Preliminary insights into the potential role of Acanthamoeba-Pseudomonas interactions in the development of antibiotic resistance
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2. Abstract Bacteria are known to develop resistance to Acanthamoeba digestion and use them to exercise virulence factors. We hypothesise that Acanthamoeba species may also play a role in promoting antimicrobial resistance (AMR) in amoeba-resistant bacteria. This study investigated whether Acanthamoeba castellanii enhanced AMR development in Pseudomonas putida under lethal ciprofloxacin concentrations. P. putida KT2440 was co-incubated with A. castellanii at ciprofloxacin concentrations starting at four times the minimum inhibitory concentration (MIC). The survival of the co-incubated Pseudomonas and the development of resistance were monitored, and antimicrobial susceptibility tests were conducted using multiple antibiotics. P. putida co-incubated with A. castellanii exhibited tolerance to ciprofloxacin, with MIC increasing from 0.5 µg/ml to 20 µg/ml after 17 days. In contrast, the naïve strain did not survive at a 2 µg/ml concentration. The co-incubated bacteria developed resistance to ciprofloxacin, chloramphenicol, azithromycin, and enrofloxacin while retaining susceptibility to streptomycin and tetracycline. Acanthamoeba significantly accelerated AMR development in P. putida exposed to ciprofloxacin. This finding highlights the need for further research on the molecular mechanisms involved to better identify strategies for combatting the emergence of antibiotic resistance in clinical and environmental settings.
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Thank you very much for submitting your manuscript to Access Microbiology. I has now been reviewed by two experts in the field, whose comment are attached at the bottom of this email. In general, both reviewers agree that this work is a valuable contribution to the field and provides interesting results on how amoebae-bacteria interactions affect antibiotic resistance. They have proposed some corrections that will need to be addressed in a revised version of the manuscript. Please pay special attention to one of the concerns by Reviewer 1, who proposes an additional control that may require further experiments. I do think that such a control added to the results already presented would conclusively support the findings. If this additional experiment was not feasible, please address it by explaining the limitations of the approach in …
Thank you very much for submitting your manuscript to Access Microbiology. I has now been reviewed by two experts in the field, whose comment are attached at the bottom of this email. In general, both reviewers agree that this work is a valuable contribution to the field and provides interesting results on how amoebae-bacteria interactions affect antibiotic resistance. They have proposed some corrections that will need to be addressed in a revised version of the manuscript. Please pay special attention to one of the concerns by Reviewer 1, who proposes an additional control that may require further experiments. I do think that such a control added to the results already presented would conclusively support the findings. If this additional experiment was not feasible, please address it by explaining the limitations of the approach in the Discussion section. A couple of other suggestions you may like to consider are: - L77: P. stutzeri is currently known as Stutzerimonas stutzeri, taken out of the Pseudomonas genus, so you may want to remove this as a Pseudomonas example - In contrast to P. aeruginosa and other Pseudomonas pathogens, P. putida is an environmental species. A couple of lines justifying the rationale for using P. putida instead of an actual Pseudomonas pathogenic species would be greatly beneficial for the understanding of the paper. Please provide a revised version of the manuscript (including a tracked-changes document), along with a point-by-point response to the reviewer comment and my suggestion within one month.
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Comments to Author
This research manuscript builds on previous work by the group identifying important phenotypic responses of the important nosocomial pathogen Pseudomonas aeruginosa when interacting with Acanthamoeba. The paper is for the most part well written, the experimental design and use of controls is robust, and the interpretation is supported by the data presented. Understanding the polymicrobial interactions that occur in natural and clinical ecosystems is an important goal, and where these are shown to promote antimicrobial resistance, steps need to be taken to counter this. Methodological rigour, reproducibility and availability of underlying data The experimental approach and inclusion of controls gives confidence on the robustness of the work presented. A minimum of three biological replicates is evident …
Comments to Author
This research manuscript builds on previous work by the group identifying important phenotypic responses of the important nosocomial pathogen Pseudomonas aeruginosa when interacting with Acanthamoeba. The paper is for the most part well written, the experimental design and use of controls is robust, and the interpretation is supported by the data presented. Understanding the polymicrobial interactions that occur in natural and clinical ecosystems is an important goal, and where these are shown to promote antimicrobial resistance, steps need to be taken to counter this. Methodological rigour, reproducibility and availability of underlying data The experimental approach and inclusion of controls gives confidence on the robustness of the work presented. A minimum of three biological replicates is evident in the data figures. Lines 107-109: If I read this correctly, 100 mg of antibiotic added to 1 ml would be 100 mg/ml. What then is the nature of the 1 mg/ml stock solution? Presumably it is a dilution of the initial preparation, but was it also prepared in acetic acid? You just need to clarify this. Line 129: Insert a space between number and unit (200 µl). Lines 137-140: Add the wavelength (and nm unit) at which optical density was measured. Lines 145-147. Gram staining would only reveal contamination with a gram positive or a gram negative cocci. Therefore, the statement "to exclude any possible contamination" is not justified by the experimental approach. I suggest you change it to "to monitor for contamination". Presentation of results The manuscript presents a clear research outcome and the presentation of the results in two figures outlines the key experimental outcomes. Line 185: The use of confluence throughout the manuscript should be considered carefully. By confluence, do you refer solely to the acanthamoeba on the bottom of the wells, or do you also refer to turbidity caused by bacterial growth in the media? Also mentioned in Figure 1 legend, the inference is that it refers to bacterial growth which would not be confluence. Also in Figure 1, is the x-axis labelled appropriately? It says "days to confluence", but given that it is an incremental cycling experiment, I suggest you relabel the x-axis. Also in this figure, please add error bars in each of the data columns. Perhaps there is no experimental variation, just need to clarify this. In Figure 2, the statistical outcomes should be checked. For example, TE Naïve vs 3 µg/ml appear to be almost identical outcomes, with variation in the Naïve sample, so it is unclear to me how this can achieve statistical significance. All statistical comparisons should be checked to ensure the correct approach is being used. How the style and organization of the paper communicates and represents key findings The paper is well written, though there are some issues with syntax in places. These are outlined below. Line 18: This needs to be revised for clarity - "and use them to exercise virulence factors". Line 26: I suggest consistency with regards to the use of "tolerance" and "resistance". These are two different phenomena. Line 38: Should this be 20 µg/ml rather than 40 µg/ml? Line 104: I suggest you use density here rather than concentration. Literature analysis or discussion The authors have presented a coherent analysis of the literature and discussed the context of their experimental outcomes in a rational and logical way. The findings are no doubt interesting, and the incremental resistance reported, building on previous work by the group using hydrogen peroxide, serves to remind us of the multifaceted nature of the AMR challenge we are facing.
Please rate the manuscript for methodological rigour
Very 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?
No: Not Applicable
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Comments to Author
The submitted manuscript presents some very interesting preliminary data showing an association between survival in Acanthamoeba and antimicrobial resistance in Pseudomonas. The manuscript is very well written and the authors reveal some interesting associations between ciprofloxacin resistance as well as resistance to other antimicrobials. The experimental design involves the incubation of the bacteria with the amoeba for an hour to allow uptake followed by exposure to ciprofloxacin and incubation over time waiting for the appearance of confluent growth and subsequent measurement of MICs. Using this method the authors show an association between exposure of acanthamoeba enclosed bacteria to this clinically important fluoroquinolone and the development of resistance to this antibiotic and others. The …
Comments to Author
The submitted manuscript presents some very interesting preliminary data showing an association between survival in Acanthamoeba and antimicrobial resistance in Pseudomonas. The manuscript is very well written and the authors reveal some interesting associations between ciprofloxacin resistance as well as resistance to other antimicrobials. The experimental design involves the incubation of the bacteria with the amoeba for an hour to allow uptake followed by exposure to ciprofloxacin and incubation over time waiting for the appearance of confluent growth and subsequent measurement of MICs. Using this method the authors show an association between exposure of acanthamoeba enclosed bacteria to this clinically important fluoroquinolone and the development of resistance to this antibiotic and others. The major limitation to the study is that the experimental design does not allow for us to discern whether the uptake of the bacteria by the protozoa in itself is enough to drive the bacteria towards a resistant phenotype or whether the presence of the antibiotic at the same time is required. At no stage is a control of just exposing the bacteria to the acanthamoeba and then subsequent MIC measurement used to examine this. This is an important question as it could be that the uptake by the amoeba is enough to reduce the exposure to the antibiotic (decreasing the actual concentration experienced by the bacteria) and thus allow time for selection of resistant clones which would not be available to planktonic bacteria or it could be that the stress of uptake by an amoeba alone drives changes in the phenotypes of the bacteria which are associated with resistance to certain antibiotics. I would not expect the authors to fully confirm the full mechanism behind the phenotypic change they report but I think the inclusion of such a control or addressing this issue/limitation of the study in a clearer way in the discussion would be very important. To be fair to the authors they touch on some of these themes but need to be clearer on the limitation of their approach. Some other minor points to be addressed below - 1) Lines 108-109 - Needs some very minor clarification here... you seem to be saying you make a stock solution by adding 100mg to 1ml but the final concentration for the stock is 1mg/ml? maybe add that you specifically dilute down as it reads a little confusing at present. 2) Page 5 or elsewhere - Given the fact that these experiments run for up to 17 days could the authors comment on whether the OD595 could be influenced by increased acanthamoeba growth or how this has been ruled out or controlled for? 3). Lines 145 - I don't think these should be referred to as cell lines.. I think "culture" would be more accurate.. I'm presuming you are talking about proving here that you are not getting contamination with a different bacteria? 4). Lines 208-209 - You say here that the inhibition zone for streptomycin was wider at every checkpoint for the coincubated strains than the naive strain... is that right? Is it not less at 2ug in Figure 2? 5) Figure 1 - The legend explains relatively well what the graph shows but could you explicitly mention "Rx MIC" in the legend and explain specifically what this means as it doesn't seem to be explained what Rx explicitly stands for? 6) Figure 2 - Again could you be explicit in what you mean by "naive" here.... Do you mean bacteria which have not been exposed to either Cip or the acanthamoeba? I presume these are just non-passaged bacteria? Needs to be clearer.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very good
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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