Rectal Colonization by Carbapenemase-Producing Enterobacterales in a Tertiary Care Hospital in Havana, Cuba

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The following suggestions are provided to improve the quality and clarity of the manuscript:

1. Please improve the study design flowchart and provide a more detailed and informative figure legend for Figure 3.

2. The resolution of Figure 2 should be enhanced to ensure better clarity and readability.

3. The Introduction section requires additional supporting evidence and should be strengthened with more relevant and up-to-date references.

4. Please clearly justify why only two antibiotics (amikacin and tigecycline) were selected for this study.

5. Provide a detailed table describing the selected patients, including their health status, lifestyle habits, and the source of the samples used in the study.

6. Please justify the suitability of the ERIC-PCR approach for this study and discuss its limitations in a dedicated paragraph.

7. The authors should expand the discussion on virulence traits, providing deeper insight and stronger supporting evidence.

Author Response

1. Please improve the study design flowchart and provide a more detailed and informative figure legend for Figure 3.

We have revised flowchart (Figure 3), and rewritten the section of “Study Design and Population” with the supplementation of inclusion and exclusion criteria. Line260-290.

2. The resolution of Figure 2 should be enhanced to ensure better clarity and readability.

We have optimized the clarity of Figure 2.

3. The Introduction section requires additional supporting evidence and should be strengthened with more relevant and up-to-date references.

We have rewritten the Introduction section, expanding on the rationale for the urgency of this study, justifying the rationality of the adopted method (active surveillance), highlighting the great significance of filling the research gap in Cuba, and incorporating 5 new references. Line40-67.

4. Please clearly justify why only two antibiotics (amikacin and tigecycline) were selected for this study.

 In the Methods section, we specify that we used the card VITEK® 2 AST-245 in the stuty that include different antibiotics (those of interest in the study are included in table 1). Amikacin and tigecycline are not included on this card and due to their importance as “salvage therapy” options, we evaluated the susceptibility by Etest method following the manufacturer instructions. 

5. Provide a detailed table describing the selected patients, including their health status, lifestyle habits, and the source of the samples used in the study.

In Supplementary Materials (Table S1. Epidemiological and clinical data of screened patients), contains some basic information of the relevant patients; however, we did not include information such as the patients’ lifestyle habits and health status during the information collection process.

6. Please justify the suitability of the ERIC-PCR approach for this study and discuss its limitations in a dedicated paragraph.

We have added the relevant content to the Limitations section in accordance with the requirements. First, we explicitly affirm that ERIC-PCR is a cost-effective and readily implementable method with substantial utility for the initial screening of outbreak investigations, especially suitable for resource-limited research settings. We then contrast it with whole-genome sequencing (WGS), explaining that while the latter constitutes the gold standard in this field, its implementation was not feasible in the context of the present study. Finally, we elaborate on the methodological limitations of ERIC-PCR, clarifying that these limitations are not inherent flaws of the study but rather honest methodological trade-offs made based on objective constraints during the research process. Line234-256.

7. The authors should expand the discussion on virulence traits, providing deeper insight and stronger supporting evidence.

While this study did not investigate virulence traits directly, the reviewer’s suggestion is highly valuable, as virulence characteristics are indeed crucial for understanding bacterial pathogenic potential and their competitive advantage in nosocomial transmission. Therefore, based on a review of the literature, we have supplemented the discussion with an emphasis on the specific roles of key virulence factors. We highlight that certain virulence determinants carried by bacterial strains not only facilitate environmental persistence but also significantly enhance resistance to antimicrobial agents and host immune clearance, while synergistically promoting mucosal colonization and tissue invasion. Lines 217-226.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript provides relevant context by linking the rise of drug resistance to the COVID-19 pandemic and citing WHO/PAHO guidance on carbapenemase surveillance. The authors emphasize the role of the gut as a reservoir and the importance of rectal screening. They correctly identify a knowledge gap in Cuba and state that this is the first study of its kind locally.

The cross-sectional surveillance of 297 patients at eight high-risk departments is appropriate for estimating colonization prevalence. Weekly follow-up cultures for up to 4 weeks also allow monitoring of hospital-acquired acquisition and progression to infection. This design is well-suited to the objectives. One limitation is that the study is single-center, which the authors acknowledge; the findings may not generalize to other hospitals. In addition, a longer follow-up period might capture late-onset infections. Overall, the design is reasonable for a preliminary study of CP-CRE carriage.

I recommend publication of the article after the following comments have been addressed.

• Carefully check that all results in the text match the tables and figures. For example, the Results text reports that 86.9% of carriers had prior hospitalization, whereas Supplementary Table S1 shows only 50.0% (23/46) of colonized patients with prior hospitalization.
• Line 86 should read “Citrobacter koseri” not “Citrobacter koserii”
• Figure 2: The font size is quite small; consider increasing.
• Table 2: It would enhance readability if the legend of Table 2 stated which statistical test was used to calculate the P values.
• In the Methods, clarify how patients and departments were selected (e.g. what are the criteria for “high-prevalence” units) and if sample size was based on prior estimates. Also describe any inclusion/exclusion criteria.

Author Response

1. Carefully check that all results in the text match the tables and figures. For example, the Results text reports that 86.9% of carriers had prior hospitalization, whereas Supplementary Table S1 shows only 50.0% (23/46) of colonized patients with prior hospitalization.

A mistake occurred during data entry for the table, and we have corrected the erroneous data in Table S1.

2. Line 86 should read “Citrobacter koseri” not “Citrobacter koserii”

We have revised to Citrobacter koseri.

3. Figure 2: The font size is quite small; consider increasing.

Due to page width constraints and the fact that each row in Figure 2 contains abundant information, increasing the font size would result in information misalignment. However, we have optimized the clarity of Figure 2,

Table 2: It would enhance readability if the legend of Table 2 stated which statistical test was used to calculate the P values.

We have added a footnote below Table 2.

4. In the Methods, clarify how patients and departments were selected (e.g. what are the criteria for “high-prevalence” units) and if sample size was based on prior estimates. Also describe any inclusion/exclusion criteria.

We have revised flowchart (Figure 3), and rewritten the section of “Study Design and Population” with the supplementation of inclusion and exclusion criteria. Line260-290.

Round 2

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Accept

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, 

your manuscript addresses an important topic and provides novel data for Cuba. However, revisions are needed to strengthen statistical analysis, improve data presentation, expand discussion on infection control strategies, and enhance clarity.

1. The risk factor analysis is limited to univariate methods. Multivariate analysis would strengthen conclusions and help identify independent predictors of infection.
2. Confidence intervals for prevalence and risk estimates should be included for clarity.
3. The study is single-center with a relatively small sample size (279 patients). Authors should acknowledge this limitation more explicitly and discuss its impact on generalizability.
4. Figures (ERIC-PCR dendrograms) are complex and lack detailed legends. Consider simplifying or providing clearer explanations for readers unfamiliar with ERIC-PCR. Figure 3 presents the study layout, so I suggest moving it to the Methods section. 
5. Table 1 is dense; summarizing key resistance trends visually (e.g., heatmap or bar chart) would improve readability.
6. The discussion compares prevalence with other countries but could better address public health implications and practical strategies for implementing surveillance in Cuban hospitals.
7. Define all abbreviations at first use
8. Why was multivariate analysis not performed for risk factors?

Reviewer 2 Report

Comments and Suggestions for Authors

This study addresses an important topic and provides novel data on intestinal colonization by carbapenemase-producing Enterobacterales in Cuba. The work has valuable epidemiological implications, and the dataset is relevant. However, the manuscript requires substantial revision before it can be considered for publication.

Below are my major concerns:

• Justify the classification of colonization detected ≤48h as “pre-admission,” acknowledging the possibility of early hospital acquisition.

• Detail the duration and uniformity of follow-up for colonized patients.

• Clarify how episodes of infection were identified and linked to colonizing isolates.

• Consider discussing the limitations of ERIC-PCR more explicitly, particularly its lower resolution compared to WGS for inferring transmission or clonal expansion.

• Several figures, including the ERIC-PCR dendrograms, are low resolution and difficult to interpret. Higher-quality images and clearer legends are needed.

• Tables would benefit from improved formatting to enhance readability.

• The flow of the Results section could be made clearer by reducing repetition and improving transitions.

• Only univariate analysis is performed. While multivariate analysis may be limited by sample size, please acknowledge this explicitly and consider including confounder-adjusted models if feasible.

• Clarify how prevalence ratios were computed and whether all assumptions for the statistical tests were met.

• Claims regarding outbreaks or inter-department transmission should be moderated and framed within the constraints of ERIC-PCR.

• Discussion could be improved by more directly comparing your findings with recent international studies on CRE colonization and infection progression. Also, regarding risk factors, speak about the importance of antibiotic duration (10.3390/ijms26146905)

• The manuscript would benefit from comprehensive language editing to improve clarity, grammar, and scientific phrasing.

• Some sections rely heavily on the authors’ previous work; incorporating a broader range of recent external literature will strengthen context and reduce redundancy.

Reviewer 3 Report

Comments and Suggestions for Authors

Following are my comments that will help the editor’s decision:

1. The introduction section requires additional supporting evidence with more relevant references.

2. Please clarify why only two antibiotics (amikacin and tigecycline) were selected for this study.

3. Provide detailed information in a table about the selected patients, including their health status, habits, and the source of the samples used in the study.