PONE-D-20-01923

Investigation of synovial fluid induced Staphylococcus aureus aggregate development and its impact on surface attachment and biofilm formation

PLOS ONE

Dear Dr. Paul Stoodley,

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: This manuscript reports a study that sheds new light on prosthetic joint infections with S. aureus biofilms. The role of synovial fluid in reducing biofilm formation through the formation of cell aggregates has not been reported previously and is somewhat unexpected. Aggregation of bacteria is often associated with biofilm formation but that relates to interactions between the cells without the involvement of extrinsic factors such as synovial fluid. The observation in this manuscript is interesting in that it suggests a natural defence mechanism to reduce biofilm formation on prosthetic joint materials. As a reviewer I am not familiar with the amount of synovial fluid present during prosthetic joint surgery but this manuscript suggests that the presence of synovial fluid should be encouraged. The effect of cell aggregation in preventing biofilm formation may be due to a number of factors including synovial fluid sequestering surface receptors or the size of the aggregates, initiating forces that prevent the binding of these aggregates to surface. The authors have discussed these possibilities. Some study of the aggregates - there structure, chemistry and physical nature will be important. This may lead to novel biofilm preventative measures in other environments.

The issue of antibiotic resistance of the aggregates is something that needs to be investigated.

The manuscript is well written. Only a few minor - recommendations and these all relate to the need for bacterial names to be italicized in the reference list. References 14, 20 and 40 all have bacterial names that have not been italicized.

Reviewer #2: The manuscript by Pestrak et al entitled “Investigation of synovial fluid induced Staphylococcus aureus aggregate development and its impact on surface attachment and biofilm formation” is a well-focused in vitro study by experts in the field. As it is current unknown how PJI biofilm formation commences, which is a significant question from both a scientific and clinical standpoint, the authors addressed this in vitro by demonstrating that fibrinogen and fibronectin promoted bacterial aggregation, while cell free DNA, serum albumin, and hyaluronic acid had minimal effect. Additionally, they used microscopy to measure bacterial attachment to implants and found that synovial fluid significantly impeded bacterial surface attachment to a variety of materials. Based on this, the authors conclude that: aggregation in synovial fluid is very rapid, has a threshold of ~60%, fibrinogen and fibronectin in synovial fluid have a crucial role in promoting bacterial aggregation and inhibiting surface adhesion during PJI, and that synovial fluid may have conflicting protective roles for the host by preventing adhesion and promoting bacterial aggregation. As their results support these novel conclusion, the manuscript is considered to be an important advance in this field. However, there are a few points that the authors should address.

1) The authors cite dated literature in the Introduction, which could be markedly improved.

Current data on the incidence and costs of PJI have recently been reported [1], and there is new consensus information of biofilm formation in PJI [2].

2) While the authors’ focused study on synovial fluid aggregates and implant biofilm formation is justified, it is now know that S. aureus utilizes four distinct mechanisms of biofilm formation during PJI [3]. Thus, the lack of studies on Staphylococcus abscess communities (SACs) and colonization of osteocytic canalicular networks in cortical bone need to be acknowledged as limitations.

3) The absence of in vivo studies should also be acknowledge as a limitation.

4) The authors conclude that the aggregation threshold of 60% in their studies is due to a size limit or an upper limit of detection with their method. However, this could also be due to Agr mediated emigration.

1. Schwarz EM, Parvizi J, Gehrke T, Aiyer A, Battenberg A, Brown SA, et al. 2018 International Consensus Meeting on Musculoskeletal Infection: Research Priorities from the General Assembly Questions. J Orthop Res. 2019;37(5):997-1006. Epub 2019/04/13. doi: 10.1002/jor.24293. PubMed PMID: 30977537.

2. Saeed K, McLaren AC, Schwarz EM, Antoci V, Arnold WV, Chen AF, et al. 2018 international consensus meeting on musculoskeletal infection: Summary from the biofilm workgroup and consensus on biofilm related musculoskeletal infections. J Orthop Res. 2019;37(5):1007-17. Epub 2019/01/23. doi: 10.1002/jor.24229. PubMed PMID: 30667567.

3. Schwarz EM, McLaren AC, Sculco TP, Brause B, Bostrom M, Kates SL, et al. Adjuvant Antibiotic-Loaded Bone Cement: Concerns with Current Use and Research to Make it Work. J Orthop Res. 2020. Epub 2020/01/31. doi: 10.1002/jor.24616. PubMed PMID: 31997412.

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Reviewer #1: Yes: Steve Flint

Reviewer #2: No

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Investigation of synovial fluid induced Staphylococcus aureus aggregate development and its impact on surface attachment and biofilm formation

PONE-D-20-01923R1

Dear Dr. Paul Stoodley,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Abdelwahab Omri, Pharm B, Ph.D

Academic Editor

PLOS ONE