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Received: 4 October 2021    Revised: 28 December 2021    Accepted: 11 February 2022

DOI: 10.1111/jam.15492

REVIEW ARTICLE

Heavy metal-­induced selection and proliferation of

antibiotic resistance: A review

Prakriti Vats  | Ujjwal Jit Kaur  | Praveen Rishi

Department of Microbiology, Panjab Abstract

University, Chandigarh, India
Correspondence
Prof. Praveen Rishi, Department of
Microbiology, BMS Block I, South
Campus, Panjab University, Sector 25,
Chandigarh, 160014, India.
Email: rishipraveen@yahoo.com
Antibiotic resistance is recognized as a global threat to public health. The selection
and evolution of antibiotic resistance in clinical pathogens were believed to be ma-
jorly driven by the imprudent use of antibiotics. However, concerns regarding the
same, through selection pressure by a multitude of other antimicrobial agents, such
as heavy metals, are also growing. Heavy metal contamination co-­selects antibiotic
and metal resistance through numerous mechanisms, such as co-­resistance and
cross-­resistance. Here, we have reviewed the role of heavy metals as antimicrobial re-
sistance driving agents and the underlying concept and mechanisms of co-­selection,
while also highlighting the scarcity of studies explicitly inspecting the process of co-­
selection in clinical settings. Prospective strategies to manage heavy metal-­induced
antibiotic resistance have also been deliberated, underlining the need to find spe-
cific inhibitors so that alternate medicinal combinations can be added to the existing
therapeutic armamentarium.

KEYWORDS
antibiotics, antimicrobials, environment health, heavy metals, resistance

I N T RO DU CT ION wherein even minor infections will be life-­threatening

The discovery of antibiotics and the exploitation of their
ability to treat infectious diseases has entirely revolution-
ized the field of public health. However, the relentless use
of antibiotics, along with the endless adaptive capabilities
of micro-­organisms, formed the basis for the selective pro-
liferation and evolution of antibiotic-­resistant bacteria.
This has ultimately triggered the advent of ‘multidrug-­
resistant bacteria (MDR)’(Aminov,  2010; Lehtinen
et al., 2019). Thus, the medical community is encounter-
ing a challenge to treat microbial infections with the cur-
rently available array of antibiotics, as resistance due to
antibiotics is spreading among pathogenic bacteria at an
unprecedented rate (Aslam et al., 2018).
Antibiotic treatment failure accounts for hundreds
of thousands of deaths each year. WHO has declared
antibiotic resistance as a global health crisis that, if not
controlled urgently, can lead us into a postantibiotic era,
(WHO, 2014). Subsequently, to control the emergence and
propagation of antibiotic-­resistant strains, various mea-
sures to promote the judicious use of antibiotics have been
established. However, these measures have encountered
limited success and the phenomenon of antibiotic resis-
tance among pathogens is still prevailing (Gillespie, 2001;
Sundqvist et al., 2009). This indicates the presence of fac-
tors, other than the antibiotics, that may govern the evo-
lution, selection and dissemination of antibiotic-­resistant
strains (Baker-­Austin et al., 2006).
Resistance in pathogens is mediated by both the in-
trinsic (chromosomal) and extrinsic (extrachromosomal)
factors. Extrachromosomal resistance is usually held re-
sponsible for antimicrobial resistance (AMR) in environ-
mental micro-­organisms. The evidence available suggests
that such resistance determinants are transferred between
the environment and the clinical disease-­causing bacte-
ria. In this context, the role of the natural environment in

| 2022 Society for Applied Microbiology.

4058    © wileyonlinelibrary.com/journal/jam J Appl Microbiol. 2022;132:4058–4076.
METAL-­INDUCED ANTIBIOTIC RESISTANCE
the selection and evolution of antibiotic resistance came
into focus. A variety of other substances, such as heavy
metals, occurring in the natural environment, may trig-
ger this process (Romero et al., 2017; Skurnik et al., 2010).
Bacteria are continuously exposed to heavy metals pres-
ent in the natural habitats due to the ungoverned release
of industrial and agricultural effluents, thereby resulting
in the survival of the metal-­tolerant cells due to muta-
tions ­(Al-­Musharafi,  2016; Masindi and Muedi,  2018).
Resistance to heavy metals in pathogens has been asso-
ciated with the acquisition of extrinsic resistance deter-
minants. Antibiotic Resistance genes (ARGs) containing
Class 1 integrons and gene cassettes in microbes have
been reported in samples collected from sewage treatment
plants, which indicates the co-­selection of metal and an-
tibiotic resistance-­associated genes (Zhang et al.,  2009;
Kotlarska et al., 2015). Such co-­selection also raises serious
concerns regarding ungoverned dumping of metal-­ridden
industrial and agricultural effluents. Once these effluents
are dumped in the open, they form waste reservoirs which
contribute towards the maintenance and transfer of ARGs
in pathogens.
The first few reports indicating the correlation between
the selection of multiple antibiotics and heavy metal resis-
tance were published as early as the 1960s (Moore, 1960;
Richmond and John, 1964). Thereafter, a significant num-
ber of such studies have been documented which suggest
the role of heavy metals in the co-­selection and proliferation
of antibiotic-­resistant bacteria (Bhattacharya et al.  2000,
Pathak & Gopal,  2005). Peltier et al.  (2010) reported en-
hancement of antibiotic resistance in the microbial com-
munity through the development of cross-­resistance via
co-­exposure to subtoxic levels of zinc and some unrelated
antibiotics, such as ciprofloxacin, oxytetracycline and
tylosin, in laboratory-­scale wastewater treatment bio-
reactors. The addition of copper to agricultural soil, in a
field-­based study, was reported to indirectly select anti-
biotic resistance along with copper resistance by encour-
aging horizontal gene transfer (HGT) of mobile genetic
elements (MGEs) (Berg et al., 2010). Furthermore, certain
reports indicate that subinhibitory concentrations of heavy
metals in the environment enhance the frequency of anti-
biotic resistance by inducing mutagenesis in bacteria (Li
et al., 2019; Li et al., 2021). Hence, several studies substan-
tiate the claims under the co-­selection theory and establish
a relation between heavy metals and antibiotic resistances.
Many of these studies direct that relatively low concentra-
tions of these heavy metals in the polluted environment
suffice to induce antibiotic resistance (Chen et al., 2015),
which is a matter of grave concern for the medical commu-
nity considering that much higher levels of heavy metals
from anthropogenic sources exist in both natural and clin-
ical environments (Stepanauskas et al., 2005).
     |  4059
Consequently, this review focuses on the current knowl-
edge regarding the indispensable role of heavy metals in
the co-­selection and dissemination of antibiotic-­resistant
bacteria. Maintenance of horizontally transferable ARG
pools and the emergence of multidrug resistance (MDR) in
natural and clinical settings are also discussed along with
potential molecular mechanisms behind the co-­selection
process and the prospective strategies to disarm it.
ROLE OF ANTIMIC ROBIAL
RESISTANCE DRIVING AGENT S :
FROM THE ENV IRONMENT TO
C LINICS
The estimated number of bacteria on earth is in the order
of magnitude of 1030 (Whitman et al., 1998). Their omni-
presence, in such a generous amount, abets their interac-
tion with a vast chemical diversity in the environment,
which may be of abiotic origin or produced by other bac-
teria, fungi, plants, or animals. Many of these chemical
compounds have an impact on the cellular processes of
bacteria in their vicinity, by which, they become the driv-
ers of natural selection and evolution of genetic diversity
of these bacteria (Martinez, 2009; Wright, 2010). The re-
sulting impact of constant exposure of environmental
chemicals on the bacteria was the development of com-
plex mechanisms and novel proteins for sensing, respond-
ing to, and metabolizing small molecules. Some of these
proteins having modest antibiotic resistance functions or
some affinity for antibiotics may evolve into a bona fide
resistance mechanism in response to antibiotic selective
pressure and thus, act as precursors to resistance pro-
teins (Kaufmann et al., 2005; Yim et al., 2006; Fajardo and
Martínez, 2008).
The environmental organisms that produce chemical
substances or secondary metabolites, such as, antibiotics,
have to protect themselves from the toxicity of these mol-
ecules also, and hence, these become the hotspots for the
evolution of resistance genes. Their neighbouring micro-­
organisms must also develop coping strategies to survive
in a toxic environment. This is done either through the
evolution of resistance mechanisms or by the HGT of re-
sistance genes from the producer organism (Benveniste
and Davies, 1973; Wright, 2007).
Genome sequencing of all bacteria, even with modest
genomes, shows the presence of specific genes responsible
for metabolizing small chemical molecules of foreign or-
igin for nutrition and defence (Fraser et al., 1995). These
genes encode outer membrane transport and efflux pro-
teins, enzymes to metabolize specific molecules, and re-
ceptor molecules. Microbes can incorporate any of these
classes of genes in response to cytotoxic molecules such
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4060       VATS et al.
as heavy metals or antibiotics. These chromosomal genes
are the reservoir for resistance genes and comprise the
environmental resistome. Therefore, it is apparent that
micro-­organisms that evolved under constant exposure
to diverse chemical molecules such as antibiotics, metal
ions and other secondary metabolites become the source
of clinically relevant ARGs and such chemical molecules
become the drivers of AMR.
The huge diversity of ARGs, fostered by the environ-
mental resistome, was neglected to a large extent by the
scientific community for a long time because the epide-
miology of antibiotic resistance was limited to clinical set-
tings. Ample studies emphasize the fact that the resistance
genes responsible for MDR can originate in nonpatho-
genic environmental microbes in nonclinical settings
(Rutgersson et al.,  2014; Xiong et al.,  2014). This is best
illustrated by the example of ARGs coding for aminogly-
coside and vancomycin whose working is almost similar
in both environmental and clinical isolates. The environ-
mental counterparts of these ARGs upon acquisition by
human pathogens would eventually evolve into explicit
antibiotic resistance determinants under specific antibi-
otic selection pressures posed by humans.
The prevalence of ARGs is narrowed down while pro-
ceeding towards clinics from environmental resistome as
they adapt to selection pressures that are specific to clini-
cal settings. Since ARGs are highly prevalent in the envi-
ronment, surveillance of natural environments should be
done regularly to monitor antibiotic resistance as it can
serve as a warning system to assess imminent antibiotic
resistance mechanisms (D’Costa et al., 2006) and also help
us understand the pattern of prevalence, distribution and
transmission of ARGs (Wright, 2010).
T H E E NV I RO N M E N TAL
RES I STO M E AN D H U M AN
PAT H O G E NS
All the ARGs circulating in the environment among
antibiotic producers and nonpathogenic environmental
bacteria constitute the environmental resistome. The
environmental resistome predates the therapeutic use
of antibiotics in humans. The widely distributed ARGs
must express in disease-­causing bacteria to pose a clini-
cal threat, establishing the point of intersection for the
environmental resistome and the resistome of patho-
genic organisms. Anthropogenic sources of antibiotics
have significantly increased in clinical settings as well as
human habitats, thus compelling the human pathogens
to acquire antibiotic resistance elements from the envi-
ronmental resistome. In the face of antibiotic selective
pressure, the MGEs rapidly became the vectors for car-
rying resistance genes. The majority of the resistance
genes found in pathogenic bacteria infecting mammals
are carried by the MGEs, such as plasmids, which are
acquired by HGT from the environmental bacteria. A
significant number of reports are available that suggest
a higher prevalence of antibiotic resistance in commen-
sal bacteria harboured by wild animals dwelling close
to human habitats (OÖsterblad et al., 2001). A report by
Skurnik et al.  (2006) demonstrated a clear correlation
between high antibiotic resistance frequency in E. coli
sampled from farm animals with increased human expo-
sure. On the same lines, higher antibiotic resistance was
also recovered from animals, such as gorillas, baboons
and rodents which lived close to human habitats and
livestock, whereas unexposed counterparts, which lived
away from human habitats showed practically no antibi-
otic resistance (Rolland et al., 1985; Gilliver et al., 1999;
OÖsterblad et al., 2001; Rwego et al., 2008). These stud-
ies imply that the enduring concentrations of antibiot-
ics present in such habitats select the ARGs and lead to
their spread and diversification among micro-­organisms
living within the same geographical boundaries.
All in all, the presence of high concentrations of an-
tibiotics serves as a selection pressure on the microbes
in the vicinity, aiding the maintenance and employment
of resistance genes in the environmental bacteria. It
should be noted that the environment is also a source
of opportunistic pathogens which may already be re-
sistant or may acquire resistance genes via HGT from
human-­associated bacteria and subsequently cause in-
fections in humans. During all these processes, a major
concern for the clinical community arises when novel
resistance genes, originally absent in the resistance
genes repertoire of pathogens, are recruited from the
environmental resistome, thus posing a challenge to
the existing therapies and severe consequences for the
infected patients. Several studies support this by ev-
idencing the existence of identical resistance genes in
human pathogens and the environmental bacterial iso-
lates. For example a metagenomic study by Forsberg
et al.  (2012) reported several ARGs present in human
pathogens, conferring resistance to antibiotics, such as
β-­lactams, sulphonamides, aminoglycosides, phenicol
and tetracyclines, which were similar to bacteria from
various U.S. soils. Reports with similar claims have also
been published by Poirel et al.  (2004, 2005). These re-
ports emphasize that the Gram-­negative bacteria from
the environmental resistome is the reservoir for future
ARGs which will ultimately transfer to human patho-
gens through HGT due to overlapped geographical
niches and cause infections in humans.
METAL-­INDUCED ANTIBIOTIC RESISTANCE
H E AV Y M ETALS: T HE AG E N T S OF
CO - ­S E L ECT ION
Microbes frequently encounter metals in their habitats.
This interaction can either be beneficial or harmful for
the microbe (Ehrlich, 1997). Based on utility, heavy met-
als may be classified as ‘essential micronutrients’ and
‘nonessential metals’. Chromium (Cr), calcium (Ca), zinc
(Zn), copper (Cu), manganese (Mn), iron (Fe), potassium
(K), magnesium (Mg), nickel (Ni), sodium (Na) and co-
balt (Co) are the ‘essential’ metals, which are involved in
the fundamental metabolic processes of micro-­organisms.
These metals play important roles in redox reactions in
cells, as cofactors of enzymes, as stabilizers, and have a
role in regulating cellular osmotic pressure. Whereas
other metals, for example gold (Au), silver (Ag), antimony
(Sb), cadmium (Cd), arsenic (As), lead (Pb), aluminium
(Al) and mercury (Hg) do not have any crucial function
in biological processes and hence, are considered as ‘non-
essential metals’. Therefore, they are extremely toxic to
micro-­organisms and are used as antimicrobials, having
a broad spectrum of activity. Even essential heavy met-
als, such as Cu, Zn and Ni, at high concentrations form
nonspecific complexes within the cells and eventuate
toxicity. Toxicity heavily depends on the environmental
conditions of the cell, for instance, the pH, redox poten-
tial and concentration of organic matter, as these affect
the valency and bioavailability of the metals (Nies, 1999;
Seiler and Berendonk, 2012). The elevation in both intra
and extra-­cellular levels of metals in bacterial cells can be
detrimental for the activity of enzymes in the cell, basic
cellular functions, may damage the cellular membranes,
and may even permanently damage the structure of
DNA. Maintaining heavy metal homeostasis is essential
for the survival of the cell and hence, the concentration
of heavy metals inside the cell is under strict regulation
(Nies, 1999). Thus, metal toxicity poses a selection pres-
sure to the bacterial community. However, utilizing their
infinite adaptive capabilities, bacteria have evolved sys-
tems for resistance against almost all toxic metals which
helps them survive and selectively proliferate in a toxic,
heavy metal-­ridden environment (Rouch et al., 1995).
H E AV Y M ETAL POLLU T ION : A
WO RL DWI DE PROB LE M
Metals are naturally present in the earth’s crust and infil-
trate the biome through natural as well as certain anthro-
pogenic activities. Natural processes, such as weathering
or erosion by wind and water, keep transmitting signifi-
cant amounts of natural metals into the ecosystem. The
aerosol formation above the sea, volcanic eruptions, and
     |  4061
forest fires are more examples of natural processes that
allow the mobilization of metals in the environment. All
these natural processes help in the cycling of metals in the
ecosystem and thus, ensure their constant background
levels in the environment (Mighall et al., 2002; Raab and
Feldmann, 2003).
In addition to the natural sources, three major anthro-
pogenic sources contribute to the heavy metal pollution
in the environment: Industries, agriculture and sewage
sludge. Lately, there has been an unprecedented increase
in ungoverned industrial activities that have intensified
the problem of environmental pollution along with the
accumulation of toxic heavy metals in air, water and soil.
Industrial activities, such as mining of ores, metal smelt-
ing, activities of metallurgical industries, refining, the
leftover waste of mining and metallurgical processes, elec-
troplating, plastics manufacturing, fertilizer-­producing
plants, thermal power plants and corrosion of metal
equipment in use, have led to an elevated concentration
of metals in different trophic levels of the ecosystem
(Zouboulis et al., 2004; Oyetibo et al., 2010).
Agriculture and aquaculture are also major burdens
to the environment in terms of metal pollution. Usage
of antimicrobial compounds, pesticides, fertilizers and
sewage sludge, all of which contain mineral nutrients
for plant and animal growth, acts as a major contributor
to the problem. Adding sewage sludge to soil serves as a
low-­cost disposal technique as well as a natural fertilizer
to the crops. Heavy metals, such as Hg, Cu, As, Zn, Cr, Pb
and Cd, are present in a wide range of concentrations in
both animal feed supplements and plant manures with Zn
and Cu being one of the most accumulated metals in ani-
mal feed as they are commonly used as growth enhancers
(Yazdankhah et al.,  2014). These metals have antimicro-
bial properties similar to antibiotics. Thus, they suppress
pathogens and fermentation processes in the gut which in
turn promotes growth by preventing the loss of nutrients.
Such high levels of Cu and Zn in the feed supplements are
poorly absorbed by the animals and hence, most of them
are excreted in the faeces. From there, these metals may
directly enter the soil environment or may be added later
to the soil through manure application. Once these nutri-
ents reach the soil, they may contaminate groundwater as
well through leaching. The addition of these nutrients to
soil has been reported to select for metal as well as indi-
rectly for antibiotic resistance (Hu et al., 2017).
Contamination of marine environments and surface
water by metals is mainly accounted for by aquaculture
practices and sewage. Fish farmers usually use antimicro-
bial metals, metal-­containing anti-­fouling agents, or fish
food enriched with micronutrients. Cu-­based paints are
the most commonly used antifouling paints in the aqua-
culture industry (Burridge et al., 2010). Cu alloys are also
 |
4062       VATS et al.
used in the netting material (Flach et al.,  2017). Zinc is
used as a supplement for salmon feed. Marine sediments
in the periphery of fish farms, thus, have high concentra-
tions of metals such as Cu and Zn. Five different drugs ap-
proved by the FDA, which are sulfamerazine, florfenicol,
oxytetracycline dihydrate, oxytetracycline hydrochloride,
chorionic gonadotropin as well as sulfadimethoxine and
ormetoprim in combination, are routinely used in aqua-
culture (Seiler and Berendonk, 2012). Thus, prospects of
selection and co-­selection of antibiotic resistance in bacte-
rial communities in aquaculture are very high as they are
repeatedly exposed to a combination of heavy metals and
antibiotics.
Continuous release of industrial and agricultural
waste, having abundant heavy metal content, into sew-
age contaminates sewage water as well. Sewage water
provides optimum conditions for the proliferation of
antibiotic-­resistant bacteria. An abundance of ARGs and
MGEs in sewage water and mining affected areas has also
been reported in many studies (Pazda et al.,  2019; Zou
et al., 2021). Hence, sewage water acts as a perfect hotspot
for HGT of MGEs containing ARGs in the presence of
metals, thereby promoting co-­selection of metal and an-
tibiotic resistance. Co-­selection can be triggered once the
metals accumulate to a critical concentration.
In the case of clinical settings, routine cleaning of
high-­touch surfaces around patients does not get rid of
contaminating pathogens, causing major healthcare-­
associated infections (HAIs) (Boyce,  2007). To mitigate
this problem, such surfaces are covered with antimicro-
bial coatings containing metals such as Cu and Ag, or
antimicrobial peptides (Page et al., 2009). However, such
metal-­ridden surfaces provide the bacterial community
with a heavy metal selection pressure and hence, may
form suitable platforms for co-­selection of antibiotic and
metal resistance (Pietsch et al., 2020). In addition to this,
hospital waste, such as damaged thermometers, barome-
ters, sphygmomanometers or other waste electrical equip-
ment and biomedical waste, is usually ridden with heavy
metals. Thus, hospitals and clinics also act as a significant
source of heavy metal pollution.
The concentration of antibiotics in aquatic (Kolpin
et al., 2002), as well as soil environments, is generally
low as they are continuously being degraded rapidly via
photolytic and other pathways (Cardoza et al., 2005). But
depending on bioavailability and chemical forms, metals
are highly prevalent in most environments and are ever-­
increasing (Stepanauskas et al., 2006; Berg et al.,  2010).
This implies that heavy metals, being highly recalcitrant,
can provide the bacterial community with a long-­lasting
selection pressure (Stepanauskas et al.,  2006). Thus,
heavy metal contamination of the environment has a
far-­reaching impact on the dissemination of ARGs and
urgent introduction of efficacious strategies and policies
is required to control it. In addition to this, heavy metals,
being nonbiodegradable, enter the food chain via contam-
inated soil and water and end up accumulating in different
trophic levels, thus, also posing a direct threat to human
health and environmental biodiversity.
CO - ­S ELECTION OF METAL AND
ANTIBIOTIC RESISTANC E
As opposed to antibiotics, metals have biologically ex-
isted since the formation of the earth and the great oxida-
tion event nearly 2.4 billion years ago (Fru et al.,  2016).
Accordingly, the genes associated with toxic metal re-
sistance are also believed to be primordial (Jackson and
Dugas, 2003; Boyd and Barkay, 2012). Even then, antibi-
otic resistance was discovered first and not metal resist-
ance. Mercury resistance transposons, having a close
resemblance to contemporary Tn21-­like transposons, but
lacking ARGs, have been isolated recently from ‘preanti-
biotic era’ bacteria (Essa et al., 2003; Kholodii et al., 2003).
Whole-­genome sequencing of ‘Preantibiotic era’ bacteria,
resuscitated from long-­term storage, has also corroborated
the presence of multiple metal resistance genes (MRGs)
(Dunne et al., 2017). Therefore, it can be concluded that
metal resistance is a fairly pre-­existing feature of heavy
metal exposed microbial communities, which might have
somehow influenced or promoted the rapid modern-­day
evolution of AMR (Pal et al., 2017).
The co-­selection of metal and antibiotic resistance is
instituted on the theoretical similarity and overlapping
between the resistance mechanism for both these an-
timicrobial agents. The first account of acquired heavy
metal resistance in bacteria was given by Moore  (1960)
after isolating mercury-­resistant S. aureus from an epi-
demic hospital infection. However, the genetic linkage of
acquired mercury resistance in S. aureus, isolated from
wound infections with high penicillinase activity was
described by Richmond and John (1964). Furthermore,
Timoney et al. (1978) recovered Bacillus strains from sew-
age sludge which showed combined resistance to mercury
and ampicillin frequently. These Bacillus strains suggest
a simultaneous selection of genes for both these resis-
tances under a mercury selection pressure in the sludge.
Decade-­old profound researches on S. aureus, E. coli, and
S. Typhimurium have also revealed a correlation of anti-
biotic and metal resistance as the ARGs carrying plasmids
of these human pathogens also carried MRGs, indicating
a co-­selection potential (Novick and Roth,  1968; Hedges
and Baumberg, 1973; Hobman and Crossman, 2015).
Since metals are ubiquitous, posing an enduring selec-
tion pressure on microbes, a positive correlation between
METAL-­INDUCED ANTIBIOTIC RESISTANCE
the presence of metals and ARGs has been described in
several environmental contexts, for example in agricul-
tural soils, mining-­affected areas, aquaculture systems,
swine and swine farms, in dairy farms and the gut mi-
crobiota of various other animals (Resende et al.,  2012;
Holman and Chénier,  2015; Zhou et al.,  2016; Ding
et al., 2019; Maurya et al., 2020; Zou et al., 2021). The use
of metals, such as Zn and Cd, as therapeutics in animal
feeds, instead of or as a supplement to antibiotics, was
shown to co-­select and increase the prevalence of meth-
icillin resistance in S. aureus. This was attributed to the
co-­located genes for metal resistance (czr) and methicil-
lin resistance (mecA) on staphylococcal cassette chromo-
some mec (SSCmec) (Cavaco et al.,  2010, 2011). Similar
co-­selection of zinc and methicillin-­resistant S. aureus
(MRSA) has been reported in the microbiota of pigs as
well (Hau et al., 2017; Slifierz et al., 2015). Environmental
isolates of E. coli frequently display multiple antibiotic re-
sistance on exposure to metals, for example resistance to
β-­lactams on exposure to copper and zinc in pig manure
(Hölzel et al., 2012), and increase in resistant isolates of
E. coli in the gut microbiota of piglets on supplementation
of zinc (Bednorz et al., 2013). The genes for streptomycin
and mercury resistance are reported to be present on Tn21
transposon in isolates of avian E. coli, thus indicating that
both these genes have a potential of co-­transferability
which may lead to the selection of streptomycin resistance
on exposure to mercuric compounds (Bass et al., 1999).
IncHI-­type plasmids, containing multiple resistance
genes, isolated from both humans and animals, and pres-
ent in Gram-­negative bacteria of the Enterobacteriaceae
family, have been linked to the propagation of metal and
antibiotic resistance. One such plasmid of the IncHI-­2
group, namely pMG101 (182  kb), conferring resistance
to ampicillin, streptomycin, sulphonamide, chloram-
phenicol, tetracycline, tellurium, mercury and silver was
isolated from Salmonella enterica serovar Typhimurium
(Larkin Mchugh et al., 1975; Gupta et al., 1999).
Various experimental as well as in situ studies, in-
vestigating the co-­selection process in the environment,
have also been carried out by several researchers (Berg
et al.,  2005; M. Zhang et al.,  2020; Silva et al.,  2021).
Microcosm study of freshly collected savannah river water
samples, using Ralstonia mannitolilytica, an opportunis-
tic human pathogen, provided experimental evidence of
co-­selection of multiantibiotic and multimetal-­resistant
micro-­organisms (Stepanauskas et al., 2006). This patho-
gen proliferated and enriched the fresh water microcosm
when Cd was added to it. The transfer of ARGs across
bacterial genera, primarily through conjugation in water
environment, was observed to increase under subinhibi-
tory concentrations of Cu and Ag nanoparticles as well as
heavy metals ions, such as Cu (II), Cr (VI), Ag(I), Zn(II)
     |  4063
(Y. Zhang et al., 2018; S. Zhang et al., 2019; Lu et al., 2020;
Wang et al.,  2020). This facilitates the transmission of
antibiotic resistance among environmental bacteria and
subsequently, the pathogenic bacteria as well. All these
studies connote that metal exposure can co-­select for an-
tibiotic resistance in clinically relevant bacterial isolates.
The primary benefit that the bacteria realize from co-­
selection is the ability to sustain a multitude of antimicro-
bial selection pressures which will help in the subsistence,
proliferation and evolution of different bacterial species
in the environment. Co-­selection makes bacteria more fit
for survival. Furthermore, in case such a bacterium, har-
bouring resistance against multiple antimicrobial agents,
including antibiotics, enters the clinical settings, it would
be difficult to eradicate it. Such drug-­resistant bacteria
have the potential to elicit hospital epidemics, critical
infections, sepsis, and the failure of antibiotic therapies.
Multidrug resistance is already prevalent in hospitals. In
addition to this, exposure to multiple antibiotics, in an
attempt to treat the existing problem, can further aid in
developing resistance to other drugs/ antibiotics which
are used clinically. Therefore, the comprehension of the
co-­selection process is imperative as it has the potential to
sustain and promote ARGs even in the dearth of antibiot-
ics, having a long-­term clinical and environmental impact.
THE CONCEPT BEHIND THE
CO - ­S ELECTION OF RESISTANC E
GENES
The ability of the micro-­organisms to undergo minute
changes that allow their survival in extreme conditions,
such as strong antimicrobial selection pressures, eventu-
ally results in the evolution of resistance. These changes
may either be chromosomal mutations or the acquisition
of MGEs, which confer resistance to the microbial cell.
Although these changes allow the cell to survive, they be-
stow a metabolic burden on the cell, which makes it less
fit to survive in the absence of selection pressure. Thus,
such changes in a bacterial cell that allow it to survive in
conditions of stress, give rise to its ‘fitness cost’ (Vogwill
and Maclean, 2015). Due to this fitness cost, the cell be-
comes biologically less efficient and only reproduces
under selection pressure. Alternatively, in case of no selec-
tion pressure, the susceptible cells will have the ability to
outgrow the biologically less fit resistant cells (Andersson
and Hughes,  2010). Thus, under no selection pressure,
the resistant cells should ideally lose the acquired MGEs
to eliminate the cost of fitness. However, under natural
circumstances, the rate of reversibility is fairly low due to
factors such as reduction in the fitness cost due to compen-
sating mutations, the absence of a fitness cost altogether,
 |
4064       VATS et al.
and genetic co-­selection between the gene under selection
and a resistance-­conferring gene. In addition to this, the
maintenance of a resistant population in the environment
has been attributed to the sub-­MIC selection which stabi-
lizes the fitness cost of resistance.
A detailed study conducted by Gullberg et al.  (2011)
suggests that the presence of a very low antibiotic con-
centration plays an essential role in the selection, prolif-
eration and maintenance of existing resistant mutants in
addition to the selection of newly evolved mutants in the
environment or infected human organs during treatment.
This minimum selective concentration (MSC), thus, can
be defined as the threshold where the fitness cost of a
resistance gene is equal to the benefit of being resistant
(Figure 1). This concept comprises two major hypotheses.
The first one being if the MSC of an antimicrobial agent is
higher than its existing concentration, then an overall de-
crease or eventual disappearance of the resistance genes
will be observed. Alternatively, keeping the concentration
of the antimicrobial agent high (≥MSC) will also keep the
existing resistance genes maintained along with selecting
for de novo resistant mutants which, in due course, will
dominate. Thus, it can be simply stated that a directly pro-
portional relationship exists between the concentration
of antimicrobial agent and the selective pressure that it
imposes. The same principle will apply to heavy metals, as
they can act as antimicrobial agents as well and are widely
distributed in the environment and the clinical settings.
This concept is supported by a report that evaluated the
MSCs of various antimicrobial agents and heavy metals
in E. coli cells harbouring a plasmid that had resistance
genes for multiple antibiotics and heavy metals. The MSCs
of CuSO4 and As were observed to be 10-­fold and 140-­fold
lower than the MICs of the susceptible strain of E. coli
(Gullberg et al.,  2014). This outcome suggests that even
Sub-MIC selecon
Growth rate
MSC MICsusc
Concentration of the Antimicrobial agent
Resistant strain Suscepble strain
limited exposure to low concentrations of antimicrobial
agents, such as heavy metals, in the environment, clinical
settings and treated animals or humans, can co-­select for
ARGs in pathogenic micro-­organisms and thus, contrib-
ute to the emergence, proliferation and dissemination of
multidrug -­esistant bacteria. Such studies highlight the
need to introduce new measures to control environmental
contamination by heavy metals and restrict the redundant
use of antibiotics in the treatment of diseases.
MEC HANISMS OF CO - ­S ELECTIO N
Co-­selection of heavy metals and antibiotic resistance is
based on overlapping between mechanisms for both these
resistances, which can either be physiological overlapping
(Cross-­resistance) or genetic overlapping (Co-­resistance/
co-­regulation) (Figure  2). These mechanisms are dis-
cussed in the following sections.
CO - ­R ESISTANCE AND CROSS -­
RESISTANC E: MOLECULAR
MEC HANISMS FOR CO - ­S ELECT IO N
Co-­resistance is established when genes for two or more
resistance phenotypes, located on a single genetic element,
are acquired by a cell under selection pressure. In such a
case, the cell ends up acquiring the genes for other resist-
ance mechanisms as well. The MGEs, such as transposons,
plasmids and integrons, which harbour the resistance
genes are involved in co-­resistance and likely undergo HGT
(Ghosh et al.,  2000; Gómez-­Sanz et al.,  2013). Mercury-­
resistant genes were demonstrated to be genetically linked
with ARGs on plasmids as they were co-­transferred during
Convenonal selecon
F I G U R E 1   The concept of minimal
selective concentration (MSC). Selection
of resistant bacteria occurs at MSC of the
antimicrobial agent which is lower than
MICres the minimum inhibitory concentration
for susceptible (MICsusc) and resistant
(MICres) cells. (adapted from Gullberg
Dead cell
et al., 2011)
METAL-­INDUCED ANTIBIOTIC RESISTANCE
F I G U R E 2   Mechanisms behind
co-­selection of antibiotic and metal
resistance. (adapted from Baker-­Austin
et al., 2006, Pal et al., 2017)
mating between Enterobacteriaceae and recipients. Ghosh
et al. (2000) reported the presence of plasmids in various
strains of S. abortis showing co-­resistance for ampicillin
and multiple heavy metals, such as As, Cd, Hg and Cr.
On removing these plasmids from the cell, the bacteria
became sensitive to these antimicrobials proving that the
genes for these agents were indeed present on plasmids.
Furthermore, in a study by Hasman and Aarestrup, the
authors reported a strong correlation between copper re-
sistance and macrolide and glycopeptides resistances. In a
set of experiments, they were able to co-­transfer plasmids
from E. facieum obtained from pigs, harbouring the tcrB
and ermB genes responsible for copper and macrolides re-
sistance, respectively, to a susceptible recipient (Hasman
and Aarestrup, 2002). Similarly, a type of IncA/C plasmid,
harbouring a mercury resistance determinant (mer operon)
and multiple ARGs was isolated from Aeromonas salmo-
nicida from aquaculture facilities (Mcintosh et al., 2008).
The use of advanced sequencing and other bioinformat-
ics platforms to analyse existing data allows an improved
understanding of natural co-­resistance mechanisms. The
examination of these reports shows that genetic elements,
through multiple recombination events, have acquired
transposase and other insertion sequences (Dennis, 2005)
and might also harbour antibiotic and metal resistance
genes. For example genetic analysis of Salmonella enteric
serovar Typhi CT18 unveiled the existence of multiple an-
tibiotic and Hg resistance genes on a conjugative plasmid
referred to as pHCM1 (Parkhill et al., 2001).
AMR genes are most commonly incorporated into
transposons which are one of the best-­documented ex-
amples of co-­resistance occurring through MGEs (Liebert
et al., 1999; Mindlin et al., 2001; Baker-­Austin et al., 2006).
Their efficient mechanism of introducing genes to a new
     |  4065
ARG
Plasmid
HMRG
vector has contributed to the unprecedented dissemina-
tion of antibiotic and metal resistance since metal resis-
tance is majorly governed by MGEs, for example, Tn21
and Tn21-­like transposons harbouring Hg resistance gene
and ARGs. This fact was supported by a report wherein a
conjugative plasmid, harboured by Enterococcus faecium,
was observed to comprise of novel mercury-­resistance op-
eron containing a Tnmer1 putative transposon as well as
streptomycin resistance gene (aadK) (Davis et al.,  2005;
Baker-­Austin et al., 2006).
Cross-­resistance is observed when different antimicro-
bial agents employ similar modes of action to kill a mi-
crobial cell. The affected cell adopts mechanisms, such
as multidrug efflux systems, alteration of the cell mem-
brane, mutations or some outer-­membrane adaptations,
that may confer resistance against more than one antimi-
crobial agent (Aendekerk et al.,  2002). Cross-­resistance
is most commonly conferred by multidrug efflux pumps
which expel antibiotics and heavy metals outside the bac-
terial cells (Martinez, 2009). The addition of vanadate to
soil due to its contamination by refinery oil was reported
to induce MDR in Enterobacter cloacae and Escherichia
hermannii through a membrane-­bound efflux system
(Hernández et al.,  1998). Many more such studies that
report cross-­resistance between antibiotics and metals
through enhanced efflux systems are available, some of
which are listed in Table 1.
CO - R
­ EGULATION AS A
MECHANISM FOR CO - ­S ELECTI O N
Heavy metal exposure, apart from triggering co-­
selection through co-­resistance and cross-­resistance,
 4066       | VATS et al.

has also been reported to enhance tolerance to antibi-

Aendekerk et al. (2002)
otics by triggering co-­regulation of ARGs. Metal ex-

Hayashi et al. (2000)

Teixeira et al. (2016)
Nishino et al. (2007)
X. Z. Li et al. (1997)
Cheng et al. (1996)
posure up-­regulates the expression of ARGs, as in the

Mata et al. (2000)
Kumar (1996)

Kaur et al. (2021)
Choudhury and
case of Enterobacteriaceae bacterium LSJC7, thereby
conferring an enhanced ability to survive antibiotic
exposure. Arsenate upregulates multiple ARG emrD,
and tetracycline resistance gene tet34 in LSJC7 strain,
a Gram-­negative member of Enterobacteriaceae (Chen
et al.,  2015). The fact that this upregulation was ob-

MexGHI–­OpmD efflux pump, RND efflux

Outer membrane proteins, efflux pumps

served at environmentally relevant concentrations of

DsbA–­DsbB multidrug efflux system

AcrD and MdtABC multidrug efflux

arsenate should be a cause of perturbation. Cu or Zn
along with the BaeR response regulator induces expres-
sion of acrD and mdtABC genes which code for AcrD
Outer membrane proteins

and MdtABC multidrug efflux pumps, responsible for

Multidrug efflux systems
Multidrug efflux pumps

Multidrug efflux pumps

antibiotic resistance in Salmonella enterica serovar
Shared mechanism

Typhimurium (Nishino et al.,  2007). Similarly, the ex-

pression of SoxS, which is a universal response regulator
Efflux pumps

protein that regulates multidrug efflux pump AcrAB in

pumps

pumps

E. coli and Salmonella sp., is upregulated in response to

oxidative stress caused by ions, such as Cd, Cu2+, and
Cr2O7− (Harrison et al., 2009; Kaur et al., 2021). Cd and
As, Cd, Cr, Cu, Hg,

Cu2+ ions also act as an inducer to derepress the multiple

As, Cr, Cu, Hg, Ni

antibiotic resistance regulatory operon marRAB in both

Zn, Co, Cd

Salmonella sp. and E. coli. The membrane-­associated

Vanadium

Cadmium
Ni, Pb
Metal(s)

copper oxidation and liberation through oxidative im-

Cu, Zn
Cd, Zn
T A B L E 1   Examples of studies that have reported cross-­resistance between metals and antibiotics

pairment of copper-­dependent envelope proteins, such

Ag
Co

as NADH dehydrogenase-­2, derepress the multiple anti-

Tetracyclines, Penicillin, Chloramphenicol, Cefotaxime
Penicillin, Quinolone, Tetracycline, Trimethoprim/

Beta Lactams, Kanamycin, Erythromycin, Novobiocin,

biotic resistance regulator (MarR), leading to increased

Ticarcillin And Clavulanic Acid; Fluoroquinolones,

antibiotic resistance (Eaves et al., 2004; Hao et al., 2014).

Tetracyclines, chloramphenicol, Beta Lactams

Increased production of SoxS and MarA subsequently

Aminoglycoside, Amphenicol, Cephalosporin,

Ampicillin, ciprofloxacin, chloramphenicol,

increases the expression of AcrAB which results in tol-

Beta-­lactams, Novobiocin, Deoxycholate
Erythromycin, Josamycin, Clindamycin

erance against antibiotics, for example, tetracycline,

novobiocin, oxacillin and chloramphenicol (Figure  3)
(Harrison et al., 2009). Co-­regulation was also reported
between carbapenem and heavy metals in Pseudomonas
aeruginosa. Initially, Conejo et al.  (2003) reported that
Some Cephalosporins

the expression of a porin protein, OprD2, responsible

Sulfamethoxazole

for the influx of carbapenem antibiotics, was observed

ceftizoxime

to be downregulated in P. aeruginosa in response to Zn

Antibiotic(s)

Ofloxacin
Tetracycline

eluted from siliconized latex urinary catheters. Zn ap-

parently had a negative effect on the expression of influx
protein OprD2, thereby rendering this bacterium resist-
ant to carbapenem. Later on, heavy metal resistance due
to Zn-­induced overexpression of CzrCBA, an RND type
E. coli, Pseudomonas aeruginosa

efflux pump, and two-­component regulator genes czcR

Salmonella enterica serovar

and czcS was also found in carbapenem-­resistant strains

Pseudomonas aeruginosa

Pseudomonas aeruginosa

of P. aeruginosa (Perron et al., 2004) suggesting a com-

Lysteria monocytogenes
Burkholderia cepacia

mon regulatory mechanism between this overexpres-

Micro-­organism

Salmonella Typhi
Typhimurium

sion and the repression of OprD2.

Bacillus subtilis

Besides these co-­selection strategies, combined re-

sistance to antibiotics and metals is aided by the forma-
E. coli

tion of biofilms also. The heavy metal or antibiotic stress

induces the production of certain substances that aid
METAL-­INDUCED ANTIBIOTIC RESISTANCE      |  4067

F I G U R E 3   Possible pathways for co-­regulation of heavy metal induced antibiotic resistance. Regulated by universal regulators SoxS or
MarA, through the acrAB operon that codes for the AcrAb-­TolC efflux pump. The transcription of MarA is repressed by a negative regulator,
MarR. The presence of metal ions derepresses the marRAB operon by catalysing the formation of MarR tetramer which has an attenuated
DNA-­binding ability. Gene clusters are represented by coloured arrows; * indicates the induced SoxR regulator protein (Harrison et al., 2009;
Hao et al., 2014)

in cellular adhesion along with the production of extra- VALIDATION OF CO - ­S ELECTIO N

cellular polymeric substances (EPS) in planktonic cells
which subsequently helps in the formation of biofilms
(Baker-­Austin et al.,  2006). Induction of biofilm due to
production of EPS as a survival strategy on exposure to
metals has been reported by García-­Meza et al. (2005) and
Kidambi et al. (1995) in Phormidium (cyanobacteria) and
Pseudomonas species respectively. Other than this, the
presence of bacterial biofilms in metal-­contaminated acti-
vated sludge has been demonstrated by other researchers
as well (Wuertz et al., 2004). Likewise, the expression of
ica, an intracellular precursor for the synthesis of poly-
saccharide adhesins, has been shown to be promoted
by subinhibitory concentrations of antibiotics, such
as tetracycline, in Staphylococcus epidermidis (Rachid
et al., 2000). Thus, antibiotic and metal exposure has been
proved to induce biofilm production in microbial commu-
nities, which in turn helps the bacteria to resist both these
antimicrobial agents.
OF METAL AND ANTIBIOTIC
RESISTANCE IN CLINIC AL
SETTINGS
Abundant studies reporting the co-­selection of metal and
antibiotic resistance in various environmental contexts
are available but there is a paucity of studies directed
solely at examining co-­selection in clinical settings. Links
of co-­selection in a clinically relevant strain were first
established in the early 1960s (Moore, 1960). In a more
recent bioinformatic study, through a systematic ap-
proach to study complete sequenced bacterial genomes
and plasmids, a high ratio of plasmids was found in clin-
ically relevant genera, including Escherichia, Klebsiella,
Staphylococcus and Salmonella (Pal et al.,  2015). The
majority of the plasmids in these bacteria carried genes
for both metal and antibiotic resistance, thus, indicat-
ing a potential for co-­selection through co-­resistance.
 |
4068       VATS et al.

This directs a pervasiveness of heavy metal-­induced
antibiotic resistance in clinical pathogens that has not
been studied enough. Correlation between metals and
antibiotic resistance in micro-­organisms was validated
in a study on clinical isolates of Salmonella enterica
serovar Typhi (Kaur et al.,  2018). This study observed
that on supplementation of Cd, the sensitive isolates of
this organism were rendered resistant and the already
resistant isolates were rendered more resistant in terms
of their MICs of antibiotics, including ampicillin, cip-
rofloxacin, chloramphenicol and ceftizoxime, through
a co-­regulatory mechanism of co-­selection. Table  2
further lists some selected studies that establish a cor-
relation between the presence of antibiotic and metal
resistance in clinical isolates or clinically relevant
micro-­organisms.
A heavy metal selection pressure exists in the clinical
settings, for example due to the use of surgical equipment,
dental fillings, metal-­containing disinfectants, thermom-
eters or other metallic apparatus. However, direct human
exposure to heavy metals through factors such as diet
(EFSA,  2004), or smoking (Ashraf,  2012) has also been
reported which may co-­select for antibiotic resistance.
This was indicated by an analysis of the national health
and nutrition examination survey where the effects of
blood lead (Pb) levels on nasal colonization of methicillin-­
resistant Staphylococcus aureus (MRSA) and methicillin-­
susceptible Staphylococcus aureus (MSSA) were studied.

T A B L E 2   Examples of studies reporting a correlation between metal and antibiotic resistance in clinical settings

Clinically relevant micro-­

organism(s) exhibiting
co-­selection
Staphylococcus aureus
Salmonella Typhimurium
Pseudomonas aeruginosa
Escherichia coli
Oral Streptococci, members of
Enterobacteriaceae
Stenotrophomonas maltophilia
Staphylococcus aureus
Pseudomonas aeruginosa,
Klebsiella pneumoniae
Pseudomonas aeruginosa
Escherichia coli, Klebsiella
pneumoniae
Staphylococcus aureus
Staphylococcus aureus
Members of Enterobacteriaceae
Salmonella Typhi
Pseudomonas sp.
Streptococcus agalactiae
Klebsiella pneumoniae
Salmonella Typhi
Antibiotic(s) Metal(s)
Penicillin Mercury Novick and Roth (1968)
Chloramphenicol, Ampicillin Silver, mercury Larkin Mchugh
et al. (1975)
Streptomycin, Kanamycin, Chloramphenicol Mercury, cadmium, Nakahara et al. (1977)
arsenic
Streptomycin, Tetracycline, Chloramphenicol, Mercury Nakahara et al. (1977)
Kanamycin
Ampicillin, Streptomycin, Tetracycline Mercury Summers et al. (1993)
Macrolide Cadmium Alonso et al. (2000)
Penicillin G, Methicillin, Oxacillin Lead, chromium Ugur and Ceylan (2003)
Kanamycin, nalidixic acid, oxacillin, Cadmium EFSA (2004)
sulphonamides
Carbapenem Zinc, copper Caille et al. (2007); Conejo
et al. (2003); Perron
et al. (2004)
Beta-­lactams, Aminoglycosides, Trimethoprim, Silver, copper, arsenic Sandegren et al. (2012)
Tetracyclines, Macrolides
Methicillin Zinc, cadmium Nair et al. (2014)
Amoxicillin/Clavulanic Acid, Co-­Trimoxazole, Lead Garhwal et al. (2014)
Cefoxitin, Amoxicillin/Clavulanic Acid
Third-­ and Fourth-­generation Cephalosporins, Lead Garhwal et al. (2014)
Gentamycin, Ciprofloxacin, Co-­Trimoxazole,
Tetracycline
Nalidixic Acid, Levofloxacin, Lead Garhwal et al. (2014)
Ampicillin-­Sulbactam
Colistin, Levofloxacin, Cefepime Lead Garhwal et al. (2014)
Macrolide, Lincosamide Copper, cadmium Rojo-­Bezares et al. (2016)
Gentamicin, Chloramphenicol, Beta-­lactams, Mercury, copper, Zhai et al. (2016)
Rifampicin, Streptomycin, Trimethoprim, Tellurite, lead, cobalt,
Aminoglycosides cadmium
Ampicillin, ciprofloxacin, chloramphenicol, Cadmium Kaur et al. (2018)
ceftizoxime
METAL-­INDUCED ANTIBIOTIC RESISTANCE
This study observed an increase in nasal MRSA coloniza-
tion in people with higher blood Pb levels, suggesting the
selection of methicillin resistance under a selection pres-
sure posed by Pb present in the blood as one of the factors
(Eggers et al., 2018). This indicates that heavy metals have
a far-­reaching impact on the co-­selection of antibiotic
resistance, so much so, that heavy metals present inside
a human body may also have a role in the selection and
propagation of antibiotic resistance. Hence, this calls for a
thorough study of the influence of heavy metals in the se-
lection of antibiotic resistance, particularly in the clinical
context; as such scientific studies are still a rarity.
STR AT EG I E S TO DISAR M T HE
CO - ­S E L ECT ION OF HE AVY METAL
A ND A N T I BIOT IC R E SISTAN CE
Since the theory of co-­selection of metal and antibiotic re-
sistance has been validated through many studies, the next
pragmatic step would be to discover strategies to curb the
emergence of MDR bacteria through it. Designing new
therapies that use biological, chemical, or artificially de-
signed inhibitors for pharmacological inhibition of active
efflux pumps are becoming attractive strategies to combat
metal-­induced antibiotic resistance (Domalaon et al., 2018).
The potency of these agents can be increased by using
them in combination with antibiotics so that they can be
used at lower concentrations to simultaneously reduce
any chances of emergence of resistance. Pathogenicity of
the micro-­organism is also reduced by these agents as they
have been reported to inhibit biofilm formation (Sabatini
et al., 2017; Anes et al., 2019; Ferrer-­Espada et al., 2019). Use
of 1-­(1-­napthylmethyl)-­piperazine or NMP, one such active
efflux pump inhibitor, was reported to reverse AcrAB ef-
flux pump mediated MDR in cadmium adapted Salmonella
Typhi cells (Kaur et al., 2021). Therefore, discovering more
such inhibitor molecules, which on administration with
antibiotics can reverse MDR in micro-­organisms, can be
very beneficial for the medical community.
Use of a metal chelator, 2, 2′-­dipyridyl diselane
(DPDS), a chemically synthesized organoselenium com-
pound, along with antibiotics has been reported as a new
combinatorial approach to managing Cd-­induced antibi-
otic resistance in clinical isolates of Salmonella enterica
serovar Typhi (Rishi et al., 2017). On the treatment of Cd-­
adapted and antibiotic-­resistant cells of S. enterica with
DPDS in combination with antibiotics, such as ampicillin
or cefotaxime, the resistant cells were rendered sensitive
to these antibiotics again. DPDS was responsible for de-
stabilizing the structural organization of cells by chelating
both essential as well as nonessential (Cd) metal ions. A
synergistic and additive effect for various combinations of
     |  4069
DPDS and the antibiotics was indicated by the fractional
inhibitory concentration (FIC) indices, which might be
due to the multipronged action of both the agents admin-
istered together.
Another study suggested the use of dandelion root
­extracts and taraxasterol for the reversal of metal (Cd, Ni,
As) induced ampicillin and kanamycin resistance in E. coli
cells (Yang and Zhang, 2020). While treatment of E. coli
cells with low concentrations of Dandelion root extract
alone did not seem to show any antimicrobial activity,
the addition of kanamycin and ampicillin along with the
heavy metals and the root extracts significantly reduced
the bacterial growth, indicating that dandelion root ex-
tracts can be effective modulators against metal-­induced
antibiotic resistance.
Since, antibiotic-­resistant pathogens of clinical rele-
vance originate from nonclinical environmental settings,
an alternate approach to disarm metal-­induced antibiotic
resistance can be direct in situ removal of metals from pol-
luted environmental sites or pretreatment of metal-­ridden
industrial effluents which form a conducive environment
for co-­selection to take place. This may be done through
processes such as bioremediation, biosorption, bioaccu-
mulation and precipitation using algae, plants or micro-­
organisms (Ahluwalia and Goyal,  2007; Parameswari
et al., 2009). Several micro-­organisms have evolved various
genetically determined mechanisms to remove toxic ions
from their vicinity (Chang et al., 1997; Al-­Musharafi, 2016;
Chellaiah, 2018). Although the use of micro-­organisms for
bioremediation has been reported as an economic alterna-
tive, the potential spread of heavy metal resistance deter-
minants in the environment also needs to be considered
as these bioremedial mechanisms make the cells metal-­
resistant as well. This is a major limitation of utilizing bac-
teria for bioremediation as exposing metal-­resistant cells
to heavy metal selection pressure might select and perpet-
uate heavy metal resistance markers in the environment.
So, a procedure that utilizes the metal removing properties
of micro-­organisms in a controlled manner, to simultane-
ously restrain the dissemination of metal-­resistant bacte-
ria into the environment, might be a suitable approach.
Biosorption is a cost-­effective and relatively safer option
as there is no risk of dissemination of metal-­resistant bac-
teria. Dead or inactive bacteria, plant and fungus-­derived
biomass obtained from various industrial fermentations
can act as a cheap and efficient source of biosorptive ma-
terial that can absorb and concentrate metal ions from the
polluted site (Chang et al.,  1997; Issazadeh et al.,  2013;
Vázquez-­Núñez et al., 2018). Removal of metal ions from
polluted sites will ensure an absence of heavy metal selec-
tion pressure for environmental microbial communities,
thereby inhibiting co-­selection of antibiotic resistance and
its subsequent transfer to pathogenic micro-­organisms.
 |
4070       VATS et al.
CON C LUS I ON AN D FU T U R E
P E R S P ECT I VE S
The challenge of antibiotic resistance, because of the ex-
tensive exploitation of antibiotics within a population, is
well recognized. Scientists, doctors, and even the general
public around the world is familiar with the implications
of the emergence of MDR in pathogenic bacteria. However,
selection pressure from other agents such as heavy metals
is also a cause of concern as they are much more prevalent
and obstinate in the environment than antibiotics. Heavy
metals induce antibiotic resistance in micro-­organisms by
selecting the resistance determinants for both antibiotic
and metal resistance through mechanisms of co-­resistance,
cross-­resistance and co-­regulation. Identifying the domi-
nant mechanism responsible for co-­selection is crucial for
elucidating possible targets for developing novel drugs.
The environment is the ultimate source of resistance
determinants, from where they are transferred to clinical
settings (Alonso et al.,  2001). Reports on metal and an-
tibiotic co-­selection in pathogenic bacteria are increasing
globally. It should be highlighted that the bacterial strains
utilized for studying co-­selection of metals and antibiotics
in almost all the reports were isolated from the environ-
ment, indicating a lack of data on co-­selection in clinical
isolates. There is a huge prospect for studies explicitly in-
vestigating the process of co-­selection in clinical settings
and assessing its impact on mammalian disease-­causing
bacteria. Comprehensive studies analysing the environ-
mental resistance gene pools and the potential of these
genes to transfer into pathogens of clinical significance
are also vital. The current advances in microbial genom-
ics and procedures for comprehensive genetic, transcrip-
tional and translational analysis can form the basis for
such studies.
In view of rapidly proliferating antibiotic resistance
and depression in the field of discovery and develop-
ment of newer antibiotic drugs, it is essential to look
for alternate therapeutic strategies. Since co-­selection
of metal and antibiotic resistance is highly predominant
and adds to the burden of selection and dissemination
of antibiotic resistance globally, it is critical to discover
novel therapeutics to combat it. The use of metal chela-
tors to sensitize metal-­adapted bacteria turned out to be
successful in vitro, but its commercialization as an anti-
bacterial therapy to treat infections in vivo would be the
next bigger challenge. More such therapies and meth-
ods need to be developed to control the metal-­induced
promulgation of antibiotic resistance. Reframing the
existing guidelines for the management of heavy metal-­
ridden waste disposal and industrial effluent treatment
is also critical as they assist in the maintenance of ARG
pools in the environment.
CONFLICT OF INTEREST
The authors have no conflict of interest to disclose.
ORCID
Praveen Rishi  https://orcid.org/0000-0001-5425-8064
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How to cite this article: Vats, P, Kaur, UJ, Rishi, P
(2022) Heavy metal-­induced selection and
proliferation of antibiotic resistance: A review.
Journal of Applied Microbiology. 132:4058–4076.
https://doi.org/10.1111/jam.15492