Pseudomonas aeruginosa shows between and within strains heterogeneity in virulence phenotype after passive exposure to zebrafish (Danio rerio) and nematodes (Caenorhabditis elegans)
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The rapid emergence of diseases and parasites in aquatic wildlife requires improved methodologies to identify and characterize new and future pathogens. While microinjection of pathogens directly into a sentinel organism such as zebrafish enables the exploration of infection and immune response in the host, such methodology focuses primarily on identifying causative agents in events of aquatic wildlife mortality due to acute infection. Here, we present an updated protocol of infection by static immersion in larval zebrafish to investigate the possible effect of prolonged environmental exposure to an opportunistic pathogen. By controlling microbial growth and monitoring mortality over five days, we show that static immersion can detect minute differences in virulence profiles between and within different strains of Pseudomonas aeruginosa, an important opportunistic pathogen of animals and humans. We then conducted two sets of passive exposure virulence assays in Caenorhabditis elegans, an alternative model. We demonstrated the virulence phenotype, while showing slight differences between experimental models, showed similar trends. We believe that passive exposure thus offers a practical host-pathogen model that simulates opportunistic infection occurring in the environment and enables the detection of minute changes in virulence between and within bacterial strains.
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Editor Decision Phrases The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. The reviewers raise concerns regarding the scientific rigour and experimental design of the work. The reviewers believe the results shown in the manuscript do not support the conclusions presented.
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Comments to Author
Summary: This study aims to establish passive infection models in zebrafish and nematodes. Current animal models of P. aeruginosa infection involve injection or nasal inoculation, both of which are not natural routes of infection by P. aeruginosa. The passive infection model that the authors are trying to establish would be more representative of environmental exposure to pathogens and would allow for better recapitulation of natural infection in the laboratory. Notably, both organisms are commonly used and accessible animal models, so their protocol for passive infection could be easily adopted. The passive infection models presented by the authors are also sensitive enough to elicit different degrees of pathogenesis of P. aeruginosa strains. However, I have some concerns about the rigor of the study …
Comments to Author
Summary: This study aims to establish passive infection models in zebrafish and nematodes. Current animal models of P. aeruginosa infection involve injection or nasal inoculation, both of which are not natural routes of infection by P. aeruginosa. The passive infection model that the authors are trying to establish would be more representative of environmental exposure to pathogens and would allow for better recapitulation of natural infection in the laboratory. Notably, both organisms are commonly used and accessible animal models, so their protocol for passive infection could be easily adopted. The passive infection models presented by the authors are also sensitive enough to elicit different degrees of pathogenesis of P. aeruginosa strains. However, I have some concerns about the rigor of the study design and data reproducibility, particularly in the zebrafish model. The paper would be strengthened if the authors presented more of their data as discussed in the methods, and address the concerns listed below. Major Comments: 1. The authors claim that the differences in lethality observed between the different P. aeruginosa strains can be attributed to bacterial virulence and not environmental factors, but the authors do not present any data to support that conclusion. This is more important for the zebrafish model rather than the nematode model, since worms were transferred to fresh media every two days. To support this statement, the authors would need to monitor the pH of the media and bacterial growth during their infection assays. The high concentration of the bacterial inoculum could also be influencing infection outcomes and the authors should address this possibility. 2. The authors mentioned that they measured growth of P. aeruginosa strains in E3 media, and these growth curves should be presented in a figure, especially because E3 is not a commonly used media and particularly if the authors are arguing against the effect of bacterial growth on the results. Likewise, microscopy data showing paramecium contamination should be included. 3. The authors only used human pathogenic P. aeruginosa strains. If the authors are establishing a model for understanding how passive exposure and infection shapes natural bacterial populations, the authors might consider including an environmental strain. It is important to establish that not all strains of P. aeruginosa are pathogenic in these model systems to strengthen their case that these models can be used to assess phenotypic diversity of emerging pathogens in animal reservoirs. 4. There is high variability in infection outcomes in the zebrafish model, particularly with strain Lw1047 (Figure 1D). The authors should address these discrepancies more thoroughly in the discussion and should consider performing a third replicate to address the variability between the infection model trials. Minor Comments: 1. Line 26: Is "sentinel" the correct word here? Should it be sentient? 2. Line 31: The authors write "…monitoring mortality over five days…" but the zebrafish experiments were 72hr and the nematode experiments were 7 days according to the methods. 3. Line 90: The authors write "…heterogeneity in virulence phenotypes…" but only one virulence phenotype is measured. 4. Line 132: Centrifuging and pelleting are the same thing, please clarify. 5. Line 159: How did the authors account for missing nematodes? Was this excluded from the survival curve data? 6. Line 198: I'm not familiar with this syntax, please clarify what the numbers in the parentheses represent. 7. Please show growth curve data for different inoculum concentrations to support the selection of 105 as the optimal static immersion conditions. 8. Lines 214-224: This paragraph is written about the trial 2 data according to the figure. Please clarify in the text which trial is discussed, and/or switch the colors on the figure. 9. Line 257: Please clarify which trial you are talking about with these statistics. I think here you are only talking about trial 1. 10. Line 274: "P. aeruginosa develops at least three virulence profiles…" Virulence profiles can't be distinguished or enumerated based on the data presented. There is only one phenotype measured: infection lethality. 11. Line 278-279: This statement is written backward. Strain A10 was virulent in zebrafish and avirulent in worms. 12. Line 280: Host-pathogen interactions also depend on the route of infection. This is an important aspect of this paper and should be discussed in-depth here. It should also be mentioned that passive exposure via ingestion is atypical for establishing a P. aeruginosa infection.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The paper "Pseudomonas aeruginosa shows between and within strains heterogeneity in virulence phenotype after passive exposure to zebrafish (Danio rerio) and nematodes (Caenorhabditis elegans)" is quite interesting, well written and organized and with data which look properly analyzed. Few notes are related to systematics. Each species when reported for the first time in the text should be written in full with Authority and systematics. some notes: L64: Pseudomonas aeruginosa add Authority and systematics L69: write Pseudomonas here. never start a sentence with a pointed letter L74: Danio rerio, each species, when reported for the first time in the text should be written in full with Authority and systematics L85: Add Authority and systematics L146: species reported for the first time in the text. Add …
Comments to Author
The paper "Pseudomonas aeruginosa shows between and within strains heterogeneity in virulence phenotype after passive exposure to zebrafish (Danio rerio) and nematodes (Caenorhabditis elegans)" is quite interesting, well written and organized and with data which look properly analyzed. Few notes are related to systematics. Each species when reported for the first time in the text should be written in full with Authority and systematics. some notes: L64: Pseudomonas aeruginosa add Authority and systematics L69: write Pseudomonas here. never start a sentence with a pointed letter L74: Danio rerio, each species, when reported for the first time in the text should be written in full with Authority and systematics L85: Add Authority and systematics L146: species reported for the first time in the text. Add Authority and systematics
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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