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Study of Mercury (II) Journal of Biotech Research [ISSN: 1944-3285] 2011; 3:72-77 Study of mercury (II) chloride tolerant bacterial isolates from Baghmati River with estimation of plasmid size and growth variation for the high mercury (II) resistant Enterobacter spp. Vivek Bhakta Mathema1, *, Bal Krishna Chand Thakuri1, Mika Sillanpää2, Reena Amatya Shrestha3 1Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal; 2Faculty of Technology, Lappeenranta University of Technology, Patteristonkatu 1, FI-50100 Mikkeli, Finland; 3Department of Civil and Environmental Engineering, Lehigh University, Bethlehem, PA, USA. A total of three mercury resistant belonging to genus Enterobacter, Streptococcus and Pseudomonas were isolated from river banks of Baghmati in Kathmandu and were further categorized on the basis of their tolerance to mercury (II) chloride. Among all these isolates Enterobacter strain expressed highest degree of resistance towards Hg (II) chloride showing distinct growth in medium with upto 80 µg/ml of HgCl2 . Excessive slime production along with delayed pattern of growth and lower viability was observed for the isolate under increasing concentrations of Hg (II) supplemented liquid culture medium. Upon investigating total genetic content of this isolate, occurrence of plasmid with approximate 18 kb size and susceptible to mercuric chloride after plasmid curing suggests a plasmid mediated tolerance. *Corresponding author: Vivek Bhakta Mathema, Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal. E-mail: [email protected] Financial Support: We are thankful to Department of Biotechnology, Tribhuvan University for providing necessary funds to conduct the research. Introduction which presents obstacle to microbial growth. Despite these antagonistic factors, microbial Frequent discharge of metallic ion containing adaptation system has evolved multiple toxic waste from industries and urban sectors mechanisms [6, 21] such as volatilization, contains high levels of arsenic, mercury, nickel, extracellular precipitation, intake exclusion and cadmium, lead and sulfur based byproducts [1, extracellular sequestration to counteract 9, 10, 26]. These wastes appear with vast adverse effects of these toxic compounds. distribution in urbanized regions in various forms and are one of the major causes of Certain environmental strains of bacteria have metallic pollution [22, 27]. Accumulation of acquired highly sophisticated resistance these heavy metals affects the microbial mechanisms for mercury detoxification. Mainly ecology [3, 14] and growth pattern of enterobacter, streptococcus, staphylococcus, microorganism in aquatic sources. Metallic and pseudomonas species extracted from toxicity and its related antibacterial effects are aquatic sources [36, 37] of urban regions show majorly due to oligodynamic property of metals, high degree of tolerance against heavy metals 72 Journal of Biotech Research [ISSN: 1944-3285] 2011; 3:72-77 [5]. Enterobacter are frequently observed in Materials and methods nosocomial infection [8] and are unambiguous risk factors for infection with multidrug- Isolation of mercury resistance bacteria resistant strains. In many bacteria, resistance to 200 ml of water sample was collected in a metallic salts is associated with plasmids [16]. sterile 250 ml glass container from banks of Both chromosomally carried genes and Baghmati River at Gausala, Kathmandu. transposable elements along with plasmids Samples were filtered using standard Whatman mediated xenobiotic tolerance have been found filter paper. Culture tubes containing 10 ml of on Enterobacter species [5, 18]. Extreme Nutrient Broth supplemented with (0-120) environmental pollution and toxic industrial µg/ml mercury (II) chloride (Qualigens©) were waste have induced variety of metal tolerance used. 500 µl of filtered sample were added as gene with reports of strain suggesting heavy inoculums in each culture tube and was metal tolerance level reaching above 100 µg/ml incubated with shaking at 120 rpm, 37oC for 24 [26]. Excess untreated chemical wastes and hours. Culture tubes showing visible growth sewages dumped into Kathmandu city‘s rivers above 10 µg/ml HgCl2 were selected for further [25] can be reflected by occurrence of such tests. Mercury (II) chloride resistant bacteria bacterial strains which is an indicator of isolation and identifications were conducted by extensive water pollution. Even though ions using standard biochemical tests and Genus of clearing mechanism like fractionation [17], the bacteria was determined using complexing electrolyte [19, 30], and activated interpretation of results with Bergey’s manual carbon treatment [24] have been used in of systematic bacteriology. treatment for removal of metal ions; adequate amount of metallic ions still seems to be Determination of Minimum inhibitory released in the water sources. Generally, concentration (MIC) and Minimum bactericidal mercury tolerant bacteria are capable of concentration (MBC) detoxifying the mercury compounds by two The MIC for the selected isolates were sequentially acting enzymes namely, determined using culture tubes containing organomercuriallyase which cleaves the nutrient agar medium supplemented using carbon–mercury bonds of typical serial twofold dilution method and maintaining organomercurials and mercuric reductase, inoculum of 1 x 106 bacteria/ml. MBC was which reducing Hg(II) into volatile and non conducted by culturing the isolates in different lethal Hg(0). The counteract mechanism can be concentrations of mercury (II) chloride broad spectrum or specific for typical supplemented Muller Hilton (MH) agar using organometallic compound. Majority of such spread plate culture technique. The tubes and resistant strains are found to be actively culture plates were incubated at 37oC for 24 expressing mer operon for mercury hours. MIC was defined as lowest concentration detoxification. This not only enables microbes of mercury (II) chloride allowing no visible to survive through toxic environment but also growth in culture tube. MBC was defined as the assists in removal [28, 35] of such compounds lowest concentration of HgCl2 that results in via detoxification or biosorption [2, 29, 33, 34]. more than 99.9% killing of the bacteria being Similar activities have been observed in tested. Enterobacter species [15] showing considerable amount of intracellular accumulation of such Growth characteristics under mercury (II) heavy metals. This research article mainly chloride stress focuses on Hg (II) chloride tolerance of The nutrient broth culture broth was Enterobacter isolate and tries to suggest a link standardized to 0.5 McFarland units by using between mercury (II) tolerance and plasmid sterile distilled water. Colony forming unit (CFU) content of the selected isolate. was calculated by carpet culture on MH agar 73 Journal of Biotech Research [ISSN: 1944-3285] 2011; 3:72-77 supplemented with different concentrations of Table 1. Bacterial isolates resistant to mercury (II) chloride. The table shows Minimum inhibitory concentration (MIC) and mercury (II) chloride (0-80 µg/ml). The MH agar Minimum bactericidal concentration (MBC) for selected mercury o plates were incubated for 18-72 hours at 37 C tolerant isolates. for observing growth. Bacterial Isolate MIC MBC Total DNA extraction and plasmid curing Enterobacter 45 µg/ml 80 µg/ml High HgCl2 plasmid was extracted by following Streptococcus 25 µg/ml 35 µg/ml Molecular cloning [23] standard extraction protocols. Alkaline lysis [23] method was Pseudomonas 20 µg/ml 30 µg/ml implemented for plasmid DNA extraction and further purification of plasmid was done using Determination of MICs and MBCs plasmid ultrapurification kit (Bangalore GeNei© Bacterial isolates were separately cultured in Kit). The DNA and plasmid extract was run in 1% nutrient broth supplemented with 0-100 µg/ml agarose gel. Genomic DNA and plasmid content of mercury (II) chloride at an increasing in the gel were visualized using Ethidium concentration interval of 5 µg/ml. Enterobacter bromide (EtBr) (Hi-Media©) mediated DNA isolates was found to show visible grow in fluorescence and UV gel documentation unit medium up to 40 µg/ml of HgCl2 whereas (LLC, Gamma Scientific© ). Plasmid curing [7] growth of Streptococcus and Pseudomonas was performed by culturing the selected isolate were not observed above 20 and 15 µg/ml of in nutrient broth supplemented with EtBr of HgCl2 MBC for Enterobacter was obtained at 90 100 µg/ml concentration followed by sub µg/ml whereas Streptococcus and Pseudomonas culturing into fresh nutrient agar plates. Isolates MBCs were observed at concentrations 35 and from fresh medium were further plated into 30 µg/ml of HgCl2, respectively. The MICs and nutrient agar with (25 µg/ml) and without MBCs results (Table 1) indicated that isolated mercury (II) chloride salt. Enterobacter was strongly resistant to mercury. This isolate was only selected for further Statistics studies. Data are provided as means ±SEM, n represents the number of independent experiments. Differences were tested using one way-ANOVA, as appropriate. GraphPad Prism 5.0 was used for generation of figures and statistical analysis of data. Results Selection and identification Selection of the bacterial isolates was purely based upon their tolerance to mercury (II) Figure 1. Effect of mercury (II) chloride on C.F.U for Enterobacter chloride in growth medium. Three distinct isolate. Isolates from various concentration of HgCl2 supplemented broth
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