Round 1

Reviewer 1 Report

The authors present SPR and phase imaging approaches for studying the effectiveness of phages therapy against bacteria cultures. The manuscript is very well written and their results are presented in an excellent manner. I found the work interesting for the scientific community especially for those who work in the bioimaging field

Author Response

We would firstly like to thank the reviewers for the care and attention that was accorded to our article. It is obvious from the points raised that you gave the article careful consideration, which is greatly appreciated. We have responded to each of the reviewer’s remarks below.

Reviewer 1

The authors present SPR and phase imaging approaches for studying the effectiveness of phages therapy against bacteria cultures. The manuscript is very well written and their results are presented in an excellent manner. I found the work interesting for the scientific community especially for those who work in the bioimaging field

The authors would like to thank reviewer 1 for their positive appraisal of our work.

Reviewer 2 Report

In this paper, “Ultrafast and multiplexed bacteriophage susceptibility testing by surface plasmon resonance and phase imaging of immobilized phage microarrays”, the authors demonstrate SPRi and phase imaging as possible candidates for rapid PST in broth phase. Based on the obtained results, the authors claimed that phage susceptibility can be revealed around 30 minutes in both the SPRi and phase imaging methods. Overall, this manuscript has a strong potential for another review round after applying the issues and addressing the shortcomings listed below:

1-The authors should polish/revise some grammatical mistakes and typos along the manuscript. I invite the authors to read their manuscript carefully and make the required changes where necessary.

2-In the Introduction section, while discussing recent developments in the SPR based sensors, the following work should also be considered and cited to give a more general view to the possible readers of the work: [(i) Photonic and plasmonic metasensors, Laser and Photonics Reviews 16, 2100328 (2022)].

3-In Figure 3, please add “a” and “b” (e.g., Figure 3a and Figure 3b). Additionally, please increase font size of the texts.

4-What is the repeatability of the proposed sensing geometry? Please explain.

5-For the proposed sensing geometry, can we talk about figure of merit? Please explain.

Author Response

We would firstly like to thank the reviewers for the care and attention that was accorded to our article. It is obvious from the points raised that you gave the article careful consideration, which is greatly appreciated. We have responded to each of the reviewer’s remarks below.

Reviewer 2

1. The authors should polish/revise some grammatical mistakes and typos along the manuscript. I invite the authors to read their manuscript carefully and make the required changes where necessary.

The article has been proof-read by a native English speaker with extensive experience in written scientific communication.

2. In the Introduction section, while discussing recent developments in the SPR based sensors, the following work should also be considered and cited to give a more general view to the possible readers of the work: [(i) Photonic and plasmonic metasensors, Laser and Photonics Reviews 16, 2100328 (2022)].

The authors have read with interest the review article proposed, finding it to be an illuminating and thorough overview of the state-of-the-art in metamaterial-based biosensors that we will surely refer to as a reference on these topics in the future. We thank the reviewer for bringing this resource to our attention.

However, in our article we propose a different form of sensor which is not based on metamaterials. Furthermore, for the sake of brevity, we did not include a discussion of recent developments in SPR sensing, since SPR is a mature technology.

The authors fear that incorporating a discussion of recent developments in SPR sensing would create an unnecessarily bloated article and furthermore to extend this discussion to a treatment of metamaterials would stray too far from the scope of our work.

For these reasons, although we would like to incorporate all suggestions from reviewers as much as possible, we have difficulty finding an appropriate section of our article in which to cite the article proposed by reviewer 2.

3. In Figure 3, please add “a” and “b” (e.g., Figure 3a and Figure 3b). Additionally, please increase font size of the texts.

Labels “A” and “B” have now been added and the font size of all labels increased in figures 2 and 3.

4. What is the repeatability of the proposed sensing geometry? Please explain.

If this question refers to the repeatability of the geometry of the sensing layer of the biosensor, we have found that the immobilized phage layer can be reliably formed with high density, good homogeneity, and little to no aggregation. We have submitted an article (currently under review) concerning the repeatable formation of the immobilized phage microarray geometry used in the present work.

Alternatively, if reviewer 2 is instead referring to the repeatability of the sensing method overall,  we would point out that the results presented in this work are extracted from two types of experiment where replication is inherent to the design of the screening method.

In the experiment presented first in the manuscript, the biosensor surface is functionalized with a 10 × 6 array of phage-functionalized regions with several replicates for each functionalization type. In this way, the response of the phage functionalized surface is assayed several times in the same experiment. In the second presented experiment, the fact of having the surface functionalized in the same way in two separate chambers allows us to validate the response of on-target regions by comparing the response to off-target regions. In this type of experiment, each functionalized region is represented by two replicates.

An alternative approach would be to compare the results of similarly fabricated biosensors on different occasions. While this approach is common in more simple bi-molecular systems that have been studied using SPR in the past, it is less pertinent to our system since we are assaying the interactions in complex media of two dynamic entities: exponentially dividing bacteria and immobilized phage. The form of this assay thus makes direct comparison between multiple biochips less relevant since large variations are to be expected. In addition, we would once again point out that the results of this assay are intended as a qualitative or semi-quantitative result and not strictly quantitative.

5. For the proposed sensing geometry, can we talk about figure of merit? Please explain.

The standard figure of merit for an SPR-based sensor is the degree of plasmon curve shift for a given change in refractive index in the solution above the sensor surface. The authors did not endeavour to establish such a value for the proposed sensor since there is a typical standard value for all SPR sensors. To quote the work of Schasfoort¹:

“The sensitivity of most SPR instruments is in the range of ∆n≈10^-5–10^-8 or 10–0.01 pg/mm² of proteinous material at the sensor surface.” where n is the refractive index of the analyte.

Aside from this typical sensitivity of SPR instruments, we would point out that the susceptibility testing method that we present is a qualitative technique rather than a quantitative one. We hope to show that our method can surface those phages which are effective at killing a given bacterial strain, rather than to directly quantify the binding affinity of the phage receptor-binding domains or to measure the quantity of bacteria (as is a common objective in biosensors).

We hope that the above modifications have improved the clarity and overall structure of the article, and adequately responded to the recommendations of the reviewers. We would once again like to thank all reviewers for the care and attention that was given to our manuscript, since it was obvious from the points raised that a thorough reading was accorded in each case.

References

1. Schasfoort, R. B. M. Handbook of Surface Plasmon Resonance. (Royal Society of Chemistry, 2017). doi:10.1039/9781788010283 doi: https://0-doi-org.brum.beds.ac.uk/10.1039/9781788010283

Reviewer 3 Report

see attached

Comments for author File: Comments.pdf

Author Response

We would firstly like to thank the reviewers for the care and attention that was accorded to our article. It is obvious from the points raised that you gave the article careful consideration, which is greatly appreciated. We have responded to each of the reviewer’s remarks below.

Reviewer 3

I’m not familiar with this term defocalized used in the first paragraph of section 2.6. Maybe they mean defocused? I’m not sure.

This term referred to the defocused operation of the phase imaging microscope setup. All mentions of the term “defocalized microscope” have now been changed to “phase imaging microscope” and we thank the reviewer for pointing out that this term lacked clarity.

The authors rely a lot on the supplementary figure showing the 1st derivative of the reflectivity. Unless there is a limitation of the number of figures it would be nice to include this in the main text.

Figure 1 Three ways of representing the first derivative of SPR sensorgrams. A: Smoothed first derivatives of SPR responses of regions exposed to S. aureus SA1. This figure is the same as Figure S1 of the supplementary material. B: An unsmoothed first derivative of the same data as in (A). It is difficult to observe any clear point of maximum rate of change. C: Smoothed first derivatives of SPR responses of regions exposed to P. putida. The gh-1 response appears to show a 5-minute interval from 10 to 15 minutes where the sensor experiences a large jump in reflectivity. However, the real duration of this jump was less than a minute, only appearing longer since the smoothed trace is the result of a convolution of a window with the raw data.

The author is entirely correct to point out that the first derivative figures may present a useful addition to the article.

The author’s themselves weighed the benefits of presenting the first derivatives in the main text of the article rather than including them in the supporting information. Our reasons for relegating them to the supplemental information were three-fold:

1. Firstly, it was our opinion that the evolution of the SPR sensorgram curves already clearly shows the difference in slope between the on- and off-target regions, and that the inclusion of the first derivatives would lead to unnecessary repetition of essentially the same information.
2. In contrast to typical SPR sensorgrams for simple bi-molecular systems the first derivative of SPR data in our experiments often has a noisy appearance (Figure 1b). In order to smooth the first derivative enough to be intelligible, we introduce artifacts that result from the averaging across a window that includes large jumps in reflectivity (Figure 1c). This is especially the case when we zoom the figure to present data between 0 and 100 minutes (Figure 1c), rather than 0 to 300 minutes. This creates the mistaken impression that the surface experienced a large change in reflectivity for several minutes, which was not the case.
3. In order to communicate our results more clearly and succinctly, the first derivative figures were smoothed with a different smoothing factor that the sensorgram figures, presented over a larger range of time (0 to 600 minutes) and included as supplementary information, since the interest in presenting them is simply to show the later peaking of the rate of change of reflectivity correlates with on-target regions.

We are left with the choice of either presenting un-smoothed first derivatives or introducing smoothing artifacts into figures of the main text. For the above reasons, we felt that including the first derivatives would interrupts the flow of the article without adding additional clarity. We hope that this explanation adequately responds to the reviewer’s recommendation.

It’s not clear to me that just because a phage is incapable of replicating in a give host that you would see no binding of the phage to the bacterial strain. It seems limited binding might explain the response seen that has been attributed to an increased bacterial concentration in solution.

The phage/host pairs were chosen to have bacteria featuring membranes with very different characteristics. While S. aureus is Gram-positive, P. putida is Gram-negative and so the two types of cell differ significantly in cell wall composition. The receptor of phage gh-1 is known to be lipopolysaccharide¹, which is present in P. putida but absent in the Gram-negative S. aureus.

In contrast, podoviridae infecting S. aureus are known to require the presence of teichoic acid in the bacterial surface in order to absorb to their host.² Teichoic acid is present in the cell wall of S. aureus but absent in that of Gram-negative P. putida. For these reasons, we consider it to be a reasonable conclusion that the observed lack of activity in off-target phage/host pairs is well correlated with an inability of phage in this study to bind to cells of the off-target bacterial strain.

We thank the reviewer for pointing out that this was unclear and we have now added a paragraph to clarify this point on lines 378-388.

The data on phase imaging shown in figure 4 is not fully developed. Why was this not repeated with the same bacteria and phage combinations used for the SPR experiments?

The phase-imaging experiments were indeed carried out with a phage-host combination that was also studied using SPR, that of gh-1 and its host P. putida.

The phase-imaging aspect of this paper is more exploratory than the SPR aspect. As such, phase-imaging was carried out in a lab dedicated to optics and experimental physics, where the manipulation of BSL2 bacteria (i.e., S. aureus) is not possible. Instead, phase-imaging was carried out on the BSL1 strain P. putida as a proof of concept of an alternative method for phage susceptibility testing. These methods are presented together in this work since the observation of phage-host interactions by two different methods are complementary, with phase-imaging observations suggesting a mechanistic explanation for the evolution of the observed SPR curves.

Are the phage immobilized the surface in this experiment or are they in solution with the bacteria?

For the SPR experiments presented in this work, the phages were immobilized on the surface while in phase-imaging experiments, phages were instead in suspension with the bacteria.

Where are the control experiments for the phase imaging? For example, where are the negative control to help validate that the lack of adhered bacteria is not due to another factor such as the conditions of the imaging?

The reviewer is entirely correct to enquire about the controls in our experiments. In order to respond to this point, we undertook a new series of experiments to better demonstrate the phase-imaging approach to PST, with the inclusion of a negative control to exclude the possibility that the observed destruction of bacteria was not due to some other factor. In these new experiments, P. putida were observed by phase-imaging with and without the addition of gh-1 phages. Figure 4 and the associated discussion (section 3.2) have now been updated to include these new experiments. In addition, Figure 5 now shows a time-lapse sequence of bacterial lysis, which reveal new insights into spheroid formation and continuing cellular division immediately prior to lysis, both of which are observed with a large FoV and with single-cell resolution.

It is not clear how the authors can attribute the increase in bacteria to division and growth

Supplementary video S3 shows individual division events of bacterial cells, which we are able to observe over a large field-of-view. An alternative explanations for the increase in bacterial numbers — sedimentation of the bacteria into the focal plane — is ruled out since we observe both the upper and lower walls of the Gene Frame chamber. Instead, the majority of cells that are detected can be seen either to be present from the beginning or to originate from division of existing cells, as shown in supplementary video S4

The discussion covers very little of the experiments that were included in the manuscript. A more in depth discussion of the data in the context of how it could be used for PST measurements would be helpful.

The data from figure 4 are no fully developed in either the results or the discussion.

We agree with the reviewer that these experiments would benefit from being discussed more thoroughly. To respond to this point, the results section has now been expanded with a greater discussion of phase-imaging observations, and an additional figure has been added (Figure 5) as described above.

We hope that the above modifications have improved the clarity and overall structure of the article, and adequately responded to the recommendations of the reviewers. We would once again like to thank all reviewers for the care and attention that was given to our manuscript, since it was obvious from the points raised that a thorough reading was accorded in each case.

References

1. Kovalyova, I. V. & Kropinski, A. M. The complete genomic sequence of lytic bacteriophage gh-1 infecting Pseudomonas putida - Evidence for close relationship to the T7 group. Virology 311, 305–315 (2003) doi: https://0-doi-org.brum.beds.ac.uk/10.1016/S0042-6822(03)00124-7
2. Li, X. et al. An accessory wall teichoic acid glycosyltransferase protects Staphylococcus aureus from the lytic activity of Podoviridae. Sci. Rep. 5, 1–10 (2015) doi: https://0-doi-org.brum.beds.ac.uk/10.1038/srep17219

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Pay attention to the self-citation issue and make the required changes.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors have thoroughly addressed my original concerns. 

Author Response

We would firstly like to thank reviewer 1 for the care and attention that was accorded to our article. It is obvious from the points raised that you gave the article careful consideration, which is greatly appreciated. The authors would like to thank reviewer 1 for its positive appraisal of our work. We are pleased that he confirmed each point has been properly addressed.