In Vitro and In Silico Analysis of the Gut Microbiome of Zebrafish for Bioremediation Approach of Zinc-Contaminated Aquatic Environments

Main Article Content

Sugitha S
Abirami G

Keywords

Bioremediation, Bioaccumulation, Gut microbiome, zinc-resistant bacteria, Zinc binding protein (ZBP)

Abstract

The discovery of bacterial organisms that aid in heavy metal bioremediation opens up new possibilities for removing radioactive compounds and heavy metals from polluted water sources. Accordingly, this study aims to the identification of Zinc resistant bacteria from the gut microbiome of zebrafish and to evaluate its bioremediation capability. The isolates, namely FG01 (Enterobacter cloacae), FG02 (Citrobacter freundii), and FG03 (Aeromonas hydrophila) identified based on 16S rRNA gene sequencing resistant to Zinc and antibiotics like Ampicillin and Amoxicillin and also selected for bioaccumulation studies. The MTC values of the three zinc-resistant bacteria from the Zebrafish gut were evaluated, and the results revealed that the growth of the isolate FG02 was better than the others, while the growth of FGO3 and FG01 decreased at the concentrations of 10 ppm and 15 ppm. By docking studies, the Zinc binding proteins (ZBPs) were discovered and beta-lactamase showed the best binding affinity compared to the other protein. In the process of treating wastewater, zinc-resistant bacteria from the gut microbiome of zebrafish are normally present and can demonstrate their capacity to adsorb heavy metals. The ZBP can be used later as a novel absorbent for heavy metal removal technology.

Abstract 199 | pdf Downloads 213

References

1. R. N. Kumar, R. Solanki, and J. I. N. Kumar, “Seasonal variation in heavy metal contamination in water and sediments of river Sabarmati and Kharicut canal at Ahmedabad, Gujarat,” Environ Monit Assess, vol. 185, no. 1, pp. 359–368, Jan. 2013, doi: 10.1007/s10661-012-2558-4.
2. A. Ramadan, “Heavy Metal Pollution and Biomonitoring Plants in Lake Manzala, Egypt,” Pakistan Journal of Biological Sciences, vol. 6, no. 13, pp. 1108–1117, Jun. 2003, doi: 10.3923/pjbs.2003.1108.1117.
3. V. Masindi and K. L. Muedi, “Environmental Contamination by Heavy Metals,” in Heavy Metals, InTech, 2018. doi: 10.5772/intechopen.76082.
4. Hussain and K. M. . Sheriff, “Status of heavy metal concentrations in groundwater samples situated in and around on the bank of Cooum river at Chennai city, Tamil Nadu.,” J Chem Pharm Res, vol. 5, pp. 73–77, 2013.
5. N. Shikazono, H. M. Zakir, and Y. Sudo, “Zinc contamination in river water and sediments atTaisyu Zn–Pb mine area, Tsushima Island, Japan,” J Geochem Explor, vol. 98, no. 3, pp. 80–88, Sep. 2008, doi: 10.1016/j.gexplo.2007.12.002.
6. M. A. Subroto, S. Priambodo, and N. S. Indrasti, “Accumulation of Zinc by Hairy Root Cultures of Solanum nigrum,” Biotechnology(Faisalabad), vol. 6, no. 3, pp. 344–348, Jun. 2007, doi: 10.3923/biotech.2007.344.348.
7. P. Andarani, H. Alimuddin, R. Suzuki, K. Yokota, and T. Inoue, “Zinc contamination in surface water of the Umeda River, Japan,” IOP Conf Ser Earth Environ Sci, vol. 623, no. 1, p. 012064, Jan. 2021, doi: 10.1088/1755-1315/623/1/012064.
8. D. L. Vullo, H. M. Ceretti, M. A. Daniel, S. A. M. Ramírez, and A. Zalts, “Cadmium, zinc and copper biosorption mediated by Pseudomonas veronii 2E,” Bioresour Technol, vol. 99, no. 13, pp. 5574–5581, Sep. 2008, doi: 10.1016/j.biortech.2007.10.060.
9. W. Maret, “Zinc and Human Disease,” 2013, pp. 389–414. doi: 10.1007/978-94-007-7500-8_12.
10. G. J. Fosmire, “Zinc toxicity,” Am J Clin Nutr, vol. 51, no. 2, pp. 225–227, Feb. 1990, doi: 10.1093/ajcn/51.2.225.
11. Y. Long, Q. Li, Y. Wang, and Z. Cui, “MRP proteins as potential mediators of heavy metal resistance in zebrafish cells,” Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, vol. 153, no. 3, pp. 310–317, Apr. 2011, doi: 10.1016/j.cbpc.2010.12.001.
12. J. Yin, A.-P. Wang, W.-F. Li, R. Shi, H.-T. Jin, and J.-F. Wei, “Time-response characteristic and potential biomarker identification of heavy metal induced toxicity in zebrafish,” Fish Shellfish Immunol, vol. 72, pp. 309–317, Jan. 2018, doi: 10.1016/j.fsi.2017.10.047.
13. J. Xia et al., “Effects of short term lead exposure on gut microbiota and hepatic metabolism in adult zebrafish,” Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, vol. 209, pp. 1–8, Jul. 2018, doi: 10.1016/j.cbpc.2018.03.007.
14. H. Qian, M. Zhang, G. Liu, T. Lu, L. Sun, and X. Pan, “Effects of different concentrations of Microcystis aeruginosa on the intestinal microbiota and immunity of zebrafish (Danio rerio),” Chemosphere, vol. 214, pp. 579–586, Jan. 2019, doi: 10.1016/j.chemosphere.2018.09.156.
15. C. I. A.- Olalusi, A. Oresegun, and E. Bernard, “Screening of Lactic Acid Bacteria from the Gut of Chrysichthys nigrodigitatus for Use as Probiotics in Aquaculture Production,” J Fish
Aquat Sci, vol. 9, no. 6, pp. 478–482, Oct. 2014, doi: 10.3923/jfas.2014.478.482.
16. López Nadal et al., “Feed, Microbiota, and Gut Immunity: Using the Zebrafish Model to Understand Fish Health,” Front Immunol, vol. 11, Feb. 2020, doi: 10.3389/fimmu.2020.00114.
17. K. Ghaima, A. Mohamed, and W. Yehia, “ Resistance and bioadsorption of Cadmium by Pseudomonas aeruginosa isolated from agricultural soil,” International Journal of Applied Environmental Sciences, vol. 12, 2017.
18. B. Yamina, B. Tahar, and F. Marie Laure, “Isolation and screening of heavy metal resistant bacteria from wastewater: a study of heavy metal co-resistance and antibiotics resistance,” Water Science and Technology, vol. 66, no. 10, pp. 2041–2048, Nov. 2012, doi: 10.2166/wst.2012.355.
19. G. Roeselers et al., “Evidence for a core gut microbiota in the zebrafish,” ISME J, vol. 5, no. 10, pp. 1595–1608, Oct. 2011, doi: 10.1038/ismej.2011.38.
20. X. Zeng, J. Tang, X. Liu, and P. Jiang, “Isolation, identification and characterization of cadmium-resistant Pseudomonas aeruginosa strain E1,” Journal of Central South University of Technology, vol. 16, no. 3, pp. 416–421, Jun. 2009, doi: 10.1007/s11771-009-0070-y.
21. E. R. Chellaiah and S. S, “ Isolation, identification and characterization of heavy metal resistant bacteria from sewage.,” 2009.
22. S. ben MILOUD et al., “First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia,” Pol J Microbiol, vol. 70, no. 2, pp. 161–174, Jun. 2021, doi: 10.33073/pjm-2021-012.
23. D. R. VanDevanter, J. M. van Dalfsen, J. L. Burns, and N. Mayer-Hamblett, “In Vitro Antibiotic Susceptibility of Initial Pseudomonas aeruginosa Isolates From United States Cystic Fibrosis Patients,” J Pediatric Infect Dis Soc, vol. 4, no. 2, pp. 151–154, Jun. 2015, doi: 10.1093/jpids/pit052.
24. G. Nitulescu et al., “Molecular Docking and Screening Studies of New Natural Sortase A Inhibitors,” Int J Mol Sci, vol. 18, no. 10, p. 2217, Oct. 2017, doi: 10.3390/ijms18102217.
25. D. Seeliger and B. L. de Groot, “Ligand docking and binding site analysis with PyMOL and Autodock/Vina,” J Comput Aided Mol Des, vol. 24, no. 5, pp. 417–422, May 2010, doi: 10.1007/s10822-010-9352-6.
26. S. Kim et al., “PubChem Substance and Compound databases,” Nucleic Acids Res, vol.44, no. D1, pp. D1202–D1213, Jan. 2016, doi: 10.1093/nar/gkv951.
27. G. Bitencourt-Ferreira and W. F. de Azevedo, “Molecular Docking Simulations with ArgusLab,” 2019, pp. 203–220. doi: 10.1007/978-1-4939-9752-7_13.
28. R. Chaudhari and Z. Li, “PyMine: a PyMOL plugin to integrate and visualize data for drug discovery,” BMC Res Notes, vol. 8, no. 1, p. 517, Dec. 2015, doi: 10.1186/s13104-015-1483-3.
29. S. A. Smith, J. M. Beaulieu, and M. J. Donoghue, “Mega-phylogeny approach for comparative biology: an alternative to supertree and supermatrix approaches,” BMC Evol Biol, vol. 9, no. 1, p. 37, 2009, doi: 10.1186/1471-2148-9-37.
30. Bergey DH and Holt JG, “Bergey’s Manual of Determinative Bacteriology,” no. 9, 2000.
31. Kalsoom et al., “Isolation and screening of chromium resistant bacteria from industrial waste for bioremediation purposes,” Brazilian Journal of Biology, vol. 83, 2023, doi: 10.1590/1519-6984.242536.
32. Wiegand, K. Hilpert, and R. E. W. Hancock, “Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances,” Nat Protoc, vol. 3, no. 2, pp. 163–175, Feb. 2008, doi: 10.1038/nprot.2007.521.
33. K. Sunda and R. Vidya, “High Chromium Tolerant Bacterial Strains from Palar River Basin: Impact ofTannery Pollution,” Research Journal of Environmental and Earth Sciences, vol. 2, pp. 112–117, 2010.
34. Vashishth and S. Khanna, “Toxic Heavy Metals Tolerance in Bacterial Isolates Based On Their Inducible Mechanism,” Jan. 2018.
35. K. Ghaima, A. Mohamed, W. Yehia, A. Meshhdany, and A. Abdulhassan, “Resistance and bioadsorption of Cadmium by Pseudomonas aeruginosa isolated from agricultural soil,” International Journal of Applied Environmental Sciences, vol. 12, Jan. 2017.
36. G. Haferburg and E. Kothe, “Microbes and metals: interactions in the environment,” J Basic Microbiol, vol. 47, no. 6, pp. 453–467, Dec. 2007, doi: 10.1002/jobm.200700275.
37. S. Sinha and S. Mukherjee, “Pseudomonas aeruginosa KUCD1, a possible candidate for cadmium bioremediation,” Braz J Microbiol, vol. 40, pp. 655–662, Jan. 2009, doi: 10.1590/S1517-838220090003000030.
38. de Vicente, M. Avilés, J. C. Codina, J. J. Borrego, and P. Romero, “Resistance to antibiotics and heavy metals of Pseudontonas aeruginosa isolated from natural waters,” Journal of Applied
Bacteriology, vol. 68, no. 6, pp. 625–632, Jun. 1990, doi: 10.1111/j.1365-2672.1990.tb05228.x.
39. G. O. Oyetibo, M. O. Ilori, S. A. Adebusoye, O. S. Obayori, and O. O. Amund, “Bacteria with dual resistance to elevated concentrations of heavy metals and antibiotics in Nigerian contaminated systems,” Environ Monit Assess, vol. 168, no. 1–4, pp. 305–314, Sep. 2010, doi: 10.1007/s10661-009-1114-3.
40. S. Hussain et al., “Zinc Essentiality, Toxicity, and Its Bacterial Bioremediation: A Comprehensive Insight,” Front Microbiol, vol. 13, May 2022, doi: 10.3389/fmicb.2022.900740.
41. V. N. Kavamura and E. Esposito, “Biotechnological strategies applied to the decontamination of soils polluted with heavy metals,” Biotechnol Adv, vol. 28, no. 1, pp. 61–69, Jan. 2010, doi: 10.1016/j.biotechadv.2009.09.002.
42. J. Scherer and D. H. Nies, “CzcP is a novel efflux system contributing to transition metal resistance in Cupriavidus metallidurans CH34,” Mol Microbiol, vol. 73, no. 4, pp. 601–621, Aug. 2009, doi: 10.1111/j.1365-2958.2009.06792.x.
43. J. Xiong et al., “Genome analysis and characterization of zinc efflux systems of a highly zinc-resistant bacterium, Comamonas testosteroni S44,” Res Microbiol, vol. 162, no. 7, pp. 671–679, Sep. 2011, doi: 10.1016/j.resmic.2011.06.002.
44. Y. Hou et al., “Biosorption of Cadmium and Manganese Using Free Cells of Klebsiella sp. Isolated from Waste Water,” PLoS One, vol. 10, no. 10, p. e0140962, Oct. 2015, doi: 10.1371/journal.pone.0140962.
45. M. Dalben et al., “Investigation of an outbreak of Enterobacter cloacae in a neonatal unit and review of the literature,” Journal of Hospital Infection, vol. 70, no. 1, pp. 7–14, Sep. 2008, doi: 10.1016/j.jhin.2008.05.003.
46. G. Delgado et al., “Genetic Characterization of Atypical Citrobacter freundii,” PLoS One, vol. 8, no. 9, p. e74120, Sep. 2013, doi: 10.1371/journal.pone.0074120.
47. J. Martinez-Murcia, S. Benlloch, and M. D. Collins, “Phylogenetic Interrelationships of Members of the Genera Aeromonas and Plesiomonas as Determined by 16S Ribosomal DNA Sequencing: Lack of Congruence with Results of DNA-DNA Hybridizations,” Int J Syst Bacteriol, vol. 42, no. 3, pp. 412–421, Jul. 1992, doi: 10.1099/00207713-42-3-412.
48. J. Thompson, “The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysistools,” Nucleic Acids Res, vol. 25, no. 24, pp. 4876–4882, Dec. 1997, doi: 10.1093/nar/25.24.4876.
49. M. Akhter, M. Tasleem, M. Mumtaz Alam, and S. Ali, “In silico approach for bioremediation of arsenic by structure prediction and docking studies of arsenite oxidase from Pseudomonas stutzeri TS44,” Int Biodeterior Biodegradation, vol. 122, pp. 82–91, Aug. 2017, doi: 10.1016/j.ibiod.2017.04.021.
50. D. K. Gahlot, N. Taheri, D. R. Mahato, and M. S. Francis, “Bioengineering of non-pathogenic Escherichia coli to enrich for accumulation of environmental copper,” Sci Rep, vol. 10, no. 1, p. 20327, Nov. 2020, doi: 10.1038/s41598-020-76178-z.
51. M. J. Gill, S. Simjee, K. Al-Hattawi, B. D. Robertson, C. S. F. Easmon, and C. A. Ison, “Gonococcal Resistance to β-Lactams and Tetracycline Involves Mutation in Loop 3 of the Porin Encoded at the penB Locus,” Antimicrob Agents Chemother, vol. 42, no. 11, pp. 2799–2803, Nov. 1998, doi: 10.1128/AAC.42.11.2799.
52. R. J. Worthington and C. Melander, “Overcoming resistance to β-lactam antibiotics,” J Org Chem, vol. 78, no. 9, pp. 4207–4213, 2013.
53. N. Ahmed et al., “Heavy Metal (Arsenic) Induced Antibiotic Resistance among Extended-Spectrum β-Lactamase (ESBL) Producing Bacteria of Nosocomial Origin,” Pharmaceuticals, vol. 15, no. 11, p. 1426, Nov. 2022, doi: 10.3390/ph15111426.