Fast Evolution of SOS-Independent Multi-Drug Resistance in Bacteria

  1. Le Zhang
  2. Yunpeng Guan
  3. Yuen Yee Cheng
  4. Nural N. Cokcetin
  5. Amy L. Bottomley
  6. Andrew Robinson
  7. Elizabeth J. Harry
  8. Antoine van Oijen
  9. Qian Peter Su
  10. Dayong Jin

  • Curated by eLife

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    eLife assessment

    This useful study examines how deletion of a major DNA repair gene in bacteria may facilitate the rise of mutations that confer resistance against a range of different antibiotics. Although the phenotypic evidence is intriguing, the interpretation of the phenotypic data presented and the proposed mechanism by which these mutations are generated are incomplete, relying on untested assumptions and suboptimal methodology. If substantially improved, this work could be of interest to microbiologists studying antibiotic resistance, genome integrity, and evolution, but as yet is of unclear significance.

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Abstract

The killing mechanism of many antibiotics involves the induction of DNA damage, either directly or indirectly, which triggers the SOS response. RecA, the master regulator of the SOS response, plays a crucial role in driving the evolution of resistance to fluoroquinolone antibiotics treated with a single dose of ciprofloxacin. However, the precise roles of RecA and SOS responses in the development of resistance under short-term β-lactam exposure remain unclear. In the present study, we observed a fast evolution of β-lactam resistance (20-fold increase in MIC in 8 hours) in E. coli after deleting RecA and exposing the bacteria to a single dose of ampicillin. Notably, once this type of resistance is established, it remains stable and can be passed on to subsequent generations. Contrary to previous findings, it is shown that this accelerated resistance development process is dependent on the hindrance of DNA repair, which is completely orthogonal to the SOS response. Additionally, we identified the rapid emergence of drug resistance associated mutations in the resistant bacterial genome, indicating the impairment of DNA repair. Through comprehensive transcriptome sequencing, we discovered that the expression of numerous antioxidative response genes is repressed in recA mutant resistant isolates, resulting in an excessive accumulation of ROS within the cells. This suggests that the induction of ROS drives the fast evolution of antibiotic resistance in RecA-deficient bacteria. Collectively, we show that the hindrance of DNA repair hampers cellular fitness, provides bacteria with genetic adaptability to survive in diverse stressful environments, and accelerates the evolution of antibiotic resistance.

Article activity feed

  1. Version published to 10.7554/elife.95058.1 on eLife
  2. Version published to 10.7554/elife.95058 on eLife
  3. eLife

    eLife assessment

    This useful study examines how deletion of a major DNA repair gene in bacteria may facilitate the rise of mutations that confer resistance against a range of different antibiotics. Although the phenotypic evidence is intriguing, the interpretation of the phenotypic data presented and the proposed mechanism by which these mutations are generated are incomplete, relying on untested assumptions and suboptimal methodology. If substantially improved, this work could be of interest to microbiologists studying antibiotic resistance, genome integrity, and evolution, but as yet is of unclear significance.

  4. eLife

    Reviewer #1 (Public Review):

    Summary:
    Jin et al. investigated how the bacterial DNA damage (SOS) response and its regulator protein RecA affect the development of drug resistance under short-term exposure to beta-lactam antibiotics. Canonically, the SOS response is triggered by DNA damage, which results in the induction of error-prone DNA repair mechanisms. These error-prone repair pathways can increase mutagenesis in the cell, leading to the evolution of drug resistance. Thus, inhibiting the SOS regulator RecA has been proposed as a means to delay the rise of resistance.

    In this paper, the authors deleted the RecA protein from E. coli and exposed this ∆recA strain to selective levels of the beta-lactam antibiotic, ampicillin. After an 8-hour treatment, they washed the antibiotic away and allowed the surviving cells to recover in regular media. They then measured the minimum inhibitory concentration (MIC) of ampicillin against these treated strains. They note that after just 8-hour treatment with ampicillin, the ∆recA had developed higher MICs towards ampicillin, while by contrast, wild-type cells exhibited unchanged MICs. This MIC increase was also observed in subsequent generations of bacteria, suggesting that the phenotype is driven by a genetic change.

    The authors then used whole genome sequencing (WGS) to identify mutations that accounted for the resistance phenotype. Within resistant populations, they discovered key mutations in the promoter region of the beta-lactamase gene, ampC; in the penicillin-binding protein PBP3 which is the target of ampicillin; and in the AcrB subunit of the AcrAB-TolC efflux machinery. Importantly, mutations in the efflux machinery can impact the resistance towards other antibiotics, not just beta-lactams. To test this, they repeated the MIC experiments with other classes of antibiotics, including kanamycin, chloramphenicol, and rifampicin. Interestingly, they observed that the ∆recA strains pre-treated with ampicillin showed higher MICs towards all other antibiotics tested. This suggests that the mutations conferring resistance to ampicillin are also increasing resistance to other antibiotics.

    The authors then performed an impressive series of genetic, microscopy, and transcriptomic experiments to show that this increase in resistance is not driven by the SOS response, but by independent DNA repair and stress response pathways. Specifically, they show that deletion of the recA reduces the bacterium's ability to process reactive oxygen species (ROS) and repair its DNA. These factors drive the accumulation of mutations that can confer resistance to different classes of antibiotics. The conclusions are reasonably well-supported by the data, but some aspects of the data and the model need to be clarified and extended.

    Strengths:
    A major strength of the paper is the detailed bacterial genetics and transcriptomics that the authors performed to elucidate the molecular pathways responsible for this increased resistance. They systemically deleted or inactivated genes involved in the SOS response in E. coli. They then subjected these mutants to the same MIC assays as described previously. Surprisingly, none of the other SOS gene deletions resulted in an increase in drug resistance, suggesting that the SOS response is not involved in this phenotype. This led the authors to focus on the localization of DNA PolI, which also participates in DNA damage repair. Using microscopy, they discovered that in the RecA deletion background, PolI co-localizes with the bacterial chromosome at much lower rates than wild-type. This led the authors to conclude that deletion of RecA hinders PolI and DNA repair. Although the authors do not provide a mechanism, this observation is nonetheless valuable for the field and can stimulate further investigations in the future.

    In order to understand how RecA deletion affects cellular physiology, the authors performed RNA-seq on ampicillin-treated strains. Crucially, they discovered that in the RecA deletion strain, genes associated with antioxidative activity (cysJ, cysI, cysH, soda, sufD) and Base Excision Repair repair (mutH, mutY, mutM), which repairs oxidized forms of guanine, were all downregulated. The authors conclude that down-regulation of these genes might result in elevated levels of reactive oxygen species in the cells, which in turn, might drive the rise of resistance. Experimentally, they further demonstrated that treating the ∆recA strain with an antioxidant GSH prevents the rise of MICs. These observations will be useful for more detailed mechanistic follow-ups in the future.

    Weaknesses:
    Throughout the paper, the authors use language suggesting that ampicillin treatment of the ∆recA strain induces higher levels of mutagenesis inside the cells, leading to the rapid rise of resistance mutations. However, as the authors note, the mutants enriched by ampicillin selection can play a role in efflux and can thus change a bacterium's sensitivity to a wide range of antibiotics, in what is known as cross-resistance. The current data is not clear on whether the elevated "mutagenesis" is driven ampicillin selection or by a bona fide increase in mutation rate.

    Furthermore, on a technical level, the authors employed WGS to identify resistance mutations in the treated ampicillin-treated wild-type and ∆recA strains. However, the WGS methodology described in the paper is inconsistent. Notably, wild-type WGS samples were picked from non-selective plates, while ΔrecA WGS isolates were picked from selective plates with 50 μg/mL ampicillin. Such an approach biases the frequency and identity of the mutations seen in the WGS and cannot be used to support the idea that ampicillin treatment induces higher levels of mutagenesis.

    Finally, it is important to establish what the basal mutation rates of both the WT and ∆recA strains are. Currently, only the ampicillin-treated populations were reported. It is possible that the ∆recA strain has inherently higher mutagenesis than WT, with a larger subpopulation of resistant clones. Thus, ampicillin treatment might not in fact induce higher mutagenesis in ∆recA.

  5. eLife

    Reviewer #2 (Public Review):

    Summary:
    This study aims to demonstrate that E. coli can acquire rapid antibiotic resistance mutations in the absence of a DNA damage response. To investigate this, the authors employed a sophisticated experimental framework based on a modified Adaptive Laboratory Evolution (ALE) workflow. This workflow involves numerous steps culminating in the measurement of antibiotic resistance. The study presents evidence that a recA strain develops ampicillin resistance mutations more quickly than the wild-type, as shown by measuring the Minimum Inhibitory Concentration (MIC) and mutation frequency. Whole-genome sequencing of 15 recA- colonies resistant to ampicillin revealed predominantly inactivation of genes involved in the multi-drug efflux pump system, whereas, in the wild-type, mutations appear to enhance the activity of the chromosomal ampC cryptic promoter. By analyzing mutants involved in the SOS response, including a lexA3 mutant incapable of inducing the SOS response, the authors conclude that the rapid evolution of antibiotic resistance occurs in an SOS-independent manner when recA is absent.

    Furthermore, RNA sequencing (RNA-seq) of the four experimental conditions suggests that genes related to antioxidative responses drive the swift evolution of antibiotic resistance in the recA- strain.

    Weaknesses:
    However, a potential limitation of this study is the experimental design used to determine the 'rapid' evolution of antibiotic resistance. It may introduce a significant bottleneck in selecting ampicillin-resistant mutants early on. A recA mutant could be more susceptible to ampicillin than the wild-type, and only resistant mutants might survive after 8 hours, potentially leading to their enrichment in subsequent steps. To address this concern, it would be critical to perform a survival analysis at various time points (0h, 2h, 4h, 6h, and 8h) during ampicillin treatment for both recA and wild-type strains, ensuring there is no difference in viability.

    The observation that promoter mutations are absent in recA strains could be explained by previous research indicating that amplification of the AmpC genes is a mechanism for E. coli resistance to ampicillin, which does not occur in a recA-deficient background (PMID# 19474201).

    The section describing Figure 3 is poorly articulated, and the conclusions drawn are apparent. The inability of a recA strain to induce the SOS response is well-documented (lines 210 and 278). The data suggest that merely blocking SOS induction is insufficient to cause 'rapid' evolution in their experimental conditions. To investigate whether SOS response can be induced independently of lexA cleavage by recA, alternative experiments, such as those using a sulA-GFP fusion, might be more informative.

    In Figure 4E, the lack of increased SulA gene expression in the wild-type strain treated with ampicillin is unexpected, given that SulA is an SOS-regulated gene. The fact that polA (Pol I) is going down should be taken into account in the interpretation of Figures 2D and 2E.

    The connection between compromised DNA repair, the accumulation of Reactive Oxygen Species (ROS) based on RNA-seq data, and accelerated evolution is merely speculative at this point and not experimentally established.

  6. eLife

    Reviewer #3 (Public Review):

    Summary:
    In the present work, Zhang et al investigate the involvement of the bacterial DNA damage repair SOS response in the evolution of beta-lactam drug resistance evolution in Escherichia coli. Using a combination of microbiological, bacterial genetics, laboratory evolution, next-generation, and live-cell imaging approaches, the authors propose short-term drug resistance evolution that can take place in RecA-deficient cells in an SOS response-independent manner. They propose the evolvability of drug resistance is alternatively driven by the oxidative stress imposed by the accumulation of reactive oxygen species and inhibition of DNA repair. Overall, this is a nice study that addresses a growing and fundamental global health challenge (antimicrobial resistance). However, although the authors perform several multi-disciplinary experiments, there are several caveats to the authors' proposal that ultimately do not fully support their interpretation that the observed antimicrobial resistance evolution phenotype is due to compromised DNA repair.

    Strengths:
    The authors introduce new concepts to antimicrobial resistance evolution mechanisms. They show short-term exposure to beta-lactams can induce durably fixed antimicrobial resistance mutations. They propose this is due to comprised DNA repair and oxidative stress. This is primarily supported by their observations that resistance evolution phenotypes only exist for recA deletion mutants and not other genes in the SOS response

    Weaknesses:
    The authors do not show any direct evidence (1) that these phenotypes exist in strains harboring deletions in other DNA repair genes outside of the SOS response, (2) that DNA damage is increased, (3) that reactive oxygen species accumulate, (4) that accelerated resistance evolution can be reversed by anything other than recA complementation. The authors do not directly test alternative hypotheses. The conclusions drawn are therefore premature.

  7. Version published to 10.1101/2022.03.29.486198v3 on bioRxiv
  8. Version published to 10.1101/2022.03.29.486198v2 on bioRxiv
  9. Version published to 10.1101/2022.03.29.486198v1 on bioRxiv