Heavy metal tolerance in association with plasmid
mediated multiple antibiotic resistances among clinical
bacterial isolates
Saumendra Nath Das
1
, Manisha Mandal
2
and Shyamapada Mandal
1
*
1
Department of Zoology, University of Gour Banga, Malda, India
2
Department of Physiology, MGM Medical College and LSK Hospital, Bihar, India
ABSTRACT
The heavy metal tolerance in association with plasmid mediated antibiotic resistance among bacteria has been reported
around the globe. This communication conducted an experiment to explore the co-existence of antibiotic resistance and
heavy metal tolerance in clinical bacteria and the involvement of R-plasmid in such phenomenon. By disc diffusion
method, 6 clinical bacteria: Escherichia coli (n=3), Pseudomonas aeruginosa (n=2) and Proteus mirabilis (n=1), utilized
in the study, displayed resistance to multiple antibiotics with MAR (multiple antibiotic resistance) indices 0.15 – 0.77;
such bacterial isolates showed tolerance to Hg
2+
, Cd
2+
, Cr
6+
and Cu
2+
at 3 – 37.5 µg/ml, 75 – 800 µg/ml, 100 – 400 µg/
ml and 600 – 900 µg/ml, respectively. The SDS treatment induced the test bacteria to mislay their resistance property
(following susceptibility test) with a parallel loss of single plasmid (following agarose gel electrophoretic analysis) con-
tained in them. This study con rms the antibiotic co-resistance with heavy metal tolerance among human pathogenic
bacteria, and underlines the regular vigilance of bacterial R-plasmid in order to combat the multiple antibiotic resist-
ances of such bacteria as well as the infection caused by them.
KEY WORDS: HUMAN PATHOGENIC BACTERIA, R-PLASMID, HEAVY METAL TOLERANCE, ANTIBIOTIC RESISTANCE, MAR INDEX
612
Microbiological
Communication
Biosci. Biotech. Res. Comm. 11(4): 612-618 (2018)
INTRODUCTION
The antibiotics, which are still the gold standard thera-
peutics against a large number of bacterial infections,
and the heavy metals, which are in use in various anthro-
pogenic activities, remain the two universal categories
of environmental pollutants, and are unsafe to public
health and biological safety (Zhu et al., 2013). Several
anthropogenic processes cause contamination of envi-
ronment with heavy metals leading to the selection and
ARTICLE INFORMATION:
Corresponding Authors: samtropmed@gmail.com
Received 21
st
Sep, 2018
Accepted after revision 10
th
Nov, 2018
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007 CODEN: USA BBRCBA
Thomson Reuters ISI ESC / Clarivate Analytics USA
Mono of Clarivate Analytics and Crossref Indexed
Journal Mono of CR
NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196
© A Society of Science and Nature Publication, Bhopal India
2018. All rights reserved.
Online Contents Available at:
http//www.bbrc.in/
DOI: 10.21786/bbrc/11.4/11
Saumendra Nath Das et al.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HEAVY METAL TOLERANCE IN ASSOCIATION WITH PLASMID MEDIATED 613
emergence of bacteria possessing the tolerance capacity
to heavy metals in the niches (Nakahara et al., 1977),
and, as such, the heavy metal accumulation in the envi-
ronment accounts for the bacterial antibiotic co-resist-
ance (Baker-Austin et al., 2006; Berg et al., 2010; Das
et al., 2016). The imprudent use of antibiotics, on the other
hand, results emergence of antibiotic resistant bacteria
having the capacity to cause life-threatening infection
to humans, around the world (Tenover, 2006; Mandal
2015). It has been reported that the exposure of heavy
metals causes an effect in the co-selection of metal tol-
erant and antibiotic resistant bacteria (Filali et al., 2000),
and such co-resistances are plasmid mediated (Smith,
1967; Das et al., 2018). Garhwal et al. (2014) observed
a signi cant change in MAR (multiple antibiotic resist-
ance) index in clinical bacterial isolates before and after
lead (Pb
2+
) exposure. Nakahara et al. (1977) studied the
frequency of antibiotic and heavy metal resistance in
clinical isolates of Escherichia coli, Klebsiella pneumo-
niae, Pseudomonas aeruginosa, and reported a similar as
well as different heavy-metal resistance frequency, when
compared to the antibiotic resistance frequency, among
the isolates, and such resistances were proved to be plas-
mid mediated. The occurrence of R-plasmid (antibiotic
resistance plasmid) conferring heavy metal tolerance in
river water isolates of E. coli and Ps. aeruginosa has
been documented earlier (Das et al., 2016). The heavy
metal induced antibiotic resistance in bacteria has also
been reported (Chen et al., 2015). A conjugative plasmid,
approximately of 56.4 kb, encoding resistance to heavy
metals (Hg
2+
, Cu
2+
, Pb
2+
, Cd
2+
) as well as antibiotics was
detected among nosocomial isolates of E. coli and K.
pneumoniae (Karbasizaed et al., 2003). Thus, an emerg-
ing concern, predominantly in the developing countries,
for the treatment of infectious disease is the acquisition
and dissemination of bacterial plasmid mediated resist-
ance to multiple antibiotics. Hence, in order to evade
the bacterial antibiotic resistance, by xing an appropri-
ate treatment ‘to-do-list’, precise and prompt detection
of resistance phenotype is an emergent and imperative
issue (Doddaiah and Anjaneya, 2014), since bacterial
antibiotic resistance has been marked as the global pub-
lic health crisis (Martinez, 2008). Therefore, the current
study has been undertaken to determine the association
between antibiotic resistance and heavy metal tolerance
among clinical bacterial isolates: E. coli, Ps. aeruginosa
and Pr. mirabilis, West Bengal state, India.
MATERIAL AND METHODS
BACTERIAL STRAIN AND MEDIA
A total of 6 randomly selected clinical bacterial isolates:
Escherichia coli (n=3), Pseudomonas aeruginosa (n=2)
and Proteus mirabilis (n=1), were considered for the
current study. The tests, in the current study, were car-
ried out by the utilization of nutrient broth (for subcul-
turing, bacterial inocula preparation and plasmid DNA
isolation) and nutrient agar (for performing antibiotic
susceptibility and heavy metal tolerance test) media
(Hi-Media, India).
ANTIBIOTIC SUSCEPTIBILITY TEST
The antibiotic susceptibility test for the bacterial isolates
were determined following Kirby-Bauer disc diffusion
(Bauer and Kirby, 1966), using tetracycline (Tc; 30-µg),
gentamicin (Gm; 10-µg), cefotaxime (Ct; 30-µg), cefpo-
doxime (Ce; 10-µg), ampicillin (Am; 10-µg), meropenem
(Mp; 10-µg), chloramphenicol (Cm: 10-µg), cipro oxa-
cin (Cp; 10-µg), cefoxitin (Cx; 30-µg), piperacillin (Pc;
100-µg), piperacillin/tazobactam (PT; 100/10-µg), ami-
kacin (Ak; 30-µg) and nalidixic acid (Nx; 30-µg). The
results, in terms of ZDI (zone diameter of inhibition)
obtained around each of the antibiotic discs, for the test
isolates were interpreted according to the CLSI criteria
(CLSI, 2011).
MAXIMUM TOLERANCE CONCENTRATION OF
HEAVY METAL
The MTC (maximum tolerance concentration) values,
for the bacterial isolates, of heavy metals: using 4 salts,
such as HgCl
2
(Hg
2+
), CdCl
2
(Cd
2+
), K
2
Cr
2
O
7
(Cr
6+
), and
CuSo
4
(Cu
2+
) were determined by agar dilution method,
using ≈10
4
CFU/spot inocula, as described earlier (Das
et al., 2016). The concentrations of heavy metals uti-
lized included: Hg
2+
(3 – 50 g/ml), Cd
2+
(25 – 1000 g/
ml), Cr
2+
(25 – 500 g/ml), Cu
2+
(200 – 1000 g/ml). The
obtained results were interpreted as described earlier
(Das et al., 2016). The bacterial isolates grown in pres-
ence of each of the heavy metals, at concentrations 3
g/ml, were considered as heavy metal tolerant.
PLASMID ANALYSIS
As mentioned earlier (Das et al., 2016), the plasmid DNA
from the test bacteria were isolated following the pro-
tocol of Kado and Liu (1981), and the agarose gel elec-
trophoresis of the isolated plasmids were done following
Maniatis et al. (1982). The plasmid DNA bands, in the
gel after ethidium bromide staining, were visualized and
documented using gel-doc system.
In order to investigate the loss of plasmid, the ran-
domly selected bacterial isolates (Pr. mirabilis CSD1,
Ps. aeruginosa CSD3, and E. coli CSD5) were subjected
to plasmid curing with SDS, following the protocol of
Anjanappa et al. (1993), as described elsewhere (Man-
dal et al., 2008; Das et al., 2016). The loss of antibiotic
Saumendra Nath Das et al.
614 HEAVY METAL TOLERANCE IN ASSOCIATION WITH PLASMID MEDIATED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
resistance and heavy metal tolerance, along with the
loss of plasmid, was determined based on the resistance
patterns of the cured bacterial strains, and absence of
plasmid in the gel following agarose gel electrophoresis
for the cured bacterial strains.
RESULTS AND DISCUSSION
The antibiotic susceptibility test results, in terms of ZDI,
are depicted in Table 1. The mounting use of antibiot-
ics, not only in health care but also in agriculture and
animal husbandry contribute to an emergent problem of
antibiotic resistant bacteria (Dhanorkar and Tambekar,
2004). Pokhrel et al. (2018) reported, among the iso-
lated environmental bacteria, 3-antibiotic resistance, 4
to 10-antibiotic resistances and more than 10-antibiotic
resistance in 6.1%, 44.89% and 48.97% isolates, respec-
tively. The fecal as well as soil isolates of Klebsiella,
Citrobacter, Shigella and Staphylococcus, showed resist-
ance to 6 – 10 antibiotics tested, and the MAR indices
for the isolates ranged 6 – 10 (Ayandele et al., 2018). The
Mahananda river water bacterial isolates had resistance
to multiple antibiotics, among Am, Cm, Ce, Cx and Tm,
as per the report of the earlier study (Das et al., 2016). In
the current study, Pr. mirabilis (n=1) had 2-drug resist-
ance “Cx-Pc”, Ps. aeruginosa isolates (n=2) had 8-drug
resistance of two different patterns “Am-Ce-Cm-Ct-Cx-
Nx-Pc-PT” and “Am-Ce-Cp-Ct-Cx-Nx-Pc-PT”, and the
E. coli isolates (n=3) showed 3 different patterns of
resistance to antibiotics: 8-drug resistance “Am-Ce-Cm-
Cp-Cx- Mp-Nx-Pc”, 9-drug resistance “Am-Ce-Cp-Ct-
Cx-Mp-Nx-Pc-PT” and 10-drug resistance “Am-Ce-Cp-
Ct-Cx-Mp- Nx-Pc-PT-Tc” (Table 2). As per the report of
Malema et al. (2018), among the 100 pathogenic E. coli
test isolates, 52% had multiple antibiotic resistance, of
which 10 showed to 9 antibiotics, and 24 different MAR
phenotypes have been identi ed.
The MAR indices for the human pathogenic bacteria
are depicted in Figure 1. As has been reported by Sandhu
et al. (2016), the majority of the clinical isolates of Aci-
netobacter had resistance to cotrimoxazole, Cp, Gm, Ak,
A/S, cefepime, Im, Mp, with overall MAR indices of 0.3
– 1.0 for the isolates. Subramani et al. (2012) reported
high MAR indices (0.64 - 0.74) among Staphylococcus
aureus isolates from clinical settings demonstrating the
origin of the bacteria from niches with high antibiotic
exposure/contamination. The MAR indices of potential
pathogenic bacteria, E. coli (MAR index: 0.44) and Ps.
aeruginosa (MAR index: 0.43-0.57), were all > 0.2, indi-
cating their origin from high risk source of antibiotic
contaminated region (Oko et al., 2016). In the previous
communication, the MAR indices have been reported to
be 0.47 in Ps. aeruginosa and zero to 0.2 in E. coli iso-
lates from Mahananda river water, Malda (India) (Das et
al., 2016). In the current study, the MAR indices for the
clinical bacteria were: 0.15 for Pr. mirabilis and 0.62
for Ps. aeruginosa, while the values ranged 0.62 – 0.77
Table 1. Antibiotic susceptibility test results for clinical bacterial
isolates (ZDI; zone diameter of inhibition)
Antibiotic ZDI (mm)
CSD1 CSD2 CSD3 CSD4 CSD5 CSD6
Tc 40 10 18 17 12 13
Gm 27 15 30 26 20 20
Ct 30 6 12 6 6 25
Ce 20 10 6 6 6 13
Am 20 6 6 6 6 6
Mp 30 12 33 34 14 18
Cm 18 22 8 22 25 8
Cp 40 8 46 15 10 6
Cx 14 6 6 6 6 11
Pc 66 6666
PT 30 15 17 6 6 20
Ak 30 20 18 30 26 15
Nx 25 6 12 10 6 6
Ak: amikacin, Am: ampicillin, Ce: cefpodoxime, Cm: chloramphenicol,
Cp: cipro oxacin, Ct: cefotaxime, Cx: cefoxitin, Gm: gentamycin, Mp:
meropenem, Nx: nalidixic acid, Pc: piperacillin, PT: piperacillin/tazobactam,
Tc: tetracycline, CSD1: Pr. mirabilis; CSD2: E. coli; CSD3: Ps. aeruginosa;
CSD4: Ps. aeruginosa; CSD5: E. coli; CSD6: E. coli.
Saumendra Nath Das et al.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HEAVY METAL TOLERANCE IN ASSOCIATION WITH PLASMID MEDIATED 615
Table 2. Antibiotic resistance and heavy metal tolerance patterns of clinical bacterial isolates and
their cured derivatives
Bacterial isolates* Resistance/tolerance patterns Resistance patterns
of cured bacteria
Antibiotic resistance Heavy metal tolerance
Pr. mirabilis CSD1 Cx-Pc Hg
2+
-Cd
2+
-Cr
6+
-Cu
2+
Pc
E. coli CSD2 Am-Ce-Cp-Ct-Cx-Mp-Nx-Pc-
PT-Tc
Hg
2+
-Cd
2+
-Cr
6+
-Cu
2+
ND
Ps. aeruginosa CSD3 Am-Ce-Cm-Ct-Cx-Nx-Pc-PT Hg
2+
-Cd
2+
-Cr
6+
-Cu
2+
Nx-Pc-PT
Ps. aeruginosa CSD4 Am-Ce-Cp-Ct-Cx-Nx-Pc-PT Hg
2+
-Cd
2+
-Cr
6+
-Cu
2+
ND
E. coli CSD5 Am-Ce-Cp-Ct- Cx-Mp-Nx-Pc-PT Hg
2+
-Cd
2+
-Cr
6+
-Cu
2+
Cp-Mp-Nx-Pc-PT
E. coli CSD6 Am-Ce-Cm- Cp-Cx- Mp-Nx-Pc Hg
2+
-Cd
2+
-Cr
6+
-Cu
2+
ND
*The clinical bacterial isolates possessed a single plasmid of ≈54 kb, and the cured bacterial strains were plasmid-less. ND:
curing not done.
Am: ampicillin, Ce: cefpodoxime, Cm: chloramphenicol, Cp: cipro oxacin, Ct: cefotaxime, Cx: cefoxitin, Gm: gentamycin, Mp:
meropenem, Nx: nalidixic acid, Pc: piperacillin, PT: piperacillin/tazobactam, Tc: tetracycline.
FIGURE 1. The MAR (multiple antibiotic resistance) index for clinical bacterial
isolates (n=6).
for E. coli isolates. Thus, considering the fact of origin
of bacterial contamination from human-fecal sources,
based on the MAR indices of >0.4 (Tambekar et al.,
2005; Kaneene et al., 2007), and from high risk zone of
contamination with antibiotics, based on the MAR indi-
ces of >0.2 (Krumperman, 1983), the currently studied
clinical bacteria (Ps. aeruginosa and E. coli) might have
been originated from niches with human-fecal contami-
nation, due to antibiotic selection pressure.
The bacterial heavy metal tolerance has been depicted
in Table 2. As has been reported by Mustapha and
Halimoon (2015), the bacterial isolates from industrial
ef uents had tolerance to Cd
2+
, Cr
6+
, Pb
2+
and Cu
2+
, at
the concentration of 50 µg/ml, while one of the isolate
showed resistance to high level of Cu
2+
(200 µg/ml), and
for one isolate the Cd
2+
MIC (minimum inhibitory con-
centration) was recorded as high as 200 µg/ml. Zhu et al.
(2013) determined the MICs of Pb
2+
, Cu
2+
, Zn
2+
, Cr
6+
and
Hg
2+
as 125, 100, 100, 100 and 25 µg/ml, respectively,
for the livestock isolate of Ps. uorescens, and recorded
the occurrence of enhancement of bacterial resistance
to antibiotics due to the presence of some heavy met-
als at certain concentrations. Ps. aerμginosa, Ps. putida
and Klebsiella pneumoniae had Cd
2+
MICs 300 – 950
µg/ml; such isolates had Zn
2+
MICs of 1150, 1100 and
2000 µg/ml, respectively, and Hg
2+
MICs of 20, 80 and
90 µg/ml, respectively (Yamina et al., 2014). Ps. aerugi-
nosa and E. coli, isolated from Mahananda river water,
Saumendra Nath Das et al.
616 HEAVY METAL TOLERANCE IN ASSOCIATION WITH PLASMID MEDIATED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
FIGURE 2. Plasmid pro le of clinical bacterial isolates; lane 1: E. coli V517 (54 kb),
lane 2: Pr. mirabilis CSD1, lane 3: Pr. mirabilis CSD1 (cured strain), lane 4: Ps.
aeruginosa CSD3, lane 5: E. coli CSD5, lane 6: Ps. aeruginosa CSD3 (cured strain),
lane 7: E. coli CSD5 (cured strain). Note the absence of plasmid in cured bacterial
strains (lane 3, 6 and 7).
Table 3. Heavy metal tolerance level for clinical bacterial
isolates (n=6)
Bacterial isolates MTC of heavy metals (μg/ml)
HgCl
2
CdCl
2
K
2
Cr
2
O
7
CuSo
4
Pr. mirabilis CSD1 12.5 75 250 700
E. coli CSD2 37.5 500 250 600
Ps. aeruginosa CSD3 3 800 400 800
Ps. aeruginosa CSD4 9 100 200 900
E. coli CSD5 25 500 250 600
E. coli CSD6 9 100 100 800
MTC: maximum tolerance concentration
Malda (India) had resistance to Cd
2+
and Hg
2+
(Das et al.,
2016). The continued usage of heavy metals since the
ancient, in medicine and other anthropogenic purposes,
select heavy metal resistant bacteria in polluted niches.
For the bacteria utilized in the current study, the level
of tolerance to Hg
2+
, Cd
2+
, Cr
6+
and Cu
2+
ranged 3 – 37.5
µg/ml, 75 – 800 µg/ml, 100 – 400 µg/ml and 600 – 900
µg/ml, respectively (Table 3). This communication is, for
the rst as we believe, to demonstrate the heavy metal
tolerance among clinical bacterial isolates of E. coli, Pr.
mirabilis and Ps. aeruginosa from our part of the globe
(West Bengal state, India).
The plasmid pro le of clinical bacterial isolates and
the cured derivatives are represented in Figure 2. Appre-
hension grows in recent times in connection with the
co-selection for antibiotic resistance among bacteria on
exposure to heavy metals, in several ecological niches
(Wales and Davies, 2015). Because the heavy metal
(pollution) acts discriminatorily as a selective agent in
the emergence and propagation of antibiotic resistance
among bacteria, wherein, along with the genes con-
ferring antibiotic resistance, metal tolerance genes are
also encoded in the same plasmids (Foster, 1983; Fang
et al., 2016). The clinical isolates of Pseudomonas spp.,
as reported by Rajasekar and Mohankumar (2016), had
resistance to multiple antibiotics and heavy metals, and
the resistance properties were shown to be plasmid (10
kb) mediated. A conjugative plasmid (≈56.4 kb), carry-
ing resistance to multiple heavy metals, such as, Hg
2+
,
Cu
2+
, Pb
2+
, Cd
2+
, and also antibiotics was detected among
the isolates of E. coli and K. pneumoniae causing noso-
comial infections (Karbasizaed et al., 2003). Das et al.
Saumendra Nath Das et al.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HEAVY METAL TOLERANCE IN ASSOCIATION WITH PLASMID MEDIATED 617
(2016) reported the occurrence of R-plasmid (antibiotic
resistance plasmid) encoding heavy metal tolerance
among E. coli and Ps. aeruginosa, isolated from river
water. Herein, we demonstrated the involvement of ≈54
kb plasmid (Figure 2) conferring heavy metal tolerance
to Hg
2+
, Cd
2+
, Cr
6+
and Cu
2+
, with associated multiple
antibiotic resistances among the clinical bacterial iso-
lates (Table 2).
The co-resistance to antibiotics and heavy metals has
been reported among bacterial food pathogens (Wales
and Davies, 2015). Wright et al. (2006) reported high-
est occurrence of heavy metal tolerance and antibiotic
resistance among bacteria isolated from the contami-
nated most location, indicating the direct selection of
heavy metal tolerant bacteria due to the exposure of
heavy metals, thereby co-selecting bacterial antibiotic
resistances. The E. coli isolates from urinary tract infec-
tion cases harbored copper/silver resistance genes, ‘pco/
sil’ with MIC of 500-µg/ml, presenting resistance to
extended spectrum -lactam antibiotics, too (Sutterlin
et al., 2018). Co-spread of antibiotic resistance (-lactams:
blaCTX-M; quinolones: oqxAB; aminoglycosides: aac-
Ib-cr; amphenicols: oR; fosfomycin: fosA3) as well
as the heavy metal resistance (Cu: pco; Ag: sil) genes,
have been shown to be plasmid mediated (Zhu et al.,
2013). The Cd
2+
resistant isolates of
Ps. aerμginosa and
Ps. putida showed multidrug resistance to kanamycin
(Km), oxacillin (Oc), Nx and sulfonamids, while K. pneu-
moniae had resistance to Ct in addition to Km, Oc, Nx
and sulfonamids resistances (Yamina et al., 2014). In the
earlier study, antibiotics (Am-Cm-Ce- Cx-Tm) and heavy
metals (Cd
2+
-Hg
2+
) co-resistances have been reported
among the river water bacteria (Das et al., 2016). The
co-resistance to heavy metals and antibiotics is, thus,
a global concern, and the phenomenon among clinical
bacteria, in our part of the globe, is not uncommon. The
plasmid mediated resistance to the test heavy metals and
to a number of antibiotics, as has been supported by
SDS curing, approved the fact of heavy metal-antibiotic
co-resistance in Ps. aerμginosa, E. coli, and Pr. mira-
bilis clinical isolates. The bacterial isolates displaying
MAR indices of >0.4 have been regarded to be derived
from human-fecal contaminated niches (Tambekar et al.,
2005; Kaneene et al., 2007) and the bacterial isolates,
with MAR indices of >0.2, deemed to be originated from
highly antibiotic polluted regions (Krumperman, 1983).
Still, the heavy metal inducing phenomenon of bacte-
rial antibiotic resistances suggests that the emergence
of multiple antibiotic resistant bacteria might be due to
either the heavy metal or the antibiotic selection pres-
sure, or both, and hence the bacterial high MAR index
does also mean their (bacteria) origin from a region with
high metal pollution, too.
CONCLUSION
The human pathogenic bacteria (Ps. aerμginosa, E. coli,
and Pr. mirabilis) had resistance to two or more antibi-
otics, which in association with heavy metal resistance
were found to be plasmid linked. This study endorses
the dissemination of bacterial antibiotic resistance under
the heavy metal as well as antibiotic selective pressure.
Therefore, regular inspection of antibiotic resistance
plasmid among human pathogenic bacteria, from our
part of the globe, is urgently needed, in order to combat
the bacterial multiple antibiotic resistance as well as the
bacterial infection to humans.
REFERENCES
Anjanappa, M., Horbola, P.C., Verma, J.C. (1993). Elimination
(curing) of R-plasmids in Salmonella gallinarum and their
transconjugants. Indian Veterinary Journal, 70, 10-13.
Ayandele, A.A., Owolabi1, L. O., Oladeinde1, A. A., Aseweje, I.
B., Oshodi, E. A. (2018). Prevalence of multi-antibiotic resistant
bacteria in birds faecal and soil samples from Poultry farms in
Ogbomoso, Oyo State, Nigeria. Journal of Advances in Medicine
and Medical Research 26(1): 1-10; Article no.JAMMR.39868.
Baker-Austin, C., Wright, M.S., Stepanauskas R., McArthur J.V.
(2006). Coselection of antibiotic and metal resistance. Trends
Microbiol, 14, 176–182.
Bauer, A.W., Kirby, W.M.M., Skerris, J.C., Turuck, M. (1966).
Antibiotic susceptibility testing by a standard single diffusion
method. American Journal Clin Pathol, 45, 494-496.
Berg, J., Thorsen, M.K., Holm, P.E., Jensen. J., Nybroe, O.,
Brandt, K.K. (2010). Cu exposure under eld conditions cose-
lects for antibiotic resistance as determined by a novel cultiva-
tion-independent bacterial community tolerance assay. Envi-
ronmental Science Technology, 44, 8724–8728.
Chen, S., Xiaomin, Li., Guoxin, Sun., Yingjiao Zhang, Jian-
qiang, Su. and Jun, Ye. (2015). Heavy metal induced antibi-
otic resistance in bacterium LSJC7. International Journal of.
Molecular Science, 16, 23390-23404.
Clinical and Laboratory Standards Institute (CLSI): Perfor-
mance standards for antimicrobial susceptibility testing (2011)
21
st
informational supplement M100S21. CLSI, Wayne, Pa.
Das, S.N., Mandal, M., Mandal, S. (2016). Plasmid mediated
antibiotic and heavy metal co-resistance in bacterial isolates
from Mahananda River Water (Malda, India). Translational
Medicine (Sunnyvale) 6, 185.
Das, S.N., Mandal, M., Mandal, S. (2018). Detection of mercury
and cadmium resistance among multiple antibiotic resistant
enteric bacteria from municipal sewage water in Malda, India.
International Research Journal of Pharmacy, 9, (in press).
Dhanorkar, D.V. and Tambekar, D.H. (2004). Studies on multid-
rug resistance pattern of clinical isolates. 45th Annual Confer-
ence of Association of Microbiologist of India, NDRL, (Karnal),
November 23-25, 2004
Saumendra Nath Das et al.
618 HEAVY METAL TOLERANCE IN ASSOCIATION WITH PLASMID MEDIATED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Doddaiah V., Anjaneya D. (2014). Prevalence of ESBL, AmpC
and Carbapenemase among Gram Negative Bacilli isolated
from clinical specimens. American Journal of Life Sciences,
Vol. 2, No. 2, 76-81.
Fang, L. et al. (2016). Co-spread of metal and antibiotic resist-
ance within ST3-IncHI2 plasmids from E. coli isolates of food-
producing animals. Sci. Rep. 6, 25312; doi: 10.1038/srep25312
(2016).
Filali, B.K., Taou k, J., Zeroual, Y., Dzairi, F.Z., Talbi, M.,
Blaghen, M. (2000). Waste water bacterial isolates resistant
to heavy metals and antibiotics. Curr. Microbiol., 41, 151–
156.
Foster, TJ. (1983). Plasmid-determined resistance to anti-
microbial drugs and toxic metal ions in bacteria.Microbiol
Rev47, 361–409.
Garhwal D., Vaghela G., Panwala T., Revdiwala S., Shah A.,
Mulla S. (2014) Lead tolerance capacity of clinical bacterial
isolates and change in their antibiotic susceptibility pattern
after exposure to a heavy metal. International Journal Med
Public Health, 4,253-6.
Kado, C.I., Liu, S.T. (1981). Rapid procedure for detection and
isolation of large and small plasmids. Jaurnal of Bacteriology,
145, 1365-1373.
Kaneene, B.J., Miller, R., Sayah, R., Johnson, Y.J., Gilliland, D.,
Gardiner, J.C. (2007). Considerations when using discrimina-
tion function analysis of antimicrobial resistance pro les to
identify sources of faecal contamination of surface water in
Michigan. Appl Environ Microbial, 73, 2878–90.
Karbasizaed, V., Naser Badami1, Giti Emtiazi. (2003) Anti-
microbial, heavy metal resistance and plasmid pro le of
coliforms isolated from nosocomial infections in a hospital
in Isfahan, Iran. African Journal of Biotechnology 2, 379-
383.
Krumperman, P.H. (1983). Multiple antibiotic resistance index-
ing of Escherichia coli to identify high-risk sources of fecal
contamination of foods. Appl Environ Microbiol 46, 165-
70.
Malema, M.S., Akebe Luther King Abia, Roman Tandlich, Bonga
Zuma, Jean-Marc Mwenge Kahinda and Eunice Ubomba-
Jaswa. (2018). Antibiotic-resistant pathogenic Escherichia coli
isolated from rooftop rainwater-harvesting t anks in the East-
ern Cape, South Africa. Int. J. Environ. Res. Public Health, 15,
892; doi:10.3390/ijerph15050892
Mandal, S. (2015). Can over-the-counter antibiotics coerce
people for self-medication with antibiotics? Asian Pac J Trop
Dis 5: S184-S186.
Mandal, S., Deb, Mandal, M., Pal, N.K. (2008). Plasmid encoded
UV resistance and UV induced cipro oxacin resistance in
Salmonella enterica serovar Typhi. Int J Integrat Biol 2: 43-
48.
Maniatis T., Fritsch E.F., Sambrook J. (1982). Molecular cloning:
A laboratory manual. Cold Spring Harbor Laboratory: New York.
Mustapha, M.U., Halimoon, N. (2015). Screening and isolation
of heavy metal tolerant bacteria in industrial Ef uent. Proce-
dia Environmental Sciences, 30, 33 – 37.
Nakahara, H., Tomoaki Ishikawa, Yasunaga Sarai, Isamu Kondo,
Susumu Mitsuhashi. (1977). Frequency of heavy-metal resist-
ance in bacteria from inpatients in Japan. Nature, 266, 165–167.
Oko, J.O., Umar, M., Akafyi, D.E., Abdullahi, M. (2016). Anti-
bacterial susceptibility of heavy metals tolerant bacteria iso-
lated from NILEST tannery ef uent. Journal of Advances in
Medical and Pharmaceutical Sciences, 8, 1-10.
Pokhrel, H., Sangipran Baishya1, Bipul Phukan1, Devika Pil-
lai and Mohd Ashraf Rather. (2018).Occurrence and distribu-
tion of multiple antibiotic resistance bacteria of public health
signi cance in backwaters and aquaculture farm. Int.J.Curr.
Microbiol.App.Sci, 7 (2), 975-987.
Rajasekar, S., Mohankumar A.(2016). Antibiotic susceptibility
and plasmid pro le of heavy metal resistant Pseudomonas spe-
cies. Biosci. Biotech. Res, Comm, 9(2), 211-215.
Sandhu, R., Shalley Dahiya, Pallavi Sayal. (2016). Evaluation
of multiple antibiotic resistance (MAR) index and Doxycy-
cline susceptibility of Acinetobacter species among inpatients.
Indian J Microbiol Res, 3(3):299-304.
Smith, DH. (1967) R Factors mediate resistance to mercury,
nickel, and cobalt. Science, 156, 1114-1116.
Subramani S., Vignesh, S. (2012). MAR Index study and MDR
character analysis of a few golden Staphylococcus isolates.
Asian Journal of Pharmacy and Life Science 2, 151-154.
Sutterlin, S., Téllez-Castillo C.J., Anselem L., Yin H., Bray J.E.,
Maiden M.C.J. (2018). Heavy metal susceptibility of Escheri-
chia coli isolated from urine samples from Sweden, Germany,
and Spain. Antimicrob Agents Chemother, 62, 209-18.
Tambekar, D.H., Hirulkar, N.B., Waghmare, A.S. (2005). MAR
indexing to discriminate the source of faecal contamination in
drinking water. Nat Environ Poll Tech, 4, 525–8.
Tenover, F.C. (2006). Mechanisms of antimicrobial resistance in
bacteria, American Journal Infect Cont, 34, S3–S10.
Wales, A.D. & Davies, R. H. (2015). Co-selection of resistance
to antibiotics, Biocides and Heavy metals, and Its relevance to
food borne pathogens. Antibiotics. 4, 567–604.
Wright, M.S., Gretchen Loef er Peltier, Ramunas Stepanaus-
kas & J Vaun McArthur. (2006). Bacterial tolerances to metals
andantibiotics inmetal-contaminated and reference streams.
FEMS Microbiol Ecol, 58, 293–302.
Yamina, B., Tahar, B., Lila, M., Hocine, H. and Laure, F.M.
(2014) Study on cadmium resistant-bacteria isolated from Hos-
pital wastewaters. Advances in Bioscience and Biotechnology,
5, 718-726.
Zhu, Y.G., Johnson, T.A., Su, J., Qiao, M.. Guo, G.X., Stedtfeld,
R.D., Hashsham, S.A., Tiedje, J.M. (2013) Diverse and abundant
antibiotic resistance genes in Chinese swine farms. Proc. Natl.
Acad. Sci. USA, 110, 3435–3440.