous resistant mutants were selected on minimal inhibitory concentration (MIC) plates and restreaked to ensure genetic resistance. These mutants were then patched on MIC plates of the other two β-lactams to test for cross-resistance. (B) Comparison of rates of spontaneous resistance on the three β-lactams on a Log10 scale. Error bars depict CI95. All comparisons were found to be significantly different from each other (likelihood ratio test; CAR vs. CEF p<0.0001, CAR vs. DOR p<0.0001, and DOR vs. CEF p<0.01). (C) Landscape of collateral effects between the three β-lactams. Fraction of cross-resistant mutants per antibiotic combination is plotted. DOR has the least cases of cross-resistance of the three. A total of 60 mutants per antibiotic were used for collateral effect testing. The following supplementary material is available for Figure 4: Figure 4—source data 1, Supplementary file 1G–I. CAR: carbenicillin; CEF: cefsulodin; DOR: doripenem.

The rate of spontaneous resistance and resulting cross-resistance determine treatment efficacy

We used the collected information to identify the critical determinant(s) of treatment efficacy in the CAR-CEF-DOR triple β-lactam experiment. We assessed the influence of either the two experimental predictors (switching rate, temporal irregularity) or the three biological predictors (hysteresis, probability of spontaneous resistance, and resulting cross-resistance) on each of the evolutionary responses extinction, rate of growth adaptation, and multidrug resistance, using separate GLM-based analyses (see Materials and methods; Supplementary file 1J–O). For the biological predictors, we calculated the levels of cumulative hysteresis, cumulative probability of spontaneous resistance, and the cumulative levels of cross-resistance in each of the 16 individual treatments up to transfer 12 (see Materials and methods). We focused our analysis on the early phase of evolution up to transfer 12 as it appeared most critical for treatment efficacy, especially for population extinctions that usually occurred early (Figure 1E). Our analysis revealed that extinction was significantly associated with both the experimental predictors, switching rate (GLM, = 14.44, p=0.0042, Figure 5B, Supplementary file 1J–M) and temporal irregularity (GLM, = 10.53, p=0.0101, Supplementary file 1M). Temporal irregularity further showed a statistical trend with multidrug resistance (GLM, = 4.19, p=0.0711, Supplementary file 1M). From our biological predictors, the cumulative cross-resistant fraction showed a significant association with extinction (GLM, = 10.42, p=0.0121, Supplementary file 1O), while cumulative probability of spontaneous resistance showed a statistical trend (GLM, = 4.14, p=0.0763, Supplementary file 1O). Indeed, the cumulative cross-resistant fraction and also the cumulative probability of spontaneous resistance are strongly correlated with extinction (Figure 5B). The cumulative cross-resistant fraction is also strongly correlated with switching rate (Figure 5—figure supplement 1), most likely explaining the latter impact on extinction. By contrast, cumulative hysteresis levels did not have a significant influence on any of the evolutionary responses (GLM, = 0.16, p=0.7015, Supplementary file 1O). Taken together, our results suggest that in our sequential CAR-CEF-DOR treatments the switching rate, temporal irregularity of antibiotics, the probability of spontaneous resistance, and especially the resulting collateral effects (maximized by switching rate) determine treatment efficacy through their effect on bacterial extinction. The limiting factor appears to be constrained evolution of resistance and low levels of cross-resistance to DOR.

Figure 5 with 1 supplement see all
Bacterial extinction is correlated to switching rate, spontaneous rate of resistance, and spontaneous cross-resistance.

(A) Variation in experimental parameters, potential biological predictors, and the measured traits up to transfer 12. The experimental parameters include switching rate and regularity of change (high irregularity in dark). Potential biological predictors are cumulative levels of hysteresis (dark indicates protective effects), cumulative probabilities of spontaneous resistance (Spont. res., dark indicates higher probability), and cumulative level of collateral effects (Cross-res., dark indicates high fraction of cross-resistance). The evolutionary response was measured for population survival (max = 12), adaptation rate (Adapt. rate, n ≤ 12, extinct lineages excluded), evolved multidrug resistance (MDR) to treatment antibiotics CAR, DOR, and CEF (MDR, n = 16). (B) Variation in extinction was best explained by collateral effects between the antibiotics (for illustrative purposes, the red line depicts linear regression and ρS the Spearman’s rank correlation coefficient). The following supplementary material is available for Figure 5: Figure 5—figure supplement 1, Figure 5—source data 1, Supplementary file 1J–O. CAR: carbenicillin; CEF: cefsulodin; DOR: doripenem.

Discussion

Treatment with multiple β-lactam antibiotics is generally avoided due to the perceived fear of therapy failure from cross-resistance. Our work now challenges this widespread belief. We characterized the ability of replicate P. aeruginosa populations to evolve de novo resistance to sequential treatments with different drug sets. To our surprise, we found that sets of three β-lactams constrained bacterial adaptation by reducing bacterial survival. We demonstrate that treatment potency was determined by variation in the spontaneous rate of resistance to the β-lactams and the resulting collateral effects across sequential treatment protocols.

Our initial screen of sequential protocols with different antibiotic triplets revealed that the triple β-lactam sequences are at least as effective at causing extinction as sequences of antibiotics with distinct modes of actions. This finding is at first sight counterintuitive, but at second sight not completely unexpected. The joint application of two β-lactam drugs was in fact tested and found effective in a few previous studies (Rahme et al., 2014). For example, the β-lactam aztreonam was shown to interact synergistically with four other β-lactam drugs against multiple resistant isolates of Enterobacteriaceae and P. aeruginosa in vitro (Buesing and Jorgensen, 1984). A combination of ticarcillin with ceftazidime produced high efficacy in a rat peritonitis model (Shyu et al., 1987). In a treatment of bacterial soft tissue infections, the combination of cefotaxime and mecillinam led to higher clinical response rates than the tested monotherapy (File and Tan, 1983). Further, the dual β-lactam combination of ceftazidime plus piperacillin was as effective as the combination of ceftazidime and tobramycin in granulocytopenic cancer patients (Joshi et al., 1993). More recent studies demonstrated that a triple combination of meropenem, piperacillin, and tazobactam successfully constrained resistance evolution in Methicillin-resistant Staphylococcus aureus (MRSA), both in vitro and in a mouse model (Gonzales et al., 2015). In addition, the combination of cefotaxime and mecillinam was effective against Salmonella enterica harboring a mutant β-lactamase in a mouse model (Rosenkilde et al., 2019). Our findings add to the high potency of treatments with multiple β-lactams. We conclude that the use of multiple β-lactams, either as a combination or sequentially, is a commonly underappreciated form of therapy and its use opens new avenues to better utilize our existing antibiotic armamentarium.

Spontaneous rate of antibiotic resistance was found to play a critical role in the success of the CAR-CEF-DOR sequential treatment. The probability of spontaneous resistance on all three β-lactams was significantly different, with the rate of DOR resistance being the lowest. These rates determined the overall probability of acquiring direct resistance in treatment, which significantly correlated with the frequency of population extinction (Figure 5B). Resistance rates were previously shown to vary towards different antibiotics, for example, in Escherichia coli (El Meouche and Dunlop, 2018) and P. aeruginosa (Oliver et al., 2004). This variation can arise from genetic factors such as mutational target space and physiological factors like activation of the bacterial SOS response (Martinez and Baquero, 2000). Such information on resistance rates has so far been used for predicting the occurrence of resistance against single drugs, and antibiotics that target multiple pathways in a cell are considered advantageous in this context (Ross-Gillespie and Kümmerli, 2014). One example of the latter are compounds against S. aureus that inhibit both DNA gyrase and topoisomerase IV (Nyerges et al., 2020). The rate of resistance emergence may also be reduced by using adjuvants that target the SOS response (Bell and MacLean, 2018), as previously shown for compounds interfering with LexA activity leading to reduced resistance rates to ciprofloxacin and rifampicin in E. coli (Cirz et al., 2005). Our study extends the role of resistance rates of antibiotics beyond this convention. We show that inclusion of an antibiotic with relatively low spontaneous resistance emergence can enhance the potency of a sequential treatment design.

What could be the underlying reasons for the particular importance of DOR compared with the other β-lactams? DOR belongs to the carbapenem subclass of the β-lactam antibiotics. Carbapenems possess broad activity against Gram-positive and Gram-negative bacteria (Papp-Wallace et al., 2011) and are active against many β-lactamase-producing microbes since their thiazolidinic ring makes them relatively resistant to β-lactamase-mediated hydrolysis (Schafer et al., 2009). In contrast, the penicillin CAR is active mostly (albeit not exclusively) against Gram-negative bacteria (Castle, 2007) while the activity of the cephalosporin CEF is restricted to P. aeruginosa (Wright, 1986). Within P. aeruginosa, all three antibiotics show high potency against a large variety of clinical isolates (Castanheira et al., 2009; Neu and Scully, 1984; Traub and Raymond, 1970). Resistance rates for β-lactam antibiotics were assessed with different approaches across P. aeruginosa strains and clinical isolates, consistently showing that DOR has a particularly low propensity to select for resistance mutations, even when compared to other carbapenems (Barbosa et al., 2017; Barbosa et al., 2021; Sakyo et al., 2006; Tanimoto et al., 2008; Mushtaq et al., 2004; Fujimura et al., 2009). Therefore, the phenotype of reduced spontaneous resistance to DOR appears to be robustly expressed across different P. aeruginosa genotypes and does not extend to other carbapenems or β-lactams. One possible reason for this pattern may be variation in the range of β-lactam target proteins, in this case the penicillin binding proteins (PBPs), and where DOR is known to bind more of these PBPs than do CAR or CEF (Davies et al., 2008; Fontana et al., 2000; Rodriguez‐Tebár et al., 1982; Rodríguez-Tebar et al., 1982; Zapun et al., 2008; Zimmermann, 1980). Thus, target resistance to DOR would likely require a larger number of mutations than that to other β-lactams. Interestingly, another carbapenem, meropenem, targets the same PBPs as DOR (Davies et al., 2008) but has a higher resistance rate, suggesting that the underlying reasons for resistance rate variation are multifactorial. Taken together, effective resistance mutations against DOR seem to be less commonly available in P. aeruginosa in comparison to that against other drugs, including the here used CEF and CAR.

A key determinant of treatment potency was the reduced level of spontaneous cross-resistance to the sequentially applied drugs (Figure 5B). This effect was maximized by the switching rate (Figure 5—figure supplement 1). Our findings are consistent with the previously and repeatedly proposed importance of collateral sensitivity for the efficacy of sequential treatment protocols (Barbosa et al., 2019; Hernando-Amado et al., 2020; Imamovic and Sommer, 2013; Kim et al., 2014; Maltas and Wood, 2019; Yen and Papin, 2017). Even though we did not measure collateral sensitivity directly, the lack of cross-resistance is related as it indicates that the mutant cells, which have become resistant to one drug, maintain at least ancestral levels of susceptibility against the second drug. Moreover, our study focused on spontaneous emergence of cross-resistance (or lack thereof). By contrast, many previous studies established collateral effects after bacteria evolved resistance to the first drug over many generations, often followed by only a single antibiotic switch to assess the impact of collateral sensitivity on therapy success (Barbosa et al., 2019; Hernando-Amado et al., 2020; Imamovic and Sommer, 2013; Yen and Papin, 2017). Surprisingly, our study revealed potentially beneficial collateral effects between antibiotics of the same class. In fact, we chose these three β-lactams because our previous work demonstrated cross-resistance between most of them, although inferred upon multigenerational adaptation to the first drug (Barbosa et al., 2017). Our current finding of a lack of cross-resistance among some of these drugs now suggests that spontaneous mutants may have different collateral profiles than the lines, which adapted over many generations. Our results further suggest that the collateral effects of spontaneous mutants are likely to be more pertinent for the design of sequential treatments with fast switches among antibiotics. This suggestion is supported by two previous studies, in which the efficacy of fast sequential treatments was optimized by considering collateral effects for either single-step mutants of S. aureus, obtained after 20 hr exposure to three distinct antibiotics for 20 hr (Kim et al., 2014), or from Enterococcus faecalis populations adapted over 2 days to four distinct antibiotics (Maltas and Wood, 2019). As a side note, it is particularly interesting that our detailed resistance analysis consistently revealed almost all treatments to cause the evolution of collateral sensitivity towards the aminoglycoside gentamicin, but not the fluoroquinolone ciprofloxacin (Figure 2B, C), possibly indicating yet another treatment option – in cases where the applied triple β-lactam sequential protocols fail.

Temporal irregularity was additionally found to constrain bacterial adaptation. When bacteria experienced the antibiotics in an irregular pattern, this caused significantly increased extinction and to some degree reduced multidrug resistance. With CAR-DOR-CEF, the lowest multidrug resistance was observed in random sequential treatments (Figure 2—figure supplement 5), as also previously observed with CIP-GEN-CAR (Roemhild et al., 2018). Environmental change anticipation has been documented in several microorganisms (Mitchell et al., 2009; Mitchell and Pilpel, 2011), indicating their capability to specifically adapt to regular environmental change. Stochastic changes can make it harder to evolve anticipation (Roemhild and Schulenburg, 2019). Stochastic changes in environmental parameters were indeed found to constrain fitness in evolving bacteria (Hughes et al., 2007) and viruses (Alto et al., 2013). We show that irregular antibiotic sequences have potential to inhibit bacterial resistance evolution.

Unexpectedly, we further identified negative hysteresis for multiple combinations of the three β-lactams. However, cumulative hysteresis levels per treatment did not significantly associate with any of our measured evolutionary responses. In our previous study (Roemhild et al., 2018), within the CAR-CIP-GEN combination, negative hysteresis was expressed for the switches from CAR to GEN and CIP to GEN. Yet, only the CAR-GEN hysteresis was significantly associated to the evolutionary responses. Thus, hysteresis interactions can exist between antibiotics from the same or different classes, but they need not impact the evolutionary outcome of a sequential treatment protocol each time. In the current study, it appears that spontaneous resistance effects and the resulting cross-resistance effects are dominant over the β-lactam hysteresis. One potential explanation could be that insensitivity to β-lactam hysteresis evolves quickly. Nevertheless, it clearly warrants further research to assess whether negative hysteresis between the β-lactam drugs is robustly shown across strains of P. aeruginosa or other bacterial species and can somehow be exploited in sequential therapy, in analogy to the previous results with antibiotics from different classes (Roemhild et al., 2018).

Taken together, our study highlights that the available antibiotics offer unexplored, highly potent treatment options that can be harnessed to counter the spread of drug resistance. It further underscores the importance of evolutionary trade-offs such as reduced cross-resistance in treatment design and introduces spontaneous resistance rates of component antibiotics as a guiding principle for sequential treatments. It is ironic that the differential cross-resistance landscape of the β-lactams was a key factor contributing to treatment potency, even though the risk of cross-resistance is usually used to reject β-lactam-exclusive treatments. The underlying reasons for differential spontaneous and long-term cross-resistance between these drugs (including the underlying molecular mechanisms) are as yet unknown and clearly deserve further attention in the future. We conclude that a detailed understanding of both spontaneous resistance rates and resulting cross-resistances against different antibiotics should be of particular value to further improve the potency of sequential protocols.

Materials and methods

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (Pseudomonas aeruginosa)PA14https://doi.org/10.1126/science.7604262UCBPP-PA14
Chemical compound, drugAZL
(azlocillin)
SigmaA7926-1G
Chemical compound, drugCAR
(carbenicillin)
Carl Roth6344.2
Chemical compound, drugCIP
(ciprofloxacin)
Sigma17850-5 G-F
Chemical compound, drugCEF
(cefsulodin)
Carl Roth4014.2
Chemical compound, drugCEZ
(ceftazidime)
SigmaC3809.1G
Chemical compound, drugDOR
(doripenem)
Sigma32138-25 MG
Chemical compound, drugGEN
(gentamicin)
Carl Roth2475.1
Chemical compound, drugSTR
(streptomycin)
SigmaS6501-5
Chemical compound, drugTIC
(ticarcillin)
SigmaT5639-1G
Software, algorithmR: A language and environment for statistical computinghttps://www.R-project.org/

Materials

All experiments were performed with P. aeruginosa UCBPP-PA14 (Rahme et al., 1995). Bacteria were grown in M9 minimal medium supplemented with glucose (2 g/L), citrate (0.58 g/L), and casamino acids (1 g/L) or on M9 minimal agar (1.5%) or Lysogeny broth (LB) agar. Antibiotics were added as indicated. Cultures and plates were incubated at 37°C. Experiments included biological replicates (initiated with independent clones of the bacteria, which were grown separately before the start of the experiment, or independent evolutionary lineages from the respective evolution treatments) and technical replicates (initiated from the same starting culture of the bacteria), as indicated below. For the experiments, treatment groups were run in parallel and randomized. Treatment names were masked in order to minimize observer bias.

Dose-response curves of ancestor

Request a detailed protocol

We used dose-response curves based on broth microdilution in order to determine antibiotic concentration causing inhibition level of 25% growth yield relative of untreated controls (inhibitory concentration 75 [IC75]) for the antibiotics azlocillin (AZL), carbenicillin (CAR), ciprofloxacin (CIP), cefsulodin (CEF), ceftazidime (CTZ), doripenem (DOR), gentamicin (GEN), and ticarcillin (TIC; see Supplementary file 1A for details on antibiotics). Briefly, bacteria were grown to exponential phase (OD600 = 0.08) and inoculated into 96-well plates (100 µL per well, 5 × 106 CFU/mL) containing linear concentration ranges close to MIC of the antibiotics in M9 medium. Antibiotic concentrations were randomized spatially. Bacteria were incubated for 12 hr after which optical density was measured in BioTek EON plate readers at 600 nm (OD600). We included six biological replicates and 1–2 technical replicates per concentration and antibiotic. Optical density was plotted against antibiotic concentration to obtain a dose-response curve. Model fitting was carried out using the package drc (Ritz et al., 2015) in the statistical environment R (R Development Core Team, 2020) and the fitted curve was used to predict IC75 values (Figure 1—figure supplement 1).

Evolution experiments

Request a detailed protocol

We carried out evolution experiments with the various combination of antibiotics according to the design described previously (Roemhild et al., 2018). A total of 16 treatments were included (Figure 1B). Treatments 1–4 were constant environments consisting of the monotherapy (#1–3) and no drug control (#4). Treatments 5–10 were the regular switching treatments. They switched between the antibiotics in a regular predictable fashion, either every transfer (fast; #5–7) or every fourth transfer (slow; #8–10). Treatments 11–16 consisted of the random treatments that switched fast in a temporally irregular fashion. The setup was designed to test the effect of switching rate and temporal irregularity.

Every treatment consisted of 12 replicate populations (initiated from six biological replicates × two technical replicates). All populations were started with an inoculum of 5 × 105 cells. Populations were propagated as 100 µL batch cultures in 96-well plates, with a transfer to fresh medium every 12 hr (transfer size 2% v/v). Antibiotic selection was applied at IC75 throughout. We monitored growth by OD600 measurements taken every 15 min through the entire evolution experiment (BioTek Instruments, USA; EON; 37°C, 180 rpm double-orbital shaking). Evolutionary growth dynamics were assessed by plotting the final OD achieved in every transfer (relative to final OD of no drug control; relative yield). Adaptation rate was calculated with a sliding window approach, where adaptation rate was the inverse of the transfer at which the mean relative yield of a sliding window of 12 transfers reached 0.75 for the first time. Cases of extinction were determined at the end of the experiment by counting wells in which no growth was observed after an additional incubation in antibiotic-free medium. Samples of the populations were frozen in regular intervals in 10% (v/v) DMSO and stored at −80°C for later analysis. The evolution experiments were carried out for a total of 96 transfers.

Resistance measurements of evolved populations

Request a detailed protocol

We characterized populations frozen at transfers 12 and 48 in detail because they represented the early and late phases of the evolution experiment. One population originating from a single biological replicate was chosen per treatment and plated onto LB agar. After incubation at 37°C, 20 colonies from each population were picked randomly and frozen in 10% (v/v) DMSO and stored at −80°C. These colonies, termed isolates, were considered to be representative biological replicates for each population. We constructed dose-response curves for the isolates using for each evolved population one technical replicate per isolate and four technical replicates of the ancestral PA14 strain, as described above, for the antibiotics CAR, CEF, DOR, GEN, and CIP. The integral of this curve for every isolate was calculated and the integral of the ancestral PA14 control subtracted. The resulting value was resistance of the isolate on the said antibiotic. We identified subpopulations in any given population by hierarchical clustering of the resistance profiles, as previously described (Roemhild et al., 2018). Resistance of a population was calculated by averaging the resistance of the isolates. Resistance of the population on CAR, CEF, and DOR was added to obtain a single value for multidrug resistance.

Whole-genome sequencing

Request a detailed protocol

From the frozen isolates at transfer 12, we chose three isolates per population (i.e., three biological replicates per population) for whole-genome sequencing to determine possible targets of selection. Each resistance cluster in the population was represented in the sequenced isolates. For the DOR monotherapy, isolates from transfer 48 were also sequenced as no phenotypic resistance was observed at transfer 12. Frozen isolates were thawed and grown in M9 medium at 37°C for 16–20 hr. We extracted DNA using a modified CTAB protocol (von der Schulenburg et al., 2001) and sequenced it at the Competence Centre for Genomic Analysis Kiel (CCGA Kiel; Institute for Clinical Microbiology, University Hospital Kiel), using Illumina Nextera DNA Flex library preparation and the MiSeq paired-end technology (2 × 300 bp). Quality control on the resulting raw reads was performed with FastQC (Andrews, 2010) and low-quality reads were trimmed using Trimmomatic (Bolger et al., 2014). We then used MarkDuplicates from the Picard Toolkit (http://broadinstitute.github.io/picard/) to remove duplicate reads and mapped the remaining reads to the P. aeruginosa UCBPP-PA14 genome (available at http://pseudomonas.com/strain/download) using Bowtie2 and samtools (Langmead and Salzberg, 2012; Li et al., 2009). Variant calling was done using the GATK suite (Poplin et al., 2018) and the called variants were annotated using SnpEFF (Cingolani et al., 2012) and the Pseudomonas Genome Database (https://www.pseudomonas.com/). We removed all variants that were detected in the no drug control as they likely represent adaptation to the medium and not the antibiotic. The fasta files of all sequenced isolates are available from NCBI under the BioProject number: PRJNA704789.

Hysteresis testing

Request a detailed protocol

The presence of cellular hysteresis was tested, following the previously developed protocol (Roemhild et al., 2018). Bacterial cells were grown to exponential phase (OD600 = 0.08), diluted 10-fold, and treated with IC75 of the first antibiotic. In the treatments where the pretreatment did not require an antibiotic, none was added. These cells were allowed to incubate for 15 min at 37°C and 150 rpm (pretreatment). After this, the first antibiotic was removed by centrifugation and fresh medium containing IC75 of a second antibiotic was added. In cases where the main treatment did not require an antibiotic, fresh medium without an antibiotic was added. Bacteria were now incubated for 8 hr at 37°C and 150 rpm (main treatment). Bacterial count was monitored through the main treatment by spotting assays. We used three biological replicates per treatment and, for CFU counting, four technical replicates per biological replicate and treatment. Log10 CFU/mL were plotted against time to obtain time-kill curves (Figure 3B). The level of hysteresis was calculated as the difference between the antibiotic switch and only main treatment curves.

Agar dilution

Request a detailed protocol

We determined the MIC on M9 agar for the antibiotics CAR, CEF, and DOR according to the EUCAST protocol (https://doi.org/10.1046/j.1469-0691.2000.00142.x) that was modified to account for inoculum effect in our fluctuation assay setup. UCBPP-PA14 was grown in M9 medium at 37°C for 20 hr. 5 × 105 cells were taken from the stationary phase cultures and spread on M9 agar plates containing doubling dilutions of the antibiotic. Plates were incubated at 37°C for 20–24 hr. MIC was read as the lowest concentration at which no growth of bacteria was seen. MIC determination for each antibiotic was done for three biological replicates (no additional technical replication).

Fluctuation assay

Request a detailed protocol

We measured resistance rates on the three β-lactams using the classic fluctuation assay (Luria and Delbrück, 1943). Briefly, a single colony of UCBPP-PA14 was inoculated to 10 mL M9 and incubated at 37°C, 150 rpm for 20 hr. This primary culture was used to start 30 parallel cultures all having a starting concentration of 102 CFU/mL. The parallel cultures were considered biological replicates and incubated at 37°C, 150 rpm for 20 hr. Thereafter, 5 × 105 cells were plated onto MIC plates of CAR, CEF, and DOR. The plates were incubated for 40 hr at 37°C. The resulting mutant colonies were taken and patched on identical antibiotic MIC plates to ensure genetic resistance. Colonies that grew after patching were counted. We used counts from all 30 cultures to estimate resistance rate on each antibiotic using the package rSalvador (Zheng, 2017) in R (R Development Core Team, 2020).

Patching assay

Request a detailed protocol

We assessed the extent of cross-resistance associated with each β-lactam using the mutants obtained from the fluctuation assay. Sixty mutants with genetic resistance to a given β-lactam were considered biological replicates and patched onto MIC plates of the two other β-lactams. The patched plates were incubated for 16–20 hr at 37°C. If the mutant grew at MIC of the second β-lactam, it was counted as resistant. If it did not grow at the MIC of the second β-lactam, it was counted as susceptible. For each switch between two drugs, the fraction of cross-resistant mutants was calculated as

NumberofmutantsthatgrewondrugBTotalmutantsisolatedondrugA

Statistical analysis for cross-resistance on secondary antibiotic

Request a detailed protocol

To test whether the secondary antibiotic had an influence on the degree of cross-resistance of the mutants obtained from the fluctuation assay, we conducted a Fischer’s exact test followed by post hoc comparisons using the R package rcompanion (Mangiafico, 2016). The obtained p-values were then corrected for multiple testing using false discovery rate.

Statistical analysis of adaptive growth dynamics

Request a detailed protocol

To test whether main treatment types were associated with altered dynamics of adaptation in non-extinct populations, we analyzed the trajectories of relative growth yield (as plotted in Figure 1E and Figure 2A) of drug-treated populations using a GLM, including sequence (##1–16) and transfer as fixed factors and preculture and replicate population as nested random factors (see Supplementary file 1B for details). Comparisons between main treatment groups were performed using pairwise post hoc tests and z statistics. All p-values were corrected for multiple testing using false discovery rate. The analysis was performed separately for the three time phases ‘early’ (transfers 2–12), ‘middle’ (transfers 13–48), and ‘late’ (transfers 49–96) of the experiment, thus fulfilling the model assumption of response linearity. All statistical analyses were carried out in the statistical environment R (R Development Core Team, 2020).

Statistical analysis of evolved multidrug β-lactam resistance

Request a detailed protocol

To test whether evolved populations displayed distinct multidrug β-lactam resistance depending on their main treatment type, we analyzed multidrug β-lactam resistance of evolved isolates – the sum of resistance values against CAR, CEF, and DOR (as plotted in Figure 3B, C) – using a GLM. The model included sequence (##1–16) as fixed factor and replicate population as nested random factor (see Supplementary file 1C for detailed information). Comparisons between main treatment groups were performed using pairwise post hoc tests and z statistics. All p-values were corrected for multiple testing using false discovery rate. The analysis was performed separately for the ‘early’ (after transfers 12) and ‘middle’ (transfer 48) time points of the evolution experiment using the R statistical environment (R Development Core Team, 2020).

Statistical analysis of treatment potency predictors

Request a detailed protocol

To test whether our experimental (switching rate and temporal irregularity) and biological predictors (hysteresis, probability of direct resistance, and cross effects) were able to explain the variability in our evolutionary responses (extinction, rate of growth adaptation, and multidrug resistance) we carried out a GLM analysis. Values per treatment protocol for the biological predictors were calculated and the GLM analysis then carried out in R (R Development Core Team, 2020). We used the lm and anova commands and the main effects model: response ~ switching rate + irregularity for the experimental predictors and response ~ hysteresis + spontaneous resistance + mutant fraction cross-resistant for the biological predictors.

Data availability

Sequencing data have been deposited at NCBI under the BioProject number: PRJNA704789. All other data is provided in the supplementary source data files.

The following data sets were generated
    1. Batra A
    (2021) NCBI BioProject
    ID PRJNA704789. Sequential beta-lactam treatment genomics.

References

  1. Book
    1. Castle SS
    (2007) Carbenicillin
    In: Enna S. J, Bylund D. B, editors. XPharm: The Comprehensive Pharmacology Reference. New York: Elsevier. pp. 1–5.
    https://doi.org/10.1016/B978-008055232-3.61381-9
  2. Book
    1. Walsh C
    (2003)
    Antibiotics: Actions, Origins, Resistance
    Washington: ASM Media & PR.
    1. Wright DB
    (1986) Cefsulodin
    Drug Intelligence & Clinical Pharmacy 20:845–849.
    https://doi.org/10.1177/106002808602001104

Decision letter

  1. Vaughn S Cooper
    Reviewing Editor; University of Pittsburgh, United States
  2. George H Perry
    Senior Editor; Pennsylvania State University, United States
  3. Vaughn S Cooper
    Reviewer; University of Pittsburgh, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study compared effectiveness of three combinations of antibiotic drug therapy regimes against the bacterial pathogen Pseudomonasaeruginosa and found that an unlikely drug combination promoted bacterial extinction. In contrast to prior studies using combinations of drugs with similar chemistries (eg. β-lactams that inhibit cell wall synthesis), which lead to cross-resistance and treatment failure, the sequential use of three β-lactams CAR-CEF-DOR (carbenicillin, cefsulodin, doripenem) was highly effective because of constraints caused by DOR. These results revitalize the potential use of similar β-lactams for treatment against bacterial pathogens and promote utility of predicting evolutionary trajectories to improve antimicrobial drug therapies.

Decision letter after peer review:

Thank you for submitting your article "High potency of sequential therapy with only beta-lactam antibiotics" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Vaughn S Cooper as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by George Perry as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential Revisions:

1) The sole strain used in this work is P. aeruginosa PA14. This is a good strain choice but the paper seems to presume that this isolate represents the species well, or even more broadly. What is known in the literature about variation within PA or related species in responses to DOR, and/or other drugs in the triple combinations? Is the hysteresis effect limited to this strain?

2) Given that much of the paper focuses on the doripenem effect, what are the relevant attributes of this drug relative to others? At face value, it's a long-lived carbapenem, so is it the fact that it's a carbapenem, its durability, or both the cause of this physiological stress that diminishes subsequent survival? Might the hysteresis effect simply be the killing of susceptible cells causing subsequent failure?

3) A primary conclusion of this paper (L 359-61) is that the spontaneous rate of resistance was a key variable dictating treatment potency. However, this conclusion does not appear to account for the diminished population size of DOR-treated populations and the high potency of this drug relative to others, which would increase the likelihood of error in DOR dosage. While Figure 4 shows that mutation rates to DOR are low, and the methods are appropriate, the results could reflect stronger selection imposed by DOR concentrations and smaller genomic targets to resistance. The authors use this to make their case, but do not adequately discuss the differences between these three β-lactam drugs. In other words, I wonder if PA14 is on a knife-edge in the presence of DOR at the concentrations used, and this reduces population sizes, strengthens selection beyond other drugs, and leads to more variable extinction probabilities. Discussion of the population-genetic consequences of evolution at 0.75x the MIC, and the role of error around this 0.75x threshold, are needed.

4) Figure 1A: I found the different dashed lines to be difficult to distinguish. Maybe place each label near each closed contour so it's easy to see? Also, consider emphasizing those from this study (perhaps with thicker contours?). The positioning of the drug classes makes it unclear what STR is. Is it DNA gyrase or Ribosome since it is in the middle of those terms? Perhaps use color coding. Also, please define what FQ, AG and BL are in Figure 1A.

5) Lines 47-50: It is unclear how competitive release occurs in this scenario. It is my understanding that competitive release occurs when one lineage acquires a highly beneficial mutation early that leads to competitive exclusion of the others. And the paper cited suggests that this phenomenon occurs due to selective pressure not strong enough to prevent this mutation. However, how it is written suggests that easily accessible genetic resistance is the source. But if it were indeed easily accessible, then there would be higher chances that other lineages would also acquire it? If my understanding is incorrect, perhaps provide a bit more of an explanation for the term competitive release.

6) Line 80-84: It is unclear how negative hysteresis was suppressed by "strong selection dynamics" with a mild increase in resistance. Can you provide a specific genetic example from the paper? This will also help conceptualize types of negative hysteresis that can occur during strong antibiotic pressures.

7) Line 91: Replace "As before" with "The new" and add "expected to contribute to collateral sensitivity…". As written, it is confusing what was previously tested and what the new design and expectations are in this study.

8) Line 97: Similar to above. Add "expected to be dominated by cross-resistance…" Helps to clarify past from present.

9) The term "season" is confusing and often used interchangeably with "transfer". I prefer the term transfer and suggest keeping this terminology throughout.

10) Figure Supplemental 1, Table1: I like the extra information about the stock concentration and storage. It would also be helpful if the drugs were ordered by class and maybe even identified using the BL, FQ, AG abbreviations.

11) Figure 2: Please describe that the dotted lines represent the three growth phases in A. For B, it is unclear what the arrows signify. It looks like they are aligned with transfers 12 and 48. Either make them more continuous or remove them altogether. Otherwise, it looks as if the populations are what is being highlighted. Also, what is the significance of the +1 to -0.5 range vs the 16x to 0.33x? The MIC column is most explanatory to me.

12) Line 192-193: To my eye, the resistance profiles between the early- and mid- phases look pretty similar. The sum of resistances is mentioned. Perhaps add the sum to each of the columns in Figure 2 to make it clearer.

13) Line 210-213: DOR monotherapy is stated to not show a noticeable amount of resistance at the early stage. However, it looks like population 1 shows 4x-8X MIC and looks similar to the mid phase. This is contradictory to what is stated. Can you please rephrase.

14) Line 210-213: It is interesting that monotherapy population one at transfer 12 showed increased resistance but resulted in no variants found. Is it possible that this was a result of heteroresistance or persistence? If you sequenced the whole population instead of isolating three clones, you might be able to distinguish variants at low frequency that would contribute to heteroresistance.

15) Figure 3C. The Blue and black are hard to distinguish as a gradient.

16) Line 361: Is it treatment potency due to cross resistance or the lack of cross-resistance. I thought that it was due to the latter because neither of the other two drugs promoted cross-resistance to DOR.

17) Line 414: Is "disadvantageous cross-resistance" the same as collateral sensitivity? If so, the double negative is confusing.

Recommendations for follow up work

1. Whole genome sequencing was limited to three clones per population. While it is understandable that the number of treatment regimens might make sequencing costly. Population dynamics and allele frequencies may help explain why the population during the DOR monotherapy struggled to acquire mutations before transfer 12.

2. One of the most interesting aspects of the study is that the principles identified for designing sequential therapies are not dependent on a unique molecular mechanism, and one could therefore envision multiple drug combinations that achieve similar evolution-slowing effects but do so in very different ways at the molecular level (where resistance has often been studied). Do the authors believe there is some inherent feature of β-lactams-for example, the fact that they compromise the cell wall and promote lysis-that may underlie the success of homogeneous sequential therapies? Or might one expect similar success with, say, triplets of fluoroquinolones or other drugs, as long as the triplet is chosen with similar qualitative features (e.g. low levels of cross-resistance, low spontaneous resistance to at least one drug). Relatedly, are there reasons to believe that certain classes of drugs will be more likely to show those qualitative features? It would be remarkable if, for example, these important features could be predicted based on, say, the known diversity of resistant mutations for the component drugs. In any event, I think one of the striking conclusions from this work is that considerable optimization may be possible without detailed knowledge of molecular mechanism.

3. The results suggest that the chosen β lactams exhibit negative hysteresis, yet the negative hysteresis does not play a dominant role in slowing resistance (unlike in the authors' previous study with heterogeneous drug combinations). Does this discrepancy arise simply because other effects (e.g. collateral effects) are dominant, so the impacts of negative hysteresis are (by comparison) negligible? Or is there some feature of the negative hysteresis-and perhaps its correlation with the collateral effects-that renders it non-beneficial? I'd venture a guess that in general, there is some underlying relationship between the hysteresis profiles, the collateral profiles, and the timescale of switching that could conceivably be tuned to optimize these therapies. It seems to be an exciting topic for future work.

Reviewer #1 (Recommendations for the authors):

The authors conducted an ambitious set of hundreds of evolution experiments with P. aeruginosa exposed to different sets of 3 antibiotics amounting to ~500 generations each, under monotherapy, fast switching, slow switching, and random switching. A primary outcome was extinction, ie failure to persist, while secondary outcomes included the extent and breadth of evolved resistance and a sampling of evolved resistance mutations. Treatments including the antibiotic doripenem led to more extinctions, likely because growth in doripenem reduced the ability to grow in alternative antibiotics, which they term negative hysteresis. This is an interesting and potentially clinically relevant discovery. The paper is well written and engaging.

I see three major limitations that the authors could reasonably address.

1. The sole strain used in this work is P. aeruginosa PA14. This is a good strain choice but the paper seems to presume that this isolate represents the species well, or even more broadly. What is known in the literature about variation within PA or related species in responses to DOR, and/or other drugs in the triple combinations? Is the hysteresis effect limited to this strain?

2. Given that much of the paper focuses on the doripenem effect, what are the relevant attributes of this drug relative to others? At face value, it's a long-lived carbapenem, so is it the fact that it's a carbapenem, its durability, or both the cause of this physiological stress that diminishes subsequent survival? Might the hysteresis effect simply be the killing of susceptible cells causing subsequent failure?

3. A primary conclusion of this paper (L 359-61) is that the spontaneous rate of resistance was a key variable dictating treatment potency. However, this conclusion does not appear to account for the diminished population size of DOR-treated populations and the high potency of this drug relative to others, which would increase the likelihood of error in DOR dosage. While Figure 4 shows that mutation rates to DOR are low, and the methods are appropriate, the results could reflect stronger selection imposed by DOR concentrations and smaller genomic targets to resistance. The authors use this to make their case, but do not adequately discuss the differences between these three β-lactam drugs. In other words, I wonder if PA14 is on a knife-edge in the presence of DOR at the concentrations used, and this reduces population sizes, strengthens selection beyond other drugs, and leads to more variable extinction probabilities. Discussion of the population-genetic consequences of evolution at 0.75x the MIC, and the role of error around this 0.75x threshold, are needed.

Reviewer #2 (Recommendations for the authors):

Batra, Aditi et al., compared the effectiveness of three in vitro antibiotic drug therapy regimes against the bacterial pathogen Pseudomonasaeruginosa and found that an unlikely drug combination promoted bacterial extinction. Previous studies have shown that using similar drug classes with similar chemistries (eg. β-lactams that inhibit cell wall synthesis) lead to increased cross resistance and therefore, treatment failure. However, the authors found that the fast and sequential use of three β-lactams CAR-CEF-DOR (carbenicillin, cefsulodin, doripenem) was the most effective treatment against P. aeruginosa. The authors further investigated the potential factors contributing to a lack of evolutionary rescue such as negative hysteresis, collateral sensitivity, and rate of spontaneous direct/indirect resistance. By conducting additional experiments to test multiple hypotheses, the authors concluded that the fast-switching three-drug series was constrained by DOR and that the cause of the constraint was due to a low rate of spontaneous resistance and cross-resistance to DOR. These results revitalize the potential use of similar β-lactams for treatment against bacterial pathogens and the utility of predicting evolutionary trajectories to improve antimicrobial drug therapies.

Strengths:

This paper introduces both physiological and evolutionary concepts as possible hypotheses for explaining antibiotic drug constraints. When discussing consequences to drug therapies, cross resistance and collateral sensitivity are often described whereas physiological constraints such as negative hysteresis is overlooked. The experiments conducted in this paper demonstrate how to test for hysteresis using short exposures to a drug and then switching to a different drug and comparing growth killing effects. Drug exposure is carried out quickly to maintain a low mutational load and reduce the probability that a beneficial mutation would arise. Mutations would interfere with a test for non-genetic physiological conditioning.

Measurements of spontaneous resistance rates is a useful phenotype to compare mechanisms of mutations for each drug. The experiments and results that compare the rate of spontaneous direct resistance and indirect (ie. Cross resistance) as well as collateral sensitivity are clear and well replicated.

Weakness:

Whole genome sequencing was limited to three clones per population. While it is understandable that the number of treatment regimens might make sequencing costly. Population dynamics and allele frequencies may help explain why the population during the DOR monotherapy struggled to acquire mutations before transfer 12.Reviewer #3 (Recommendations for the authors):

The manuscript by Batra and colleagues investigates the impact of sequential antibiotic treatments on evolving laboratory populations of P. aeruginosa, a clinically important microbial pathogen. A growing body of work suggests that cycling between different antibiotics can slow resistance in the lab, though the conventional view is that successful treatments would require drugs from different classes, limiting the propensity for cross resistance and potentially harnessing sensitizing collateral effects or negative hysteresis. This study provides a large-scale investigation of the efficacy of sequential treatments comprised of different 3-drug treatments. The drugs are chosen to span different classes, with triplicates including drugs targeting the ribosome, DNA gyrase, or the cell wall (β lactams) in some combination. Previous work from the same group showed that fast switching between a heterogeneous set (3 different classes) of drugs promoted population extinction at sub-MIC concentrations, with selection favoring genetic suppression of negative hysteresis. In this work, they investigate the response to different 3-drug sequences (both homogeneous and heterogeneous) in a total of 756 (!!) replicate populations. Treatments included monotherapy, fast or slow switching, and random switching. Interestingly, they found that switching among two different sets of β lactams produced high levels of extinction-comparable to that previously observed with a heterogeneous set-while switching between a new heterogeneous set did not produce extinction. Extinction tended to occur early in the treatment and was significantly reduced when the period of treatment switching was extended. The authors focus on one drug set (CAR-CEF-DOR) for an in-depth characterization. Using general linear models (GLM) to evaluate growth adaptation and total β lactam resistance in the three distinct growth phases, they showed that early phase adaptation is decelerated in the fast-switching group relative to monotherapy and slow-switching group; interestingly, the fast and random treatments showed relatively similar rates of extinction, suggesting that precise ordering is not the primary driver of the effects. They also observed generally increased β lactam resistance in the second phase, but the different treatment types led to largely distinct resistance profiles. Population diversity increased between early (transfer 12) and later (transfer 48) days of adaptation, and resistance in the early phase was often constrained by susceptibility to a particular drug (DOR) (and whole-genome sequencing found DOR resistance mutations only at later times). The authors go on to show that negative hysteresis is common among the three β-lactams (Figure 3), while DOR exhibits the lowest rates of direct and cross resistance (as measured by classic fluctuation tests and a patching assay). Finally, they used another GLM to quantify the impacts of different features on slowing resistance; they found that switching rate, temporal irregularity, spontaneous resistance probability, and (most prominently) the collateral effects dominate, with low levels of spontaneous resistance to DOR a primary constraint.

This study is timely, important, extremely thorough, and perhaps most importantly, it helps to identify a number of new principles to guide the rational design of sequential antibiotic therapies. It represents a monumental effort to thoroughly characterize adaption to 3-drug cycles in several representative examples, an achievement in itself. But as impressively, the authors draw on GLM's to extract meaningful patterns from the web of complex phenotypic data, and in doing so they uncover features of these treatments (e.g. surprising cross-resistance landscapes between drugs from the same class; potential importance of having one drug with low rates of spontaneous resistance) that may guide general design of new therapies. As with all good studies, it raises a number of questions for future work, and I list a few that come to mind below (that authors may wish to respond, perhaps in the discussion, at their discretion). But in general, I have no major concerns with the study at a technical or conceptual level (a rarity!). I believe this is an excellent contribution to the literature that will interest a broad range of readers in evolutionary biology and microbiology.

1) To me, one of the most interesting aspects of the study is that the principles identified for designing sequential therapies are not dependent on a unique molecular mechanism, and one could therefore envision multiple drug combinations that achieve similar evolution-slowing effects but do so in very different ways at the molecular level (where resistance has often been studied). Do the authors believe there is some inherent feature of β-lactams-for example, the fact that they compromise the cell wall and promote lysis-that may underlie the success of homogeneous sequential therapies? Or might one expect similar success with, say, triplets of fluoroquinolones or other drugs, as long as the triplet is chosen with similar qualitative features (e.g. low levels of cross-resistance, low spontaneous resistance to at least one drug). Relatedly, are there reasons to believe that certain classes of drugs will be more likely to show those qualitative features? It would be remarkable if, for example, these important features could be predicted based on, say, the known diversity of resistant mutations for the component drugs. In any event, I think one of the striking conclusions from this work is that considerable optimization may be possible without detailed knowledge of molecular mechanism.

2) The results suggest that the chosen β lactams exhibit negative hysteresis, yet the negative hysteresis does not play a dominant role in slowing resistance (unlike in the authors' previous study with heterogeneous drug combinations). Does this discrepancy arise simply because other effects (e.g. collateral effects) are dominant, so the impacts of negative hysteresis are (by comparison) negligible? Or is there some feature of the negative hysteresis-and perhaps its correlation with the collateral effects-that renders it non-beneficial? I'd venture a guess that in general, there is some underlying relationship between the hysteresis profiles, the collateral profiles, and the timescale of switching that could conceivably be tuned to optimize these therapies. It seems to be an exciting topic for future work.

https://doi.org/10.7554/eLife.68876.sa1

Author response

Essential Revisions:

1) The sole strain used in this work is P. aeruginosa PA14. This is a good strain choice but the paper seems to presume that this isolate represents the species well, or even more broadly. What is known in the literature about variation within PA or related species in responses to DOR, and/or other drugs in the triple combinations? Is the hysteresis effect limited to this strain?

Many thanks for this comment. We agree that the strain PA14 does not necessarily capture the phenotypes present within the species Pseudomonas aeruginosa and it is also possible that other bacterial species may show different evolutionary responses to the treatments tested. We here use PA14 as an established, tractable pathogen model system. In response to the reviewer’s comment, we now emphasize this point at the end of the introduction. Moreover, some information is indeed available on the response of other P. aeruginosa strains to the three antibiotics used. We now summarized this information in a new paragraph in the discussion. In contrast, hysteresis tests with the considered three antibiotics have thus far only been done by us, and the results are presented in the manuscript. It would indeed be of particular value to assess such hysteresis also in other strains, as we now point out in the final part of the discussion. In fact, this is something which we will address in a new project in my lab.

2) Given that much of the paper focuses on the doripenem effect, what are the relevant attributes of this drug relative to others? At face value, it's a long-lived carbapenem, so is it the fact that it's a carbapenem, its durability, or both the cause of this physiological stress that diminishes subsequent survival? Might the hysteresis effect simply be the killing of susceptible cells causing subsequent failure?

This is indeed a very interesting point. We now added more information on the characteristics of Doripenem in comparison with the other drugs in the first part of the discussion. Moreover, as indicated above, we now also provide a focused summary of the available information on resistance rate evolution of P. aeruginosa to Doripenem and the other two drugs. As to hysteresis: The used assay is set up to minimize any killing or differential replication of cells during the pretreatment, in order to ensure that any responses seen in the main treatment are based on physiological changes and not selection. This aspect is now highlighted in the Results section in the chapter on hysteresis.

3) A primary conclusion of this paper (L 359-61) is that the spontaneous rate of resistance was a key variable dictating treatment potency. However, this conclusion does not appear to account for the diminished population size of DOR-treated populations and the high potency of this drug relative to others, which would increase the likelihood of error in DOR dosage. While Figure 4 shows that mutation rates to DOR are low, and the methods are appropriate, the results could reflect stronger selection imposed by DOR concentrations and smaller genomic targets to resistance. The authors use this to make their case, but do not adequately discuss the differences between these three β-lactam drugs. In other words, I wonder if PA14 is on a knife-edge in the presence of DOR at the concentrations used, and this reduces population sizes, strengthens selection beyond other drugs, and leads to more variable extinction probabilities. Discussion of the population-genetic consequences of evolution at 0.75x the MIC, and the role of error around this 0.75x threshold, are needed.

Many thanks for this interesting point. However, we politely disagree that a stronger reduction in population size through DOR in comparison to the other drugs could have influenced the results.

Firstly, the dose response curves in the first supplementary figure (Figure 1—figure supplement 1) indicate very little variation in yield upon DOR treatment, especially at the IC75 concentration used to initiate the evolution experiments. Please note the 2nd panel in the top row and the right panel in the middle row; error bars are standard deviation of the mean (using 6 biological replicates) and hardly visible for the IC75 point because of the lack of variation (see red data points). Much more variation was for example found for CAR.

Secondly, the main evolution experiments were initiated using specifically standardized IC75 drug concentrations. Therefore, we do not expect initial variation in population size across treatments. Such variation should only arise thereafter, as a consequence of hysteresis effects or spontaneously evolved resistances and collateral effects.

Thirdly, the same argument holds for the fluctuation assays, which were performed with specifically standardized antibiotic concentrations, leading to identical selection being imposed to capture the spontaneous mutants.

Therefore, we consider it most likely that the lower adaptation to DOR in the monotherapies and the DOR transfer periods within the sequential treatments and also the reduced resistance rate within the fluctuation assays are due to a lower rate of DOR-specific resistance mutations (i.e., smaller genomic targets to DOR resistance) and also a lower rate of cross-resistance to DOR. These arguments are now presented in the revised Results section – in the chapter on the dynamics of resistance for the triple β-lactam treatment, and the chapter on spontaneous resistance rates.

4) Figure 1A: I found the different dashed lines to be difficult to distinguish. Maybe place each label near each closed contour so it's easy to see? Also, consider emphasizing those from this study (perhaps with thicker contours?). The positioning of the drug classes makes it unclear what STR is. Is it DNA gyrase or Ribosome since it is in the middle of those terms? Perhaps use color coding. Also, please define what FQ, AG and BL are in Figure 1A.

Many thanks for your suggestions. We hope that the revised figure is more accessible.

5) Lines 47-50: It is unclear how competitive release occurs in this scenario. It is my understanding that competitive release occurs when one lineage acquires a highly beneficial mutation early that leads to competitive exclusion of the others. And the paper cited suggests that this phenomenon occurs due to selective pressure not strong enough to prevent this mutation. However, how it is written suggests that easily accessible genetic resistance is the source. But if it were indeed easily accessible, then there would be higher chances that other lineages would also acquire it? If my understanding is incorrect, perhaps provide a bit more of an explanation for the term competitive release.

We agree that the sentence was not entirely clear. We have now removed the concept of competitive release from the sentence, as it is not important for the particular argument given and also not for the focus of our paper.

6) Line 80-84: It is unclear how negative hysteresis was suppressed by "strong selection dynamics" with a mild increase in resistance. Can you provide a specific genetic example from the paper? This will also help conceptualize types of negative hysteresis that can occur during strong antibiotic pressures.

Many thanks for helping us to clarify this section. We now refer to a genetic example from our previous publication in the revised text.

7) Line 91: Replace "As before" with "The new" and add "expected to contribute to collateral sensitivity…". As written, it is confusing what was previously tested and what the new design and expectations are in this study.

Many thanks. This is now corrected.

8) Line 97: Similar to above. Add "expected to be dominated by cross-resistance…" Helps to clarify past from present.

Corrected.

9) The term "season" is confusing and often used interchangeably with "transfer". I prefer the term transfer and suggest keeping this terminology throughout.

Many thanks for this suggestion, which we now followed throughout the revised manuscript.

10) Figure Supplemental 1, Table1: I like the extra information about the stock concentration and storage. It would also be helpful if the drugs were ordered by class and maybe even identified using the BL, FQ, AG abbreviations.

We followed the advice of the reviewer and changed the table accordingly.

11) Figure 2: Please describe that the dotted lines represent the three growth phases in A. For B, it is unclear what the arrows signify. It looks like they are aligned with transfers 12 and 48. Either make them more continuous or remove them altogether. Otherwise, it looks as if the populations are what is being highlighted. Also, what is the significance of the +1 to -0.5 range vs the 16x to 0.33x? The MIC column is most explanatory to me.

Many thanks for your suggestions.

First, we now describe that the dotted lines indicate the three growth phases in the text (line 165-166) and in the figure legend.

Second, we have replaced the misleading arrows by curled brackets to more clearly indicate that all adjacent columns characterize the same transfer (see revised Figure 2).

Third, we completely agree with the reviewer that the MIC column of the resistance scale is more intuitive. However, the visualized resistance scale that ranges from -0.5 to +1 is a better representation of the observed biological variation as it captures not only the x-axis intercept, but the entire shape of the dose-response curve. Thus, the used parameter will differentiate sub-populations that have equivalent MIC, but differ in dose-sensitivity or metabolic fitness cost. Still, it is important to know the approximate range of effect sizes, for which we indicated approximate change of MIC corresponding to resistance value. In the revised manuscript, we now provide more information about the double scaling of the resistance quantification in the new Figure 2—figure supplement 3.

12) Line 192-193: To my eye, the resistance profiles between the early- and mid- phases look pretty similar. The sum of resistances is mentioned. Perhaps add the sum to each of the columns in Figure 2 to make it clearer.

Many thanks for this comment. The reviewer is correct that there is no general increase in resistance across the two considered time points. Instead, resistance changes vary and depend on the exact treatment. In response to the reviewer’s comments, we now adjusted our description of the results, where we now point out that “Resistance to the used β-lactams increased across the two time points only in some treatments, but not all (Figure 2B and C, Figure 2—figure supplement 4, Figure 2—figure supplement 5, Supplementary File 1F), suggesting treatment-dependent evolutionary responses to the antibiotics.”. We also adjusted our conclusions of this part of the result, where we now state that “the population analysis of resistance profiles indicates that resistance evolution depends on the exact treatment protocol and that the dynamics of resistance emergence to DOR may be key for the observed deceleration of beta-lactam adaptation in the fast-regular treatments.”. To enhance clarity, we further added two supplementary figures.

Figure 2—figure supplement 4 shows the early- and mid-phase resistance values side-by-side for each antibiotic in a box plot design. Please see also the related new statistical analysis in Supplementary File 1F that shows which population resistance profiles changed over time.

The sum of resistances is now presented in Figure 2—figure supplement 5, separately for the two considered transfer time points in panels A and B and also separately for three β-lactams (left columns) and the two collateral antibiotics CIP and GEN (right columns). We now refer to this figure to further support our observation of the comparatively high efficacy of irregular sequential protocols in the discussion.

13) Line 210-213: DOR monotherapy is stated to not show a noticeable amount of resistance at the early stage. However, it looks like population 1 shows 4x-8X MIC and looks similar to the mid phase. This is contradictory to what is stated. Can you please rephrase.

Many thanks for this comment. It is based on a misunderstanding of the design of panels B and C of Figure 2, which were apparently not sufficiently clear. In detail, Mono 1 is monotherapy with CAR, mono 2 is monotherapy with DOR, and mono 3 is monotherapy with CEF. For the early time point, the considered isolates from DOR treatment (=2nd row) did not produce any resistances against DOR (=2nd column) and almost no resistance against any of the other drugs. In the revised manuscript, we now describe the design of the figure in more detail in order to prevent any misunderstandings.

14) Line 210-213: It is interesting that monotherapy population one at transfer 12 showed increased resistance but resulted in no variants found. Is it possible that this was a result of heteroresistance or persistence? If you sequenced the whole population instead of isolating three clones, you might be able to distinguish variants at low frequency that would contribute to heteroresistance.

Many thanks for this comment. It shows that our figure was not sufficiently clear, as the comment is based on the same misunderstanding as mentioned above for comment 13. In detail, the results for the DOR monotherapy are shown in the second row of panels B and C of Figure 3 (not the first row). Thus, the isolates from the DOR treatment at the early time point do not show increases in resistance. This is then entirely consistent with our finding that the sequenced genomes for this treatment at the early time point do not bear any resistance mutations. This is something, which we now emphasize in the revised manuscript. These consistent results indicate that evolution of resistance to DOR is constrained, possibly because of a smaller range of genomic targets for DOR resistance. We apologize that the design of Figure 2 panels B and C led to a misunderstanding. As outlined above, we adjusted the design and description of the two panels, in order to prevent such misunderstandings.

We further agree with the reviewer that a more detailed population genomic analysis could have provided further insights into adaptation to DOR monotherapy. The conducted sequencing does not have the resolution to detect low-frequency variants or potential gene amplifications that could confer heteroresistance. However, we are also convinced that such additional genomics data is not essential for our conclusions. Firstly, the phenotypic and genomic results are consistent, as outlined above. Secondly, the indicated lower mutation rate towards DOR resistance was independently validated by fluctuation assays. And this test did indeed confirm the initial finding. This is a point, which we now emphasize more clearly in the Results section. Therefore, we are convinced that our conclusion of a constrained rate of DOR resistance evolution is fully valid and justified and would not receive further support by population sequencing.

Nevertheless, we agree that an understanding of the very early steps of antibiotic adaptation is very important. We are therefore considering to perform a detailed population genomic sequence analysis in the future. Unfortunately, we are currently constrained by consequences of the Corona pandemic, because our sequencing center cannot promise results in the close future. Hence, we are grateful that the eLife guidelines suggest to perform such interesting further analyses separate from the current manuscript with the possibility of linking them in the future. Most importantly, as outlined above, the suggested population genome analyses would be a useful addition, but they are not essential for the final conclusions drawn.

15) Figure 3C. The Blue and black are hard to distinguish as a gradient.

The color scheme has been adjusted to improve clarity.

16) Line 361: Is it treatment potency due to cross resistance or the lack of cross-resistance. I thought that it was due to the latter because neither of the other two drugs promoted cross-resistance to DOR.

Many thanks for this comment. We agree that our previous statement was not sufficiently clear. We now point out that treatment potency was determined by variation in the spontaneous rate of resistance to the β-lactams and by variation in the resulting collateral effects across sequential treatment protocols.

17) Line 414: Is "disadvantageous cross-resistance" the same as collateral sensitivity? If so, the double negative is confusing.

This expression is indeed not very clear. We deleted the word “disadvantageous”, because it is not really needed here.

Recommendations for follow up work

1. Whole genome sequencing was limited to three clones per population. While it is understandable that the number of treatment regimens might make sequencing costly. Population dynamics and allele frequencies may help explain why the population during the DOR monotherapy struggled to acquire mutations before transfer 12.

Many thanks for this recommendation. We agree that a more detailed population genomic analysis of the evolving bacteria could yield important insights into the exact dynamics of resistance evolution. We are indeed planning to perform such analyses in the future – as soon as this is reliably possible again, and we would then be happy to link the results to the current manuscript. We would still like to point out that these population genomic analyses will be insightful, yet they are not essential for our conclusion on the constrained rate of DOR resistance evolution, as explained in detail in our above reply.

2. One of the most interesting aspects of the study is that the principles identified for designing sequential therapies are not dependent on a unique molecular mechanism, and one could therefore envision multiple drug combinations that achieve similar evolution-slowing effects but do so in very different ways at the molecular level (where resistance has often been studied). Do the authors believe there is some inherent feature of β-lactams-for example, the fact that they compromise the cell wall and promote lysis-that may underlie the success of homogeneous sequential therapies? Or might one expect similar success with, say, triplets of fluoroquinolones or other drugs, as long as the triplet is chosen with similar qualitative features (e.g. low levels of cross-resistance, low spontaneous resistance to at least one drug). Relatedly, are there reasons to believe that certain classes of drugs will be more likely to show those qualitative features? It would be remarkable if, for example, these important features could be predicted based on, say, the known diversity of resistant mutations for the component drugs. In any event, I think one of the striking conclusions from this work is that considerable optimization may be possible without detailed knowledge of molecular mechanism.

Many thanks for these comments. We agree that the raised questions would be extremely fascinating to study in more detail. They cannot really be answered based on the current data. In fact, we are in the process of setting up new projects that address for example the last point and also the molecular basis of interactions between different β-lactam drugs. We will hopefully be able to present interesting results on at least these points in the future.

3. The results suggest that the chosen β lactams exhibit negative hysteresis, yet the negative hysteresis does not play a dominant role in slowing resistance (unlike in the authors' previous study with heterogeneous drug combinations). Does this discrepancy arise simply because other effects (e.g. collateral effects) are dominant, so the impacts of negative hysteresis are (by comparison) negligible? Or is there some feature of the negative hysteresis-and perhaps its correlation with the collateral effects-that renders it non-beneficial? I'd venture a guess that in general, there is some underlying relationship between the hysteresis profiles, the collateral profiles, and the timescale of switching that could conceivably be tuned to optimize these therapies. It seems to be an exciting topic for future work.

Again, very interesting points. And again, we cannot answer the raised questions in consideration of the current data. There is clearly a need for an even more systematic analysis on the relationship between hysteresis, collateral effects, and the rate of antibiotic changes. As part of the above mentioned new projects, we do indeed plan to study the molecular basis of hysteresis so that this information could be linked to known mechanisms of collateral sensitivity. So again, we hope to be able to answer at least some of the raised questions with our new projects in the future.

Reviewer #1 (Recommendations for the authors):

The authors conducted an ambitious set of hundreds of evolution experiments with P. aeruginosa exposed to different sets of 3 antibiotics amounting to ~500 generations each, under monotherapy, fast switching, slow switching, and random switching. A primary outcome was extinction, ie failure to persist, while secondary outcomes included the extent and breadth of evolved resistance and a sampling of evolved resistance mutations. Treatments including the antibiotic doripenem led to more extinctions, likely because growth in doripenem reduced the ability to grow in alternative antibiotics, which they term negative hysteresis. This is an interesting and potentially clinically relevant discovery. The paper is well written and engaging.

I see three major limitations that the authors could reasonably address.

1. The sole strain used in this work is P. aeruginosa PA14. This is a good strain choice but the paper seems to presume that this isolate represents the species well, or even more broadly. What is known in the literature about variation within PA or related species in responses to DOR, and/or other drugs in the triple combinations? Is the hysteresis effect limited to this strain?

Many thanks for this comment. We agree that the strain PA14 does not necessarily capture the phenotypes present within the species Pseudomonas aeruginosa and it is also possible that other bacterial species may show different evolutionary responses to the treatments tested. We here use PA14 as a tractable model system. In response to the reviewer’s comment, we now emphasize this point at the end of the introduction. Moreover, some information is indeed available on the response of other P. aeruginosa strains to the three antibiotics used. We now summarized this information in a new paragraph in the discussion. In contrast, hysteresis tests with the considered three antibiotics have thus far only been done by us, and the results are presented in the manuscript. It would indeed be of particular value to assess such hysteresis also in other strains, as we now point out in the final part of the discussion.

2. Given that much of the paper focuses on the doripenem effect, what are the relevant attributes of this drug relative to others? At face value, it's a long-lived carbapenem, so is it the fact that it's a carbapenem, its durability, or both the cause of this physiological stress that diminishes subsequent survival? Might the hysteresis effect simply be the killing of susceptible cells causing subsequent failure?

This is indeed a very interesting point. We now added more information on the characteristics of Doripenem in comparison with the other drugs in the first part of the discussion. Moreover, as indicated above, we now also provide a focused summary of the available information on resistance rate evolution of P. aeruginosa to Doripenem and the other two drugs. As to hysteresis: The used assay is set up to minimize any killing or differential replication of cells during the pretreatment, in order to ensure that any responses seen in the main treatment are based on physiological changes and not selection. This aspect is now highlighted in the Results section in the chapter on hysteresis.

3. A primary conclusion of this paper (L 359-61) is that the spontaneous rate of resistance was a key variable dictating treatment potency. However, this conclusion does not appear to account for the diminished population size of DOR-treated populations and the high potency of this drug relative to others, which would increase the likelihood of error in DOR dosage. While Figure 4 shows that mutation rates to DOR are low, and the methods are appropriate, the results could reflect stronger selection imposed by DOR concentrations and smaller genomic targets to resistance. The authors use this to make their case, but do not adequately discuss the differences between these three β-lactam drugs. In other words, I wonder if PA14 is on a knife-edge in the presence of DOR at the concentrations used, and this reduces population sizes, strengthens selection beyond other drugs, and leads to more variable extinction probabilities. Discussion of the population-genetic consequences of evolution at 0.75x the MIC, and the role of error around this 0.75x threshold, are needed.

Many thanks for this interesting point. However, we politely disagree that a stronger reduction in population size through DOR in comparison to the other drugs could have influenced the results.

Firstly, the dose response curves in the first supplementary figure (Figure 1—figure supplement 1) indicate very little variation in yield upon DOR treatment, especially at the IC75 concentration used to initiate the evolution experiments. Please note the 2nd panel in the top row and the right panel in the middle row; error bars are standard deviation of the mean (using 6 biological replicates) and hardly visible for the IC75 point because of the lack of variation (see red data points). Much more variation was for example found for CAR.

Secondly, the main evolution experiments were initiated using specifically standardized IC75 drug concentrations. Therefore, we do not expect initial variation in population size across treatments. Such variation should only arise thereafter, as a consequence of hysteresis effects or spontaneously evolved resistances and collateral effects.

Thirdly, the same argument holds for the fluctuation assays, which were performed with specifically standardized antibiotic concentrations, leading to identical selection being imposed to capture the spontaneous mutants.

Therefore, we consider it most likely that the lower adaptation to DOR in the monotherapies and the DOR transfer periods within the sequential treatments and also the reduced resistance rate within the fluctuation assays are due to a lower rate of DOR-specific resistance mutations (i.e., smaller genomic targets to DOR resistance) and also a lower rate of cross-resistance to DOR. These arguments are now presented in the revised Results section – in the chapter on the dynamics of resistance for the triple β-lactam treatment, and the chapter on spontaneous resistance rates.

Reviewer #2 (Recommendations for the authors):

Batra, Aditi et al., compared the effectiveness of three in vitro antibiotic drug therapy regimes against the bacterial pathogen Pseudomonas aeruginosa and found that an unlikely drug combination promoted bacterial extinction. Previous studies have shown that using similar drug classes with similar chemistries (eg. β-lactams that inhibit cell wall synthesis) lead to increased cross resistance and therefore, treatment failure. However, the authors found that the fast and sequential use of three β-lactams CAR-CEF-DOR (carbenicillin, cefsulodin, doripenem) was the most effective treatment against P. aeruginosa. The authors further investigated the potential factors contributing to a lack of evolutionary rescue such as negative hysteresis, collateral sensitivity, and rate of spontaneous direct/indirect resistance. By conducting additional experiments to test multiple hypotheses, the authors concluded that the fast-switching three-drug series was constrained by DOR and that the cause of the constraint was due to a low rate of spontaneous resistance and cross-resistance to DOR. These results revitalize the potential use of similar β-lactams for treatment against bacterial pathogens and the utility of predicting evolutionary trajectories to improve antimicrobial drug therapies.

Strengths:

This paper introduces both physiological and evolutionary concepts as possible hypotheses for explaining antibiotic drug constraints. When discussing consequences to drug therapies, cross resistance and collateral sensitivity are often described whereas physiological constraints such as negative hysteresis is overlooked. The experiments conducted in this paper demonstrate how to test for hysteresis using short exposures to a drug and then switching to a different drug and comparing growth killing effects. Drug exposure is carried out quickly to maintain a low mutational load and reduce the probability that a beneficial mutation would arise. Mutations would interfere with a test for non-genetic physiological conditioning.

Measurements of spontaneous resistance rates is a useful phenotype to compare mechanisms of mutations for each drug. The experiments and results that compare the rate of spontaneous direct resistance and indirect (ie. Cross resistance) as well as collateral sensitivity are clear and well replicated.

Weakness:

Whole genome sequencing was limited to three clones per population. While it is understandable that the number of treatment regimens might make sequencing costly. Population dynamics and allele frequencies may help explain why the population during the DOR monotherapy struggled to acquire mutations before transfer 12.

Many thanks for this comment. We agree with the reviewer that a more detailed population genomic analysis could have provided further insights into the absence of resistance changes in the bacteria from the DOR monotherapy treatment. However, such additional genomics data is not essential for our main conclusions. Firstly, the phenotypic and genomic results are consistent, as both demonstrate a lack of resistance to the DOR treatment. Secondly, the phenotypic and current genomic data only provide an indication of a lower mutation rate towards DOR resistance. Additional population genomic data could confirm it, but not validate it. Such a validation may best be achieved by an independent experimental approach, which directly assesses the rate of DOR resistance mutations. This is the reason why we performed such an independent experimental test, using fluctuation assays. This test indeed confirmed the initial finding. Therefore, we consider our conclusion of a constrained rate of DOR resistance evolution to be valid and justified.

Reviewer #3 (Recommendations for the authors):

[…]

This study is timely, important, extremely thorough, and perhaps most importantly, it helps to identify a number of new principles to guide the rational design of sequential antibiotic therapies. It represents a monumental effort to thoroughly characterize adaption to 3-drug cycles in several representative examples, an achievement in itself. But as impressively, the authors draw on GLM's to extract meaningful patterns from the web of complex phenotypic data, and in doing so they uncover features of these treatments (e.g. surprising cross-resistance landscapes between drugs from the same class; potential importance of having one drug with low rates of spontaneous resistance) that may guide general design of new therapies. As with all good studies, it raises a number of questions for future work, and I list a few that come to mind below (that authors may wish to respond, perhaps in the discussion, at their discretion). But in general, I have no major concerns with the study at a technical or conceptual level (a rarity!). I believe this is an excellent contribution to the literature that will interest a broad range of readers in evolutionary biology and microbiology.

1) To me, one of the most interesting aspects of the study is that the principles identified for designing sequential therapies are not dependent on a unique molecular mechanism, and one could therefore envision multiple drug combinations that achieve similar evolution-slowing effects but do so in very different ways at the molecular level (where resistance has often been studied). Do the authors believe there is some inherent feature of β-lactams-for example, the fact that they compromise the cell wall and promote lysis-that may underlie the success of homogeneous sequential therapies? Or might one expect similar success with, say, triplets of fluoroquinolones or other drugs, as long as the triplet is chosen with similar qualitative features (e.g. low levels of cross-resistance, low spontaneous resistance to at least one drug). Relatedly, are there reasons to believe that certain classes of drugs will be more likely to show those qualitative features? It would be remarkable if, for example, these important features could be predicted based on, say, the known diversity of resistant mutations for the component drugs. In any event, I think one of the striking conclusions from this work is that considerable optimization may be possible without detailed knowledge of molecular mechanism.

We share the enthusiasm of the reviewer that the available drugs offer many currently unsuspected options to optimize antibiotic therapy. We agree that the raised questions would be extremely fascinating to study in more detail. They cannot really be answered based on the current data. They do require future additional studies that systematically assess the ability of bacteria to adapt to a variety of multi-drug sequential therapy as well as the underlying mechanisms. Further work is also required to understand the exact molecular basis of interactions between different β-lactam drugs or the general influence of variation in spontaneous resistance to different antibiotics on the efficacy of treatments with these drugs.

2) The results suggest that the chosen β lactams exhibit negative hysteresis, yet the negative hysteresis does not play a dominant role in slowing resistance (unlike in the authors' previous study with heterogeneous drug combinations). Does this discrepancy arise simply because other effects (e.g. collateral effects) are dominant, so the impacts of negative hysteresis are (by comparison) negligible? Or is there some feature of the negative hysteresis-and perhaps its correlation with the collateral effects-that renders it non-beneficial? I'd venture a guess that in general, there is some underlying relationship between the hysteresis profiles, the collateral profiles, and the timescale of switching that could conceivably be tuned to optimize these therapies. It seems to be an exciting topic for future work.

Many thanks for these interesting comments. Again, the raised questions cannot really be answered in consideration of the current data. There is clearly a need for a more systematic analysis of the relationship between hysteresis, collateral effects, and additionally the rate of antibiotic switches. Our earlier work found a two-component regulator cpxS to contribute to negative hysteresis, but only mildly to resistance, possibly suggesting that hysteresis relies on molecular processes that are distinct to those mediating resistance or collateral sensitivity. However, this is only a single case and more examples need to be characterized in detail before general conclusions can be drawn

https://doi.org/10.7554/eLife.68876.sa2

Article and author information

Author details

  1. Aditi Batra

    1. Department of Evolutionary Ecology and Genetics, University of Kiel, Kiel, Germany
    2. Max Planck Institute for Evolutionary Biology, Ploen, Germany
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Roderich Roemhild
    Competing interests
    No competing interests declared
  2. Roderich Roemhild

    1. Department of Evolutionary Ecology and Genetics, University of Kiel, Kiel, Germany
    2. Max Planck Institute for Evolutionary Biology, Ploen, Germany
    3. Institute of Science and Technology, Klosterneuburg, Austria
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Aditi Batra
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9480-5261
  3. Emilie Rousseau

    Borstel Research Centre, National Reference Center for Mycobacteria, Borstel, Germany
    Contribution
    Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Sören Franzenburg

    Competence Centre for Genomic Analysis Kiel, University of Kiel, Kiel, Germany
    Contribution
    Investigation, Project administration, Whole genome sequencing
    Competing interests
    No competing interests declared
  5. Stefan Niemann

    Borstel Research Centre, National Reference Center for Mycobacteria, Borstel, Germany
    Contribution
    Supervision, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6604-0684
  6. Hinrich Schulenburg

    1. Department of Evolutionary Ecology and Genetics, University of Kiel, Kiel, Germany
    2. Max Planck Institute for Evolutionary Biology, Ploen, Germany
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    hschulenburg@zoologie.uni-kiel.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1413-913X

Funding

Deutsche Forschungsgemeinschaft (SCHU 1415/12-2)

  • Hinrich Schulenburg

Deutsche Forschungsgemeinschaft (EXC 2167-390884018)

  • Stefan Niemann
  • Hinrich Schulenburg

Deutsche Forschungsgemeinschaft (GRK 2501)

  • Stefan Niemann
  • Hinrich Schulenburg

Max-Planck-Gesellschaft (IMPRS Stipend)

  • Aditi Batra

Max-Planck-Gesellschaft (Fellowship)

  • Hinrich Schulenburg

Leibniz-Gemeinschaft (EvoLUNG)

  • Stefan Niemann
  • Hinrich Schulenburg

Deutsche Forschungsgemeinschaft (407495230)

  • Sören Franzenburg

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank Leif Tueffers and João Botelho for discussions and suggestions as well as Kira Haas and Julia Bunk for technical support. We acknowledge financial support from the German Science Foundation (grant SCHU 1415/12-2 to HS, and funding under Germany’s Excellence Strategy EXC 2167-390884018 as well as the Research Training Group 2501 TransEvo to HS and SN), the Max-Planck Society (IMPRS scholarship to AB; Max-Planck fellowship to HS), and the Leibniz Science Campus Evolutionary Medicine of the Lung (EvoLUNG, to HS and SN). This work was further supported by the German Science Foundation Research Infrastructure NGS_CC (project 407495230) as part of the Next Generation Sequencing Competence Network (project 423957469). NGS analyses were carried out at the Competence Centre for Genomic Analysis Kiel (CCGA Kiel).

Senior Editor

  1. George H Perry, Pennsylvania State University, United States

Reviewing Editor

  1. Vaughn S Cooper, University of Pittsburgh, United States

Reviewer

  1. Vaughn S Cooper, University of Pittsburgh, United States

Publication history

  1. Received: March 29, 2021
  2. Accepted: July 22, 2021
  3. Accepted Manuscript published: July 28, 2021 (version 1)
  4. Version of Record published: August 31, 2021 (version 2)

Copyright

© 2021, Batra et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,365
    Page views
  • 179
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Evolutionary Biology
    2. Epidemiology and Global Health
    3. Microbiology and Infectious Disease
    4. Genetics and Genomics
    Edited by George H Perry et al.
    Collection

    eLife is pleased to present a Special Issue to highlight recent advances in the growing and increasingly interdisciplinary field of evolutionary medicine.

    1. Evolutionary Biology
    2. Microbiology and Infectious Disease
    Emily R Ebel et al.
    Research Article

    The replication of Plasmodium falciparum parasites within red blood cells (RBCs) causes severe disease in humans, especially in Africa. Deleterious alleles like hemoglobin S are well-known to confer strong resistance to malaria, but the effects of common RBC variation are largely undetermined. Here we collected fresh blood samples from 121 healthy donors, most with African ancestry, and performed exome sequencing, detailed RBC phenotyping, and parasite fitness assays. Over one third of healthy donors unknowingly carried alleles for G6PD deficiency or hemoglobinopathies, which were associated with characteristic RBC phenotypes. Among non-carriers alone, variation in RBC hydration, membrane deformability, and volume was strongly associated with P. falciparum growth rate. Common genetic variants in PIEZO1, SPTA1/SPTB, and several P. falciparum invasion receptors were also associated with parasite growth rate. Interestingly, we observed little or negative evidence for divergent selection on non-pathogenic RBC variation between Africans and Europeans. These findings suggest a model in which globally widespread variation in a moderate number of genes and phenotypes modulates P. falciparum fitness in RBCs.