Mobilizable antibiotic resistance genes are present in dust microbial communities


Autoři: Sarah Ben Maamar aff001;  Adam J. Glawe aff001;  Taylor K. Brown aff001;  Nancy Hellgeth aff001;  Jinglin Hu aff001;  Ji-Ping Wang aff002;  Curtis Huttenhower aff003;  Erica M. Hartmann aff001
Působiště autorů: Department of Civil and Environmental Engineering, Northwestern University, Evanston, Illinois, United States of America aff001;  Department of Statistics, Northwestern University, Evanston, Illinois, United States of America aff002;  Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, United States of America aff003
Vyšlo v časopise: Mobilizable antibiotic resistance genes are present in dust microbial communities. PLoS Pathog 16(1): e32767. doi:10.1371/journal.ppat.1008211
Kategorie: Research Article
doi: 10.1371/journal.ppat.1008211

Souhrn

The decades-long global trend of urbanization has led to a population that spends increasing amounts of time indoors. Exposure to microbes in buildings, and specifically in dust, is thus also increasing, and has been linked to various health outcomes and to antibiotic resistance genes (ARGs). These are most efficiently screened using DNA sequencing, but this method does not determine which microbes are viable, nor does it reveal whether their ARGs can actually disseminate to other microbes. We have thus performed the first study to: 1) examine the potential for ARG dissemination in indoor dust microbial communities, and 2) validate the presence of detected mobile ARGs in viable dust bacteria. Specifically, we integrated 166 dust metagenomes from 43 different buildings. Sequences were assembled, annotated, and screened for potential integrons, transposons, plasmids, and associated ARGs. The same dust samples were further investigated using cultivation and isolate genome and plasmid sequencing. Potential ARGs were detected in dust isolate genomes, and we confirmed their placement on mobile genetic elements using long-read sequencing. We found 183 ARGs, of which 52 were potentially mobile (associated with a putative plasmid, transposon or integron). One dust isolate related to Staphylococcus equorum proved to contain a plasmid carrying an ARG that was detected metagenomically and confirmed through whole genome and plasmid sequencing. This study thus highlights the power of combining cultivation with metagenomics to assess the risk of potentially mobile ARGs for public health.

Klíčová slova:

Antibiotic resistance – Antibiotics – Antimicrobial resistance – Metagenomics – Mobile genetic elements – Staphylococcus – Tetracyclines – Water resources


Zdroje

1. WHO. Antimicrobial resistance: Global Health Report on Surveillance [Internet]. Bulletin of the World Health Organization. 2014. doi: 10.1007/s13312-014-0374-3

2. Hu Y, Yang X, Li J, Lv N, Liu F, Wu J, et al. The bacterial mobile resistome transfer network connecting the animal and human microbiome. Appl Environ Microbiol. 2016;82: 6672–6681. doi: 10.1128/AEM.01802-16 27613679

3. Karkman A, Do TT, Walsh F, Virta MPJ. Antibiotic-Resistance Genes in Waste Water. Trends Microbiol. Elsevier Ltd; 2018;26: 220–228. doi: 10.1016/j.tim.2017.09.005 29033338

4. Ma L, Li B, Jiang X-T, Wang Y-L, Xia Y, Li A-D, et al. Catalogue of antibiotic resistome and host-tracking in drinking water deciphered by a large scale survey. Microbiome. Microbiome; 2017;5: 154. doi: 10.1186/s40168-017-0369-0 29179769

5. Marston HD, Dixon DM, Knisely JM, Palmore TN, Fauci AS. Antimicrobial resistance (AMR). J Am Med Assoc. 2016;316: 1193–1204. doi: 10.1136/ejhpharm-2018-001820

6. de Kraker MEA, Stewardson AJ, Harbarth S. Will 10 Million People Die a Year due to Antimicrobial Resistance by 2050? PLOS Med. 2016;13: 1–6. doi: 10.1371/journal.pmed.1002184 27898664

7. Fitzpatrick D, Walsh F. Antibiotic resistance genes across a wide variety of metagenomes. FEMS Microbiol Ecol. 2016;92: 1–8. doi: 10.1093/femsec/fiv168 26738556

8. Hartmann EM, Hickey R, Hsu T, Betancourt Román CM, Chen J, Schwager R, et al. Antimicrobial Chemicals Are Associated with Elevated Antibiotic Resistance Genes in the Indoor Dust Microbiome. Environ Sci Technol. 2016;50: 9807–9815. doi: 10.1021/acs.est.6b00262 27599587

9. Fahimipour AK, Ben Maamar S, McFarland AG, Blaustein RA, Chen J, Glawe AJ, et al. Antimicrobial Chemicals Associate with Microbial Function and Antibiotic Resistance Indoors. mSystems. 2018;3: 1–13. doi: 10.1128/mSystems.00200-18 30574558

10. European Centre for Disease Prevention and Control. The bacterial challenge: time to react. 2009.

11. Uyaguari-Díaz MI, Croxen MA, Luo Z, Cronin KI, Chan M, Baticados WN, et al. Human activity determines the presence of integron-associated and antibiotic resistance genes in Southwestern British Columbia. Front Microbiol. 2018;9: 1–20. doi: 10.3389/fmicb.2018.00001

12. Zhu Y-G, Zhao Y, Li B, Huang C-L, Zhang S-Y, Yu S, et al. Continental-scale pollution of estuaries with antibiotic resistance genes. Nat Microbiol. Nature Publishing Group; 2017;2: 1–7. doi: 10.1038/nmicrobiol.2016.270 28134918

13. Gibson MK, Wang B, Ahmadi S, Burnham C-AD, Tarr PI, Warner BB, et al. Developmental dynamics of the preterm infant gut microbiota and antibiotic resistome. Nat Microbiol. Nature Publishing Group; 2016;1: 1–10. doi: 10.1038/nmicrobiol.2016.24 27572443

14. Su J-Q, An X-L, Li B, Chen Q-L, Gillings MR, Chen H, et al. Metagenomics of urban sewage identifies an extensively shared antibiotic resistome in China. Microbiome. Microbiome; 2017;5: 84. doi: 10.1186/s40168-017-0298-y 28724443

15. Finley RL, Collignon P, Larsson DGJ, Mcewen SA, Li XZ, Gaze WH, et al. The scourge of antibiotic resistance: The important role of the environment. Clin Infect Dis. 2013;57: 704–710. doi: 10.1093/cid/cit355 23723195

16. Gat D, Mazar Y, Cytryn E, Rudich Y. Origin-Dependent Variations in the Atmospheric Microbiome Community in Eastern Mediterranean Dust Storms. Environ Sci Technol. 2017;51: 6709–6718. doi: 10.1021/acs.est.7b00362 28422476

17. Rosas I, Salinas E, Martínez L, Calva E, Cravioto A, Eslava C, et al. Urban dust fecal pollution in Mexico City: Antibiotic resistance and virulence factors of Escherichia coli. Int J Hyg Environ Health. 2006;209: 461–470. doi: 10.1016/j.ijheh.2006.03.007 16762593

18. Ludden C, Cormican M, Austin B, Morris D. Rapid environmental contamination of a new nursing home with antimicrobial-resistant organisms preceding occupation by residents. J Hosp Infect. Elsevier Ltd; 2013;83: 327–329. doi: 10.1016/j.jhin.2012.11.023 23369466

19. Gandara A, Mota LC, Flores C, Perez HR, Green CF, Gibbs SG. Isolation of Staphylococcus aureus and antibiotic-resistant Staphylococcus aureus from residential indoor bioaerosols. Environ Health Perspect. 2006;114: 1859–1864. doi: 10.1289/ehp.9585 17185276

20. Klepeis NE, Nelson WC, Ott WR, Robinson JP, Tsang AM, Switzer P, et al. The National Human Activity Pattern Survey (NHAPS): A resource for assessing exposure to environmental pollutants. J Expo Anal Environ Epidemiol. 2001;11: 231–252. doi: 10.1038/sj.jea.7500165 11477521

21. Chen CH, Lin YL, Chen KH, Chen WP, Chen ZF, Kuo HY, et al. Bacterial diversity among four healthcare-associated institutes in Taiwan. Sci Rep. Springer US; 2017;7: 1–11. doi: 10.1038/s41598-017-08679-3 28811583

22. Yano R, Shimoda T, Watanabe R, Kuroki Y, Okubo T, Nakamura S, et al. Diversity changes of microbial communities into hospital surface environments. J Infect Chemother. Elsevier Ltd; 2017;23: 439–445. doi: 10.1016/j.jiac.2017.03.016 28431935

23. Poza M, Gayoso C, Gómez MJ, Rumbo-Feal S, Tomás M, Aranda J, et al. Exploring Bacterial Diversity in Hospital Environments by GS-FLX Titanium Pyrosequencing. PLoS One. 2012;7: 1–10. doi: 10.1371/journal.pone.0044105 22952889

24. ElRakaiby MT, Gamal-Eldin S, Amin MA, Aziz RK. Hospital Microbiome Variations As Analyzed by High-Throughput Sequencing. 2019;23(9):426–38.

25. Hsu T, Joice R, Vallarino J, Abu-Ali G, Hartmann EM, Shafquat A, et al. Urban Transit System Microbial Communities Differ by Surface Type and Interaction with Humans and the Environment. mSystems. 2016;1: 1–18. doi: 10.1128/mSystems.00018-16 27822528

26. Be NA, Avila-Herrera A, Allen JE, Singh N, Checinska Sielaff A, Jaing C, et al. Whole metagenome profiles of particulates collected from the International Space Station. Microbiome. Microbiome; 2017;5: 1–19. doi: 10.1186/s40168-016-0209-7

27. Blaustein RA, Mcfarland AG, Ben Maamar S, Lopez A, Castro-Wallace S, Hartmann EM. Pangenomic Approach To Understanding Microbial Adaptations within a Model Built Environment, the International Space Station, Relative to Human Hosts and Soil. mSystems. 2019;4: 1–16. doi: 10.1128/mSystems.00281-18 30637341

28. Roberts JA, Paul SK, Akova M, Bassetti M, De Waele JJ, Dimopoulos G, et al. DALI°: De fi ning Antibiotic Levels in Intensive Care Unit Patients°: Are Current β -Lactam Antibiotic Doses Sufficient for Critically Ill Patients? Clin Infect Dis. 2014;58: 1072–1083. doi: 10.1093/cid/ciu027 24429437

29. Shapiro DJ, Hicks LA, Pavia AT, Hersh AL. Antibiotic prescribing for adults in ambulatory care in the USA, 2007–09. J Antimicrob Chemother. 2014;69: 234–240. doi: 10.1093/jac/dkt301 23887867

30. Karanika S, Paudel S, Grigoras C, Kalbasi A, Mylonakis E. Systematic Review and Meta-analysis of Clinical and Economic Outcomes from the Implementation of Hospital-Based Antimicrobial. Antimicrob Agents Chemother. 2016;60: 4840–4852. doi: 10.1128/AAC.00825-16 27246783

31. Wong D, Nielsen TB, Bonomo RA, Pantapalangkoor P, Luna B, Spellberg B. Clinical and Pathophysiological Overview of Acinetobacter Infections°: a Century of Challenges. Clin Microbiol Rev. 2017;30: 409–447. doi: 10.1128/CMR.00058-16 27974412

32. Arciola CR, Campoccia D, Montanaro L. Implant infections: adhesion, biofilm formation and immune evasion. Nat Rev Microbiol. Springer US; 2018;16: 387–409. doi: 10.1038/s41579-018-0019-y 29720707

33. Pulcini C, Binda F, Lamkang AS, Trett A, Charani E, Goff DA, et al. Developing core elements and checklist items for global hospital antimicrobial stewardship programmes°: a consensus approach. Clin Microbiol Infect. Elsevier Ltd; 2019;25: 20–25. doi: 10.1016/j.cmi.2018.03.033 29625170

34. Mahnert A, Moissl-Eichinger C, Zojer M, Bogumil D, Mizrahi I, Rattei T, et al. Man-made microbial resistances in built environments. Nat Commun [Internet]. 2019;10(968):1–12. Available from: http://dx.doi.org/10.1038/s41467-019-08864-0

35. Gupta S, Arango-Argoty G, Zhang L, Pruden A, Vikesland P. Identification of discriminatory antibiotic resistance genes among environmental resistomes using extremely randomized tree algorithm. Microbiome. 2019;7(123):1–15.

36. Martinez JL, Fajardo A, Garmendia L, Hernandez A, Linares JF, Martínez-Solano L, et al. A global view of antibiotic resistance. FEMS Microbiol Rev. 2009;33: 44–65. doi: 10.1111/j.1574-6976.2008.00142.x 19054120

37. Martínez JL, Coque TM, Baquero F. What is a resistance gene? Ranking risk in resistomes. Nat Rev Microbiol. 2015;13: 116–123. doi: 10.1038/nrmicro3399 25534811

38. Klümper U, Riber L, Dechesne A, Sannazzarro A, Hansen LH, Sørensen SJ, et al. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community. ISME J. 2015;9: 934–945. doi: 10.1038/ismej.2014.191 25333461

39. Beaulaurier J, Zhu S, Deikus G, Mogno I, Zhang XS, Davis-Richardson A, et al. Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation. Nat Biotechnol. 2018;36: 61–69. doi: 10.1038/nbt.4037 29227468

40. Bengtsson-Palme J, Larsson DGJ, Kristiansson E. Using metagenomics to investigate human and environmental resistomes. J Antimicrob Chemother. 2017;72: 2690–2703. doi: 10.1093/jac/dkx199 28673041

41. Kaminski J, Gibson MK, Franzosa EA, Segata N, Dantas G, Huttenhower C. High-Specificity Targeted Functional Profiling in Microbial Communities with ShortBRED. PLoS Comput Biol. 2015;11: 1–22. doi: 10.1371/journal.pcbi.1004557 26682918

42. Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, et al. CARD 2017: Expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 2017;45: D566–D573. doi: 10.1093/nar/gkw1004 27789705

43. Franzosa EA, McIver LJ, Rahnavard G, Thompson LR, Schirmer M, Weingart G, Lipson KS, Knight R, Caporaso JG, Segata N, Huttenhower C. Species-level functional profiling of metagenomes and metatranscriptomes. Nat Methods. 2018;15: 962–968. doi: 10.1038/s41592-018-0176-y 30377376

44. Suzek BE, Huang H, McGarvey P, Mazumder R, Wu CH. UniRef: comprehensive and non-redundant UniProt reference clusters. Bioinformatics. 2007;23: 1282–1288. doi: 10.1093/bioinformatics/btm098 17379688

45. Arkin AP, Cottingham RW, Henry CS, Harris NL, Stevens RL, Maslov S, et al. KBase: The United States Department of Energy Systems Biology Knowledgebase. Nature Biotechnology. 2018;36: 566. doi: 10.1038/nbt.4163 29979655

46. Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, et al. RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep. 2015;5: 1–6. doi: 10.1038/srep08365 25666585

47. Niu B, Zhu Z, Fu L, Wu S, Li W. FR-HIT, a very fast program to recruit metagenomic reads to homologous reference genomes. Bioinformatics. 2011;27: 1704–1705. doi: 10.1093/bioinformatics/btr252 21505035

48. Quinlan AR, Hall IM. BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26: 841–842. doi: 10.1093/bioinformatics/btq033 20110278

49. Wang Y, Ghaffari N, Johnson CD, Braga-Neto UM, Wang H, Chen R, et al. Evaluation of the coverage and depth of transcriptome by RNA-Seq in chickens. BMC Bioinformatics. BioMed Central Ltd; 2011;12: 1–7. doi: 10.1186/1471-2105-12-1

50. Rodriguez-R LM, Konstantinidis KT. Nonpareil: A redundancy-based approach to assess the level of coverage in metagenomic datasets. Bioinformatics. 2014;30: 629–635. doi: 10.1093/bioinformatics/btt584 24123672

51. Deng M, Jiang R, Sun F, Zhang X. Research in Computational Molecular Biology. 17th Annual International Conference, RECOMB 2013 Beijing, China, April 2013 Proceedings. 2013. pp. 1–345.

52. Arkin AP, Cottingham RW, Henry CS, Harris NL, Stevens RL, Maslov S, et al. KBase: The United States Department of Energy Systems Biology Knowledgebase. Nat Biotechnol. 2018;36: 566–569. doi: 10.1038/nbt.4163 29979655

53. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k -mer weighting and repeat separation. Genome Res. 2017;27: 722–736. doi: 10.1101/gr.215087.116 28298431

54. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement. PLoS One. 2014;9: 1–14. doi: 10.1371/journal.pone.0112963 25409509

55. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 9.0, 2019. http://www.eucast.org. 2019. Available from: http://www.eucast.org.

56. Nishino K, Nikaido E, Yamaguchi A. Regulation and physiological function of multidrug efflux pumps in Escherichia coli and Salmonella. Biochim Biophys Acta—Proteins Proteomics. Elsevier B.V.; 2009;1794: 834–843. doi: 10.1016/j.bbapap.2009.02.002 19230852

57. Huffman JL, Brennan RG. Prokaryotic transcription regulators: more than just the helix-turn-helix motif. Curr Opin Struct Biol. 2002;12: 98–106. Available: https://ac.els-cdn.com/S0959440X02002956/1-s2.0-S0959440X02002956-main.pdf?_tid=195744fc-3286-4878-993a-3e3a1fc78f87&acdnat=1524963742_98c8e58e00e437f3a7b1d0b365cc79e6 doi: 10.1016/s0959-440x(02)00295-6 11839496

58. Mitchell BA, Brown MH, Skurray RA. QacA multidrug efflux pump from Staphylococcus aureus: Comparative analysis of resistance to diamidines, biguanidines, and guanylhydrazones. Antimicrob Agents Chemother. 1998;42: 475–477. 9527814

59. Rice LB. Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE. J Infect Dis. 2008;197:1079–81. doi: 10.1086/533452 18419525

60. O’Halloran T, Walsh C. Metalloregulatory DNA-Binding Protein Encoded by the merR Gene: Isolation and Characterization. Science (80-). 1987;235: 211–214.

61. Brown NL, Stoyanov J V, Kidd SP, Hobman JL. The MerR family of transcriptional regulators. FEMS Microbiol Rev. 2003;27: 145–63. doi: 10.1016/S0168-6445(03)00051-2 12829265

62. Anton A, Grosse C, Reissmann J, Pribyl T, Nies DH. CzcD is a heavy metal ion transporter involved in regulation of heavy metal resistance in Ralstonia sp. strain CH34. J Bacteriol. 1999;181: 6876–6881. 10559151

63. Fu HL, Meng Y, Ordóñez E, Villadangos AF, Bhattacharjee H, Gil JA, et al. Properties of arsenite efflux permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum. J Biol Chem. 2009;284: 19887–19895. doi: 10.1074/jbc.M109.011882 19494117

64. Ordóñez E, Letek M, Valbuena N, Mateos LM. Analysis of Genes Involved in Arsenic Resistance in Corynebacterium glutamicum ATCC 13032. Appl Environ Microbiol. 2005;71: 6206–6215. doi: 10.1128/AEM.71.10.6206-6215.2005 16204540

65. Lou Y, Liu H, Zhang Z, Pan Y, Zhao Y. Mismatch between antimicrobial resistance phenotype and genotype of pathogenic Vibrio parahaemolyticus isolated from seafood. Food Control. Elsevier Ltd; 2015;59: 207–211. doi: 10.1016/j.foodcont.2015.04.039

66. Hughes D, Andersson DI. Environmental and genetic modulation of the phenotypic expression of antibiotic resistance. FEMS Microbiol Rev. 2017;41: 374–391. doi: 10.1093/femsre/fux004 28333270

67. Sommer MOA, Dantas G, Church GM. Functional characterization of the antibiotic resistance reservoir in the human microflora. Science (80-). 2009;325: 1128–1132. doi: 10.1126/science.1176950 19713526

68. Chambers HF, DeLeo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol. Nature Publishing Group; 2009;7: 629–641. doi: 10.1038/nrmicro2200 19680247

69. Roberts MC, Soge OO, No D. Comparison of multi-drug resistant environmental methicillin-resistant Staphylococcus aureus isolated from recreational beaches and high touch surfaces in built environments. Front Microbiol. 2013;4: 1–8. doi: 10.3389/fmicb.2013.00001

70. Coughenour C, Stevens V, Stetzenbach LD. An Evaluation of Methicillin-Resistant Staphylococcus aureus Survival on Five Environmental Surfaces. Microb Drug Resist. 2011;17: 457–461. doi: 10.1089/mdr.2011.0007 21612512

71. Hu J, Ben Maamar S, Glawe AJ, Gottel N, Gilbert JA, Hartmann EM. Impacts of indoor surface finishes on bacterial viability. Indoor Air. 2019;29(January 3rd):551–62. doi: 10.1111/ina.12558 30980566

72. Conlan S, Mijares LA, Becker J, Blakesley RW, Bouffard GG, Brooks S, et al. Staphylococcus epidermidis pan-genome sequence analysis reveals diversity of skin commensal and hospital infection-associated isolates. Genome Biol. 2012;13: 1–13. doi: 10.1186/gb-2012-13-7-r64 22830599

73. Byrd AL, Deming C, Cassidy SKB, Harrison OJ, Ng W-I, Conlan S, et al. Staphylococcus aureus and Staphylococcus epidermidis strain diversity underlying pediatric atopic dermatitis. Sci Transl Med. 2017;9: 1–12. doi: 10.1126/scitranslmed.aal4651 28679656

74. Aziz RK, Breitbart M, Edwards RA. Transposases are the most abundant, most ubiquitous genes in nature. Nucleic Acids Res. 2010;38: 4207–4217. doi: 10.1093/nar/gkq140 20215432

75. Zhang AN, Li LG, Ma L, Gillings MR, Tiedje JM, Zhang T. Conserved phylogenetic distribution and limited antibiotic resistance of class 1 integrons revealed by assessing the bacterial genome and plasmid collection. Microbiome. Microbiome; 2018;6: 1–14. doi: 10.1186/s40168-017-0383-2

76. O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, et al. Reference sequence (RefSeq) database at NCBI: Current status, taxonomic expansion, and functional annotation.

77. Su F, Tian R, Yang Y, Xu X, Chen D, Zhang J, et al. Pan-genome analysis reveals the molecular basis of niche adaptation of Staphylococcus epidermidis strains. bioRxiv. 2019; 1–17. doi: 10.1101/604629

78. Shintani M, Sanchez ZK, Kimbara K. Genomics of microbial plasmids: Classification and identification based on replication and transfer systems and host taxonomy. Front Microbiol. 2015;6: 1–16. doi: 10.3389/fmicb.2015.00001


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