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

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Abstract: 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.
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  Journal of Biotech Research [ISSN: 1944-3285]   2011; 3:72-77 72 Study of mercury (II) chloride tolerant bacterial isolates from BaghmatiRiver with estimation of plasmid size and growth variation for the highmercury (II) resistant  Enterobacter  spp. Vivek Bhakta Mathema 1, * , Bal Krishna Chand Thakuri 1 , Mika Sillanpää 2 , Reena AmatyaShrestha 3   1 Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal; 2 Faculty of Technology,Lappeenranta University of Technology, Patteristonkatu 1, FI-50100 Mikkeli, Finland; 3 Department of Civil and Environmental Engineering, Lehigh University, Bethlehem, PA, USA.   A total of three mercury resistant belonging to genus Enterobacter  , Streptococcus and Pseudomonas wereisolated from river banks of Baghmati in Kathmandu and were further categorized on the basis of their toleranceto mercury (II) chloride. Among all these isolates Enterobacter  strain expressed highest degree of resistancetowards Hg (II) chloride showing distinct growth in medium with upto 80 µg/ml of HgCl 2  . Excessive slimeproduction along with delayed pattern of growth and lower viability was observed for the isolate underincreasing concentrations of Hg (II) supplemented liquid culture medium. Upon investigating total geneticcontent of this isolate, occurrence of plasmid with approximate 18 kb size and susceptible to mercuric chlorideafter plasmid curing suggests a plasmid mediated tolerance. *Corresponding author: Vivek Bhakta Mathema, Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal. E-mail:vivek_mathema@hotmail.comFinancial Support: We are thankful to Department of Biotechnology, Tribhuvan University for providing necessary funds to conduct theresearch. Introduction Frequent discharge of metallic ion containingtoxic waste from industries and urban sectorscontains high levels of arsenic, mercury, nickel,cadmium, lead and sulfur based byproducts [1,9, 10, 26]. These wastes appear with vastdistribution in urbanized regions in variousforms and are one of the major causes of metallic pollution [22, 27]. Accumulation of these heavy metals affects the microbialecology [3, 14] and growth pattern of microorganism in aquatic sources. Metallictoxicity and its related antibacterial effects aremajorly due to oligodynamic property of metals,which presents obstacle to microbial growth.Despite these antagonistic factors, microbialadaptation system has evolved multiplemechanisms [6, 21] such as volatilization,extracellular precipitation, intake exclusion andextracellular sequestration to counteractadverse effects of these toxic compounds.Certain environmental strains of bacteria haveacquired highly sophisticated resistancemechanisms for mercury detoxification. Mainlye nterobacter, streptococcus, staphylococcus, and p seudomonas species extracted fromaquatic sources [36, 37] of urban regions showhigh degree of tolerance against heavy metals  Journal of Biotech Research [ISSN: 1944-3285]   2011; 3:72-77 73 [5]. Enterobacter  are frequently observed innosocomial infection [8] and are unambiguousrisk factors for infection with multidrug-resistant strains. In many bacteria, resistance tometallic salts is associated with plasmids [16].Both chromosomally carried genes andtransposable elements along with plasmidsmediated xenobiotic tolerance have been foundon Enterobacter  species [5, 18]. Extremeenvironmental pollution and toxic industrialwaste have induced variety of metal tolerancegene with reports of strain suggesting heavymetal tolerance level reaching above 100 µg/ml[26]. Excess untreated chemical wastes and sewages dumped into Kathmandu city‘s rivers [25] can be reflected by occurrence of suchbacterial strains which is an indicator of extensive water pollution. Even though ionsclearing mechanism like fractionation [17],complexing electrolyte [19, 30], and activatedcarbon treatment [24] have been used intreatment for removal of metal ions; adequateamount of metallic ions still seems to bereleased in the water sources. Generally,mercury tolerant bacteria are capable of detoxifying the mercury compounds by twosequentially acting enzymes namely,organomercuriallyase which cleaves thecarbon  – mercury bonds of typicalorganomercurials and mercuric reductase,which reducing Hg(II) into volatile and nonlethal Hg(0). The counteract mechanism can bebroad spectrum or specific for typicalorganometallic compound. Majority of suchresistant strains are found to be activelyexpressing mer  operon for mercurydetoxification. This not only enables microbesto survive through toxic environment but alsoassists in removal [28, 35] of such compoundsvia detoxification or biosorption [2, 29, 33, 34].Similar activities have been observed in Enterobacter  species [15] showing considerableamount of intracellular accumulation of suchheavy metals. This research article mainlyfocuses on Hg (II) chloride tolerance of  Enterobacter isolate and tries to suggest a linkbetween mercury (II) tolerance and plasmidcontent of the selected isolate. Materials and methodsIsolation of mercury resistance bacteria  200 ml of water sample was collected in asterile 250 ml glass container from banks of Baghmati River at Gausala, Kathmandu.Samples were filtered using standard Whatmanfilter paper. Culture tubes containing 10 ml of Nutrient Broth supplemented with (0-120)µg/ml mercury (II) chloride (Qualigens © ) wereused. 500 µl of filtered sample were added asinoculums in each culture tube and wasincubated with shaking at 120 rpm, 37 o C for 24hours. Culture tubes showing visible growthabove 10 µg/ml HgCl 2 were selected for furthertests. Mercury (II) chloride resistant bacteriaisolation and identifications were conducted byusing standard biochemical tests and Genus of the bacteria was determined using interpretation of results with Bergey’s manual of systematic bacteriology. Determination of Minimum inhibitoryconcentration (MIC) and Minimum bactericidalconcentration (MBC)  The MIC for the selected isolates weredetermined using culture tubes containingnutrient agar medium supplemented usingserial twofold dilution method and maintaininginoculum of 1 x 10 6 bacteria/ml. MBC wasconducted by culturing the isolates in differentconcentrations of mercury (II) chloridesupplemented Muller Hilton (MH) agar usingspread plate culture technique. The tubes andculture plates were incubated at 37 o C for 24hours. MIC was defined as lowest concentrationof mercury (II) chloride allowing no visiblegrowth in culture tube. MBC was defined as thelowest concentration of HgCl 2 that results inmore than 99.9% killing of the bacteria beingtested. Growth characteristics under mercury (II)chloride stress  The nutrient broth culture broth wasstandardized to 0.5 McFarland units by usingsterile distilled water. Colony forming unit (CFU)was calculated by carpet culture on MH agar  Journal of Biotech Research [ISSN: 1944-3285]   2011; 3:72-77 74 supplemented with different concentrations of mercury (II) chloride (0-80 µg/ml). The MH agarplates were incubated for 18-72 hours at 37 o Cfor observing growth. Total DNA extraction and plasmid curing High HgCl 2 plasmid was extracted by followingMolecular cloning [23] standard extractionprotocols. Alkaline lysis [23] method wasimplemented for plasmid DNA extraction andfurther purification of plasmid was done usingplasmid ultrapurification kit (Bangalore GeNei © Kit). The DNA and plasmid extract was run in 1%agarose gel. Genomic DNA and plasmid contentin the gel were visualized using Ethidiumbromide (EtBr) (Hi-Media © ) mediated DNAfluorescence and UV gel documentation unit(LLC, Gamma Scientific © ). Plasmid curing [7]   was performed by culturing the selected isolatein nutrient broth supplemented with EtBr of 100 µg/ml concentration followed by subculturing into fresh nutrient agar plates. Isolatesfrom fresh medium were further plated intonutrient agar with (25 µg/ml) and withoutmercury (II) chloride salt. Statistics  Data are provided as means ±SEM, n representsthe number of independent experiments.Differences were tested using one way-ANOVA,as appropriate. GraphPad Prism 5.0 was usedfor generation of figures and statistical analysisof data. ResultsSelection and identification Selection of the bacterial isolates was purelybased upon their tolerance to mercury (II)chloride in growth medium. Three distinctisolates were obtained in nutrient agarsupplemented with 10 µg/ml of HgCl 2 .Morphological analysis and biochemical tests(Table 1) along with interpretation of results using Bergey’s manual of systematic bacteriology identified three isolates as Enterobacter  , Streptococcus , and Pseudomonas . Table 1. Bacterial isolates resistant to mercury (II) chloride. Thetable shows Minimum inhibitory concentration (MIC) andMinimum bactericidal concentration (MBC) for selected mercurytolerant isolates. Bacterial Isolate   MIC   MBC   Enterobacter    45 µg/ml   80 µg/ml   Streptococcus   25 µg/ml   35 µg/ml   Pseudomonas   20 µg/ml   30 µg/ml   Determination of MICs and MBCs  Bacterial isolates were separately cultured innutrient broth supplemented with 0-100 µg/mlof mercury (II) chloride at an increasingconcentration interval of 5 µg/ml. Enterobacter   isolates was found to show visible grow inmedium up to 40 µg/ml of HgCl 2 whereasgrowth of  Streptococcus and Pseudomonas  were not observed above 20 and 15 µg/ml of HgCl 2 MBC for Enterobacter  was obtained at 90µg/ml whereas Streptococcus and Pseudomonas MBCs were observed at concentrations 35 and30 µg/ml of HgCl 2, respectively. The MICs andMBCs results (Table 1) indicated that isolated Enterobacter  was strongly resistant to mercury.This isolate was only selected for furtherstudies. Figure 1. Effect of mercury (II) chloride on C.F.U for Enterobacter isolate.   Isolates from various concentration of HgCl 2 supplemented broth were cultured by using spread plate methodand C.F.U was calculated. Arithmetic mean ± SD were calculated,the figure represents average results of (n=3) independentmeasurement. Enterobacter  isolate showed   HgCl 2 concentra-tion   dependent growth inhibition Growth pattern of  Enterobacter  isolateindicates that the lag phase of growth was  Journal of Biotech Research [ISSN: 1944-3285]   2011; 3:72-77 75 prolonged when cultured at nutrient agarsupplemented with successively increasingconcentrations of mercury (II) chloride 0-80µg/ml in the medium. Effect of higherconcentrations of HgCl 2 was clearly evident(Figure 1) in terms of reduction in CFU.Interestingly, presence of mercury (II) in themedium induced excessive slime productionwhich maintained its abundance even whennutrient broth was centrifuged and washedseveral times with normal saline. On thecontrary, growth in normal nutrient brothproduced little or no slime. Figure 2. Extraction of total genomic DNA and plasmid from Enterobacter  isolates . Plasmid and genomic extract was run in 1%agarose gel electrophoresis and visualized by EtBr staining. Lane 1(L1) to Lane 8 (L8) from left to right represents Ruler DNA (M), λ /Mlu I digest (LD), Genomic DNA extract G1, G2, G3 along with Enterobacter    spp. Plasmid extract P1, P2, and P3 respectively(Indicated by White arrow). Plasmid size is approximately 18 Kbwith reference to LD. The white arrows indicate plasmids. Total DNA extraction of  Enterobacter  isolateand plasmid curing Extraction of total genetic content revealedpresence of at least one plasmid common in Enterobacter  isolates (Figure 2). Experimentswere carried out in triplicates for genomic DNAand plasmid extracts. Computation of theplasmid fragment size with standard λ /Mlu Idigest estimates it to be around 18 Kb bycomputing with the control of 50 Kb. Averagesize for the plasmid (Figure 3) was furtherconfirmed using standard 50 kb λ / Mlu I Digest(Bangalore GeNei © Kit). Plasmid curingsignificantly decreased viability of  Enterobacter   isolates in HgCl 2 supplemented medium. Viableculture of isolates subsequent to plasmid curingwas limited to 5 µg/ml of HgCl 2, whereas normalgrowth was observed in standard nutrientbroth. Figure 3. Plasmid extraction and estimation of size . Purifiedplasmid was run in 1% agarose gel electrophoresis and visualizedby EtBr staining. Control DNA 50 Kb (L1), Enterobacter  isolate Plasmid (L2), λ /Mlu I Digest (L3) *fragment size in Bp+ . Discussion Pollution of water sources as a result of variousmetal salts contamination [11, 12] shown to berelated to development of strains capable of tolerating such metal ions [1, 4, 10, 32]. Amongthese contaminants mercury is one of theleading chemicals in water pollution [5] inindustrialized zones. In most cases theevolutionary pattern of mutagenesis and slowgenetic adaptation leads to production of resistant strains. However, unusually high levelof tolerance to heavy metals ions [20] suggest aplasmid mediated tolerance strains bacteria [7].Previous studies indicate that Pseudonmonas  strains are presumably more competent toforeign genome [6]   for their flexible geneticadaption. During the study, both streptococcus and  pseudomonas isolates were found to betolerant to mercury (II) chloride. Both of thesebacteria have been known to carry plasmidsand transposable elements for their mode of 
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