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Additional Editor Comments:

Dear author

Revise the whole manuscript in light of all reviewer's comments and submit the revised version.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Title:

ln 1-3; is too long and academically clichéd. The study is limited to a single tertiary care center in KSA; therefore, extending the title to cover all of Saudi Arabia is incorrect. Rephrase the title to be more scientifically precise and to indicate the specific regions of KSA it surveils.

Abstract

ln 45-68, the abstract has several typographical errors and methodological issues, especially the inappropriate application of statistical methods to a large longitudinal dataset. A thorough revision is therefore required. The "Results" section should not be vague; instead, it should provide actual percentages, proportions, or confidence intervals for the MRSA and MSSA groups. How many of the total isolates in the samples were MRSA, and how many MSSA? Their distribution among inpatients and outpatients is also not given. The abstract should provide p-values, odds ratios or chi-square metrics for the comparisons in which the authors claim to find "significant differences" in antibiotic profiles. The authors also describe their work as a "retrospective cross-sectional study," but a study that spans more than a decade of tracking shifts in microbial AMR profiles is a longitudinal surveillance or a retrospective cohort study. I think the author's style of concluding by stating that "Continuous surveillance and tailored antimicrobial stewardship are imperative" is too generic and banal. It can be made meaningful if it discusses specific findings from the Aseer region data, local resistance trends and their impact on clinical treatments.

Keywords

ln 69; The current list is generic, which does not add value to the search engine optimization (SEO) of the paper. Terms in the title and abstract should be avoided, as they reduce the manuscript's discoverability.

Introduction

ln 71-73; This statement needs to be rewritten for clarity and scientific accuracy. Too many concepts—including anatomy, statistics, and pathogenic potential have been lumped together into a single awkwardly made sentence. I don't think the authors are correct in stating that the bacteria colonize 25–30% of the population at any given time, since their prevalence in the nasal area fluctuates with distinct patterns of permanent, transient and non-carriers. Rephrase.

ln 78-80; the authors' tendency to use the terms 'pathogenicity' and 'virulence' interchangeably is also incorrect, since they describe distinct aspects of disease. The toxins and surface proteins the authors discuss relate to the bacterium's virulence, not its pathogenicity. The authors should also avoid terms with anthropomorphic or militaristic connotations, such as 'arsenal' for describing bacterial aspects, and replace them with objective, academic terms.

ln 83-85; I think the narrative here is grossly reductive, reminiscent of the 'one gene, one phenotype' paradigm in the context of the clinical challenge of MRSA. A biochemical explanation of resistance does not account for the pathogen's epidemiological success or clinical virulence. It would be incorrect to state that the threat of MRSA is exclusively due to the presence of PBP2a; it is the manifestation of a complex interplay of several virulence factors. The authors ought to separate how bacteria gain resistance from how this is a clinical concern, against a backdrop of the genomics of MRSA pathogenesis.

ln 87-91; this section should be completely revamped to make the clinical and geographic relevance clearer, as well as to correct referencing errors. I don't think the authors should rely on generalities like the global WHO initiatives and ignore the national and regional contexts of KSA. There are several recent studies that the authors should cite to justify the scope of their study. Several cited references are academically below par for instance, reference # 7) Meet WHO's dirty dozen: The 12 bacteria for which new drugs are most urgently needed. AAAS Articles DO Group. 2021. doi:10.1126/science.aal0829

This is an obsolete 2017 science journalism news article and not a peer-reviewed scientific report! Similarly, reference #8) Guidelines on data disaggregation for SDG Indicators using survey data. FAO, 2021. 285 doi:10.4060/cb3253en is a manual published by FAO that has nothing to do with clinical microbiology! Can the authors justify including them here?

ln 92-94; "The authors have been abrupt in their use of terminology and regional context. A comparison between MRSA and MSSA requires a sound clinical context. The authors should clarify what MSSA represents and its clinical significance for KSA and the wider MENA region. The term 'resistance rates' is epidemiologically inaccurate, since it requires a time denominator, which is not the case here. Replace with a statistically accurate term.

ln 98-100; I think the authors have cited questionable studies to support their argument. The reference (Alghoribi et al., 2020) discusses OXA-48 carbapenemase-producing Salmonella enterica and has nothing to do with S. aureus or MRSA. The other reference (Alhazmi et al., 2025) has been grossly misinterpreted, as it does not report a 30–50% prevalence of S. aureus isolates anywhere. Either reanalyze or remove these citations.

ln 101-106, I think the authors' claim is factually incorrect, as it ignores several recent large-scale retrospective cohort studies conducted in KSA. Similarly, the distinction between HA-MRSA and CA-MRSA has been studied in this region. The authors must provide an accurate justification for the novelty of their work in light of the high-quality surveillance data publicly available in KSA.

ln 107-110; in light of my above comment, the authors' stated objectives are also redundant. Local treatment guidelines, antibiotic protocols for MRSA and MSSA are well established in Saudi healthcare facilities. One cannot claim that its objective is to 'recognize appropriate empiric choices' when the country has functional stewardship programs. The authors should refrain from framing it as a pioneering study for making empirical treatment strategies from scratch. The following are examples of recent works on antibiotic stewardship in KSA, which can help the authors frame their objectives.

1. Al-Oman A, et al. Antimicrobial Stewardship Programs in Saudi Hospitals. Antibiotics (Basel). 2021;10(2):193. doi:10.3390/antibiotics10020193.

2. Al-Garni MA, et al. Antimicrobial utilization among hospitalized patients according to WHO AWaRe Classification: results from a multicentre point prevalence survey in Saudi Arabia. Journal of Global Antimicrobial Resistance. 2025/2026. doi:10.1016/j.jgar.2025.xx.xxx.

Methods

ln 113-121; The authors dropped 932 pediatric and neonatal isolates (nearly 20% of the cohort) without providing any scientific justification. This exclusion introduces severe selection bias and degrades the value of this data for developing evidence-based clinical options. I find the authors' claim implausible that they were able to access a decade's worth of lab data in a single day, which brings to light issues like data integrity and quality control. I also find the author's claim tenuous that 'patient details were not revealed to the research team while in the same breath also claiming to have identified and excluded 932 pediatric and neonatal samples! Surely the authors cannot be ignorant of the particulars of these patients since they used age-based demographic criteria for screening. The detached reference to 'the research team' is also a concern, as it raises issues of authorship and data ownership. The exact roles of the researchers in the data extraction, cleaning, and analysis processes should be clearly defined in line with ICMJE authorship requirements.

ln 123-133; I think the use of two completely different automated platforms—BD Phoenix 100 and VITEK 2—interchangeably across an 11-year study period is a serious concern. These platforms use different detection principles and proprietary algorithms to calculate MICs. Additionally, the breakpoints specified by CLSI have undergone multiple revisions during the timeframe of 2013 to 2024, so how this 'methodological consistency and comparability were ensured' should be made clear! The authors' claim of using the Xpert® MRSA NxG kit on the GeneXpert® system to confirm the SCCmec gene is also technically flawed, as this system cannot perform SCCmec chromosomal typing. It is a basic diagnostic PCR kit and not a molecular typing tool. It is unclear to me when the authors state they 'randomly selected' 100 MRSA isolates for molecular testing. How did they evaluate the scientific validity of this sample without knowing the total number of MRSA isolates it was drawn from? The distribution of these 100 isolates across the 12-year study period and across inpatient/outpatient categories should also be made clear.

ln 134-140; I find the 'Statistical Analysis' subsection grossly unsuitable for a long-term, large-scale retrospective epidemiological study. The authors state they used Fisher's exact test due to 'the presence of small, expected cell counts,' which is mathematically infeasible given the large sample size and the fact that Fisher's exact test is meant for small-sample contingency tables, not for thousands of points across demographic data. Also, reducing a decade of dynamic epidemiological data into cumulative frequencies masks tracking of resistance trends over time, which was the goal of this study. The dataset contains multiple clinical and demographic variables, and univariate cell comparisons do not account for confounding, a glaring deficiency.

Reviewer #2: My analysis of this research study: This is a cross-sectional study aimed to evaluate and compare the antimicrobial resistance pattern for MRSA and MSSA, in outpatient and inpatient settings. Results showed increased resistance to antimicrobials including macrolides, flouroquinolones, aminogylcosides among MRSA as compared to MSSA. No resistance to vancomycin or daptomycin found.

COMMENTS:

1.Abstract page 3 and 4: Briefly add existing literature on your topic.

2. Background page 5: Briefly mention the cause of MRSA's resistance to non lactam antibiotics.

3.Microbiological methods page 7: Please provide the reference to exact CLSI guidelines used to interpret susceptibility.

4. Results page 12: If antimicrobial resistance pattern was analyzed separately by specimen type, mention here.

5.Discussion page 13: Compare your results with existing literature regarding antimicrobials' resistance in terms of percentages if available.

Discuss the efficacy of susceptible antimicrobials with respect to site of infection.

6.Limitations page 14: Mention the other limitations like linezolid response was not evaluated.

Reviewer #3: This manuscript addresses an important public health issue and presents a substantial dataset comprising 4,194 non-duplicate Staphylococcus aureus isolates collected over more than a decade. The study provides valuable local antimicrobial resistance surveillance data and may contribute to antimicrobial stewardship efforts in the region. However, several methodological and reporting issues require clarification before the conclusions can be fully supported.

1. Major Concerns

Study duration inconsistency

The manuscript refers to an 11-year study period in the title and abstract, whereas the study period extends from January 2013 to June 2024. In addition, the Microbiological Methods section refers to a 12-year study period. The study duration should be described consistently throughout the manuscript.

Title emphasizes "trends" but trend analyses are not presented

The title highlights antimicrobial resistance trends from 2013–2024; however, the results present pooled resistance data across the entire study period. No year-wise resistance trends, temporal analyses, or trend statistics are provided. The authors should either include appropriate temporal trend analyses or revise the title to better reflect the study design and findings.

Potential inconsistency in Table 3

Table 3 reports a total of 2,231 MRSA isolates, stratified into inpatient and outpatient groups. The authors should carefully verify that the sample sizes, percentages, and corresponding narrative descriptions are correctly labeled and internally consistent. Any discrepancy in the assignment of inpatient versus outpatient isolates could influence interpretation of the observed erythromycin resistance patterns and the subsequent discussion regarding community-associated resistance dynamics.

Limited molecular characterization

Only 100 MRSA isolates underwent molecular confirmation using GeneXpert. The rationale for selecting these isolates, the sampling strategy, and their representativeness of the overall MRSA population should be clarified. This limitation should also be discussed in greater detail.

2. The statistical approach is generally acceptable for categorical comparisons; however, several points require clarification.

Comments

Table 1 statistical testing

Table 1 contains footnotes referring to both Chi-square and Fisher’s exact tests. The manuscript should clearly indicate which statistical test was applied to each comparison.

Multiple comparisons

Numerous antibiotic-specific comparisons were performed across Tables 2 and 3. The authors should indicate whether adjustment for multiple testing was considered or provide justification for not performing such adjustment.

Confidence intervals

Reporting 95% confidence intervals alongside resistance estimates would improve interpretability and allow readers to assess the precision of estimates.

3. The authors indicate that all relevant data are contained within the manuscript. However, provision of supplementary datasets or summary tables would further support transparency and reproducibility in accordance with PLOS ONE data-sharing principles.

4. The manuscript is generally understandable and logically organized. However, several grammatical and formatting issues should be corrected.

Examples include:

• Line 90: “Recent statistic” should be revised to “Recent statistics.”

• Lines 119–121 require grammatical revision.

• Line 133: “all hospital sitting” should be revised to “all hospital settings.”

• Ensure consistent formatting of p-values throughout the manuscript.

5. Detailed Review Comments and Feedback

Major Revisions

1. Clarify study duration

The manuscript alternately describes the study as 11 years, 11.5 years, and 12 years. A single consistent description should be used throughout the manuscript.

2. Reconcile Table 3 and related interpretation

Please carefully audit Table 3 and the accompanying text to ensure that inpatient and outpatient sample sizes, percentages, resistance rates, and narrative descriptions are correctly reported and interpreted.

3. Address the “trends” claim

The title suggests temporal trend analysis, yet no annual trend data are presented. Either include trend analyses or revise the title accordingly.

4. Clarify molecular confirmation strategy

Please explain:

• Why only 100 isolates underwent molecular confirmation.

• How these isolates were selected.

• Whether they are representative of the broader MRSA collection.

5. Microbiological methodology

The manuscript reports use of both BD Phoenix and VITEK 2 systems during the study period. Please specify:

• When the transition occurred.

• Whether CLSI breakpoints changed during the study period.

• How methodological consistency was maintained across the surveillance period.

Minor Revisions

1. Abstract

The Methods section begins with the fragment:

“A retrospective cross-sectional study to investigate and compare the antimicrobial resistance patterns…”

This should be revised to a complete sentence.

2. Reference verification

Reference 11 appears to concern Salmonella enterica serovar Kentucky and does not appear to support the statement regarding MRSA prevalence in Saudi Arabia. Please verify and replace this citation if necessary.

3. Vancomycin and daptomycin susceptibility

No resistance was detected to vancomycin or daptomycin, which is reassuring. If available, inclusion of MIC distributions or MIC trend data would strengthen the surveillance value of the study.

4. Discussion

Some interpretations regarding community prescribing practices and demographic explanations are speculative and should be presented more cautiously unless supported by additional data.

Overall Assessment

This study addresses a clinically important topic and is strengthened by its large sample size and extended surveillance period. However, clarification of methodological details, verification of data presentation, resolution of title-versus-analysis inconsistencies, and correction of citation issues are necessary before the manuscript can be considered for publication.

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Reviewer #1: No

Reviewer #2: Yes:  Fizza Khalid

Reviewer #3: Yes:  Dr. Godfred Antony Menezes

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Methicillin resistance and antimicrobial non-susceptibility trends among Staphylococcus aureus clinical isolates in Aseer, Saudi Arabia (2013–2024): a retrospective laboratory-based surveillance study

PONE-D-25-66618R1

Dear Dr. ,Abdulah J. Alqahtani, MD

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

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Additional Editor Comments (optional):

Dear author

The reviewer is satisfied with all the changes made by author in revised manuscript, so I recommend that paper is now acceptable to be published in PLoS ONE.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #4: Yes

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Reviewer #4: The revised manuscript is substantially improved and addresses many of the concerns raised in the previous round. The title, abstract, methods, statistical analysis, and discussion are much clearer and more scientifically appropriate, and the longitudinal trend analysis adds genuine value. Please make a final editorial pass to correct minor formatting and terminology issues, including italicizing gene names (e.g. mecA and mecC) and S. aureus throughout.

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what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #4: No

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