Isolation of Fluoride Tolerant Bacillus spp (KT201599, KT201600) from the Midgut of Drosophila melanogaster: Their Probable Role in Fluoride Removal

  • Moumita Dutta
  • Bappaditya Pan
  • Koushik Ghosh
  • Pradipta Saha
  • Sumedha RoyEmail author
Research Article


This is for the first time that two fluoride tolerant bacterial strains of Bacillus spp (KT201599 and KT201600) have been isolated from the mid-gut of third instar larvae of Drosophila melanogaster. They are characterized and identified using phenotypic characters and 16S rRNA gene based molecular phylogenetic analysis. Interestingly, KT201599 and KT201600 are able to survive at 2000–2500 µg/mL sodium fluoride (NaF) concentrations. The chronic lethal concentration 50 (LC50) for NaF in Drosophila larvae is 125 µg/mL, whereas, the above-mentioned Bacillus strains isolated from their mid-gut are found to be more tolerant to fluoride (F) in comparison to their host. In search for biological relevance of KT201599 and KT201600, we further deciphered that these strains not only survived at high F concentrations, but also were efficient in removing the fluoride ions from the medium by 16.66 and 24.71% respectively. Thus, the study indicates that resident gut flora of Drosophila having the property of reducing fluoride load from the medium, might be helping the host to thrive in a fluoridated environment.


Bacteria Bacillus Bioremediation Drosophila melanogaster Fluoride 



The authors are grateful to the Head, DST-FIST and UGC-DRS sponsored Department of Zoology, the University of Burdwan for providing the infrastructural facilities. Thanks are due to Prof. Abhijit Mazumdar and Dr. Niladri Hazra and Dr. S Chakraborty, USIC (University Science Instrumentation Centre for kindly providing microscopic facilities and BOD incubator for bacterial growth. Authors are grateful to Prof. Apurba Ratan Ghosh and Dr. Shrimanta Gupta, Dept of Environmental Science, The University of Burdwan for their kind help during fluoride estimation.


  1. Aoba, T., and O. Fejerskov. 2002. Dental fluorosis: Chemistry and biology. Critical Reviews in Oral Biology and Medicine 13: 155–170.CrossRefPubMedGoogle Scholar
  2. Apidianakis, Y., and L.G. Rahme. 2011. Drosophila melanogaster as a model for human intestinal infection and pathology. Disease Models and Mechanism 4: 21–30.CrossRefGoogle Scholar
  3. Arulananthan, A., T.V. Ramakrishna, and N. Balasubramanian. 1992. Studies on fluoride removal by coconut shell carbon. Indian Journal of Environmental Protection 12: 531–536.Google Scholar
  4. Bagge, E., M. Persson, and K.E. Johansson. 2010. Diversity of spore forming bacteria in cattle manure, slaughterhouse waste and samples from biogas plants. Journal of Applied Microbiology 109: 1549–1565.PubMedGoogle Scholar
  5. Banerjee, G., A. Sengupta, T. Roy, P.P. Banerjee, A. Chattopadhyay, and A.K. Ray. 2016. Isolation and characterization of fluoride resistant bacterial strains from fluoride endemic areas. Fluoride 49: 414–425.Google Scholar
  6. Beighton, D., and W.A. McDougll. 1977. The effects of fluoride on the percentage bacterial composition of dental plaque, of carries incidence, and on the in vitro growth of Streptococcus mutants, Actinomyces viscous, and Actinobacillus sp. Journal of Dental Research 56: 1185–1191.CrossRefPubMedGoogle Scholar
  7. Broderick, N.A., and B. Lemaitre. 2012. Gut-associated microbes of Drosophila melanogaster. Gut Microbes 3: 307–321.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Bronckers, A.L.J.J., D.M. Lyaruu, and P.K. Denbesten. 2009. The impact of fluoride on ameloblasts and mechanisms of enamel fluorosis. Journal of Dental Research 88: 877–893.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Calomiris, J.J., J.L. Armstrong, and R.J. Seidler. 1984. Association of metal tolerance with multiple antibiotic resistances of bacteria isolated from drinking water. Applied and Environmental Microbiology 47: 1238–1242.PubMedPubMedCentralGoogle Scholar
  10. Campbell, C., D. Mummey, and E. Schmidtmann. 2004. Culture-independent analysis of midgut microbiota in the arbovirus vector Culicoiessonorensis (Diptera: Ceratopogonidae). Journal of Medical Entomology 41: 340–348.CrossRefPubMedGoogle Scholar
  11. Chouhan, S., U. Tuteja, and S.J.S. Flora. 2012. Isolation, identification and characterization of fluoride resistant bacteria: Possible role in bioremediation. Applied Biochemistry and Microbiology 48: 51–58.CrossRefGoogle Scholar
  12. Chun, J., J.H. Lee, Y. Jung, M. Kim, S. Kim, B.K. Kim, and Y.W. Lim. 2007. Ez Taxon: A web- based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. International Journal of Systematic and Evolutionary Microbiology 57: 2259–2261.CrossRefPubMedGoogle Scholar
  13. Creamer, B. 1976. The turnover of the epithelium of the small intestine. British Medical Bulletin 23: 226–230.CrossRefGoogle Scholar
  14. Dey, U., S.N. Chatterjee, and N.K. Mondal. 2016. Isolation and characterization of arsenic-resistant bacteria and possible application in bioremediation. Biotechnology Reports 10: 1–7.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Dhar, V., and M. Bhatnagar. 2009. Physiology and toxicity of fluoride. Indian Journal of Dental Research 20: 350–355.CrossRefPubMedGoogle Scholar
  16. Dillon, R.J., and V.M. Dillon. 2004. The gut bacteria of insects: Nonpathogenic interactions. Annual Review of Entomology 49: 71–92.CrossRefPubMedGoogle Scholar
  17. Duc, L.H., H.A. Hong, T.M. Barbosa, A.O. Henriques, and S.M. Cutting. 2004. Characterization of Bacillus probiotics available for human use. Applied Environmental Microbiology 70: 2161–2171.CrossRefGoogle Scholar
  18. Dutta, M., P. Rajak, S. Khatun, and S. Roy. 2017. Toxicity assessment of sodium fluoride in Drosophila melanogaster after chronic sub-lethal exposure. Chemosphere 166: 255–266.CrossRefGoogle Scholar
  19. Eren, E., M. Ozturk, E.F. Mumcu, and D. Canatan. 2005. Fluorosis and its hematological effects. Toxicology and Industrial Health 21: 255–258.CrossRefPubMedGoogle Scholar
  20. Filali, B.K., J. Taoufik, Y. Zeroual, F.Z. Dzairi, M. Talbi, and M. Blaghen. 2000. Waste water bacterial isolates resistant to heavy metals and antibiotics. Current Microbiology 41: 151–156.CrossRefPubMedGoogle Scholar
  21. Gupta, A., L.T. Phung, D.E. Taylor, and S. Silver. 2001. Diversity of silver resistance genes in IncH incompatibility group plasmids. Microbiology 147: 3393–3402.CrossRefPubMedGoogle Scholar
  22. Harrison, J.J., M. Rabie, R.J. Turner, E.A. Badry, K.M. Sproule, and H. Ceri. 2006. Metal resistance in Candida biofilms. FEMS Microbiology Ecology 55: 479–491.CrossRefPubMedGoogle Scholar
  23. He, L.F., and J.G. Chen. 2006. DNA damage, apoptosis and cell cycle changes induced by fluoride in rat oral mucosal cells and hepatocytes. World Journal of Gastroenterology 12: 1144–1148.CrossRefPubMedPubMedCentralGoogle Scholar
  24. Holt, J.G., N.R. Krieg, and P.H.A. Sneath. 1994. Bergey’s Manual of Determinative Bacteriology. Baltimore: Williams & Wilkins.Google Scholar
  25. Hongoh, Y., P. Deevong, T. Inoue, S. Moriya, S. Trakulnaleamsai, M. Ohkuma, C. Vongkaluang, N. Noparatnaraporn, and T. Kudo. 2005. Intra and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Applied and Environmental Microbiology 71: 6590–6599.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Hughes, M.N., and R.K. Poole. 1991. Metal speciation and microbial growth-the hard (and soft) facts. Journal of General Microbiology 137: 725–734.CrossRefGoogle Scholar
  27. Hussein, H., H. Moawad, and S. Farag. 2004. Isolation and characterization of Pseudomonas resistant to heavy metals contaminants. Arab Journal of Biotechnology 7: 13–22.Google Scholar
  28. Hutchens, E., E. Valsami-Jones, N. Harouiya, C. Chairat, E.H. Oelkers, and S. McEldoney. 2006. An experimental investigation of the effect of Bacillus megaterium on apatite dissolution. Geomicrobiology Journal 23: 177–182.CrossRefGoogle Scholar
  29. Johnson, J.L. 1994. Methods for general and molecular bacteriology. In Similarity analysis of DNAs, ed. P. Gerhardt, R.G.E. Murray, W.A. Wood, and N.R. Krieg, 658–660. Washington DC: American Society for Microbiology Press.Google Scholar
  30. Joshi-Tope, G., and A.J. Francis. 1995. Mechanisms of biodegradation of metal-citrate complexes by Pseudomonas fluorescens. Journal of Bacteriology 177: 1989–1993.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Jukes, T.H., and C.R. Cantor. 1969. Evolution of protein molecules. In Mammalian protein metabolism, ed. H.N. Munro, 21–132. New York: Academic Press.CrossRefGoogle Scholar
  32. Khatun, S., P. Rajak, M. Dutta, and S. Roy. 2017. Sodium fluoride adversely affects ovarian development and reproduction in Drosophila melanogaster. Chemosphere 186: 51–61.CrossRefPubMedGoogle Scholar
  33. Kim, M., H.S. Oh, S.C. Park, and J. Chun. 2014. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology 64: 346–351.CrossRefPubMedGoogle Scholar
  34. Lane, D.J. 1991. 16S/23S rRNA sequencing. In Nucleic acid techniques in bacterial systematic, ed. E. Stackebrandt and M. Goodfellow, 115–175. New York: Wiley.Google Scholar
  35. Marcano, I.E., C.A. Díaz-Alcántara, B. Urbano, and F. González-Andrés. 2016. Assessment of bacterial populations associated with banana tree roots and development of successful plant probiotics for banana crop. Soil Biology & Biochemistry 99: 1–20.CrossRefGoogle Scholar
  36. Mergeay, M. 1991. Towards an understanding of the genetics of bacterial metal resistance. Trends in Biotechnology 9: 17–24.CrossRefPubMedGoogle Scholar
  37. Mukhopadhyay, D., and A. Chattopadhyay. 2014. Induction of oxidative stress and related transcriptional effects of sodium fluoride in female zebrafish liver. Bulletin of Environmental Contamination and Toxicology 93: 64–70.CrossRefPubMedGoogle Scholar
  38. Mullen, M.D., D.C. Wolf, F.G. Ferri, T.J. Beveridge, C.A. Flemming, and G.W. Bailey. 1989. Bacterial sorption of heavy metals. Applied and Environmental Microbiology 55: 3143–3149.PubMedPubMedCentralGoogle Scholar
  39. Nies, D.H. 1992. Resistance to cadmium, cobalt, zinc, and nickel in microbes. Plasmid 27: 17–28.CrossRefPubMedGoogle Scholar
  40. Ohlstein, B., and A. Spradling. 2006. The adult Drosophila posterior midgut is maintained by pluri potent stem cells. Nature 439: 470–474.CrossRefPubMedGoogle Scholar
  41. Pal, K.C., N.K. Mondal, S.N. Chatterjee, T.S. Ghosh, and J.K. Datta. 2014. Characterization of fluoride-tolerant halophilic Bacillus flexus NM25 (HQ875778) isolated from fluoride-affected soil in Birbhum District, West Bengal, India. Environmental Monitoring and Assessment 186: 699–709.CrossRefPubMedGoogle Scholar
  42. Pas, M., R. Milacic, K. Draslar, N. Pollak, and P. Raspor. 2004. Uptake of chromium (III) and chromium (VI) compounds in the yeast cell structure. BioMetals 17: 5–33.CrossRefGoogle Scholar
  43. Podder, S., and S. Roy. 2015. Study of the changes in life cycle parameters of Drosophila melanogaster exposed to fluorinated insecticide, cryolite. Toxicology and Industrial Health 31: 1341–1347.CrossRefPubMedGoogle Scholar
  44. Rajbhansi, A. 2008. Study on heavy metal resistant bacteria in guheswori sewage treatment plant. Our Nature 6: 52–57.Google Scholar
  45. Reddy, D.R. 2009. Neurology of endemic skeletal fluorosis. Neurology India 57: 7–12.CrossRefPubMedGoogle Scholar
  46. Saha, P., and T. Chakrabarti. 2006. Emticiciaoligorophica gen nov., a new member of the family Flexibacteraceae, phylum Bacteroidetes. International Journal of Systematic and Evolutionary Microbiology 56: 991–995.CrossRefPubMedGoogle Scholar
  47. Stackebrandt, E., and B.M. Goebel. 1994. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. International Journal of Systematic and Evolutionary Microbiology 44: 846–849.CrossRefGoogle Scholar
  48. Tang, Q.Q., J. Du, H.H. Ma, S.J. Jiang, and X.J. Zhou. 2008. Fluoride and children intelligence: A meta-analysis. Biological Trace Element Research 126: 5–20.CrossRefGoogle Scholar
  49. Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmouqin, and D.G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882.CrossRefPubMedPubMedCentralGoogle Scholar
  50. Van de Peer, Y., and R.D. Wachter. 1997. Construction of evolutionary distance trees with TREECON for Windows: accounting for variation in nucleotide substitution rate among sites. Computer Applications in the Biosciences 13: 227–230.PubMedGoogle Scholar
  51. Wasi, S., G. Jeelani, and M. Ahmad. 2008. Biochemical characterization of a multiple heavy metal, pesticides and phenol resistant Pseudomonas fluorescens strain. Chemosphere 71: 1348–1355.CrossRefPubMedGoogle Scholar
  52. Wayne, L.G., D.J. Brenner, R.R. Colwell, P.A.D. Grimont, O. Kandler, M.I. Krichevsky, L.H. Moore, W.E.C. Moore, R.G.E. Murray, E. Stackebrandt, M.P. Starr, and H.G. Truper. 1987. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. International Journal of Systematic Bacteriology 37: 463–464.Google Scholar

Copyright information

© Zoological Society, Kolkata, India 2018

Authors and Affiliations

  • Moumita Dutta
    • 1
  • Bappaditya Pan
    • 2
  • Koushik Ghosh
    • 3
  • Pradipta Saha
    • 4
  • Sumedha Roy
    • 1
    Email author
  1. 1.Toxicology Research Unit (Cytogenetics Laboratory), Department of ZoologyThe University of BurdwanBurdwanIndia
  2. 2.Malda Medical College and HospitalMaldaIndia
  3. 3.Aquaculture Research Unit, Department of ZoologyThe University of BurdwanBurdwanIndia
  4. 4.Department of MicrobiologyThe University of BurdwanBurdwanIndia

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