First Report of IMI-2-Producing Enterobacter bugandensis and CTX-M-55-Producing Escherichia coli isolated from Healthy Volunteers in Tunisia

Round 1

Reviewer 1 Report

1. The PCR sequencing results and raw images of PFGE should be present in supplementary materials.
2. Line 112-114, the ANI value over 95% doesn't mean they are the same species. I compared the genome of 9i strain with other E. bugandensis strains. The average of ANI value between 9i and other E. bugandensis strains is about 95% while ANI values among the other E. bugandensis are 98%. The authors need more strong evidences such as the phylogenetic tree. The authors can download the genome of Enterobacter spp. from NCBI or EBI as a reference genome and build a phylogenetic tree to see which clade I9 is in?
3. Line 115-117, the sequencing depth of E. bugandensis9i (NZ_JAMYWA000000000.1) is not consistent with the description in NCBI's BioProject (https://www.ncbi.nlm.nih.gov/nuccore/JAMYWA000000000.1/). The sequencing depth in the manuscript is 80-fold, while the description from NCBI shows that i9 the isolate was sequenced at only 12-fold depth. Theoretically, it is impossible to yield a high quality genome at such a low depth. I would like to know how the authors assembled a genome with an N50 of over 221 kb using such low depth data?
4. Line 211-212, which software or tool is used for MLST typing and clonal complex?
5. Line 212, the url http://mlst.ucc.ie/ is dead.
6. Line 217, '…subsequent sequencing.8', does the "8" refer to reference 8? If so, please fix it, otherwise delete it.
7. Line 233-236, the description of the assembly procedure is not accurate. Velvet is a genome assembler which can't do adaptor removal and trimming. I don't understand why the authors assembled the genome with two assemblers but only kept the results of one? Why not just use Spades?? What's the purpose or benefit of using velvet here?
8. Line 235-236, the usage of Spades. In our experience, using the Spades to automatically pick the k-mer size produces a better quality genome than manually picking the k-mer size. This can be done by setting '-k auto'.

 

Author Response

Reviewer1:

We appreciate the time and effort that you dedicated to our manuscript and are grateful for the insightful comments on and valuable improvements to our paper.

We have incorporated all the suggestions made . Those changes are highlighted within the manuscript. Please see below, in blue, for a point-by-point response to your comments and concerns.

1- In methods, the authors did not mention how they identify two bacterial species from fecal? just by VITEK? 

2-why they did not detection by genotypic method?

Answer: Thank you very much for pointing that out. We apologize for the lack of clarity in the original version. All ESBL producing strains in our collection were first identified by VITEK as belonging to the E.coli species,  and then further confirmed  by species-specific PCR (amplification of the uidA gene) The text has been amended accordingly. we invite the reviewer to see our revised manuscript page 6 “Resistant isolates were identified by the VITEK 2 Compact system (bioMérieux, Mar-cy-l’Étoile, France) and by species-specific polymerase chain reaction (PCR) (amplification of the uidA gene)”.

3-The Isolates and susceptibility testing in methods needs rearrange. 

Answer: According to your suggestion, the Isolates and susceptibility testing section has been divided into two parts: (i) Isolation and identification, (ii) Antibiotic sensitivity testing

4- In Detection and characterization of beta-lactamase genes and other resistance genes: please explain how many genes exactly detection by PCR? please mention them in seperat table 

Answer: Suggestion has been followed and details of the primers have been presented in Supplementary Tables 1 and 2.  Tables have been mentioned in the text.

5- Did the authors registered the WGS of two bacterial species in NCBI and get accession number?

Answer: We have submit our WGS data and an accession number has been assigned, Accession Enterobacter bugandensis 9i (JAMYWA000000000 ; BioSample SAMN28405492). This information is included in the revised manuscript page 3: “E. bugandensis 9i (NZ_JAMYWA000000000.1) consists of 5.12 Mbp, distributed over 103 contigs and organized in 67 scaffolds with fold coverage of 80X, having an N50 contig size of 221,597 kb and an average G+C content of 56 %”.

6-From the finding aim, why the healthy carried these genes in bacterial species?also did the authors think these bacterial species normal flora?? if yes why NF carry resistance genes?I think (in my opinion) this urban area my be contaminated with weast materials or water, what did you think?

Answer: Thank you for highlighting this important point, which we have developed in the discussion section (page 5 )

 7- Please mention at least to 5 reference from meddle east.

Answer: As suggested by the reviewer,  6 references have been added to the revised manuscript(page 5).

1. Kader, A. A.; Kamath, K. A. Faecal carriage of extended-spectrum beta-lactamase-producing bacteria in the community. East Mediterr  Health  2009,15, 1365–1370.
2. Bassyouni, R. H.; Gaber, S. N.; Wegdan, A. A. Fecal carriage of extended-spectrum β-lactamase- and AmpC- producing Escherichia coli among healthcare workers. J.Infect.Dev.Ctries. 2015, 9, 304-308.  https://doi.org/10.3855/jidc.5633
3. Moghnia, O. H.; Rotimi, V. O.; Al-Sweih, N. A. Monitoring antibiotic resistance profiles of faecal isolates of Enterobac-teriaceae and the prevalence of carbapenem-resistant isolates among food handlers in Kuwait. J Glob Antimicrob Re-sist. 2021, 25, 370–376. https://doi.org/10.1016/j.jgar.2021.04.009 
4. Eltai, N. O.; Yassine, H. M.; Al Thani, A. A.; Abu Madi, M. A.; Ismail, A.; Ibrahim, E.; Alali, W. Q. Prevalence of antibi-otic resistant Escherichia coli isolates from fecal samples of food handlers in Qatar. Antimicrob Resist Infect Control. 2018, 7, 78. https://doi.org/10.1186/s13756-018-0369-2
5. Jallad, M. A.; Naoufal, R.; Irani, J.; Azar, E. Extended spectrum beta-lactamase carriage state among elderly nursing home residents in beirut. ScientificWorldJournal. 2015, 2015:987580. https://doi.org/10.1155/2015/987580 
6. Dandachi, I.; Salem Sokhn, E.; Najem, E.; Azar, E.; Daoud, Z. Carriage of beta-lactamase-producing enterobacteriaceae among nursing home residents in north Lebanon. Int. J. Infect. Dis.2016, 45, 24–31. https://doi.org/10.1016/j.ijid.2016.02.007

 

Reviewer 2 Report

I am interest to review manuscript (First report of IMI-2-producing Enterobacter bugandensis and 2 CTX-M-55-producing Escherichia coli isolated from healthy 3 volunteers in Tunisia) in Antibiotics, I have a lot of comments needs clarify from authors

1- In methods, the authors did not mention how they identify two bacterial species from fecal? just by VITEK? 

2-why they did not detection by genotypic method?

3-The Isolates and susceptibility testing in methods needs rearrange. 

4- In Detection and characterization of beta-lactamase genes and other resistance genes: please explain how many genes exactly detection by PCR? please mention them in seperat table 

5- Did the authors registered the WGS of two bacterial species in NCBI and get accession number?

6-From the finding aim, why the healthy carried these genes in bacterial species?

also did the authors think these bacterial species normal flora?? if yes why NF carry resistance genes?

I think (in my openion) this urban area my be contaminated with weast materials or water, what did you think?

7- Please mention at least to 5 reference from meddle east.

 

Author Response

Reviewer 2

We appreciate the time and effort that you dedicated to our manuscript and are grateful for the insightful comments on and valuable improvements to our paper.

We have incorporated all the suggestions made . Those changes are highlighted within the manuscript. Please see below, in blue, for a point-by-point response to your comments and concerns.

Major issues:

Line 51: "and ESC for some of them (KPC, NDM…)." – These three dots are unprofessional. Please edit

Answer: Suggestion has been followed and modified as follows “(KPC, NDM, OXA, VIM, etc)”. Thank you!

Line 168: "Through horizontal gene transfer" – I think it will improve the text if you named the tools of this HGT – transposons? Plasmids? It would add to this paragraph

Answer: We thank reviewer for pointing out these important points and we agree that it is important to further discuss them. – We invite the reviewer to see our revised manuscript page 6.

Line 171: "…among others." – I think it better to delete these two words.

Answer: As suggested, « among others » has been removed from the text. Thank you!

Line 241-245: "Comparative genomic analysis was based on the average nucleotide identity using both best hits (one-way ANI) and reciprocal best hits (two-way ANI) between the Genome sequence and closest relative species E. bugandensis (NZ_AP022508.1). Typically, the ANI values between genomes of the same 244 species are above 95% [20]." – Please add the tool used for ANI calculations. Also, please write Genome with a small g.

Answer: The suggestions have been followed: (i) The ANI calculator (http://enve-omics.ce.gatech.edu/ani/index) has been added in the text as recommended. (ii) The genome has been modified with a small g. Thank you!

Minor issues:

Line 31 – bla ACT – please style as blaACT instead.

Answer: Done, thanks

Line 39: "they constitute a highly populated ecosystem... These AMR bacteria may spread to other hosts or transfer genetic resistance elements to other members of the microbiota including pathogens " – this statement is a little ambiguous – I would put the two parts into one sentence and not divide it into two for clarity.

Answer: As suggested the two parts were combined into one sentence: “Antimicrobial-resistant bacteria in the gut microbiota of humans and animals may pose a serious threat, as they can spread to other hosts or transfer genetic resistance elements to other members of the microbiota, including pathogens”. Thank you!

 

 

 

 

Reviewer 3 Report

 

This is a descriptive study that aimed at characterizing extended-pectrum beta-lactamases and carbapenemase-producing Gram-negative bacteria in of fecal samples among healthy humans in Tunisia. 51 samples were grown on suitable plates with antibiotics followed by analysis of resistance genes, integrons and phylogroup typing by PCR and also PFGE and MLST typing.

 Sixteen ESBL-21 producing E.coli isolates and one carbapenem-resistant E. bugandensis were detected and several antibiotic genes indicated; CTX-M-15, CTX-M-1, CTX-23 M-27 and CTX-M-55. Also, 6 isolates contained class 1 integrons with four gene cassette arrangements. Most importantly, these results revealed an alarming rate of ESBL-E. coli in healthy humans in Tunisia. Also, this is the first description of IMI-2 in E. bugandensis

 

Major issues:

Line 51: "and ESC for some of them (KPC, NDM…)." – These three dots are unprofessional. Please edit

Line 168: "Through horizontal gene transfer" – I think it will improve the text if you named the tools of this HGT – transposons? Plasmids? It would add to this paragraph.

Line 171: "…among others." – I think it better to delete these two words.

Line 241-245: "..Comparative genomic analysis was based on the average nucleotide identity using both best hits (one-way ANI) and reciprocal best hits (two-way ANI) between the Genome sequence and closest relative species E. bugandensis (NZ_AP022508.1). Typically, the ANI values between genomes of the same 244 species are above 95% [20]." – Please add the tool used for ANI calculations. Also, please write Genome with a small g.

 

Minor issues:

Line 31 – bla ACT – please style as blaACT instead.

Line 39: "they constitute a highly populated ecosystem... These AMR bacteria may spread to other hosts or transfer genetic resistance elements to other members of the microbiota including pathogens " – this statement is a little ambiguous – I would put the two parts into one sentence and not divide it into two for clarity.

 

Author Response

Reviewer3:

 We appreciate the time and effort that you dedicated to our manuscript and are grateful for the insightful comments on and valuable improvements to our paper.

We have incorporated all the suggestions made. Those changes are highlighted within the manuscript. Please see below, in blue, for a point-by-point response to your comments and concerns.

1. The PCR sequencing results and raw images of PFGE should be present in supplementary materials.

Answer: As suggested, the raw image of the PFGE has been included in the supplementary material. Regarding sequences, there are so many sequences that it is difficult to include them in the paper. However, following your suggestion, we sent all the sequences to the editor.

2. Line 112-114, the ANI value over 95% doesn't mean they are the same species. I compared the genome of 9i strain with other E. bugandensis strains. The average of ANI value between 9i and other E. bugandensis strains is about 95% while ANI values among the other E. bugandensis are 98%. The authors need more strong evidence such as the phylogenetic tree. The authors can download the genome of Enterobacter spp. from NCBI or EBI as a reference genome and build a phylogenetic tree to see which clade I9 is in?

Answer: Thank you very much for pointing this out to us. We agree with the reviewer on this interesting remark and the changes have been made as recommended:

We first performed a phylogenomic analysis by the type strain genome server (TYGS) (at https://tygs.dsmz.De). The results (Figure S2) showed that strain 9i is close to E. bugandensis EB-247 (NZ_AP022508.1). For in-depth analysis, we calculated the ANI values of strain 9i with all genome assemblies of bugandensis species deposited in the GenBank database (as of December 2022) and found that the closest species is E. bugandensis strain 153_ECLO (NZ_JVSD00000000.1) (ANI 95.23%) (Table S5).   

The text has been updated accordingly: we invite the reviewer to consult our revised manuscript: Materials and methods: Page 7 / Results section: Page3

3. Line 115-117, the sequencing depth of E. bugandensis9i (NZ_JAMYWA000000000.1) is not consistent with the description in NCBI's BioProject (https://www.ncbi.nlm.nih.gov/nuccore/JAMYWA000000000.1/). The sequencing depth in the manuscript is 80-fold, while the description from NCBI shows that i9 the isolate was sequenced at only 12-fold depth. Theoretically, it is impossible to yield a high quality genome at such a low depth. I would like to know how the authors assembled a genome with an N50 of over 221 kb using such low depth data?

Answer: Thank you for the interesting comment, we fully agree with you. In fact, an error was made while inserting the data into the NCBI database, and the depth will be corrected in the NCBI database. We have already sent a request to the NCBI staff for the correction.

4. Line 211-212, which software or tool is used for MLST typing and clonal complex?
5. Line 212, the url http://mlst.ucc.ie/ is dead.

Answer: Thank you very much for pointing this out. We apologize for the error. Nucleotide sequences of the housekeeping genes were submitted to the Escherichia coli MLST Database : (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli) this information has been corrected in the text, thank you again!

6. Line 217, '…subsequent sequencing.8', does the "8" refer to reference 8? If so, please fix it, otherwise delete it.

Answer: Done thanks

7. Line 233-236, the description of the assembly procedure is not accurate. Velvet is a genome assembler which can't do adaptor removal and trimming. I don't understand why the authors assembled the genome with two assemblers but only kept the results of one? Why not just use Spades?? What's the purpose or benefit of using velvet here?

Answer: The reviewer’s comment is correct concerning the trimming step. Trimmomatic software was added in the paragraph as follows:

Page7 : Quality filtering was performed with the Trimmomatic program (Bolger et al 2014).

- We totally agree with the fact that only one software is enough for the genome assembly, in fact, we made the assembly with both software separately and combined. We noticed a little improvement in the quality of the assembling if we used the output of Velvet results as input for a second assembly with the spades.

8. Line 235-236, the usage of Spades. In our experience, using the Spades to automatically pick the k mer size produces a better quality genome than manually picking the k-mer size. This can be done by setting '-k auto'.

Answer: We agree with the reviewer’s interesting comment, that using the Spades to automatically pick the k-mer size produces a better-quality genome. Here, we tried to provide the different parameters used in this work that gives better results.

Round 2

Reviewer 1 Report

The authors have responded to all comments.

 

Reviewer 2 Report

Thank you for your respons and complete comments to improve your paper