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1 Ecological Indicators xxx (2006) xxx–xxx

This article is also available online at:
www.elsevier.com/locate/ecolind

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3 Characterization of marine bacteria highly resistant to mercury
exhibiting multiple resistances to toxic chemicals
4
De Jaysankar*, N. Ramaiah

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5
6 National Institute of Oceanography, Dona Paula, Goa 403004, India
7 Received 24 December 2005; received in revised form 23 March 2006; accepted 20 May 2006
89

10
Abstract
11
12
13
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Several strains of bacteria unusually highly resistant to mercury were isolated from seawater and marine sediment samples
and identified by 16S rDNA sequencing and were also characterized by a battery of biochemical and morphological tests. The
14 bacterial isolates were identified to belong to the genera Pseudomonas, Alcaligenes, Brevibacterium and Bacillus. Many of the
15 chosen isolates were tested for growth in the presence of different heavy metals and a variety of xenobiotics. Growth curves of all
16 six bacteria highly resistant to mercury examined for growth at different concentrations of Hg exhibited prolonged lag phase,
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17 during which time necessary physiological adaptations to toxic milieu were undergone. All the strains tested for antibiotic
18 resistance showed little to no effect of antibiotics on their normal growth. Results of this study demonstrate the occurrence of
19 diverse groups of marine prokaryotes capable of high tolerance to mercury with a potential to degrade a variety of toxic heavy
20 metals and xenobiotics.
21 # 2006 Published by Elsevier Ltd.
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22 Keywords: Marine; Mercury-resistant bacteria; Characterization; Heavy metals; Xenobiotics; Plasmid

23
24 30
1. Introduction environment (Fukuda et al., 1999; Horvat et al., 1999). 31
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25 Elemental mercury, its ore cinnabar and several of its 32

26 Mercury is the most toxic of the heavy metals compounds enter the aquatic environment through 33
27 (Gerlach, 1981) and occupies the sixth position in the leaching and washing of soils, rocks, and the 34
28 list of hazardous compounds (Nascimento and atmosphere by rain (Pahan et al., 1990). In addition, 35
29 Chartone-Souza, 2003). Worldwide many areas con- anthropogenic contributions through which high 36
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30 taminated with mercury pose threat to people and concentration of mercury can reach the aquatic 37
environment include chloro-alkali production, instru- 38
ment manufacturing, chemical laboratories and 39
* Corresponding author. Present address: Graduate School of dentistry (Osborn et al., 1995). The dilution and 40
Kuroshio Science (GRAKUS), Kochi University, Nankoku, Kochi
783-8502, Nippon, Japan. Tel.: +81 88 864 5152;
dispersion of dissolved mercury is continuous and 41
fax: +81 88 864 5157. both natural and manmade causes lead to increased 42
E-mail address: jaysankarde@yahoo.com (J. De). concentrations of mercury in the marine environment. 43

1470-160X/$ – see front matter # 2006 Published by Elsevier Ltd.

doi:10.1016/j.ecolind.2006.05.002

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 2 J. De, N. Ramaiah / Ecological Indicators xxx (2006) xxx–xxx
43 91
44 Further, mercury has an affinity to bind with organics 2. Materials and methods
45 forming generally recalcitrant and highly toxic 92

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46 organomercurial complexes in marine sediments 2.1. Bacterial isolates
47 (Gerlach, 1981; Misra, 1992) and is bioaccumulated 93
48 through food chain. Mercury-resistant bacteria in water and sediment 94
49 Certain environmental strains of bacteria have complexes were isolated from various locations along 95
50 acquired highly specific resistance mechanisms for the Indian Coast during 1999–2002 using seawater 96

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51 mercury. There is considerable evidence about nutrient agar (SWNA: 5.0 g peptone, 1.5 g beef 97
52 mercury resistance among common microbial species extract, 1.5 g yeast extract, 500 ml aged seawater, 98
53 (Canstein et al., 1999; Macalady et al., 2000; Barkay 500 ml deionized water and 15 g agar) amended with 99
54 et al., 2003). Mercury-resistant bacteria are extremely 10 ppm (
50 mM Hg) HgCl2 (Merck, Germany). 100
55 important in detoxifying the mercury compounds by After enumerating MRB several single colonies were 101

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56 two sequentially acting enzymes namely, organomer- picked and streaked onto SWNA plates containing 102
57 curial lyase that cleaves the carbon–mercury bonds of 25 ppm mercury for further purification. These 103
58 certain organomercurials and mercuric reductase, isolates were found to have obligate requirement for 104
59 which reduces Hg2+ to volatile mercury (Nakamura sodium for their growth suggesting their marine origin 105
60 et al., 1990). Some bacteria have broad-spectrum (Baumann et al., 1972). Based on colony character- 106
61 resistance, i.e., resistance to both Hg2+ and certain istics, 13 isolates originating mainly from coastal 107
62
63
64
organomercurial compounds whereas some are resis-
tant only to Hg2+. Under anaerobic conditions some
bacteria are not harmed by mercury as it is changed
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samples collected off Goa on the West Coast and off
Chennai and Gopalpur on the East Coast of India were
selected for 16S rDNA sequencing and further
108
109
110
65 directly into apparently non-toxic mercury sulfide detailed experimentation were carried out involving 111
66 (Gerlach, 1981). some of the most potential isolates. 112
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67 Although the plasmid-mediated resistance to Hg
68 has been linked to resistance to antibiotics and also 2.2. 16S rDNA sequencing
69 to 2,4-dichlorophenoxyacetic acid (Ka et al., 1994), 113
70 few previous studies have examined mercury- Overnight grown cultures were used for extraction 114
71 resistant bacteria (MRB) and their potential to of the genomic DNA and the extraction was done 115
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72 catabolize toxic xenobiotics (Barbieri et al., 1989, using the Necleospin Extract (Macherey Nagel, 116
73 1996; De et al., 2003). In the recent years, there has Germany) and the quality of the product was checked 117
74 been an unusual increase in the numbers of mercury- on 0.8% TAE agarose gel. The sequencing was done 118
75 resistant bacteria (Ramaiah and De, 2003). In this using forward primer (27f: 50 -AGAGTTT- 119
76 study, we aimed at characterizing bacterial isolates GATCCTGG CTCAG-30 ; Weisberg et al., 1991) and 120
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77 that grew well in the presence of 25 ppm reverse primer (1492r: 50 -GGTTACCTTACGACTT- 121
78 (
125 mM) Hg. We have designated them as 30 ; Reysenbach et al., 1992). The PCR was done in a 122
79 bacteria highly resistant to mercury (BHRM; De thermocycler (PE, Applied Biosystem) using a cyclic 123
80 et al., 2003). We have also examined their ability to program of 94 8C (1 min) followed by 35 cycles of 124
81 grow in a range of toxic heavy metals and 94 8C (0.10 min), 54 8C (0.20 min), 68 8C (1.30 min) 125
82 xenobiotics. This was done mainly to evaluate their with a final extension temperature of 68 8C (4.30 min). 126
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83 physiologic response and their potential to degrade The PCR products were electrophoresed on 0.8% TAE 127
84 an assortment of randomly chosen toxicants (which agarose gel stained in ethidium bromide solution 128
85 reach the marine environment through one or the (1 mg ml1) and were photographed using a transillu- 129
86 other route) that have been reported to cause serious minator. The PCR products were cleaned using the 130
87 harm to biota including human. Presence of QIAquick purification kit (Qiagen, USA). The cleaned 131
88 plasmids that may mediate Hg detoxification, confer PCR products were extended further using big dye 132
89 antibiotic resistance and enable the BHRM to grow sequencing kit (version 1.1) in a thermocycler using a 133
90 in presence of various heavy metals and xenobiotics program of 95 8C followed by 25 cycles of 55 8C 134
91 were also checked. (0.20 min) and 60 8C (3.30 min). The final extension 135

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 J. De, N. Ramaiah / Ecological Indicators xxx (2006) xxx–xxx 3
135 177
136 products were cleaned further using ethanol–sodium nosa), GP08 (B. pumilus), GP13 (Brevibacterium 178
137 acetate (EtOH–NaAc) and speedvac system. The iodinium), GO02 (A. faecalis), GP16 (A. faecalis), 179

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138 sequencing was done using ABI sequencing instru- GP17 (A. faecalis), GP14 (B. iodinium), GP06 (A. 180
139 ment (ABI PRISOM model no. 3700). The sequence faecalis), CH13 (B. pumilus), 3C (B. pumilus; an MRB 181
140 was compared online, checked for the match using but not BHRM), one mercury-sensitive (unidentified) 182
141 FASTA program (http://www.ebi.ac.uk/) for identifi- and Pseudomonas putida KT2442<mer73 (positive 183
142 cation and were submitted to the gene bank (accession control; Deckwer et al., 2004) were grown in marine 184

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143 numbers: DQ377441–DQ377468). broth for 24 h and the cells from 2 ml broth culture 185
were pelleted by centrifugation at 10,000 rpm. The 186
2.3. Biochemical characterization cells were washed with phosphate buffer and placed in 187
144 wells of microtitre plates. Mercury stock solution was 188
145 Several biochemical tests were carried out to added to the phosphate buffer to a final concentration 189

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146 characterize strains of BHRM. Presence of various of 10 ppm and then 1 ml mercurated phosphate buffer 190
147 enzymes such as lipase, gelatinase, amylase, oxidase, was added to wells containing the cells. The whole 191
148 catalase, urease, decarboxylases (arginine and plate was covered with Kodak XAR film and the plate 192
149 ornithine), utilization of different carbon sources such was incubated at 30 8C in dark for 4 h. After 193
150 as gluconate, pyruvate, citrate, cellobiose, glucose, incubation, the XAR film was removed and developed. 194
151 sucrose, mannitol, arabinose, rhamnose, nitrate reduc-
152
153
154
tion, Methyl Red (MR), Voges–Proskaeur (VP)
reaction, H2S production and oxidation–fermentation
were checked as described by MacFaddin (1980).
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2.6. Resistance of BHRM to other toxicants

The ability of BHRM to grow in the presence of

195
196
different toxic xenobiotics was tested by adding these 197
2.4. Growth characteristics of BHRM chemicals at different concentrations into the growth 198
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155 media. Cultures were incubated at 28 8C for a 199
156 The growth kinetics of BHRM in the presence and maximum of 14 days and inspected regularly for 200
157 absence of mercury singly and combination with DDT growth. The presence of growth was confirmed by 201
158 (Bayer, Germany) and Phenol (Qualigens, India) was streaking a loopful of liquid culture on to SWNA as in 202
159 determined in seawater nutrient broth (SWNB). some cases growth could not be discerned due to 203
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160 Twenty-four hour old cultures of six BHRM isolates turbidity produced by some xenobiotics in SWNB. 204
161 (CH07: Pseudomonas; GP08: Bacillus pumilus; GO01, The changes in growth of BHRM in presence of 205
162 GP15, GP16 and GP17: Alcaligenes faecalis) were different toxicants were monitored. A reference strain, 206
163 inoculated in replicates of three SWNB flasks with Hg Photobacterium leognathi (ATCC 25521: PL3; 207
164 concentrations of 0, 10 and 50 ppm. The flasks were Ramaiah, 1989) unable to grow in medium containing 208
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165 incubated on a rotary shaker (maintained at 180 rpm) at 1.0 ppm Hg was included as a control to ascertain 209
166 room temperature (28 8C) for 120 h. The growth from the Hg tolerance of environmental isolates studied. 210
167 all the flasks was monitored at a regular interval and log-
168 transformed cell counts (number ml1) were plotted to 2.7. Antibiotic sensitivity
169 draw growth curves. The growth patterns of two isolates 211
170 (CH07 and GP16) capable of tolerating either 75 ppm Sensitivity of all the BHRM to kanamycin, 212
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171 Hg in SWNA, 100 ppm DDT in SWNB or 1000 ppm streptomycin, chloramphenicol, neomycin, tetracy- 213
172 phenol in SWNB individually were determined in cline and ampicillin was examined by antibiotic disc 214
173 SWNB with 20 ppm Hg + 20 ppm phenol + 10 ppm sensitivity assay. The isolates were determined to be 215
174 DDT in combination. sensitive or resistant to antibiotics based on the 216
sensitivity chart supplied by the manufacturers 217
2.5. Mechanism of detoxification of mercury (Himedia, India) beside of comparing to an Escher- 218
175 ichia coli strain. Results are shown for three strains of 219
176 Eleven BHRM isolates, viz. GP15 (A. faecalis), A. faecalis (GP06, GP15 and GP16) and one P. 220
177 CM10 (Bacillus sp.), CH07 (Pseudomonas aerugi- aeruginosa (CH07) which showed comparatively 221

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Table 1
16S rDNA identity of BHRM (compared with FASTA)

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Code Sequence match (%), Sequence match (%), Identity on the basis of
forward strand reverse strand 16S rDNA sequences
GO02 99.59 100 Alcaligenes faecalis
GP06 99.58 99.78 Alcaligenes faecalis
GP08 100 99.74 Bacillus pumilus

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GP13 98.54 99.02 Brevibacterium iodinium
GP14 98.94 100 Alcaligenes faecalis
GP15 99.56 99.79 Alcaligenes faecalis
GP16 99.65 100 Alcaligenes faecalis
GP17 99.56 100 Alcaligenes faecalis
CH07 100 100 Pseudomonas aeruginosa
CH13 100 99.75 Bacillus pumilus

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CM10 96.08 99.48 Bacillus sp.
S3 99.77 99.77 Bacillus pumilus
GO01 100 100 Alcaligenes faecalis
221 247
222 higher resistance to toxic heavy metals and xenobio- isolates (GP08, CH13 and S3) as B. pumilus and one 248
223 tics and were used in more detailed experiments. each of P. aeruginosa (CH07), B. iodinium (GP13) and 249
224
225
226
Antibiotic sensitivity of all three isolates after plasmid
curing assays was also examined to investigate
whether the antibiotic resistance was associated with
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Bacillus sp. (CM10) from 16S rDNA sequencing
(Table 1). Biochemical characteristics of the BHRM,
i.e., those capable of growth in SWNB with 25 ppm
250
251
252
227 any plasmid that might confer mercury resistance in Hg are listed in Table 2. 253
228 these strains.
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3.2. Growth characteristics
2.8. Detection of plasmids 254
229 Growth curves of six isolates of BHRM, including 255
230 DNA was extracted from all the BHRM and one representative of all the genera identified during 256
231 checked for plasmid using Nucleospin Plasmid this study, suggest that there is a prolonged lag phase 257
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232 isolation kit (Macherey Nagel, Germany) and agarose in the presence of Hg (Fig. 1). Once cells enter the log 258
233 gel electrophoresis. To further confirm the presence/ phase, in some cases a steep increase follows a long 259
234 absence of plasmid, two different plasmid curing gap (e.g. GP16); in others there is exponential growth 260
235 assays were performed to note the loss, if any, of without any lag (e.g. GP17); in others there is initial 261
236 mercury resistance (for details please, see De et al., rise in numbers. Generation times were far lower in 262
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237 2003). In brief, three strains of BHRM (CH03, CH07 the presence of 50 ppm Hg than 10 ppm Hg at least in 263
238 and CH12) were subjected to two different plasmid the initial 48 h. For example, in the case of isolate 264
239 curing assays involving very high nutrient rich (four- GP16, an A. faecalis, there was only one doubling 265
240 strength) SWNB and acriflavine (final concentration during the first 48 h whereas stationary phase was 266
241 of 2.5 mg ml1). The treated cells were checked for attained within 48 h in the flasks without added Hg. 267
242 their resistance to different heavy metals and Growth of a pseudomonad isolated from Chennai 268
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243 xenobiotics after the curing experiments. harbor (CH07) was much faster, i.e., two generations 269
within the first 6 h at 50 ppm Hg concentration. After 270
a minor lag, its generation was rapid and attained 271
3. Results nearly 161 generations between 54 and 72 h before 272
244 entering stationary phase at 108 h. One of the A. 273
3.1. Identification faecalis GP15 had the slowest growth rate in the 274
245 SWNB with 50 ppm Hg. The growth patterns of the 275
246 Seven isolates (GO01, GO02, GP06, GP14, GP15, two isolates tested in the presence of Hg, DDT and 276
247 GP16 and GP17) were identified as A. faecalis, three phenol also showed similar trend. When compared 277

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Table 2
Biochemical characteristics of BHRM

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Identitya AF PA BI BP BS
GO01b GO02b GP06b GP14b GP15b GP16b GP17b CH07b GP13b GP08b CH13b S3b CM10 b
Gelatinase +  +     + +   + +
Lipase   + + + + +  +  +  
Amylase   +     + + +   +

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Arginine  +      +     
Ornithin       +  +    
Oxidase  + +      + +   +
NO32 reduction +  + +  + + + +   + 
Catalase  + + + + + + ++ + + ++ + +++
Urease  + +  + + + + + +  + +
           

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VP +
MR   +      +   + 
H2S    + + + +    +  
Indole + + + + + + +   + +  
OF NR NR F O O O F NR F F NR NR NR
Utilization of
Citrate  + + +  + + +   +  
Gluconate
Pyruvate
Cellobiose



+
+
+

+
+









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ND
ND
ND



+
+

ND
ND
ND
ND
ND
ND
ND
ND
ND
Sucrose NR AL A A A A A AL A A NR A NR
Mannitol AL AL A AL AL AL A A A A NR A A+
Rhamnose AL AL AL AL AL AL AL AL+ AL A NR A A
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Arabinose AL A AL AL AL AL AL A AL A AL AL+ NR
a
Identity of isolates derived from 16S rDNA sequencing. AF, Alcaligenes faecalis; PA, Pseudomonas aeruginosa; BI, Brevibacterium
iodinium; BP, Bacillus pumilus; BS, Bacillus sp.; A, acidic reaction; AL, alkaline reaction; O, oxidative; F, fermentative; VP, Voges–Proscaeur;
MR, Methyl Red; NR, no reaction; ND, not done; +, growth; , no growth.
b
Code.
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277 293
278 their growth in SWNB with 50 ppm Hg, the growth 3.4. Resistance of BHRM to other toxicants
279 rate of both the strains was lower in the SWNB 294
280 containing all three toxicants (Fig. 1). Analyses of The ability of the BHRM to grow in the presence 295
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281 variance (ANOVA) showed F values far above the of various toxic chemicals was highly variable 296
282 tabulated values at p = 0.001 level indicating that the (Tables 3 and 4). For example, not all isolates of 297
283 effect of Hg or other toxic chemicals on the growth A. faecalis were able to grow in SWNB containing 298
284 rate of bacteria were highly significant. 100 ppm DDT or 1000 ppm phenol or 10% 299
(v/v) trichloroethane (TCE). Similarly, there were 300
3.3. Detoxification of Hg differences in the growth response of these 301
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285 A. faecalis towards toxic pesticides (for example, 302

286 The foggy areas on the XAR plate denoted the in case of penconazole). All three plasmid-cured 303
287 reaction of silver (Ag) reduction by the Hg vapour as isolates were able to grow in 100 ppm DDT. None of 304
288 seen in the photograph (Fig. 2). All isolates tested the strains of BHRM tested during this study was 305
289 during this study (viz. GP15, CM10, CH07, GP08, able to tolerate and grow in the presence of 306
290 GP13, GO02, GP16, GP17, GP14, GP06 and CH13) formaldehyde (final concentration, 2%, v/v) or 307
291 volatilized mercury from the assay medium. The formic acid (2%, v/v). However, two of the A. 308
292 negative control showed no indication of Hg vapour faecalis isolates could grow in the presence of 309
293 (Fig. 2). 10% TCE. 310

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Fig. 1. Growth curves of different bacteria highly resistant to mercury (BHRM) in seawater nutrient broth (SWNB) with no added Hg (open
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circles), 10 ppm Hg (open triangles) and 50 ppm Hg (open squares). The isolates are Pseudomonas aeruginosa (CH07); Alcaligenes faecalis
(GP15, GP16, GP17 and GO01); Bacillus pumilus (GP08). Isolates with asterisks represent the growth curves (filled triangles) when Hg
(20 ppm), phenol 20 ppm and DDT 10 ppm were added together to SWNB.

310 314
3.5. Antibiotic sensitivity tested. Except for GP15 all other isolates were 315
311 sensitive to gentamicin. CH07 was sensitive also to 316
312 All the BHRM strains (tested along with a known kanamycin and chloramphenicol whereas GP06 was 317
313 sensitive strain E. coli) examined for antibiotic sensitive to neomycin besides being sensitive to 318
314 sensitivity were resistant to most of the antibiotics gentamicin as evidenced from the inhibition zone 319

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329
containing several other toxicants tested during this 330
study was not lost in any of these strains. 331

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4. Discussion
332
Many bacterial species have been shown to develop 333

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resistance to mercury and other heavy metals (Silver 334
and Phung, 1996; Osborn et al., 1997; Nies, 1999; 335
Barkay et al., 2003). Several genera have been 336
reported to possess resistance to mercury mostly at 337
10 ppm or lower levels (Robinson and Tuovinen, 338
Fig. 2. Mercury volatilization by different bacteria highly resistant

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to mercury (BHRM) as visualized on Kodak XAR film. (Upper row, 1984; Baldi et al., 1989; Osborn et al., 1997; 339
from left to right) GP15 (A. faecalis), CM10 (Bacillus sp.), CH07 (P. Nascimento and Chartone-Souza, 2003). During this 340
aeruginosa) and GP08 (B. pumilus); +ve control (P. putida study we have found four different genera (Pseudo- 341
KT2442<mer73); (middle row) GP13 (B. iodinium), GO02 (A. 342
faecalis), GP16 (A. faecalis), GP17 (A. faecalis) and GP14 (B.
monas, Alcaligenes, Bacillus and Brevibacterium).
Microorganisms in the marine environment are 343
iodinium); (lower row) mercurated PBS used in the experiment, non-
MRB isolate (ve control), 3C (B. pumilus), GP06 (A. faecalis) and suggested to undergo selection pressures in the 344

319
CH13 (B. pumilus). TE
presence of toxic pollutants and develop resistance
(Hideomi et al., 1977). Such organisms become
important in continuing the basic biological processes
345
346
347
320 (Table 5). All three isolates subjected to plasmid in contaminated habitats. Although the pathways of 348
321 curing assay were able to grow in the presence of Hg modifications have not completely been under- 349
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322 chloramphenicol and neomycin before and after the stood, the isolates of BHRM investigated during this 350
323 curing assays. study have many unique characteristics. 351
The BHRM strains studied during this investigation 352
3.6. Absence of plasmid exhibited resistance to variety of toxic heavy metals 353
324 and xenobiotics. Such environmental strains are of 354
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325 No plasmid was detected from DNA of any BHRM. practical interest to microbiologists not only to 355
326 In the overall, there was no indication of’ loss of revalidate present concepts of Hg resistance by native 356
327 resistance in any of the three strains tested owing microflora but also to understand the evolution and 357
328 probably to the absence of plasmids. Also, resistance significance of Hg resistance. Further, from the 358
329 to antibiotics and the capability to grow in SWNB samples collected from Chennai harbour area for this 359
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Table 3
Growth response of BHRM in presence of heavy metals
Isolates groups PA AF BI BP
Heavy metals Concentration (ppm)a CH07 GO01 GO02 GP06 GP14 GP15 GP16 GP17 GP13 GPO8
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Mercury 25 + + + + + + + + + +
Mercury 50 + +   + + + + + 
Mercury 55          
Mercury 75 b + NT NT NT + + NT NT + 
Cadmium 100 +  + + + + + + + 
Copper 100 + + + + + + + + + +
Zinc 100 + + + + + + + + + +
Lead 100 + + + + + + + + + +
a
Parts per million (mg ml1) spiked concentrations; +, positive growth; , no growth; NT, not tested; PA, Pseudomonas aeruginosa; AF,
Alcaligenes faecalis; BI, Brevibacterium iodinium; BP, Bacillus pumilus.
b
In SWNA; in all other cases it was in SWNB.

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Table 4
Growth response of BHRM in presence of xenobiotics

OF
Isolate groups Concentration (ppm)a PA AL BI BP
CH07b GO01b GO02b GP06b GP14b GP15b GP16b GP17b GP13b GPO8b
DDTc 100 + +   + + + +  
Penconazolec 93     + +  + + 
Propiconazolec 95 + + + + + + + + + +

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Metolachlorc 95 + + + + + + + + + +
Pretilachlorc 96 + + + + + + + + + +
Profenofosc 91 + + + + + + + + + +
Phenol 50 +  + + + + + + + +
Phenol 1000 NT NT NT + NT NT  NT + NT
Phenanthrene UN +  +       
       

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Benzene UN + +
Crude oil UN +  +       
TCE 10% NT NT NT NT + NT + NT NT NT
Parts per million (mg ml1) spiked concentrations.
a
b
Code.
c
Stock solutions prepared using hexane: +, positive growth; , no growth; NT, not tested; UN, unknown concentration; PA, Pseudomonas
aeruginosa; AF, Alcaligenes faecalis; BI, Brevibacterium iodinium; BP, Bacillus pumilus.
359
360
361
study, Hg concentrations (determined by cold vapour
atomic absorption spectrophotometer) ranged from
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(Silver and Phung, 1996). In addition, many moieties
of chromosomal DNA have been shown to be
373
374
375
362 100 to 2100 ng l1 in seawater and from 110 to important in resistance to heavy metals. For example, 376
363 230 ng g1 in sediment (De et al., 2003). It is Cànovas et al. (2003) reported that the genome 377
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364 interesting to note that all the examined strains were sequence of P. putida KT2440 has 61 open reading 378
365 resistant to much higher concentration of mercury frames (ORFs) involved in different metal tolerance/ 379
366 than the in situ concentrations. Environmental factors resistance. Pain and Cooney (1998) reported that most 380
367 responsible for such high levels of mercury resistance of the TBT-resistant bacteria are also resistant to six 381
368 need to be identified for a better understanding of the heavy metals (Hg, Cd, Zn, Sn, Cu and Pb), which 382
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369 ecology of MRB including the BHRM described in suggest that resistance to many types of toxicants may 383
370 this study. be present in the same organism. Moreover, the 384
371 Unlike the mercury and arsenic resistance systems combined toxicity from different toxicants may 385
372 that are highly homologous in all bacteria studied, adversely affect the growth rates of native bacteria. 386
373 other resistance systems have evolved several times Environmental isolates of BHRM that grew in the 387
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Table 5
Response of BHRM to antibiotics in terms of inhibition zone in mm diameter
Antibiotics Concentration (mg/disc) Sensitivity limit Isolates of BHRM Reference culture (E. coli)
CH07 GP06 GP15 GP16
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Kanamycin 30 18 22 (S) 17 16 11 20 (S)

Streptomycin 10 15 13 15 12 14 20 (S)
Neomycin 30 17 12 20 (S) 15 16 19 (S)
Tetracycline 30 19 7 11 9 6 21 (S)
Ampicillin 10 17 14 10 14 15 18 (S)
Chlorampheniol 10 18 19 (S) 17 13 14 29 (S)
Nalidixic acid 30 19 17 17 11 18 19 (S)
Gentamicin 10 15 22 (S) 25 (S) 12 16 (S) 28 (S)
Penicillin G (U) 10 29 0 11 4 6 20
(S) sensitive; rests are resistant.

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 J. De, N. Ramaiah / Ecological Indicators xxx (2006) xxx–xxx 9
387 429
388 presence of 50 ppm Hg, due probably to genetically Dr. Upal Roy, Goa University, is acknowledged for 430
389 conferred resistance, were also able to grow at very providing the E. coli strain. De acknowledges CSIR- 431

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390 high concentrations of other heavy metals like Cd, Pb, SRF grant 31/26/75/2002 EMR-I and UGC-DAAD 432
391 Zn, Cu and xenobiotics including TCE (considered as short-term scholarship. This is NIO contribution #. . .. 433
392 an environmental hormone), DDT or other pesticides,
393 phenols and PCBs warrants further detailed studies on
394 the mechanism of multiple resistance at the molecular References

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395 level. 434
396 From the results on plasmid curing it is most likely Baldi, F., Filippelli, M., Olson, G.J., 1989. Biotransformation of 435
mercury by bacteria isolated from a river collecting cinnabar 436
397 that mercury resistance is chromosomally encoded in
mine waters. Microbial. Ecol. 17, 263–274. 437
398 these tested strains. Thus, the resistance to Hg and the Barbieri, P., Bestetti, G., Reniero, D., Galli, E., 1996. Mercury 438
399 potential to tolerate a variety of xenobiotics and resistance in aromatic compound degrading Pseudomonas 439

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400 various antibiotics is possibly conferred chromoso- strains. FEMS Microbiol. Ecol. 20, 185–194. 440
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