Identification and Characterization of Heavy Metal Resistant Bacteria from the Klip River

Authors: P. Chihomvu, P. Stegmann, M. Pillay

Abstract:

Pollution of the Klip River has caused microorganisms inhabiting it to develop protective survival mechanisms. This study isolated and characterized the heavy metal resistant bacteria in the Klip River. Water and sediment samples were collected from six sites along the course of the river. The pH, turbidity, salinity, temperature and dissolved oxygen were measured in-situ. The concentrations of six heavy metals (Cd, Cu, Fe, Ni, Pb and Zn) of the water samples were determined by atomic absorption spectroscopy. Biochemical and antibiotic profiles of the isolates were assessed using the API 20E® and Kirby Bauer Method. Growth studies were carried out using spectrophotometric methods. The isolates were identified using 16SrDNA sequencing. The uppermost part of the Klip River with the lowest pH had the highest levels of heavy metals. Turbidity, salinity and specific conductivity increased measurably at Site 4 (Henley on Klip Weir). MIC tests showed that 16 isolates exhibited high iron and lead resistance. Antibiotic susceptibility tests revealed that the isolates exhibited multitolerances to drugs such as Tetracycline, Ampicillin, and Amoxicillin.

Keywords: Klip River, heavy metals, resistance.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1096695

References:

[1] V. Vermaak, A geomorphological Investigation of the Klip River Wetland, South of Johannesburg. MSc. Dissertation, Johannesburg, SA: University of Witwatersrand, 2009.
[2] T. S. McCarthy, V. Arnold, J. Venter, and, W. N. Ellery, “The collapse of the Johannesburg Klip River Wetland,” SAJS, vol. 104, pp. 391-397, Oct. 2007.
[3] A. Ackil and, S. Kolda, “Acid mine drainage (AMD): causes, treatment and case studies,” J. of Clean Prod., vol 14, pp 1136–1145. Apr. 2005.
[4] D. N. Muruven, An Evaluation of the cumulative surface water pollution within the consolidated Main Reef Area, Roodeport. SA: University of South Africa, 2011.
[5] G. Ozer, A. Ergene, and, B. Icgen, “Biochemical and molecular characterization of strontium resistant environment isolates of Pseudomonas flourescens and Sphingomonas paucimobilis,” Geomicrobiol. J., vol. 30, no. 5, pp. 381-390, Mar. 2013.
[6] T. Aktan, S. Tan, and B. Icgen, “Characterization of lead-resistant river isolate Enterococcus faecalis and assessment of its multiple metal and antibiotic resistance,” Environ. Monit. Assess., vol. 185, pp. 5285-5293, Oct. 2013.
[7] S. Silver, “Plasmid determined metal resistance mechanisms, Range and Overview,” Plasmid, vol. 27, no. 1, pp. 1-3. Jan. 1992.
[8] A. Hynninen, T. Touze, L. Pitkanen, D. Mengin-Lecreulx, and M. Virta, “An efflux transporter PbrA and a phosphatase PbrB cooperate in a leadresistance mechanism in bacteria,” Mol. Microbiol., vol. 74, pp. 384– 394. Oct. 2009.
[9] B. Rajkumar, G. D and, A. K. Paul, “Isolation and characterization of heavy metal resistant bacteria from Barak River contaminated with pulp paper mill effluent, South Assam,” B. Environ. Contam. Tox., vol. 89, pp. 263-268, May 2012.
[10] S. Koc, B. Kabatas, and, B. Icgen, “Multidrug and Heavy Metal- Resistant Raoultella planticola Isolated from Surface Water,” B. Environ. Contam. Tox., vol 91, pp. 177-183 Jun. 2103.
[11] M. K. Chattopadhay, and H. P. Grossart, “Antibiotic and heavy metal resistance of bacterial isolates obtained from some lakes in northern Germany,” NSHM J. Phar. and Health Manag., vol. 2, pp. 74-75, Feb. 2011.
[12] C. Pires, Bacteria in heavy metals contaminated soil, diversity, tolerance and use in remediation system. PhD Thesis, UK: Cranfield University, 2010.
[13] E. C. Raja, G. S. Selvam, and K. Ominie, “Isolation, identification and characterization of heavy metal resistant bacteria from sewage” in International Joint Symposium on Geodisaster Prevention and Geoenvironment in Asia. 2009, pp. 205-211.
[14] T. Maniatis, E. Fritisch, and J. Sambrook, Molecular cloning a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor, Laboratory. 1982.
[15] M. J. Pelczer, and R. D. Reid, “Pure cultures and growth characteristics,” in: Microbiology, New York: McGraw Hill Book Company, 1958, pp 76-84.
[16] J. P. Duguid, “Staining methods,” in Mackie & McCartney Practical Medical Microbiology, J. G. Collee, A. G. Frazer, B. P. Marmion, and A. Simmons, Eds. Edinburgh, New York: Churchill Livingstone, 1996, pp. 41-51.
[17] A. W. Bauer, W. M. Kirby, J. C. Sherris, and M. Turck, “Antibiotic Susceptibility Testing by a standardized single disc method” AJCP, vol. 36, pp. 493-496, Apr. 1966.
[18] K. Phillips, F. Zaidan, O. R. Elizond, and K. L. Lowe, “Phenotypic characterization and 16SrDNA identification of culturable non-obligate halophilic bacterial communities from a hypersaline lake, La Sal del Rey, in extreme South Texas (USA)” Aquatic Biosystems, vol. 8, no. 5, pp. 1-11, 2012.
[19] S. Giri, Isolation and biochemical characterization of Mercury Resistant Bacteria (MRB) from soil samples of industrially contaminated areas of Rourkela, Orissa. MSc. Dissertation, Rourkela, Orissa: National Institute of Technology, 2011.
[20] J. Zhou, M. Bruns, and J. M. Tiedje, “DNA recovery from Soils of Diverse Composition,” Appl. Environ. Microbiol., vol. 62, no. 2, pp. 316-322, Feb. 1996.
[21] S. F. Altschul, T. L. Maddan, A. A.Schaffer, J. Jang, Z. Zang, W. Miller, and D. J. Lipman, “Gapped BLAST and PSI-BLAST a generation of protein database search programs,” Nucl. Acids Res., vol. 25, pp. 3389- 3402, Jul. 1997.
[22] K. Katoh, and D. S Standley, “MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability,” Mol. Biol. Evol., vol. 30, no. 4, pp. 772-780, Jan. 2013.
[23] In-Stream Water Quality Guidelines for the Klip River Catchment. 2003. (www.reservoir.co.za). Accessed 4 October 2013
[24] M. L. De La Torre, D. Sanchez Rhodas, J. A. Grande, and T. Gomez, “Relationships between pH, colour and heavy metal concentrations in the Tinto and Odiel Rivers (Southwest Spain),”. Hydrol. Res., vol. 41, no. 5, pp. 406-413, 2010.
[25] C. Davidson, Catchment Diagnostic Framework for the Klip River Catchment, Vaal Barrage. MSc Dissertation, SA: University of Witwatersrand. 2003.
[26] Minnesota Pollution Control, Low Dissolved Oxygen in water. Water Quality/Impaired Waters 3.24. Minesota: 2009.
[27] T. S. McCarthy, “The impacts of Acid Mine Drainage in South Africa,” SAJS, vol. 107, no. 5/6, pp. 1-7, June 2011.
[28] J. Willey, L. Sherwood, and C. Woolverton, “Microbial Growth,” in Presscotts's Microbiology, 1st ed, J. Willey, L. Sherwood, and C. Woolverton, Eds. New York: McGraw-Hill Custom Publishing, 2004, pp. 126-151.
[29] D. H. Nies, and S. Silver, “Ion efflux systems involved in bacterial metal resistance,” J. Ind. Microbiol., vol. 14, pp. 186-199. Nov.1994.
[30] T. M. Roane, “Lead Resistance in Two Bacterial Isolates from heavy metal contaminated soils,” Microbial Ecol., vol 37, pp. 218-224. Dec 1998.
[31] P. V. Bramachari, P. B. Kavi Kishor, R. Ramadevi, R. Kumar, B. Rao, and S. K. Dubey, “Isolation and characterization of mucous exoplysaccharide produced by Vibrio furnissii VB0s3” J. Microbiol. and Biotechn., vol. 17, pp. 44-51. Jan. 2007.
[32] J. S. Chang, R. Law, and C. C. Chang, “Biosorption of lead, copper and cadmium by biomass of Pseudomonas aeruginosa PU21,” Water Res., vol. 31, pp. 1651–1658. July 1997.
[33] S. M. Hossain, and N. Anantharaman, “Studies on bacterial growth and lead biosorption using Bacillus subtilis,” IJCT, vol. 13, pp. 591–596, Nov. 2006.
[34] M. M. Naik, and S. K. Dubey, “Lead resistant bacteria: Lead resistance mechanisms, their applications in lead bioremediation and biomonitoring,” Ecotoxicol. Environ. Saf., vol. 98, pp. 1-7. 2013.
[35] J. T. Scahntz, and N. Kee-Woel, “A manual for primary cell culture,” World Scientific, pp. 89, Dec. 2004.
[36] R. N. Brogden, R. C. Heel, T. M. Seight, and G. S. Avery, “Amoxicillin injectable: a review of its antibacterial spectrum, pharmacokinetics and therapeutic use,” Drugs, vol. 18, no. 3, pp. 169-184, Sep. 1979.
[37] S. K. Sharma, L. Singh, and S. Singh “Comparative Study between Penicillin and Ampicillin,” SJAMS, vol. 1, no. 4, pp. 291-294, 2013.
[38] B. Zakeri, and G, D. Wright, “Chemical biology of Tetracycline antibiotics,” Biochem. Cell Biol., vol. 86, no. 2, pp. 124-136, Apr. 2008.
[39] I. Stock, “Natural Antibiotic Susceptibility of Proteus spp. with special reference to P. mirabilis and P. penneri strains,” J. Chemother., vol. 15, no. 1, pp. 12-26. Feb. 2003.
[40] M. P. Reyes, J. J. Zhao, and J. A. L. Buensalido, “Current Perspectives: Therapeutic Uses of Tobramycin,” J. Pharmacovigilance, vol. 2, no. 2, pp. 1-5, Feb. 2014
[41] E. J. Kuijper, M. F. Peeters, B. S. Schoenmakers, and H. C. Zanen, “Antimicrobial susceptibility of sixty human fecal isolates of Aeromonas species,”. Eur. J. Clin. Microbiol. Infect. Dis., vol. 8, pp. 248-250, Mar.1989.
[42] M. P. Wilhem, and L. Estes, “Vancomycin,” Mayo Clinic Proceedings, vol. 74, no. 9, pp. 928-935, Sep 1999.
[43] A. O. Odeyemi, A. Asmat, and G. Usup, “Antibiotic resistance and putative vitulence factors of Aeromonas hydrophila isolated from estuary,” JMBFS, vol. 1, no. 6, pp. 1339-1357, Jun. 2012.
[44] R. N. N. Abskharon, S. H. A. Hassan, S. M. F. Gad-el-rab, and A. A. M. Shoreit, “Heavy Metal Resistant of E. coli Isolated from wastewater sites in Assiut City, Egypt,”. Bull. Environ. Contamin. Tox., vol. 81, pp. 309-315. 2008
[45] P. Bohwmik, P. K. Bag, T. K. Hajra, P. S. Ritupama, and T. Ramamurthy, “Pathogenic potential of Aeromonas hydrophila isolated from surface waters in Kolkata, India,” J. Med. Microbiol., vol. 58, pp. 1549-1558, Dec. 2009.
[46] A. Alperi, A. J. Martiinez-Murcia, K. O Wechien, M. Arturo, J. S. Maria, and J. F. Maria, “Aeromonas fluvialis sp. nov., isolated from a Spanish river,” IJSEM, vol. 60, pp. 2048-2055, Jan. 2010.
[47] S. Virender, P. K. Chauchan, R. Kanta, T. Phewa, and V. Kumar, “Isolation and characterization of Pseudomonas resistant to heavy metal contaminants,” IJPSRR, vol. 3, no. 2, pp. 164-167, Aug. 2010.
[48] K. K. Sharma, and U. Kalawat, “Emerging infections: Shewanella, A series of Five Cases,” J. Lab. Physicians, vol. 2, no. 2, pp. 61-65, July 2010.
[49] A. C. M. Toes, J. S. Geelhoed, J. G. Kuenen, and, G. Muyzer, “Characterization of Heavy Metal Resistance of Metal-Reducing Shewanella Isolates from Marine Sediments,” Geomicrobiol. J., vol. 25, no. 6, Aug. 2008.
[50] S. T. Odonkor, and J. K. Ampofo “E. coli as an inidcator of bacteriological quality of water: an overview,” Microbiol. Res., vol. 4, no. e2, pp. 5-11, Nov. 2012.
[51] P. Setlow, “I will survive: DNA protection in bacterial spores,” Trends Microbiol, vol. 15, pp. 172–180. Apr. 2007.
[52] S. Panadey, P. Saha, S. Biswas, and T. Maiti, “Characterization of two metal resistant Bacillus strains isolated from slag disposal site at Burnpur, India,” J. Environ. Biol., vol. 32, pp. 773-779, Nov. 2011.
[53] S. Lüders, F. David, M. Steinwand, E. Jordan, M. Hust, S. Dubel, and E.Franco-Lara, “Influence of the hydromechanical stress and temperature on growth and antibody fragment production with Bacillus megaterium,” Appl. Microbial. Biotechn., vol. 91, pp. 81-90, July 2011.
[54] M. Hookom, and D. Puchooa, “Isolation and identification of Heavy Metals Tolerant Bacteria from Industrial and Agricultural Areas in Mauritius,” CRMB, vol. 1, no. 3, pp. 119-123. May 2013.
[55] T. D. Pena-Montenegro, and J. Dussan, “Genome sequence and description of the heavy metal tolerant bacterium Lysinibacillus sphaericus strain OT4b,”.31. Stand.Genomic, vol. 9, no. 1, pp. 42-56, Oct. 2013
[56] R. Margesin, and F. Schiner, “Bacterial heavy metal-tolerance- extreme resistance to nickel in Arthrobacter spp. strains,” JBM, vol. 36, no. 4, pp. 269-282, Jan. 2007