Download Characterization of metal and antibiotic resistance in a

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.
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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.
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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
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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.
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