Evolution of Pandemic Cholera at its Global Source

  1. Amber Barton
  2. Mokibul Hassan Afrad
  3. Alyce Taylor-Brown
  4. Nisha Singh
  5. Chetan Thakur
  6. Taufiqul Islam
  7. Sadia Isfat Ara Rahman
  8. Marjahan Akhtar
  9. Yasmin Ara Begum
  10. Taufiqur Rahman Bhuiyan
  11. Ashraful Islam Khan
  12. Neelam Taneja
  13. Nicholas R. Thomson
  14. Firdausi Qadri

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Abstract

The seventh pandemic of cholera, caused by the 7 th Pandemic El Tor Lineage (7PET) Vibrio cholerae , was previously shown to have emanated in three global waves from the Bay of Bengal, bordering Bangladesh and India. However, the respective roles of the Ganges delta and basin regions in seeding these global pandemic waves were not known. We find that while there are transmission events between Bangladesh and India, V. cholerae within the two countries has largely evolved separately over the past 20 years, contained by national borders rather than following hydrological features such as the Ganges delta and basin. Evolution within Bangladesh was distinct from that seen in India, involving rapid gain and loss of genes and mobile genetic elements, particularly those involved in phage defence. The loss/gain of these anti-phage elements mirrored loss/gain of anti-defence systems in lytic phage ICP1. Importantly, the loss of these systems was associated with increased risk of severe disease and transmission outside of Bangladesh. Here we show that the Ganges basin, falling across Bangladesh and Northern India, rather than the Ganges delta, acts as a global launch pad for pandemic disease. This completely shifts our understanding of Bangladesh as the purported global source of cholera, and the role of phage in controlling spread of lineages within the current seventh pandemic.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/15376379.

    Summary and overall impression

    This fascinating and data-rich paper tackles a long-held assumption: Bangladesh, particularly the Ganges Delta, is the epicenter of global cholera outbreaks. With support from an impressive dataset of over 2,300 samples from Bangladesh and Northern India, the authors argue that we may need to shift our understanding that the upper Ganges basin, especially parts of India, might be playing a bigger role in seeding global cholera strains.

    The sections dealing with the evolution of sub-lineages, the gain/loss of phage defence elements, and their potential links to disease severity are all exciting. However, some claims feel a bit stronger than the data alone can carry, and the writing is dense or hard to follow in places. This can be an impactful piece of work with clearer framing and a bit more caution in the interpretations.

    Abstract

    •  Minor Flaw: The abstract lacks a focused framing around the key research question.

    Suggestion: Reframe the abstract around the central hypothesis: "Does the upper Ganges basin in India play a larger role than Bangladesh in global cholera spread?"

    •  Minor Flaw: Phrases like "mirrored gain/loss of anti-defence systems" are difficult for non-specialist readers.

    Suggestion: Use simpler, clearer wording. Example: "We found repeating patterns in the presence or absence of genes that help cholera bacteria evade viruses, which may affect how strains spread and evolve."

    Introduction

    • Major flaw: The introduction does not clearly state the paper's key argument that India's upper Ganges basin may be more central to global cholera spread than Bangladesh. This weakens the framing of the study.

    Suggestion: Add a clear sentence early in the introduction, such as: "This study explores whether India's upper Ganges basin, rather than the Ganges delta in Bangladesh, may be the primary source of pandemic cholera lineages."

    • Major Flaw: The terms Ganges delta and Ganges basin are used without explanation, though they are crucial to the paper's geographic argument. Readers unfamiliar with the region may not grasp the difference.

    Suggestion: Briefly define both terms early in the introduction. For example: "The Ganges delta refers to the coastal region in Bangladesh and West Bengal, while the Ganges basin spans a broader area upstream in northern India."

    • Minor Flaw: The introduction is packed with background information in long paragraphs, which can overwhelm the reader and dilute the paper's focus.

    Suggestion: Break the introduction into shorter paragraphs, each focused on one theme:

    -Global burden of cholera

    - Importance of genomic surveillance

    -The Ganges region as a historical hotspot

    -Hypothesis about India's upstream role

    Methods

    Strength:

    • The authors use an impressive number of genomes (~2,300), which provides strong

    statistical power and broad representation across regions.

    • Tools like Snippy, Gubbins, IQ-TREE, and BEAST are widely accepted and standard

    for microbial genomic epidemiology.

    • The authors go beyond phylogenies to look at functional genes and structural elements,

    adding depth to the evolutionary interpretation.

    Major Flaw: The methods are described in long, text-heavy blocks that are hard to follow. This reduces clarity, especially for readers who want to understand or replicate the study.

    Suggestion: Use a visual flowchart or a clear step-by-step breakdown to show the analytical process. This will help readers quickly grasp the sequencing, filtering, alignment, and phylogenetic methods.

    Minor Flaw: The paper mentions "high-quality genomes" but does not define the criteria used to include or exclude them. This limits transparency.

    Suggestion: Briefly explain what filtering thresholds or quality control steps were applied (e.g., coverage depth, assembly metrics, contamination filtering).

    Results

    Major Flaw: Some claims in the Results appear to draw causal inferences from associational data.

    For example, links between gene deletions (e.g., phage defense islands) and disease severity or spread are discussed as they drive these outcomes. However, the paper does not present statistical models (e.g., regression analysis, adjusted comparisons) to support causality, only temporal and genomic associations.

    Suggestion: Reframe these claims as hypotheses rather than conclusions unless statistical tests directly support causation. For instance, clarify that observed patterns are correlations, and highlight the need for functional studies or adjusted models to test causal mechanisms.

    Minor Flaw: While the genomic analysis is impressive, the section contains too much information without enough structure. Key findings get lost in the detail.

    Suggestion: Break the results into smaller, clearly labelled sections. Add summary sentences at the end of each key point (e.g., "This suggests X sub-lineage may have emerged independently in Y region"). This will help readers follow the significance of each result.

    Discussion

    Major Flaw: The paper strongly frames India as the main source of global cholera spread, but the data do not fully support this. It's a problem as there has been no adjustment for sampling bias or differences in surveillance systems across regions.

    Suggestion: Soften the language and add nuance. Example edit: "These findings suggest the upper Ganges basin may play a significant role in cholera spread, though differences in sampling and surveillance must be considered."

    Minor flaw: The discussion overlooks key social and ecological drivers such as human mobility, trade, water systems, and healthcare access.

    Suggestion: Add a short paragraph acknowledging these influences.

    Example: "Cholera spread is also shaped by human behaviour and infrastructure, which should be considered in future analyses."

    Minor flaw: The discussion jumps between ideas without a clear structure.

    Suggestion: Use short sub-sections or clearer paragraph breaks by topic:

    -Phage pressure

    -Regional comparisons

    - Spread/export

    -Limitations

    End each theme with a summary sentence. Example: "Cholera spread is also shaped by human behaviour and infrastructure, which should be considered in future analyses."

    Conclusion

    Minor Flaw: The conclusion summarises the paper well, but doesn't clearly state what should happen next.

    Suggestion: Add 2–3 specific priorities. Example: "To improve future cholera preparedness, countries should expand local sequencing infrastructure, invest in phage surveillance and prioritise regional data sharing networks"

    Minor Flaw: The ending feels soft. A sharper final statement could reinforce the paper's significance.

    Suggestion: End with a strong call to action or takeaway message. Example: "Cholera's future spread may hinge not only on pathogens but also on how quickly we act to track, share, and respond."

    Final Note: This is an important study with the potential to shift how we think about the global spread of cholera. The data is impressive, the methods are strong, and the big-picture questions are worth asking. However, the paper would benefit from pulling back a bit in its conclusions, simplifying some dense sections, and sharpening its main message.

    Competing interests

    The authors declare that they have no competing interests.

  6. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/15043068.

    Does the introduction explain the research objective?

    The introduction does a good job of explaining that the study is about tracking how Vibrio cholera evolves using genomic data. However, it doesn't fully explain why this research is different from previous studies. The authors should highlight what new information they are providing that hasn't already been studied. They also need to explain why they chose these specific cholera samples and why their research focuses on certain regions. This will help readers understand the significance of their work.

    Are the methods appropriate?

    The methods seem appropriate, but they need more details. The study uses whole-genome sequencing and phylogenetic analysis, which are standard techniques for studying bacterial evolution. However, the authors don't clearly explain how they selected their samples or if their dataset might be biased toward certain regions. It's also unclear which method they used to build their evolutionary tree, which makes it difficult to assess how reliable their conclusions are.

    Are the conclusions supported by the data?

    The conclusions are mostly supported by the data, but some claims need stronger evidence. The study suggests that cholera's evolution follows a clear timeline, but without a detailed time-based analysis, it's hard to confirm this. The authors also discuss antibiotic resistance, but they don't explain what pressures caused these resistance genes to develop. The paper states that genomic surveillance can help track pandemic cholera, which is true, but this is already well known. The authors should be more specific about what new insights their study provides and how it can improve cholera prevention efforts.

    Are the figures and visualizations effective?

    The figures are useful but need some improvements. The phylogenetic trees, which show how different cholera strains are related, do not include statistical support values, making it unclear how confident we should be in the results. The genomic maps should have clearer labels to highlight important genetic features, such as antibiotic resistance genes. Some figures are difficult to read due to small font sizes. Improving these aspects will make the data more accessible and easier to interpret.

    Do the authors explain their findings and suggest next steps?

    The discussion section could be clearer and more detailed. The authors summarize their results well, but they don't explore other possible explanations for their findings. Could environmental factors, human behavior, or other influences play a role in cholera's evolution? The study also lacks clear next steps. What additional research should be done based on their findings? Should more samples be collected? Should different genetic markers be used?

    Does this study contribute something new to the field?

    Yes, but the novelty isn't clearly stated. The research has potential value, but without a stronger discussion on how it builds on previous studies, it may not stand out. The authors should compare their findings to past genomic studies on cholera and highlight what is new. They should also explain how their research could be applied in real-world cholera control efforts.

    Does the writing need editing?

    The writing is generally clear, and there are no major grammar issues. However, some sections contain unnecessary background information that could be condensed. The authors should focus on making their key findings more direct and easier to understand.

    Would I recommend this preprint?

    Yes, but only after major revisions. The study covers an important topic, but it needs improvements in methodology, explanation of findings, and clarity.

    Is it ready for journal submission?

    Not yet, major revisions are needed. Before submitting to a journal, the authors should clarify their methodology, strengthen their conclusions with better evidence, and improve their discussion by addressing alternative explanations and future research directions.

    Competing interests

    The author declares that they have no competing interests.

  7. Version published to 10.1101/2025.02.03.25321585v1 on medRxiv