Review Reports - <i>Mycobacterium avium</i> Subspecies <i>paratuberculosis</i> and Colorectal Cancer: Putting MAP on the Map

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article discusses a possible link between Mycobacterium avium subsp. paratuberculosis
and colorectal cancer. The author suggests that MAP may contribute to cancer development through
chronic inflammation, epithelial damage, immune modulation, and possible genomic instability. The
manuscript is mainly built around recent evidence showing MAP detection in precancerous and
cancerous colorectal lesions. Overall, it is an interesting and provocative perspective, but the
argument based just on a hypothesis and would benefit from a more balanced tone regarding
causality.
Before further processing, I have the following major queries:
Query 1: The manuscript suggests that MAP may play a role in CRC development, but the
current evidence mainly shows an association, not causation. The authors should make this
distinction clearer and discuss more carefully whether MAP could also be present because of tumor
related tissue changes, rather than being a cause of them.
Query 2: The perspective tends to support the MAP hypothesis rather strongly and gives
relatively limited attention to contradictory findings, methodological controversies, false positives,
contamination risks, or negative studies. A more balanced discussion is needed. Please address this
problem.
Query 3: One Health section (8) is too weak. Please increase the quality and impact of this
section by adding important scientific data regarding this particularly important concept.
Query 4: The topic is interesting and potentially original. However, the novelty of the
manuscript could be highlighted more clearly. The authors are encouraged to better emphasize what
distinguishes this perspective from previous hypotheses and to more explicitly articulate the added
value of integrating recent evidence linking MAP to precancerous and cancerous colorectal lesions.
Maybe this is the most important aspect that is mandatory to be addressed before further
processing of the manuscript.
Minor: Please be sure that all species names are written in italics (e.g. title).

Author Response

Response to Reviewer 1
Thank you for your valuable suggestions to improve the manuscript.

Query 1: The manuscript suggests that MAP may play a role in CRC development, but the
current evidence mainly shows an association, not causation. The authors should make this distinction clearer and discuss more carefully whether MAP could also be present because of tumor related tissue changes, rather than being a cause of them.

New text:

-Assertions are “softened” throughout:

105: …consistent with possible early involvement in tumorigenesis.

228: Accordingly, the present Perspective should be viewed as hypothesis-generating rather than confirmatory.

-Section 10: An important alternative explanation of the Tehrani study is that evolving neoplastic tissue may create a permissive microenvironment favoring persistence or enrichment of MAP rather than MAP initiating disease.

-The conclusion: At present, available evidence supports association rather than causation

Query 2: The perspective tends to support the MAP hypothesis rather strongly and gives
relatively limited attention to contradictory findings, methodological controversies, false positives, contamination risks, or negative studies. A more balanced discussion is needed. Please address this problem.

The new version contains an expanded “Limitations and Counterarguments” section

211: detection methodologies vary substantially across studies, including PCR, culture, serology and phage amplification assays, each with differing sensitivity and specificity.

Contamination concern addressed

213: MAP’s environmental ubiquity also raises concerns regarding laboratory contamination and false-positive molecular detection.

Negative studies acknowledged

215: Some investigations have failed to detect MAP consistently in Crohn’s disease or other proposed MAP-associated conditions…

Query 3: One Health section (8) is too weak. Please increase the quality and impact of this
section by adding important scientific data regarding this particularly important concept.

This is strengthened.

There is explicit zoonotic ecosystem framing:

163: MAP exists within a complex zoonotic ecosystem involving livestock, wildlife, agricultural runoff, water systems and food production.

Added environmental persistence point:

165: Large-scale surveillance studies demonstrate substantial MAP prevalence in dairy herds worldwide…

168: documented survival in water biofilms and some pasteurized dairy products…

174: CRC may therefore represent not solely an individual disease process, but potentially an additional downstream consequence of long-term ecological interactions among agricultural practices, food systems, microbial exposure and host susceptibility.

Query 4: The topic is interesting and potentially original. However, the novelty of the
manuscript could be highlighted more clearly. The authors are encouraged to better emphasize what distinguishes this perspective from previous hypotheses and to more explicitly articulate the added value of integrating recent evidence linking MAP to precancerous and cancerous colorectal lesions.

50: While a prior hypothesis has proposed a relationship between MAP and colorectal neoplasia, the recent demonstration of MAP across the histologic continuum from normal mucosa to precancerous lesions and carcinoma provides a substantially stronger biologic framework for considering this association.

Minor: Please be sure that all species names are written in italics (e.g. title). Done.

I have also added Figure 4 to move the discussion from conceptual to experimentally actionable.

Reviewer 2 Report

Comments and Suggestions for Authors

This article reflects author’s opinion regarding the association of Mycobacterium avium subspecies paratuberculosis with colorectal cancer.

I have the following comments:

Line 36: Please, replace MAP with the full name of the organism, as it is the first time it is mentioned in the text.
Line 58: The term “extended periods” is general and should be defined more thoroughly.
Lines 71-72: The author should add in the text that Koch's postulates have not yet been fully fulfilled for MAP.
Lines 123-125: The author should mention that further studies are needed to support the latter finding.
Lines 150-152: The meaning of this sentence is general; it should be better supported by the findings mentioned in the literature.
Lines 168-172: The limited number of studies associating MAP with colorectal cancer should be included as a limitation. Additionally, the geographic origin of these studies should be discussed.
Lines 175-176: The author should mention whether there are any clinical trials demonstrating controversial or conflicting responses to anti-MAP antibiotic therapy in Crohn’s disease.
Lines 184-185: It should be noted that more studies are required to precisely identify the underlying drivers.
Figure 1: The growth of MAP in cultures retrieved from cattle specimens of cattle is a challenging process, as it requires an extended incubation periods and specialized media. Furthermore, MAP is difficult to be isolated from the feces in the first stages of infection. The author should make the necessary corrections in the figure and, if necessary in the main text to accurately reflect these diagnostic limitations.
Figure 3: The author mentions in the text that Fusobacterium nucleatum, Bacteroides fragilis and Escherichia coli are microbial contributors of CRC. However, Bacteroides fragilis is missing from the figure and should probably be included for consistency between the text and the illustration.
The author should state clearly in the text that a cause-and-effect relationship has not yet been established, although existing data support the association of MAP.

Author Response

Reply to Reviewer 2

Line 36: Please, replace MAP with the full name of the organism:

done, thank you.

Line 58: The term “extended periods” is general and should be defined more thoroughly: replaced with: …

can persist in soil and water environments for months to years under favorable conditions [13,14]

Lines 71-72: The author should add in the text that Koch's postulates have not yet been fully fulfilled for MAP.

Added:

Importantly, Koch’s postulates have not yet been fully satisfied for MAP in Crohn’s disease or colorectal cancer, owing in part to the organism’s slow growth, intracellular persistence and diagnostic complexity. Nevertheless, accumulating molecular, microbiologic and epidemiologic findings continue to strengthen the biologic association.

Lines 123-125: The author should mention that further studies are needed to support the latter finding.

Added:

MAP’s association with Crohn’s disease provides a plausible indirect pathway via chronic inflammation and raises the possibility of a more direct contribution to carcinogenesis, although additional mechanistic and longitudinal studies are required.

Lines 150-152: The meaning of this sentence is general; it should be better supported by the findings mentioned in the literature.

Changed to: MAP must be considered within a One Health framework; unlike many candidate oncogenic microbes restricted primarily to human transmission, MAP exists within a complex zoonotic ecosystem involving livestock, wildlife, agricultural runoff, water systems and food production. Large-scale surveillance studies demonstrate substantial MAP prevalence in dairy herds worldwide, resulting in persistent environmental shedding into soil and watershed systems. The organism’s resistance to environmental degradation and documented survival in water biofilms and some pasteurized dairy products raises the possibility of chronic low-level human exposure at population scale [12–14].

Lines 168-172: The limited number of studies associating MAP with colorectal cancer should be included as a limitation. Additionally, the geographic origin of these studies should be discussed.

Addressed and augmented substantially.

Lines 175-176: The author should mention whether there are any clinical trials demonstrating controversial or conflicting responses to anti-MAP antibiotic therapy in Crohn’s disease.

Changed to: Although several studies have reported favorable responses to anti-MAP therapy in Crohn’s disease [37,38], the broader literature remains mixed, and MAP’s role in human pathology continues to be hotly debated.

Lines 184-185: It should be noted that more studies are required to precisely identify the underlying drivers.

Rewritten: At present, available evidence supports association rather than causation. MAP should therefore be regarded as a candidate infectious cofactor in CRC, with its precise pathogenic role remaining to be defined through mechanistic, longitudinal and multinational studies using standardized detection methodologies. Importantly, proposed investigations using the preclinical IL-10⁻/⁻ model may help clarify whether MAP directly contributes to inflammation-driven tumorigenesis and provide mechanistic insight into its potential role in colorectal cancer.

Figure 1: The growth of MAP in cultures retrieved from cattle specimens of cattle is a challenging process, as it requires an extended incubation periods and specialized media. Furthermore, MAP is difficult to be isolated from the feces in the first stages of infection. The author should make the necessary corrections in the figure and, if necessary in the main text to accurately reflect these diagnostic limitations.

Figure 1 completely redone and reflects the important suggestions.

Figure 3: The author mentions in the text that Fusobacterium nucleatum, Bacteroides fragilis and Escherichia coli are microbial contributors of CRC. However, Bacteroides fragilis is missing from the figure and should probably be included for consistency between the text and the illustration.

Done thank you.

The author should state clearly in the text that a cause-and-effect relationship has not yet been established, although existing data support the association of MAP.

This has been done throughout the manuscript; eg, responses to comments 3 and 4.

In addition, I have added Figure 4 which describes a protocol for testing the IL-10 ko model of colitis and dysplasia.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Mycobacterium avium subspecies paratuberculosis and colorectal cancer: putting MAP on the map” presents a Perspective on a compelling and timely topic. It is very comprehensive and focuses on the potential role of MAP as a driver of colorectal cancer, while also considering its nature as a pleiotropic pathogen able to influence multiple disease processes.

The manuscript is well written, and the figures significantly improve the clarity and impact of the proposed mechanisms.

Author Response

Thank you to reviewer 3. The other reviewers have made recommendations that improve the manuscript. I hope that you find them agreeable.