Drug Resistance Updates 15 (2012) 162–172

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Drug Resistance Updates

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Evolution of antibiotic resistance at non-lethal drug concentrations

Dan I. Andersson ∗ , Diarmaid Hughes
Department of Medical Biochemistry and Microbiology, Box 582, SE-75123 Uppsala, Sweden

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

Article history:
Received 28 January 2012
Received in revised form 22 March 2012
Accepted 26 March 2012
We would like to dedicate this paper to
Prof. Fernando Baquero, who has pioneered
ideas about the importance of low levels of
antibiotics for antibiotic resistance
development.
Human use of antimicrobials in the clinic, community and agricultural systems has driven selection for
resistance in bacteria. Resistance can be selected at antibiotic concentrations that are either lethal or
non-lethal, and here we argue that selection and enrichment for antibiotic resistant bacteria is often a
consequence of weak, non-lethal selective pressures – caused by low levels of antibiotics – that operates
on small differences in relative bacterial fitness. Such conditions may occur during antibiotic therapy or
in anthropogenically drug-polluted natural environments. Non-lethal selection increases rates of mutant
appearance and promotes enrichment of highly fit mutants and stable mutators.
© 2012 Elsevier Ltd. All rights reserved.

Keywords:
Antibiotic resistance
Biocide
Minimal inhibitory concentration
Selective window

1. Antibiotic resistance is an ecological problem
The extensive human use and misuse of antimicrobials in the
clinic, community, animal husbandry and agriculture has resulted
in strong selection pressures for the emergence, enrichment and
spread of various resistance mechanisms in pathogenic bacteria.
The temporal and spatial dynamics and driving forces of these
processes are complex and we are only slowly beginning to under-
stand how resistance genes, resistant bacteria and the selective
agents (antibiotics, biocides, environmental pollutants, etc.) move
between different ecosystems and exert their effect. In this review,
resistance is used in the broadest sense to indicate any reduction
in susceptibility in comparison to the wild type population.
With regard to the dynamics of antibiotic resistance develop-
ment it is essential to distinguish between (at least) three levels
of description, namely (i) where did the resistance mechanism
itself originate, (ii) where did the resistant pathogenic bacteria
of relevance to human and animal infections emerge and (iii)
where were these resistant pathogens enriched and transmitted?
In cases where the resistance is conferred by a mutational mecha-
nism these three levels often converge on one particular bacterial
species and environment. For example, for Escherichia coli resistant
to fluoroquinolones the de novo emergence of the resistance mech-
anism and the subsequent enrichment might occur in the same
∗ Corresponding author. Tel.: +46 18 4714175.
E-mail address: Dan.Andersson@imbim.uu.se (D.I. Andersson).
environment and bacterial species (e.g. in E. coli bacteria present
in urine/feces in antibiotic-treated individuals) but for many other
types of resistances, in particular for those that are horizontally
transferred, these three levels might be fully separated tempo-
rally and spatially. Thus, the resistance mechanism could have
originated pre-therapeutic use of antibiotics in non-pathogenic
environmental bacteria, and recent evidence strongly supports the
notion that many types of resistance mechanisms and resistant bac-
teria were present long before human production, use and spread of
antibiotics expanded rapidly in the second half of the 1900s (Smith,
1967; Hughes and Datta, 1983; Aminov and Mackie, 2007; Mindlin
et al., 2008; Allen et al., 2009; D’Costa et al., 2011). The emergence of
resistant pathogens could have followed later when the resistance
was horizontally transferred from an environmental bacterium to
a human/animal pathogen (Patel et al., 2000; Poirel et al., 2002,
2005; Hong et al., 2004; Canton, 2009; Wright, 2010), possibly
in an environment where these bacteria only transiently resided
together. Finally, these resistant human pathogens were enriched
due to selective pressures present in the clinic, community or agri-
cultural setting. Often it is difficult to distinguish between these
levels but from the point of view of trying to restrict the emergence
and spread of resistance it is important that we attempt to do so
since the design and efficiency of our strategies will vary consider-
ably depending on whether we try to prevent the initial emergence
of resistance, or try to prevent the enrichment of already existing
resistant pathogens that have spread into clinical/community set-
tings. For example, as suggested by increasing amounts of data,
clinically relevant plasmid/ICE mediated resistances may emerge

1368-7646/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.drup.2012.03.005
 D.I. Andersson, D. Hughes / Drug Resistance Updates 15 (2012) 162–172 163

Fig. 1. Schematic representation of flows of resistant bacteria (blue) and antibiotics (red) between different environments and areas of use.

by HGT from environmental bacteria to pathogens in the out- various aquatic environments and soil and exert a weaker selective
side environment (e.g. in waste water treatment facilities, manure, pressure. Even though antibiotic concentrations in these environ-
sludge, soils or similar environments) (Patel et al., 2000; Poirel et al., ments are several orders lower than where the drugs were initially
2002, 2005; Hong et al., 2004; Canton, 2009; Wright, 2010; Taylor used they could still, as argued below, be of great importance for
et al., 2011). With this knowledge in hand, an important control the evolution of resistance.
measure to reduce the rate of resistance emergence might be active
stewardship in environmental resistance reservoirs (e.g. killing of
2. Which are the significant selectors of resistance?
bacteria and destruction/removal of antibiotic pollutants in waste
water, sludge, manure, etc.) in addition to clinical control programs
With regard to the selective pressures that enrich for resistance,
that are aimed at reducing antibiotic use and selective pressures
it is probable that antibiotics represent the main selector. Thus,
near the patients. Conceivably it might be more efficient to prevent
there exist convincing correlations between amount of antibiotic
the initial emergence of resistant pathogens rather than to later
used at the hospital, community and country level and the fre-
try to control their enrichment and spread in clinical and human
quency of resistance observed (van de Sande-Bruinsma et al., 2008;
community settings by restrictive antibiotic use.
Bergman et al., 2009; Goossens, 2009). However, it is likely that
As outlined schematically in Fig. 1, resistance genes, resistant
other types of selective pressures are involved as well. For example,
bacteria and antibiotics will flow between different compartments
the amount (in metric tons) of human-made biocides that bacteria
and environments, including humans, animals, soil and water,
are exposed to on a global scale is many orders of magnitude higher
etc., creating situations where resistant bacteria may emerge,
than that for antibiotics (SCENIHR, 2009). As the increasing use of
become enriched and spread between hosts and various natural
antibiotics and biocides has been temporally and spatially linked it
environments. Resistance might be selected in any of the above
is difficult to entangle their relative contributions to the resistance
environments but it is difficult to assess the relative contributions
problem but based on the wide use of biocides in numerous types
of various environments and selections to the total problem. For
of environments (for example, in health care, household prod-
example, it is commonly assumed that hospitals represent signifi-
ucts, textiles, food industry and many other environments) and
cant foci for resistance evolution because of the high use (per capita)
fundamental evolutionary principles, it is expected that biocides
of antibiotics that generate a strong local selection pressure, and the
will exacerbate the problem (Maillard, 2007; Tumah, 2009). Bio-
high host population density that facilitates spread of the resistant
cides could contribute to the problem by co-selection mechanisms
bacteria. This notion is most likely true for certain pathogens but
where the biocide resistance conferring genes (e.g. qac, heavy metal
with regard to the total selective pressure asserted globally due
resistance genes, etc.) are present on the same genetic element
to anthropogenic influences, hospital use of antibiotics probably
(e.g. a plasmid or ICE) as the antibiotic resistance genes, thereby
represents only a few percent of the total volume of antibiotic use
and it is conceivable that the weaker selective pressures present
in other environments are equally important breeding grounds for Table 1
the emergence of resistance (Baquero, 2001a,b), and possibly high Fraction of antibiotic dose for different antibiotic classes that is excreted in active
antibiotic-use environments like hospitals mostly act as enrich- form in human urine (Bryskier, 2005). The fractions will vary between different
compounds from the same antibiotic class and these values should therefore be
ment and transmission centers of already existing resistant clones. taken as approximate.
It is also notable that antibiotics exert their effect with widely
different active concentrations during their movement through Antibiotic class Fraction of dose excreted in
human urine in active form
various environments and ecosystems. When antibiotics are used
therapeutically in humans, animals and agriculture they generally Fluoroquinolones 40%
Aminoglycosides 80–90%
are present at high local concentrations and exert strong selection
Tetracycline 40%
pressures in these environments. However, about half of all antibi- Macrolides 20–30%
otics given to humans and animals are excreted in an unchanged Penicillins 50%
active form mainly via urine (Table 1) into waste water, manure and Cephalosporins 70–90%
run-off water, where they will ultimately end up in a diluted form in Trimetoprim 50%
164 D.I. Andersson, D. Hughes / Drug Resistance Updates 15 (2012) 162–172
indirectly driving enrichment for antibiotic resistance (Laraki et al.,
1999; Bjorland et al., 2001; Sidhu et al., 2001, 2002; Cookson, 2005;
Noguchi et al., 2005; Sekiguchi et al., 2005, 2007; Dumitrescu et al.,
2007; Levings et al., 2007). Another mechanism might be by cross-
resistance where the biocide and the antibiotic share a common
resistance mechanism, as has been shown for up-regulation of
efflux pumps (Paulsen et al., 1996; Brown et al., 1999; Putman et al.,
2000; Borges-Walmsley and Walmsley, 2001; Poole, 2001, 2002;
Levy, 2002; McKeegan et al., 2003; Piddock, 2006) alterations of
the cell wall (Denyer and Maillard, 2002; Nikaido, 2003; Tkachenko
et al., 2007) and modification of target enzymes (McMurry et al.,
1999; Heath et al., 2000; Parikh et al., 2000). Several studies have
shown that cross-resistance to antibiotics can result from selection
for biocide resistance in the laboratory, strongly suggesting that
biocides in natural settings would have similar effects (Pomposiello
et al., 2001; Braoudaki and Hilton, 2004; Codling et al., 2004;
Sanchez et al., 2005; Karatzas et al., 2007; Randall et al., 2007; Fraud
et al., 2008; Huet et al., 2008).
3. Where are resistant bacteria selected?
It is commonly assumed that resistant bacteria are selected in
antibiotic-treated humans or animals in response to the strong
selective pressures that are generated when antibiotics are used
therapeutically and the concentrations reached are well above the
minimal inhibitory concentration (MIC) of the susceptible popu-
lation of bacterial cells. This notion is certainly true for certain
types of infections, antibiotic regimens and resistance mechanisms.
For example, in Mycobacterium tuberculosis examples exist where
mutationally resistant bacilli were selected de novo from an ini-
tially susceptible population during inadequate treatment regimes
(Burman et al., 2006; Cox et al., 2007; Hu et al., 2011; Mariam
et al., 2011; Skrahina et al., 2012). Similarly, mutational resistance
to fluoroquinolones has been shown to emerge during treatment of,
for example, E. coli/Salmonella enterica (Cattoir et al., 2006; Corvec
et al., 2008; de Toro et al., 2010; Jeong et al., 2011) as well as fluoro-
quinolone and ␤-lactam resistance in Pseudomonas aeruginosa (Le
Thomas et al., 2001; Leotard et al., 2001; Nakano et al., 2001). Other
examples where resistance emerged de novo in treated patients
as a result of horizontal gene transfer of resistance genes include
the appearance of vancomycin resistant MRSA (Chang et al., 2003;
Weigel et al., 2003) and carbapenem resistant E. coli (Richter et al.,
2011) where the resistant donor bacteria were either co-infecting
with resistant Enterococcus faecalis or Klebsiella pneumoniae.
On the other hand, many types of plasmid/ICE-mediated resis-
tances (which represent the largest proportion of all resistance
mechanisms) are unlikely to emerge de novo during treatment.
Thus, the patient/animal is either infected with the susceptible
bacteria (and no resistance development occurs during treatment)
or they are infected with the resistant strain and the antibi-
otic treatment mainly causes an enrichment of a pre-existing
mutant. As described above, it is likely that for many of the
resistance mechanisms found today in human pathogens, their
origin was in environmental bacteria and anthropogenic changes
of the environment via the use of selective antimicrobials and
ecological opportunity for gene transfer allowed for transfer of
resistance genes into previously susceptible populations of bacte-
rial pathogens.
4. The power of small selective differences
In Section 1 we argued that the wider environment contains lev-
els of antibiotics and other agents with the potential to enrich for
antibiotic-resistant pathogens. To understand quantitatively how
environmental contamination influences resistance development
we must determine the lowest concentrations of different antibi-
otics that selectively enrich for resistant variants. A complication
is that the interplay between the relative antibiotic susceptibility
and growth fitness of different bacterial variants will determine
how each is selectively enriched at different drug concentrations.
At an antibiotic concentration that we term the minimum selective
concentration (MSC) the growth rate of the susceptible strain will
be reduced to a level where it equals the growth rate of the resis-
tant strain, and by definition any antibiotic concentration higher
than the MSC will selectively enrich the resistant strain. Thus,
paradoxically, relative to a particular susceptible strain, two dif-
ferent resistant mutants with identical MICs but different fitness
values would have different MSCs. MSC will be lowest for strains
with the smallest fitness costs. Experiments have shown that in
general resistant strains have a lower fitness in drug-free envi-
ronments than susceptible strains (Bjorkman et al., 1998; Nagaev
et al., 2001), but the range of fitness values can extend up to a level
where it can prove very difficult to distinguish it experimentally
from that of an isogenic susceptible strain (Marcusson et al., 2009;
Andersson and Hughes, 2010). In addition, the relative fitness dif-
ference between isogenic resistant and susceptible strains is not
a constant but instead changes continuously as a function of the
antibiotic concentration to which they are exposed. The question
boils down to how small a selective effect can be, and still influence
the outcome of a competition?
We argue here that extremely small differences in relative fit-
ness can be effectively selected in nature. An extreme example that
supports this argument is the widespread evolution of the pref-
erential use of some synonymous codons, so-called codon usage
bias, in many free-living microorganisms. The particular set of
synonymous codons that are preferentially used differs between
microorganisms, showing that codon usage bias evolved indepen-
dently in different genetic lineages. Codon usage bias is thought
to be the result of selection for translationally optimal codons
(Ikemura, 1985; Sharp et al., 2010). Based on this model the growth
advantage and selection coefficient for an optimal versus a non-
optimal codon has been calculated to be in the range of 10−5
(Bulmer, 1991) to 10−8 (Sharp et al., 2010) per codon. The wide
range in these values reflects different assumptions of effective
population size (Ne ) for bacterial species like E. coli (Berg, 1996)
but the major conclusion from the widespread occurrence of bias
in synonymous codon usage is that miniscule differences in fitness
(≤10−5 ) can be effectively selected over evolutionary time scales.
This example provides strong support for the idea that even an
extremely weak selective advantage can drive the genome-wide
evolution of DNA sequences in bacteria.
A relevant clinical example with respect to antibiotic expo-
sure and selection is the situation that arises each time a patient
is treated with an antibiotic. It is expected that, because of the
complexity of the human body, the antibiotic will reach differ-
ent concentrations in different body compartments, and that these
concentrations will change over time (Baquero et al., 1997, 1998).
Thus the exposure of different bacteria in the patient to the antibi-
otic will vary temporally and spatially. If there are within the
patient sub-populations of less susceptible bacteria these could
then be enriched in particular body compartments and may explain
for example, the apparent incremental step-wise evolution of ␤-
lactamases (Baquero et al., 1998). Similar variation in antibiotic
selective pressures is also predicted to occur in the highly struc-
tured environments of soil.
There are examples where small differences in fitness of antibi-
otic resistant strains have been shown experimentally to be
selectable in the absence of the drug both in vitro and in vivo
(Marcusson et al., 2009). The concept that small differences in
fitness could also be selected in a drug-concentration-dependent
window was tested experimentally by competing isogenic strains
 D.I. Andersson, D. Hughes / Drug Resistance Updates 15 (2012) 162–172
carrying variant ␤-lactamases, TEM-1 and TEM-12 that differed
only slightly in MIC for cefotaxime, 0.008 and 0.012 ␮g/mL, respec-
tively (Negri et al., 2000). Isogenic strains carrying the variant
TEM genes were competed in vitro (laboratory liquid medium)
and in vivo (mouse thigh model) at a range of cefotaxime con-
centrations. The TEM-12 variant was preferentially enriched in
a selection lasting several hours in a narrow drug concentra-
tion range, both in vitro and in vivo, supporting the concept of a
drug-concentration selective window (Negri et al., 2000) in which
specific resistant mutants would be enriched. Interestingly, when
the competition between the two TEM variants was continued for a
longer period of several days and with higher drug concentrations,
a variant with a secondary mutation in an outer membrane pro-
tein, OmpF, was selected. The mutant selection window hypothesis,
that different drug concentrations will selectively enrich specific
mutant sub-populations, has also been extensively explored and
validated in the context of the multi-step evolution of resistance
to fluoroquinolones (Dong et al., 1999; Marcusson et al., 2005;
Drlica and Zhao, 2007). The TEM-1, TEM-12 experiment described
above (Negri et al., 2000) provides a model framework that poten-
tially explains the step-wise evolution of novel ␤-lactamases in
response to changing antibiotic selective pressures (Petrosino et al.,
1998; Salverda et al., 2010). However, because specific spontaneous
mutations are generally very infrequent (typical eubacterial muta-
tion rates are 10−9 to 10−11 per nucleotide per generation) this
particular mode of resistance evolution is expected to be depen-
dent on large bacterial populations to supply the specific mutations
required for the selected phenotype. One way this limitation can
be overcome is that several antibiotics increase mutation rates, at
least within a particular concentration range (Phillips et al., 1987;
Ren et al., 1999; Perez-Capilla et al., 2005; Kohanski et al., 2010).
Another way in which small bacterial populations can bypass the
mutation-supply bottleneck is by high frequency gene amplifica-
tion events, that alter the phenotype of the selected strain, and
simultaneously amplify the genetic target for spontaneous muta-
tions (Andersson and Hughes, 2009). The potential importance of
this mechanism for ␤-lactamase evolution was shown experimen-
tally when the TEM-1 gene was evolved in vitro to successively
higher levels of resistance to a cephalosporin (cephalothin). Note
that TEM-1 is associated with a very small increase in MIC to
this antibiotic that provides a weak resistance phenotype that
can be amplified under selection. It was found that the most
frequent response to cephalosporin selection in independent lin-
eages was amplification of the copy number of the TEM-1 gene
(Sun et al., 2009). The amplification of the TEM-1 gene by itself
provided a higher level of resistance (amplifying the very weak
cephalosporinase activity of TEM-1) but importantly it also allowed
the population of bacteria to survive and expand sufficiently to real-
ize additional rare secondary mutations in the genome, including in
OmpF (Sun et al., 2009). These experiments illustrate the potential
interplay between the diversity of bacterial genotypes present in
large populations, the possibility for small populations to undergo
high-frequency gene amplification, and the diversity of selective
pressures that can be produced along the antibiotic concentration
gradients formed in human body compartments and other natu-
ral environments. These antibiotic gradients will potentially result
in differential growth rates of resistant bacterial variants (Baquero
and Negri, 1997; Baquero et al., 1998; Baquero, 2001a,b), regardless
of the specific mechanisms by which the variants arise and evolve.
We can thus safely assume that antibiotic selection, especially
if repeated over many generations, can amplify the effects of
extremely small differences in fitness, resulting in an increased fre-
quency of resistant variants. In an experimental setting however, a
practical concern is the resolution that can be achieved in measur-
ing small selective differences on a short time scale (if we assume
that we do not have millions of years at our disposal to complete
165
an experiment). Below we discuss different methods of measuring
and selecting small differences in fitness and resistance.
5. Detection of selection
To get a quantitative understanding of the problems posed by
antibiotics in the environment we need to determine the lowest
levels of antibiotics that confer a measurable selective advantage
under experimental conditions. Some standard measures of fitness,
with a discriminatory power that is at best ±3% per generation, such
as growth rate measurements in the presence of different concen-
trations of antibiotic, are too crude to be useful. In addition, such
measurements quantify only the exponential part of a bacterial
growth cycle and may completely miss fitness parameters that are
important in the presence of growth inhibiting antibiotics. A bet-
ter approach is to compete isogenic strains differing only in some
resistance determinant in mixed cultures where the competition
involves the complete growth cycle (Andersson and Hughes, 2010).
A small difference in relative fitness can be amplified by cycling the
competing mixture of strains, with successive rounds of inoculation
into fresh media, over several days. The change in the ratio of one
strain relative to the other can be monitored as a function of time
and the slope of the line generated can be used to calculate a selec-
tion coefficient (Dean et al., 1988). We have used this method to
measure relative fitness costs in isogenic strains marked with dif-
ferent antibiotic resistance markers where the two populations are
counted on separate agar plates (Bjorkman et al., 1998). However,
drawbacks include the tedium of counting colonies, the statistical
uncertainty introduced by the relatively small numbers counted,
and the possibility that growth of colonies on different antibiotic-
containing media introduces a systematic error into the ratio. A
better method, which avoids using antibiotics to distinguish strains,
is to compete isogenic strains where one is marked with a neutral
mutation (for example araB), which allows the two populations
to be distinguished on the same agar plate based on differential
colony colour (Marcusson et al., 2009). This has the advantage
that the competing populations are measured as colonies in the
same environment but a drawback is that the quantitative range
over which the two populations can be measured is limited in
practice to about three orders of magnitude. Each of these com-
petition methods discriminates fitness differences in the range of
∼
=1% per generation. An elegant chromogenic assay has been devel-
oped that uses bacterial strains expressing either of the proteins
amilCP and amilGFP, and has the advantage that it permits a quali-
tative discrimination between competing strains in liquid medium
(Liu et al., 2011). Although the method is not designed for accu-
rate quantification of small differences in selection coefficients, it
may prove useful for assaying environmental contamination by
antibiotics.
Our aim was to measure small differences in selection coeffi-
cients and so we developed an assay that is both sensitive and
quantitative (Gullberg et al., 2011). The assay is based on growth
competition between isogenic strains genetically tagged with vari-
ants of the green fluorescent protein gene (yfp and cfp, encoding
yellow- and cyan-fluorescent proteins, respectively). The compet-
ing strains, susceptible and resistant, were competed for up to 80
generations by serial passage in batch cultures. After each cycle
of growth (10 generations per cycle) large populations of cells
(approximately 105 cells) were counted by fluorescent activated
cell sorting (FACS), thereby significantly reducing any experimental
errors associated with counting small cell populations. This exper-
imental set-up allows detection of growth rate differences of 0.2%
per generation (Lind et al., 2010; Gullberg et al., 2011). This level of
discrimination approaches the limit of sensitivity set by the inter-
ference caused by periodic selection events (Atwood et al., 1951;
166 D.I. Andersson, D. Hughes / Drug Resistance Updates 15 (2012) 162–172
Koch, 1974; Berg, 1995) that are associated with mutational adap-
tation to the growth media/conditions unrelated to the antibiotic
selective pressure. We have also begun using pre-adapted strains
for these assays; i.e. strains grown for at least 1000 generations
in the growth medium to be used for competition, before the
introduction of the fluorescent and resistance markers. The use of
pre-adapted strains permits extended competition runs without
interference from periodic selection, thus increasing the discrimi-
natory power of the assay. This highly sensitive fluorescence-based
assay can be adapted for use in natural environments or in in vivo
animal models, and we have recently initiated such experiments.
In conclusion, we have developed a robust assay that can reliably
and quantitatively measure selective differences of ∼ =10−3 per gen-
eration, in a competition experiment that can be completed in 1–2
weeks. We have used this experimental system to measure the MSC
of several different antibiotic classes in relation to several clinically
relevant antibiotic resistance determinants (Gullberg et al., 2011).
6. Sub-MIC selection
We measured, under controlled laboratory conditions, the MSC
for three different classes of antibiotic: ciprofloxacin, a fluoro-
quinolone; streptomycin, an aminoglycoside; and tetracycline,
using the growth competition method described in the previous
section (Gullberg et al., 2011). The aim was to determine the
lowest antibiotic concentration that discriminated between com-
peting isogenic wild type and mutant strains carrying any one
of six different resistance determinants that are clinically rele-
vant for resistance to these antibiotics. The mutations tested were:
two point mutations affecting the fluoroquinolone target DNA
gyrase (gyrA Ser83Leu, and Asp87Asn); two deletion mutations of
regulators of drug efflux (marR, and acrR); a point mutation
in ribosomal protein S12 that causes resistance to streptomycin
(rpsL105 Lys42Arg); and the presence in the chromosome of a
transposon causing resistance to tetracycline (Tn10). We made the
experiments as cyclic growth competitions in laboratory medium
using two different bacterial species, E. coli and S. enterica serovar
Typhimurium. Each strain (wild-type and isogenic mutants) was
constructed in two variant forms. One form was tagged with a cfp
gene inserted at the galK locus while the other form was tagged
with a yfp gene inserted at the same locus. This allowed us to make
parallel competition experiments in which either the wild type
or the mutant strain was tagged with either of the fluorescence-
producing genes, to ensure that the tagging itself did not alter the
outcome of the competition. Control experiments showed that the
small difference in fitness costs between the cfp and yfp markers
had a negligible impact on growth rates. For the competition exper-
iments wild-type and mutant cultures were mixed in a 1:1 ratio
and grown in liquid medium containing different antibiotic con-
centrations, as well as in the absence of antibiotic, for up to 80
generations. After each cycle of growth (10 generations) the ratio
of resistant to susceptible bacteria in the population was scored
by FACS analysis. Over this time scale (40–80 generations) peri-
odic selection was not a significant problem. The data on changing
ratios were then plotted to calculate a selection coefficient for each
mutant strain as a function of antibiotic concentration. For all six
of the competition experiments the data showed that the MSC of
the mutant strain was significantly below the MIC of the isogenic
wild-type strain. The MSC values ranged from (1/4) MIC (strepto-
mycin; rpsL105), through 1/10 MIC (ciprofloxacin; gyrA Asp87Asn,
marR, and acrR), to 1/100 MIC (tetracycline; Tn10). The lowest
MSC was 1/230 MIC measured for gyrA Ser83Leu (ciprofloxacin),
a mutation that is almost always found in clinical isolates of E.
coli that are fluoroquinolone-resistant (Komp Lindgren et al., 2003;
Christiansen et al., 2011; Nazir et al., 2011). There was also a
correlation between the magnitude of the reduced fitness of the
mutant strains in the absence of antibiotic and the magnitude of
the measured MSC. Thus, the fitness deficits were smallest for the
gyrA Ser83Leu mutation and the Tn10 insertion and this correlated
with them having the smallest MSC values. Because each of the
above competition experiments had been made with a 1:1 ratio of
wild type and mutant we asked whether this experimental set-up
could have influenced the outcome of the competition. To test this
we repeated a series of competition experiments (with wild-type
and isogenic Tn10 mutant) but this time used initial strain ratios of
1:1, 10:1, 100:1, 1000:1 and 10,000:1 (wild type:mutant). The data
were collected and analyzed as before and for each of the competi-
tions, regardless of the initial ratio, the calculated MSC was 1/100.
This confirms that the measured MSC values are robust and are not
significantly influenced by the initial ratio of wild type to mutant.
The conclusion from these competition experiments is that in every
case tested the MSC is significantly below the MIC of the susceptible
wild type, and in some cases it is very far below MIC (1/100, 1/230).
Results from an independent study agree qualitatively with those
from our study and show that antibiotic concentrations that are sig-
nificantly below the wild-type MIC can selectively enrich resistant
mutant populations (Liu et al., 2011). The MSC values measured
in our experiments correspond to absolute antibiotic concentra-
tions of 1 ␮g/ml (streptomycin), 15 ng/ml (tetracycline), and from
2.5 ng/ml down to 100 pg/ml (ciprofloxacin).
Having shown that very low concentrations of antibiotic could
selectively enrich resistant mutants in competition experiments
we next asked whether sub-MIC concentrations could also select
and enrich de novo mutants. For that experiment, 20 indepen-
dent lineages each of the S. enterica and E. coli wild-types were
grown in batch cultures in the presence of sub-MIC concentra-
tions of streptomycin (S. typhimurium, (1/4) MIC, 700 generations)
or ciprofloxacin (E. coli, 1/10 MIC, 600 generations). Each lineage
was monitored periodically throughout the experiment to quan-
tify the frequency of cells with increased levels of resistance. In
each case we observed a significant and continuous increase in the
frequency of more resistant sub-populations within the lineages.
For example, for streptomycin, after 400 generations all 20 lineages
had sub-populations (at least 1% frequency) that were 8 times the
wild-type MIC, and after 600 generations 14 lineages had subpop-
ulations that were 16 times MIC. Similarly for E. coli and sub-MIC
selection with ciprofloxacin: after 500 generations five of the lin-
eages had sub-populations (≥1% frequency) with MICs from two-
to six-fold higher than the wild-type (Gullberg et al., 2011). Thus,
sub-MIC concentration of antibiotics can select for both low- and
high-level resistance mutants de novo from a susceptible popula-
tion. We draw two important conclusions from these evolution
experiments. First, sub-MIC antibiotic concentrations can select
resistant mutants de novo from a susceptible bacterial population.
Second, de novo-selected mutants often had MICs that were much
higher than the selective concentration of antibiotic. Thus, both
high- and low-level antibiotic-resistant mutants could potentially
be selected by exposure to low levels of antibiotics.
We also developed a mathematical model to calculate how
rapidly a de novo resistance mutation could take over (at least 50%
of the population) a susceptible population under selection at sub-
MIC antibiotic concentrations. The calculations took into account
the mutation rate (), the population size (N), and the selective
advantage of the mutant (s) as a function of antibiotic concentra-
tion based on the data from the competition experiments (Gullberg
et al., 2011). There are two terms that dominate the equations: the
stochastic waiting time for the first mutation to appear, and the
subsequent time for the mutant to grow to 50% of the population.
For populations where the mutant is already present (N > 1) the
time to fixation can be rapid (100–1000 generations) for s values
between 0.1 and 0.01. For populations where N < 1 (for example
 D.I. Andersson, D. Hughes / Drug Resistance Updates 15 (2012) 162–172 167

Table 2
Differences in outcome for selection at lethal and non-lethal drug concentrations.

Lethal (>MIC) Non-lethal (<MIC)

Selection for rare mutations of big Selection for common

effects
Mutations need to pre-exist
selection
Selective agent (i.e. antibiotic)
cannot modulate the rate of
formation of mutants since
bacteria die (or do not grow)
The enrichment for mutators is
Fig. 2. Growth rates as a function of antibiotic concentrations. The concentration weak for rare one-step
interval where the susceptible strain (blue line) will outcompete the resistant strain mutations of large effect
(red line) is indicated in green. Sub-MIC selective window (orange) and traditional
mutant selective window (red) indicate concentration intervals where the resistant
strain will outcompete the susceptible strain. MICsusc = minimal inhibitory concen-
tration of the susceptible strain, MICres = minimal inhibitory concentration of the
resistant strain and MSC = minimal selective concentration. Fitness of mutant less important
Figure reproduced from PLoS Pathogens (Gullberg et al., 2011).
very rare mutations, or small bacterial populations) the stochastic
waiting time for a mutation to appear will dominate. However, it
mutations of small
effect → Higher rates of
appearance of resistant mutants
Mutations can form during
growth after selection is
applied → Mutation supply
increased
Selective agent can modulate
rates of mutation,
recombination, and horizontal
gene transfer → Rates of mutant
formation increased
For step-wise selection of
mutations of small effect the
enrichment for mutators is
strong → Enrichment for
mutators with increased
propensity for further resistance
development
Fitness of mutant important
since only resistant mutants
with a fitness reduction less
than that caused by the
antibiotic in the susceptible
bacteria are selected → Mutants
with higher fitness selected

is worth noting here that many antibiotics, including the fluoro-

quinolones, have been shown to increase mutation rates (Kohanski
et al., 2010) which could potentially reduce waiting times and shift
the balance toward N > 1 where takeover is dominated by the
growth selective advantage of the resistant mutant as a function of
antibiotic selective pressure. Thus, from the mathematical model
we can infer that, especially in large populations where N > 1,
and at antibiotic concentrations where 0.01 < s < 1.0, that resistant
mutants will rapidly appear and take over the population in less
than 1000 generations of growth.
7. Problems generated by non-lethal (sub-MIC) resistance
selection
Clearly resistant mutants can be selected by antibiotic concen-
trations both in the traditional selective window and the sub-MIC
window ranges (Fig. 2) and an important question is whether these
different concentration intervals will generate different endpoints
with regard to which mutants are selected? As outlined below we
would argue that non-lethal selections for resistance, where the
antibiotics do not kill the susceptible bacteria but only slow down
their growth, are more problematic with regard to both the rate
by which mutants emerge and the types of resistance mechanisms
that are selected (Table 2). The reasons for this are several.
First, in a lethal selection (i.e. in the classical selective window),
selection only detects rare pre-existing mutations that provide suf-
ficiently high levels of resistance, and if they are not present when
the selective pressure is applied the susceptible population will
die and go extinct (e.g. the infection is cured). In contrast, in the
sub-MIC window selection is not lethal but only detects small dif-
ferences in growth rate between susceptible and resistant bacteria
(Fig. 2). A consequence is that under conditions when the mutation
supply rate (i.e. population size times mutation rate) is limiting
the rate of resistance evolution, sub-MIC concentrations of antibi-
otics will allow the population to grow until a (rare) resistance
mutation appears. In effect, sub-MIC selections allow an increase
in population size and mutation supply.
Second, as shown by recent studies certain antibiotic classes
can at sub-MIC increase the mutation rate by conferring a muta-
genic effect. For example, fluoroquinolones, aminoglycosides and
beta-lactams can by induction of the SOS response (Ysern et al.,
1990; Miller et al., 2004; Perez-Capilla et al., 2005; Cirz et al., 2007;
Baharoglu and Mazel, 2011; Thi et al., 2011) and translational mis-
reading (Ren et al., 1999; Balashov and Humayun, 2002) as well as
by generation of mutagenic oxygen radicals (Kohanski et al., 2010)
increase the mutation rate up to 15-fold, an effect that might be
relevant under certain selective conditions (Cirz et al., 2005). For
example, by experimentally increasing the mutation rate by as lit-
tle as two-fold, the rate of evolution to fluoroquinolone-resistance
in E. coli is significantly increased (Örlén and Hughes, 2006). In addi-
tion, non-lethal concentrations of antibiotics also increase rates of
homologous recombination and horizontal gene transfer, includ-
ing in the biofilms present in many infections (Lopez et al., 2007;
Couce and Blazquez, 2009; Canton and Morosini, 2011). Antibiotics
at sub-MIC levels have been shown to induce the SOS response
and activate mobilization of genetic elements and increase rates
of recombination. For example, tetracycline stimulates by up to
1000-fold horizontal transfer rates of both conjugative and inte-
grative genetic elements in several significant human pathogens,
including Staphylococcus aureus (Barr et al., 1986), E. faecalis and
Listeria monocytogenes (Doucet-Populaire et al., 1991; Bahl et al.,
2004) and various SOS-inducing classes of antibiotics can increase
integron recombination in Vibrio cholerae and E. coli (Guerin et al.,
2009).
Third, another problematic aspect of exposure to sub-lethal
levels of drug is the increased potential for enrichment of sta-
ble genetic mutators. It has long been understood that antibiotics
exert a second-order selection for mutator clones, i.e. clones of bac-
teria with increased mutation rates, because increased mutation
rates speed up the rate of adaptation to the selective conditions,
especially when stepwise selection of different resistance muta-
tions occurs (Mao et al., 1997; Taddei et al., 1997; Shaver et al.,
2002). For example, among fluoroquinolone-resistant clinical iso-
lates of E. coli there is a positive correlation between the number
of resistance-associated mutations and an increased mutation rate
(Komp Lindgren et al., 2003). Similarly, natural isolates of E. coli
with intermediate mutator phenotypes have been found to be
168 D.I. Andersson, D. Hughes / Drug Resistance Updates 15 (2012) 162–172
Fig. 3. Schematic representation of how selection at high and low antibiotic con-
centrations influences the characteristics of resistant mutants with regard to their
fitness and level of resistance. The blue area indicates that high levels of antibi-
otic select high-level resistant mutants, with either high or low fitness costs. The
yellow area indicates that low levels of antibiotic select low-fitness cost mutants,
with either high- or low-level resistance. The overlapping box outlined in red indi-
cates that mutants with high-level resistance and low fitness costs can potentially
be selected at both high and low levels of antibiotic.
more likely to be multidrug resistant (Denamur et al., 2005).
These data strongly suggest that low levels of antibiotic in the
environment could have significant effects on resistance evolu-
tion by secondary selection of increased mutation rates. This is in
contrast to selection at a high level of drug where typically resis-
tance is conferred by acquiring in one step a specific mutation
of large effect, e.g. rpsL and rpoB mutations causing streptomycin
and rifampicin resistance, respectively, or HGT of plasmid/ICE that
carries a specific resistance gene(s) and the enrichment effect is
low.
Finally, one of the conclusions we can draw from the exper-
iments that measure MSC is that there is a positive correlation
between small fitness costs in the absence of antibiotic, and low
MSC values (Gullberg et al., 2011). The correlation between small
fitness costs and low MSC is expected on theoretical grounds
because a fitness cost must first be overcome by the negative
effect of antibiotics on the susceptible strain before the resis-
tant mutant can be selectively enriched. Thus, only resistance
mechanisms with a fitness cost smaller than the growth reduc-
tion caused by the antibiotic in the susceptible bacteria will be
competitive, and resistance mutations that confer a too high fit-
ness costs will not be enriched at low levels of antibiotic (even
though they might confer high level of resistance). An important
implication from this reasoning is that non-lethal selective con-
ditions will enrich for resistant mutants with higher fitness, and
these mutants may express either a low level or a high level of
resistance (Fig. 3). In contrast, when the selection is lethal (above
MIC) it is only required that the acquired mechanism (mutation
or HGT) increases antibiotic resistance above the applied drug
concentration, and the effect of the resistance mechanism on bac-
terial fitness is less important because even resistance mechanisms
with a very high fitness cost will be selected as long as sus-
ceptible competitor bacteria are eliminated. In conclusion, from
the point of view of persistence and potential for reversibility
of resistance mechanisms that are selected at sub-MIC might be
especially problematic since the low fitness cost means that such
bacteria will remain in the population longer when the antibi-
otic use and selective pressure is reduced (Andersson and Hughes,
2010).
8. Which resistance mechanisms are most likely to be
selected at low antibiotic concentrations?
A priori one might think that selection at low antibiotic con-
centrations preferentially select for mutations that confer low
resistance, but the key factor is that any mechanism that con-
fers a sufficiently high increase in resistance and concomitantly
does not decrease fitness too much can potentially be selected.
However, it has been empirically observed that most studied, high-
level resistances whether mutational or HGT based do confer a
substantial (i.e. >1%) fitness cost and at very low antibiotic concen-
trations (i.e. well below the MIC) these mutants will accordingly
not be selected because the antibiotic selective effect is smaller
than the fitness cost. Thus, many of the presently known and
well-described resistance mechanisms are not expected to enrich
under sub-MIC conditions. One illustrative example of this is pro-
vided by selection for streptomycin resistance in S. typhimurium
where at high streptomycin concentrations (>100 mg/L) the classi-
cal rpsL mutations are almost exclusively isolated. These mutations
provide a high level of resistance (>1024 mg/L) but they are also
generally associated with fitness costs ranging between 3 and
27% (Bjorkman et al., 1998; Maisnier-Patin et al., 2002; Gullberg
et al., 2011). Instead, if selection occurs at sub-MIC of strepto-
mycin for the susceptible wild type strain, rpsL mutations are not
selected (because they are too costly) and instead a new class of
low cost mutations (in the gidB gene) and with moderate increases
in MIC is found (Okamoto et al., 2007). Note that this does not
exclude the possibility that sub-MIC could select high-level resis-
tance. The tendency will be that sub-MIC selects mutants with
high fitness (regardless of resistance level), whereas greater-than-
MIC selects mutants with high resistance (regardless of fitness
level).
Few studies have been aimed at identifying resistance mutations
at low antibiotic concentration regimes, especially under condi-
tions of long-term exposure to sub-MIC levels. Several types of
mutants are known to provide low-level resistance but at present
we cannot predict their occurrence, mainly because of limited
knowledge of which mutations confer no or very small fitness costs.
Thus, as discussed above the likelihood that low-level resistance
will become enriched at sub-lethal concentrations of drug is largely
determined by the magnitude of the fitness reduction as compared
to the strength of the selection. Below are outlined some examples
of potential mechanisms to be considered:
1. Resistance conferred by activation of efflux systems (either by
mutation or regulation) can confer an unspecific and gener-
ally low-level type of resistance. For example, activation of the
mar system in E. coli results in a several-fold increase in resis-
tance to a number of different antibiotics (e.g. chloramphenicol,
fluoroquinolones, tetracycline) and biocides (e.g. triclosan and
quaternary ammonium compounds). Although these mutations
occur at a high frequency (by inactivation of repression), they
often have a considerable fitness cost and they are therefore
unlikely to be selected during prolonged exposure at very low
antibiotic concentrations (Gullberg et al., 2011).
2. Another class of mutations increasingly recognized as being
important for low-level resistance is mutation in porins, proteins
that form water channels in the outer membrane of Gram-
negative bacteria. Again these mutations typically only cause a
few-fold increase in resistance to for example ␤-lactams, and
their fitness costs are often quite low. Thus, at least certain types
of porin mutations are predicted to appear at sub-MIC selections.
3. Increased dosage of the antibiotic target molecule or an inef-
ficient enzymatic antibiotic-detoxifying mechanism can also
provide low-level resistance. The latter type of mechanism have
been observed for ␤-lactam resistance where successive gene
 D.I. Andersson, D. Hughes / Drug Resistance Updates 15 (2012) 162–172
amplification of the ampC/bla genes conferred increasing resis-
tance from a very low basal level (Normark et al., 1977; Edlund
and Normark, 1981; Sun et al., 2009). Interestingly, a change in
the opposite direction, by a mutation that reduced drug-target
enzyme expression, confers low-level resistance to quinolones
(Ince and Hooper, 2003).
4. Low-level resistance can also occur as a byproduct of a resis-
tance mechanism that provides a high-level of resistance to
a specific antibiotic but a low level of resistance to a related
antibiotic of the same structural or chemical class. Examples of
this includes certain gyrA mutations in E. coli that confer high-
level resistance to nalidixic acid but only low level resistance to
ciprofloxacin (Yoshida et al., 1990; Gullberg et al., 2011), CTX-
M, TEM and AmpC genes that confer high-level resistance to
penicillins, but only miniscule increases in resistance to car-
bapenems (Ardanuy et al., 1998; Martinez-Martinez et al., 1999;
Mammeri et al., 2008; Girlich et al., 2009; Yang et al., 2009) and
the aminoglycoside modifying enzyme aminoglycoside (AG) 3 -
phosphotransferase II that provide full resistance to kanamycin
but only low-level resistance to amikacin (Perlin and Lerner,
1986).
5. Furthermore, it is likely that there exist a number of resistance
mechanisms that are yet to be identified. It is hard to predict
which these functions might be but as shown by a recent screen
for low-level resistance conferred by over-expression of E. coli
genes, increased dosage of 61 ORFs (from a library containing
over 4000 genes) could confer partial resistance to 86 of 237
antibiotic/toxin-containing environments. Of these ORFs, most
conferred small but significant increases in MIC against tetracy-
clines, ␤-lactams, antifolates, aminoglycosides, and macrolides
(Soo et al., 2011). This experiment demonstrates that there exists
a considerable reservoir of genes in the so-called intrinsic resis-
tome that could confer low-level resistance when increased in
dosage. It is also conceivable that amino acid changes in exist-
ing ORFs could confer low resistance. A still unanswered key
question is how such genetic changes would affect fitness but if
they are not too costly they could potentially be selected during
sub-MIC antibiotic exposure.
6. Apart from genetic changes in the bacterium, low-level resis-
tance can also be conferred by physiological and regulatory
responses that activate intrinsic resistance mechanisms or gen-
erate a physiological state where the bacteria are less susceptible
to the drugs. One example of the former is the activation of a
cryptic aminoglycoside resistance gene, aadA, which when acti-
vated by certain growth conditions provides a moderate level of
resistance (Koskiniemi et al., 2011). Another well-studied exam-
ple is the occurrence of tolerant cells that are refractory to drugs
(Jayaraman, 2008; Lewis, 2008; Allison et al., 2011) or the for-
mation of biofilms in which the bacteria are less susceptible
(Anderson and O’Toole, 2008; Hoiby et al., 2010). In contrast
to genetic mechanisms, these physiological states are rapidly
reversible if environmental conditions change but they could still
potentially serve as stepping-stones that provide transient low-
levels of resistance that allow bacteria to subsequently acquire
stable, high-level resistance mechanisms.
9. What are the drug concentrations in different
environments?
When examining the actual concentrations of antibiotics that
are found in different environments there is obviously an enor-
mous variation. Generally the concentrations observed in treated
humans, animals and in farming are relatively high (even though
there might exist compartments or time intervals where concen-
trations are low, i.e. below MICs of the drugs against susceptible
169
bacteria) whereas levels in the outside environments are typi-
cally substantially lower. Thus, antibiotic concentrations in rivers,
lakes and soils are usually in the ␮g/L to ng/L range, i.e. they
would act as non-lethal selections (Thiele-Bruhn, 2003; Chander
et al., 2005; Kummerer, 2009). However, high antibiotic concen-
trations can be observed in connection with wastewater outlets
from, for example, pharmaceutical industries (Larsson et al., 2007;
Fick et al., 2009) and hospitals (Kummerer and Henninger, 2003;
Gomez et al., 2006; Thomas et al., 2007; Duong et al., 2008;
Seifrtova et al., 2008; Verlicchi et al., 2010). High antibiotic con-
centrations are also associated with wastewater from agricultural
and aquacultural activities (Pena et al., 2010; Thuy et al., 2011;
Wei et al., 2011; Zou et al., 2011) and detectable levels are some-
times found in tap water for human consumption (Fick et al., 2009;
Yiruhan et al., 2010). In one extreme case the concentration of
the fluoroquinolone ciprofloxacin found in river water (31 mg/L)
downstream of a region with several antibiotic-producing indus-
tries well exceeded the antibiotic concentration measured in the
serum of ciprofloxacin-treated patients (about 2–3 mg/L), indicat-
ing an extremely strong selective pressure (Larsson et al., 2007).
10. Approaches to reduce the presence of sub-lethal
selective conditions
Even though antibiotic levels can be at sub-MIC in treated
humans, animals and plants in clinical, community and agricultural
settings, it is likely that the most common environments where
low levels are consistently present over long time periods is in
animals treated with antibiotics for growth promotion and in vari-
ous aquatic environments (Baquero et al., 2008; Kummerer, 2009;
Martinez, 2009; Hoa et al., 2011) and in soils (Rooklidge, 2004;
Pico and Andreu, 2007; Sukul and Spiteller, 2007; Martinez, 2008)
where a big part of the therapeutically used drugs will ultimately
end up if they have not been degraded. It is hard to obtain accurate
estimates of global antibiotic use but it is clearly in excess of several
hundred thousands of tons per year and about half of these antibi-
otics will enter natural environments in active form after they have
exerted their therapeutic or growth-promoting action in humans
and animals.
Is there any reasonable way we may deal with these phar-
maceutical pollutants and prevent them exerting selection for
antimicrobial resistance in the environment? Obviously a general
reduction of antibiotic use will reduce antibiotic release into the
environment. Thus, a more prudent therapeutic use of antibiotic
for infections will have beneficial effects. Similarly, it is possible
to stop the use of antibiotics for growth promotion without major
negative effects on animal production, as shown by bans in Sweden
and Denmark introduced in 1986 and 1997, respectively (Aarestrup
et al., 2001; Bengtsson and Wierup, 2006; Grave et al., 2006) and the
recent ban, applicable within all countries of the European Union,
that was introduced by the European Parliament and the Council of
Europe in 2006 (2003; Cogliani et al., 2011). As the use of antibiotics
for growth promotion represents a substantial part of total antibi-
otic consumption a global ban would drastically reduce the overall
selective pressure. With regard to release of antibiotics into the
environment one could either prevent antibiotic release into water
from humans (mainly urine) or inactivate the drugs downstream
from the release site. The latter is clearly achievable and several
methods already exist that are technically feasible and economi-
cally viable (Esplugas et al., 2007; MistraPharma, 2011; Wahlberg
et al., 2011). For example, ozone treatment of sewage water is an
effective and relatively cheap method for destruction of pharma-
ceuticals, including antibiotics. An additional beneficial effect of
ozone treatment is that essentially all types of pathogenic infec-
tious agents will be efficiently inactivated. Thus, ozone treatment
170 D.I. Andersson, D. Hughes / Drug Resistance Updates 15 (2012) 162–172
will not only remove the selective agents but also break transmis-
sion cycles of both susceptible and resistant microbes.
Acknowledgements
This work was supported by grants from the Swedish Research
Council Medicine, EU project Predicting Antibiotic Resistance (PAR,
grant no. 241476), Swedish Strategic Research Foundation (SSF),
Swedish Innovation Agency (Vinnova) to DIA and DH. We thank
Christina Greko and Linus Sandegren for helpful discussion and
comments on the manuscript.
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