Methods for detecting and monitoring Salmonella infection and chronic carriage in living mice using bioluminescent in vivo imaging
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Salmonella enterica serovar Typhi primarily persists in chronic carriers by forming biofilms on gallstones in the gallbladder. We have developed a gallstone mouse model to study chronic carriage. To better understand the infection timeline and differentiate between mice that have maintained long-term gallbladder carriage from those that have cleared infection, we utilized bioluminescent S. Typhimurium and in vivo imaging to detect and track the organ-specific presence of bacteria in living mice. The mice infected with our bioluminescent S. Typhimurium showed luminescence in the abdomen as early as three days by comparison to the mice infected with non-luminescent wild type S. Typhimurium. With our methods, we achieve image resolution such that we can confidently identify the presence of S. Typhimurium in the gallbladder at >60 days post-infection. Using these methods we have determined that the minimum number of bacteria necessary to detect luminescence in the mice is 103 colony forming units (CFUs), and that 1 out of 6 initially infected mice will remain persistently infected for greater than 60 days, with gallbladder bacterial loads reaching upwards of 103 per mg of tissue. Given that our limit of detection of luminescence is 103 CFUs, our sensitivity is robust enough to identify the bacterial loads present in the average chronically infected mouse. Quantification of individual organs’ bacterial CFUs and comparison of luminescence between WT and luminescent S. Typhimurium validates that our technique is specific and sensitive enough to detect organ specific infection in our model of typhoidal chronic carriage.
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Thank you very much for submitting your revised manuscript to Access Microbiology, addressing the reviewers' comments and suggestions and performing additional experiments. This has really improved the quality of the manuscript and will be useful for any prospective researcher interested in using this method. I am please to let you know that this current version of the manuscript has been accepted for publication. Congratulations!
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Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comments you will find at the bottom of this email. They both agree that this is a valuable contribution and an important methodological advancement in the study of typhoid fever. However, they both have concerns that need to be addressed and suggestions for improvement of the manuscript. Please amend the text according to their suggested corrections and provide clarifications or discuss their specific points, specially to those regarding strain construction methodology, animal numbers and cfu standardisation. Please provide a revised version of the manuscript (including a tracked-changes document) along with a point-by-point response to the reviewer comments within 2 months.
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Comments to Author
In this article, Bennett et al describe a method involving the use of a luminescent reporter (luxCDABE operon from Photorhabdus luminescens) integrated into the chromosome of Salmonella Typhimurium to monitor the infection kinetics of the pathogen in a long-term asymptomatic carriage model (>60 days) and gallbladder colonization. The authors use an in vivo imaging system and a combination of innovative computational techniques to model and identify, without the need to euthanize each animal, the pathogen's persistence sites. Remarks : ● Bacterial Strain : The strain of interest, 14028lux, used in this study was designed based on S. Typhimurium ATCC 14028, transduced with the luxCDABE operon from Photorhabdus luminescens, originally provided by Perkin Elmer (xen33). Although I trust the authors and …
Comments to Author
In this article, Bennett et al describe a method involving the use of a luminescent reporter (luxCDABE operon from Photorhabdus luminescens) integrated into the chromosome of Salmonella Typhimurium to monitor the infection kinetics of the pathogen in a long-term asymptomatic carriage model (>60 days) and gallbladder colonization. The authors use an in vivo imaging system and a combination of innovative computational techniques to model and identify, without the need to euthanize each animal, the pathogen's persistence sites. Remarks : ● Bacterial Strain : The strain of interest, 14028lux, used in this study was designed based on S. Typhimurium ATCC 14028, transduced with the luxCDABE operon from Photorhabdus luminescens, originally provided by Perkin Elmer (xen33). Although I trust the authors and the suppliers (Perkin Elmer) of the parental strain (xen33) regarding the reliability of the strain, the reference provided do not give details about the construction. It would be necessary to add some clarification about the chromosomal location of the lux operon, and possibly how it was inserted into the Xen33 strain. ● Figure 2 - The competition experiment is critical and the base of the paper, but the results of the competition index are not fully convincing. Although non-significant, one can see a trend for the Gallbladder in comparison with the Liver or the Spleen. 1) Is the power enough, meaning is n=4 enough to be able to see a difference? If not, it might be useful to redo the experiment with a few more animals. 2) What was the p value for the gallbladder? 3) Additionally, why is there 1 datapoint missing from the cecum (only n=3)? ● L148-151 - "The ROIs drawn around the gallbladder region of the mice indicate that some mice had bacteria in their gallbladder and this is supported by CFU enumeration that shows that all 14028lux infected mice with gallbladder CFUs >103 also had ROI radiances measured at above that of the background (Fig 3b.).". Given the colonization rates of organs adjacent to the gallbladder, and the luminescent signals measured across the entire abdominal regions of mice 4922#2, #7, #13, #20, #21, and #22, it seems complicated, at this stage, to link the luminescence observed in the "whole" animal specifically to the gallbladder. Since the animal were euthanized for CFU counting, why not measuring the radiance of the organs (as in Fig 5)? ● L162-163 - "In contrast, some mice (i.e. 4922 #4 and #5) maintain detectable luminescence throughout the experiment, with periodic reductions below the limit of detection (Fig 4.)." - Mouse 4922 #4 was euthanized at 15 dpi, but the authors give no reason why. In any case, the figure would be clearer if the mice were annotated directly on the figure rather than on the side. Again, given the distribution of the signals, the link with the gallbladder specifically, without data concerning bacterial loads, is unclear and may be premature. ● L168-177 : I am much more convinced by the reasoning and conclusions (Fig 5A-C). The 3D imaging of individual 4922#3 is convincing, and the subsequent verifications of the origin of the signals are relevant. Living Image allows for adjusting the contrast, which would be interesting to do in order to standardize the images showing the isolated organs. In the legend of Figure 5, the authors specify, "The CT images of mice 4922 #3 and #5 are shown with the DLIT analysis of 4922 #3 shown in cross-sectional panels," but no image of individual #5 is shown. Unless it is the image on the right with no signals (which is not annotated?). Therefore, what about the location of the signals measured on individual #5 in Fig 5A? These signals are not found on the individual during the acquisition of the image shown in Fig 5B or in Fig 5C (top-right panel). Given the location of the signals and the bacterial loads, one could think of a cecal source, for example, which does not appear in the CT/DLIT ? ● L177-179 - "We enumerated the CFUs in the organs of all mice and compared the gallbladder CFUs to the "gallbladder" ROIs drawn on the live mouse images. The presence of >100 CFUs of 14028lux correlated with a radiance above background (Fig 5d.).". To be more precise, from their data the authors cannot really state that >100CFU/mL means radiance. The only positive point is for mouse #3 (which show 10e5 CFU/mL). It could be that the radiance can be measured at 1.000 CFU/mL or 10.000 CFU/mL or even 100.000 CFU/mL. Additional mice would be needed to affirm this. ● Clarity : Several times, the bacterial loads of the different organs are described in CFU in the text but are expressed in CFU/mg in the figures. Please standardizing the units in CFU/mg throughout the manuscript. Minor remarks : The authors use S. Typhimurium as a model for the host-restricted S. Typhi infection. A few lines in the introduction explaining that Typhimurium display the same symptoms in mice as Typhi in Human would be useful. To the risk of being pedantic, the word "data" is plural (data are..). Please correct across the manuscript Figure 1: Where is the electron microscopy picture coming from ? L141 - Typo : « 1.1x104 » instead of "1.1x104". L188 - "overexpression" ?
Please rate the manuscript for methodological rigour
Satisfactory
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
This manuscript is well written and presents an important advancement in the analysis of carriage of typhoid fever. There are several minor modifications that would improve clarity: Methods: The Evans Blue-Uranine detection of p22 needs a brief explanation and a reference. Why is the infection delivered IP and not oral or IV? This needs a reference and/or explanation. In the Image Analysis, the background normalization needs a bit more detail. It should be explained that the signal is normalized to area and that the units allow areas of different size to be compared. Figures are generally not cited in the Methods section. Figure 1 should be cited in the Results, as an explanation of the overall method. Results: Actual p-values of Figure 2b would help to allay concerns that the bioluminescent …
Comments to Author
This manuscript is well written and presents an important advancement in the analysis of carriage of typhoid fever. There are several minor modifications that would improve clarity: Methods: The Evans Blue-Uranine detection of p22 needs a brief explanation and a reference. Why is the infection delivered IP and not oral or IV? This needs a reference and/or explanation. In the Image Analysis, the background normalization needs a bit more detail. It should be explained that the signal is normalized to area and that the units allow areas of different size to be compared. Figures are generally not cited in the Methods section. Figure 1 should be cited in the Results, as an explanation of the overall method. Results: Actual p-values of Figure 2b would help to allay concerns that the bioluminescent strain is out-competing the wild type in the gallbladder. In Figures 3, BKG needs to be briefly explained in the legend. This explanation would not be necessary for Figure 5 if it was in the Figure 3 legend.
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
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