Comparative Genomics of Three Hybrid-Pathogen Multidrug-Resistant Escherichia coli Strains Isolated from Healthy Donors’ Feces

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I think this manuscript is logical and informative. This manuscript uses computer simulation to conduct a genetic comparative study at the genome level. It sequenced and analyzed the genomes of three multidrug-resistant E. coli strains isolated from feces. Then the resistance and virulence characteristics of the bacteria were analyzed by bioinformatics, and a phylogenetic tree based on UPGMA was constructed. Ultimately, the authors concluded that the three bacterial genomes belonged to heteropathogenic hybrid E. coli. Although the manuscript is coherent overall, there are some areas that need to be improved. Therefore, I think the manuscript should be slightly revised before publication in this journal.

Main comments:

First, the introduction should briefly explain the types and hazards of diseases caused by "heteropathogenic" strains and "hybrid pathogenic" strains. Second, the content of the Materials and Methods needs to reduce the number of conjunctions and make the expression as simple as possible. And the figures in the supplementary information should also be described in the text as much as possible. In addition, this manuscript should indicate the page numbers.

Small comments:

1. Page 2, lines 68-73 "We selected ... the same donor samples." This statement is not clear and needs to be expressed in a clearer way.

2. Page 5, line 180 "Computer simulation sequence type analysis ... ETEC in the United States." This paragraph requires the inclusion of analysis results images in the article to support the conclusion.

3. Page 9, Figure 2 The layout of this figure needs to be adjusted, and the figure explanation should be placed on the same page.

Finally, the summary and significance of this manuscript should be added at the end of the manuscript as a conclusion.

Author Response

Comments 1: First, the introduction should briefly explain the types and hazards of diseases caused by "heteropathogenic" strains and "hybrid pathogenic" strains.

Response 1: Thanks a lot for your valuable suggestions, we have added the next pargraphs: 

The conflict arises when these virulence factors are common in different E. coli pathogenic strains, which can cause a severe disease expanding their sites of colonization, along with other adaptative features. They can also harbor similar resistance genes present in mobile genetic elements such as plasmids, facilitating their spread and making the disease more difficult to treat. Many studies related to the occurrence of these hybrids have been reported. The most reported cases of “hybrid-pathogenic” have been ExPEC / EAEC and ExPEC / EPEC, because it is proposed to of the homology between the different genes coding for the fimbriae that allow adhesion to the epithelium [6-9] but also the finding of the different toxins of the ETEC pathotype in samples of patients with UTI (ExPEC) [10].

The best-documented example of “hetero-pathogenic” was a severe acute gastroenteritis outbreak (EAEC) and Hemolytic Uremic Syndrome (EHEC) [11]. Another common pathotype reported more recently in clinical samples from countries such as Sweden and South Korea has been EHEC/ETEC [12, 13].

Are now between lines 60-76

Comments 2: Second, the content of the Materials and Methods needs to reduce the number of conjunctions and make the expression as simple as possible. And the figures in the supplementary information should also be described in the text as much as possible. In addition, this manuscript should indicate the page numbers.

Response 2: We have revised the manuscript and paraphrased it, more information was added regarding the supplementary information as well as the page numbers.

Comments 3: Page 2, lines 68-73 "We selected ... the same donor samples." This statement is not clear and needs to be expressed in a clearer way.

Response 3: We have revised the manuscript and paraphrased it in order to improve it.

Comments 4: Page 5, line 180 "Computer simulation sequence type analysis ... ETEC in the United States." This paragraph requires the inclusion of analysis results images in the article to support the conclusion.

Response 4: A supplementary Figure 2 was added. We decided to include it as Supplementary material since it implied the inclusion of new 23 references.

Comments 5: Page 9, Figure 2 The layout of this figure needs to be adjusted, and the figure explanation should be placed on the same page.

Response 5: It was corrected and formatted according to your suggestions.

Comments 6: Finally, the summary and significance of this manuscript should be added at the end of the manuscript as a conclusion.

Response 6: We have added the next paragraph

This work contributes to understanding the genetic diversity and adaptability of hybrid-pathogenic E. coli strains. The findings highlight the potential public health risks posed by these strains, particularly in regions with high migration rates. By identifying key resistance and virulence determinants, the study underscores the necessity for continuous monitoring and development of effective treatment protocols to manage infections caused by such multidrug-resistant pathogens. Moreover, this comparative genomics approach provides a valuable framework for future research on the evolution and spread of pathogenic E. coli strains. The data generated can inform public health policies and health devise strategies to mitigate the spread of these bacteria. Overall, this report contributes significantly to the field of microbiology and epidemiology understanding the dynamics of multidrug-resistant E. coli in human populations.   It is now  between lines 406-419

Reviewer 2 Report

Comments and Suggestions for Authors

The article presents a comprehensive study on the genetic and phylogenetic analysis of three Escherichia coli strains, highlighting their hybrid pathotypes. The identification of specific virulence genes and mobile genetic elements, as well as the construction of a phylogenetic tree, provides novel insights into the genetic diversity and pathogenic potential of these E. coli strains. The significance of the study is evident as it addresses the genetic complexity of E. coli strains that pose significant public health risks. The quality of the presentation is generally high, with clear and detailed descriptions of the methods and results. The use of tables and figures, such as the phylogenetic tree, effectively supports the textual content and enhances the reader’s understanding of the data. However, there are several instances of grammatical and typographical errors that need correction to improve readability and coherence.

Minor Comments:

Lines 43-44. "Enterotoxigenic (EHEC)" correct to "Enterotoxigenic (ETEC)".

Line 51. "bacteriemia" correct to "bacteremia".

Line 107. "executed" should be "identified".

Line 108. Consider specifying the full name of the program, "MobileElementFinder," instead of just "MGE".

Line 184. "tract urine infections" correct to "urinary tract infections".

Lines 194-196. The sentence ”In a previous study, Barrios-Villa et al., in 2020 report the fimH27 allele in ExPEC strains belonging to the AIEC pathotype, as well as in EIEC and K12 genomes.” should include a citation to the referenced study.

Line 199. "diarrhoeagenic" correct to “diarrheagenic”.

Line 240. “Herterotrimer” correct to "Heterotrimer".

Line 250. "non fimbrial-adhesins" should be "non-fimbrial adhesins".

Line 255. "homologue genes" to "homologous genes".

Line 262. “(MGE)” correct to “(MGEs)”

Line 267. “Poultry” should be "poultry".

Line 281. “kpb” correct to “kbp”.

Line 308. “The full term “Miniature inverted-repeat transposable elements (MITEs)” should be used instead of “Miniature Inverted Repeat (MITEs)”.

Lines 318-319. “like Salmonella” correct to "such as Salmonella".

Line 331-332. The sentence “pathotypes previously associated with (Mendez-Moreno et al., 2022)” appears incomplete. Please verify and complete the sentence as necessary.

Author Response

We would like to thank you for te time taken to revise our manuscript as for your valuable suggestions. 

Comments 1: Lines 43-44. "Enterotoxigenic (EHEC)" correct to "Enterotoxigenic (ETEC)".

Response 1: It was corrected (line 44)

Comments 2: Line 51. "bacteriemia" correct to "bacteremia".

Response 2: It was corrected (line 51)

Comments 3: Line 107. "executed" should be "identified".

Response 3: It was corrected (line 125)

Comments 4: Line 108. Consider specifying the full name of the program, "MobileElementFinder," instead of just "MGE".

Response 4: It was corrected (line 126)

Comments 5: Line 184. "tract urine infections" correct to "urinary tract infections".

Response 5: It was corrected (line 165)

Comments 6: Lines 194-196. The sentence ”In a previous study, Barrios-Villa et al., in 2020 report the fimH27 allele in ExPEC strains belonging to the AIEC pathotype, as well as in EIEC and K12 genomes.” should include a citation to the referenced study.

Response 6: The reference was added (line 218)

Comments 7: Line 199. "diarrhoeagenic" correct to “diarrheagenic”.

Response 7: It was corrected (line 221)

Comments 8: Line 240. “Herterotrimer” correct to "Heterotrimer".

Response 8: It was corrected (line 262)

Comments 9: Line 250. "non fimbrial-adhesins" should be "non-fimbrial adhesins".

Response 9: It was corrected (line 272)

Comments 10: Line 255. "homologue genes" to "homologous genes".

Response 10: It was corrected (lines 277-278)

Comments 11: Line 262. “(MGE)” correct to “(MGEs)”

Response 11: It was corrected (line 285)

Comments 12: Line 267. “Poultry” should be "poultry".

Response 12: It was corrected (line 290)

Comments 13: Line 281. “kpb” correct to “kbp”.

Response 13: It was corrected (line 304)

Comments 14: Line 308. “The full term “Miniature inverted-repeat transposable elements (MITEs)” should be used instead of “Miniature Inverted Repeat (MITEs)”.

Response 14: It was corrected (line 331)

Comments 15: Lines 318-319. “like Salmonella” correct to "such as Salmonella".

Response 15: It was corrected (line 342)

Comments 16: Line 331-332. The sentence “pathotypes previously associated with (Mendez-Moreno et al., 2022)” appears incomplete. Please verify and complete the sentence as necessary.

Response 16: It was corrected rephrasing the paragraph (lines 351-355)