Review Reports - Distribution and Influence on the Microbial Ecological Relationship of Antibiotic Resistance Genes in Soil at a Watershed Scale

Round 1

Reviewer 1 Report

Hao et al. studied the Taipu River Basin for the distribution of antibiotic resistance genes in soil on the microbial ecological relationship at a watershed scale. Further, the influencing factors associated with ARG distribution are also discussed. Overall, the results are interesting, and their discussion is logical. However, before considering the manuscript for publication, the authors are suggested to revise the manuscript based on the following comments:

Improve the quality of the figures.
Provide statistical analysis of the data.
Consult a native English to improve the language.
Provide some quantitative information in the conclusions.
References should be formatted in an appropriate style, consistent for all cited references.

Author Response

General comment: Hao et al. studied the Taipu River Basin for the distribution of antibiotic resistance genes in soil on the microbial ecological relationship at a watershed scale. Further, the influencing factors associated with ARG distribution are also discussed. Overall, the results are interesting, and their discussion is logical. However, before considering the manuscript for publication, the authors are suggested to revise the manuscript based on the following comments:

Improve the quality of the figures.

Response: Thanks for your good suggestion, we have improved and uploaded the updated high-resolution figures in the manuscript and attachment.

Provide statistical analysis of the data.

Response: We have added the statistical analysis method in section 2.6 (Line 192-194), and supplemented the statistical results, such as Pearson’s r. (Line 220)

The original sentence

“ Principal coordinate analysis (PCoA) was performed using R software 'vegan' package” has been changed to the revised one “

has been changed to the revised one

“ Principal coordinate analysis (PCoA),distance-based redundancy analysis (db-RDA) and variation partitioning analysis (VPA) were performed using R software 'vegan' package”.

Consult a native English to improve the language.

Response: Thanks, we have asked a native speaker to revise the language of the manuscript.

Provide some quantitative information in the conclusions.

Response: We have provided the quantitative information in both the results and the conclusions.

References should be formatted in an appropriate style, consistent for all cited references.

Response: We have double checked the format of the references to make them consistent.

Author Response File: Author Response.docx

Reviewer 2 Report

I have examined the manuscript contents, which relate to antibiotic resistance genes distribution in soil at a watershed scale.

In my opinion, the data are potentially valuable, but the conclusions provided may be negatively affected by the large dispersion of concepts due to massive statistical analysis and poor discussion.

Moreover, English used makes the manuscript difficult to read, especially in the discussion section. I honestly could not mark each point and it needs a major revision most probably by a native/proficient speaker. Furthermore, the results and discussion are quite inadequate, and some relevant references in this field are missing. Some other comments are given below:

Please format the name of all genes in italic.

Line 27: Antibiotics have not been analysed in this work, please remove them from the abstract.

Line 37: Please change Int1 with intI1.

Lines 111-112: Please format NO3- and NH4+ correctly (NO₃^-and NH₄⁺)

Line 117: Please specify the meaning of metals (i.e., Iron: Fe; Nickel: Ni; …)

Line 149: This is not Real Time PCR, but quantitative PCR (qPCR).

Line 185: Please describe RDA method as well.

Line 194: What does it mean positively? What is the reference?

Lines 203-205: What does this sentence mean? I do not understand.

Line 205: Please remove “obviously”, this is a Result section.

Figure 2: Please increase the dimension of all the text inside the images. I cannot read the legend.

Lines 212-213. This sentence is too much vague.

Line 220: The gene czcA is not an ARG.

Line 222: S8? Isn’t it S11? Please check it out.

Lines 230-231: Please indicate the corresponding figure.

Line 233: Once again, please remove “obviously”, this is a Result section.

Figure 4: Same as figure 2. Please change colours to have more contrast in the graph. Moreover, increase the text size of the legend. I cannot read it.

Line 242: RDA and VPA are not described in the method section.

Line 271: The gene czcA is not an ARG.

Figure 7: Same as figure 2 and 4.

Discussion and Conclusion section.

The discussion is very poor. It is almost a repetition of the Results section. The comparison with the existing literature is inadequate. The data presented here are very valuable, but the poor elaborated discussion is holding back the entire manuscript.

For example, you found the gene IS1247 to be the main responsible of the diffusion of ARGs. This result is very fresh, compared to literature, but it is not discussed enough by the authors. I feel like all the statistical analysis and the amount of data presented in this work deceived the authors.

I suggest to re-elaborate the discussion section. Maybe try to eliminate some sub-chapter to make a more comprehensive discussion.

Moreover, I strongly recommend adding several update references, especially in the discussion section.

Author Response

General comment: We have examined the manuscript contents, which relate to antibiotic resistance genes distribution in soil at a watershed scale.

Overall, the topic and the outcomes are interesting. Although the research line on the distribution of ARGs has been excessively documented in the literature, the fact that ARGs, MGEs, soil properties, toxic metals, PAHs and microbial community structure have been considered all together is very attractive.In my opinion, the data are potentially valuable, but the conclusions provided may be negatively affected by the large dispersion of concepts due to massive statistical analysis and poor discussion.Moreover, English used makes the manuscript difficult to read, especially in the discussion section. We honestly could not mark each point and it needs a major revision most probably by a native/proficient speaker. Furthermore, the results and discussion are quite inadequate, and some relevant references in this field are missing. Some other comments are given below: Please format the name of all genes in italic.

Response: Thanks for your valuable comments. We have addressed all of the issues raised by the reviewer. We have improved the English language and rephrased the results and discussion. And we also keep the name of all genes consistent and doble check the other inconsistent content.

Line 27: Antibiotics have not been analysed in this work, please remove them from the abstract.

Response: We have analyzed antibiotics and will add the analysis method in Materials and Methods (Line 119-123).

Line 37: Please change Int1 with

Response: Thanks, we have changed all “Int1” to “intI 1” and keep it consistent all through the manuscript.

Lines 111-112: Please format NO3- and NH4+ correctly (NO₃^-and NH₄⁺)

Response: We have changed “NO3-” and “NH4+” to “NO₃^-” and “NH₄⁺” (Line 114).

Line 117: Please specify the meaning of metals (i.e., Iron: Fe; Nickel: Ni; …)

Response: We have indicated the full name of the elements when they appear for the first time (Line 124-127).

The original sentence The original sentence”

A total of fourteen toxic metals (i.e.,Be, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Cd, Sb, Ba, Pb, Bi) in soil were detected by inductively coupled plasma mass spectrometry (iCAPQ, ThermoFisher, USA).”

has been changed to the revised one

“ A total of fourteen toxic metals (e.g.Beryllium(Be), Chromium(Cr), Manganese(Mn), Iron(Fe), Cobalt(Co), Nickel(Ni), Copper(Cu), Zinc(Zn), Arsenic(As), Cadmium(Cd), Antimony(Sb), Barium(Ba), Lead(Pb), Bismuth(Bi)) in soil were detected by inductively coupled plasma mass spectrometry (iCAPQ, ThermoFisher, USA).”

Line 149: This is not Real Time PCR, but quantitative PCR (qPCR).

Response: Thanks for pointing the misused term, we have changed “Real Time PCR” to “Quantitative PCR (qPCR)” (Line 159)

Line 185: Please describe RDA method as well.

Response: We have added the RDA method in section 2.6. (Line 197-198)

The original sentence

“Principal coordinate analysis (PCoA) was performed using R software 'vegan' package,”

has been changed to the revised one

“Principal coordinate analysis (PCoA),distance-based redundancy analysis (db-RDA) and variation partitioning analysis (VPA) were performed using R software 'vegan' package”

Line 195: What does it mean positively? What is the reference?

Response: Sorry for the confusion, we have deleted the “positively” (Line 205)

The original sentence

“The ARGs and MGEs were positively detected in all sampling sites.”

has been changed to the revised one

“The ARGs and MGEs were detected in all sampling sites.”

Lines 203-205: What does this sentence mean? We do not understand.

Response: The sentence means that the number of ARGs detected in some sites was similar to that of nearby sites, but differed greatly from sites with the same landuse. We have added the summary into that paragraph. (Line 212-213)

The original sentence

“The numbers of ARGs detected in the aquaculture area were in the order of S4> S12; in a residential area, the number of detected ARGs in S9 were much higher than that of S7 and S11; in farmland, the number of detected ARGs in S10 were significantly higher than that of S13 and S14.”

has been changed to the revised one

“The number of ARGs detected in some sites was similar to that of nearby sites, but differed greatly from sites of the same landuse. The numbers of ARGs detected in the aquaculture area were in the order of S4 > S12; in a residential area, the number of detected ARGs in S9 were much higher than that of S7 and S11; in farmland, the number of detected ARGs in S10 were significantly higher than that of S13 and S14, but the number of detected ARGs in S9 was similar to that in S10.”

Line 205: Please remove “obviously”, this is a Result section.

Response: We have removed “obviously” according to your suggestion.

Figure 2: Please increase the dimension of all the text inside the images. We cannot read the legend.

Response: We have updated the high-resolution figure in manuscript and attachment.

Lines 212-213. This sentence is too much vague.

Response: We have rephrased the sentence to make it more clear.

The original sentence

“There was a strong positive correlation (R² = 0.67, p= 0.05) between the absolute abundance of MGEs and ARGs (Figure 2e). The normalized abundance represented the degree of ARGs enrichment in soil bacteria (Figure 2f)”

has been changed to the revised one

“ There was a strong positive correlation (Pearson’s r = 0.82, p= 0.05) between the absolute abundance of MGEs and ARGs (Figure 2e). The normalized copy number represented the degree of ARGs enrichment in soil bacteria (Figure 2f)”

Line 220: The gene czcA is not an ARG.

Response: Sorry for confusion, we have revised the sentence. (Line 234-235)

The original sentence

“of which aac-Via and czcA were the most abundant ARGs subtypes”

has been changed to the revised one

“of which aac-Via was the most abundant ARGs subtypes.”

Line 222: S8? Isn’t it S11? Please check it out.

Response: We have double checked the results, it is S8. (Line 236)

Lines 230-231: Please indicate the corresponding figure.

Response: Sorry for confusion, we have added the detailed figure number corresponding to the related results (Line 244-252).

The original sentence

“Microbial diversity (chao1 index) in aquafarm and petroleum storage depot was lower than in other land uses. Overall, the alpha diversity of microbial community was not significantly different, whether it was indifferent land uses or regions, which contributed 26.7% and 8.34% of the total variance. Proteobacteria and Acidobacteria were the dominant species in the soil microbial community. Proteobacteria occupied a higher proportion of the microbial community in the petroleum storage depot than in other land uses, while Acidobacteria occupied a smaller proportion. Compared with Proteobacteria, the microbial composition of other phyla had more obvious differences in different land uses or regions (Figure 4).

has been changed to the revised one

“Microbial diversity (chao1 index) in aquafarm and petroleum storage depot was lower than in other land uses (Figure 4a). Overall, the alpha diversity of bacterial bacterial community was not significantly different, whether it was indifferent land uses or regions (Figure 4b), which contributed 26.7% and 8.34% of the total variance (Figure 4c, 4d). Proteobacteria and Acidobacteria were the dominant species in the soil bacterial bacterial community. Proteobacteria occupied a higher proportion of the bacterial bacterial community in the petroleum storage depot than in other land uses (Figure 4c), while Acidobacteria occupied a smaller proportion. Compared with Proteobacteria, the microbial composition of other phyla had more differences in different land uses or regions (Figure 4d).

Line 233: Once again, please remove “obviously”, this is a Result section.

Response: We have removed “obviously” according to your suggestion.

Figure 4: Same as figure 2. Please change colours to have more contrast in the graph. Moreover, increase the text size of the legend. We cannot read it.

Response: We have revised and upload the high-resolution figure in manuscript and attachment to make it more clear.

Line 242: RDA and VPA are not described in the method section.

Response: We have added the detailed description in section 2.6. Please refer to comment 6 too. (Line 197-198)

Line 271: The gene czcA is not an ARG.

Response: Thanks for valuable suggestion, we have revised the sentence according to your suggestion (Line 289-290).

The original sentence

“Among them, int1 was significantly correlated with the higher abundance of ARGs subtypes (czcA, qnrD, mepA, ttgA, and ttgB).”

has been changed to the revised one

“Among them, intI 1 was significantly correlated with the higher abundance of ARGs subtypes (qnrD, mepA, ttgA, and ttgB).”

Figure 7: Same as figure 2 and 4.

Response: We have uploaded the high-resolution figures and made the revision.

Discussion and Conclusion section.

The discussion is very poor. It is almost a repetition of the Results section. The comparison with the existing literature is inadequate. The data presented here are very valuable, but the poor elaborated discussion is holding back the entire manuscript. For example, you found the gene IS1247 to be the main responsible of the diffusion of ARGs. This result is very fresh, compared to literature, but it is not discussed enough by the authors. We feel like all the statistical analysis and the amount of data presented in this work deceived the authors. We suggest to re-elaborate the discussion section. Maybe try to eliminate some sub-chapter to make a more comprehensive discussion. Moreover, We strongly recommend adding several update references, especially in the discussion section.

Response: Thanks for valuable suggestion, we have made the following adjustments as suggested: (1) We have eliminated some sub-chapter title, combined section 4.1.1 and 4.1.2 into section 4.1, divided section 4.2 into two separate parts. The parts of the discussion section that are similar to the results were integrated. (2) We have added the literature on IS1247 and further discussed IS1247 as an indicator of mesuring ARGs ecological hazards. (Line 316-405)

The original sentence

“4.2. Factors affecting ARGs distribution and transmission

4.2.1. Antibiotics and MGEs

Antibiotics and MGEs were the drivers of ARGs distribution as documented in the previous studies [37, 38]. A typical aminoglycoside ARGs subtype (aac3Via), was identified with the highest absolute abundance, while, there was no significant correlation was observed with MGEs. The same was true for aph4ib (the co-occurrence hub of ARGs and MGEs), which implied that MGEs were not the main driving force for the enrichment of aminoglycoside ARGs in the environment. In addition, correlation analysis showed a positive correlation between MGEs and ARGs (except aminoglycoside; Figure S4). Aminoglycoside ARGs revealed a significant positive correlation with antibiotics but not MGEs, indicating that the enrichment of aminoglycoside ARGs were related to the antibiotic application. In contrast, multi-drug ARGs and other ARGs were linked with MGEs. Some ARG subtypes were even specifically related to MGEs. For example, tet44 was only significantly associated with IS1133, while IS3 was only related to erm (36).

4.2.2. Environmental factors

On the contrary, the influence of soil properties and PAHs on ARGs distribution was generally not observed at the watershed scale. However, PAHs can provide selective pressure for ARGs enrichment by selecting microorganisms [41]. The mechanism of soil properties (such as pH) providing selective pressure was similar to that of PAHs [42]. The investigation of the Taipu River Basin did not reveal the correlation of soil properties and PAHs with ARGs, MGEs, and microorganisms. Compared with toxic metals, soil properties and PAHs had a close association with the microbial communities, while, less to MGEs (Table S2). Therefore, selective pressure may be the main mechanism for the soil properties and PAHs affecting ARGs distribution.

4.3. The effect of ARGs enrichment on microbial ecological relationship

The results suggested that the effect of ARGs enrichment on the microbial community structure did not extend to the watershed scale, but existed. For land use with a low abundance of ARGs, such as residential areas and farmland, the microbial community was relatively independent in the co-occurrence network and had little connection with ARGs. But in land use with a high abundance of ARGs, the microbial community was not only closely related to ARGs and MGEs, but also divided into multiple modules in a co-occurrence network. To some extent, the structural differences of networks in various land use reflected the changes in the influence of ARGs enrichment on the microbial community. In other words, the structural differences of the network indicated the feasibility of ARGs enrichment as a pollutant affecting the microbial ecological relationship. The reason why pollution had not risen to the watershed scale was that the ARGs shown in the co-occurrence network were not highly enriched in the area. However, due to the significant correlation between ARGs and MGEs, the ecological risk caused by ARGs migration may appear within the watershed scale.

4.4. The indicator of ARGs transmission and pollution risk

Int1 has been proved to be a common integron closely related to multidrug resistance [43-46]. The previous studies also showed that the int1 played a key role in the distribution of high-abundance ARGs and HRGs [47]. However, the ARGs that were significantly related to int1 revealed a non-significant impact on the ecological relationship of soil microbial community in the Taipu River Basin. IS1247 was a common transposase in the co-occurrence network of ARGs and microbial communities under different land use, and it had been found to be a close correlation with ARG subtypes that affected the structure of the microbial communities. The abundance of IS1247 was strongly related to the co-occurrence network properties (average degree, density, average clustering coefficient) of ARGs, MGEs, and microbial community (Figure 8), but not related to environmental factors. Thus, int1 can be regarded as an indicator of MGEs-mediated ARGs transmission under the influence of toxic metals, while IS1247 was more suitable as an indicator of the influence of ARGs on microbial community structure.”

has been changed to the revised one

“4.1. The characteristics of ARGs distribution

The results showed that ARGs and MGEs were widely enrichedin aquafarm, pharmaceutical factory, and sewage treatment with massive application of antibiotics [34-36]. In this study, the abundance of ARGs and MGEs within these land uses were higher than other areas (except petroleum storage areas) (Figure 2). In addition, the distribution characteristics of ARGs in petroleum storage areas were extremely different from other sites, which further showed the important impact of land uses on ARGs distribution.

Although the land-use types had a certain influence on the distribution of ARGs, the regional features were more obvious within the scope of the watershed scale. There was little difference in the number of ARGs detected in different land uses (Figure 2d). In the case of the same land use type, the distribution of ARGs and MGEs at various locations were quite dissimilar. Moreover, no apparent regional variations were found in the microbial distribution at the order level (Figure S2). Differences in the changes of ARGs and bacterial communities under the spatial geography gradient were compared. Spatial geography difference had a higher contribution to the total variation and was more obvious in ARGs distribution (Figure S3). The regional correlation to some extent represented the ARGs transmission at the macro scale, which was also supported by the strong positive correlation between the abundance of ARGs and MGEs in the soil of the Taipu River Basin.

4.2. Driving factors of ARGs distribution

Antibiotics and MGEs were the drivers of ARGs distribution as documented in the previous studies [37, 38]. The correlation analysis showed the role of two driving factors in the spatial pattern of ARGs. A typical aminoglycoside ARGs subtype (aac3Via), was identified with the highest absolute abundance, while, there was no significant correlation was observed with MGEs. The same was true for aph4ib (the co-occurrence hub of ARGs and MGEs), which implied that MGEs were not the main driving force for the enrichment of aminoglycoside ARGs in the environment. In addition, correlation analysis showed a positive correlation between MGEs and ARGs (except aminoglycoside; Figure S4). Aminoglycoside ARGs revealed a significant positive correlation with antibiotics but not MGEs, indicating that the enrichment of aminoglycoside ARGs were related to the antibiotic application. In contrast, multi-drug ARGs and other ARGs were linked with MGEs. Some ARG subtypes were even specifically related to MGEs. For example, tet44 was only significantly associated with IS1133, while IS3 was only related to erm(36). That is to say, the distribution of aminoglycoside ARGs was related to the input of antibiotics, and the transmission of ARGs mediated by MGE was the main factor affecting the distribution pattern of other ARGs.

4.3. The effects of environmental factors on ARGs distribution

Toxic metals were the key factors affecting the distribution of ARGs and MGEs [39]. The abundance of multi-drug ARGs was significantly related to Cr, Co and As (Figure S5). A significant positive correlation was found between macrolide ARGs and Cr, Co, Pb, Bi. In addition, tetracycline, sulfonamide and rifamycin ARGs were positively correlated with one or several toxic metals. CzcA, a typical HRG, was confirmed to have a positive coupling relationship with ARGs and class 1 integron [22, 40]. CzcA was highly enriched in the soil along the Taipu River and was strongly related to intI 1 at the watershed scale, indicating the compound pollution risk of toxic metals and ARGs in the Taipu River Basin. On the contrary, the influence of soil properties and PAHs on ARGs distribution was generally not observed at the watershed scale. However, PAHs can provide selective pressure for ARGs enrichment by selecting microorganisms [41]. The mechanism of soil properties (such as pH) providing selective pressure was similar to that of PAHs [42]. The investigation of the Taipu River Basin revealed soil properties and PAHs contributed more to bacterial communities and less to differences in MGEs than toxic metals(Table S2). Therefore, selective pressure may be the main mechanism for the soil properties and PAHs affecting ARGs distribution.

4.4. The effect of ARGs enrichment on microbial ecological relationship

The results suggested that the effect of ARGs enrichment on the bacterial community structure did not extend to the watershed scale, but existed. For land use with a low abundance of ARGs, such as residential areas and farmland, the bacterial community was relatively independent in the co-occurrence network and had little connection with ARGs. But in land use with a high abundance of ARGs, the bacterial community was not only closely related to ARGs and MGEs, but also divided into multiple modules in a co-occurrence network. To some extent, the differences in networks characteristics in various land use reflected the changes in the influence of ARGs enrichment on the bacterial community. In other words, the structural differences of the network indicated the feasibility of ARGs enrichment as a pollutant affecting the microbial ecological relationship. The reason why pollution had not risen to the watershed scale was that the ARGs shown in the co-occurrence network were not highly enriched in the area. However, due to the significant correlation between ARGs and MGEs, the ecological risk caused by ARGs migration may appear within the watershed scale.

4.5. The indicator of ARGs transmission and pollution risk

Intl1 has been proved to be a common integron closely related to multidrug resistance [43-46]. The results and previous studies also showed that the intI 1 played a key role in the distribution of high-abundance ARGs and HRGs [47]. However, the ARGs that were significantly related to intI 1 revealed a non-significant impact on the ecological relationship of soil bacterial community in the Taipu River Basin. Actually, the dominant MGE will be taken over as the selective pressure changes or new genes enter the pool [48]. IS1247 was a common transposase in the co-occurrence network of ARGs and bacterial communities under different land use, and can spread between different bacteria and pathogens [49, 50]. It had been found to be a close correlation with ARG subtypes in water, guts of insects, fish and humans [51-53]. IS insertions can move regions adjacent to alter the ARGs expression, enhance the niche adaptability of bacteria, and affect the structure of the bacterial communities [54-56]. Jose etc suggested that the co-occurrence network of integrons and microorganisms reflects the specific hosts of integrons , the dominant bacteria and their relationship under resistance pressure [57]. This study showed IS1247 was widespread in Taipu River Basin, and strongly related to the co-occurrence network properties (average degree, density, average clustering coefficient) of ARGs, MGEs, and bacterial community (Figure 8), but not environmental factors, which indicated that IS1247 was a common and stable element that can be used to measure the impact of ARGs on the properties of bacterial co-occurrence networks. In other words, intI 1 can be regarded as an indicator of MGE-mediated ARGs transmission under the influence of toxic metals, while IS1247 was more suitable as an indicator of the influence of ARGs on bacterial community structure.”

Author Response File: Author Response.docx

Reviewer 3 Report

Line 34: MGE should be singular (MGE, not MGEs)

Line 64: research “objectives”

Please define what the authors mean by “macro-scale” the first time it is mentioned.

Lines 79-83: This is confusing, please rephrase it for clarification.

Line 97: 45 samples collected (no comma)

Lines 97-98: Should 2, 3, and 4 be spelled out since they are under 10?

Line 102: Units are not consistently spaced. Please make sure there is consistent formatting of the units. Also, what does <2mm refer to?

Line 104: “air dried (<2mm) and used for….” This sentence is very long so I suggest separating it by adding a separate sentence: “Samples were stored at 4°C.” at the end.

Legend Fig 1: sample sites should have a space between them, not “samplesites”

Line 115: Germany is missing the y.

Line 117: should be e.g., not i.e.

Line 122: Is this R supposed to be ®? The letter looks transposed, but it could be a pdf conversion issue. Please check that your ® are clear in the text.

Line 125: Please change to 10 ng/µL.

Line 129, intI-1 and other integrase genes need to be italicized. Please check with the journal standards to review formatting guidelines for genes. Please use consistent formats; you have intI-1 but then intI2 and intI3.

Line 130: nuclease-free Master Mix (Manufacturer?)

Line 131: Do the authors mean forward and reverse instead of pre and post primer?

Line 152: Please use copy/µL

Line 155: nuclease-free

Line 158: Each sample in the study was in triplicate

Line 166: Bovine serum albumin. Please define it the first time it is used.

Line 167: You could say “cycled 35 times” or “for 35 cycles”

Line 173: Illumina HiSeq. Make sure all trademarks and restricted symbols are used when appropriate.

Line 176: The wording is confusing, please rephrase for clarification.

Line 179: If the authors only amplified bacterial rRNA (16S) then how do they have fungal reads?

Line 194: Just say “detected”, remove positively

Line 200: Please move (99) to behind S4 for consistency.

Line 201: Put a space between S4 and >. Please make sure your spacing is consistent.

Fig 2: There is no horizontal axis for a, c, or f. Also, landuse should be land use. The text is small, and it is difficult to read the legend, axes vertical numbers, and land use labels. I suggest making the figure larger and bolding all text. Also, the font for e graph is different. Please check for consistency.

Line 211: The authors stated an R²correlation is strong, but is 0.67 a strong correlation? It is a positive correlation, but I am not certain it can be stated as a “strong” positive correlation. Please either remove strong or provide some evidence/explanation of why this can be considered “strong”. Also, make sure throughout the test that R², not R2 is used. I have found this throughout the text and please check for consistency.

Line 220: Verify if these genes need to be italicized.

Fig 4: I cannot read anything on this graph. Please modify it to be clearer. Also, what are the Divisions (JS, QP, WJ)? There is no x axis for graph f

Line 243: “to show”, not “for showing”

Fig 5: again, I can’t read anything. These figures must be improved.

Lines 266-267: Confusing language, please rephrase. Also check R²

Line 283: When you say microorganisms, the authors are referring to bacteria, correct? The study did not analyze fungi.

Fig 8: Make sure capitalization is consistent. (a) there aren’t any labels or legends to know what is an ARG, MGE, or bacteria. Does the reader need to know this much information?

Lines 299-308: There wasn’t much discussion here; it seemed like the results were just repeated.

Lines 311-313: This statement is confusing, please rephrase for clarification.

Lines 317-318: Is “interpretation” the right word? Please check.

Line 320: Again, is an R²value of 0.6 a strong correlation, especially since it is on the border of statistical significance? Please remove “strong” or provide supporting information.

Line 335: Is (36) a reference? If so, it should be in brackets.

Line 343: There shouldn’t be a space between int and 1. Please be consistent.

Note: The authors discuss class 1 integrons, which have been abbreviated as int1. However, they also refer to the class 1 integron-integrase gene (intI-1), which is a common gene used to identify class 1 integrons. Throughout the text, the authors use “int1” to refer to the gene they amplified and class 1 integrons in general. The authors need to be consistent with the abbreviations for class 1 integrons (int1) and the class 1 integron integrase gene (intI-1). Please see the articles below for further information/examples.

https://journals.asm.org/doi/10.1128/jcm.39.1.8-13.2001?permanently=true

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4438328/

Line 351: Only bacteria were analyzed, correct? The authors need to be clear that they did not analyze all microorganisms, and change microorganisms to bacteria (or bacterial communities/populations/etc.) throughout the text.

Line 351. “Compared with toxic metals, soil properties and PAHs had a close association with the microbial communities, while, less to MGEs.”

In the previous sentence, the authors stated “did not reveal the correlation of soil properties and PAHs with ARGs, MGEs, and microorganism” How can soil prop and PAH have a close association to microbial (bacterial) communities and then say that data “did not reveal the correlation of soil prop and PAHs with….and microorganism (bacteria)”? These statements seem to contradict each other, so please clarify them.

Line 353: ARG should be singular (not ARGs)

Line 361: What does “structural difference” refer to?

Line 371: Again, make sure it is clear that “int1” refers to class 1 integrons, not the gene you selected to identify class 1 integrons (intI-1)

Line 389: MGE-mediated, not MGEs-mediated.

Line 393: “affected”

Lines 393-394: This is confusing, please rephrase for clarification.

Overall, please ensure all figures are clear, consistent, and sufficiently describe what is being presented so the reader does not need to go searching through the text to understand the figures.

Comments for author File: Comments.pdf

Author Response

Line 34: MGE should be singular (MGE, not MGEs)

Response: “MGEs” is more commonly abbreviation of mobile genetic genes. So it is often used in plural.

Line 64: research “objectives” Please define what the authors mean by “macro-scale” the first time it is mentioned.

Response: Sorry for the confusion, we have changed “macro scale” to “watershed scale" (line 67), and added further explanation. (Line 77)

The original sentence

“The coupling relationship between ARGs and environmental factors or pollutants indicates possibility of large-scale migration of ARGs. It is necessary to break through the limitations of previous research objects and explore the ARGs pollution of riverside soils at a macro scale.”

has been changed to the revised one

“The coupling relationship between ARGs and environmental factors or pollutants indicates possibility of large-scale migration of ARGs.Watershed is the most ideal spatial scale for macro-scale research on resistance genes, because the spatial pattern of pollutants at this scale is affected by multiple factors such as natural environment, land use, and human disturbance. It is necessary to break through the limitations of previous research objects and explore the ARGs pollution of riverside soils at a watershed scale.”

Lines 79-83: This is confusing, please rephrase it for clarification.

Response: Sorry for confusion, we have corrected the grammatical error and further explained the reasons for choosing the study area (lines 82-86).

The original sentence

”However, studies on soil resistome and risks along the Yangtze riverside at the macro-scale are still lacking. The Taipu River, a young artificial river in the Yangtze River Delta, the existence of which coincides with the urbanization development of the Yangtze River Delta, and along which a variety of typical antibiotic pollution sources and other pollution sources are distributed, was selected in this study is the representative area for the Yangtze River Delta.”

has been changed to the revised one

“However, studies on soil resistome and risks along the Yangtze riverside at the watershed-scale are still lacking. As a young artificial river in the Yangtze River Delta, the existence of which Taipu River coincides with the urbanization development of the Yangtze River Delta, and along which there are a variety of typical antibiotic pollution sources and other pollution sources are distributed along the river, in which can fully demonstrate the impact of human activities on the existence of ARGs. Therefore, Taipu River was selected in this study is as the representative area for the Yangtze River Delta.”

Line 97: 45 samples collected (no comma)

Response: Thanks for suggestion, we have removed the comma. (line 101)

Lines 97-98: Should 2, 3, and 4 be spelled out since they are under 10?

Response: Thanks for suggestion, we have spelled out the number under 10 (lines 101-103)

Line 102: Units are not consistently spaced. Please make sure there is consistent formatting of the units. Also, what does <2mm refer to?

Response: We have adjusted the unit spacing according to your comment. “<2mm” refer to “soil particle size < 2mm”, and added “soil particle size” before “< 2mm”. (lines 106-109)

Line 104: “air dried (<2mm) and used for….” This sentence is very long so We suggest separating it by adding a separate sentence: “Samples were stored at 4°C.” at the end.

Response: We have reduced the conjuctions in accordance with your suggestion (lines 108-109)

The original sentence

”and the other part was air-dried (<2mm) used for physical and chemical properties determination and stored at 4°C.”

has been changed to the revised one

“the other part was air-dried (soil particle size < 2mm) and stored at 4°C for physical and chemical properties determination.”

Legend Fig 1: sample sites should have a space between them, not “samplesites”

reponse: Thanks for the reminding, we havechanged “samplesites” to “sample sites”. (Line 110)

Line 115: Germany is missing the y.

Response: We have changed “German” to “Germany”. (Line 118)

Line 117: should be e.g., not i.e.

Response: We have changed “i.e.” to “e.g.”. (Line 124)

Line 122: Is this R supposed to be ®? The letter looks transposed, but it could be a pdf conversion issue. Please check that your ® are clear in the text.

Response: Thanks for reminding, we have double checked it and it is clear now in manuscript.

Line 125: Please change to 10 ng/µL.

Response: We have changed “10 ng·uL-1” to “10 ng/uL” and keep consistent all through the manuscript. (Line 134)

Line 129, intI-1 and other integrase genes need to be italicized. Please check with the journal standards to review formatting guidelines for genes. Please use consistent formats; you have intI-1 but then intI2 and intI3.

Response: We modified the formats according to your advice. (Line 138)

The original sentence

“The MGEs included 8 transposons, 4 common integrons (intI-1, intI-1LC, intI2, intI3) and a 16S rRNA gene.”

has been changed to the revised one

“The MGEs included 8 transposons, 4 common integrons (intI 1, intI 1LC, intI 2, intI 3) and a 16S rRNA gene.”

Line 130: nuclease-free Master Mix (Manufacturer?)

Response: We added the manufacturer information. (Line 139)

Line 131: Do the authors mean forward and reverse instead of pre and post primer?

Response: Yes, We replaced “pre and post primer” with “forward and reverse primer”. (Line 139)

The original sentence

”The PCR system consisted of nuclear-free PCR grade water, 1×LightCycler 480 SYBR Green We Master, 20 mg/mL BSA, 500 nM pre and post primer, and 20 ng/μL DNA templates.”

has been changed to the revised one

“The PCR system consisted of nuclease-free PCR grade water (Promega, USA), 1×LightCycler 480 SYBR Green We Master, 20mg/mL Bovine Serum albumin (BSA), 500nM forward and reverse primer, and 20 ng/μL DNA templates.”

Line 152: Please use copy/µL

Response: We changed “copy·uL-1” to “copy/uL”. (Line 162)

Line 155: nuclease-free

Response: We have revised the word.

The original sentence

“GreenWe MasterMix, 0.8 uL 16S rRNA gene primer (10 uM), 2 uL DNA template, 0.5 uL BSA solution (20 ug/uL) and nuclear-free water.”

has been changed to the revised one

“GreenWe MasterMix, 0.8 uL 16S rRNA gene primer (10 uM), 2 uL DNA template, 0.5 uL BSA solution (20 ug/uL) and nuclease-free water.”

Line 158: Each sample in the study wasin triplicate

Response: We have changed “is” to “was”. (Line 169)

The original sentence

“Each sample in the study is in triplicate”

has been changed to the revised one

“Each sample in the study was in triplicate”

Line 166: Bovine serum albumin. Please define it the first time it is used.

Response: We have defined it in Line 139.

Line 167: You could say “cycled 35 times” or “for 35 cycles”

Response: We have changed “cycle 35 times” to “cycled 35 times”.

Line 173: Illumina HiSeq. Make sure all trademarks and restricted symbols are used when appropriate.

Response: We have changed “Hiseq2500” to “HiSeq 2500”

Line 176: The wording is confusing, please rephrase for clarification.

Response: We have rephrased it. Pre-processing and denoising are the processes of sequence processing

Line 179: If the authors only amplified bacterial rRNA (16S) then how do they have fungal reads?

Response: Sorry for confusion, we have removed fungal reads. (line 189)

The original sentence

“The average length of the remaining high-quality amplicon reads was 392 bps (bacterial) and 300 bps (fungal).”

has been changed to the revised one

“The average length of the remaining high-quality amplicon reads was 392 bps (bacterial).”

Line 194: Just say “detected”, remove “positively”

Response: We have removed “positively”. (line 205)

Line 200: Please move (99) to behind S4 for consistency.

Response: We have modified the sentence according to your comment. (line 210)

Line 201: Put a space between S4 and >. Please make sure your spacing is consistent.

Response: We have revised the sentence according to your comment. (line 214)

Fig 2: There is no horizontal axis for a, c, or f. Also, landuse should be land use. The text is small, and it is difficult to read the legend, axes vertical numbers, and land use labels. We suggest making the figure larger and bolding all text. Also, the font for e graph is different. Please check for consistency.

Response: We have added the horizontal axis label and unified format. In order to facilitate the checking, we have added a 300dpi image to the document. (Figure 2)

Line 211: The authors stated an R²correlation is strong, but is 0.67 a strong correlation? It is a positive correlation, but We am not certain it can be stated as a “strong” positive correlation. Please either remove strong or provide some evidence/explanation of why this can be considered “strong”. Also, make sure throughout the test that R², not R2 is used. We have found this throughout the text and please check for consistency.

Response: Sorry for confusion. Judging whether there is a strong correlation should be based on the correlation coefficient, we have added the Pearson correlation coefficient. (Line 224)

The original sentence

“R2=0.67, P = 0.05”

has been changed to the revised one

“Pearson’s r = 0.82, p = 0.05”

Line 220: Verify if these genes need to be italicized.

Response: We have modified the genes’ format and kept them consistent all through the manusript. (Line 234-239).

Fig 4: We cannot read anything on this graph. Please modify it to be clearer. Also, what are the Divisions (JS, QP, WJ)? There is no x axis for graph f

Response: We have modified the figures and added the x axis label. (Line 253)

Line 245: “to show”, not “for showing”

Response: We have changed “for showing” to “to show”. (line 260)

Lines 266-267: Confusing language, please rephrase. Also check R²

Response: Sorry for cofusion, we have changed “r2” to “Pearson’r”. (line 284)

Line 283: When you say microorganisms, the authors are referring to bacteria, correct? The study did not analyze fungi.

Response: Yes, “microorganisms” in this study refers to bacteria.

Fig 8: Make sure capitalization is consistent. (a) there aren’t any labels or legends to know what is an ARG, MGE, or bacteria. Does the reader need to know thismuch information?

Response: Thanks for your suggestion. We have double checked the problems and made the revision.

Lines 299-308: There wasn’t much discussion here; it seemed like the results were just repeated.

Response: Thanks for valuable suggestions, we made the following adjustments: (1) We have eliminated some sub-chapter title, combined section 4.1.1 and 4.1.2 into section 4.1, divided section 4.2 into two separate parts. The parts of the discussion section that are similar to the results were integrated. (2) We have added the literature on IS1247 and further discussed IS1247 as an indicator of mesuring ARGs ecological hazards. (Line 316-403)

The original sentence

“4.2. Factors affecting ARGs distribution and transmission

4.2.1. Antibiotics and MGEs

4.2.2. Environmental factors

4.3. The effect of ARGs enrichment on microbial ecological relationship

4.4. The indicator of ARGs transmission and pollution risk

has been changed to the revised one

“4.1. The characteristics of ARGs distribution

4.2. Driving factors of ARGs distribution

4.3. The effects of environmental factors on ARGs distribution

4.4. The effect of ARGs enrichment on microbial ecological relationship

4.5. The indicator of ARGs transmission and pollution risk

Lines 311-313: This statement is confusing, please rephrase for clarification.

Response: We have rephrased the sentence. (lines 323-325)

The original sentence

“Although, the land-use types had a certain influence on the distribution of ARGs and the regional features were more obvious within the scope of the watershed scale.”

has been changed to the revised one

“Although the land-use types had a certain influence on the distribution of ARGs, the regional features were more obvious within the scope of the watershed scale.”

Lines 317-318: Is “interpretation” the right word? Please check.

Response: We have revised the sentence for better understanding.(Line 329-330)

The original sentence

“Differences in the changes of ARGs and microbial communities under the spatial geography gradient were compared. Spatial geography difference showed higher interpretation for the total variation and was more obvious in ARGs distribution (Figure S3).”

has been changed to the revised one

“Spatial geography difference had a higher contribution to the total variation and was more obvious in ARGs distribution (Figure S3).”

Line 320: Again, is an R²value of 0.6 a strong correlation, especially since it is on the border of statistical significance? Please remove “strong” or provide supporting information.

Response: We have added “Pearson’s r = 0.82, p= 0.05”, We believe it is a strong supporting information. (Line 224)

Line 335: Is (36) a reference? If so, it should be in brackets.

Response: No, erm(36) is an ARG.

Line 343: There shouldn’t be a space between int and 1. Please be consistent. Note: The authors discuss class 1 integrons, which have been abbreviated as int1. However, they also refer to the class 1 integron-integrase gene (intI-1), which is a common gene used to identify class 1 integrons. Throughout the text, the authors use “int1” to refer to the gene they amplified and class 1 integrons in general. The authors need to be consistent with the abbreviations for class 1 integrons (int1) and the class 1 integron integrase gene (intI-1). Please see the articles below for further information/examples.

https://journals.asm.org/doi/10.1128/jcm.39.1.8-13.2001?permanently=true

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4438328/

Response: We have revised the name of genes according to the website you provide.

Line 351: Only bacteria were analyzed, correct? The authors need to be clear that they did not analyze all microorganisms, and change microorganisms to bacteria (or bacterial communities/populations/etc.) throughout the text.

Response: Sorry for confusion, we have changed all “microorganisms” to “bacteria” in results.

Line 352-353. “Compared with toxic metals, soil properties and PAHs had a close association with the microbial communities, while, less to MGEs. In the previous sentence, the authors stated “did not reveal the correlation of soil properties and PAHs with ARGs, MGEs, and microorganism” How can soil prop and PAH have a close association to microbial (bacterial) communities and then say that data “did not reveal the correlation of soil prop and PAHs with….and microorganism (bacteria)”? These statements seem to contradict each other, so please clarify them.

Response: We have revised the sentence for better understanding. (Line 362-364)

The original sentence

“Compared with toxic metals, soil properties and PAHs had a close association with the microbial communities, while, less to MGEs (Table S2).”

has been changed to the revised one

“The investigation of the Taipu River Basin revealed soil properties and PAHs contributed more to bacterial communities and less to differences in MGEs than toxic metals(Table S2).”

Line 353: ARG should be singular (not ARGs)

Response: “ARGs” is often used for abbreviation for antibiotic resistance genes.

Line 361: What does “structural difference” refer to?

Response: The “structural difference” refers to the difference on the characteristics of co-occurrence network, such as density, modularity. We have revised the sentence for preventing confusion. (Line 374-376)

The original sentence

“To some extent, the structural differences of networks in various land use reflected the changes in the influence of ARGs enrichment on the microbial community. In other words, the structural differences of the network indicated the feasibility of ARGs enrichment as a pollutant affecting the microbial ecological relationship.”

has been changed to the revised one

“To some extent, the differences in networks characteristics in various land use reflected the changes in the influence of ARGs enrichment on the bacterial community. In other words, the structural differences of the network indicated the feasibility of ARGs enrichment as a pollutant affecting the microbial ecological relationship.”

Line 371: Again, make sure it is clear that “int1” refers to class 1 integrons, not the gene you selected to identify class 1 integrons (intI-1)

Response: We have revised all gene names in manusript.

Line 389: MGE-mediated, not MGEs-mediated.

Response: We have revised “MGEs-mediated” to “MGE-mediated” (Line 401)

Line 393: “affected”

Response: We have changed “affect” to “affected”.

Lines 393-394: This is confusing, please rephrase for clarification.

Response: Sorry for confusion, we have revised the conclusion for better understanding.

The original sentence

“Differed to toxic metals, the host bacteria and the selected ARGs were most likely affect the ARGs distribution with respect to PAHs and soil properties but had no direct influence on the soil ARGs distribution in the Taipu River Basin.”

has been changed to the revised one

“In contrast, PAHs and soil properties affected the ARG distribution mainly through the selection of host bacteria. In general, the contribution of toxic metals to the ARG distribution was greater than PAHs and soil properties in the Taipu River Basin.”

Overall, please ensure all figures are clear, consistent, and sufficiently describe what is being presented so the reader does not need to go searching through the text to understand the figures.

Response: We uploaded the high-resolution figure in the manuscript and attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I have examined the manuscript contents after the revision. In my opinion, the data now are well presented and discussed.

I suggest some minor spell-check (i.e. spacing after comma) and to add the reference of Jose etc (line 389).