Extra-intestinal pathogenic lineages of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are associated with prolonged ESBL gene carriage
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Objectives. Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-Ec) are frequently acquired during international travel, contributing to the global spread of antimicrobial resistance. Human-adapted ESBL-Ec are predicted to exhibit increased intestinal carriage duration, resulting in a higher likelihood of onward human-to-human transmission. Yet, bacterial determinants of increased carriage duration are unknown. Previous studies analysed small traveller cohorts, with short follow-up times, or did not employ high-resolution molecular typing, and were thus unable to identify bacterial traits associated with long-term carriage after recent acquisition. We aimed to identify which ESBL-Ec lineages are associated with increased carriage duration after return from international travel.
Methods. In a prospective cohort study of 2001 international travellers, we analysed 160 faecal ESBL-Ec isolates from all 38 travellers who acquired ESBL-Ec during travel and subsequently carried ESBL-Ec for at least 12 months after return, by whole-genome sequencing. For 17 travellers, we confirmed the long-term carriage of ESBL-Ec strains through single nucleotide variant typing. To identify determinants of increased carriage duration, we compared the 17 long-term carriers (≥12 months of carriage) with 33 age-, sex- and destination-matched short-term carriers (<1 month of carriage). Long-read sequencing was employed to investigate long-term ESBL plasmid carriage.
Results. We show that in healthy travellers with very low antibiotic usage, extra-intestinal pathogenic lineages of E. coli (ExPEC) are significantly more likely to persist than other E. coli lineages. The long-term carriage of E. coli from ExPEC lineages is mainly driven by sequence type 131 and phylogroup D E. coli .
Conclusions. Although ExPEC lineages frequently cause extra-intestinal infections such as bloodstream infections, our results indicate that ExPEC lineages are also efficient intestinal colonizers, which potentially contributes to their onward transmission.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. Thank you for your revisions. I believe your responses have satisfactorily addressed the reviewers concerns. However, the reason for the major revisions this time is due to our duplication/plagiarism assessment using iThenticate. It appears there is substantial self plagiarism to the thesis document for the first author with section having been entirely copied. The report is available to the authors and I ask that the section of concern be addressed. While additions have already reduced this score, rewriting these copied sections is necessary.
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This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Thank you for your submission, while the work appears well carried out, presentation of this in the manuscript could be improved. Reviewer 1 has highlighted that much of the data is in the supplementary sections. I encourage you to address both reviewers concerns and consider including more in the main body of the manuscript as well as additional analysis. As it stands, assessment of the data relies heavily on the reader.
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Comments to Author
Manuscript number: ACMI-D-22-00175 Title: Extraintestinal pathogenic Escherichia coli (ExPEC) are associated with prolonged carriage of extended-spectrum β-lactamase-producing E. coli acquired during travel. The authors of this manuscript present pertinent research that is needed in tackling the antimicrobial resistance (AMR) problem. The manuscript reads well, and its primary emphasis is on the role that travel plays in the acquisition of ESBL E. coli (ESBL-Ec) among travellers since this may have implications for how these strains spread across different geographic contexts and result in an increase in antibiotic resistance in countries with low prevalence. In the manuscript, the authors highlight that EXPEC E. coli is more likely to persist than other lineages and that phylogroup D and sequence …
Comments to Author
Manuscript number: ACMI-D-22-00175 Title: Extraintestinal pathogenic Escherichia coli (ExPEC) are associated with prolonged carriage of extended-spectrum β-lactamase-producing E. coli acquired during travel. The authors of this manuscript present pertinent research that is needed in tackling the antimicrobial resistance (AMR) problem. The manuscript reads well, and its primary emphasis is on the role that travel plays in the acquisition of ESBL E. coli (ESBL-Ec) among travellers since this may have implications for how these strains spread across different geographic contexts and result in an increase in antibiotic resistance in countries with low prevalence. In the manuscript, the authors highlight that EXPEC E. coli is more likely to persist than other lineages and that phylogroup D and sequence type 131 are associated with long-term carriage. The author provides sound interpretation of the results in the discussion section and an emphasis on the study's inherent limitations. However, there are minor changes that need to be addressed as indicated in the sections below. Minor revision Methodological rigour, reproducibility, and availability of underlying data The authors applied the appropriate bioinformatics tools and microbiology techniques to analyse the data and have made the data readily accessible to the readers. Things that need to be rectified are as follows: Introduction * The author highlights the role that travel plays in ESBL-Ec transmission and long-term carriage. However, the authors need to present literature and statistics from a geographical perspective in order to effectively establish the gap in transmission of ESBL-Ec among travellers. * The author states in lines 107 to 109 that the emphasis is on reporting host and bacterial characteristics associated with long-term carriage of ESBL genes as well as the mechanism by which the ESBL genes persisted. The word "host characteristics" is somewhat ambiguous, and it is unclear which host traits the author is emphasising. Additionally, I did not see any analysis that specifically addressed that element in the methods section. * Line 101 - 105: The section describing the study subjects needs to be included under materials and methods * Line 109 - 110: This is a result and needs not to be in the introduction section. * The author draws attention to the gap in the literature but omits providing the aim of the study in the abstract and the introduction. Materials and methods * Since the author's primary area of interest is EXPEC, the methodology should include a special section describing how EXPEC strains were identified in comparison to commensals. Commensal E. coli rather than EXPEC are more likely to mediate long-term carriage. The authors appear to have employed solely VFDB in their pipeline for virulence characterization, which is insufficient as of right now to distinguish an EXPEC from a UPEC, from an ETEC, from a commensal, etc. I am aware that Flemming created a new E. coli categorization in CGE (https://cge.food.dtu.dk/services/VirulenceFinder/). This is worth checking further. There seems to be a strong focus on EXPEC, but the data presented does not support this * The author makes no mention of the time when travellers were screened before travel, despite the authors' assertion that 38 travellers contracted ESBL- Ec. The author also needs to provide additional information about the study subjects. They need to clearly outline the attributes of the study subjects, including the geographical destinations of the travellers. * The text lacks crucial information on study subject characteristics presenting a reproducibility challenge. Presentation of results * This manuscript has good data, and the results are satisfactorily interpreted, however the lack of a logical flow of information makes it difficult to conceptualise the project. * The sentence "We included a median of 1... each timepoint (range: 1-5 isolates)" in lines 183-184. Is a misplaced statement. It belongs in the methods section. * Lines 184-186 should be in the section dedicated to SNP analysis (a logical flow issue) * The author mentions discovering ESBL genes on plasmids, but there is no discussion on the types of plasmids identified or any illustrations of any in this section. Moreover, there are no accession numbers provided of those plasmids. * The author asserts in lines 220 to 222 that two ESBL-Ec MLST types that were derived from a single traveller and collected at two separate timepoints, T0 and T12, shared a plasmid with 99.8% nucleotide identity. However, the author does not mention whether the nucleotide identity relates to the entire plasmid or only a portion of it. How the style and organization of the paper communicates and represents key findings * Despite the authors' assessment of published literature and quality data, the manuscript lacks a logical flow of information, as noted in the earlier text. Literature analysis or discussion * The author demonstrated satisfactory interpretation of the data using relevant literature while clearly highlighting the limitations of the study, thereby allowing subsequent research to make careful use of the findings.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The authors of "Extraintestinal pathogenic Escherichia coli (ExPEC) are associated with prolonged carriage of extended-spectrum β-lactamase-producing E. coli acquired during travel" have written an interesting manuscript but they have put most of their actual data in supplementary Tables and in such a way that it is impossible to understand which long-term ESBL-producing ExPEC carriers they are actually talking about in the manuscript. For example Lines 231-232 mention "a comparison of 17 isolates from 17 long-term carriers and 42 isolates from 33 matched short-term carriers, which were sequenced (Table S1)". Table S1 contains 56 isolates from 17 long-term carriers and 41 isolates from 33 short-term carriers. The STs and phylogroups of the isolates are missing in Table S1, while the authors talk about …
Comments to Author
The authors of "Extraintestinal pathogenic Escherichia coli (ExPEC) are associated with prolonged carriage of extended-spectrum β-lactamase-producing E. coli acquired during travel" have written an interesting manuscript but they have put most of their actual data in supplementary Tables and in such a way that it is impossible to understand which long-term ESBL-producing ExPEC carriers they are actually talking about in the manuscript. For example Lines 231-232 mention "a comparison of 17 isolates from 17 long-term carriers and 42 isolates from 33 matched short-term carriers, which were sequenced (Table S1)". Table S1 contains 56 isolates from 17 long-term carriers and 41 isolates from 33 short-term carriers. The STs and phylogroups of the isolates are missing in Table S1, while the authors talk about them in the manuscript. Consequently the result in lines 241-242 "This difference appears to be driven mostly by ST131 and phylogroup D strains." cannot be checked. Moreover the Materials and Methods section describes the identification of resistance genes, plasmid replicons and virulence genes, but the results of these analyses are nearly only presented in the supplementary Table S1 and hardly described in the manuscript itself not even for the long-read sequencing data. To my surprise the manuscript only mentions CTX-M seven times while it concerns ESBL-EC and it does not contain any ExPEC virulence factors. In addition, Table S1 and S2 are mentioned before Table 1, that is rather strange to me. It is also unclear what thresholds the authors used for the AMRfinderplus and ABRicate looking for the AMR genes, plasmid replicons and virulence genes. In addition, the title is confusing, it now reads that two entities ExPEC and ESBL-producing E. coli are associated but it actually concerns the ExPEC virulence factors being part of the AMR resistant bacteria. Figure 1 can be skipped, it has no added value for me, since the text is explanatory enough. Table S1 in my view has many inconsistencies and is confusing, for example; isol016 misses the CTX-M variant, isol014 has "blaEC|blaEC" in column T, why? In column AA five cells contain aadA2|aadA1 and three aadA1|aadA2, why the difference in order of the aadA variants? In column AC 25 cells contain aph(3'')-Ib|aph(6)-and 22 aph(6)-Id|aph(3'')-Ib, why the difference in order? In column AD two cells contain dfrA12|dfrA1 and one dfrA1|dfrA12, why the difference in order of the dfrA variants? Several of the ExPEC virulence genes are mentioned twice for a certain isolate, for example fepB in cell BK69, gspM in cell BS8 and BS24, gspL in B24, iucA and iucB in CB27 etc etc what do the authors mean with that. fyuA, irp1 and irp2 are part of a high pathogenicity island why are they mentioned in two separate columns. Minor comments Throughout the manuscript the CTX-M variant should not be written in italic only in subscript after bla. Either choose long-read or long read but not both. References are not consistent, some with capital letters in the title and others not. And bacterial names are not written in italic. Line 96; Please alphabetize the STs Lines 126-127; What read length were chosen for the library ? Line 150; Table S2 Line 210; included instead of not excluded Line 245; ESBL-Ec
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Not at all
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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