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Antibiotic and metal resistant-bacteria Research International Journal of Integrative Biology A journal for biology beyond borders ISSN 0973-8363 Characterization of metal and antibiotic resistance in a bacterial population isolated from a Copper mining industry Pankaj Kumar Jain, S Ramachandran, Vibhava Shukla, Deepti Bhakuni, Sanjay Kumar Verma* Centre for Biotechnology, Birla Institute of Technology and Science, Pilani (Raj.), India Submitted: 16 Mar. 2009; Revised: 28 Apr. 2009; Accepted: 5 May. 2009 Abstract The emergence of multiple metal/antibiotic resistance among bacterial populations poses a potential threat to human health. The co-existence of metal/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 is to characterize multiple metal/antibiotic resistant bacteria from the soil and water samples of a copper mining industry. A total of 24 strains (12 Gram-positive and 12 Gram-negative bacteria) were identified by the 16s rRNA gene sequencing. Out of the 24 bacterial isolates, 9 isolates show multiple metal and antibiotics resistance. These strains were screened to find the presence of an endogenous plasmid DNA. One strain, Bacillus sp. (PRS3) was found to have a plasmid DNA of 4.2 kb that could replicate in both Gram positive and Gram negative bacteria. Thus, it can be predicted that metal pollution results in selective pressure that leads to the development of multiple metal/antibiotic resistance among bacterial populations, probably through horizontal gene transfer. Keywords: metal resistance, antibiotic resistance, copper mining, metal pollution, soil bacteria. INTRODUCTION The heavy metal and radionuclide pollution from nuclear power plants, mining industries, electroplating industries and agricultural runoffs is a major cause of concern to public health, animals and ecosystem (Rehman et al., 2007). 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 (Austin et al., 2006). 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 (Summers et al., 1993; Alonso et al., 2001; Summers, 2002). Resistance to antibiotics can be conferred by chromosomal or mobile genetic elements (e.g. plasmids) and achieved using four main strategies: reduction of membrane permeability to * Corresponding author: Sanjay Kumar Verma,, Ph.D. Centre for Biotechnology, Biological Sciences Group Birla Institute of Technology & Science Pilani -333031, Rajasthan, India Email: [email protected] International Journal of Integrative Biology ©OmicsVista Group, All rights reserved antibiotics, drug inactivation, rapid efflux of the antibiotic and mutation of the cellular target (Krulwich et al., 2005). 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 plasmids (Novick et al., 1968; Foster, 1983). A few Bacillus sp. have been explored for the presence of endogenous plasmid DNA of size 2-200kb (Lars et al., 2008). The advantages of using an endogenous plasmid DNA for the expression of foreign genes into Bacillus sp. includes, recognition of the promoter by the host RNA polymerase, protection from the host methylation and restriction system, easy replication and ease of transformation. Bacillus sp. plasmids have successfully been used for the construction of shuttle expression vectors, which can function in both Grampositive as well as in Gram-negative bacteria (Noriyuki et al., 1992; Mesrati et al., 2005). In the present study we have explored a number of bacterial isolates from a metal polluted environment. Among these is a Bacillus sp. which is multiple metal and antibiotic resistant and possesses an endogenous plasmid DNA. This plasmid may have potential applications in molecular biology for cloning and expression of foreign genes. IJIB, 2009, Vol. 6, No. 2, 57 Antibiotic and metal resistant-bacteria MATERIALS AND METHODS Isolation and purification of the microorganisms Soil and water samples were collected from the metal polluted sites of Khetri copper complex near Pilani, Rajasthan, India. A quantity of 1.0 gm of soil and 1ml of water from each of the collected samples was dissolved in 9 ml sterile distilled water and serial dilutions were made. Each dilution was plated on nutrient agar plate without antibiotics and incubated at 37oC. 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 (Aneja, 2001). Each bacterial culture was then inoculated in nutrient broth, incubated and glycerol stocks were made and frozen at -70oC. Strain characterization: Gram’s staining and 16s rRNA sequencing Gram’s staining was performed to confirm a Grampositive/Gram-negative bacterial strain (Godkar, 1994). 16s rRNA sequencing from genomic DNA with universal primers was performed (Therese et al., 1998). To know the identity of organism, obtained sequences were compared with nucleotide databases like GenBank (Benson et al., 2000), EMBL (Kanz et al., 2005) and the Ribosomal Database Project (RDP) (Cole et al., 2003) through BLAST programme. Nucleotide sequence accession All the sequences were submitted to GenBank and accession numbers obtained (Table 1). Antibiotic and metal resistance profile Overnight grown cultures of bacterial strains were used for antibiotic resistance/susceptibility. Bacillus cereus and E. coli K12 were used as a reference for gram positive and negative bacterial strain, respectively. Disc diffusion method was used to check the resistance/sensitivity of bacterial strain towards given antibiotics (Bauer et al., 1966). The bacterial cultures were incubated for 14-16h at 37oC and the zone of inhibition was measured in millimeter. The resistance/sensitivity of the bacterial strains toward any antibiotic was calculated as per the guidelines of the manufacturer (Himedia Laboratories Pvt. Limited, Mumbai, India). Antibiotics used in this study were Amikacin, Ampicillin/Sulbactam, Metronidazole, Cloxaicillin, Oxacillin, Meropenem, Bacitracin, Vancomycin, Erythromycin, Clindamycin, Gentamycin, Oxytetracycline, Ampicillin, Tetracycline, Chloramphenicol, Kanamycin and Streptomycin. International Journal of Integrative Biology ©OmicsVista Group, All rights reserved For metal resistance profile, overnight grown cultures of bacterial strains, Bacillus cereus and E. coli K12 were inoculated and incubated at 37oC under shaking conditions (150rpm). The cultures were incubated for 14-16h and growth in each concentration was recorded and Minimum Inhibitory Concentration (MIC) was determined at millimolar level (Vajiheh et al., 2003.). Metals used in this study were copper(II), cadmium(II), zinc(II), chromium(VI), arsenic(II), manganese(II) and uranium(II). All metal solutions were prepared in milliQ water from respective metal salts. Table 1: A preliminary identification of bacterial isolates by 16s rRNA sequencing. Identification was done based on comparison of 16s rRNA sequences in different data bases, using bioinformatics tool BLAST and submitted to GenBank. Bacterial strain PRS-1 PRS-2 PRS-3 PRS-4 PRS-5 PRS-6 PRS-7 PRS-8 PRS-9 PRS-10 PRS-11 PRS-12 PRS-13 PRS-14 PRS-15 PRS-16 PRS-17 PRS-18 PRS-19 PRS-20 PRS-21 PRS-22 PRS-23 PRS-24 Name Bacillus thuringiensis Bacterium Bacillus sp. Bacterium Bacillus megaterium Bacillus sp. Lysinibacillus sphaericus Pseudomonas aeruginosa Bacillus cereus Acinetobacter junii Bacillus megaterium Comamonas sp. Alcaligenes sp. Stenotrophomonas sp. Alcaligenes sp. Stenotrophomonas maltophilia Achromobacter sp. Stenotrophomonas sp. Bacillus pumilus Pseudomonas stutzeri Bacillus flexus Bacillus thuringiensis Acinetobacter junii Bacillus benzoevorans NCBI Accession EU723845 EU723846 EU744603 EU744601 EU744602 EU744604 EU744605 EU744606 EU744607 EU744608 EU744609 EU744610 EU744611 EU744612 EU744613 EU744614 EU744615 EU744616 EU744617 EU744618 EU744619 EU744620 EU744621 EU744622 Plasmid DNA isolation Plasmid DNA from bacterial strains was isolated by the method of Alkaline lysis (Birnboim et al., 1979) with minor modifications. The cultures were harvested and washed twice with PBS (pH 7.4) and EDTA (pH 8.0). Finally, pellet was resuspended in buffer P-I (50 mM Tris.Cl, 10 mM EDTA) and lysozyme added to the final concentration of 2mg/ml and incubated at 37oC for 30min. Finally, DNA precipitation was done by isopropanol followed by two washes with 70% ethyl alcohol. Plasmid DNA were electrophoresed and separated on a 1.0% agarose gel. Transformation and restriction endonuclease digestion Isolated Plasmid DNA from PRS-3 (named as pPRS3a) was transformed into E. coli DH5α by calcium chloride IJIB, 2009, Vol. 6, No. 2, 58 Antibiotic and metal resistant-bacteria method as per standard molecular biology protocol. From E. coli DH5α-pPRS3a, plasmid DNA was isolated by alkaline lysis method and subjected to restriction enzyme digestion (Sambrook et al., 2001). Restriction enzymes used in this study were: BamHI, BglII, SalI, XhoI, PstI, PvuII, SmaI, NcoI, EcoRI, EcoRV, NotI (MBI Fermentas). Digested Plasmid DNA was electrophoresed and separated on a 1.0% agarose gel. RESULTS Isolation and purification of the microorganisms A total of 24 strains were isolated from soil and water samples. They were distinguished on the basis of their colony morphology on nutrient agar plates. Strain characterization: Gram’s staining and 16s rRNA sequencing All isolated bacterial strains were subjected to Gram’s staining. Based on the results of Gram’s staining, 24 strains (12 Gram-positive and 12 Gram-negative) were found to be unique in shape, size and morphology from one another. These 24 bacterial strains were used for 16s rRNA sequencing with universal primers. The obtained sequences were matched with the existing sequences using BLAST programme in various databases like GenBank (Benson et al., 2000), EMBL (Kanz et al., 2005), Ribosomal Database Project (RDP) (Cole et al., 2003) and finally submitted to GenBank. The putative identification of bacterial strains and GenBank accession nos. have been shown in Table 1. Figure 1: Plasmid DNA profile of bacterial strains. Lane 1, Lambda DNA marker (double digest of EcoR1 & HindIII); lane 2-7, plasmid DNA of PRS-2, PRS-8, PRS-3, PRS-10, PRS-13 and PRS-18 respectively; lane 8, DNA marker (MBI Fermentas, SM0331, 1kb ladder). Arrows show multiple bands of PRS-3. The DNA samples were separated on a 1.0% agarose gel. International Journal of Integrative Biology ©OmicsVista Group, All rights reserved Figure 2: Restriction enzyme digestion profile of pPRS3a. Lane 1, DNA marker*; lane 2, pPRS3a from E. coli DH5α-pPRS3a; lane 3-13, pPRS3a digested with BamHI, BglII, SalI, XhoI, PstI, PvuII, SmaI, NcoI, EcoRI, EcoRV, NotI respectively (4.2kb); lane 14, DNA marker (*MBI Fermentas, SM0331, 1kb ladder). Arrows show a 4.2kb band. The DNA samples were separated on a 1.0% agarose gel. Antibiotic and metal resistance profile For antibiotic resistance/susceptibility profiling, the disc diffusion method was used. Bacillus cereus and E. coli K12 were used as a reference bacterial strain for Gram-positive and Gram-negative respectively. The zone of inhibition was measured in millimeter and the resistance and sensitivity of the bacterial strains toward any antibiotic was calculated as per the guidelines of the manufacturer (Himedia Laboratories Pvt. Limited, Mumbai, India). Most of the strains were resistant to Metranidazol, Meropenem, Bacitracin, Vancomycin and Ampicillin. Some of the strains like PRS-8, PRS10, PRS-12, PRS-13, PRS-17 and PRS-18 were resistant to most of the antibiotics. These results have been shown in Table 2 [Supplementary data]. For metal resistance profile, minimum inhibitory concentration (MIC) was determined at millimolar (mM) level. In the varying concentration of metals, the growth of the bacterial strains was observed as compared to control bacterial strain of the same. Here also Bacillus cereus and E. coli K12 were used as a reference bacterial strain for Gram-positive and Gramnegative respectively. Most of the strains were found to have higher MIC as compared to Bacillus cereus and E. coli K12 at millimolar level for metals like copper(II) and uranium(II). Some of the bacterial strains like PRS2, PRS-3, PRS-8, PRS-13 and PRS-18 had significantly higher MICs for most of the metals as compared to Bacillus cereus and E. coli K12. These MIC results have been shown in Table 3 [Supplementary data]. Plasmid DNA Most of the strains were found to have a good degree of resistance for metals and antibiotics. It is now wellknown that these properties of resistance generally reside on extra chromosomal DNA molecule like plasmid. So, from highly resistant bacterial strains IJIB, 2009, Vol. 6, No. 2, 59 Antibiotic and metal resistant-bacteria plasmid DNA was isolated and all the strains were found to have plasmid DNA of size ranging from 50200kb or mega plasmids, except PRS-3, as shown in gel picture (Fig. 1). Multiple bands in PRS-3 confirm the presence of plasmid DNA which possesses various forms like covalently closed circular, open circular, linear, etc. Transformation and restriction endonuclease digestion The presence of covalently closed circular, open circular, linear, etc. forms of plasmid DNA in PRS-3 makes it a suitable candidate for transformation studies. Therefore, plasmid DNA of PRS-3 (designated as pPRS3a) was transformed into E. coli DH5α, selected on ampicillin and it was found that it can replicate efficiently. This confirms the replication ability of pPRS3a in both Gram-positive/Gram-negative organisms (as PRS-3 is a Bacillus sp.) which may be due to the presence of the origin of replication that can be recognized by both Gram-positive as well as Gramnegative bacterial replication system. This plasmid DNA isolated from DH5α-pPRS3a was subjected to restriction endonuclease digestion (Sambrook et al., 2001) for the confirmation of its size and a single band of 4.2 kb was obtained by using BamHI, SalI, PvuII, SmaI, NcoI, EcoRI, NotI but not by BglII, XhoI, PstI, EcoRV, as shown in the gel picture (Fig. 2). DISCUSSION 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 24 bacterial strains were isolated and first identified by Gram’s staining, followed by 16s rRNA sequencing. Although the sequences were compared in different databases but results obtained were quite ambiguous. Some strains are Gram-positive in nature but during 16s rRNA analysis using databases [GenBank (Benson, et al., 2000), EMBL (Kanz, et al., 2005) and Ribosomal Database Project (RDP) (Cole et al., 2003)] they were found to be a Gram-negative organism and vice versa. Also, some of them exhibited different morphological characters, including different shape and size. This has also been supported by Kevin et al., (2005), who states that “At least 1 in 20 16s rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies”. Therefore, further identification of these strains will be done by FAME (Fatty Acid Methyl Ester) analysis, which have so far been proved to be a better tool for identification (Lawrence et al., 1984; Brown et al., 1996; Kim et al., 2000). There is a clear association between heavy antimicrobial consumption within a population and the International Journal of Integrative Biology ©OmicsVista Group, All rights reserved frequent recovery of antibiotic resistant bacteria (Enne et al., 2001). 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 (Calomiris et al., 1984). Multiple metal resistance bacterial isolates have exhibited high resistance towards a group of antibiotics (Vajiheh et al., 2003) but another study has confirmed that, when the concentration of heavy metal increases, it leads, to a decrease in antibiotic resistance (Tuckfield et al., 2008). 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 previous studies by Vajiheh et al., (2003). It has been found that large plasmids are responsible for encoding resistance to antibiotics and heavy metals (Mergeay et al., 1985; Vajiheh et al., 2003; Sebastien et al., 2003). Also, transferable plasmid encoding resistance to various heavy metals and antibiotics from Salmonella abortus equi (Vajiheh et al., 2003) were reported. Large plasmid DNA from Bacillus spp. [B. thuringiensis (350 kb & 105 kb), B. anthracis (208 kb) and B. cereus (208 kb)] have also been reported (Lars et al., 2008.). To the best of our knowledge, the presence of a 4.2 kb plasmid DNA in Bacillus sp. have not been reported so far. Because of the smaller size (4.2 kb) and presence of the ampicillin resistance marker as confirmed by transformation in E. coli DH5α and the selection on ampicillin, pPRS3a 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 & shuttle sequences analysis, etc. This plasmid DNA can be used for cloning and expression of foreign genes in Bacillus sp. and may have its application in fermentation and other industrial processes. CONCLUSION It was found that metal pollution results in selective pressure and leads to the development of multipleantibiotic resistance among bacterial population, probably through horizontal gene transfer. To overcome this problem, this study strongly emphasizes the need for metal laden waste treatment and safe disposal, thus preventing the transfer of such potential pathogens to human population. Acknowledgement Authors are thankful to the BITS-Pilani for providing laboratory facilities. The financial assistance in the form of Senior Research Fellowship to Pankaj Kumar Jain (09/719/(0027)2008-E.M.R.-I) and S. Ramachandran (09/719/(0013)2003-E.M.R.-I) by the Council of Scientific IJIB, 2009, Vol. 6, No. 2, 60 Antibiotic and metal resistant-bacteria and Industrial Research (CSIR), New Delhi is gratefully acknowledged. Kim WY, Song TW, et al. (2000) Analysis of cellular fatty acid esters (FAMEs) for the identification of Bacillus anthracis. J. Kor. Soc. Microbiol., 35(1): 31-40. References Krulwich TA, Lewinson O, et al. (2005) Do physiological roles foster persistence of drug/multidrug- efflux transporters? A case study. Nat. Rev. Microbiol., 3: 566-572. Alonso A, Sanchez P, et al. (2001) Environmental selection of antibiotic resistance genes. Environ. Microbiol., 3(1): 1-9. Aneja KR (2001) Experiments in microbiology, plant pathology, tissue culture and mushroom production technology, New Age International (P) Limited, New Delhi, India. ISBN: 81-224-1308-0, pp: 69-207. Austin CB, Wright MS, et al. (2006) Co-selection of antibiotics and metal resistance. Trends Microbiol., 14(4): 176-182. Bauer AW, Kirby WWM, et al. (1966) Antibiotic susceptibility tests by standard single disc method. Am. J. Clin. Pathol., 45(4): 493-496. Benson DA, Karsch-Mizrachi I, et al. (2000) GenBank. Nucleic Acids Res., 28(1):15-18. Brown BJ and Leff LG (1996) Comparison of Fatty Acid Methyl Ester Analysis with the Use of API 20E and NFT Strips for Identification of Aquatic Bacteria. Appl. Environ. Microbiol., 62(6): 2183-2185. Birnboim HC and Doly J (1979) A rapid alkaline procedure for screening recombinant plasmid DNA. Nucleic Acids Res., 7(6): 15131523. Calomiris JJ, Armstrong JL, et al. (1984) Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking water. Appl. Environ. Microbiol., 47(6): 1238-1242. Cole J, Chai B, et al. (2003) The Ribosomal Database Project (RDPII): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res., 31(1):442-443. Enne V, Livermore D, et al. (2001) Persistence of sulphonamide resistance of Escherichia coli in the UK despite national prescribing restriction. Lancet, 357(9265): 1325-1328. Foster TJ (1983) Plasmid-determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol. Rev., 47(3): 361409. Godkar BP (1994) Text book of medical laboratory technology, Bhalani Publishing house, Mumbai, India. ISBN: 81-85578-10-9, pp: 311-313. Kanz C, Aldebert P, et al. (2005) The EMBL nucleotide sequence database. Nucleic Acids Res., 33: D29-D33. Kevin EA, Nadia AC, et al. (2005) At least 1 in 20 16s rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies. Appl. Environ. Microbiol., 71(12): 7724-7736. International Journal of Integrative Biology ©OmicsVista Group, All rights reserved Lars A, Kenneth KB, et al. (2008) Detection of large plasmids from the Bacillus cereus group. Plasmid, 59(2): 139-143. Lawrence MM and Gary SS (1984) Application of FAME (fatty acid methyl ester) analysis in the numerical taxonomic determination of bacterial guild structure. Microb. Ecol., 10(3): 283-296. Mergeay M., Nies D, et al. (1985) Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol., 162(1): 328-334. Mesrati LA, Karray MD, et al. (2005) Construction of a new highcopy number shuttle vector of Bacillus thuringiensis. Lett. Appl. Microbiol., 41(4): 361-366. Noriyuki N, Issay N, et al. (1992) A new shuttle vector for Bacillus stearothermophilus and Escherichia coli. Biotechnol. Lett., 14(8): 649-652. Novick RP and Roth C (1968) Plasmid-linked resistance to inorganic salts in Staphylococcus aureus. J. Bacteriol., 95(4): 1335-1342. Rehman A, Shakoori FR, et al. (2007) Multiple metal resistance and uptake by a ciliate, Stylonychia mytilus, isolated from industrial effluents and its possible use in wastewater treatment. Bull. Environ. Contam. Toxicol., 79(4): 410-414. Sambrook J, Fritsch EF, et al. (2001) Molecular cloning-A laboratory manual, 3rd Ed., Cold Spring Harbour Laboratory Press, New York, USA, ISBN: 978-087969577-4. Sébastien M, Tatiana V, et al. (2003) Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. FEMS Microbiol Rev., 27(2-3): 385-410. Summers AO (2002) Generally overlooked fundamentals of bacterial genetics and ecology. Clin. Infect. Dis., 34: S85-S92. Summers AO, Wireman J, et al. (1993) Mercury released from dental silver fillings provokes an increase in mercury-resistant and antibiotic resistant bacteria in oral and intestinal floras of primates. Antimicrob. Agents Chemother., 37(4): 825-834. Therese KL, Anand AR, et al. (1998) Polymerase chain reaction in the diagnosis of bacterial endophthalmitis. Br. J. Ophthalmol., 82(9): 1078-1082. Tuckfield RC and McArthur JV (2008) Spatial analysis of antibiotic resistance along metal contaminated streams. Microb. Ecol., 55(4): 595-607. Vajiheh K, Naser B, et al. (2003) Antimicrobial, heavy metal resistance and plasmid profile of coliforms isolated from nosocomial infections in a hospital in Isfahan, Iran. African J. Biotechnol., 2(10): 379-383. IJIB, 2009, Vol. 6, No. 2, 61