Discharge of urban stormwater containing organic matter, heavy metals and sometime human feces, to the natural aquatic reservoirs without any treatment is not only an environmental problem. It can lead to prevalence of antibiotic resistant bacteria in stormwater systems and transmission of antibiotic resistance genes to the environment. We performed antibiotic resistome identification and virus detection in stormwater samples from Stockholm, using publicly available metagenomic sequencing MinION data. A MinION platform offers low-cost, precise environmental metagenomics analysis. 37 groups of antibiotic resistant bacteria (ARB), 11 resistance types with 26 resistance mechanisms - antibiotic resistance genes (ARGs) giving tolerance to the aminoglycoside, beta-lactams, fosmidomycin, MLS, multidrug and vancomycin were identified using ARGpore pipeline. The majority of the identified bacteria species were related to the natural environment such as soil and were not dangerous to human. Alarmingly, human pathogenic bacteria carrying resistance to antibiotics currently used against them (Bordetella resistant to macrolides and multidrug resistant Propionibacterium avidum) were also found in the samples. Most abundant viruses identified belonged to Caudovirales and Herpesvirales and they were not carrying ARGs. Unlike the virome, resistome and ARB were not unique for stormwater sampling points. This results underline the need for extensive monitoring of the microbial community structure in the urban stormwater systems to assess antimicrobial resistance spread.

Department of Cancer Biology Institute of Biology Warsaw University of Life Sciences Nowoursynowska 159 02 776 Warsaw Poland; Department of Physiological Sciences Faculty of Veterinary Medicine Warsaw University of Life Sciences Nowoursynowska 159 02 776 Warsaw Poland

Department of Water Technology and Environmental Engineering University of Chemistry and Technology Prague Technická 5 166 28 Prague 6 Czech Republic

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Achermann Y., Goldstein E.J., Coenye T., Shirtliff M.E. Propionibacterium acnes: from commensal to opportunistic biofilm-associated implant pathogen. Clin. Microbiol. Rev. 2014;2014(27):419–440. PubMed PMC

Achermann Y., Liu J., Zbinden R., Zingg P.O., Anagnostopoulos A., Barnard E. Propionibacterium avidum: a virulent pathogen causing hip periprosthetic joint infection. Clin. Infect. Dis. 2018;2018(66):54–63. PubMed PMC

Agga G.E., Arthur T.M., Durso L.M., Harhay D.M., Schmidt J.W. Antimicrobial-resistant bacterial populations and antimicrobial resistance genes obtained from environments impacted by livestock and municipal waste. PloS One. 2015;10 PubMed PMC

Almakki A., Jumas-Bilak E., Marchandin H., Licznar-Fajardo P. Science of the total environment antibiotic resistance in urban runoff. Sci. Total Environ. 2019;667:64–76. doi: 10.1016/j.scitotenv.2019.02.183. PubMed DOI

Alonso A., Rojo F., Martínez J.L. Environmental and clinical isolates of Pseudomonas aeruginosa show pathogenic and biodegradative properties irrespective of their origin. Environ. Microbiol. 1999;1:421–430. PubMed

Altunaiji S., Kukuruzovic R., Curtis N., Massie J. Antibiotics for whooping cough (pertussis) Cochrane Database Syst. Rev. 2007;3 doi: 10.1002/14651858.CD004404.pub3.PMID17636756. CD004404. PubMed DOI PMC

Araújo C., Torres C., Silva N., Carneiro C., Gonçalves A., Radhouani H., Correia S., da Costa P.M., Paccheco R., Zarazaga M., Ruiz-Larrea F., Poeta P., Igrejas G. Vancomycin- resistant enterococci from Portuguese wastewater treatment plants. J. Basic Microbiol. 2010;50:605–609. PubMed

Ashton P.M., Nair S., Dallman T., Rubino S., Rabsch W., Mwaigwisya S. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. 2015. 33. PubMed DOI

Baker-Austin C., Wright M.S., Stepanauskas R., McArthur J.V. Co-selection of anti- biotic and metal resistance. Trends Microbiol. 2006;14:176–182. PubMed

Brown A.C., Parish T. Dxr is essential in Mycobacterium tuberculosis and fosmidomycin resistance is due to a lack of uptake. BMC Microbiol. 2008;8:1–9. doi: 10.1186/1471-2180-8-78. PubMed DOI PMC

Buffet-Bataillon S., Tattevin P., Maillard J.-Y., Bonnaure-Mallet M., Jolivet-Gougeon A. Efflux pump induction by quaternary ammonium compounds and fluoroquinolone resistance in bacteria. Future Microbiol. 2016;11:81–92. doi: 10.2217/fmb.15.131. PubMed DOI

Cacace D., Fatta-Kassinos D., Manaia C.M., Cytryn E., Kreuzinger N., Rizzo L., Karaolia P., Schwartz T., Alexander J., Merlin C., Garelick H., Schmitt Daisy, de Vries, Carsten U Schwermer, Sureyya Meric, Can Burak Ozkal, Marie H., Pons N., Kneis D., Berendonk T.U. Water Research; 2019. Antibiotic Resistance Genes in Treated Wastewater and in the Receiving Water Bodies: A Pan-European Survey of Urban Settings. PubMed DOI

Chang W., Wickham H. 2018. Ggvis: Interactive Grammar of Graphics.

Tortoli E., Chianura L., Fabbro L., Mariottini A., Martín-Casabona N., Mazzarelli G. Infections due to the newly described species Mycobacterium parascrofulaceum. J. Clin. Microbiol. 2005;43:4286–4287. doi: 10.1128/JCM.43.8.4286-4287.2005. PubMed DOI PMC

Colombo S., Arioli S., Guglielmetti S., Lunelli F., Mora D. Virome-associated antibiotic-resistance genes in an experimental aquaculture facility. FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Ecol. 2016;ume 92(3) doi: 10.1093/femsec/fiw003. March 2016, fiw003. PubMed DOI

Eggen T., Vogelsang C. Vol. 67. Elsevier; 2015. Occurrence and fate of pharmaceuticals and personal care products in wastewater. Compr. Anal. Chem. DOI

Falkinham J.O., Hilborn E.D., Arduino M.J., Pruden A., Edwards M.A. Epidemiology and ecology of opportunistic premise plumbing pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa. Environ. Health Perspect. 2015;123:749–758. doi: 10.1289/ehp.1408692. PubMed DOI PMC

Fang Z., Tan J., Wu S., Li M., Xu C., Xie Z., Zhu H. PPR-Meta: a tool for identifying phages and plasmids from metagenomic fragments using deep learning. GigaScience. 2019;8(6):giz066. PubMed PMC

Ferreira da Silva M., Vaz-Moreira I., Gonzalez-Pajuelo M., Nunes O.C., Manaia C.M. Antimicrobial resistance patterns in Enterobacteriaceae isolated from an urban waste water treatment plant. FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Ecol. 2007;60:166e176. PubMed

Figueira V., Serra E., Manaia C.M. Differential patterns of antimicrobial resistance in population subsets of Escherichia coli isolated from waste- and surface waters. Sci. Total Environ. 2011;409:1017–1023. PubMed

Freilich S., Kreimer A., Meilijson I., Gophna U., Sharan R., Ruppin E. The large-scale organization of the bacterial network of ecological co-occurrence interactions. Nucleic Acids Res. 2010;38:3857–3868. doi: 10.1093/nar/gkq118. PubMed DOI PMC

Hahn M.W., Kasalický V., Jezbera J., Brandt U., Jezberova J., Šimek K. Limnohabitans curvus gen. nov., sp. nov., a planktonic bacterium isolated from a freshwater lake. Int. J. Syst. Evol. Microbiol. 2010;2010(60):1358–1365. doi: 10.1099/ijs.0.013292-0. PubMed DOI PMC

Hamilton K.A., Garner E., Joshi S., Ahmed W., Ashbolt N., Medema G., Pruden A. Antimicrobial resistant microorganisms and their genetic determinants in stormwater: a systematic review. Curr. Opin. Environ. Sci. Health. 2020 doi: 10.1016/j.coesh.2020.02.012. DOI

Happi C.T., Gbotosho G.O., Folarin O.A., Akinboye D.O., Yusuf B.O., Ebong O.O., Sowunmi A., Kyle D.E., Milhous W., Wirth D.T., Oduola A.M.J. Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfaxine-pyrimethamine resistance in malaria-infected patients from Nigeria. Acta Trop. 2005;95:183–193. PubMed

Hu Y., Ndegwa N., Alneberg J., Johansson S., Logue J., Huss M. Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systems. Sci. Rep. 2018;8:11970. doi: 10.1038/s41598-018-29920-7. PubMed DOI PMC

Huovinen P., Sundstrom L., Swedberg G., Skold O. Trimethoprim and sulfonamide resistance. Antimicrob. Agents Chemother. 1995;39:279–289. PubMed PMC

Huson D.H., Albrecht B., Bagci C., Bessarab I., Gorska A., Jolic D. MEGAN-LR: new algorithms allow accurate binning and easy interactive exploration of metagenomic long reads and contigs. Biol. Direct. 2018;13(1):6. PubMed PMC

Jia B., Raphenya A.R., Alcock B., Waglechner N., Guo P., Tsang K.K. Card 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 2017;45:D566–D573. PubMed PMC

Kamathewatta K., Bushell R., Young N., Stevenson M., Billman-Jacobe H., Browning G. Exploration of antibiotic resistance risks in a veterinary teaching hospital with Oxford Nanopore long read sequencing. PloS One. 2019;14 PubMed PMC

Kanehisa M., Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30. 2000. PubMed PMC

Karkman A., Pärnänen K., Larsson D.G.J. Fecal pollution can explain antibiotic resistance gene abundances in anthropogenically impacted environments. Nat. Commun. 2019;10 Article number: 80 (2019) PubMed PMC

Krishna S., Ciraj A.M., Bairy I., Shobha K.L. Sphingomonas paucimobilis urinary tract infection in a renal transplant recipient: a rare case. Int. J. Med. Publ. Health. 2011;1(No. 1):47–49. 2011.

Kwak Y.-K., Colque P., Byfors S., Giske C.G., Möllby R., Kühn I. Surveillance of antimicrobial resistance among Escherichia coli in wastewater in Stockholm during 1 year: does it reflect the resistance trends in the society? Int. J. Antimicrob. Agents. 2015;45:25–32. doi: 10.1016/j.ijantimicag.2014.09.016. PubMed DOI

Lambert P.A. Bacterial resistance to antibiotics: modified target sites. Adv. Drug Deliv. Rev. 2005;57:1471–1485. PubMed

Leinonen R., Sugawara H., Shumway M. The sequence read archive. Nucleic Acids Res. 2011;39:D19–D21. doi: 10.1093/nar/gkq1019. 2011. PubMed DOI PMC

Li B., Yang Y., Ma L., Ju F., Guo F., Tiedje J.M. Metagenomic and network analysis reveal wide distribution and co-occurrence of environmental antibiotic resistance genes. ISME J. 2015;9:2490–2502. doi: 10.1038/ismej.2015.59. PubMed DOI PMC

Loman N.J., Quick J., Simpson J.T. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat. Methods. 2015;12:733–735. doi: 10.1038/nmeth.3444. PubMed DOI

Map of Stockholm (Map Data © Google). Available at: https://www.google.se/maps/place/Sztokholm,+Szwecja/@59.3258414,17.7018721,10z/data=!3m1!4b1!4m5!3m4!1s0x465f763119640bcb:0xa80d27d3679d7766!8m2!3d59.3293235!4d18.0685808.

Marti E., Jofre J., Balcazar J.L. Prevalence of antibiotic resistance genes and bacterial community composition in a river influenced by a wastewater treatment plant. PloS One. 2013;8 PubMed PMC

Martínez J.L. Ecology and evolution of chromosomal gene transfer between environmental microorganisms and pathogens. Microbiol. Spectr. 2017;6(1):1–16. doi: 10.1128/microbiolspec.MTBP-0006-8862016. PubMed DOI PMC

Mattoo S., Cherry J.D. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin. Microbiol. Rev. 2005;18:326–382. PubMed PMC

McIlroy S.J., Kirkegaard R.H., McIlroy B., Nierychlo M., Kristensen J.M., Karst S.M., Albertsen M., Nielsen P.H. 2017. MiDAS 2.0: an ecosystem-specific taxonomy and online database for the organisms of wastewater treatment systems expanded for anaerobic digester groups. Database 2017. PubMed PMC

Meroueh S.O., Minasov G., Lee W., Shoichet B.K., Mobashery S. Structural aspects for evolution of beta-lactamases from penicillin-binding proteins. J. Am. Chem. Soc. 2003;125:9612–9618. PubMed

Morris D., Galvin S., Boyle F., Hickey P., Mulligan M., Cormican M. Enterococcus faecium of the vanA genotype in rural drinking water, effluent, and the aqueous environment. Appl. Environ. Microbiol. 2012;78:596–598. PubMed PMC

Moura A., Pereira C., Henriques I., Correia A. Novel gene cassettes and integrons in antibiotic- resistant bacteria isolated from urban wastewaters. Res. Microbiol. 2012;163:92–100. PubMed

Nesme J., Cecillon S., Delmont T.O., Monier J.M., Vogel T.M., Simonet P. Large-scale metagenomic-based study of antibiotic resistance in the environment. Curr. Biol. 2014;24(2014):1096–1100. PubMed

Nigro O.D., Jungbluth S.P., Lin H.T., Hsieh C.C., Miranda J.A., Schvarcz C.R., Rappe M.S., Steward G.F. Viruses in the oceanic basement. mBio. 2017;2017(8) e02129-16. PubMed PMC

Ondov B.D., Bergman N.H., Phillippy A.M. Interactive metagenomic visualization in a Web browser. BMC Bioinf. 2011;12(1):385. 2011 Sep. 30. PubMed PMC

Parmar K., Dafale N., Pal R., Tikariha H., Purohit H. An insight into phage diversity at environmental habitats using comparative metagenomics approach. Curr. Microbiol. 2018;2018(75):132–141. PubMed

Piddock L.J.V. Multidrug-resistance efflux pumps - not just for resistance. Nat. Rev. Microbiol. 2006;4:629–636. doi: 10.1038/nrmicro1464. PubMed DOI

Pruden A., Alvarez P.J.J., Ashbolt N., Bischel H., Alcalde R.E., Capiro N.L., Riquelme M.V. An environmental science and engineering framework for combating antimicrobial resistance an environmental science and engineering framework for combating antimicrobial resistance, (December) 2018. DOI

Roberts M.C. Antimicrobial Resistance in the Environment. 2011. Mechanisms of bacterial antibiotic resistance and lessons learned from environmental tetracycline-resistant bacteria; pp. 93–121. DOI

Rosenberg Goldstein R.E., Micallef S.A., Gibbs S.G., George A., Claye E., Sapkota A., Joseph S.W., Sapkota A.R. Detection of vancomycin-resistant enterococci (VRE) at four U.S. wastewater treatment plants that provide effluent for reuse. Sci. Total Environ. 2014;466(67):404–411. PubMed PMC

Segata N., Waldron L., Ballarini A., Narasimhan V., Jousson O., Huttenhower C. Metagenomic microbial community profiling using unique clade-specific marker genes. Nat. Methods. 2012;9:811–814. PubMed PMC

Subirats J., Sanchez-Melsio A., Borrego C.M., Balcazar J.L., Simonet P. Metagenomic analysis reveals that bacteriophages are reservoirs of antibiotic resistance genes. Int. J. Antimicrob. Agents. 2016;48(2):163–167. 2016. PubMed

Szczepanowski R., Linke B., Krahn I., Gartemann K., Gu T., Eichler W., Pu A., Schlu A. Detection of 140 clinically relevant antibiotic- resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics. Microbiology. 2009;155:2306–2319. doi: 10.1099/mic.0.028233-0. PubMed DOI

Trias J., Jarlier V., Benz R. Porins in the cell wall of mycobacteria. Science. 1992;1992(258):1479–1481. doi: 10.1126/science.1279810. PubMed DOI

Vaz-Moreira I., Nunes O., Manaia C. Diversity and antibiotic resistance patterns of Sphingomonadaceae isolates from drinking water. Appl. Environ. Microbiol. 2011;77(16):5697–5706. 2011 Published by American Society for Microbiology. PubMed PMC

Wang H., Pryor M.A., Edwards M.A., Falkinham J.O., Pruden A. Effect of GAC pre- treatment and disinfectant on microbial community structure and opportunistic pathogen occurrence. Water Res. 2013;47:5760–5772. PubMed

Wang Y., Jiang X., Liu L., Li B., Zhang T. High-resolution temporal and spatial patterns of Virome in wastewater treatment systems. Environ. Sci. Technol. 2018;2018(52):10337–10346. PubMed

Wildeman P., Brüggemann H., Scholz C.F.P., Leimbach A., Söderquist B. Propionibacterium avidum as an etiological agent of prosthetic hip joint infection. PloS One. 2016;11(6) PubMed PMC

Wood D.E., Salzberg S.L. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 2014;15:R46. doi: 10.1186/gb-2014-15-3-r46. PubMed DOI PMC

Wright G.D. Bacterial resistance to antibiotics: enzymatic degradation and modification. Adv. Drug Deliv. Rev. 2005;57:1451–1470. PubMed

Xia Y., Li A.D., Deng Y., Jiang X.T., Li L.G., Zhang T. MinION Nanopore sequencing enables correlation between resistome phenotype and genotype of coliform bacteria in municipal sewage. Front. Microbiol. 2017;8(OCT):1–13. doi: 10.3389/fmicb.2017.02105. PubMed DOI PMC

Yang Y., Li B., Zou S., Fang H.H.P., Zhang T. Fate of antibiotic resistance genes in sewage treatment plant revealed by metagenomic approach. Water Res. 2014;62:97–106. doi: 10.1016/j.watres.2014.05.019. PubMed DOI

Yeom J.R., Su Y., Kim C.G. Quantification of residual antibiotics in cow manure being spread over agricultural land and assessment of their behavioral effects on antibiotic resistant bacteria. Chemosphere. 2017;182:771–780. PubMed

Zhang X.X., Zhang T., Fang H.H.P. Antibiotic resistance genes in water environment. Appl. Microbiol. Biotechnol. 2009;82(3):397–414. doi: 10.1007/s00253-008-1829-z. PubMed DOI