Silencing of Salmonella typhimurium Pathogenesis: Atenolol Acquires Efficient Anti-Virulence Activities

Round 1

Reviewer 1 Report

In this manuscript the authors try to examine the effect of atenolol on virulence and virulence factors of E. coli and S. typhimurium. It makes for an interesting story, but I have great reservations about some of the data presented. I feel this data should be better controlled to allow conclusions to be drawn. There are also several examples where the writing could be improved and I have provided some examples below.

Line 71: "of the spy" should be "of the spying"

Line 120 "treated or treated"- one of these should be untreated?

Methods: in general the methods are missing some key points. What was atenolol dissolved in? What concentrations of this solvent were present in the experiments? Do the controls have the same solvent present?

It was also not always clear when during an experiment was atenolol present- one example in the cell invasion assay- were bacteria grown in atenolol? Or was atenolol only added when cells were used for infection? More detail is needed.

In the statistical analysis section it states "The tests were conducted in triplicate..." Which tests? Presumably not the statistical tests, but the experiments upon which the tests were done. But which experiments? Were all experiments done in triplicate? More detail needed.

Results:

Line 198 "Atenolol inhibited the growth of S. typhimurium and E. coli K-12 at 2 mg/mL." Where is this data? 

Using 1/5 of this concentration means around 0.4 mg/ml. This still seems very high- what is the typical concentration that can be given to a patient? This should be included. It does not invalidate the study, but might mean that this treatment might need to be altered before use in humans?

Figure 1: this is not a graph showing an effect or not on growth, rather on survival. Do you have growth curves in the presence/absence of the drug?

Line 212: "cove" should be cover

Figure 3: are all the micrographs on the same scale- there is only one scale bar. However, the cells in the treated panel look much smaller than the untreated one.

Figure 3D legend: "fold changes were calculated" - fold changes from what?

RT qPCR: this is an experiment I think requires more controls before it can be interpreted or published. The use of a single housekeeping gene is not good practice. I would use at least two reference genes. 

Is gyrB a good gene to use as your housekeeping gene? Why choose this? Does gyrase expression alter due to stress that atenolol is inducing. One explanation for all these results is that in fact gyrB expression is up, so your DDCt values are off. I would suggest using a second housekeeping gene which has been previously validated to either compare to gyrB or to the whole set of genes. The use of a single reference is very unreliable.Preferably use a couple of extra housekeeping genes and compare the relative expression of each.

Line 258 and elsewhere: use test rather than attest.

Line 262: norepinephrine deaths- is this a significant difference? I don't think it is.

Figure 5 data: the data shown has 70% survival for the atenolol treated cells at 5 days not the only 2 deaths described in the text. This discrepancy needs to be addressed and the results re-written and the significance re-tested using the 70% survival rate.

Discusison

Line 347: "As an obvious demonstration.." I don't think this is a demonstration at all?

Line 352-53. This does not flow on at all from the previous lines?

Line 358 "bacterial resistance"- do you mean virulence?

Line 358 "confers"- do you mean uses?

Line 363 "legibility" - do you mean legitimacy?

Line 367- as above- this assay showed bacterial survival not bacterial growth.

Line 368 "Besides its clinical importance," wasn't sure this was appropriate given you are still talking about virulance.

Line 376 "virtually"- do you mean during in silico testing?

Author Response

Dear Reviewer,

We are very thankful for your valuable comments and suggestions. All the raised points are addressed, please find an attached reply.

Again we are grateful for the fruitful discussion that improved our manuscript.

Best Regards,

Wael

Author Response File: Author Response.pdf

Reviewer 2 Report

The studies presented for review are very interesting and have great scientific value; the manuscript is well written, the results are clearly presented. The manuscript needs some minor corrections.

The studies presented for review are very interesting and have great scientific value; the manuscript is well written, the results are clearly presented. The manuscript needs some minor corrections.

 - Serotype names (Typhimurium) should be capitalized and not italicized - this should be corrected throughout the manuscript.

- Systematic names of microorganisms (Salmonella) and "in vitro", "in vivo", "in silico" as well as gene names (sdiA, qseC etc., eg. L246, 253) should be written in italics;

- Table 1 - sifB, not SifB

- Authors should provide the approval number of their local ethics committee for animal testing.

-L120 - method description should be completed; in what medium were the bacteria grown? Was it a microplate culture? Was the measurement taken in a spectrophotometer?

-L171 - studies, not Studies

-L164 - How long have the bacteria been incubated with atenolol and norepinephrine and under what conditions?

- Figure 1, 2 4 and 4 - I think the figure will be clearer if instead of "Treated Salmonella" the authors write "Salmonella treated with atenolol"

L262-264 - six out of 10 ?; 2 deaths aout of 10 ?; add also percentage please

L343-353 - I suggest that you delete the first paragraph of the discussion as it contains repeated information

- Information on atenolol is missing in the manuscript; what is known about this substance? what is it currently used for? Has anyone noticed the anti-pathogenic effect of this substance before?

- L384 - Literature reports indicate that Salmonella enterica has 22 islands of pathogenicity (https://pubmed.ncbi.nlm.nih.gov/24405577/)

-L390 - Why did the authors study the expression of only genes encoded within SPI-2? SPI-1 genes also play a key role in the pathogenicity of Salmonella.

Author Response

Dear Reviewer,

We are very thankful for your valuable comments and suggestions. All the raised points are addressed, please find an attached reply.

Again we are grateful for the fruitful discussion that improved our manuscript.

Best Regards,

Wael

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I appreciate the many changes the authors have made to improve the amnuscripr and it is generally much better for it.

I have a few major concerns remaining.

1)    Figure 3: I still am unsure why the cells look so different between A and B- the cells in A seem to be around 10 microns in length if the scale bar is correct? I think the scale bar in A must be wrong or else those cells are huge for Salmonella? Similarly I do’t think the scale bar in C can be correct either. I would urge the authors to recheck these scale bars as they do make make any sense to me.

2)    Figure 4. As stated previously I would never rely on just a single housekeeping gene for RTqPCR. You may be looking at upregulation of gyrase here, which might skew your results significantly. A second (or more) different housekeeping gene should be used to validate this.

Author Response

Dear Reviewer,

We are very thankful for your valuable comments and suggestions. Please find the attached reply.

Best Regards,

Wael

Author Response File: Author Response.pdf