Oxygen-Independent Antimicrobial Photoinactivation: Type III Photochemical Mechanism?

Round 1

Reviewer 1 Report

The manuscript "Oxygen-independent antimicrobial photoinactivation: Type III photochemical mechanism?" by Michael R Hamblin and Heidi Abrahamse is written in well English and contains sufficient pictures, which makes it easy reading. The manuscript contains 78 references (17 of them with Michael R Hamblin co-authorship). 

The main hypothesis about "Type III photochemical pathway” is supported with references to relevant studies, including some papers published by the Hamblin laboratory.

The manuscript looks like a high-quality review paper, which will be interesting for many researchers working in this area.

There are only a few minor questions:

1. The manuscript is focused on inactivation of different bacteria. But what about viruses and fungi? Is it possible to include some references and brief descriptions of such studies?

2. The manuscript contains plenty references to high-quality microbiological and biochemical studies, but what about ultrastructural aspects (electron microscopy, etc) of the oxygen-independent antimicrobial photoinactivation? Do such studies exist?

Author Response

The manuscript is focused on inactivation of different bacteria. But what about viruses and fungi? Is it possible to include some references and brief descriptions of such studies?

 

Response: This section has been added “Although the studies that have so far been conducted have concentrated on bacteria, the technique could in principal be applied to viruses and fungi. Psoralens in particular would be expected to allow photoinactivation of viruses in the absence of oxygen.”

 

The manuscript contains plenty references to high-quality microbiological and biochemical studies, but what about ultrastructural aspects (electron microscopy, etc) of the oxygen-independent antimicrobial photoinactivation? Do such studies exist?

 

Response: Not as yet.

 

Reviewer 2 Report

This manuscript reviews the concept of oxygen-independent photo-inactivation of microbes. The manuscript introduces the common oxygen-dependent mechanisms, then focuses on the more unusual oxygen-independent mechanism, dubbed type III by the authors. The evidence for oxygen-independence is clearly outlined, and the manuscript is easy to follow and well-written for the most part. This is interesting work, although, as the authors admit in the conclusion, it is not clear that this mechanism can be applied to actual clinical treatment of disease. One problem that is not particularly emphasized, but common to phototherapy, presumably lies in the delivery of light deep into tissues. Nonetheless, the review is timely and thoroughly referenced, and as such should be of interest to those involved in novel approaches to antimicrobial treatments.

There are a few minor points that should be addressed:

Chemical structures are incorrect: Fig. 2 and Fig. 3: one double bond missing in the central ring, should be a benzene ring in all the structures throughout both figures. Fig. 4A: doxycycline structure: it is very odd to include wedges within a ring, should show the ring with single bonds and the stereochemistry just for the substituents. Line 20: tetracycline mis-spelled; line 177 demeclocycline Line 94 “ref” missing reference Lines 203-204: sentence unclear: “While….” Line 211: define DMCT at first occurrence, it doesn’t seem to be defined until the next paragraph. 5: what is the y-axis, survival fraction? “Dark” clearly is not the control since the survival fraction is less than 1. But it is unclear to me what the control is. Line 283: “Ki” means KI, potassium iodide, not the inhibition constant Ki Line 350 “the restored killing” means “restored the killing” Line 383-4: not two sentences Line 396-399: this sentence is unclear, were the individuals treated for sterilization of platelets, etc.? Or was that the blood that was treated?

Overall, this is an interesting manuscript that will inform readers of an unusual aspect of phototherapy. I recommend publication after the above points have been addressed.

Author Response

Chemical structures are incorrect: Fig. 2 and Fig. 3: one double bond missing in the central ring, should be a benzene ring in all the structures throughout both figures.

 

Response: I apologize for the error. The structures have been corrected

 

Fig. 4A: doxycycline structure: it is very odd to include wedges within a ring, should show the ring with single bonds and the stereochemistry just for the substituents.

 

Response: This has been changed as requested

 

 

Line 20: tetracycline mis-spelled;

 

Response: corrected

 

line 177 demeclocycline

 

Response: corrected

 

Line 94 “ref” missing reference

 

Response: the “ref” has been deleted. Since the Endnote formatting has been lost it is impossible to add the missing reference

 

Lines 203-204: sentence unclear: “While….”

 

Response: sentence has been rewritten “While photoactivated MB effectively eradicated bacteria incubated in phosphate buffered saline, this activity was completely lost in growth medium.”

 

 

Line 211: define DMCT at first occurrence, it doesn’t seem to be defined until the next paragraph.

 

Response: The abbreviations DOTC and DMCT have been added in Figure 4.

 

 

5: what is the y-axis, survival fraction? “Dark” clearly is not the control since the survival fraction is less than 1. But it is unclear to me what the control is.

 

Response: we have added “Values are calculated based on no treatment control. “

 

 

Line 283: “Ki” means KI, potassium iodide, not the inhibition constant Ki

 

Response:

 

 

Line 350 “the restored killing” means “restored the killing”

 

Response: sentence has been rewritten “In the absence of oxygen, the addition of azide substantially restored the killing, and in the case of LC15 brought it back to the levels found with oxygen present.”

 

Line 383-4: not two sentences

 

Response: the criticism is unclear, but we believe the text is correct

 

 

Line 396-399: this sentence is unclear, were the individuals treated for sterilization of platelets, etc.? Or was that the blood that was treated?

 

Response: We apologize for the confusion. It has been rewritten “In some cases the patients’ skin has been treated for dermatological disorders, but in other cases they have received platelets, plasma and other blood products that have been treated ex vivo for antimicrobial sterilization.”