-->PONE-D-25-60195-->-->Fresh fruit surface: a potential source for multidrug-resistant biofilm-forming Enterococcus faecalis having potential public health significance-->-->PLOS One

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6. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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1. A reviewer has proposed additional articles for possible inclusion in the manuscript. It is essential to carefully review each recommendation and determine whether it aligns with the scope of the section where these references are to be added. If any suggested articles are found to be outside the defined scope, please provide a clear explanation and justification for their exclusion. Articles that do not fit within the intended context should not be incorporated into the manuscript.

2. The current presentation of the information on lines 231 to 239 lacks a supporting table. Please organise this content into a table to enhance clarity and ease of interpretation. Additionally, ensure that the statistical methods section explicitly describes the process used to calculate and interpret the correlations, thus promoting reproducibility and transparency in the analysis.

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Reviewer #1: The manuscript described molecular detection and characterization of MDR Enterococcus faecalis associated with fresh fruit surface sold in Mymensingh area. In addition to AMR, biofilm formation was also observed in this study. Considering it as the first such study in Bangladesh and the public health importance of biofilm forming MDR E. faecalis the study carries significant and crucial message for the food sellers/vendors, consumers and policy makers.

Specific Comments.

1. How much DNA was used in each PCR as template?

2. Congo Red assay was used for biofilm detection. Describe the principle/mechanism of Cogo Red test.

3. Several virulence genes were used, please mention the functions of these genes, many are not mentioned in the manuscript.

4. What was the rationale of selecting those 9 antibiotics in the AST?

5. Please remove the CI from the values e.g, in line 180 instead of 47 (61.84%, CI95: 50.60–71.94), simply write 47 (61.84%)

6. There are different types of fruits available in the market. Why were these particular fruits were selected?

7. Line 2412, Moreover, Strong biofilm-forming E. faecalis isolates exhibited higher resistance to certain antibiotics compared to intermediate and non-biofilm formers, in discussion explain this observed phenomenon.

8. Line 252, Furthermore, all the patterns showed resistance to a similar number of antibiotics and classes. any explanation why similar patterns were observed??

Reviewer #2: The manuscript entitlted ‘’ Fresh fruit surface: a potential source for multidrug-resistant biofilm-forming Enterococcus faecalis having potential public health significance’’ presented a well-designed and timely investigation into the occurrence, AMR, biofilm formation and virulence gene profiles of E. faecalis from fresh fruit surfaces in Bangladesh. The study addressed an important gap in food safety and One Health research, particularly in low- and middle income country where surveillance data on antimicrobial-resistant bacteria in fresh produce remain scarce.

The methodology is clearly described, results are logically structured, and discussion is well contextualized within the existing literature. Overall, the manuscript makes a valuable contribution to understanding the public health implication of antimicrobial-resistant enterococci in the food chain.

Besides its novelty and strength, several limitation should be acknowledged more explicitly to make it scientifically sound.

Limitations and Suggesstions

1. This study analyzed 76 fruit samples of different types exclusively from Mymensingh city, which is not adequate for an exploratory investigation, and restricts generalizability. Moreover, seasonal variation was not explicitly assessed. Thus, results should be interpred as region-specific baseline data rather than national prevalence estimates

2. Only 18 isolates were tested for biofilm formation and 12 isolates for virulence gene profiling, despite 47 PCR-confirmed isolates. The subsampling approach is acceptable for preliminary characterization but limits inferential depth. The authors are suggested to examine all the 47 isolates to make the result scientifically robust.

3. Biofilm formation was assessed using only Congo Red Agar (CRA) method, which is qualitative and phenotype-dependent. CRA provides rapid screening but may underestimate or overestimate true biofilm biomass.

4. Among resistance genes only blaTEM was screened. Key enterococcal resistance genes (e.g. vanA, vanB, ermB tetM, optrA, postA) were not investigated, despite high phenotypic resistance to vancomycin and linezolid. Phenotype-genotype discordance cannot be fully resolved without broader resistance gene profiling.

5. The study relies on PCR-based detection rather than whole-genome sequencing. Consequently, clonal relationship, plasmid content, mobile genetic elements, and resistome structure remain unexplored. WGS in recommended for future studies to confirm transmission dynamics and One Health linkages.

Reviewer #3: Dear Editor,

This manuscript introduces a unique concept in an area that is both intriguing and important for medical research. The topic is timely and is likely to engage the journal's readers. Additionally, the manuscript is well-written, presenting ideas clearly and logically.

Dear Authors,

You are doing excellent work, and this manuscript reflects a great deal of effort and thoughtful research. The study is promising and contributes meaningfully to the field. However, there are a few important points that require further clarification and revision to enhance the overall quality and impact of the manuscript.

Line 44: The Introduction covers several important themes (enterococci biology, public health relevance, fruit contamination, and antibiotic resistance). However, the narrative would benefit from improved coherence and a clearer logical flow. The authors are encouraged to better integrate these topics, explicitly linking Enterococcus faecalis ecology with fruit-associated transmission and public health implications.

Line 46: It is preferred to use the following reference to support your updated citations: Alduhaidhawi AHM, AlHuchaimi SN, Al-Mayah TA, Al-Ouqaili MTS, Alkafaas SS, Muthupandian S, Saki M. Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater. Infect Drug Resist. 2022 Mar 19;15:1143-1154. Doi: 10.2147/IDR.S358248. PMID: 35340673; PMCID: PMC8942119.

Statements suggesting that consumption of fresh fruits may increase the “baseline level of antibiotic resistance” in humans (lines 69–71) are potentially overstated. These claims should be more carefully qualified and supported with stronger evidence or rephrased to avoid causal implications not fully substantiated by current data.

Lines 76–79 repeat nearly identical statements regarding the intrinsic resistance mechanisms of E. faecalis. This redundancy should be eliminated or consolidated into a single, concise description to improve readability and conciseness.

Line 76: I recommend that the authors add the following sentences in their introduction using the mentioned studies:

"The increase in multidrug-resistant (MDR) microorganisms triggering infections is growing worldwide and becoming more serious in developing countries."

In line 76: It is important to use the update and so related reference according to the above comment:

Owaid HA, Al-Ouqaili MTS. Molecular and bacteriological investigations for the co-existence of CRISPR/Cas system and β-lactamases of types extended-spectrum and carbapenemases in Multidrug, extensive drug and Pandrug-Resistant Klebsiella pneumoniae. Saudi J Biol Sci. 2024 Jul;31(7):104022. doi: 10.1016/j.sjbs.2024.104022. Epub 2024 May 19. PMID: 38817398; PMCID: PMC11137337.

Rawaa A. Hussein, Shaymaa H. AL-Kubaisy, Mushtak T.S. Al-Ouqaili. The influence of efflux pump, outer membrane permeability and β-lactamase production on the resistance profile of multi, extensively and pandrug-resistant Klebsiella pneumoniae. Journal of Infection and Public Health, Volume 17, Issue 11, 2024. https://doi.org/10.1016/j.jiph.2024.102544.

Lines 94–101: The manuscript provides detailed geographic coordinates and fruit types; however, the sampling design is not clearly defined. It remains unclear whether samples were collected using random, convenience, or systematic sampling. The authors should clarify the sampling strategy, inclusion and exclusion criteria, and whether the total sample size (n = 76) was statistically justified.

Lines 98–101:The number of samples per fruit type is highly uneven (e.g., apples n = 18 vs. dates n = 4). The authors should justify this disparity or discuss its potential impact on prevalence estimates, comparative analyses, and statistical power.

Lines 105–108, 111: The enrichment step involves transferring 100 µL of PBS wash into 1 mL of nutrient broth; however, the rationale for this volume selection is not provided, nor is it clear whether selective enrichment media were considered. Additionally, the term “enriched culture” (line 111) appears to be a typographical error and should be corrected.

Lines 154–160: The manuscript's claim that nine antibiotics correspond to eight groups requires clarification. Additionally, since rifampin (an ansamycin) is usually used as an adjuvant against Enterococcus spp., a justification for its inclusion in susceptibility testing should be provided.

Lines 189–190, 197–198, 248: For prevalence across fruit types and biofilm formation categories, the authors report non-significant differences (p > 0.05) but do not provide test statistics or clarify which tests were used. Including the test type and exact p-values would enhance transparency.

Similarly, differences in antibiotic resistance among biofilm categories are described qualitatively, but statistical comparisons are lacking (Lines 242–248).

Lines 192–196, 205–208, 220–225: Biofilm formation was tested in only 18 out of 47 PCR-positive isolates (Lines 192–196), virulence genes in 12 isolates (Lines 205–208), and antibiotic resistance in 40 isolates (Lines 220–225). The rationale for selecting these subsets is not provided. The authors should justify the sample sizes for these analyses and discuss potential limitations regarding representativeness and statistical power.

Lines 212–214, 231–239: Spearman and Pearson correlations between virulence genes and between antibiotics are reported, but the biological interpretation is not clearly explained. The authors should specify the rationale for these correlations and discuss potential confounding factors.

Some correlations are repeated or redundant (Lines 235–237: Linezolid and Vancomycin are reported twice). The manuscript should remove duplicates to avoid confusion.

Lines 250–253: The MDR prevalence and MAR index are reported, but methods for calculating MAR for each isolate are not linked to the results clearly. The authors should provide a table listing MAR values per isolate and clarify how multidrug resistance was defined (≥3 classes, consistent with Methods, Lines 160–162).

Lines 261–269: The first paragraph repeats information about E. faecalis as a commensal organism and nosocomial pathogen, which was already described in the Introduction. Consider condensing these statements to focus on the relevance of your current study.

Lines 270–288: While prevalence data are restated (Lines 271–273), the discussion relies heavily on speculation (e.g., poor hygiene, cross-contamination, dust) without citing study-specific observations. The authors should explicitly link environmental or vendor practices observed during sampling to the high prevalence findings.

Line 293: It is preferred to use the following reference to support your updated citations: Al-Ouqaili, M.T.S., Al-Kubaisy, S.H.M., Al-Ani, N.F.I. Biofilm antimicrobial susceptibility pattern for selected antimicrobial agents against planktonic and sessile cells of clinical isolates of staphylococci using MICs, BICs and MBECs. Asian Journal of Pharmaceutics, volume 12, Issue 4, October-December 2018, Pages S1375-S1383.

Line 358: The Conclusion should be objective, with further perspective, or should add at least a few sentences about future study/future perspective of it

Reviewer #4: Global comment

This manuscript investigates the presence of E. faecalis from fruit surfaces and characterizes the antibiotic resistance profile of these bacteria, detect its virulence genes and biofilm-forming ability, using selective media for bacterial identification and PCR assay. Phenotypic profile was obtained through the antibiotic susceptibility testing, and the genotypic profile was performed by PCR. Notably, the study highlights the frequency of multidrug-resistant (MDR) E. faecalis in fruits, a finding of global relevance due to the growing concern surrounding antimicrobial resistance.

The topic is timely and relevant, and the manuscript is scientifically supported. However, the language throughout the manuscript requires corrections for grammar, clarity, and style to ensure precision and readability. Moreover, some areas, require clarification and expansion to improve scientific rigor and reproducibility. Below are detailed comments and suggestions:

Comments

Introduction

Line 49. The sentence was rewritten as follows: “Approximately 80 to 90 percent of human enterococcal infections are caused by E. faecalis, while most of the remaining cases are attributable to E. faecium.”

Line 73. Rephrase. Multidrug resistance (MDR) typically refers to bacteria resistant to three or more distinct antibiotic classes.

Line 76. Please check the entire manuscript. The names of genera, species, and genes should be written in italics.

Material and Methods

Line 105. Please clarify how the fruits were processed. Were they washed before analysis, or were they immersed directly in PBS inside sterile bags?

Line 110. From bacterial growth in nutrient broth, please specify the volume or concentration used to inoculate the Enterococcosel agar base.

Line 156-160. The terms “access”, “watch”, and “reserve” are unclear in this context and make the procedure confusing. Please revise this section for clarity.

Line 165. This information would be more appropriate in the section “Detection of Antibiotic Resistance and Virulence Genes”.

Results

Line 180. Please rephrase. The entire manuscript requires revision for clarity and accuracy.

Line 186. It is incorrect to state that the PCR is positive. Instead, the isolates obtained from different fruits should be described as positive or negative for E. faecalis.

Line 193. Replace the term “samples” with “isolates”.

Figure 1, The text in the figure is not legible and should be improved.

Line 200-202, the sentence should be moved to a table.

Line 205. Please explain why only 12 E. faecalis isolates were selected for further analysis.

Figure 2. This figure does not present new data. Please add the total number of E. faecalis isolates. In addition, include information about the source of the isolates in Figures 2 and 3, and describe these data clearly in the Results section.

Throughout the text, the names of antibiotics should not begin with a capital letter.

Discussion

Line 261-269 This section repeats information already presented in the Introduction and should be revised.

The Discussion should not be divided into subsections.

Line 281. According to a study, the prevalence 'of E. faecalis?’’

Reviewer #5: Thank you for an interesting and potentially important manuscript reporting the detection of multidrug-resistant, biofilm-forming Enterococcus faecalis on fresh fruit surfaces, a first report for Bangladesh. The study has merit, but the authors should address several important methodological and interpretive issues before the manuscript can be finalized. My specific comments and suggestions are as follows-

1. The manuscript uses different numbers of samples for different fruit types, but does not explain why sample sizes vary. This raises concerns about sampling bias and how representative the results are across fruit types. Add a clear explanation of the sampling design and justify the unequal sample sizes. If the unequal sizes were unavoidable (availability, seasonality, logistics), state this explicitly and discuss the limitation. If possible, include a short statistical justification.

2. The manuscript lists antibiotic susceptibility results but does not explain the rationale for selecting the particular antibiotic discs used. Provide a clear justification for each antibiotic tested: link each to its clinical relevance in human medicine and/or veterinary use in Bangladesh, and to commonly used classes for enterococci.

3. The discussion should be revised to interpret resistance findings in the local epidemiological and antimicrobial-use (AMU) context in Bangladesh. Summarize which antibiotics are commonly prescribed/used in human clinical practice in Bangladesh for infections where enterococci are relevant (e.g., urinary tract infections, bloodstream infections) and which antibiotics are used in livestock/aquaculture/horticulture in the country.

Reviewer #6: The manuscript addresses an important and underexplored public health issue and is well aligned with the One Health framework. It effectively integrates microbiological, molecular, and epidemiological approaches and provides novel regional data relevant to food safety and antimicrobial resistance (AMR) surveillance. Overall, the study has scientific merit and is potentially suitable for publication.

However, several revisions are required to strengthen methodological transparency, statistical rigor, and language quality. The key issues and recommended revisions are outlined below.

Major Comments

1. Sampling Design and Representativeness

The overall sample size (n = 76) is relatively small and unevenly distributed across fruit types. In addition, the sampling strategy appears to be convenience-based and limited to a single city.

Recommendations:

• Clearly acknowledge this as a limitation of the study.

• Avoid generalizing the findings to all fruit types or to the entire country of Bangladesh.

• Where appropriate, reframe conclusions to reflect the localized nature of the data.

2. Subsampling for Biofilm Formation and Virulence Gene Analysis

Only 18 isolates were examined for biofilm-forming ability and 12 isolates for virulence gene detection, without sufficient justification for this subsampling approach.

Recommendations:

• Provide a clear rationale for the random selection of isolates (e.g., logistical or resource constraints).

• Explicitly state that the reported prevalence of biofilm formation and virulence genes may be underestimated.

• If feasible, expand the number of isolates tested or provide a statistical justification for the chosen subsample size.

3. Interpretation of Antibiotic Resistance Findings

The reported high resistance rates to vancomycin and linezolid are concerning and atypical for food-derived Enterococcus isolates.

Recommendations:

• Provide a more detailed discussion of potential methodological limitations, including the use of disc diffusion for glycopeptides and oxazolidinones.

• Discuss possible environmental, anthropogenic, or ecological drivers that could explain these findings.

• Cite relevant CLSI cautions and limitations regarding susceptibility testing for these antibiotic classes.

4. Statistical Analysis

Some of the reported correlations (e.g., Linezolid–Vancomycin, ρ = 1.000) suggest potential artefacts related to small sample sizes. In addition, no correction for multiple testing is mentioned.

Recommendations:

• Acknowledge the exploratory nature of the correlation analyses.

• Include a statement addressing the increased risk of type I error due to multiple comparisons.

• Interpret correlation results with appropriate caution.

5. Language and Editorial Quality

The manuscript contains repetitive statements, overly long sentences, minor grammatical errors, and occasional inconsistencies in terminology (e.g., use of “weak” versus “intermediate” biofilm categories).

Recommendation:

• Professional English language editing is strongly recommended prior to resubmission to improve clarity, consistency, and readability.

Section-by-Section Comments

Abstract

The abstract is well structured and data-rich. However, it would benefit from:

• Reducing excessive numerical detail.

• More clearly highlighting the key findings and their policy or public health relevance.

Introduction

The introduction is scientifically sound and well referenced. Improvements are recommended to:

• Reduce redundancy, particularly regarding antimicrobial resistance mechanisms.

• Improve conciseness and narrative flow.

Materials and Methods

This section is generally robust. The ethics statement marked as “N/A” is acceptable; however:

• The authors should explicitly clarify that the study did not involve human or animal subjects.

• Additional justification should be provided for partial molecular testing of isolates.

Results

The results are clearly presented; however:

• Tables require harmonization to ensure consistency in numbering and formatting.

• Figure captions should be expanded to be fully self-explanatory.

Discussion

The discussion is well contextualized within the existing literature. To further strengthen this section, the authors should:

• Avoid speculation beyond what is directly supported by the data.

• Strengthen comparisons with studies from South Asia and similar settings.

• Include a clearly defined Limitations subsection.

Conclusions

The conclusions are appropriate and supported by the data. The section could be further strengthened by:

• Adding a brief statement on policy or regulatory implications, such as market hygiene practices, food safety monitoring, or routine AMR surveillance.

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: Yes:  Sukumar Saha

Reviewer #6: Yes:  Lawan Adamu

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-->PONE-D-25-60195R1-->-->Fresh fruit surface: a potential source for multidrug-resistant biofilm-forming Enterococcus faecalis having potential public health significance-->-->PLOS One

Dear Dr. Rahman,-->-->

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.-->-->

Please submit your revised manuscript by Jul 12 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Leonard Ighodalo Uzairue, PhD

Academic Editor

PLOS One

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Reviewer #6: (No Response)

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Reviewer #6: Yes

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Reviewer #6: Yes

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Reviewer #6: Yes

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-->6. Review Comments to the Author

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Reviewer #6: Thank you for your careful revision. The manuscript has improved substantially in clarity, organization, and transparency. The study is relevant and contributes useful baseline data on antimicrobial-resistant Enterococcus faecalis in fresh fruits.

However, the following issues remain that need to be addressed

1. The study confirms 47 isolates of E. faecalis, yet:

• Biofilm analysis: 18 isolates

• Virulence genes: 12 isolates

This represents a significant reduction in analytical coverage.

While you have acknowledged this limitation, the manuscript still states that virulence genes and biofilm traits are “widely distributed”, which is not fully supported by the subsampled data.

To address this the author should:

• Rephrase conclusions to indicate findings are preliminary or indicative

• Avoid generalizing to all isolates

• Explicitly state that prevalence may be over- or under-estimated

2. The reported resistance levels: Vancomycin: 47.5% and Linezolid: 47.5% are unexpectedly high for food-derived isolates and require stronger caution. Although you mention CLSI limitations, this is not sufficiently emphasized.

The Critical issue here is that: Disk diffusion is not the preferred method for these antibiotics in enterococci, and may lead to inaccurate classification.

To address this the author is Required to:

• Add a strong statement that:

o These results may overestimate resistance

o MIC-based confirmation is required

• Avoid implying confirmed presence of:

o Vancomycin-resistant enterococci (VRE)

o Linezolid-resistant strains

• Tone down conclusions accordingly, especially in the Conclusion section

3. The manuscript reports correlation values (e.g., ρ = 1.000) that are statistically questionable, especially given small sample sizes. The concerns here are:

• Likely artefacts of small sample size

• No correction for multiple comparisons

• Limited biological interpretability

The required revisions in this circumstances is for the author to:

• Clearly state that:

o Correlation analysis is exploratory

o Results are hypothesis-generating only

• Add a statement on risk of Type I error

• Interpret correlations cautiously or move to supplementary material

4. The study includes: 76 fruit samples from a single city (Mymensingh), Uneven distribution across fruit types, This limits generalizability.

The author should:

• Clearly describe sampling as:

o “localized” or “site-specific”

• Emphasize findings as:

o baseline data, not national estimates

• Avoid general statements about broader populations

5. The manuscript suggests that fruit surfaces pose a “significant risk of transmission to humans”

This statement is too strong given that there is:

• No exposure assessment

• No transmission pathway analysis

• No genomic linkage to human infections

Required revisions:

• Replace with cautious phrasing such as:

o “may pose a potential risk”

o “could contribute to exposure”

• Avoid causal or definitive public health claims

6. The following minor improvements in language and presentation are also recommended:

a. The manuscript Language and Grammar has improved but still contains:

o Typographical errors

o Awkward phrasing

Professional language editing is recommended

b. MAR index formula is included but should be clearly linked to results

c. Clarify how isolates were selected for subsampling (randomization process)

d. CRA is correctly described

e. However, emphasize more clearly that it is:

o qualitative

o less precise than quantitative assays

f. Ensure all figures and tables:

o Include sample sizes (n)

o Have self-explanatory captions

g. Ensure consistency in formatting

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Reviewer #6: Yes:  Lawan Adamu

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Fresh fruit surface: a potential source for multidrug-resistant biofilm-forming Enterococcus faecalis having potential public health significance

PONE-D-25-60195R2

Dear Dr. Rahman,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Guadalupe Virginia Nevárez-Moorillón, Ph.D.

Academic Editor

PLOS One

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