Antibiotic Resistance and Molecular Characterization of Staphylococcus aureus Strains Colonizing the Nose and Pharynx

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting article which adds to the knowledge on the asymptomatic carriage of Staphylococcus aureus and the antimicrobial resistance profiles of these bacteria. The study has strengths in that it uses a large number of people from a variety of different age groups which is relatively unusual in these studies

Although generally well written, there are some areas which need some clarification, not least that MDR is the resistance to three classes of antimicrobial not just three different antimicrobials. The results also show a lot of repetition, with the same data presented in the text, in a table and then in a graph, when it only needs to be presented once.

There are a few minor comments below.

 

Line 38- are these not usually referred to as classes of antimicrobials?

Please include reagent manufacturers in the methodology

Lines 61-62- not a complete sentence so please reword

Line 65-68- please include concentrations of antimicrobials

Line 72-73- please include the year that the CLSI standards were used from

Line 87-99- is it worth putting this in a table, gene, primers, cycling conditions and amplicon size? May just be clearer and easier to read?

Line 101- how were sequences QC checked?

Line 112-117- the data here doesn’t need to be in a table and the graph

You alternate between pharynx and throat, please be consistent throughout

Line 142-149 -  please check this- MDR is not resistance to 3 or more antimicrobials but to 3 or more antimicrobial classes.

Table 4 maybe better as supplementary as you have the heat map which shows the information? And most of it is in the text

Similar is true for table 5- although having a % in here maybe useful

Line 262-270 is largely just a repeat of the results

Line 352- please can you state some of these prevalences, and how they are similar?

You also tend to include quite a lot of long sentences which would benefit from being split into several separate sentences for ease of reading

There are also lots of repetition of the results in discussion which would benefit from being removed and replaced with how the results from this study links with what is currently known in the literature .

Author Response

This is an interesting article which adds to the knowledge on the asymptomatic carriage of Staphylococcus aureus and the antimicrobial resistance profiles of these bacteria. The study has strengths in that it uses a large number of people from a variety of different age groups which is relatively unusual in these studies

Although generally well written, there are some areas which need some clarification, not least that MDR is the resistance to three classes of antimicrobial not just three different antimicrobials. The results also show a lot of repetition, with the same data presented in the text, in a table and then in a graph, when it only needs to be presented once.

Thank you very much for your comments that allow us to improve the manuscript.

You're right about the term MDR; the text was changed to antibiotic resistance. The discussion also included information about the MDR strains found.

To avoid repetitions, the graphs and tables were reduced, and two tables were placed as complementary tables. 

Line 38- are these not usually referred to as classes of antimicrobials?

The term was changed to classes of antimicrobials

Please include reagent manufacturers in the methodology

Manufacturers were included in the methodology

Lines 61-62- not a complete sentence so please reword

The sentence was rewritten

Line 65-68- please include concentrations of antimicrobials

Concentrations were included

Line 72-73- please include the year that the CLSI standards were used from

The year was added

Line 87-99- is it worth putting this in a table, gene, primers, cycling conditions and amplicon size? May just be clearer and easier to read?

This is included in Supplementary Table S1 with the corresponding reference.

Line 101- how were sequences QC checked?

The electropherogram was reviewed. In addition, if the sequence had errors, the program indicated that there was an error and could not provide the spa-type. When the sequences showed errors in identification, they were re-sequenced to eliminate the errors.

Line 112-117- the data here doesn’t need to be in a table and the graph

Following your recommendation, Figure 1 was removed and only the description in the text and in Table 1 remained.

You alternate between pharynx and throat, please be consistent throughout

Everything was homogenized to pharynx

Line 142-149 -  please check this- MDR is not resistance to 3 or more antimicrobials but to 3 or more antimicrobial classes.

According to your observation, it was corrected only to resistance, and the phrase was changed. MDR strains were analyzed in the discussion.

Table 4 maybe better as supplementary as you have the heat map which shows the information? And most of it is in the text

Following your recommendation, Table 4 was changed to Supplementary Table TS2.

Similar is true for table 5- although having a % in here maybe useful

Table 5 was changed to supplementary table S3 and the percentages of the spa-types were put into the new Figure 7.

Line 262-270 is largely just a repeat of the results

The sentence was rewritten and shortened.

Line 352- please can you state some of these prevalences, and how they are similar?

The information is complete.

You also tend to include quite a lot of long sentences which would benefit from being split into several separate sentences for ease of reading

Sentences were separated for better reading.

There are also lots of repetition of the results in discussion which would benefit from being removed and replaced with how the results from this study links with what is currently known in the literature .

Results data were removed from the discussion and comparisons were made with other research.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review the manuscript "Antibiotic resistance and molecular characterization of Staphylococcus aureus strains colonizing the nose and pharynx" by González-García et al. The study addresses a relevant topic; however, I have several concerns regarding both the methodology and the interpretation of the results.

I am not fully convinced that the tests performed are sufficient to conclusively identify the isolates as Staphylococcus aureus. While the host source supports the likelihood of this identification, it is important to acknowledge that other coagulase-positive and mannitol-fermenting staphylococcal species may present similar phenotypic characteristics. Additional confirmatory tests (e.g., molecular identification or MALDI-TOF) would strengthen the reliability of the results.

Line 73: Was a control strain used during the identification process?

Regarding the CA-MRSA vs. HA-MRSA classification: The classification of strains as community-associated (CA) or hospital-associated (HA) MRSA based solely on SCCmec typing is highly controversial. I do not believe that such a classification is appropriate in this context, especially without epidemiological data to support it. I recommend that the authors refer instead to the general association of SCCmec types with CA- or HA-MRSA in the literature, rather than assigning their isolates to these categories directly.

Almost all figures in this manuscript need substantial revision: Figures 1 and 2: Percentages should be included directly in the pie charts rather than only in the figure legends. Figure 3: I recommend simplifying the figure by presenting only resistance data as bars to reduce visual clutter. Figure 4: This figure is particularly confusing. It would be clearer to group the number of strains with 0, 1, and 2 resistance genes into a single bar chart. Also, the data should be presented as percentages rather than absolute numbers, as the latter alone is not informative. Figure 5: The same suggestion applies—percentages should be provided to make the data more meaningful.

Table 5 would be better presented as a pie chart highlighting the most common types, with the less frequent ones grouped under "Others." The full table could be included as a supplementary file.

The discussion is disorganized and often difficult to follow. It needs careful revision to improve clarity and coherence. For example, in lines 283–290, the sentences are overly long and at times it is unclear whether the authors are referring to their own findings or those from the literature.

Comments on the Quality of English Language

Needs revision, some parts are very confusing.

Author Response

Thank you for the opportunity to review the manuscript "Antibiotic resistance and molecular characterization of Staphylococcus aureus strains colonizing the nose and pharynx" by González-García et al. The study addresses a relevant topic; however, I have several concerns regarding both the methodology and the interpretation of the results.

Thank you very much for your comments that allow us to improve the manuscript.

I am not fully convinced that the tests performed are sufficient to conclusively identify the isolates as Staphylococcus aureus. While the host source supports the likelihood of this identification, it is important to acknowledge that other coagulase-positive and mannitol-fermenting staphylococcal species may present similar phenotypic characteristics. Additional confirmatory tests (e.g., molecular identification or MALDI-TOF) would strengthen the reliability of the results.

You're right, we made a mistake and didn't notice the missing identification via 16S rRNA gene sequencing, which was performed to verify the strain identification. It's been added to the text.

Line 73: Was a control strain used during the identification process?

1. aureus strains ATCC 43300 and ATCC 29213 were used. Added to the text.

Regarding the CA-MRSA vs. HA-MRSA classification: The classification of strains as community-associated (CA) or hospital-associated (HA) MRSA based solely on SCCmec typing is highly controversial. I do not believe that such a classification is appropriate in this context, especially without epidemiological data to support it. I recommend that the authors refer instead to the general association of SCCmec types with CA- or HA-MRSA in the literature, rather than assigning their isolates to these categories directly.

You are right, SCCmec alone cannot be used to determine whether a strain is HA-MRSA or CA-MRSA; other markers are needed. In the case of CA-MRSA strains, in addition to SCCmec IV, IVa, or V, they must also present the Pantone Valentine Leukocidin gene, as indicated in the literature. That is why the manuscript only mentions strains that presented both markers for CA-MRSA strains. The other strains could no longer be classified as HA-MRSA, since more epidemiological information is indeed required. For this reason, the sentence was corrected. In addition, the epidemiological component was added to the discussion.

Almost all figures in this manuscript need substantial revision: Figures 1 and 2: Percentages should be included directly in the pie charts rather than only in the figure legends. Figure 3: I recommend simplifying the figure by presenting only resistance data as bars to reduce visual clutter. Figure 4: This figure is particularly confusing. It would be clearer to group the number of strains with 0, 1, and 2 resistance genes into a single bar chart. Also, the data should be presented as percentages rather than absolute numbers, as the latter alone is not informative. Figure 5: The same suggestion applies—percentages should be provided to make the data more meaningful.

Following your comments on Figure 2, the percentage was included in the graph; Figure 1 was removed at the suggestion of another reviewer.

In Figure 3, only the resistance bars were included.

In Figure 4, the bar for total strains was removed, leaving only the bars for pharyngeal and nasal strains. The bars are also presented as percentages. Only strains resistant to three or more antibiotics are shown. No strains sensitive to all antibiotics were found; all strains were resistant to at least one antibiotic. The percentages of strains resistant to one and two antibiotics were added to the text.

In Figure 5, the percentages were added.

Table 5 would be better presented as a pie chart highlighting the most common types, with the less frequent ones grouped under "Others." The full table could be included as a supplementary file.

Based on your comments, a pie chart was created and Table 5 was added as a supplementary table (TS3).

The discussion is disorganized and often difficult to follow. It needs careful revision to improve clarity and coherence. For example, in lines 283–290, the sentences are overly long and at times it is unclear whether the authors are referring to their own findings or those from the literature.

The discussion was reviewed and changed for greater clarity and coherence.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes the prevalence, antibiotic resistance, and molecular characterization of Staphylococcus aureus strains, including MRSA, colonizing the nose and pharynx in a healthy Mexican population. The sample size (n = 1777) is commendable, and the inclusion of molecular typing (SCCmec, spa-typing) and virulence gene profiling give new scientific infirmation. Overall, the work contributes meaningfully to the surveillance of antibiotic resistance and supports the inclusion of both nasal and pharyngeal sampling in public health protocols.

Please see specific comments:

Abstract:

Lines 12–13: Please specify the population (country, region)

Line 20–21: The phrase “no significant differences” changed to “no statistically differences”

 

Introduction

Lines 43–45: The term "parallel sampling" should be explained, and specific studies cited to support the statement.

 

Materials and Methods

Line 62: In my opinion identification of S. aureus based only on mannitol fermentation and coagulase test is insufficient. You should make molecular confirmation or MALDI-TOF MS if possible.

Lines 103–108: Statistical analysis is only briefly mentioned. The specific tests used for each type of data should be clarified.

 

Results

Figure 3: please explain  the aberrations antibiotics in figure

You check the presence a lot of number of virulence factor genes S. aureus –you need to explain what these genes are responsible for eg. In intrudation or discussion section

 

Lines 208–224: Statistical comparisons between pharyngeal and nasal isolates for gene prevalence should be included, maybe in table 4

 

Discussion:

Lines 226–228: The CA-MRSA classification is based solely on molecular markers. Consider referencing recent epidemiological criteria or studies supporting this approach and add some  information in discussion section

 

Lines 326–336: Discuss the clinical relevance of high lukE-D and tst prevalence. Are these associated with increased transmission or infection risk?

 

Lines 366–371: The implication of spa-type t-189 as the most frequent type should be discussed in terms of geographical distribution, known virulence, or resistance associations.

 

This manuscript presents a valuable epidemiological and molecular dataset regarding S. aureus colonization in a healthy population. The dual-site sampling, large sample size, and molecular analysis strengthen the impact of the study. However, improvements in methodological clarity and discussion of certain findings are needed. In my opinion it should be considered for publication after minor corrections.

Author Response

This manuscript describes the prevalence, antibiotic resistance, and molecular characterization of Staphylococcus aureus strains, including MRSA, colonizing the nose and pharynx in a healthy Mexican population. The sample size (n = 1777) is commendable, and the inclusion of molecular typing (SCCmec, spa-typing) and virulence gene profiling give new scientific infirmation. Overall, the work contributes meaningfully to the surveillance of antibiotic resistance and supports the inclusion of both nasal and pharyngeal sampling in public health protocols.

Thank you very much for your comments that help make the manuscript better.

Please see specific comments:

Abstract:

Lines 12–13: Please specify the population (country, region)

Information added

Line 20–21: The phrase “no significant differences” changed to “no statistically differences”

The sentence was changed

Introduction

Lines 43–45: The term "parallel sampling" should be explained, and specific studies cited to support the statement.

 The sentence was corrected

Materials and Methods

Line 62: In my opinion identification of S. aureus based only on mannitol fermentation and coagulase test is insufficient. You should make molecular confirmation or MALDI-TOF MS if possible.

You're right, we made a mistake and didn't notice the missing identification via 16S rRNA gene sequencing, which was performed to verify the strain identification. It's been added to the text.

Lines 103–108: Statistical analysis is only briefly mentioned. The specific tests used for each type of data should be clarified.

 The text has been completed and updated.

Results

Figure 3: please explain  the aberrations antibiotics in figure

Abbreviations were added to the text

You check the presence a lot of number of virulence factor genes S. aureus –you need to explain what these genes are responsible for eg. In intrudation or discussion section

The function of the genes studied was added in both Materials and Methods and Discussion.

Lines 208–224: Statistical comparisons between pharyngeal and nasal isolates for gene prevalence should be included, maybe in table 4

The statistical comparison was performed, but a statistically significant difference was only found for the tst gene, and the phrase was added to the results.

Discussion:

Lines 226–228: The CA-MRSA classification is based solely on molecular markers. Consider referencing recent epidemiological criteria or studies supporting this approach and add some  information in discussion section

Epidemiological criteria were added to the Discussion.

Lines 326–336: Discuss the clinical relevance of high lukE-D and tst prevalence. Are these associated with increased transmission or infection risk?

The clinical importance of the lukE-D and tst genes was added to the discussion. 

Lines 366–371: The implication of spa-type t-189 as the most frequent type should be discussed in terms of geographical distribution, known virulence, or resistance associations.

The importance of the spa-type t-189 was added to the discussion.

This manuscript presents a valuable epidemiological and molecular dataset regarding S. aureus colonization in a healthy population. The dual-site sampling, large sample size, and molecular analysis strengthen the impact of the study. However, improvements in methodological clarity and discussion of certain findings are needed. In my opinion it should be considered for publication after minor corrections.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I think the paper has improved, but I am still concerned about its quality. So it is up to the editors to decide.

Author Response

I think the paper has improved, but I am still concerned about its quality. So it is up to the editors to decide.

Thank you very much for your comments. I would also like to point out that this work was carried out over several years and was always conducted with the highest quality standards, both methodologically and academically. We follow the same quality principles as all the manuscripts published by our laboratory over the years.

Please be assured that we always ensure the quality of our research projects.