The Role of Selected Bacteria in Breast Cancer Initiation and Development

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The reviewed article is initially attractive based on its title; however, unfortunately, the remaining content is of very poor quality and does not meet the expected scientific standard.

1. A substantial part of the manuscript consists of general, textbook-style descriptions that are not directly related to breast cancer initiation or progression. These include classifications of Fusobacteriaceae, serotypes and phylogroups of Escherichia coli, descriptions of toxigenic bacterial strains, cytokine responses, drug toxicity, as well as virulence factors and pathogenic mechanisms of Staphylococcus aureus and Clostridium species, including Clostridioides difficile-associated diarrhea. The paper reads more like a general microbiology review rather than a focused discussion on the relationship between bacteria and breast cancer development.
2. The majority of the cited studies are based on in vitro experiments (cell cultures) or in vivo animal models, mainly murine systems. It is well established that introducing bacteria at high concentrations into experimental systems can induce inflammatory responses and cytokine production in both animal and human cells. The most critical limitation of the manuscript is the lack of direct evidence from human clinical samples, particularly the absence of data demonstrating the presence, localization, or activity of bacteria within breast tumor tissues from actual patients. Without such clinical validation, the conclusions remain largely hypothetical and speculative.
3. The authors further propose potential mechanisms of bacterial transmission, for example from the gut microbiota to breast tissue, which could hypothetically contribute to tumor development. However, these proposed pathways are not convincingly supported by empirical evidence. The manuscript does not adequately explain how such translocation would occur under physiological conditions. For instance, if systemic dissemination were involved, one would expect clinical manifestations such as bacteremia or sepsis, which are not addressed. Alternatively, suggestions that bacteria may be transferred via breastfeeding remain purely speculative and are not supported by clinical or mechanistic data. As such, this section of the manuscript appears highly conjectural.
4. The language quality of the manuscript is suboptimal. Several sentences are grammatically incorrect, lack verbs, or appear incomplete, which reduces overall readability and scientific clarity. In some sections, the structure of the text is difficult to follow, and the narrative does not maintain a consistent logical flow. This further weakens the impact of the scientific content.
5. If the authors aim to strengthen the manuscript, it would be highly beneficial to include well-designed figures or schematic diagrams illustrating the proposed mechanisms linking bacterial presence to breast cancer development, based on human clinical data. The existing patient-based studies, would significantly improve the scientific value and credibility of the work.

Comments on the Quality of English Language

The language quality of the manuscript is suboptimal. Several sentences are grammatically incorrect, lack verbs, or appear incomplete, which reduces overall readability and scientific clarity. In some sections, the structure of the text is difficult to follow, and the narrative does not maintain a consistent logical flow. This further weakens the impact of the scientific content.

Author Response

Please, find the attached response.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present a comprehensive review of the emerging role of the microbiome in breast cancer pathogenesis, focusing specifically on five prominent bacterial taxa: Fusobacterium nucleatum, Escherichia coli, Bacteroides fragilis, Staphylococcus spp., and Clostridium spp. The manuscript is timely, as the intersection of microbial dysbiosis and oncology is a rapidly evolving and highly relevant field.

However, the authors treat the presence of intratumoral bacteria as an absolute, undisputed fact. They cite high-profile papers to support the existence of a tumor microbiome, but they completely ignore the seismic pushback the field has faced over the last three years. There is a highly active, ongoing debate regarding false positives in low-biomass microbiome sequencing. Recent landmark critiques (such as those by the Salzberg group and the re-evaluations published in Nature in 2023/2024) have demonstrated that many "cancer microbiome signatures" are actually artifacts of reagent contamination (the "kitome"), cross-contamination during batch processing, or misalignment of human genomic sequences to bacterial databases. The authors must acknowledge this controversy. One cannot discuss the translocation of E. coli or Clostridium into breast tissue without addressing the stringent contamination controls required to prove these bacteria are biologically active in the tumor microenvironment, rather than just sequencing noise.

The manuscript discusses dysbiosis and the "gut-breast axis" entirely in a vacuum. When evaluating pathogenesis and infectious diseases in an oncology setting, understanding the selective pressures at play is non-negotiable. Breast cancer patients are frequently subjected to intensive antibiotic regimens. The manuscript completely ignores how this routine clinical intervention drives the very dysbiosis it describes. Furthermore, the absence of any discussion regarding antimicrobial resistance (AMR) is a major blind spot. If opportunistic pathogens like S. aureus or E. coli are driving metastasis or chemotherapy resistance, how do multidrug-resistant strains compound this issue? The authors must incorporate the clinical reality of antibiotic pressure. They should address how antimicrobial stewardship programs might intersect with microbiome-targeted oncological therapies. Treating the breast microbiome as if it exists outside the reality of a modern hospital environment severely limits the paper's translational value.

While the paper lists several virulence factors (e.g., Fap2 binding to Gal-GalNAc, FadA binding to E-cadherin), the mechanistic depth is lacking. The authors state that these interactions happen, but they omit recent advancements in structural microbiology that explain how they happen. There is no discussion of the conformational changes, thermodynamics, or protein-protein interfaces that govern these host-pathogen interactions. For instance, how do the structural variations in F. nucleatum subspecies alter binding affinities at the molecular level? The authors need to expand on the precise molecular mechanisms and structural interactions driving these phenomena. If the goal is "microbiome-targeted therapies" as their conclusion states, the molecular targets must be thoroughly characterized.

The conclusion is somewhat brief and generic. It would be stronger if it briefly reiterated which specific bacteria are associated with which primary mechanisms (e.g., E. coli with genotoxicity, Clostridium with metabolic alteration) rather than solely providing a high-level summary.

Author Response

Please, find the attached response.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have replied to all the concerns and questions. The text has been modified and improved. The manuscript can be accepted in present form.