Genome-centric analyses of 165 metagenomes show that mobile genetic elements are crucial for the transmission of antimicrobial resistance genes to pathogens in activated sludge and wastewater

ABSTRACT Wastewater is considered a reservoir of antimicrobial resistance genes (ARGs), where the abundant antimicrobial-resistant bacteria and mobile genetic elements facilitate horizontal gene transfer. However, the prevalence and extent of these phenomena in different taxonomic groups that inhabit wastewater are still not fully understood. Here, we determined the presence of ARGs in metagenome-assembled genomes (MAGs) and evaluated the risks of MAG-carrying ARGs in potential human pathogens. The potential of these ARGs to be transmitted horizontally or vertically was also determined. A total of 5,916 MAGs (completeness >50%, contamination <10%) were recovered, covering 68 phyla and 279 genera. MAGs were dereplicated into 1,204 genome operational taxonomic units (gOTUs) as a proxy for species ( average nucleotide identity >0.95). The dominant ARG classes detected were bacitracin, multi-drug, macrolide-lincosamide-streptogramin (MLS), glycopeptide, and aminoglycoside, and 10.26% of them were located on plasmids. The main hosts of ARGs belonged to Escherichia, Klebsiella, Acinetobacter, Gresbergeria, Mycobacterium, and Thauera. Our data showed that 253 MAGs carried virulence factor genes (VFGs) divided into 44 gOTUs, of which 45 MAGs were carriers of ARGs, indicating that potential human pathogens carried ARGs. Alarmingly, the MAG assigned as Escherichia coli contained 159 VFGs, of which 95 were located on chromosomes and 10 on plasmids. In addition to shedding light on the prevalence of ARGs in individual genomes recovered from activated sludge and wastewater, our study demonstrates a workflow that can identify antimicrobial-resistant pathogens in complex microbial communities. IMPORTANCE Antimicrobial resistance (AMR) threatens the health of humans, animals, and natural ecosystems. In our study, an analysis of 165 metagenomes from wastewater revealed antibiotic-targeted alteration, efflux, and inactivation as the most prevalent AMR mechanisms. We identified several genera correlated with multiple ARGs, including Klebsiella, Escherichia, Acinetobacter, Nitrospira, Ottowia, Pseudomonas, and Thauera, which could have significant implications for AMR transmission. The abundance of bacA, mexL, and aph(3")-I in the genomes calls for their urgent management in wastewater. Our approach could be applied to different ecosystems to assess the risk of potential pathogens containing ARGs. Our findings highlight the importance of managing AMR in wastewater and can help design measures to reduce the transmission and evolution of AMR in these systems.

1.Line 2 and 58: please make sure to use "antimicrobial resistance genes" instead of "antibiotic resistance genes".2.Line 24: contamination <10%.3.Line 40: the names of genes need to be uniformly italicized in context, such as aph(3')-I.Please carefully check for other genes.4.Line 82: such research methods have also been done in previous studies , for example in the field of fermented foods (such as sufu) in 2021.It is appropriate to supplement this literature here.5.Line 108: 14 locations?The figure 1 shows 13 positions.6.Line 122: 2660 should be expressed as 2,660.Pay attention to other numerical expressions in manuscript.7.Line 129: should be Fig.S1 and Table S4.8.Line 132: change GTDB-tk to GTDB-Tk.9.Line 141: why is the number of MAGs at the genus level in Table 2 not 5916?10.Line 170: change Figure 1 to Fig. 1. 11.Line 197: change Fig. 3b to Fig. 3B.The lowercase letters after the numerical value need to be changed to uppercase letters.Please note that the entire text should be expressed in this way.12.Line 296: 94%? 13.Line 450: there are many mobile genetic elements (MGEs), such as plasmids, bacteriophages, transposons, and insertion sequences.Why did this article only consider plasmids?14.Line 549-550: pay attention to uppercase or lowercase letters of the titles in the references.Many similar errors occur in the references.Please check carefully in context.15.Line 810: 14 different locations?16.Line 846: plasmids and chromosomes.17.Line 860: activated sludge or wastewater.18.Line 864: change GTDB-tk to GTDB-Tk.

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1 Manuscript Spectrum02918-23 Dear Dr. Adriana Rosato, We appreciate the reviewers' comments and yours for the essential remarks and suggestions to improve our manuscript.
We have considered all the comments in the revised version of the manuscript and indicated the changes in the point-by-point reply.In short, the introduction to the manuscript has been updated with comprehensive literature on antimicrobial studies.We have also identified the shortcomings and suggested future research in the field.We also supplemented the introduction with studies using similar approaches to ours in other environmental compartments, such as fermented food, which were added to indicate the advancement of genome-centric analysis in antibiotic resistance genes studies.
We hope the point-by-point replies are clear and address all reviewer's comments, and we are looking forward to your reply.

Point-by-point reply to the reviewers' comments:
Reply to comments by Reviewer #1: Reviewer #1 (Comments for the Author): Intro: This study determined the presence of ARGs in metagenome-assembled genomes (MAGs) recovered from AS and WW treatment plants and evaluated the risks of MAGs-carrying ARGs in potential human pathogens.This study also analyzed whether ARGs and virulence factor genes (VFGs) in MAG are present on chromosomes or plasmids.This manuscript is original and generally well written.This article has advanced experimental methods and rigorous writing.

General comments by the reviewer:
The main issue is the "INTRODUCTION", which requires a detailed review of previous research progress and existing shortcomings.

Reply to general comments:
We thank the reviewer for the kind comments and understanding of the issues in the introduction, which lacks a detailed review of the previous research and challenges in antimicrobial research.
We added a detailed review of the literature and identified the challenges in the AMR research.
The previous research progress added in the introduction of the revised version of the manuscript is as follows: 1. WWTPs are considered an environmental hotspot for disseminating ARGs due to the presence of mobile genetic elements and diverse ARBs.The high abundance of bacterial communities in the WW facilitates the direct route for the distribution of ARGs in the environment.This addition of previous research progress appeared in the revised version of the manuscript in lines 66 -67.Other specific comments are as follows: Comment 1: Line 2 and 58: please make sure to use "antimicrobial resistance genes" instead of "antibiotic resistance genes".
Reply 1: We thank the reviewer for the comment.We use the term "antimicrobial resistance genes as suggested.The revised version of the manuscript is indicated changes in lines 2 and 58.
Reply 2: We thank the reviewer for the comment.We use the less than sign (<10%) to indicate the level of contamination in the recovered metagenome-assembled genomes (MAGs).In the updated version of the manuscript, the changes were reflected in line 24.
Comment 3: Line 40: the names of genes need to be uniformly italicized in context, such as aph(3')-I.Please carefully check for other genes.
Reply 3: We thank the reviewer for the comment.We checked and italicized the names of resistance genes in places where they were not italicized before throughout the manuscript.In the revised version of the manuscript, such changes can be seen in lines 40, 240-241 and 256.

Comment 4:
Line 82: such research methods have also been done in previous studies, for example in the field of fermented foods (such as sufu) in 2021.It is appropriate to supplement this literature here.
Reply 4: Thank you for the information regarding the relevance of supplementing our manuscript with previous studies conducted in fermented foods, such as sufu, using genome-centric analysis.
We appreciate your input, and we addressed this comment.We agree that supplementing relevant studies in the introduction can provide a broader context for our research.This literature would help establish a more comprehensive understanding of the complexity of antimicrobial research and show the continuity of antimicrobial studies.We carefully read the manuscript and supplement the literature in the introduction in lines 128 -134.We apologize for any confusion in the figure's description and appreciate your diligence in pointing out this discrepancy.We have also thoroughly reviewed the data and ensure the accuracy of this classification at the genus level.We apologize for any confusion this may have caused.We have now rectified this issue, and the revised Table 2 accurately reflects 4,182 as the number of MAGs with unknown classification at the genus level.As expected, the total number of MAGs with known and unknown taxonomic classification are 5,916 at the genus.We sincerely appreciate your valuable input, as it has significantly improved the quality and accuracy of our manuscript.Reply 11: We have changed the lowercase letters after the numerical value to uppercase in all the places it appeared in the manuscript.In the revised version of the manuscript, such changes can be seen in lines 227, 238, 243, 246, 249, 263, 265, 269, 274, 297, 302-305 and 413.Comment 12: Line 296: 94%?
Reply 12: We have carefully checked the data and noticed that the percentage of MAGs with unknown phylogeny is 94%, not 92%.The text has been corrected in the revised version of the manuscript in lines 334 and 342.
Comment 13: Line 450: there are many mobile genetic elements (MGEs), such as plasmids, bacteriophages, transposons, and insertion sequences.Why did this article only consider plasmids?
2. Previous studies showed that WWTPs are the most important sources of resistance genes due to the secretion of antibiotic residues from human waste, veterinary and hospitals.Therefore, surveillance of ARGs is necessary in WWTP as part of the effort to diminish the emergence and distribution of resistance in the ecological environment and the possibility of detecting new bacterial resistance mechanisms.It is also essential to understand the various mechanisms by which bacterial species develop resistance to antibiotics for the establishment of policies to fight resistance.This addition of previous research progress appeared in the revised version of the manuscript in lines 71 -76. 3. Additionally, previous studies reported the occurrence of ARGs in the treated WW and their distribution into the receiving environment, which further revealed that continuous discharge of poorly treated WW could enable the transfer of resistance genes to pathogenic bacteria and spread ARBs in the environments.The existence of ARGs and ARBs in the activated sludge, effluents and influents of WWTPs from various countries shows the global distribution of resistance genes in the environment.This addition of previous research progress appeared in the revised version of the manuscript in lines 77 -81. 4. Recently, Nava and co-workers revealed that multi-resistance bacteria are present in WW and distributed into the environment through effluent discharge, which may lead to the development of "Superbug" species.The existence of multi-resistance species in WW is supported by the co-association of antibiotics, heavy metals, ARBs, and ARGs in WWTPs.Therefore, an urgent need to monitor heavy metal resistance in the WWTP and design adequate strategies for assessing the risks of ARGs and heavy metal resistance in ecological settings is necessary.This addition of previous research progress appeared in the revised version of the manuscript in lines 82 -87. 5. Studies on metagenomics analysis unveiled the presence of important clinical ARG classes in activated sludge, including penicillin, tetracycline, sulfonamides and others that remain in treated wastewater.A study by Cacace showed the abundance of ARGs in treated WW and receiving bodies from WWTP effluent in ten euro European countries.The study showed the presence of ARGs in all effluents and river water samples, demonstrating a complex method of acquiring ARGs in different bacterial communities.Recently, Talat and co-workers provided a comprehensive overview of ARGs in hospital wastewater using metagenomics.The study uncovered many important clinical resistance gene classes, including beta-lactam, aminoglycosides, macrolide carbapenem and sulfonamides, which were hosted by human pathogens, such as Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, evinced the peril associated with ARGs transmission in the environments.This addition of previous research progress appeared in the revised version of the manuscript in lines 106 -116.6.Recently, genome-centric approaches have been used to determine the distribution patterns of resistance genes in urban, fermented foods and sewage to manage ARGs as a public health problem.A previous study by Tan and colleagues identified the distribution patterns of ARGs in sulfur bacterial communities.Further, it revealed that ARGs were enriched in opportunistic pathogens.The study revealed the presence of the most critical clinical drug classes used to treat diseases.It showed that MAGs uncovered significant pathways of human resistance gene consumption due to the uptake of ready-to-eat food.The study by Junya Zhang revealed the profile of ARGs in combined sewage overflows and recovered MAGs conferring resistance to many ARGs, including bacA, acrA, rsmA and mexK OmpA, among others.This addition of previous research progress appeared in the revised version of the manuscript in lines 128 -136.The existing shortcomings that were added to the revised version of the manuscript are: a) One of the challenges of AMR research is the lack of a standard method for quantification and surveillance of ARG acquisition, despite the richness of ARGs in the WW effluents and transmission of genes from pathogens to commensal species in the environment facilitated by mobile genetic elements (MGEs) MGE via horizontal gene transfer (HGT) or vertical gene transfer (VGT).This addition of the existing shortcomings in antimicrobial research appeared in the revised version of the manuscript in lines 88 -91.b) The studies of Nava et al., and Larsson and Flach, further revealed no standard method for removing ARG in the environment, including WWTPs.Therefore, strategies involving biotic and microbial remediation are needed to mitigate the evolutionary selection of ARGs.To tackle the menace of ARGs in the environment, public awareness about the reasonable usage and pernicious upshot of antibiotic misuse and abuse should be implemented to reduce antibiotic dissipation.This addition of the existing shortcomings in antimicrobial research appeared in the revised version of the manuscript in lines 91 -96.
Thank you for your attention to detail and comment regarding Figure1.We appreciate your thorough review of our work.You are correct; Figure1indeed depicts 13 positions, not 14.
We have changed Figure 1 to Fig. 1.In the revised version of the manuscript, such changes can be seen in line 216.Comment 11: Line 197: change Fig. 3b to Fig. 3B.The lowercase letters after the numerical value need to be changed to uppercase letters.Please note that the entire text should be expressed in this way.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred