Environmental Pollution 157 (2009) 2893–2902

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Review

Environmental pollution by antibiotics and by antibiotic resistance determinants

Jose Luis Martinez*
Departamento de Biotecnologı́a Microbiana, Centro Nacional de Biotecnologı́a, Consejo Superior de Investigaciones Cientı́ficas, Darwin 3, Cantoblanco,
28049 Madrid, and CIBERESP, Spain
The article reviews the current knowledge on the effects that pollution by antibiotics and antibiotic resistance genes
may have for the microbiosphere.

a r t i c l e i n f o a b s t r a c t

Article history: Antibiotics are among the most successful drugs used for human therapy. However, since they can
Received 2 December 2008 challenge microbial populations, they must be considered as important pollutants as well. Besides being
Received in revised form used for human therapy, antibiotics are extensively used for animal farming and for agricultural
21 May 2009
purposes. Residues from human environments and from farms may contain antibiotics and antibiotic
Accepted 25 May 2009
resistance genes that can contaminate natural environments. The clearest consequence of antibiotic
release in natural environments is the selection of resistant bacteria. The same resistance genes found at
Keywords:
clinical settings are currently disseminated among pristine ecosystems without any record of antibiotic
Antibiotic resistance
Antibiotic pollution contamination. Nevertheless, the effect of antibiotics on the biosphere is wider than this and can impact
Aquaculture the structure and activity of environmental microbiota. Along the article, we review the impact that
pollution by antibiotics or by antibiotic resistance genes may have for both human health and for the
evolution of environmental microbial populations.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction consigned to medical limbo. The repercussions are almost

unimaginable’’ (WHO, 2000).
Antibiotics are probably the most successful family of drugs so It is important to remark that several antibiotics are produced
far developed for improving human health. Besides this funda- by environmental microorganisms (Waksman and Woodruff,
mental application, antibiotics (antimicrobials at large) have also 1940). Conversely, antibiotic resistance genes, acquired by patho-
been used for preventing and treating animals and plants infections genic bacteria trough Horizontal Gene Transfer (HGT) have been
as well as for promoting growth in animal farming (McManus et al., originated as well in environmental bacteria (Davies, 1997),
2002; Smith et al., 2002; Singer et al., 2003; Cabello, 2006). All although they can evolve later on under strong antibiotic selective
these applications made antibiotics to be released in large amounts pressure during the treatment of infections (Martinez and Baquero,
in natural ecosystems. Little is known on the overall effects of 2000; Martinez et al., 2007). To understand in full the development
antibiotics on the population dynamics of the microbiosphere of resistance, we will thus need to address the study of antibiotics
(Sarmah et al., 2006). However, the effect of antibiotics used for and their resistance genes, not just in clinics but in natural
treating infections or for farming purposes in the selection of non-clinical environments also (Martinez, 2008). The situation
antibiotic-resistant microorganisms, which can impact human concerning antibiotics and their resistances resembles in some
health has been studied in more detail (Witte, 1998; Ferber, 2003; aspects to heavy metal contamination. Like antibiotics, heavy
Singer et al., 2003). As stated by the World Health Organization, the metals are natural compounds present in different ecosystems.
increasing emergence of antibiotic resistance in human pathogens However, their utilization by humans has increased their
is a special concern, not only for treating infectious disease, but also bioavailability, leading to dramatic changes in polluted ecosystems.
for other pathologies in which antibiotic prophylaxis is needed for Differing to heavy metals that challenge all forms of life, antimi-
avoiding associated infections. In this regard, the spread of antibi- crobials mainly alter the microbiosphere and probably because of
otic-resistant bacteria ‘‘.means that commonplace medical this, the consequences of antibiotic pollution on the biodiversity
procedures once previously taken for granted could be conceivably have received less attention.
Understanding heavy metal resistance in natural ecosystems
may help as well to understand antibiotic resistance in the envi-
* Tel.: þ39 91 5854542; fax: þ34 91 5854506. ronment. The elements involved in the resistance to heavy metals
E-mail address: [email protected] are encoded in the chromosomes of bacteria like Ralstonia

0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2009.05.051
2894 J.L. Martinez / Environmental Pollution 157 (2009) 2893–2902

metallidurans (Mergeay et al., 2003), which are well adapted for One example of this situation is a study in which the effect of the
surviving in naturally heavy metals-rich habitats (e.g. volcanic antibiotic ciprofloxacin on natural algal freshwater communities
soils). However, strong selective pressure due to anthropogenic was tested upstream and downstream a wastewater treatment
pollution has made that these chromosomally-encoded determi- plant. Significant differences were observed in the final biomass
nants are now present in gene-transfer units, so that they can yield, in the structure of suspended and attached algae, in the
efficiently spread among bacterial populations (Silver and Phung, nutrient processing capacity and in the natural food web of the
1996, 2005; Nies, 2003). Similarly, antibiotic resistance genes that ecosystems (Halling-Sorensen et al., 1998; Hirsch et al., 1999). A
were naturally present in the chromosomes of environmental similar study has demonstrated that tetracycline have a negative
bacteria (D’Acosta et al., 2006; Wright, 2007; Fajardo et al., 2008) impact on the functional diversity of soil microbial communities
are now present in plasmids that can be transferred to human (Kong et al., 2006).
pathogens. It has been highlighted that the contact of bacteria from Antibiotics at much higher concentrations that usually found in
human-associated microbiota with environmental microorganisms natural ecosystems can be found in water (e.g. sewage waters) and
in sewage plants or in natural ecosystems is an important feature soils (e.g. soils treated with manure and farm soils). However, these
to understand the emergence of novel mechanisms of resistance in high concentrations are usually concentrated to areas of human
human pathogens (Baquero et al., 2008). A key issue for this activity, whereas pristine environments usually have low concen-
emergence will be the integration of antibiotic resistance genes in trations of antibiotics. Risk assessments might thus take into
gene-transfer elements (e.g. plasmids), a feature that is favoured by consideration mainly those areas with high antibiotic load and
the release of antibiotics in natural ecosystems (Cattoir et al., containing human-associated microorganisms (reactors for evolu-
2008). tion of resistance, see Baquero et al., 2008) for analyzing the effect
of antibiotic pollution on natural ecosystems.
2. Functional role of antibiotics and antibiotic resistance A different situation occurs for antibiotic resistance genes. It has
elements in bacterial natural ecosystems been stated that acquisition of an antibiotic resistance phenotype
produces a metabolic burden (Andersson and Levin, 1999; Morosini
Since antibiotics are efficient inhibitors of bacterial growth et al., 2000; Andersson, 2006), and it was predicted that in the
produced by environmental microorganisms, it has been widely absence of selective pressure, resistance would disappear. Unfor-
accepted that their role in nature will be to inhibit microbial tunately, this situation is not always true. Acquisition of antibiotic
competitors. Conversely, antibiotic resistance determinants should resistance may produce specific changes in the bacterial metabo-
serve to avoid the activity of antibiotics, in such a way that they lism that can be even beneficial for bacterial growth in some
would be a good example of the Darwinian struggle for life. habitats (Sanchez et al., 2002b; Alonso et al., 2004; Linares et al.,
Although this can be true in some occasions, an alternative 2005; Luo et al., 2005). Antibiotic resistance genes are found
hypothesis stating that antibiotics could be signal molecules that worldwide, as can be predicted due to their origin in environmental
shape the structure of microbial communities has been proposed bacteria (Davies, 1994; Alonso et al., 2001). However, the wide
(Linares et al., 2006; Yim et al., 2007; Fajardo and Martinez, 2008). dissemination of genes frequently present in human pathogens in
Under this view, the antibiotics will have a hormetic effect, bene- places without a high antibiotic load (Pallecchi et al., 2008) indi-
ficial at low concentrations likely found in most natural ecosystems, cates that, once those elements are present in gene-transfer plat-
and harmful at the high concentrations used for therapy (Calabrese, forms, the probability for their maintenance in natural ecosystems
2004, 2005; Davies et al., 2006). can be high. For this reason, antibiotic resistance genes are being
Similarly, it has been stated that some elements that serve to considered as pollutants themselves. Since antibiotic resistance
resist high concentrations of antibiotics, have disparate functional genes are naturally located in the chromosomes of environmental
roles (e.g. cell homeostasis, signal trafficking, metabolic enzymes, .) bacteria (D’Acosta et al., 2006; Wright, 2007; Martinez, 2008), only
in their original hosts (Martinez et al., 2007, 2009). The strong those elements that are present in gene-transfer elements and thus
increase of antibiotic concentrations in natural ecosystems as can be transferred and enriched under antibiotic selection, should
the consequence of human activities (human therapy, farming) shifts be considered as bona fide pollutants (see below).
the original functions of antimicrobials and resistance elements to Contamination by antibiotics is relevant, not just for its impact
the weapon/shield roles they play in hospitals or farms (Martinez, on bacteria that can infect humans. As discussed along the review,
2008). These changes might influence, not just the selection of the release of antibiotics together with antibiotic resistance
antibiotic-resistant microorganisms, but also the structure of the bacteria can impact as well the environmental microbiota.
natural microbial populations and may alter the physiology of
microorganisms as well. 3. Different consequences of antibiotic pollution
Besides selecting antibiotic-resistant mutants and favouring the and antibiotic resistance pollution
acquisition of antibiotic resistance determinants by gene-transfer
elements that can spread among the environmental microbiota, Antibiotic utilization for clinical or farming purposes selects
antibiotic pollution can enrich the population of intrinsically resistant microorganisms (Teuber, 2001; Livermore, 2005). It is
resistant microorganisms, and reduce the population of susceptible thus predictable that residues from hospitals or farms will contain
microbiota. For instance Cyanobacteria, which are responsible of both types of pollutants: antibiotics and resistance genes. Never-
more than a third of total free O2 production and CO2 fixation, are theless, the fate of both types of pollutants is likely different.
susceptible to antibiotics. There is not at the moment any indication Several antibiotics are natural compounds that have been in contact
that the Cyanobacteria population is suffering the impact of anti- with environmental microbiota for millions of years and are thus
biotic pollution, and the risks for this situation are likely very low. biodegradable, an even serve as a food resource for several
However, the dramatic effect that eliminating Cyanobacteria as the microorganisms (Dantas et al., 2008). Synthetic antibiotics (e.g.
consequence of antibiotic pollution might have for the biosphere quinolones) can be more refractory to biodegradation. However,
reinforce the idea that the release of antibiotics in natural envi- they are still degraded at different rates in natural environments. It
ronments have relevant consequences not just in terms of resis- has been shown that ciprofloxacin present in river water samples is
tance but for the maintenance of the global activity of the completely degraded after 3 months, whereas only 20% of oxolinic
microbiosphere also. acid present in these samples is degraded after five months
 J.L. Martinez / Environmental Pollution 157 (2009) 2893–2902 2895

(Turiel et al., 2005). Recent work has shown that the binding of
quinolones to soil and sediments delays their biodegradation.
Nevertheless, wastewater treatment of quinolones-polluted waters
efficiently removes these antibiotics by processes that include, not
just biodegradation, but photodegradation also (Sukul and Spiteller,
2007). Consistent with these results, it has been shown that most
antibiotics are usually below detectable limits in ground water
samples, although they are more stable upon adsorption to sedi-
ments (Halling-Sorensen et al., 1998; Hirsch et al., 1999). For this
reason, sediment samples from antibiotic-polluted environments
have higher antibiotic concentrations than water samples from the
same site (Kim and Carlson, 2007).
The fact that antibiotics are degraded in natural ecosystems
does not mean that they are not relevant pollutants. For instance
the degradation process is slow at low temperatures in winter
(Dolliver and Gupta, 2008), and the composition and moisture of
the soil clearly impact antibiotic degradation (Stoob et al., 2007).
More important, some ecosystems suffer a constant release of
antibiotics (e.g. hospital effluents, farms residues), so that they are
constantly polluted irrespectively of antibiotic degradation. As
stated by Lindberg et al. ecotoxicity tests are usually performed
using very high concentrations of antibiotics for short periods of
time, whereas in these types of environments, the organisms are
continuously exposed to antibiotics at sub-therapeutic levels
(Lindberg et al., 2007). Since sub-inhibitory concentrations of
antibiotics trigger specific transcriptional responses in bacteria
(Tsui et al., 2004; Davies, 2006; Davies et al., 2006; Linares et al.,
2006; Yim et al., 2007; Fajardo and Martinez, 2008), the presence of
antibiotics will necessarily modify the metabolic activity of the
microbiota present in these polluted environments. Finally, the
impact on the structure of bacterial populations due to the presence
of antibiotics might remain even when the antimicrobials have
been mineralized (Fig. 1). In any case, the fate of antibiotics in
natural ecosystems is their degradation (Pei et al., 2006) in such
a way that if the utilization of a given antibiotic is banned, it will
disappear as a pollutant from natural ecosystems. As we will see
later on, this justifies the rational implementation of methodolo-
gies for alleviate antibiotic pollution, even by reducing the utiliza-
tion of antimicrobials or by enhancing their degradation.
Differing to this situation, resistance determinants present in
gene-transfer units are auto-replicative elements that can be
maintained in microbial populations unless they confer a fitness
cost to the recipient bacteria. Some works have shown that Fig. 1. Differential fate of polluting antibiotics and antibiotic resistance determinants
reducing the antibiotic load in natural ecosystems may reduce as in natural ecosystems. As shown in panel 1, antibiotics are degraded (more or less
rapidly), so that they will disappear unless new events of pollution occur. If antibiotics
well the amount of pollutant antibiotic resistance genes. It has
are released in soils, their dissemination is low, and they can disseminate more when
been described that sewage dilution in river waters reduces the released in water. In the case of antibiotics, dissemination implies a reduction of their
amount of plasmid-encoded antibiotic resistance genes in Escher- concentration. Differing to that situation, antibiotic resistance genes (panel 2) can be
ichia coli (Gonzalo et al., 1989). In another well-known example, integrated into genetic platforms that may spread in bacterial populations. Since these
the ban in the utilization of some antibiotics in farming has clearly populations replicate and move among different ecosystems, resistance genes do not
necessarily disappear once their release is discontinued. Furthermore, they can
reduced antibiotic resistance in animals and its transfer to humans eventually spread without reducing their concentration (a). Finally, the amount of
(Aarestrup et al., 2001). In these cases, it seems that antibiotic resistance genes can increase in other allocations or some time after their release in
resistance should be eliminated once the selective pressure is the environment (b), if they are selected by new events of antibiotic pollution or are
released. co-selected by an ecologically favourable determinant present in the same genetic
element (see text for more details). In both panels, darker regions represent higher
However, the situation is not that simple. It has been described
levels of contamination.
that even though the incidence of resistance declines, this decline is
slow and part of the resistant population remains (Andersson,
2003), a situation that agrees with current predictions based in (Salyers and Amabile-Cuevas, 1997), this indicates that antibiotic
mathematical models (Levin, 2002). Furthermore, the presence of resistance genes are resistant to elimination even in the absence of
the same resistance genes currently present in human pathogens antibiotic selective pressure. As we will discuss later on, several
has been reported in environments without a history of antibiotic mechanisms allow the maintenance, and eventually spread of
contamination (Pallecchi et al., 2008). These environments include antibiotic resistance genes in natural ecosystems, so that differing
remote human and animal populations without known antibiotic to antibiotic contamination, pollution by antibiotic resistance
exposure, which might present a high prevalence of resistance determinants would not be necessarily disappear even if the
despite not receiving any antibiotic (Gilliver et al., 1999; Livermore release of antibiotic resistance genes in the environment is
et al., 2001; Grenet et al., 2004; Bartoloni et al., 2009). As stated in discontinued (Fig. 1).
2896 J.L. Martinez / Environmental Pollution 157 (2009) 2893–2902

4. Release, behaviour and effects of antibiotics consideration that 25–75% of the antibiotics administered to
in natural ecosystems feedlot animals are excreted unaltered in faeces, it is clear that the
antimicrobial use in livestock is an important source of antibiotics
Most antibiotics used for preventing or treating infections in release into the environment (Dolliver and Gupta, 2008).
humans or animals as well as for promoting faster growth of live- Although some effects of antibiotics on plants’ growth have
stock are only partially metabolized and are then discharged along been reported (Jjemba, 2002), the main impact of these pollutants
the excreta, either to sewage treatment plants or straightforward in will be on the environmental microbiota. This impact has different
waters or soils (Dolliver and Gupta, 2008). Besides, antimicrobial levels and consequences.
compounds used in intensive fish farming are added directly to the First, utilization on antibiotics can select for antibiotic-resistant
water rendering high local concentrations both in water and in bacteria within the treated host. In the case of antibiotics used for
adjoining sediments (Cabello, 2006). In addition, antibiotics are farming purposes, selection of resistance can be important for both
topically added to the aerial organs of infected plants, although the the treatment of animal infections and for human health. Several
amount of antibiotics used in plant agriculture is low comparing evidences support an association between the use of antimicrobial
with human and veterinary medicine and animal production agents in food animals and antimicrobial resistance among bacteria
(McManus et al., 2002). isolated from humans (Angulo et al., 2004). However, some con-
To alleviate the effect that the release of antibiotics for non- flicting issues concerning the strategies for avoiding this problem
human use may have for the selection of resistance in human remain. Whereas some authors state that banning the use of anti-
pathogens, the European Union banned the feeding of those anti- biotics might increase the number of foodborne pathogens (Cox
biotics, which are valuable in human medicine, to livestock for and Ricci, 2005, 2008; Mathew et al., 2007), others indicate that the
growth promotion in 1998. In 2006, the ban was expanded to all removal of growth promoting antibiotics have reduced the inci-
antibiotics and related drugs. Similarly, many countries have dence of resistant bacteria isolated from animals (Aarestrup et al.,
restricted the use of antibiotics in aquaculture, including strong 2001; Smith et al., 2002).
restrictions in the use of antibiotic prophylaxis, and proscription of The effect of antibiotics used for farming in human health has
the utilization of antibiotics that are still useful in the therapy of mainly focused on foodborne pathogens. These bacteria are
human infections. For instance, the utilization of quinolones for present in the animals and can infect humans. Examples of food-
aquaculture has been banned in several industrialized countries, borne pathogens are Campylobacter jejuni, E. coli, Salmonella or
because resistance to one member of the family usually confers Enterococcus faecium among others. For those pathogens, both
simultaneously resistance to all quinolones (Martinez et al., 1998; mutation-driven antibiotic resistance and the acquisition of anti-
Hooper, 1999; Piddock, 1999) and because those antibiotics can biotic resistance genes (Garofalo et al., 2007) are important
remain active in sediments for long periods. concerns for human health, because the same strain can colonize
As stated by the World Health Organization, the amount of both animals and humans, and antibiotic resistance genes can
antimicrobials used in animals is not known precisely because easily spread among bacterial species (or clones) that are closely
national statistics on the amount and pattern of use of antimicro- phylogenetically related (Sundsfjord et al., 2001).
bials exist in only a few countries (WHO, 2002). This is an important Another issue concerns the enrichment of resistance genes in
drawback to evaluate the impact of antibiotic utilization in veteri- gene-transfer units that can be interchanged between different
nary on the selection of resistance and the release of antibiotics in organisms. For instance, it has been found that the dissemination of
the environment. Overall, the World Health Organization estimates the beta-lactamase gene CMY-2 in Salmonella strains from dairy
that about half of the total amount of produced antibiotics is used in farms is not due to the spread of a ceftiofur-resistance Salmonella
food animals (WHO, 2002). It is important to remark however that clone, but to the multiple acquisition of the same CMY-2 gene by
the total amount of antibiotics used in animals is not known with different Salmonella lineages (Alcaine et al., 2005). Similarly, the
certainty. Cabello reported in 2006 that ‘‘in Chile, statistics indicate same vanA elements have been found not just in vancomycin-
that annually 10–12 metric tons of quinolones are used in human resistant enterococci isolates from the same clonal complex, but in
medicine and approximately 100–110 metric tons of these antibi- other isolates from both humans and chickens (van den Bogaard
otics are used veterinary medicine per year, most of them in et al., 2002). Given that about 180 million dry tons of livestock and
aquaculture’’ (Cabello, 2006). Concerning Europe, the International poultry waste are generated every year at the USA, the finding of
Federation for Animal Health Europe (www.fedesa.be) states that animal-derived antibiotic-resistant organisms in ground water,
between 1997 and 1999, the total volume of antibiotic consumption surface water and crops is not surprising (Dolliver and Gupta,
increased by 10%, from 12 752 Tm to 13 216. Human use repre- 2008). As we will discuss below, in these occasions, increasing
sented 60% of this consumption in 1997 and increased to 65% in overall resistance may impact human health even if the resistance
1999. The utilization of antimicrobials for veterinary therapy also genes are originally selected in microorganisms’ lineages that are
increased from 27.5% to 29%, whereas their utilization as growth specific of the animals and cannot infect humans. For instance,
promoters decreased from 12.5% to 6%. The estimations of the it has been recently described that horizontal gene-transfer
percentage of the 22.7 million kg of antibiotics produced in the USA events link human methicillin-resistant Staphylococcus aureus
that are used in animal production ranges from 17.8% to 70% human pathogens to contagious bovine mastitis bacteria (Brody
(Mathew et al., 2007). Whereas the Union of Concerned Scientists et al., 2008).
reported in 2001 that the usage of antibiotics for animal growth Second, antibiotics can remain in the tissues of the animals, so
promotion and for therapeutic purposes was eight time higher than that they can be considered as food pollutants. The effect of these
for human therapy (Mellon et al., 2001), the International Federa- compounds in the human host has not been studied in detail.
tion for Animal Health (www.ifahsec.org) reported than in 1999, However, it has been suggested that they might trigger in occasions
65% of antibiotics were used in humans, 29% for veterinary thera- allergic reactions and contribute to select for antibiotic-resistant
peutic and just 6% for growth promotion. Given these strong bacteria in the human microbiota (Cabello, 2006).
differences in the estimations and the lack of reliable information Third, antibiotics released to soils or waters can modify the local
on antibiotic consumption in most countries, it is not possible at the environmental microbiota producing changes in their composition
moment to present a global view of antibiotic consumption in or activity that are not fully understood. The alterations in the
humans and animals around the world. Nevertheless, taking into bacterial populations include selection of resistant mutants in
 J.L. Martinez / Environmental Pollution 157 (2009) 2893–2902 2897

susceptible species, changes in the distribution of antibiotic 5. When an antibiotic resistance gene is a pollutant
resistance genes present in gene-transfer units, and selection of
resistant species in such a way that the overall composition of the Several works highlight the presence of antibiotic resistance
microbiota is modified. For instance, the exposition to ciprofloxacin genes in pristine, isolated or extreme environments that are
of salt marsh sediment microbial communities, favours selection of unlikely contaminated with antibiotics used by humans. These
sulphate-reducing and Gram-negative bacteria (Cordova-Kreylos include the deep terrestrial subsurface (Brown and Balkwill, 2009),
and Scow, 2007). Selection of resistance and reduction of the non-contaminated Antarctic waters (De Souza et al., 2006) or deep
complexity of environmental microbiota is not however the unique Greenland ice core (Miteva et al., 2004) among others. These works
consequence of antibiotic pollution. Antibiotics can produce tran- constitute a good example of the ubiquity of genes that might
sient changes in the activity of microbial populations (Linares et al., confer resistance upon expression in a heterologous host, inde-
2006; Fajardo and Martinez, 2008) that might be relevant for their pendently on whether or not their primary function is resistance,
productivity even at sub-inhibitory concentrations. It has been but should not be considered as a surprise given the high number of
described that the pollution of manure with sulfadiazine reduces potential resistance determinants present in environmental
microbial activity, mainly some processes in nitrogen turnover bacteria (D’Acosta et al., 2006; Wright, 2007). It is thus important to
(Kotzerke et al., 2008), besides increasing resistance in soil (Ghosh remark that the finding of resistant organisms in a given environ-
and LaPara, 2007; Heuer and Smalla, 2007). ment should not necessarily be considered as an evidence of
Although the overall utilization of antibiotics for farming pollution by antibiotics or by resistance genes.
purposes might be decreasing as the consequence of the imple- What can be considered as an evidence of pollution is an
mentation of politics for reducing antibiotic resistance, the removal increase in the fraction of resistant organisms above the normal
of antibiotics as growth promoters has been followed by an value. Even in this case, contamination by resistance genes would
increase of therapeutic antibiotics used in animals (e.g. in Denmark not be necessarily involved. Under antibiotic pollution, intrinsi-
this increase was 49.7% between 1999 and 2001, Singer et al., 2003). cally resistant bacterial species and resistant mutants of the
On the other hand, although national programmes to control susceptible ones can be selected without involvement of pollutant
antimicrobial resistance and to improve the rational use of antibi- antibiotic resistance genes. There are however two situation in
otics in humans are reducing the amount of antibiotics used for which an antibiotic resistance determinant can be considered as
human therapy (Muller et al., 2007), this use of antibiotics will a pollutant.
obviously remain. It is thus predictable that the amount of antibi- First, antibiotic selective pressure in natural ecosystems may
otics released in the environment from farms and humans’ residues select the integration and further dissemination of antibiotic
will likely keep at rather high levels in the future. This means that resistance genes in gene-transfer units, which can be then
besides control policies in the use of antibiotics, studies for considered as contaminants. A good example of this situation is the
improving their degradation are needed. quinolone-resistance gene qnr, which is chromosomally encoded in
Treatment of water, sewage and in general residues contami- several water-borne bacteria (Sanchez et al., 2008). It has been
nated with antibiotics should be implemented before their release shown that contamination of river waters by quinolones favours
to natural ecosystem or its transformation in manure to be used as the integration of the qnr gene into plasmids and its further
organic fertilizer in agriculture (Dolliver and Gupta, 2008), because dissemination among natural ecosystems that can be geographi-
antibiotics present in manure antibiotics are incorporated in soil cally distant (Cattoir et al., 2008; Picao et al., 2008).
(Martinez-Carballo et al., 2007; Aust et al., 2008). Water chlorina- Second, residues from hospitals, houses and farms contain
tion helps to degrade antibiotics such as beta-lactams or trimeth- bacteria that can carry antibiotic resistance determinants. Search of
oprim (Dodd and Huang, 2007; Li et al., 2008) and wastewater specific antibiotic resistance genes in the sediments of Cache La
treatment might eliminate 80% of fluoroquinolones or tetracyclines, Poudre River, which have high concentrations of antibiotics related
being the removal of macrolides less efficient (Shellie et al., 2003; to urban and agricultural activities showed the presence of resis-
Sukul and Spiteller, 2007; Gulkowska et al., 2008). Several tance genes in all sites, although impacted sites presented higher
techniques such as coagulation, activated carbon filtration, ionic concentration of those genes than pristine environments (Pei et al.,
treatment or micelle-clay systems are effective for the removal of 2006). The finding of specific antibiotic resistance genes, which are
different antibiotics (Choi et al., 2008). However in all cases already disseminated among human, animals or plants bacterial
a variable percentage of the antibiotics usually remain after pathogens (or commensals) will be an indication of a history of
wastewater treatment (Brown et al., 2006) and can challenge contamination. Differing to the situation with antibiotics, this
bacterial populations downstream the wastewater processing plant contamination is not necessarily local neither dependant on
(Watkinson et al., 2007). the constant release of residues, because once those genes are in
It is important to remark that treatment of water and residues the environment, they can disseminate among different bacterial
from human activity is far to be performed worldwide, so that in species and distinct habitats. It has been demonstrated that anti-
most occasions antibiotic-polluted residues are released in the biotic resistance genes can migrate between connected aquatic
environment without further processing. As stated above, antibi- systems. It is unclear whether the presence of antibiotic resistance
otics are naturally degraded by processes that include photo- genes is the result of the migration of antibiotic-resistant bacteria
degradation, chemical degradation and biodegradation. These or the transmission of resistance genes by HGT (Koike et al., 2007).
processes are dependent on temperature, moisture, chemical Worldwide transport and commercial activities are helping as well
composition of the environment (e.g. pH, ionic strength) and the the dissemination of bacteria even between different oceans and
microbiota that can contribute to biodegradation. Under this scope, continents (Ruiz et al., 2000). For this reason, antibiotic resistance
different habitats will render different paths of antibiotic degra- genes firstly reported in human pathogens, are found as well in
dation. For instance, binding of antibiotics to clay and sediments several different habitats including those in which antibiotic
delays their degradation, but simultaneously remove antibiotics pollution is very low or even null. The fact that remote human
from water, in such a way that particulate matter present in rivers populations with minimal antibiotic exposure carry antibiotic-
may reduce antibiotic pollution in waters at long distance of waste resistant commensal bacteria further support the worldwide
drainage, at the cost of increasing locally antibiotic concentration at dissemination of resistance genes (Grenet et al., 2004; Bartoloni
sediments (Baquero et al., 2008). et al., 2009).
2898 J.L. Martinez / Environmental Pollution 157 (2009) 2893–2902

6. Mechanisms for the maintenance of antibiotic plasmids were found, two of which confer fitness advantage to
resistance genes a new E. coli host (Yates et al., 2006). This indicates that antibiotic
resistance plasmids can be maintained and their transfer to new
A key issue for predicting the fate of antibiotic resistance genes hosts favoured even in the absence of antibiotics.
in natural environments is the understanding of their effects on Antibiotic resistance genes can be clustered in the same unit
bacterial physiology. It has been assumed that antibiotic resistance with other elements that may provide an ecological advantage. The
confers a metabolic burden for the resistant bacteria so that they integrons (Stokes and Hall, 1989) are modular structures with
will be out-competed by their wild-type counterparts in the a major role in the development of antibiotic resistance (Fig. 2).
absence of antibiotic selective pressure (Andersson and Levin, Their structures allow the formation of arranges of antibiotic
1999; Andersson, 2006). Although in some occasions this is likely resistance genes that are transferred simultaneously in such a way
true, some examples demonstrate that antibiotic resistance can that the selection by one antibiotic co-selects other resistance
remain in the absence of antibiotic selection. Among the different genes present in the integron. The same types of integrons are
mechanisms involved, some concerns bacteria physiology and found in samples from humans, animals and natural habitats,
some others the structure of the genetic platforms involved in the indicating that those gene-recruiting elements can traverse
transfer of the antibiotic resistance determinants. through different ecosystems (Rosser and Young, 1999; Roe et al.,
The fitness cost of antibiotic resistance is not always the same. It 2003a,b; Biyela et al., 2004; Nandi et al., 2004; Murinda et al., 2005;
has been described that some resistance mechanisms have no cost Mukherjee and Chakraborty, 2007; Barlow et al., 2008). Since
for bacteria (Balsalobre and de la Campa, 2008), in such a way that several integrons contain a conserved gene that encodes for resis-
resistant microorganisms are not out-competed by their wild-type tance to quaternary amines (qac), antibiotic resistance can also be
counterparts. Furthermore, some mechanisms of resistance may maintained as the consequence of antiseptics contamination. For
render a higher fitness to the resistant strain as compared with the instance, it has been demonstrated that the amount of class I
susceptible counterpart. For instance, a quinolone resistant mutant integrons in samples from a reed bed system used to remediate
C. jejuni with a single point quinolone-resistance mutation in the waste from a wool mill with a high use of quaternary amines is
gyrA gene presents enhanced persistence in chickens (Luo et al., higher that their amount in control samples, including samples
2005). This quinolone resistant mutant is thus better adapted for from different agricultural soils (Gaze et al., 2005).
surviving in chicken than the wild-type counterpart survives and Cross-resistance to heavy metals and to antibiotics or linkage of
resistance will be maintained. antibiotic resistance genes with heavy metal resistance determi-
It is important to remark that bacterial fitness is dependent on nants in the same plasmid (co-resistance) can also allow the
the environment where bacteria are growing. For instance, Steno- maintenance of antibiotic resistance in the absence of antibiotics in
trophomonas maltophilia resistant mutants that overproduce the heavy metals polluted environments (Hernandez et al., 1998;
multidrug efflux pump SmeDEF (Alonso and Martinez, 2000; Stepanauskas et al., 2006). Plasmids can also contain genes
Sanchez et al., 2002a) are less competitive in a Dyctiostelium conferring adaptive advantages to their hosts besides resistance.
discoideum infection model, but are more proficient in the utiliza- This includes resistance to toxic compounds such as other antibi-
tion of sugars such as gentibiose, dextrin or mannose (Alonso et al., otics, heavy metals or biocides, and other determinants, like side-
2004). Some specific mutations in the rpoB gene that provide rophores, microcins or toxins that can favour the survival of
resistance to rifampin can increase the fitness of Bacillus subtilis in bacteria carrying these plasmids (de Lorenzo et al., 1984; Delgado-
specific habitats. It has been shown that some rifampin-resistant Iribarren et al., 1987; Martinez et al., 1987; Martinez and
mutants, but not others are capable of using b-glucosides more Perez-Diaz, 1990). Co-selection of the resistance determinant in the
efficiently than their susceptible counterparts (Perkins and Nich- absence of the corresponding antibiotic is a compelling possibility
olson, 2008). These nutrients, produced from decomposing plant for explaining the persistence of some antibiotic resistance
matter through the breakdown of celluloses and hemicelluloses
(Zhang et al., 2007), are present in soil, so that the resistant mutants
might be more fit than the wild-type rifampin-susceptible
B. subtilis strains for growing in soil. Finally, even in the case of
strong fitness costs, compensatory mutations that reduce the
bacterial metabolic burden are easily selected (Bjorkman et al.,
2000; Paulander et al., 2007; Lofmark et al., 2008). All this justifies
the idea that fitness costs are not always enough for eliminating
resistant bacteria from the environment and that once established,
these resistant bacteria can invade different ecosystems. For
instance, resistant bacteria are now established in wild animal
populations, despite the absence of any deliberate antibiotic
prescribing (Gilliver et al., 1999; Livermore et al., 2001).
Since antibiotic resistance genes can be present in gene-transfer
units, the structure of those elements will also impact the possi-
bility of maintenance of resistance genes. For instance, several
plasmids encode toxin-antitoxin systems, making plasmid loss Fig. 2. Structure of an integron. Integrons are formed by a gene coding for a site-
specific recombinase known as integrase (intI1), an attachment site (attI), and
unsuitable (Hayes, 2003). If those plasmids contain antibiotic
a promoter (P) that drives the expression of the genes located downstream from the
resistance genes, the probability for the maintenance of those attI site. The figure shows a Type I integron containing qac, which encodes quaternary
genes will thus be high. This probability is even higher if the ammonium resistance, sul1 that encodes sulphonamide resistance, and orf5 which has
plasmid-encoded genes display low fitness costs. A search of no known function. Genes can be recruited by site-specific recombination driven by
apramycin resistance plasmids from commensal bacterial of calves the integrase between the attI site and the attC site present in the gene-cassettes. After
gene recruitment, the recombination sites are reconstructed, allowing the incorpora-
has been made on a farm that had not used apramycin or related tion of new gene-cassettes. This structure allows the formation of long arrangements
aminoglycoside antibiotics for more than 20 months before of antibiotic resistance genes that can be transferred simultaneously among bacterial
sampling. In spite of lack of selection, three apramycin resistance populations.
 J.L. Martinez / Environmental Pollution 157 (2009) 2893–2902 2899

determinants. This might be the resistance to sulphonamides in the outcome (Rysz and Alvarez, 2004). If antibiotic resistance genes,
UK. This family of antimicrobial agents was widely used for treating already located in gene-transfer units, are present as pollutants in
human infections until 1990. Nevertheless, sulphonamide resis- the ecosystem, the presence of antibiotics will favour their
tance is still widespread in the UK despite national prescribing dissemination and the chances for the mitigation of antibiotic
restrictions (Enne et al., 2001). One possibility for explaining this resistance after eliminating the release of antibiotics will be lower.
persistence consists on the location of genes coding for sulphona-
mide resistance in widespread integrons containing other resis- 8. Concluding remarks
tance genes.
The analysis of multiple antibiotic-resistant bacteria isolated The release of high concentrations of antibiotics and resistance
from a mariculture farm in China, demonstrated a high prevalence genes in natural ecosystems is a recent event in evolutionary terms.
of chloramphenicol resistant isolates carrying different types of cat Nevertheless, both types of pollution can impact the structure and
gene. In all cases, the resistant isolates also harboured one or two of the activity of environmental microbial populations. Given that
the tetracycline resistance genes tetD, tetB or tetA. Since chloram- environmental microorganisms are the original source of resistance
phenicol has never been used in this farm, the most suitable genes acquired through HGT by human pathogens (Davies, 1997),
hypothesis for explaining the prevalence of the cat genes should be these changes are relevant for the future of human health. It has
co-selection, likely by oxytetracycline (Dang et al., 2006). Similarly, been reported that the same antibiotic resistance genes and the
co-selection of antibiotic resistance by heavy metals in freshwater same genetic platforms (plasmids, integrons, .) are currently
microcosms and industrially polluted streams has been demon- present in human pathogens, in pristine environments and in pop-
strated (McArthur and Tuckfield, 2000; Stepanauskas et al., 2006). ulations (humans and animals) that are isolated and have not been in
contact with antibiotics. This indicates that, once they are integrated
7. Consequences of pollution by antibiotic resistance genes in successful gene-transmission elements, antibiotic resistance
genes can persist and spread even in the absence of antibiotics.
Pollution by antibiotic resistance genes can increase the chances Available data support the notion that the utilization of antibi-
of human pathogens for acquiring resistance. The release of resi- otics for other purposes than therapy can enrich the population of
dues containing human microbiota into environments containing resistant bacteria capable to infect humans. However, few reliable
bacteria enriched in resistance elements increases the possibility of quantitative data on the amount of antibiotics used for different
acquiring novel resistance determinants by human-linked bacteria. purposes (from growth promotion to human therapy) are available.
For this reason, it has been proposed that the release of residues The availability of these data form national and international
from hospitals that contain human commensal and infective agencies is an urgent need for evaluating the risks associated with
bacteria (resistant and susceptible) as well as antibiotics, should be the release of antibiotics as recognized by the World Health Orga-
reduced to a minimum to avoid interchange of genetic material nization (WHO, 2002).
(even in wastewater treatment plants, Pauwels and Verstraete, Different measurements can be taken to reduce the impact of
2006). As stated by Baquero et al. the contact of human microbiota antibiotic/resistance genes pollution. A judicious utilization of
with other types of microbiota in different ecosystems (from soil antibiotics in non-therapeutic purposes is needed, but even if the
sediments of ground water to animal microbiota) will increase the antibiotics are not used for growth promotion, it is unclear that the
possibility of genetic variation and the possible emergence of novel total amount of antibiotics used for all purposes and released in
mechanisms of resistance that are re-introduced in the human the environment will decrease in the near future. The reduction of
environment (Baquero et al., 2008). the impact of antibiotic contamination thus requires the imple-
The spread of resistance genes in natural ecosystems can chal- mentation of methods for waste treatment that favour the degra-
lenge the population dynamics and the physiology of natural dation of these toxic compounds (Baquero et al., 2008). Concerning
microbial populations. Several reports indicate that the resistance resistance genes, the situation is more complex given that they are
genes currently present in human or animal associated microbiota not ‘‘degradable pollutants’’ but auto-replicative elements. Even
are found in environments without antibiotic pollution. In several though a reduction in the prevalence of resistance has been
occasions, the genes are located in bacteria with human/animal reported after discontinuation of a given antibiotic, some works
origin. However, although not analyzed in detail, it is also possible indicate that the total restoration of the population to its previous
that those genes are spread among environmental bacterial pop- antibiotic-susceptible situation is unlikely (Salyers and Amabile-
ulations. The question here is whether the incorporation of anti- Cuevas, 1997; Andersson, 2003). To reduce the impact of resistance
biotic resistance genes may produce relevant changes in the genes containment measurements avoiding as much as possible
recipient organisms. Some works indicate that this can be the case. the contact of human-linked and environmental bacteria should be
For instance, resistance to either glycopeptides or beta-lactam evaluated (Baquero et al., 2008).
antibiotics strongly modifies the structure of the peptidoglycan in
Gram-positive bacteria (Mainardi et al., 2008), and it has been Acknowledgements
described that antibiotic resistance of small colony variants of
S. aureus is associated with the rewiring of bacterial metabolism Author’s laboratory is supported by grants BIO2005-04278 and
(Heinemann et al., 2005). This indicates that acquisition of resis- BIO2008-00090 from the Spanish Ministerio de Ciencia e Innovación,
tance may have unpredictable consequences for the bacterial and LSHM-CT-2005-518152 and LSHM-CT-2005-018705 from
metabolism, and thus for the evolution of the environmental European Union. Thanks are given to Helen Green for language
microbiosphere. editing.
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