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Summary

This paper introduces a novel technology using chromosome-free, non-replicating bacterial cells called SimCells for targeted pathogen elimination. This technology is very relevant as it addresses antimicrobial resistance which is projected cause millions of deaths annually. SimCells and mini-SimCells are E. coli derivatives, 1-2 micrometers and 100-400 nanometers respectively, that integrate a three-part killing system: Nanobodies that selectively bind to target bacteria, a Type VI Secretion System needle that injects toxic proteins into bad bacteria, and the NahG enzyme that generates hydrogen peroxide from aspirin. The researchers tested SimCells in various in vitro environments and found that SimCells killed 94% of target E. coli within 24 hours and 99% by 48 hours, all while leaving non-target bacteria mostly alone. This technology was developed in 2020 by Oxford scientists (the same authors of this paper) as a live drug that could bind and kill cancer cells. It is important to note that the technology led to the creation of a spin-out company called Oxford SimCell, which could implicate potential private interests.

Major Concerns

• While the potential application for this technology is vast, the approach relies on having clearly identified surface markers on pathogens. Since some bacteria change or lose surface antigens, it could be possible for some to escape targeting. In clinical applications, this would be limiting because you cannot compete with broad-spectrum antibiotics that you can administer while waiting for cultures.

• A major aspect of the safety of this new technology is the fact that SimCells must be non-replicative to avoid potential danger to hosts. The paper repeatedly claims that these cells are chromosome-free, but the evidence does not prove that with 100% certainty. DNA spectroscopy exhibits a definite decrease in DNA presence but doesn’t guarantee 100% elimination.

  • The authors should address this limitation in their manuscript using more sensitive techniques like qPCR, which could significantly strengthen the claim, even though no method can guarantee absolute elimination.

• The language surrounding the potential for DNA escape is slightly misleading. The paper observed no DNA survival and growth out of 100 million cell plates, which would mean the escape frequency is less than 1-in-100-million. While the paper repeatedly states that this meets the NIH-recommended escape frequency threshold, it’s important not to equate that replication rate to 0 – the authors should include a statistical test for near absolute elimination within a confidence interval rather than implying a sense of certainty.

• For the NahG enzyme mechanism, the paper doesn’t truly explore the presence of hydrogen peroxide to kill bacteria. It seems that the brown color from catechol was used to infer that the conversion of aspirin to h202 worked, since catechol is an intermediary.

  • The authors should include a standard H2O2 detection assay like Amplex Red fluorescence Assay or Borinic Acid-Based Fluorogenic Probes (2).

• The synergy between Type 6 SS and the NahG mechanism wasn’t thoroughly mathematically quantified. While the beneficial relationship of both bacteria killing mechanisms acting simultaneously was evident, the authors should perform additional statistical tests to strengthen the dual-mechanism concept which is a huge advantage for this new platform.

• While this paper functions more as a proof-of concept work, I think the lack of in vivo data is a huge gap because we have no idea how the immune system would respond and the realistic penetration of these non-replicative cells. Also, the NahG mechanism would require significant aspirin presence but could limit use in certain portions of the population (children, pregnancy, etc), which are limitations the authors should address in their discussion section.

Strengths

• Application of this relatively new technology to achieve 97% elimination of a clinically relevant strain of E. coli is quite promising. The results in this experiment prove that SimCells can have an immediate beneficial impact in the real world, rather than generic lab strains.

• The paper uses appropriate negative controls with inactive T6SS, non-engineered mini-SimCells, and antigen-negative prey strains. This promotes good science by separating correlation from causation.

• The strong selective capability of SimCells using surface nanobodies is a huge bonus because most antimicrobial technologies kill even the good bacteria which can cause host complications. This 1000-fold reduction in target bacteria without affecting non-target strains is impressive.

Overall Thoughts

This is well-written paper that provides compelling proof-of-concept for a groundbreaking technology with much immediate clinical potential. I appreciated how every claim is backed by some sort of quantitative measurement. The detailed and specific methods sections contribute to readers’ understanding of the paper and reproducibility of the science. With precise targeting, dual antimicrobial mechanisms, and low risk for replication or further resistance, SimCells are a strong candidate for future antimicrobial therapies. While further research addressing the gaps I outlined would strengthen the case for clinical translation, the current work lays a strong foundation. Overall, I believe this paper merits publication to facilitate continued advancement of the technology.

References:

1.     “Reprogrammable Cells May Be the Future of Cancer Treatment.” Revolutionizing Cancer Treatment: Reprogrammable Cells Unlock New Possibilities, 12 June 2020, eng.ox.ac.uk/news/reprogrammable-cells-may-be-the-future-of-cancer-treatment.

2.     Pucher M, Makenthirathasan K, Jalaber H, LeSaux T, Nüsse O, Doisneau G, Bourdreux Y, Gatin-Fraudet B, Jullien L, Vauzeilles B, Guianvarc'h D, Erard M, Urban D. Borinic Acid-Based Fluorogenic Probes as an Alternative to the Amplex Red Assay for Real-Time H₂O₂ Monitoring in Live Cells. ACS Chem Biol. 2025 Jul 18;20(7):1574-1583. doi: 10.1021/acschembio.5c00156. Epub 2025 Jun 25. PMID: 40560655.

3.     C. Fan, P.A. Davison, R. Habgood, H. Zeng, C.M. Decker, M. Gesell Salazar, K. Lueangwattanapong, H.E. Townley, A. Yang, I.P. Thompson, H. Ye, Z. Cui, F. Schmidt, C.N. Hunter, & W.E. Huang, Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology, Proc. Natl. Acad. Sci. U.S.A. 117 (12) 6752-6761, https://doi.org/10.1073/pnas.1918859117 (2020).

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.