Molecular characterization of metal and antibiotic

International Journal of Biotechnology and Bioengineering Research. ISSN 2231-1238 Volume 4, Number 1 (2013) pp. 21-30 © Research India Publications http://www.ripublication.com/ijbbr.htm

Molecular characterization of metal and antibiotic resistance activities in a bacterial population isolated from waste water sample Diptendu Sarkar * and Goutam Paul © Environmental Physiology division, Microbiology unit, Dept. Of Physiology, University Of Kalyani, Nadia, West Bengal, Pin-712435 ©

Corresponding author, working as Professor, Dept of Physiology, University of Kalyani, Nadia, West Bengal, Pin-712435 e-mail:[email protected] * Working as Assistant Professor, PG Dept of Biotechnology, Acharya’s Bangalore B- school (ABBS), Lingerdhnahalli, Magadi Road, Bangalore-91 e-mail:[email protected] Abstract: The incidence of multiple metal and antibiotic resistance among bacterial populations poses a potential threat to human health. The co-existence of metal and antibiotic resistance in bacterial strains suggests the role of metals as a factor, which can also contribute to such a phenomenon. The objective of this study was to characterize multiple metal and antibiotic resistance bacteria from the water samples of a main drainage system of water purifying plant in Bangaluru, Karnataka and to transform the plasmid DNA into another species to confirm about their horizontal gene transfer . A total of 5 strains designated as DSGPM1-5 [3 are Gram- negative bacteria (GenBank Accn No JN228116, JN245880 and JN245881 respectively) and 2 are Gram-positive bacteria] were identified by physico-chemical methods. All the 5 bacterial isolates showed different antibiotic resistance activity (Ampicillin, Chloramphenicol, Erythromycin, Streptomycin, Tetracyclin). All the isolates showed different metal resistance activity too (Zn2+,Mn2+, Ni2+, Cd2+ ,Cu2+ ,Hg2+ , Pb2+), but among them only two isolate (DSGPM3 and DSGPM4) showed mercury resistance activity. These strains were screened to find the presence of an endogenous plasmid DNA. One Gram-negative strain (Bacillus sp.) DSGPM4 was found to have a plasmid DNA of different restriction cutting site and that could replicate and express in other Gram-negative bacteria (E.coli DH5α).

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Diptendu Sarkar and Goutam Paul Thus, from our result it can be concluded that multiple metal and antibiotic resistance among bacterial populations probably through horizontal gene transfer. The transformed DH5α-DSGPM4 showed increase MIC values for metals, 5 fold for Zn2+, 4 fold of Mn2+ and 2 fold of Hg2+, where as for Ni2+, Cd2+ and Pb2+ the MIC values were not significantly changed after the transformation. This was the prove that this plasmid may have potential applications in molecular biology for cloning and expression of foreign genes. Keywords: metal resistance, metal pollution, antibiotic, transform

INTRODUCTION Heavy metals are stable and persistent contaminants since they cannot be degraded or destroyed. Therefore, they tend to accumulate in the soils and sediments. Environmental pollution with heavy metals is spreading throughout industrial progress [1]. The important toxic metals cadmium, nickel and lead find its way to the water bodies through waste water [2]. Metal contamination, therefore, represents a long standing, widespread and recalcitrant selection pressure with both environmental and clinical importance, that potentially contributes to the maintenance and spread of antibiotic resistance factors [3]. A substantial number of reports suggest that metal contamination in natural environments could have an important role in the maintenance and proliferation of antibiotic resistance [4,2,5]. Resistance to antibiotics can be conferred by chromosomal or mobile genetic elements (e.g.plasmids), using four main strategies: reduction of membrane permeability to antibiotics, drug inactivation, efflux of the antibiotics and mutation of the cellular target [6]. In addition, antibiotic sequestration has also been suggested as a potential resistance strategy. It has been known for several decades that metal and antibiotic resistance genes are linked, particularly on plasmid [7,8]. A few Bacillus sp. have been explored for the presence of endogenous plasmid DNA of size 2-200kb [9]. The advantages of using an endogenous plasmid DNA for the expression of foreign genes in to Bacillus sp. includes, recognition of the promoter by the host RNA polymerase, protection from the host methylation and restriction system, easy replication and easy of transformation. Bacillus sp. plasmids have successfully been used for the construction of shuttle expression vectors, which can function both in gram positive as well as gram negative bacteria [10,11]. In the present study we have screened out a number of bacterial isolates from a metal polluted environment and designated them as DSGPM1-5.Among the isolated strains, all the Bacillus sp. (DSGPM3-5), are having multiple metal and antibiotic resistance activity as well as endogenous plasmid DNA. This plasmid may have potential applications in molecular biology for cloning and expression of foreign genes.

Molecular characterization of metal and antibiotic resistance activities

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MATERIALS AND METHODS Waste water samples were collected from the main water purifying plant of Bangaluru, Karnataka, South India. A quantity of 1.0 ml of water from each of the collected samples was dissolved in 9.0 ml of sterile distilled water and serial dilution was plated on nutrient agar plate without antibiotics and incubated at 37°C. After the growth of different microorganisms on the plate, each bacterial colony on the basis of its morphological characteristics was picked up and further purified by repeated streaking on nutrient agar plate [12]. Each bacterial culture was then inoculated in nutrient broth, incubated and glycerol stock were made and frozen at -70°C. Strain were characterized by Gram’s staining and 16s rRNA sequencing. Gram’s staining was performed to confirm a Gram positive or Gram negative bacterial strain [12]. 16s rRNA sequencing from genomic DNA with universal primers was performed [13]. To know the identity of organism, obtained sequences were compared with nucleotide databases like GenBank [14] EMBL [15 ]and the Ribosomal Database Project (RDP) [16] through BLAST programme. All the sequences (DSGPM3-5) were submitted to GenBank and accession numbers obtained. Overnight grown cultures of bacterial strains were used for antibiotic resistance or susceptibility. Disc diffusion method was used to check the resistance or sensitivity of bacterial strain towards given antibiotics [17, 18] .The bacterial cultures were incubated for 14-16hr at 37oC and the zone of inhibition was measured in millimeter. The resistance or sensitivity of the bacterial strains towards any antibiotic was calculated as per the guidelines of the manufacturer (Himedia Laboratories Pvt. Limited,Mumbai,India). Antibiotics used in this study were Ampicillin, Chloramphenicol, Erythromycin, Streptomycin and Tetracyclin. The MIC of the metal for each isolate was determined by the plate dilution method [4,5]. The metals, Hg, Cd, Cu, Pb, Ni, Mn and Zn were used as HgCl2, CdCl2, CuCl2, PbCl2, NiCl2 MnCl2 and ZnCl2 in different concentrations ranging from 5 to 1000 ppm. Stock solutions of the metal solutions of the metal salts were prepared in doubled distilled water and were added to sterilized nutrient agar plates. In each test, 5µl aliquid of overnight broth culture (containing approximately 2-4 x 105 CFU) was applied onto duplicate agar plates containing the appropriate heavy metal salts and incubation was at 30°C for 3 days. The lowest concentration of the metals, which inhibit the bacterial growth, was considered as MIC. Since there is no defining concentration of metal ions which can be used to distinguish metal resistant bacteria and metal sensitive bacteria, the concentration used in this study has been employed in similar studies reported on soil bacteria [19, 4]. Plasmid DNA from bacterial strains was isolated by the method of Alkaline lysis [20] with minor modification. The cultures were harvested and washed twice with PBS (pH7.4) and EDTA (pH8.0). Finally, the pellets were resuspended in bufferP-I(50mM Tris HCl,10mM EDTA) and lysozyme was

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Diptendu Sarkar and Goutam Paul

added to the final concentration at the 2mg/ml and incubated at 37oC for 30 min. Finally, the DNA precipitation was done by isopropanol followed by two washes with 70% ethyl alcohol. Plasmid DNA was electrophorised and separated on a 1.0% agarose gel [21]. Isolated plasmid DNA from DSGPM4 (named as pDSGPM4) was transformed into E.coli DH5α by calcium chloride method as per standard protocol of molecular biology . From E.coli DH5α-p DSGPM4, plasmid DNA was isolated by alkaline lysis method and subjected to restriction enzyme digestion. Restriction enzymes used in this study were: Hind III, EcoRI , PstI and BamHI. Digested plasmid DNA was electrophorised and separated on a 1.5% agarose gel [21].

RESULTS AND DISCUSSION: A total of 5 strains were isolated from water samples. They were distinguished on the basis of their colony morphology on nutrient agar plates. All isolated bacterial strains were subjected to Gram’s staining. Based on the result of Gram’s staining, 3 bacterial strains were found Gram-negative and 2 were found Gram-positive. All 5 isolates were found unique in shape, size and morphology from one another. These 5 bacterial strains were used for the further biochemical characterization [table 1,2]. For antibiotic resistance or susceptibility profiling, the disc diffusion method was used for the Ampicillin, Chloramphenicol, Erythromycin, Streptomycin and Tetracyclin. The zone of inhibition was measured in millimeter and the resistance and sensitivity of the bacterial strains towards any antibiotics was calculated as per the guidelines of the manufacturer (Himedia Laboratories Pvt.Limited, Mumbai, India). Most of the strains were resistant to Ampicillin, Chloramphenicol, Erythromycin, Streptomycin, Tetracyclin .For metal resistance profile, minimum inhibitory concentration (MIC) was determined at microgram/ml level [Table 3 & 4]. Most of the strains were found to have a good degree of resistance for metals and antibiotics. It is now well known that these properties of resistance generally reside on extra chromosomal DNA molecule like plasmid. So, from highly resistant bacterial strains plasmid DNA was isolated and all the strains were found to have two plasmid DNA. Multiple bands in DSGPM3 and DSGPM4 confirm the presence of plasmid DNA which possesses various forms like covalently closed circular, open circular, linear etc [Fig. 1]. The presence of covalently closed circular, open circular, linear, etc. forms of plasmid DNA in DSGPM4makes it a suitable candidate for transformation studies. Therefore, plasmid DNA of DSGPM4 was transformed into E.coli DH5α, selected on Ampicillin, Tetracycline, Chloremphenicol and Erythromycin and it was found that it could replicate and express efficiently in E.coli DH5α. This confirms the replication ability of DSGPM4 in another Gram-negative organisms, which may be due to the presence of the origin of replication that can be recognized by Gram-negative bacterial replication, transcription and

Molecular characterization of metal and antibiotic resistance activities translation system. This plasmid DNA isolated from DH5αcell and was subjected to restriction endonuclease digestion blot analysis [Fig. 2 & 3] The restriction digestion pattern Hind III has1, Eco RI has 2, Pst I has 2 and BamHI has site.

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DSGPM4transform [21] and Southern was indicating that 1 restriction cutting

Table 1 : Morphological and Biochemical identification of the isolated bacterial strains Morphological and Biochemical characteristics Cell shape Size Gram’s reaction Capsule Spore

DSGPM1

DSGPM2

Cocci Diplococci 2.5µm 4.5µm Gram Gram positive positive Present Present Non spore Non spore forming forming Aerobic Aerobic Non motile Non motile _ _

Growth Motility Growth in NaCl (20%) Growth temperature 30-35 (°C) Catalase + Oxidase + Glucose Acid Sucrose Acid/Gas Lactose Acid Fructose Acid Arabinose Galactose Acid Mannitol Raffinose Xylose Methyl red VP-test + Indole Citrate Nitrate + Urease Casein Hydrolysis Starch Gelatin + Identification Azomonas sp.

30-37

DSGPM3

DSGPM4

DSGPM5

Rod 6.75µm Gram negative

Rod 5.25 µm Gram negative

Rod 6.54 µm Gram negative

Present Non spore forming Aerobic Non motile +

Present spore forming

Present Spore forming

Aerobic Motile +

Aerobic Motile +

25-30

35-37

30-37

+ + + + + + Acid (Weak) Acid Acid Acid Acid Acid/Gas Acid Acid (Weak) Acid Acid Acid Acid Acid/Gas Acid Acid/Gas Acid Acid/Gas Acid Acid Acid/Gas Acid/Gas Acid (weak) Acid Acid Acid Acid/Gas Acid Acid/Gas + + + + + + _ + + + + + + + + + + + + + + + + Deinococcus Bacillus sp. Pseudomonas Pseudomonas sp. (GenBank Accn aurogenosa. putida. NoJN228116) (GenBank Accn (GenBank Accn NoJN245880) NoJN245881)

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Diptendu Sarkar and Goutam Paul Table 2: Shows exoenzyme activity result: Strain No. Oxidase test DNase test Catalase test Lipase assay Protease assay DSGPM1 +ve +ve +ve -ve +ve DSGPM2 -ve +ve -ve -ve +ve DSGPM3 +ve +ve +ve -ve +ve DSGPM4 +ve +ve -ve -ve +ve DSGPM5 -ve +ve +ve +ve +ve Table 3 : Shows MICs values against the different metals

Strain No

DSGPM1 DSGPM2 DSGPM3 DSGPM4 DSGPM5

Zn2+ Mn2+ Ni2+ Cd2+ Cu2+ Hg2+ Pb2+ Resistant Resistant Resistant Resistant Resistant Resistant Resistant pattern pattern pattern pattern pattern pattern pattern (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) 50 50 100 400 75 ……… 1000 40 50 350 200 250 …….. 1200 25 50 300 200 200 10 1200 30 25 300 200 200 10 1000 25 50 300 200 300 ……… 1200

Table 4: Showing the antibiotic susceptibility patterns of isolates Serial no. Ampicillin Chloramphenicol Erythromycin Streptomycin Tetracycline DSGPM1 Resistant Not resistant Resistant Resistant Not resistant DSGPM2 Resistant Resistant Resistant Not resistant Not resistant DSGPM3 Resiatant Resiatant Not resiatant Resiatant Resistant DSGPM4 Resistant Resistant Resistant Resistant Resistant DSGPM5 Resistant Not resistant Resistant Not resistant Resistant

Photo 1: Gel electrophoresis pattern of plasmid DNA isolated from different heavy metal resistance bacterial strain. 1-DNA digested with Hind III (,mol. weight marker), 2- DSGPM1, 3 -DSGPM2, 4-DDGPM3, 5-DSGPM4, 6DSGPM5 and 7 -pBR 322 (mol. weight marker)


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Photo 2: Restriction enzyme digestion pattern of bacterial strain DSGPM4. 1.λ DNA digested with Hind III, 2. DSGPM4 untreated, 3. DSGPM4digested with Hind III, 4. DSGPM4digested with ECoR I, 5. DSGPM4digested with Pst I, 6. DSGPM4digested with Bam H I

Photo 3: A. Agarose gel electrophoresis pattern of plasmid DNA. B. Southern blot of DNA transferred from A (hybridized with [α-32p]-labeled pDSM4 ). Legend: Lane 1- λDNA digested with Hind III (Mol. weight marker), Lane2Crude pDSGPM4, Lane 3&4-Purified pDSGPM4, Lanes5- Crude DSGPM4plasmid DNA isolated from transform DH5α, Lane6&7- Purified pDSGPM4plasmid DNA isolated from transform DH5α.

Table 5: Shows MICs values against the different metals after transformation Strain No

Zn2+ Mn2+ Ni2+ Cd2+ Resistant Resistant Resistant Resistant pattern pattern pattern pattern (µg/ml) (µg/ml) (µg/ml) (µg/ml) E.coliDH5α150 100 300 200 pDSGPM4

Cu2+ Hg2+ Pb2+ Resistant Resistant Resistant pattern pattern pattern (µg/ml) (µg/ml) (µg/ml) 200 20 1000

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Now-a-days, metal and antibiotic resistance among bacterial population is becoming a major global concern. To carry out the present study, a total of 5 bacterial strains were isolated and identified by Gram’s staining, followed by different other staining techniques. Also, some of them exhibited different morphological characters, including different shape and size. Further 16S rRNA sequencing (DSGPM4,5 and 6) and phylogenetic analysis were done. There is a clear association between heavy antimicrobial consumption within a population and the frequent recovery of antibiotic resistant bacteria [22]. Also, a correlation between the resistance to high level of Cu(II), Pb(II), Zn(II) and antibiotic in bacterial species found in drinking water has long been established [23] . Multiple metal resistance bacterial isolates has exhibited high resistance towards a group of antibiotics [24] but another study has confirmed that, when the concentration of heavy metals increases, it leads to a decrease in antibiotic resistance [25]..In our results, we have reported that if a bacterial isolate has high MIC value for a set of metals, it also exhibits high resistance pattern towards a group of antibiotics and this is supported by Vajiheh et al. [24]. It has been found that large plasmids are responsible for encoding resistance to antibiotics and heavy metals [26,24,27]. Also, transferable plasmid encoding resistance to various heavy metals and antibiotics from Salmonella abortus equi [24] were reported. Large plasmid DNA from Bacillus sp. (B.thuringiensis)(350kb & 105kb),B.anthracis (208kb) and B.cereus(208kb) have also been reported[9] .To the best of our knowledge, the presence of multiple plasmid DNA in DSGPM4 have not been reported so far. The presence of the Ampicillin , Chloramphenicol ,Ttetracycline and Erythromycin resistance marker as confirmed by transformation in E.coli DH5α and the selection on Ampicilin, Chloramphenicol, Tetracycline and Erythromycin .Here, we found after transforming the plasmid of DSGPM4 to the recipient DH5α strain of E.coli, the recipient cell increase MIC values for metals 5 fold for Zn2+, 4 fold of Mn2+ and 2 fold of Hg2+, where as for Ni2+, Cd2+ and Pb2+ the MIC values were not significantly changed. Similar type of result was reported by Vajiheh K et.al in 2003 [24]. This DSGPM4 can have significant applications in molecular biology. This plasmid DNA needs to be further characterized by sequencing through primer walking, ORF finding, promoter prediction, origin of replication and shuttle sequences analysis, etc. This plasmid DNA can be used for cloning and expression of foreign genes in any Gram-negative bacteria and may have its application in fermentation and other industrial processes.

CONCLUSION The development of metal resistant population in a water sample can result from vertical gene transfer, including transposons and broad host range plasmids spontaneous mutation due to the presence of heavy this metal resistance gene suggests that horizontal gene

metal contaminated waste horizontal gene transfer, and selection pressure on metals. Transformation of transfer may be an


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important factor for the development of metal resistant populations in the environment. Indeed, further studies have to be performed to elucidate bioremediation of heavy metals and detoxification processes.

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