PONE-D-22-23137Quantitative evaluation of Mycobacterium abscessus clinical isolate virulence using a silkworm infection modelPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: A nice study, easy to follow. I have only two text addition requests for the discussion and one statistical recalculation:

How accurate is hemolymph for assessing MABC burden? Presumably these bacteria can invade the tissue like they do in other model systems so there might be unaccounted burden missed by a more thorough method such as homogenisation. Please elaborate discussion to speculate if tissue invasion is expected and if the silk worm in amenable to histological study.

Line 308: zebrafish models can evaluate virulence < 7 days (Kremer lab) in embryos, or the 14-15 day time period is from an Oehlers lab paper using adult zebrafish. It is worth commenting around this paragraph on the temperature differences, the silk worm allows the study of virulence at 37 while zebrafish studies are in the 28-32 degree range.

Figure 7: please replace T test with ANOVA

Reviewer #2: Virulence of seven clinical isolates of Mycobacterium abscessus subspecies abscessus was investigated using silkworm infection model. Additional, using a standard/validated protocol, virulence of these isolates was also studied in human macrophage cell line THP-1. Overall, the studies were straightforward and systematically performed. Data were interpreted in a rigorous manner and conclusions are justified by the results. Below I have included recommendations for revisions to improve on clarity and accuracy. Also, I have recommended to include additional discussion on the limitations of these M. abscessus infection model.

MINOR COMMENTS:

1. Abstract: It is redundant to use M. abscessus subspecies M. abscessus throughout the abstract as only supspecies abscessus is used in the study. So, please delete ‘subspecies abscessus’ from the following lines (Line 25, another one in line 25, line 26, 27, 29).

2. Line 28: add ‘in humans’ after ‘infection’.

3. Line 30: replace ‘were determined using’ with ‘in’.

4. Line 33: delete ‘with’.

5. Line 46: replace ‘effects’ with ‘efficacies’.

6. Line 46-48: It is stated that ‘mice infected with MABC dies over several months’. This is completely untrue. Even the cited references show that it requires immunesuppression or immunecompromised mice to maintain MABC infection. So this sentence is factually incorrect and should be deleted. Replace it with the following sentence: ‘Existing mouse models of MABC infection require several weeks to complete a single study, which is not convenient especially for MABC virulence screening purposes, and thus the development of a model that permits a more rapid evaluation of MABC virulence is highly desirable.’

7. Animal models of a human disease have their strengths and limitations. A good and objective manuscript should include limitations of the model. The authors should include the fact that most of the Mycobacterium abscessus infections are acquired via aerosol and this model does not take that into account. Most of the Mycobacterium abscessus disease is pulmonary and this model cannot recapitulate that aspect. The formation of lesion in the lungs and immune response are distinct from that in the silkworm. Therefore, it is important to include limitations of the silkworm model. Then describe the unique strengths of this model, which is the rapid time in which virulence of M. abscessus can be studied. Also, a large number of silkworms can be used, which is important for screening studies.

8. NOTE that the study assesses ‘virulence’ and not ‘pathogenecity’ of M. abscessus isolates.

9. Figure 1: what is n? n should be described as the number of independent M. abscessus isolates so that it is not confused with the number of silkworms used. Also, the number of silkworms used should be included in the figure legend. Pathogenecity study should include progression of disease in terms of tissue pathology and disease phenotypes in the host as the disease evolves. Therefore, ‘virulence’ is the correct terms and ‘pathogenecity’ should not be used. Line 31: replace ‘pathogenic’ with ‘virulent’. Line 224: replace ‘pathogenecity’ with ‘virulence’. Line 233: replace ‘pathogenecity’ with ‘virulence’. Line 295: replace ‘pathogenic’ with ‘virulent’.

10. Figure 2. In the figure legend ‘n’ should be defined as the number of silkworms used.

11. Figure 3. What is ‘n’? Is it the number of silkworms used or M. abscessus isolates?

12. Figure 4: Please define ‘n’. It represents the number of what?

13. Figure 5: Please define ‘n’.

14. Figure 6: Please define ‘n’.

15. Line 97: insert ‘were’ after ‘silkworms’.

16. Line 98: insert ‘ , and’ after ‘larva)’.

17. Line 99: Briefly describe how silkworms were injected with M. abscessus. What type of instrument/device was used to deliver M. abscessus?

18. Line 200: Insert ‘in humans’ after ‘infections’.

19. Line 288: replace ‘severe’ with ‘severity’.

20. Line 299: replace ‘was identified as a highly pathogenic strain using the’ with ‘is highly virulent in’

21. Line 303: delete ‘avirulent’. This term is unnecessary here.

22. Line 305: The cited reference #33 by Foreman et al is not appropriate here as random transposon mutagenesis is described in the publication rather than targeted gene deletion in MABC. This reference should be removed. More relevant references are [Nessar R, Reyrat JM, Davidson LB, Byrd TF. Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response. Microbiology (Reading). 2011 Apr;157(Pt 4):1187-1195. doi: 10.1099/mic.0.046557-0. Epub 2011 Feb 3. PMID: 21292749.] and [Galanis C, Maggioncalda EC, Kumar P, Lamichhane G. Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild β-Lactamase Activity. J Bacteriol. 2022 May 17;204(5):e0004622. doi: 10.1128/jb.00046-22. Epub 2022 Apr 5. PMID: 35380462; PMCID: PMC9112878.].

23. Line 308: insert ‘mice’ after ‘Danio rerio’. Mice should be included in this list, otherwise this sentence will be factually incorrect.

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Reviewer #2: No

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Quantitative evaluation of Mycobacterium abscessus clinical isolate virulence using a silkworm infection model

PONE-D-22-23137R1

Dear Dr. Matsumoto:

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