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Review 2: "Engineering Probiotic Escherichia Coli Nissle 1917 to Block Transfer of Multiple Antibiotic Resistance Genes by Exploiting a Type I CRISPR-Cas System"

The reviewers found the study reliable to strong, highlighting its innovative approach to engineering probiotic E. coli Nissle 1917 to block antibiotic resistance gene transfer using a type I CRISPR-Cas system.

Published onJul 16, 2024
Review 2: "Engineering Probiotic Escherichia Coli Nissle 1917 to Block Transfer of Multiple Antibiotic Resistance Genes by Exploiting a Type I CRISPR-Cas System"
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key-enterThis Pub is a Review of
Engineering probiotic Escherichia coli Nissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I CRISPR-Cas system
Engineering probiotic Escherichia coli Nissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I CRISPR-Cas system
Description

Abstract Many multidrug-resistant (MDR) bacteria evolved through accumulation of antibiotic-resistance genes (ARGs). Although the potential risk of probiotics as reservoirs of ARGs has been recognized, strategies for blocking transfer of ARGs while using probiotics have rarely been explored. The probiotic Escherichia coli Nissle 1917 (EcN) has long been used for treating intestinal diseases. Here, we showed frequent transfer of ARGs into EcN both in vitro and in vivo, raising its potential risk of accumulating antibiotic resistance. Given that no CRISPR-Cas system is found in natural EcN, we integrated the endogenous type I-E CRISPR-Cas system derived from E. coli BW25113 into EcN, and showed that the engineered EcN was able to efficiently cleave multiple ARGs (i.e., mcr-1, blaNDM-1 and tet(X)). By co-incubation of EcN expressing Cas3-Cascade and that expressing Cas9, we showed that the growth of the former strain outcompeted the latter strain, demonstrating better clinical application prospect of EcN expressing the type I-E CRISPR-Cas system. Finally, the engineered EcN exhibited immunity against transfer of targeted ARGs in the intestine of a model animal (i.e. zebrafish). Our work provides a new strategy for restricting transfer of ARGs in EcN, paving the way for safe use of this probiotic and development of probiotics as living therapeutics.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review:Β The manuscript presents an innovative approach to engineering a probiotic E. coli strain to combat antibiotic resistance gene transfer. However, several aspects of the experimental design, results interpretation, and potential implications require clarification and further investigation. Addressing these points will strengthen the manuscript and its contributions to the field.

Major concerns:

  1. The use of two plasmids induced by the arabinose promoter in Figure 4 raises concerns about potential gene expression selectivity issues. The reviewer recommends testing different concentrations of arabinose to mitigate this.

  2. The manuscript employs a type I CRISPR-Cas system, which is more complex due to the requirement of multiple Cas protein encoding. The reviewer suggests comparing this system with Cas12, which is smaller and less toxic, and to highlight the innovative aspects of the chosen system.

  3. Although the CRISPR system is said to have minimal impact on the gut microbiome, a systematic study of its effects on the gut microbiota's structure and function is recommended.

  4. It is recommended that the rationale for the deletion of the hns gene be elucidated, along with additional details about this gene to enhance the comprehension of the experimental procedures.

  5. Appreciation is expressed for the simplification of the CRISPR system through the reduction of plasmid numbers. However, it would be beneficial to discuss why a plasmid-free system was not considered, and to address the potential adaptive costs associated with integrating large genetic elements into the genome.

Minor comments:

  1. The manuscript would benefit from specifying the number of times each experiment was replicated to ensure the reliability and reproducibility of the results.

  2. The authors have chosen pHG101 as the study plasmid without providing a rationale for its selection. A brief background description of this plasmid and the reasons for its choice would be valuable.

  3. The application of the GFP plasmid seems redundant since the donor strain is auxotrophic. The reviewer suggests considering the integration of the GFP fluorescence directly into the chromosome of the recipient strain to avoid complications from multiple plasmid maintenance.

  4. It is suggested that key colonization data of Nissle 1917 in the gut be presented to validate the assertion that effective ARG immunity is predicated on successful colonization.

  5. An explanation is called for regarding the upward trend in conjugation frequencies depicted in Figure 6. Additionally, the effectiveness of ARG blocking should be scrutinized, particularly if the donor strain WM3064 demonstrates inadequate colonization capabilities.

  6. Clarification is sought on the capacity of the CRISPR array to incorporate multiple spacer sequences and to understand the maximum number that could be supported.

  7. The use of resistance genes as selection markers within the ARG defense strategy is highlighted as a paradox. It would be advantageous to outline how this issue is intended to be resolved to facilitate seamless delivery.

  8. The observed effectiveness of the endogenous CRISPR system in blocking ARGs is acknowledged. Suggestions include further optimization of the system or the development of an activator to enhance in situ ARG blocking.

Comments
1
Sarah John:

To stop the spread of antibiotic resistance, it is essential to stop the spread of superbugs. By taking this method, the spread of resistant bacteria can be considerably inhibited, maintaining the efficacy of currently available medicines. Resources for online dissertation writing can offer insightful analysis and helpful guidance to students who are investigating this issue. Innovative approaches in medical research may be made possible by comprehending the mechanisms underlying resistance transfer.