Intensive care unit sinks are persistently colonized with multidrug resistant bacteria and mobilizable, resistance-conferring plasmids

ABSTRACT Contamination of hospital sinks with microbial pathogens presents a serious potential threat to patients, but our understanding of sink colonization dynamics is largely based on infection outbreaks. Here, we investigate the colonization patterns of multidrug-resistant organisms (MDROs) in intensive care unit sinks and water from two hospitals in the USA and Pakistan collected over 27 months of prospective sampling. Using culture-based methods, we recovered 822 bacterial isolates representing 104 unique species and genomospecies. Genomic analyses revealed long-term colonization by Pseudomonas spp. and Serratia marcescens strains across multiple rooms. Nanopore sequencing uncovered examples of long-term persistence of resistance-conferring plasmids in unrelated hosts. These data indicate that antibiotic resistance (AR) in Pseudomonas spp. is maintained both by strain colonization and horizontal gene transfer (HGT), while HGT maintains AR within Acinetobacter spp. and Enterobacterales, independent of colonization. These results emphasize the importance of proactive, genomic-focused surveillance of built environments to mitigate MDRO spread. IMPORTANCE Hospital sinks are frequently linked to outbreaks of antibiotic-resistant bacteria. Here, we used whole-genome sequencing to track the long-term colonization patterns in intensive care unit (ICU) sinks and water from two hospitals in the USA and Pakistan collected over 27 months of prospective sampling. We analyzed 822 bacterial genomes, representing over 100 different species. We identified long-term contamination by opportunistic pathogens, as well as transient appearance of other common pathogens. We found that bacteria recovered from the ICU had more antibiotic resistance genes (ARGs) in their genomes compared to matched community spaces. We also found that many of these ARGs are harbored on mobilizable plasmids, which were found shared in the genomes of unrelated bacteria. Overall, this study provides an in-depth view of contamination patterns for common nosocomial pathogens and identifies specific targets for surveillance.

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Line 87: Numbers 1-10 should be spelled out, so 2 should be two.Line 317: I suggest changing "particularly in taxa like S. marcescens, P. aeruginosa, and C. freundii" to "particularly in S. marcescens, P. aeruginosa, and C. freundii taxa" to avoid the phrase "taxa like."Line 324: "as well as" to and Line 336: "or [they] were present" Line 341: Drop the 's' from infections or change the 'is' to 'are' Line 359: Drop the 's' from contains Line 369: 370: "could illuminate HGT [events?] in non-Enterobacterales isolates.
Line 403: If a sentence starts with a number, it needs to be spelled out.Supplementary figure 7: "strains the were recovered" needs to be fixed.

Reviewer #2 (Comments for the Author):
The authors present a detailed analysis of a culture-based prospective study of sink drain colonisation patterns from intensive care units, private homes and office buildings in the United States and Pakistan.The analysis presented is of high quality and will be of broad interest to journal's readership.I commend the author's application of suitable state of the art methods and especially their presentation of high quality figures which greatly enhance the manuscript.While it is unfortunate that metagenomics was not employed in this analysis, this is nevertheless an interesting and high quality piece of work.
I wish to draw attention to two concerns: Firstly, the authors state that the sequence data is publicly available, however the accession PRJNA868296 contains no publicly available sequences.Submitting papers for review containing false statements about data availability is unacceptable, and I cannot recommend acceptance of this article until the both assemblies and sequence data are deposited and available as has been claimed.Secondly (minor): in light of the relatively small body of existing work about hospital sink drain colonisation with MDROs, perhaps more of the existing works with similar conclusions to this manuscript could have been cited.

Typos: Line 448: "refereces"
This manuscript characterizes MDROs collected from sink surfaces and water in hospital ICUs found in Pakistan and the United States.These samples were compared to those collected from sinks found in homes and workplaces located in the local community.Individual bacterial strains were isolated and assessed for antibiotic resistance profiles, including the identification of plasmids that are likely to have been shared between taxa during horizontal gene transfer events.The authors recovered more isolates from sinks in Pakistan than from the US, at a disparity that was not observed between community sites, and ARG abundance and diversity was greater on hospital sink surfaces than in home and work sites.Additionally, they were able to identify strains that were persistent in the environment over the two-year period, and they presented evidence that horizontal gene transfer events are likely to play an important role in the maintenance of ARGs in these environments over time.
Overall, I believe this to be a well-written, well-planned, and interesting manuscript.The comparison between hospitals in different countries is a unique and meaningful contribution to the literature.The lab-based methods are rigorous (e.g., the recovery of >800 isolates and the use of multiple agar types with appropriate controls), and bioinformatic methods are both sound and thoroughly considered.The conclusions are supported by their findings, and the manuscript, more generally, is presented in an organized and logical format.Figures and tables are a good addition to the text -I particularly appreciate that color palettes are color-blind friendly -and the length is appropriate for the results presented.
I have no major comments for the authors, with some minor comments below.
Minor comments: 'antibiotic resistance genes' is not hyphenated.Should either be 'antibiotic resistance genes' or 'antibiotic-resistant organisms.'Same is true of 'built environment.'Lines 77, 79, 97, 173, etc: 'which' should be 'that' -'that' indicates an essential part of the sentence needed to convey the meaning.'which' is an add-on to the concept presented and should be proceeded by a comma.
Line 87: Numbers 1-10 should be spelled out, so 2 should be two.Supplementary figure 7: "strains the were recovered" needs to be fixed.

Reviewer #1 (Comments for the Author):
This manuscript characterizes MDROs collected from sink surfaces and water in hospital ICUs found in Pakistan and the United States.These samples were compared to those collected from sinks found in homes and workplaces located in the local community.Individual bacterial strains were isolated and assessed for antibiotic resistance profiles, including the identification of plasmids that are likely to have been shared between taxa during horizontal gene transfer events.The authors recovered more isolates from sinks in Pakistan than from the US, at a disparity that was not observed between community sites, and ARG abundance and diversity was greater on hospital sink surfaces than in home and work sites.Additionally, they were able to identify strains that were persistent in the environment over the two-year period, and they presented evidence that horizontal gene transfer events are likely to play an important role in the maintenance of ARGs in these environments over time.
Overall, I believe this to be a well-written, well-planned, and interesting manuscript.The comparison between hospitals in different countries is a unique and meaningful contribution to the literature.The lab-based methods are rigorous (e.g., the recovery of >800 isolates and the use of multiple agar types with appropriate controls), and bioinformatic methods are both sound and thoroughly considered.The conclusions are supported by their findings, and the manuscript, more generally, is presented in an organized and logical format.Figures and tables are a good addition to the text -I particularly appreciate that color palettes are color-blind friendly -and the length is appropriate for the results presented.
We thank the reviewer for their positive assessment of our work!I have no major comments for the authors, with some minor comments below.

Minor comments:
'antibiotic resistance genes' is not hyphenated.Should either be 'antibiotic resistance genes' or 'antibiotic-resistant organisms.'Same is true of 'built environment.'Fixed Lines 77, 79, 97, 173, etc: 'which' should be 'that' -'that' indicates an essential part of the sentence needed to convey the meaning.'which' is an add-on to the concept presented and should be proceeded by a comma.

Fixed
Line 87: Numbers 1-10 should be spelled out, so 2 should be two.The sampling period between the two studies was about 27 months and did not occur in exact one-month intervals, so we tried to simplify this using a common minimum round number.Rounded to the nearest whole month and added "about".The exact collection dates are provided as supplementary data.Thank you for submitting the revisions and I am pleased to tell you that your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.For your reference, ASM Journals' address is given below.Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication.They 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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Line 92: It's not clear what '2 two' means.Two paired?Line 107: A comma is needed after 'In this study' Line 108: If Pakistan is presented first in the introduction, it should be listed before the US throughout.Line 111: I would suggest removing 'We used WGS to determine that' to make the sentence flow better.Line 118: 'commonly' to 'common' Line 123: ' geography' should be switched to 'country', 'geographic location', or something similar In paragraph starting at line 159: Why the switch to >24 months, when the sampling period was 25 months.Why not say 25 months, or a minimum of 25 months.Line 225: "We also observed a pair of near-identical [plasmids]" Line 283: 'a' needs to be un-italicized.Line 296: "recovery [of]"

Fixed
Line 92: It's not clear what '2 two' means.Two paired?Fixed, that was a typo.Line 107: A comma is needed after 'In this study' Fixed Line 108: If Pakistan is presented first in the introduction, it should be listed before the US throughout.Fixed Line 111: I would suggest removing 'We used WGS to determine that' to make the sentence flow better.Done Line 118: 'commonly' to 'common' Fixed Line 123: ' geography' should be switched to 'country', 'geographic location', or something similar Done In paragraph starting at line 159: Why the switch to >24 months, when the sampling period was 25 months.Why not say 25 months, or a minimum of 25 months.
Diorio-Toth Washington University in St Louis School of Medicine Edison Family Center for Genome Sciences and Systems Biology 4515 McKinley Avenue 5th Floor, Room 5121 St. Louis, MO 63110 Re: mSystems00206-23R1 (Intensive care unit sinks are persistently colonized with multi-drug resistant bacteria and mobilizable, resistance-conferring plasmids) Dear Dr. Luke Diorio-Toth: Thank you for submitting your paper to mSystems.