Genome sequence-based species classification of Enterobacter cloacae complex: a study among clinical isolates

ABSTRACT Accurate species-level identification of Enterobacter cloacae complex (ECC) is crucial for related research. The classification of ECC is based on strain-to-strain phylogenetic congruence, as well as genomic features including average nucleotide identity (ANI) and digitalized DNA-DNA hybridization (dDDH). ANI and dDDH derived from whole-genome sequencing have emerged as a reliable metric for assessing genetic relatedness between genomes and are increasingly recognized as a standard for species delimitation. Up to now, there are two different classification methods for ECC. The first one categorizes E. hormaechei, a species within ECC, into five subspecies (E. hormaechei subsp. steigerwaltii, subsp. oharae, subsp. xiangfangensis, subsp. hoffmannii, and subsp. hormaechei). The second classifies E. hormaechei as three species: E. hormaechei, “E. xiangfangensis,” “E. hoffmanii.” While the former is well-accepted in the academic area, the latter may have a greater ability to distinguish different species of ECC. To assess the suitability of these identification criteria for clinical ECC isolates, we conducted a comprehensive analysis involving phylogenetic analysis, ANI and dDDH value alignment, virulence gene identification, and capsule typing on 256 clinical ECC strains isolated from the bloodstream. Our findings indicated that the method of categorizing E. hormaechei into five subspecies has better correlation and consistency with the molecular characteristics of clinical ECC isolates, as evidenced by phylogenetic analysis, virulence genes, and capsule typing. Therefore, the subspecies-based classification method appears more suitable for taxonomic assignments of clinical ECC isolates. IMPORTANCE Standardizing taxonomy of the Enterobacter cloacae complex (ECC) is necessary for data integration across diverse studies. The study utilized whole-genome data to accurately identify 256 clinical ECC isolated from bloodstream infections using average nucleotide identity (ANI), digitalized DNA-DNA hybridization (dDDH), and phylogenetic analysis. Through comprehensive assessments including phylogenetic analysis, ANI and dDDH comparisons, virulence gene, and capsule typing of the 256 clinical isolates, it was concluded that the classification method based on subspecies exhibited better correlation and consistency with the molecular characteristics of clinical ECC isolates. In summary, this research contributes to the precise identification of clinical ECC at the species level and expands our understanding of ECC.


Review of: Genome Sequence-Based Species Classification of Enterobacter cloacae complex: a Study among Clinical Isolates
Qiu and colleagues compared two taxonomic approaches for Enterobacter cloacae complex genomes.These two were based on Average Nucleotide Identity (ANI) but the thresholds applied for the species definition could be: ANI > 95% and subspecies >98% or ANI > 96% for species definition and no data for subspecies.
To test the two thresholds, they used a collection of 256 clinical strains isolated from blood cultures between 2010 and 2022.A global phylogenetic approach was performed using the kSNP.For each genome, several typing methods were applied such as sequence typing, determination of virulence encoding genes and capsule locus.
According to the authors, results are convergent for all the species regardless the ANI threshold used expect for Enterobacter hormaechei genomes.One threshold defined one species divided into five subspecies, the other one three species.The authors argued that the approach species ANI > 95% and subspecies >98% should be preferred to the other one for two reasons.First, results of typing methods and phylogenetic approach suited best to the five subspecies classification.Then two strains were associated to two different species using the ANI > 96% for species definition which is not acceptable.This article is interesting and well written.There are some comments/limitation which need to be corrected.

Major comments:
Lines 50 -53, a very important taxonomic method among Enterobacter cloacae strains is based on the sequence of the gene hsp60 described by Hoffman et al in 2003: Hoffmann, H., & Roggenkamp, A. (2003).Population genetics of the nomenspecies Enterobacter cloacae.Applied and environmental microbiology, 69(9), 5306-5318.This approach needs to be present in this section.Lines 53-54: "While MS has been commonly used in clinical laboratories, it has been 54 proven inaccurate for ECC classification".Please add a citation to this affirmation.
Line 69: I don't find any information about the Granger et al publication in the reference section.
Line 87: Is it possible to have a few clinical data about the demography of the patients?Thank you.Line 76 -77: "Zong et al. reclassified ECC species of ECC based on the threshold of ANI > 96% and 77 dDDH > 70.0% (36)."What were their conclusion?How many species in the complex?What were the consequences for E. hormaechei classification?Line 88: Strains were isolated from 2010 to 2022.How were they conserved?What was the conservation medium?What temperature?What were the MALDI-TOF results for these strains?Lines 92 -98: Sequencing, assembly, and annotation of bacterial genomes: The absence of hsp60 Hoffman cluster assignation is a limitation as this classification is widespread among Enterobacter cloacae complex studies and publications.Line 96 -97: "Virulence genes were identified using VFDB databases ( 27)" What blast parameters thresholds did you used for identity and coverage to consider a gene as present in the genome?Line 100: "the type strains of Enterobacter species and subspecies were collected from NCBI Refseq Database ( 24)".Could you please provide a list of the reference genomes you used as the reference for species and subspecies definition?Line 103: The whole-genome phylogenetic analysis was performed kSNP3.Did you used a reference genome to annotate your SNP?If so, what was the reference chosen?What was the kmer size used?Did you run Kchooser first?Line 107: BioProject accession PRJNA1046721.Data are not available at the moment of the review, could you please release at least the bioproject and the biosamples?Thank you.Lines 126 -133: Phylogenetic analysis and sequence types (STs).Is it possible to inform the genomic distance based on SNP between the five clades and within each clades using the results of kSNP3 ?You highlighted some outbreaks.What were the genomic distances between the outbreak strains?Lines 135 -148: Virulence genes analysis.The association between iroBCDEN genes clusters and E. hormaechei subsp.steigerwaltii is very interesting.Did you look at the outcome of the patients or the clinical presentation of the bacteraemia?You discussed the association with E. bugandensis and neonate's mortality lines 200 -201.This is why is it important to provide some clinical data associated with you strains.
Lines 163 -169: ANI, interesting examples of taxonomy confusion.Did this problem occurred for more than two strains?Table I is informative.Lines 172 -175: "Accurate subspecies and species identification of important pathogens is pivotal for effective clinical decision-making in clinical practice (6,7).With the tools of species classification continually improving (33).and a significant reduction in costs, ANI emerged as a widely utilized and recommended method for determining species boundaries."These statements should be moderate.In everyday practical practice, all the strains are not sequenced.As the consequence "effective clinical decision" are not based on ANI results even if the cost of sequencing decreased significantly.Line 185 -189: "In contrast to the alignment-based approaches, like ANI, the k-mer-based kSNP approach exhibited a high contrast enhancement effect in analyzing highly similar sequences and identifying small differences between highly similar sequences, thereby providing a valuable perspective for constructing evolutionary trees with higher accuracy (11)."I did not see any comparison of clustering approaches in your study i.e. trees or distance matrix obtained by kSNP approach versus ANI approach.As far as I'm concerned you did not provide results to discuss the ability of the two methods to cluster genomic sequences.

Minor comment:
Line 46: The order of the bibliography is not appropriate.The number of the first article cited is 28 Line 51: please put a capital on the g of Gram staining Line 63 and in diverse part of the manuscript: the expression "et al" needs to be written in italic.
Line 117 -118: The most common species in clinical samples was E. hormaechei (Fig. 1 We hope that our thoroughly revised manuscript is now acceptable for publication in the Microbiology Spectrum.

Reviewers' Comments Reply to Comments
The manuscript entitled "Genome Sequence-Based Species Classification of Enterobacter cloacae complex: a Study among Clinical Isolates" reports a study, which is genome sequence-driven identification of 200+ clinical strains within the ECC.
1) However, the ANI values were used as the only deciding parameter for species and subspecies level identification of the bacterial strains.
2) The unavailability of sequence accession numbers makes it more difficult to convince with the overall conclusions of the study.
3) The study included ECC strains collected from the year 2010 to 2022, which is a big positive, but no information on the related metadata, like the origin of strains is missing.The initial identification was done by MALDI-TOF MS but the results were not discussed at all.
1) We thank the reviewer for pointing out this issue.Digital DNA: DNA hybridization (dDDH) was the major criterion used to justify ANI implementations and thresholds for species delineation, in accordance with the recommendations by the ad hoc committee for the re-evaluation of the species definition in bacteriology.For the ECC strains in our study, we also used dDDH for species classification, and the results were consistent with the results of species and subspecies classification using ANI value.We indeed should present this result in the study.We have revised it in METHOD section, Line134-135, in RESULT section, Line 160-162 and in DISCUSSION section, Line 264-269.
2)We have revised the release date and the sequence accession number is now available (BioProject accession: PRJNA1046721).
3) The isolates were stored in 30% glycerol at -80°C prior to further analysis.The duplicate samples were removed after the strain was resuscitated.256 bloodstream isolated ECC was distributed at all ages, and the largest number of strains were middle-aged (41 to 65 years old) and elderly (>66 years old We have revised it in the Line 112-115.9) L100-101: I think more information is required on how the dataset has been generated for type strains because in some instances NCBI RefSeq database can also have taxonomic errors.It is advised to define the whole process of dataset creation, which was further used for ANI calculations and phylogenetic analysis.
I do not understand why the authors ignored the dDDH calculations, as it is still very relevant.
The following type strains were used for the ANI analysis: E. The dDDH among 256 clinical isolates were determined using the web-service https://tygs.dsmz.de/(23).We have revised it in the Line 123-135.
10) L135-137: No information is provided on the sequence similarity cutoff used for virulence gene assignments.
Virulence genes were identified using VFDB databases ( 19), the coverage ≥75% and identity ≥50%.We have added it in the Line 117.
11) L164-169: Authors are making an overstatement here, such situations are likely to happen, as there will be always a few strains that are almost on the border of a species, and in such cases the high closest similarity with enough dissimilarity with the second closest need to be considered before making conclusions.Secondly, there is a need to look into other parameters, like dDDH and phylogenetic position in core-gene trees or species trees.
Thanks for the reviewer's suggestions and we agree with your statements.We also mentioned this in the DISCUSSION section, Line 260, but it is still not comprehensive enough.We added our statements in Line 274-277.
Reviewer 2: Applied and environmental microbiology, 69(9), 5306-5318.This approach needs to be present in this section.

Major points Reply to Comments
We thank the reviewer for pointing out this issue.We have revised it in the Line 62.
2) Lines 53-54: "While MS has been commonly used in clinical laboratories, it has been 54 proven inaccurate for ECC classification".Please add a citation to this affirmation.
We have added it in the Line 63.
3) Line 69: I don't find any information about the Granger et al publication in the reference section.
We have revised it in the Line 79.We thank the reviewer for pointing out this issue.Zong et al. found that all Enterobacter subspecies assignments were incorrect.In this study, they updated the taxonomy of Enterobacter from species (n=19, including 6 subspecies) as of April 2020 to species (n=22).In other words, E. hormaechei were classified into three species (E. xiangfangensis, E. hoffmanii, and E. hormaechei).We have added it in the Line 87-89.Thank you for your advice.There is no doubt that hsp60 Hoffman cluster assignation is very useful and convenient.It can be divided into ECC clusters by pcr method.In this paper, we conducted whole genome sequencing of strains, focusing on species classification by using the whole genome of clinical strains.Therefore, we did not include the hsp60 Hoffman cluster assignation in this paper.

5)
8) Line 96 -97: "Virulence genes were identified using VFDB databases ( 27)" What blast parameters thresholds did you used for identity and coverage to consider a gene as present in the genome?
We have added it in the Line 117. 9) Line 100: "the type strains of Enterobacter species and subspecies were collected from NCBI Refseq Database ( 24)".
Could you please provide a list of the reference genomes you used as the reference for species and subspecies definition?
We have added it in the Line 123-134.The collected strains have been identified as ECC by MS, and the sequence similarity between strains is very high, so we did not use a reference genome.We ran the Kchooser to calculate the appropriate k-mer size, which is 19.We have added it in the Line 138.We thank the reviewer for pointing out this issue.We have revised the release date and the sequence accession number is now available.All The genomic distance has showed in the figure as 3 decimals kept.The outbreaks genomic distance is smaller than 0.0003.We have added it in the Line 168-170.
steigerwaltii is very interesting.Did you look at the outcome of the patients or the clinical presentation of the bacteraemia?
You discussed the association with E. bugandensis and neonate's mortality lines 200 -201.This is why is it important to provide some clinical data associated with you strains.
We looked at the clinical outcomes of patients with bacteremia in different subspecies of Enterobacter and found no significant differences in disease severity and mortality, regardless of whether this iroBCDEN virulence gene cluster was present.
Our findings may have some implications, but there are many factors that influence the clinical presentation of patients with bacteremia.
Thank you for your suggestion, which is of great clinical value and significance, and we will also pay attention to this aspect in the future.
We have added it in the Line 188-190.and a significant reduction in costs, ANI emerged as a widely utilized and recommended method for determining species boundaries."These statements should be moderate.In everyday practical practice, all the strains are not sequenced.
As the consequence "effective clinical decision" are not based on ANI results even if the cost of sequencing decreased significantly.
The statements have been corrected, these statements should be moderate.We have revised it in the Line 213-214.
16) Line 185 -189: "In contrast to the alignment-based approaches, like ANI, the k-mer-based kSNP approach exhibited a high contrast enhancement effect in analyzing highly similar sequences and identifying small differences between highly similar sequences, thereby providing a valuable perspective for constructing evolutionary trees with higher accuracy (11)."I did not see any comparison of clustering approaches in your study i.e. trees or distance matrix obtained by kSNP approach versus ANI approach.As far as I'm concerned you did not provide results to discuss the ability of the two methods to cluster genomic sequences.
We thank the reviewer for pointing out this issue.There was some inappropriateness in our presentation.We did ANI clustering analysis and the results of 162 E.hormaechei clinical isolates were consistent with kSNP approach.Tree matrix obtained by ANI approach are shown in the figure below.
We just wanted to provide a multi-dimensional approach to species classification in addition to ANI clustering analysis, so we did a k-mer-based kSNP phylogenetic analysis.We have revised our statements in the Line 225, Line 229-231.Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.

Minor points Reply to Comments
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Reviewer #1 (Comments for the Author): I believe the authors still do not agree that the recent classifications within ECC were not solely based on ANI values; instead, they are based on phylogenetic congruence and various genomic features, including ANI.However, if there is something wrong with one or the other ECC taxonomy, it should not influence this study.As far as I understood, this study does not aim to define the species and subspecies within E. hormaechei, but rather to report on the identification of ECC isolates.If the authors consider subspecies-based taxonomy to be accurate, then they should discuss their results accordingly.Defining E. hormaechei falls outside the scope of this study, and the authors have not conducted any tests to confirm which taxonomy is better or more suitable.If authors, aim to define this species, or the whole ECC, they need to generate scientific arguments based on strong experimental data.Furthermore, linking iroBCDEN genes clusters and E. hormaechei subsp.steigerwaltii, was not done appropriately like this linkage was noticed only for the genomes sequenced during this study, and authors do not include genomes of other E. hormaechei subsp.steigerwaltii strains.Secondly, the criteria for the identification of virulence factors (genes) was quite relaxed (the coverage {greater than or equal to}75% and identity {greater than or equal to}50%).Additionally, authors mentioned that kSNP (k-mers-based) phylogenetic tree is better, and I also agree with it, however, the choice depends on your specific research questions.If authors, just want to represent the genetic diversity within the closely related strains.In this case, kSNP might capture sufficient variation, but for species-level variations, a core-gene phylogeny might be more informative.Overall, I suggest that the authors need to change the focus of this study and establish a direct link between the methods used and the results obtained, else authors should use more robust methods and obtain new results, which provide strong evidence for their conclusions.
Reviewer #2 (Comments for the Author): Thank you very much for your article.Its improved a lot from the first version.I still have three major comments, two from the results and the last one from the discussion part.
Review of: Genome Sequence-Based Species Classification of Enterobacter cloacae complex: a Study among Clinical Isolates Qiu and colleagues compared two taxonomic approaches for Enterobacter cloacae complex genomes.
These two were based on Average Nucleotide Identity (ANI) but the thresholds applied for the species definition could be: ANI > 95% and subspecies >98% or ANI > 96% for species definition and no data for subspecies.
To test the two thresholds, they used a collection of 256 clinical strains isolated from blood cultures between 2010 and 2022.A global phylogenetic approach was performed using the kSNP.For each genome, several typing methods were applied such as sequence typing, determination of virulence encoding genes and capsule locus.
According to the authors, results are convergent for all the species regardless the ANI thresholds used expect for Enterobacter hormaechei genomes.One threshold defined one species divided into five subspecies, the other one three species.The authors argued that the approach species ANI > 95% and subspecies >98% should be preferred to the other one for two reasons.First, results of typing methods and phylogenetic approach suited best to the five subspecies classification.Secondly, two strains were associated to two different species using the ANI > 96% for species definition which is not acceptable.
This article is interesting and well written and improved a lot from the first version.
There are some comments/limitations which need to be corrected: The important part needs to be rewrite, this part should explain why the article is important.In its present form, the text looks like a small abstract of the article.

Major comment:
In the figure 1: Previously unknown sequence types are now labelled "novel"; this is not enough.News sequence types must be declared into PubMLST database to obtain proper attribution numbers.This is an important limitation for readers, they can't easily compare their genomes with those from the publication without proper ST declaration.
Lines 168 -170: "The genomic distance between the five clades and within each clade has showed in the figure as 3 decimals kept (Fig. 1).The outbreaks genomic distance is smaller than 0.0003."I'm not familiar with this distance metric and didn't see any information about it in the methods part.Could you please explain how it was calculated?SNP counts were unavailable from the kSNP3 output files?
Line 245 -246 "This underscores the clinical significance of distinguishing strains with and without specific virulence gene clusters, such as iroN gene clusters, in clinical pathogenicity analyses."But according to the results section, you didn't find any difference in outcome for your patient according to the ECC species i.e the presence of iroN genes clusters.This should be discussed.

Minor comments:
Line 63, genes need to be written in italic: hsp60.
Lines 76 -77 "Chavda et al. employed single nucleotide polymorphisms 76 (SNPs) from whole genome alignments to establish groups within ECC (13).".What were their conclusions?How many groups did they obtained?
hoffmanii" (15,16)."Could you please explain the scientific reasons why these authors changed the ANI threshold?Why didn't they accept the classification of Sutton et al?
Lines 104 -105 "because MALDI-TOF MS method is inadequate for distinguishing ECC".Seems to me unnecessary.
Line 112: The SPAdes version is unknown.
Line 120: ANI and digital DNA:DNA hybridization (dDDH) analysis.It is possible to put the data into a data frame?
Line 174-176: "We annotated siderophores-related genes, including entABESfepABCDG (enterobactin), iutAiucABCD (aerobactin), and iroBCDEN (salmochelin), across the 162 clinical isolates".This should be in the methods part.The non-siderophores-related genes are not mentioned in the text which is surprising.Does it mean that the genomes only contained siderophores-related genes as virulence genes?
Line 231: E. hormaechei needs to be in italic.As far as I understood, this study does not aim to define the species and subspecies within E. hormaechei, but rather to report on the identification of ECC isolates.If the authors consider subspecies-based taxonomy to be accurate, then they should discuss their results accordingly.Defining E. hormaechei falls outside the scope of this study, and the authors have not conducted any tests to confirm which taxonomy is better or more suitable.If authors, aim to define this species, or the Thank you for your careful review and valuable suggestions on this research work.As you pointed out, our study is aiming to discuss that the taxonomy based on subspecies is more suitable and valuable for the clinical isolates, but not to evaluate the accuracy of the classification criteria to identify species/subspecies.However, some expressions in the manuscript may be ambiguous and prone to misunderstanding.We have modified the manuscript thoroughly.
1.In our study, we found that taxonomy based on subspecies is more suitable for clinical isolates.It can differentiate strains with multiple shared characteristics, including virulence genes and capsule types.
Our results indicate that the identification result of clinical strains based on subspecies is more suitable for subsequent analysis such as molecular features, pathogenicity and virulence characteristics.The species-level identification may obscure the differences in pathogenicity and virulence characteristics among different whole ECC, they need to generate scientific arguments based on strong experimental data.
Furthermore, linking iroBCDEN genes clusters and E.
hormaechei subsp.steigerwaltii, was not done appropriately like this linkage was noticed only for the genomes sequenced during this study, and authors do not include genomes of other E. hormaechei subsp.
steigerwaltii strains.Secondly, the criteria for the identification of virulence factors (genes) was quite relaxed (the coverage {greater than or equal to}75% and identity {greater than or equal to}50%).
Additionally, authors mentioned that kSNP (k-mers-based) phylogenetic tree is better, and I also agree with it, however, the choice depends on your 2. We fully agree that the classifications within ECC were based on many dimensions including ANI, dDDH and phylogenic analysis.
The point of this manuscript is to discuss the application of the Furthermore, we adjusted the thresholds for the coverage and identity of virulence genes to 80% and 90%, respectively.Our analysis revealed that the comparison results for the iroBCDEN genes cluster exhibit the coverage > 99.8% and the identity > 91%.
It has been shown in METHODS section, Line 121-122.
4. We agree with your perspective that for variation at the species level, the core-gene phylogenetic tree could provide more information.kSNP is a software that simultaneously identifies SNPs based on both the core genome and the whole genome across a set of sequenced genomes.These tree files are then reported in the output files.In our study, we sought to characterize the species-level variation, thus we constructed phylogenetic trees using core SNPs.
It has been modified in METHODS section, Line 131-132, in DISCUSSION section, Line 217-222.

Major points Reply to Comments
The important part needs to be rewrite, this part should explain why the article is important.In its present form, the text looks like a small abstract of the article.
We have modified it in the Line 42-49.
In the figure 1 The genomic distance based on SNP between the five clades and within each clade are illustrated in the figure (Fig. 1).
The identification of outbreak strains should be based on clinical please explain how it was calculated?SNP counts were unavailable from the kSNP3 output files?epidemiological analysiss, which includes considerations of isolate time, location, and phylogenetic distance.We previously assumed that strains with a very close genetic distance on the evolutionary tree represented an outbreak, which may cause controversy.Therefore, we have removed the relevant content from the manuscript to avoid potential ambiguity..
We have revised it in the Line 161-162.It has been modified.
It has been modified in Line 118.
The data has been put into Supplemental Material Table S1.
Virulence genes were identified using VFDB databases, it has been shown in the methods part.We found virulence genes (encoding fimbriae, curli fibers, capsules, and flagella) were widely distributed in 162 strains with no significant differences.Siderophore-related genes (entABESfepABCDG (enterobactin) and iutAiucABCD Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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Figure 1 :
Figure 1: Why some Sequence-type are missing?You need to declare all the new MLST into PubMLST.The figure cannot be acceptable with such missing data.If I understand correctly, Only the E. hormaechei are represented in this figure.This should be precise in the figure tittle.

)
Line 145: (update to Septerber 2023) --> September Lines 150 -161: Capsule typing.Maybe this paragraph should be shortened.Dear editor, We have revised the article (Genome Sequence-Based Species Classification of Enterobacter cloacae complex: a Study among Clinical Isolates [Manuscript ID: Spectrum04312-23]) according to the major concerns of the reviewers.Detailed descriptions of the changes being made in our current manuscript are given in the point-by-point account below.

1)
Lines 50 -53, a very important taxonomic method among Enterobacter cloacae strains is based on the sequence of the gene hsp60 described by Hoffman et al in 2003: Hoffmann, H., & Roggenkamp, A. (2003).Population genetics of the nomenspecies Enterobacter cloacae.

4)
Line 76 -77: "Zong et al. reclassified ECC species of ECC based on the threshold of ANI > 96% and 77 dDDH > 70.0% (36)."What were their conclusion?How many species in the complex?What were the consequences for E. hormaechei classification?
10) Line 103: The whole-genome phylogenetic analysis was performed kSNP3.Did you used a reference genome to annotate your SNP?If so, what was the reference chosen?What was the kmer size used?Did you run Kchooser first?
figure.This should be precise in the figure tittle.
new MLST have been uploaded to PubMLST.Data submitted the website will go in to a queue for handling by a curator.New STs identified in this study are indicated in the figure using novel.The missing data in the figure has been added and the figure title has been modified.12)Lines 126 -133: Phylogenetic analysis and sequence types (STs).Is it possible to inform the genomic distance based on SNP between the five clades and within each clades using the results of kSNP3?You highlighted some outbreaks.What were the genomic distances between the outbreak strains?

1)
Line 46: The order of the bibliography is not appropriate.The number of the first article cited is 28 Line 51: please put a capital on the g of Gram staining Line 63 and in diverse part of the manuscript: the expression "et al" needs to be written in italic.2) Line 117 -118: The most common species in clinical samples was E. hormaechei (Fig. 1) Line 145: -23R1 (Genome Sequence-Based Species Classification of Enterobacter cloacae complex: a Study among Clinical Isolates) Dear Dr. Jiyong Yang: the article (Genome Sequence-Based Species Classification of Enterobacter cloacae complex: a Study among Clinical Isolates [Manuscript ID: Spectrum04312-23R1]) according to the major concerns of the reviewers.Detailed descriptions of the changes being made in our current manuscript are given in the point-by-point account below.We hope that our thoroughly revised manuscript is now acceptable for publication in the Microbiology Spectrum.authors still do not agree that the recent classifications within ECC were not solely based on ANI values; instead, they are based on phylogenetic congruence and various genomic features, including ANI.However, if there is something wrong with one or the other ECC taxonomy, it should not influence this study.
specific research questions.If authors, just want to represent the genetic diversity within the closely related strains.In this case, kSNP might capture sufficient variation, but for species-level variations, a core-gene phylogeny might be more informative.Overall,I suggest that the authors need to change the focus of this study and establish a direct link between the methods used and the results obtained, else authors should use more robust methods and obtain new results, which provide strong evidence for their conclusions.subspecies.All current classification criteria are undisputable and have their application scenarios.The aim of this study is not to establish classification criteria, but rather to evaluate the practicality of various taxonomy in the current application scenario (clinical strains isolated from bloodstream).Utilizing established molecular identification techniques including ANI, dDDH and phylogenetic analysis, we sought to find the most appropriate classification criteria for our clinical strains.Corresponding content has been modified in taxonomy in clinical scenario but not to questioning the classification criterion itself.It has been modified in ABSTRACT sectionorder to solidify the relationship between iroBCDEN genes and E. hormaechei subsp.steigerwaltii, we also analyzed 6221 genomes of E. hormaechei from the NCBI database (update to September 2023).The results revealed that iroBCDEN genes were only found in 2722 E. hormaechei subsp.steigerwaltii genomes but not in other four subspecies.The 3 rd generation sequencing results indicated that the iroBCDEN located in the chromosome of the E. hormaechei subsp.steigerwaltii, which is not easily transferable as plasmid.It has been shown in RESULT section, Line 177-181.

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Previously unknown sequence types are now labelled "novel"; this is not enough.News sequence types must be declared into PubMLST database to obtain proper attribution numbers.This is an important limitation for readers, they can't easily compare their genomes with those from the publication without proper ST declaration.New alleles were identified and assigned new allele numbers by comparison against the pubMLST database.All new allele sequences are deposited at the public databases for molecular typing and microbial genome diversity (pubMLST).The allelic profiles of strains are marked in red in Fig 1.Lines 168 -170: "The genomic distance between the five clades and within each clade has showed in the figure as 3 decimals kept (Fig. 1).The outbreaks genomic distance is smaller than 0.0003."I'm not familiar with this distance metric and didn't see any information about it in the methods part.Could you As you pointed out, the genomic genetic distance between species calculated based on the SNP counts and the nucleotide substitution model.The kSNP3 output files include SNP count files for branches.
-23R2 (Genome Sequence-Based Species Classification of Enterobacter cloacae complex: a Study among Clinical Isolates) Dear Dr. Jiyong Yang:

have added it in the Line 101-102 and Line 103-105.
Line 87: Is it possible to have a few clinical data about the demography of the patients?Thank you.Bloodstream isolated ECC was distributed at all ages, and the largest number of strains were middle-aged (41 to 65 years old) and elderly (>66 years old), with 116 strains (45.31%) and 94 strains (38.67%) respectively.We have added it in the Line 106-108.6)Line88: Strains were isolated from 2010 to 2022.How were The isolates were stored in 30% glycerol at −80°C until further they conserved?What was the conservation medium?What temperature?What were the MALDI-TOF results for these strains?analysis.The isolates were identified as E. asburiae, E. hormaechei, E. kobei, E. ludwigii or E. cloacae because MALDI-TOF MS method is inadequate for distinguishing ECC.We ? Table I is informative.species of ECC, the ANI values varied greatly, and the genetic isolation between species was obvious.
14) Lines 163 -169: ANI, interesting examples of taxonomy confusion.Did this problem occurred for more than two In our study, the problem of taxonomy confusion due to different ANI thresholds only occurred in these two strains of E. hormaechei.In other strains

has been modified in Line 239-243.
S, et al.Using average nucleotide identity to improve taxonomic assignments in prokaryotic genomes at the NCBI [J].Int J Syst Evol Microbiol) Zong et al. used cutoff of AN≥96% and isDDH≥70.0%for the type strain of Enterobacter (E.hormaechei subsp.oharae DSM 16687, E. hormaechei subsp.steigerwaltii DSM 16691, E. xiangfangensis LMG 27195).The ANI of three type strains were ≥ 96.62%, and the isDDH were ≥75.8%.Consequently, they classified the three type strains as one species.It Siderophore can promote the survival and reproduction of bacteria in the host.We will further investigate the impact of this gene cluster on the survival and transmission of this strains.It SHARMA