Cadmium resistant Enterobacter cloacae and ... - Springer Link

World Journal of Microbiology & Biotechnology 15: 283±290, 1999. Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Cadmium resistant Enterobacter cloacae and Klebsiella sp. isolated from industrial e‚uents and their possible role in cadmium detoxi®cation Riazul Haq, Sayyed Kaleem Zaidi and A.R. Shakoori* Cell and Molecular Biology Laboratory, Department of Zoology, University of the Punjab, Lahore, Pakistan *Author for correspondence: Tel.: 92-42-5864028, Fax: 92-42-5868376, E-mail: [email protected] Received in revised form 8 August 1998; accepted 15 January 1999

Keywords: Cadmium detoxi®cation, cadmium resistant bacteria, Enterobacter cloacae, heavy metal resistance, industrial e‚uent, Klebsiella

Summary Three bacterial strains, two of Klebsiella sp. and one Enterobacter cloacae were isolated from industrial e‚uents of chemical and textile industries. They showed high eciency in removing cadmium (Cd2+) from the medium. When 100 lg/ml of Cd was added to the medium, the three isolates namely CMBL-Cd1, CMBL-Cd2 and CMBL-Cd3 removed or accumulated 86%, 87% and 85% of Cd, respectively, from the medium within 24 h. Plasmids were detected in all the three strains. Plasmids of E. cloacae (pCBL1) and Klebsiella sp. (pCBL2 and pCBL3), estimated to be 6.6 kb, were used to transform Escherichia coli C600. The transformed E. coli cells showed elevated resistance to Cd. Ethidium bromide curing indicated the presence of the Cd resistance gene on the plasmid. Resistance of the isolated strains against other metals like chromium (cr6+) and lead (pb2+) and a number of antibiotics was also checked. Cured strains showed lowered resistance against Cr and some antibiotics. This again supported the indication of the presence of Cd, Cr and some antibiotics resistance genes on plasmids. Introduction Cadmium (Cd) is a heavy metal contaminant in the environment. Extensive data suggest Cd as the most toxic heavy metal and it is included on the black list of several international agreements established to regulate the input of Cd into the environment (Jeanthon & Prieur 1990; Ray et al. 1993; Pacheco et al. 1995). It is extensively used in the industry for a number of applications, including electroplating, protection against corrosion and stabilizing plastic (Lebrun et al. 1994). The waste waters of these industries may contain Cd ranging from 10 mM to 100 mM (Shuttleworth & Unz 1988). Cd can enter the human food chain through plants, smoking materials and diet (Dabeka & Mckenzie 1992; Kalcher et al. 1993). Cd is carcinogenic, embryotoxic, teratogenic and

mutagenic and may cause hyperglycemia, reduced immunopotency and anaemia, due to its interference with iron metabolism (Sanders 1986). The toxicity of Cd has also been well documented in selective types of almost all major phyla of eukaryotes (Rainbow 1995; Unger & Roesijadi 1996). Among microorganisms, bacteria, yeast and protozoa are generally the ®rst category to be exposed to heavy metals present in the environment (Gelmi et al. 1994). Microorganisms have acquired a variety and array of mechanisms to remove or detoxify toxic metal ions. They remove toxic metal ions via adsorption to cell surfaces (Mullen et al. 1989), complexation by exopolysaccharides (Scott & Palmer 1988), binding with bacterial cell envelopes (Flatau et al. 1987), intracellular accumulation (Laddaga & Silver 1985), biosynthesis of

284 metallothioneins and other proteins that trap metals (Higham et al. 1984), precipitation (Aiking et al. 1985) and transformation to volatile compounds (Robinson & Tuovinen 1984). Bacteria are the microorganisms most exposed to Cd toxicity. Cd ions are bactericidal resulting in exponential killing that starts immediately after exposure (Inbar & Ron 1993). Cd is also reported to induce single-stranded breaks in DNA with coordinated e€ect of free radical generation (Din & Ahmed 1997). Cd-resistant bacteria have been isolated in a number of studies which explain the mechanism of Cd uptake in bacteria (Nies & Silver 1995). Moreover, it is also indicated that Gram negative bacteria are highly resistant to Cd2+ ions and accumulate great amounts of Cd during growth. Gram positive bacteria on the other hand, show a heterogenous behaviour and the intracellular pool reaches a plateau on increasing Cd concentration (Gelmi et al. 1994). An ecient waste water treatment strategy which has emerged as a result of a large number of studies on the ability of microorganisms to detoxify industrial e‚uents is bioremediation. This natural environment clean-up is reported to be superior to and safer than mechanical or chemical clean-up strategies (Grasius et al. 1997). Many di€erent assays such as molecular assay, DNA probing assay, microtox assay, biosensors and bioassays are nowadays in practice to assess the heavy metal contamination of the environment and its remediation (Beath 1992; Kong et al. 1995). Of these, bioassay involving the use of microorganisms to monitor and tackle the heavy metal pollution in the environment was the objective of this study. Three Cd-tolerant bacterial strains were isolated from industrial e‚uents. Their growth conditions, biochemical and molecular characters were studied to exploit these bacteria for clean-up of industrial waste waters containing Cd. Materials and Methods Sample collection and isolation of strains Industrial e‚uent samples were collected from the waste waters of Ittehad Chemicals and Ravi Rayon Food and Plastic Industries situated 20 miles from Lahore city on Sheikhupura road and

R. Haq et al. from the textile industry of Faisalabad city. A total of eight samples were taken from both localities in sterilized screw-capped bottles and brought to the laboratory. Luria Bertani (LB) agar plates (tryptone 10 g, yeast extract 5 g, sodium chloride 5 g added to 1 l distilled water, pH adjusted to 7±7.5, 15 g agar was added, and autoclaved) containing CdCl2 with Cd2+ concentration 10 lg/ml of the medium were used to select Cd-resistant bacteria. Bacterial colonies growing on the plates were puri®ed by streaking and re-streaking. Identi®cation and characterization of the bacterial strains Bacterial isolates were grown on blood agar (Merck) and MacConkey agar (Difco) media. The shape and colour of colonies were observed and bacterial cells were examined under the microscope after Gram staining. The isolates were biochemically analysed for the activities of oxidase, catalase, b-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease and tryptophan deaminase. Besides that, the isolates were also tested for motility, H2S production, indole production and utilization of glucose, mannitol, sorbitol, rhamnose, sucrose, melibiose, arabinose, inositol and amygdalin. These tests were undertaken to identify and characterize bacterial isolates according to procedures described in Holt et al. (1994). Determination of optimal growth conditions The optimal growth conditions with reference to pH and temperature were determined. The isolates were grown in LB liquid medium with nine different pH values i.e. 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 & 9 and incubations done at temperatures i.e. 25 °C, 30 °C, 37 °C and 42 °C. O.D. of the log phase growing cultures (8±10 h) in all the above-mentioned conditions was recorded at 600 nm to determine the optimum growth. All the experiments were performed three times. Determination of heavy metal tolerance Maximum resistance of the bacterial isolates against Cd2+ was determined by gradually increasing the concentration of Cd2+ as cadmium

Cadmium resistant bacteria chloride (10 lg/ml each time) in LB agar plates until the strains were unable to give colonies on the plates. The initial concentration used was 10 lg/ ml. The culture growing on the last concentration was transferred to the higher concentration by streaking on the plates. Minimum inhibitory concentration (MIC) was noted when the isolates failed to grow on plates even after ten days of incubation. Resistance of the isolated strains against Cr6+ and Pb2+ was also checked by observing growth of the strains on LB agar plates containing various concentrations of heavy metals. The starting concentration of Cr6+ (K2Cr2O7) and Pb2+ (lead acetate) was 50 lg/ml, which was gradually increased 20 lg/ml each time until MICs were achieved. Determination of antibiotic resistance The various bacterial isolates were tested for antibiotic sensitivity by growing these bacteria on LB agar plates with antibiotic discs (Difco) of Enoxocin, Optochin, Erythromycin, Minocyclin, Augmentin, Ceptazimide, Gentamicin, Ampicillin, Doxicyclin and Septron (25 lg on each disc), at 37 °C for 24 h. Formation of an inhibition zone around the discs was taken as the indicator of bacterial sensitivity to antibiotics. Plasmid isolation and transformation Plasmids from bacterial isolates were isolated according to Holmes (1984). The size of the plasmids was determined by agarose gel electrophoresis. k-HindIII cut was used as a marker for comparison. The plasmids were used to transform E. coli C600. The transformation procedure adopted was as described by Sambrook et al. (1989). The transformants were selected on selective medium plates containing 100 lg Cd/ml. Curing of plasmids Curing of the plasmids was done by using di€erent concentrations of ethidium bromide ranging from 50 lg/ml to 300 lg/ml (Lakshmi et al. 1988; Crosa et al. 1994). Ethidium bromide was ®ltered sterilized and added in the autoclaved medium after cooling down. Bacteria were able to grow at 300 lg/ml. The ethidium bromide-containing me-

285 dium was inoculated with bacterial isolates and incubated for 48 h with vigorous shaking. Samples were taken out of the medium, diluted with LB broth and spread on plates to get well-separated colonies. Isolated colonies were tooth-picked and transferred simultaneously onto grids made on LB agar medium plates with and without 100 lg Cd/ ml. The colonies appearing on LB agar plates without Cd and not on plates containing Cd were counted as the ones with their plasmids cured. The original bacterial isolates as well as cured and transformed bacteria were checked for their antibiotic resistance to ascertain curing of the plasmids and transformation. Sensitivity or resistance of bacteria against antibiotics was checked by using sensi discs of antibiotics i.e. Enoxocin, Optochin, Erythromycin, Minocyclin, Augmentin, Ceptazimide, Gentamicin, Ampicillin, Doxicyclin and Septron. Cd2+, Cr6+ and Pb2+ at a concentration of 100 lg/ml each, were used to check resistance of uncured and cured cultures. Change in resistance against antibiotics and Cr6+ was used as an indicator of curing and transformation. Cd estimation Bacterial cultures were grown in LB liquid medium containing 100 lg Cd2+/ml of the medium for di€erent time intervals viz. 4, 8, 12, 16, 20 and 24 h. Each time the cultures were spun at 6000 rev/ min to remove bacterial cells, and the supernatant used to determine Cd2+ concentration by atomic absorption spectrophotometry at 228.8 nm using a Cd lamp (Wilson & Walker 1994). The amount of Cd in samples after various intervals of time was estimated by using a standard curve, which was prepared by taking various known concentrations of CdCl2 in the medium. Reduction in the amount of Cd in the medium after growth of bacteria was taken as the Cd processing ability of the isolates. Results Isolation and characterization of Cd-tolerant bacteria Three Cd-tolerant bacterial strains (CMBL-Cd1, CMBL-Cd2, CMBL-Cd3) were isolated from eight di€erent industrial water samples and MIC of Cd

286

R. Haq et al.

for these strains was checked. The strain CMBLCd1 tolerated 220 lg of Cd/ml in the medium, while strain CMBL-Cd2 and CMBL-Cd3 tolerated 110 lg of Cd/ml in the medium at the maximum. On the basis of microbiological and biochemical tests performed, the strain CMBL-Cd1 was identi®ed as Enterobacter cloacae, while CMBL-Cd2 and CMBL-Cd3 were identi®ed as belonging to the Klebsiella genus (Holt et al., 1994). All the three isolates formed creamy white colonies on blood agar and pink colonies on MacConkey agar, were Gram negative motile bacilli, showing positive catalase and b-galactosidase activities; and they could utilize glucose, mannitol, sorbitol, rhamnose, sucrose, arabinose and amygdalin. E. cloacae showed positive arginine dihydrolase and ornithine decarboxylase activities and utilize citrate and melibiose. The Klebsiella spp, on the other hand, showed positive urease activity and could utilize inositol. E. cloacae and one of the Klebsiella strains (CMBL-Cd2) showed optimum growth at 30 °C and pH 7.0, while the second Klebsiella strain (CMBL-Cd3) showed optimum growth at 37 °C and pH 7.0. Resistance to other heavy metals and antibiotics The resistance of E. cloacae (CMBL-Cd1) and Klebsiella spp. (CMBL-Cd2 and CMBL-Cd3) was also checked against Cr6+ and Pb2+. CMBL-Cd1 showed resistance against Cr and Pb up to

a maximum concentration of 800 lg/ml and 1400 lg/ml, respectively. CMBL-Cd2 showed resistance against 600 lg Cr/ml and 1200 lg Pb/ml, whereas CMBL-Cd3 showed resistance against maximum concentration of 500 lg Cr/ml and 900 lg Pb/ml. All the three isolates were sensitive to Enoxocin, Minocyclin, Doxicyclin and Septron, and resistant to Optochin, Gentamicin and Ampicillin. Klebsiella spp. were resistant to Erythromycin, Augmentin and Ceptazimide, whereas E. cloacae was sensitive to all these three antibiotics (Table 1). Heavy metal resistance gene on plasmid DNA The plasmid isolated from E. cloacae was designated as pCBL1, while plasmids of the two Klebsiella spp. were named as pCBL2 and pCBL3 for strain CMBL-Cd2 and CMB-Cd3, respectively. The estimated size of the plasmids was 6.6 kb. The location of heavy metal resistance genes on plasmid DNA was determined by plasmid curing and transformation experiments. Plasmid curing. The loss of resistance to Cd in cured strains was used as an indicator of curing (Table 1). Out of 260 colonies transformed each for E. cloacae and Klebsiella sp. 1 (CMBL-Cd2), 203 and 198 colonies were cured, respectively, showing thereby 78% and 76% curing for the two bacterial isolates, respectively. For Klebsiella sp. 2

Table 1. Antibiotic and heavy metal resistance of three bacterial isolates (Enterobacter cloacae, CMBL-Cd1; Klebsiella sp., CMBLCd2, Klebsiella sp., CMBL-Cd3) before (uncured) and after treatment (cured) with ethidium bromide.

Enoxocin Optochin Erythromycin Minocyclin Augmentin Ceptazimide Gentamicin Ampicillin Doxicyclin Septron Cd (100 lg/ml) Cr (100 lg/ml) Pb (100 lg/ml)

CMBL-Cd1

CMBL-Cd1 (cured)

CMBL-Cd2

CMBL-Cd2 (cured)

CMBL-Cd3

CMBL-Cd3 (cured)

S R S S S S R R S S R R R

S R S S S S S* R S S S* S* R

S R R S R R R R S S R R R

S R R S S* S* ± R S S S* S* R

S R R S R R R R S S R R R

S S* S* S S* S* ± S* S S S* S* R

R, resistant; S, sensitive; *, changed from resistant to sensitive; ±, not determined.

Cadmium resistant bacteria (CMBL-Cd3), 154 colonies were cured out of a total of 230 transformed, showing 67% curing eciency. The heavy metal resistance of cured and uncured strains was compared to determine the location of genes for metal resistance. The results showed that while the E. cloacae resisted Cd2+, Cr6+ and Pb2+ at concentrations of 220 lg/ml, 800 lg/ml and 1400 lg/ml of the medium respectively, it could not grow on medium containing Cd2+ and Cr6+ concentrations of 100 lg/ml each after curing. Similarly, Klebsiella strain CMBLCd2 resisted Cd2+, Cr6+ and Pb2+ concentrations of 110 lg/ml, 600 lg/ml and 1200 lg/ml of the medium, respectively, while the cured Klebsiella strain could not grow in medium containing Cd2+ and Cr6+ concentrations of 100 lg/ml each. Klebsiella strain CMBL-Cd3 resisted Cd2+, Cr6+ and Pb2+ concentrations of 110 lg/ml, 500 lg/ml and 900 lg/ml of the medium, respectively, while it could not grow in medium containing Cd2+ and Cr6+ 100 lg/ml each of the medium after curing. All the three strains showed growth on medium containing Pb2+ before curing and after curing. The antibiotic resistance of uncured and cured cultures of the isolates was compared to determine the location of antibiotic resistance genes. The comparison of resistance before and after curing is shown in Table 1. This was taken as an indication of curing of the plasmids.

287 medium after removal of bacterial cells at the end of the growth and processing period. The results showed that all the three strains were ecient in removal of Cd (Figure 1). Di€erences were observed in the eciency of the three strains. E. cloacae and Klebsiella strain 1 (CMBL-Cd2) were more ecient during the late log phase and less ecient during the early log phase, while Klebsiella strain 2 (CMBL-Cd3) was less ecient in the late log phase and more ecient in the early log phase. Within 4 h of inoculation, the culture medium showed 52% reduction in the presence of CMBLCd3 as against 9% decrease in the presence of CMBL-Cd2 and 17% decrease in the presence of CMBL-Cd1. After 8 h of incubation, this reduction was 65% for CMBL-Cd1 and 2 and 52% for CMBL-Cd3. After 24 h, this reduction ranged between 85±87% in the three strains. Discussion Bacteria have been so continuously exposed to heavy metal contaminants of the environment that they have developed genetically determined

Transformation. The plasmid preparations were used to transform E. coli C600 cells. Success of transformation was determined by growth of transformants on medium containing 100 lg Cd/ ml of the medium. E. coli C600 and competent cells before transformation were also spread on plates containing Cd as control. E. coli C600 and competent cells showed no growth on medium containing Cd at a concentration of 100 lg/ml. Competent cells transformed with the three plasmids (i.e. pCBL1, pCBL2, pCBL3), separately, showed growth after 24 h on plates containing Cd (100 lg/ml). Reduction in amount of Cd caused by the bacterial isolates Cd processing ability of the bacterial isolates was checked by estimation of the amount of Cd in the

Figure 1. Reduction in amount of Cd after inoculation of the three bacterial strains, checked by atomic absorption spectrophotometric estimation of cadmium in the medium after various time intervals. The medium containing the same original amount of Cd but without inoculation was taken as control. The initial concentration of Cd2+ in the medium was 100 lg/ml.

288 resistance systems against heavy metal toxicity (Ji & Silver 1995; Parsek et al. 1995). These systems may be (1) enhanced expression of genes involving regulation of operons, acquisition of more copies of genes on plasmids and gene transfer through plasmids (Suen & Spain 1993), (2) coordinated expression of pre-existing genes (Daubaras & Chakrabarty 1992), (3) integrated regulation of ancestral and newly evolved genes through promoter regions of the gene (Parsek et al. 1995), and (4) a continuous alteration in DNA sequences that may lead to a favorable adaptation (Roelof Van der Meer 1994). Contaminated environments like those in the vicinity of industries or industrial dump grounds accumulate a heavy burden of toxic metal ions, organic wastes and antibiotics. A large number of bacterial isolates from these areas have depicted a pronounced capability of processing and resisting toxic industrial wastes. Cadmium has no known biological role in animals and metallothioneins against Cd has been reported in eukaryotic cells (Tamai et al. 1993). The bacteria isolated in this study showed relatively high resistance against Cd. This capability gave an insight into the potential of processing this toxic metal through the use of bacteria. In this study pH, temperature and inoculum size for the optimum growth of the isolates were checked. These information would help in better exploitation of the bacterial strains for detoxi®cation of Cd and related environmental clean-up. Resistance of the strains against other metal ions i.e. Pb2+ and Cr6+ shows versatility of the strains in resistance against heavy metals. All the three bacteria isolated in this study show a high eciency in removal of Cd from the medium. They accumulated a signi®cant amount of Cd within 24 h. Cadmium uptake is reported to be through an ATPase pump in Gram positive bacteria and by the action of proton-cation antiporters in Gram negative bacteria (Nies & Silver 1995). The removal of Cd from the medium is indicative of Cd accumulation inside the Gram negative cells. This capability of the strains render them appropriate for bioremediation of contaminated environment. Microbes used for detoxi®cation of wastes containing heavy metals would not produce secondary wastes which are inevitable by-products of chemical treatment plants.

R. Haq et al. Resistance against metals is frequently coded by the plasmids present in bacteria (Ohman 1988). Curing of the plasmids in the isolated strains was done by using ethidium bromide in order to determine the presence of metal ion resistance genes on plasmid or chromosomal DNA (Crosa et al. 1994). This study also revealed the high eciency of ethidium bromide in curing the plasmids (Fig. 2). Cured and uncured cultures were compared for their resistance against metal ions and antibiotics. The comparison clearly showed the di€erence in resistance and it was concluded that plasmids harboured the genes for antibiotic resistance and metal ion resistance. Cadmium resistance gene was present on all the three plasmids as well as in the gene for Cr resistance. Lead resistance gene was shown to be present on the chromosomal DNA rather than the plasmid DNA as the cured and uncured cultures remained similar in Pb resistance. The study gives clues towards multidimensional strategies which can be adopted for environmental cleanup. The bacterial isolates may be suitable for Cd removal from industrial wastes. Optimized growth conditions would help in ecient growth

Figure 2. Agarose gel electrophoresis picture showing plasmids isolated from various cultures; wild type (lane 1, Plasmid of Enterobacter cloacae pCBL1; lanes 2 and 3, plasmids of Klebsiella sp. pCBL2, pCBL3, respectively), kHindIII cut DNA (lane 4), E. coli C600 transformants (lane 5, pCBL1; lane 6, pCBL2; lane 7, pCBL3) and cured cultures (lane 8, preparation from Enterobacter cloacae CMBL-Cd1; lane 9, Klebsiella sp. CMBL-Cd2; lane 10, Klebsiella sp. CMBL-Cd3).

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