Virulence Characteristics of mecA-Positive Multidrug-Resistant Clinical Coagulase-Negative Staphylococci

Round 1

Reviewer 1 Report

The mauscript by Chon et al. is a decent description of selected molecular and phenotypic characteristic of multidrug-resistant coagulase-negative (CoN) staphylococci, that carry the mecA allele. Initial characteristic concerns 29 clinical isolates of CoN staphylococci, of which some have been characterized in more detail with respect to their ability to form biofilms. Most of the methods are described properly and many features of tested strains have been analysed. However, the manuscript is rather purely descriptive and this is its major weak point. Additionally, a rationale of selecting particular strains for a more detailed analysis (e.g. for FESEM an NS-TEM analysis of biofilms) is not clearly presented.

Fig. 1. Authors should provide the information concerning the DNA size marker used and the conditions of gel electrophoresis to separate plasmids. One can assume that it was PFGE, but it should be stated in the figure legend. Additionally, it cannot be verified base on the data provided.

L. 202-203: The size of plazmids in their CCC form cannot be estimated based on the migration of plasmid CCC forms in a gel, as the rate of CCC forms migration strongly depends on topology.

For easier comparison of data presented in Figure 2, 3 and 4 the results of biofilm assays by different methods should be presented in one figure

L. 265-271 and Figures 6 and 7: Author should provide a rationale for their choice of strains for imaging of their biofilm with the use of FESEM and NS-TEM.

L. 282: Do the values in parentheses represent the percentage of sensitive or resistant strains. Please specify. From the present form of text it is unclear.

L. 287: Remove "strains"

Author Response

Reviewer 1

The mauscript by Chon et al. is a decent description of selected molecular and phenotypic characteristic of multidrug-resistant coagulase-negative (CoN) staphylococci, that carry the mecA allele. Initial characteristic concerns 29 clinical isolates of CoN staphylococci, of which some have been characterized in more detail with respect to their ability to form biofilms. Most of the methods are described properly and many features of tested strains have been analysed. However, the manuscript is rather purely descriptive and this is its major weak point. Additionally, a rationale of selecting particular strains for a more detailed analysis (e.g. for FESEM an NS-TEM analysis of biofilms) is not clearly presented.

Response: Authors highly appreciate the reviewer’s thorough reading of the manuscript and helpful remarks that helped us to improve the manuscript. We have seriously considered and addressed your valuable comments by point-to-point responses in the following context. The reviewer’s concern of the weak point was reflected in the revised manuscript. We hope our revision has improved the paper to a level of your satisfaction.

Fig. 1. Authors should provide the information concerning the DNA size marker used and the conditions of gel electrophoresis to separate plasmids. One can assume that it was PFGE, but it should be stated in the figure legend. Additionally, it cannot be verified base on the data provided.

Response: Authors are very grateful to the reviewer’s great suggestion. Based on the reviewer’s suggestion, a supercoiled DNA ladder and agarose gel electrophoresis running conditions were provided in Line 129-131 and Fig. 1. legend (Line 221-223) was revised.

Revised manuscript:

Line 129-131

DNA profiles were revealed by 0.8% agarose gel electrophoresis at 100 V for 4 hrs and a supercoiled DNA from Agilent Technologies (Santa Clara, CA) was employed as a molecular size marker.

Line 221-223

Fig. 1. Plasmid DNA profiles of Staphylococcus species. Agarose gel electrophoresis (0.8%) was run at 100 V for 4 hrs and a supercoiled DNA ladder from Agilent Technologies was used as a molecular size marker.

1. 202-203: The size of plazmids in their CCC form cannot be estimated based on the migration of plasmid CCC forms in a gel, as the rate of CCC forms migration strongly depends on topology.

Response: Authors appreciate and absolutely agree with the reviewer’s sharp point. We removed plasmid sizes that were mentioned and revised Line 212-214 of the manuscript.

Revised manuscript:

Although most S. sciuri isolates carried only megaplasmids, other CoNS strains carried multiple plasmids with various sizes (Fig. 1)

For easier comparison of data presented in Figure 2, 3 and 4 the results of biofilm assays by different methods should be presented in one figure

Response: Authors are very thankful for the reviewer’s critical advice. Figure 2, 3, and 4 was renamed as Figure 2A, 2B, and 2C (Line 262, 266, 270) and the changes were applied to Line 246, 249, 250 of the manuscript. In addition, we will ask an assistant editor of Microorganisms Editorial Office for putting Figures 2A, 2B, and 2C in one figure.

1. 265-271 and Figures 6 and 7: Author should provide a rationale for their choice of strains for imaging of their biofilm with the use of FESEM and NS-TEM.

Response: Authors really appreciate the reviewer’s great comment. Among good biofilm formers, S. sciuri strains 10SC and 70SC were chosen since they had only a single (eno) or two (eno, icaA) biofilm-associated genes. S. simulans strain 297SI and S. lugdunensis strain 266LU were selected as intermediate and poor biofilm formers. The manuscript was revised in the Line 279-281.

1. 282: Do the values in parentheses represent the percentage of sensitive or resistant strains. Please specify. From the present form of text it is unclear.

Response: Authors are very sorry to make the reviewer confuse and revised Line 296-299 of the manuscript based on the reviewer’s advice.

Revised manuscript:

Most CoNS were resistant to β-lactam antibiotics and their resistance rates of PEN, OXA and CEF were 89.7%, 75.9%, and 38.0%, respectively. On the other hand, the resistance rates of VAN and RIF were very low, showing 3.5% and 13.8%, respectively.

1. 287: Remove "strains"

Response: Authors are very thankful for that the reviewer found a misspelling. We removed “strains” and revised Line 302-305 of the manuscript.

Revised manuscript:

Similarly, we observed that S. sciuri strains 40SC, 50SC, 60SC, and S. epidermidis strain 224EP carried aac(6')-Ie-aph(2')-Ia, aph(3')-IIIa, and aac(6')-aph(2"), they were susceptible to aminoglycoside antibiotics, including gentamicin, kanamycin, and streptomycin.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors aim to detect the virulence characterizes of a set of clinical CoNS. The manuscript is well written, and the experiments are well performed, and the results are presented in a uniform manner. However, the experiments show that this is a preliminary screening tests performed on the isolates (targeting a set of determined genes);

• Representative isolates based on the PCR screening results should be selected for WGS.
• Plasmid sequencing should be performed as well since it may carry the antibiotic resistance/ virulence genes and investigate the conjugatively of these plasmids.
• Table 1 should include the treatment and the patient outcome
• Figure 1 is of low quality; again this can be used as screening experiment to help with the closing of the plasmid when sequenced.
• The wgs will reveal much more regarding the virulence characteristics especially with the presence of huge databases that may better improve the manuscript

Author Response

Reviewer 2 

Comments and Suggestions for Authors

The authors aim to detect the virulence characterizes of a set of clinical CoNS. The manuscript is well written, and the experiments are well performed, and the results are presented in a uniform manner. However, the experiments show that this is a preliminary screening tests performed on the isolates (targeting a set of determined genes);

Response: Authors highly appreciate the reviewer’s thorough reading of the manuscript and helpful remarks that helped us to improve the manuscript. We have seriously considered and addressed your valuable comments by point-to-point responses in the following context.

• Representative isolates based on the PCR screening results should be selected for WGS. 
• Response: Authors are very grateful to the reviewer’s keen suggestion. We planned to perform the whole genome sequencing of the representative isolates, but, unfortunately, US FDA budget is currently very limited for supporting the sequencing due to the urgent expenditure priorities for unprecedented coronavirus outbreak in USA. When the budget is available, we will perform the whole genome sequencing and their comparative genome analysis.
•  
• Plasmid sequencing should be performed as well since it may carry the antibiotic resistance/ virulence genes and investigate the conjugatively of these plasmids. 
• Response: Authors really appreciate the reviewer’s great comment. Plasmid DNA sequencing and conjugation experiment will be performed when the whole genome sequencing will be done, and the results will be prepared for the next manuscript.
•  
• Table 1 should include the treatment and the patient outcome 
• Response: Authors are very thankful for the reviewer’s sharp advice. Since some CoNS strains were donated from the other institution, the donor didn’t want to provide the detailed strain information, and the treatment and the patient outcome data are available in only thirteen among twenty-nine strains, authors are very sorry for not including that additional information in the Table 1.
•  
• Figure 1 is of low quality; again this can be used as screening experiment to help with the closing of the plasmid when sequenced. 
• Response: Authors are very grateful for the reviewer’s great advice and very sorry for not providing the good quality image of agarose gel electrophoresis due to long running time (4 hrs) of low concentration (0.8%) of agarose gel at 100 Voltage. We are also very grateful for your comments that plasmid DNA agarose gel can be used as screening experiment to help with the closing of the plasmid when sequenced.
•  
• The wgs will reveal much more regarding the virulence characteristics especially with the presence of huge databases that may better improve the manuscript
• Response: Authors really appreciate and absolutely agree with the reviewer’s point. The whole genome sequencing including the plasmid DNA sequencing will reveal very useful information associated with virulence, biofilm, antibiotic resistance, and other genetic characteristics. When the budget is available, the sequencing project will be continued, and the results will be reported in the next manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

The present work, titled “Virulence characteristics of mecA-positive multidrug-resistant clinical coagulase-negative staphylococci”, by Jung-Whan Chon, Un Jung Lee, Ryan Bensen, Stephanie West, Angel Paredes, Jinhee Lim, Saeed Khan, Mark E. Hart, K. Scott Phillips and Kidon Sung, constitutes a good account for the study of mecA-positive coagulase-negative staphylococci. In this work genotypical and phenotypical studies were done regarding genes responsible for antimicrobial resistance, biofilm formation and pathogenicity. The study was well performed, with a good experimental design and it is also well written and clearly structured, however the discussion could be improved.

Line 86: Indicate the date and time span when the samples were obtained and also how was the presence of mecA determined in the isolates?

Line 178: How many strains were studied by microscopy and what were the criterium used

Lines 206-207: Why did you choose not the study the staphylococcal cassette chromosome mec (SCCmec)? Please see:Antimicrob Agents Chemother. 2017 Jun; 61(6): e02302-16. Published online 2017 May 24. Prepublished online 2017 Apr 3. doi: 10.1128/AAC.02302-16;  Ann Clin Microbiol Antimicrob. 2017; 16: 57. Published online 2017 Aug 22. doi: 10.1186/s12941-017-0231-z; Malays J Med Sci. 2017 Oct; 24(5): 7–18. Published online 2017 Oct 26. doi: 10.21315/mjms2017.24.5.2

Line 293-299: It would be nice to perform some conjugation and/or transformation experiments to see if the plasmids could in fact be mobilized. Why wasn’t this done?

Line 218-320: It might be important to deposit the sequences of these genes in databases, since

Lines 333-340: Is there a relationship between the presence of any antibiotic(s) resistance genes and the formation of biofilms? Why wasn’t this determined?

Author Response

Reviewer 3

Comments and Suggestions for Authors

The present work, titled “Virulence characteristics of mecA-positive multidrug-resistant clinical coagulase-negative staphylococci”, by Jung-Whan Chon, Un Jung Lee, Ryan Bensen, Stephanie West, Angel Paredes, Jinhee Lim, Saeed Khan, Mark E. Hart, K. Scott Phillips and Kidon Sung, constitutes a good account for the study of mecA-positive coagulase-negative staphylococci. In this work genotypical and phenotypical studies were done regarding genes responsible for antimicrobial resistance, biofilm formation and pathogenicity. The study was well performed, with a good experimental design and it is also well written and clearly structured, however the discussion could be improved.

 Response: Authors highly appreciate the reviewer’s thorough reading of the manuscript and helpful remarks that helped us to improve the manuscript. We have seriously considered and addressed your valuable comments by point-to-point responses in the following context. The discussion was improved as the reviewer suggested. We hope our revision has improved the paper to a level of your satisfaction.

Line 86: Indicate the date and time span when the samples were obtained and also how was the presence of mecA determined in the isolates?

Response: Authors are very thankful for the reviewer’s great suggestion. CoNS strains were isolated from 01/01/2010 to 10/01/2011 and the manuscript was revised in Line 94-96. The presence of mecA gene was determined by PCR method, which Schnellmann et al. used in their research article (Schnellmann, C.; Gerber, V.; Rossano, A.; Jaquier, V.; Panchaud, Y.; Doherr, M.G.; Thomann, A.; Straub, R.; Perreten, V. Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission. J Clin Microbiol 2006, 44, 4444-4454). The amplified PCR products were sequenced, and the sequences were BLAST searched against the existing GenBank database to confirm their identity. Primer sequences and cited journal information are available in the supplementary Table S1.

Revised manuscript:

Line 94-96

Twenty-nine mecA-positive clinical CoNS isolated from various sources, including nasal, catheter, blood, urine, perirectal, and wound, during January 2010 to October 2011 were used in this study (Table 1).

Line 178: How many strains were studied by microscopy and what were the criterium used?

Response: Authors really appreciate the reviewer’s keen comment. We took images of total 6 strains. Among good biofilm formers, S. sciuri strains 10SC and 70SC were chosen since they had only a single (eno) or two (eno, icaA) biofilm-associated genes. S. simulans strain 297SI and S. lugdunensis strain 266LU were selected as intermediate and poor biofilm formers. The manuscript was revised in the Line 279-281.

Lines 206-207: Why did you choose not the study the staphylococcal cassette chromosome mec (SCCmec)? Please see:Antimicrob Agents Chemother. 2017 Jun; 61(6): e02302-16. Published online 2017 May 24. Prepublished online 2017 Apr 3. doi: 10.1128/AAC.02302-16;  Ann Clin Microbiol Antimicrob. 2017; 16: 57. Published online 2017 Aug 22. doi: 10.1186/s12941-017-0231-z; Malays J Med Sci. 2017 Oct; 24(5): 7–18. Published online 2017 Oct 26. doi: 10.21315/mjms2017.24.5.2

Response: Authors are very grateful to the reviewer’s sharp comment. We determined antimicrobial susceptibility of ampicillin, bacitracin, cefazolin, ciprofloxacin, erythromycin, gentamicin, kanamycin, lincomycin, novobiocin, oxacillin, penicillin, polymyxin B, rifampicin, streptomycin, tetracycline, and vancomycin. Then, in order to see the relationship between phenotype and genotype of antimicrobial resistance, the presence of representative antibiotic resistant gene for each antibiotic was investigated. Accordingly, authors did not perform SCCmec typing.

Line 293-299: It would be nice to perform some conjugation and/or transformation experiments to see if the plasmids could in fact be mobilized. Why wasn’t this done?

Response: Authors are very thankful for the reviewer’s great suggestion and absolutely agree that it would be nice to include conjugation and/or transformation experiments to see if the plasmids could be transferred between CoNS strains. In this study, we investigated the three major aspects including biofilm, antibiotic resistance, and pathogenicity. Since we focused more on biofilm of CoNS strains, the works associated with biofilm were carried out more than the other two aspects, including conjugation and/or transformation experiments.  

Line 218-320: It might be important to deposit the sequences of these genes in databases, since

Response: Authors very much appreciate the reviewer’s great advice. As the reviewer recommended, we will deposit the gene sequences to GenBank. 

Lines 333-340: Is there a relationship between the presence of any antibiotic(s) resistance genes and the formation of biofilms? Why wasn’t this determined?

Response: Authors are very grateful to the reviewer’s keen point. Previously, there were reports to see relationship between the presence of antimicrobial resistance gene and the formation of biofilms in CoNS strains. Dr. Un Jung Lee, statistician and one of the coauthors, analyzed initially the relationship but found it was not statistically significant.

Round 2

Reviewer 2 Report