Hollow-fibre infection model: adaptations for the culture and assessment of fastidious organisms
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
The hollow-fibre infection model (HFIM) is a valuable in vitro platform for emulating antimicrobial drug pharmacokinetic profiles. Despite its potential, standardized protocols for HFIM operation, especially concerning fastidious organisms, are lacking. This study addresses this gap by examining challenges in culturing Pasteurella multocida and Actinobacillus pleuropneumoniae , two fastidious organisms, in the HFIM. Our findings reveal effective strategies to prevent system clogging, involving multiple freeze–thaw cycles of horse blood, centrifugation and cell straining to enhance the clarity of the Mueller-Hinton fastidious medium defined by the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute. Additionally, we propose that the provision of a CO 2 atmosphere, along with the utilization of gas-permeable tubing and gas vent filters, significantly facilitates the growth of fastidious organisms. Remarkably, both P. multocida and A. pleuropneumoniae were sustained for a period of up to 10 days under these optimized conditions. This study provides crucial insights into the modifications necessary to successfully culture fastidious organisms in the HFIM, paving the way for more accurate and representative in vitro models for antimicrobial drug testing. These advancements hold promise for advancing research in the field of antimicrobial pharmacokinetics and efficacy against challenging pathogens.
Article activity feed
-
-
You have addressed the raised concerns of the reviewers.
-
-
The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments. Please clarify the points raised by the reviewers about the methods (in particular CO2 measurements and whether or not it was a fresh isolate each time or the same isolate for each species). Please also address the concern about Figure 6 and the wording of your abstract and impact statement in regards to the experimental design.
-
Comments to Author
This is a methodology paper which expands the use of Hollow-fibre Infection Modelling (HFIM) to result in sustained culture of two chosen fastidious microbial species. Hitherto, it has not been possible to cultivate these species using HFIM under the standard (EUCAST/CLSI, the international benchmark) conditions used routinely in 'classical' methods of culture. The advance reported here permits in vitro assessment of pharmacokinetic and pharmacodynamic metrics for these exemplar species - with the inference that this new methodology will be applicable to other fastidious species. Such a system provides invaluable in vitro data to incorporate into pre-clinical development of new drugs and new treatments. 1. Methodological rigour etc At L188 is is not clear whether a fresh isolate from ECO was used for …
Comments to Author
This is a methodology paper which expands the use of Hollow-fibre Infection Modelling (HFIM) to result in sustained culture of two chosen fastidious microbial species. Hitherto, it has not been possible to cultivate these species using HFIM under the standard (EUCAST/CLSI, the international benchmark) conditions used routinely in 'classical' methods of culture. The advance reported here permits in vitro assessment of pharmacokinetic and pharmacodynamic metrics for these exemplar species - with the inference that this new methodology will be applicable to other fastidious species. Such a system provides invaluable in vitro data to incorporate into pre-clinical development of new drugs and new treatments. 1. Methodological rigour etc At L188 is is not clear whether a fresh isolate from ECO was used for each experiment OR whether a single isolate was employed and used throughout for each species. This is important in interpretation of data. Given that each experiment was conducted only once (or reported here only once), this question relates to how reproducible a single experiment is. ** I see that in L286 you do introduce this caveat. That you realise the limitations of this work (variation in inocula and insufficient investigation of reproducibility) does diminish the impact. At L193, it is stated that the system was inoculated at 5 x 10⁵ /mL and A. pleuropneumoniae grew to 1 x 10⁹ /mL in just 4 hours. That is a remarkable 11 doublings if no lag. Is this compatible with your expectation of the Mean Generation Time for this species in this medium? If so, it's worthy of note. 2. Presentation of results Fig. 7 is an excellent illustration that compares MH-F and CAMHB for P. multocida. Fig.6 depicts both species in MH-F, but surely the point missed here is the performance of A. pleuropneumoniae in CAMHB? This omission weakens the impact of the paper as you only have data presented for comparison of one species. In Figs 6 & 7, 'LoQ' needs to be defined in the Legends. Yes, they are defined elsewhere but a Legend should be self-explanatory. L273-275. Initially I was curious about this tangential referral to 'laked horse blood' and inclusion of haematin. However I have realized subsequently that there is other literature that investigates these. You should quote them! L283. 'static growth phase' may not be accessible to someone whose first language is not English because it appears paradoxical. Is 'stationary phase of growth' more appropriate? It's the classical way of describing this part of the cycle. 3. Style and key Figures I refer to concern about Fig 6 above. I suspect that the text in Fig. 1 will not reproduce well. $. Literature analysis and Discussion L290-298 I do not see evidence of any controls for requirement for CO₂ or gas-permeable tubing. This appears to be an important omission in experimental design to support the statements made in both the Abstract and the Impact Statement where a reader might be duped into thinking such controls had been done to support the dogmatic conclusions inferred in the Abstract and Impact Statement. I have no doubt that these fastidious microbes need a partial CO₂ atmosphere but your wording implies that you have done the appropriate control to establish this in HFIM. You haven't. So word your Abstract and Impact Statement appropriately. Likewise, to make the claim about gas-permeable tubing, you need to show that non-permeable tubing gives a significantly different outcome. Or word your statements more appropriately.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
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
-
Comments to Author
Mead et al. present a modification of the hollow fibre infection model to allow investigation of growth and antibiotic activity against fastidious organisms. The setup is well described and the results are important for future research in this area. I have only one comment: It is not clear from the text, whether sampling from the system, which takes place numerous times over the 9-10 days, can be performed without opening the CO2 incubator ? If not, there must be a dip in the CO2 concentration and may influence the growth e.g., the falling curve for A. pleuropneumoniae. Is it possible to measure the CO2 content inside the fluid in the model ?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Comments to Author
Mead et al. present a modification of the hollow fibre infection model to allow investigation of growth and antibiotic activity against fastidious organisms. The setup is well described and the results are important for future research in this area. I have only one comment: It is not clear from the text, whether sampling from the system, which takes place numerous times over the 9-10 days, can be performed without opening the CO2 incubator ? If not, there must be a dip in the CO2 concentration and may influence the growth e.g., the falling curve for A. pleuropneumoniae. Is it possible to measure the CO2 content inside the fluid in the model ?
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
-
Comments to Author
The presented manuscript entitled "Hollow-fibre infection model: adaptations for the culture and assessment of fastidious organisms" is matching the scope of Access Microbiology and deserves full consideration. The manuscript deals with an important issue for researchers working with the Hollow-fibre models (HFIM) and provides new solutions for problems with fastidious organisms. The authors did a great job, and their results will help other researchers to avoid multiple challenges working with HFIM. The manuscript is clearly written (results reproducible if needed), adequately presented illustrated, concise and, thus, highly valuable for the scientific community. It is recommended for publication in Access Microbiology.
Please rate the manuscript for methodological rigour
Very …
Comments to Author
The presented manuscript entitled "Hollow-fibre infection model: adaptations for the culture and assessment of fastidious organisms" is matching the scope of Access Microbiology and deserves full consideration. The manuscript deals with an important issue for researchers working with the Hollow-fibre models (HFIM) and provides new solutions for problems with fastidious organisms. The authors did a great job, and their results will help other researchers to avoid multiple challenges working with HFIM. The manuscript is clearly written (results reproducible if needed), adequately presented illustrated, concise and, thus, highly valuable for the scientific community. It is recommended for publication in Access Microbiology.
Please rate the manuscript for methodological rigour
Very 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?
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
-