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Oxytetracycline and heavy metals promote the migration of resistance genes in the intestinal microbiome by plasmid transfer

The ISME Journal volume 17, pages 2003–2013 (2023)Cite this article

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Abstract

Horizontal gene transfer (HGT) has been considered the most important pathway to introduce antibiotic resistance genes (ARGs), which seriously threatens human health and biological security. The presence of ARGs in the aquatic environment and their effect on the intestinal micro-ecosystem of aquatic animals can occur easily. To investigate the HGT potential and rule of exogenous ARGs in the intestinal flora, a visual conjugative model was developed, including the donor of dual-fluorescent bacterium and the recipient of Xenopus tropicalis intestinal microbiome. Some common pollutants of oxytetracycline (OTC) and three heavy metals (Zn, Cu and Pb) were selected as the stressor. The multi-techniques of flow cytometry (FCM), scanning electron microscopy (SEM), atomic force microscopy (AFM), single-cell Raman spectroscopy with sorting (SCRSS) and indicator analysis were used in this study. The results showed that ARG transfer could occur more easily under stressors. Moreover, the conjugation efficiency mainly depended on the viability of the intestinal bacteria. The mechanisms of OTC and heavy metal stressing conjugation included the upregulation of ompC, traJ, traG and the downregulation of korA gene. Moreover, the enzymatic activities of SOD, CAT, GSH-PX increased and the bacterial surface appearance also changed. The predominant recipient was identified as Citrobacter freundi by SCRSS, in which the abundance and quantity of ARG after conjugation were higher than those before. Therefore, since the diversity of potential recipients in the intestine are very high, the migration of invasive ARGs in the microbiome should be given more attention to prevent its potential risks to public health.

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Fig. 1: Schematic depicting experimental design and conjugation results of ARGs induced by OTC and heavy metals.
Fig. 2: Effects of OTC and heavy metals on the cell viability of donors and recipients.
Fig. 3: Effects of mRNA expression in different exposures.
Fig. 4: Effects of antibiotics and heavy metals on reactive oxygen species in mating systems of each group.
Fig. 5: Effects of antibiotics and heavy metals on the characteristics of the cell membrane surface in conjugation solution.
Fig. 6: Transconjugant sorting and ARG analysis.
Fig. 7: A model depicting the mechanisms underlying the RP4 plasmid-mediated conjugation promoted by OTC and heavy metals.

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References

  1. Sanchez-Cid C, Guironnet A, Keuschnig C, Wiest L, Vulliet E, Vogel TM. Gentamicin at sub-inhibitory concentrations selects for antibiotic resistance in the environment. ISME Commun. 2022;2:29.

    PubMed Central  Google Scholar 

  2. Zhu YG, Zhao Y, Li B, Huang CL, Zhang SY, Yu S, et al. Continental-scale pollution of estuaries with antibiotic resistance genes. Nat Microbiol. 2017;2:16270.

    CAS  PubMed  Google Scholar 

  3. Yao Y, Maddamsetti R, Weiss A, Ha Y, Wang T, Wang S, et al. Intra- and interpopulation transposition of mobile genetic elements driven by antibiotic selection. Nat Ecol Evol. 2022;6:555.

    PubMed  Google Scholar 

  4. Aminov RI. Horizontal gene exchange in environmental microbiota. Front Microbiol. 2011;2:158.

    PubMed  PubMed Central  Google Scholar 

  5. Huddleston JR. Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes. Infect Drug Resist. 2014;7:167.

    PubMed  PubMed Central  Google Scholar 

  6. Lerminiaux NA, Cameron A. Horizontal transfer of antibiotic resistance genes in clinical environments. Can J Microbiol. 2019;65:34.

    CAS  PubMed  Google Scholar 

  7. Yu Z, Wang Y, Lu J, Bond PL, Guo J. Nonnutritive sweeteners can promote the dissemination of antibiotic resistance through conjugative gene transfer. ISME J. 2021;15:2117.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Ding C, Yang D, Ma J, Jin M, Shen Z, Shi D, et al. Effects of free antibiotic resistance genes in the environment on intestinal microecology of mice. Ecotoxicol Environ Saf. 2020;204:111119.

    CAS  PubMed  Google Scholar 

  9. Jandhyala SM, Talukdar R, Subramanyam C, Vuyyuru H, Sasikala M, Nageshwar RD. Role of the normal gut microbiota. World J Gastroenterol. 2015;21:8787.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Smillie CS, Smith MB, Friedman J, Cordero OX, David LA, Alm EJ. Ecology drives a global network of gene exchange connecting the human microbiome. Nature 2011;480:241.

    CAS  PubMed  Google Scholar 

  11. Anthony WE, Burnham CD, Dantas G, Kwon JH. The gut microbiome as a reservoir for antimicrobial resistance. J Infect Dis. 2021;223:S209.

    CAS  PubMed  Google Scholar 

  12. Jacoby GA, Griffin CM, Hooper DC. Citrobacter spp. as a source of qnrB alleles. Antimicrob Agents Chemother. 2011;55:4979.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Larsson D, Flach CF. Antibiotic resistance in the environment. Nat Rev Microbiol. 2022;20:257.

    CAS  PubMed  Google Scholar 

  14. Pal C, Asiani K, Arya S, Rensing C, Stekel DJ, Larsson D, et al. Metal resistance and its association with antibiotic resistance. Adv Micro Physiol. 2017;70:261.

    CAS  Google Scholar 

  15. Wales AD, Davies RH. Co-selection of resistance to antibiotics, biocides and heavy metals, and its relevance to foodborne pathogens. Antibiotics (Basel). 2015;4:567.

    PubMed  Google Scholar 

  16. Yao Y, Doijad S, Falgenhauer J, Schmiedel J, Imirzalioglu C, Chakraborty T. Co-occurrence of dual carbapenemases KPC-2 and OXA-48 with the mobile colistin resistance gene mcr-9.1 in Enterobacter xiangfangensis. Front Cell Infect Microbiol. 2022;12:960892.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Fu J, Yang D, Jin M, Liu W, Zhao X, Li C, et al. Aquatic animals promote antibiotic resistance gene dissemination in water via conjugation: role of different regions within the zebra fish intestinal tract, and impact on fish intestinal microbiota. Mol Ecol. 2017;26:5318.

    CAS  PubMed  Google Scholar 

  18. Liu C, Li B, Wu B, Lin H, Jiang L, Qiu Y. How heavy metal stress promotes dissemination of antibiotic resistance genes in the activated sludge process. J Hazard Mater. 2022;437:129279.

    CAS  PubMed  Google Scholar 

  19. Obayashi Y, Kadoya A, Kataoka N, Kanda K, Bak SM, Iwata H, et al. Tetracycline resistance gene profiles in red seabream (Pagrus major) intestine and rearing water after oxytetracycline administration. Front Microbiol. 2020;11:1764.

    PubMed  PubMed Central  Google Scholar 

  20. Zhang YL, Li J, Hu Z, Li JL, Lu H. Oxytetracycline stress stimulates antibiotic resistance gene proliferation and quorum sensing response of marine anammox bacteria in seawater-based wastewater treatment. Chem Eng J. 2022;447:137539.

  21. Komijani M, Shamabadi NS, Shahin K, Eghbalpour F, Tahsili MR, Bahram M. Heavy metal pollution promotes antibiotic resistance potential in the aquatic environment. Environ Pollut. 2021;274:116569.

    CAS  PubMed  Google Scholar 

  22. Gupta S, Graham DW, Sreekrishnan TR, Ahammad SZ. Effects of heavy metals pollution on the co-selection of metal and antibiotic resistance in urban rivers in UK and India. Environ Pollut. 2022;306:119326.

    CAS  PubMed  Google Scholar 

  23. Zhou Q, Wang MZ, Zhong XX, Liu P, Xie XY, Wangxiao JY, et al. Dissemination of resistance genes in duck/fish polyculture ponds in Guangdong Province: correlations between Cu and Zn and antibiotic resistance genes. Environ Sci Pollut Res. 2019;26:8182.

    CAS  Google Scholar 

  24. Komijani M, Eghbalpour F, Lari E, Shaykh-Baygloo N. Developing erythromycin resistance gene by heavy metals, Pb, Zn, and Co, in aquatic ecosystems. Sci Rep. 2022;12:20797.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Su HC, Liu S, Hu XJ, Xu XR, Xu WJ, Xu Y, et al. Occurrence and temporal variation of antibiotic resistance genes (ARGs) in shrimp aquaculture: ARGs dissemination from farming source to reared organisms. Sci Total Environ. 2017;607:357.

    PubMed  Google Scholar 

  26. Zhang Y, Gu AZ, He M, Li D, Chen J. Subinhibitory concentrations of disinfectants promote the horizontal transfer of multidrug resistance genes within and across genera. Environ Sci Technol. 2017;51:570.

    CAS  PubMed  Google Scholar 

  27. Wang Q, Liu L, Hou Z, Wang L, Ma D, Yang G, et al. Heavy metal copper accelerates the conjugative transfer of antibiotic resistance genes in freshwater microcosms. Sci Total Environ. 2020;717:137055.

    CAS  PubMed  Google Scholar 

  28. Li W, Zhang G. Detection and various environmental factors of antibiotic resistance gene horizontal transfer. Environ Res. 2022;212:113267.

    CAS  PubMed  Google Scholar 

  29. Wang Y, Lu J, Zhang S, Li J, Mao L, Yuan Z, et al. Non-antibiotic pharmaceuticals promote the transmission of multidrug resistance plasmids through intra- and intergenera conjugation. ISME J. 2021;15:2493.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Huang Q, Wu H, Cai P, Fein JB, Chen W. Atomic force microscopy measurements of bacterial adhesion and biofilm formation onto clay-sized particles. Sci Rep. 2015;5:16857.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Liu Z, Liu Q, Qi X, Li Y, Zhou G, Dai M, et al. Evolution and resistance of a microbial community exposed to Pb(II) wastewater. Sci Total Environ. 2019;694:133722.

    CAS  PubMed  Google Scholar 

  32. Yu Z, Rabiee H, Guo J. Synergistic effect of sulfidated nano zerovalent iron and persulfate on inactivating antibiotic resistant bacteria and antibiotic resistance genes. Water Res. 2021;198:117141.

    CAS  PubMed  Google Scholar 

  33. Di Cesare A, Eckert EM, D’Urso S, Bertoni R, Gillan DC, Wattiez R, et al. Co-occurrence of integrase 1, antibiotic and heavy metal resistance genes in municipal wastewater treatment plants. Water Res. 2016;94:208.

    PubMed  Google Scholar 

  34. Lin H, Sun WC, Jin DF, Yu QG, Yang YY, Zhang ZL, et al. Effect of composting on the conjugative transmission of sulfonamide resistance and sulfonamide-resistant bacterial population. J Clean Prod. 2021;285:125483.

    Google Scholar 

  35. Lin X, Xu Y, Han R, Luo W, Zheng L. Migration of antibiotic resistance genes and evolution of flora structure in the Xenopus tropicalis intestinal tract with combined exposure to roxithromycin and oxytetracycline. Sci Total Environ. 2022;820:153176.

    CAS  PubMed  Google Scholar 

  36. Sun S, Korheina D, Fu H, Ge X. Chronic exposure to dietary antibiotics affects intestinal health and antibiotic resistance gene abundance in oriental river prawn (Macrobrachium nipponense), and provokes human health risk. Sci Total Environ. 2020;720:137478.

    CAS  PubMed  Google Scholar 

  37. Zhang P, Lu G, Sun Y, Yan Z, Dang T, Liu J. Metagenomic analysis explores the interaction of aged microplastics and roxithromycin on gut microbiota and antibiotic resistance genes of Carassius auratus. J Hazard Mater. 2022;425:127773.

    CAS  PubMed  Google Scholar 

  38. Zhou B, Wang C, Zhao Q, Wang Y, Huo M, Wang J, et al. Prevalence and dissemination of antibiotic resistance genes and coselection of heavy metals in Chinese dairy farms. J Hazard Mater. 2016;320:10.

    CAS  PubMed  Google Scholar 

  39. Wu T, Zhang Y, Wang B, Chen C, Cheng Z, Li Y, et al. Antibiotic resistance genes in Chishui River, a tributary of the Yangtze River, China: Occurrence, seasonal variation and its relationships with antibiotics, heavy metals and microbial communities. Sci Total Environ. 2022;846:157472.

    CAS  PubMed  Google Scholar 

  40. Tang T, Chen Y, Du Y, Yao B, Liu M. Effects of functional modules and bacterial clusters response on transmission performance of antibiotic resistance genes under antibiotic stress during anaerobic digestion of livestock wastewater. J Hazard Mater. 2023;441:129870.

    CAS  PubMed  Google Scholar 

  41. Xu Y, Xu J, Mao D, Luo Y. Effect of the selective pressure of sub-lethal level of heavy metals on the fate and distribution of ARGs in the catchment scale. Environ Pollut. 2017;220:900.

    CAS  PubMed  Google Scholar 

  42. Ji H, Cai Y, Wang Z, Li G, An T. Sub-lethal photocatalysis promotes horizontal transfer of antibiotic resistance genes by conjugation and transformability. Water Res. 2022;221:118808.

    CAS  PubMed  Google Scholar 

  43. Rebelo JS, Domingues C, Nogueira T, Dionisio F. Plasmids increase the competitive ability of plasmid-bearing cells even when transconjugants are poor donors, as shown by computer simulations. Microorganisms 2023;11:1238.

    PubMed  PubMed Central  Google Scholar 

  44. Bojer MS, Frees D, Ingmer H. SosA in Staphylococci: an addition to the paradigm of membrane-localized, SOS-induced cell division inhibition in bacteria. Curr Genet. 2020;66:495.

    CAS  PubMed  Google Scholar 

  45. Jones C, Holland IB. Role of the SulB (FtsZ) protein in division inhibition during the SOS response in Escherichia coli: FtsZ stabilizes the inhibitor SulA in maxicells. Proc Natl Acad Sci USA. 1985;82:6045.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang Q, Mao D, Luo Y. Ionic liquid facilitates the conjugative transfer of antibiotic resistance genes mediated by plasmid RP4. Environ Sci Technol. 2015;49:8731.

    CAS  PubMed  Google Scholar 

  47. Low WW, Wong J, Beltran LC, Seddon C, David S, Kwong HS, et al. Mating pair stabilization mediates bacterial conjugation species specificity. Nat Microbiol. 2022;7:1016.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Achouak W, Heulin T, Pages JM. Multiple facets of bacterial porins. Fems Microbiol Lett. 2001;199:1.

    CAS  PubMed  Google Scholar 

  49. Samuels AL, Lanka E, Davies JE. Conjugative junctions in RP4-mediated mating of Escherichia coli. J Bacteriol. 2000;182:2709.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Achtman M, Morelli G, Schwuchow S. Cell-cell interactions in conjugating Escherichia coli: role of F pili and fate of mating aggregates. J Bacteriol. 1978;135:1053.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Sutherland KM, Ward LM, Colombero CR, Johnston DT. Inter-domain horizontal gene transfer of nickel-binding superoxide dismutase. Geobiology 2021;19:450.

    CAS  PubMed  Google Scholar 

  52. Martins D, McKay G, Sampathkumar G, Khakimova M, English AM, Nguyen D. Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase Pseudomonas aeruginosa. Proc Natl Acad Sci USA. 2018;115:9797.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Lynch M, Kuramitsu H. Expression and role of superoxide dismutases (SOD) in pathogenic bacteria. Microbes Infect. 2000;2:1245.

    CAS  PubMed  Google Scholar 

  54. Reddy S, Kaur K, Barathe P, Shriram V, Govarthanan M, Kumar V. Antimicrobial resistance in urban river ecosystems. Microbiol Res. 2022;263:127135.

    CAS  PubMed  Google Scholar 

  55. Ding J, An XL, Lassen SB, Wang HT, Zhu D, Ke X. Heavy metal-induced co-selection of antibiotic resistance genes in the gut microbiota of collembolans. Sci Total Environ. 2019;683:210.

    CAS  PubMed  Google Scholar 

  56. Loftie-Eaton W, Bashford K, Quinn H, Dong K, Millstein J, Hunter S, et al. Compensatory mutations improve general permissiveness to antibiotic resistance plasmids. Nat Ecol Evol. 2017;1:1354.

    PubMed  PubMed Central  Google Scholar 

  57. Grilo ML, Sousa-Santos C, Robalo J, Oliveira M. The potential of Aeromonas spp. from wildlife as antimicrobial resistance indicators in aquatic environments. Ecol Indic. 2020;115:106396.

    Google Scholar 

  58. Shi Y, Zhang Y, Wu X, Zhang H, Yang M, Tian Z. Potential dissemination mechanism of the tetC gene in Aeromonas media from the aerobic biofilm reactor under oxytetracycline stresses. J Environ Sci. 2021;105:90.

    CAS  Google Scholar 

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Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 42277260, 41977340) and the Guangdong Basic and Applied Basic Research Foundation (No. 2019A1515110937). The authors acknowledge the help of the Guangdong University of Technology analysis and test center.

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Authors and Affiliations

  1. School of Environmental Science and Engineering, Guangdong University of Technology, No. 100 Wai Huan Xi Road, Guangzhou Higher Education Mega Center, Panyu District, Guangzhou, 510006, PR China

    Xiaojun Lin, Chaonan Zhang, Ruiqi Han, Huishi Peng & Yanbin Xu

  2. Analysis and Test Center, Guangdong University of Technology, No. 100 Wai Huan Xi Road, Guangzhou Higher Education Mega Center, Panyu District, Guangzhou, 510006, PR China

    Shoupeng Li, Xiao Zhou & Lu Huang

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YX conceived and supervised the project. XL conceived and designed the experiments. XL, CZ, RH, SL, HP, XZ and LH performed the experiments. XL analyzed data and wrote the manuscript.

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Correspondence to Yanbin Xu.

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Lin, X., Zhang, C., Han, R. et al. Oxytetracycline and heavy metals promote the migration of resistance genes in the intestinal microbiome by plasmid transfer. ISME J 17, 2003–2013 (2023). https://doi.org/10.1038/s41396-023-01514-w

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