Accepted Article 1 2
Interplay between intrinsic and acquired resistance to quinolones in
Stenotrophomonas maltophilia1
3
Guillermo García-León, Fabiola Salgado1, Juan Carlos Oliveros, María Blanca
4 5
Sánchez*, José Luis Martínez*
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Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología,
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CSIC, Darwin 3, Cantoblanco, 28049 Madrid, Spain.
8
1
9
Horno 777, Talcahuano, Chile.
10
Present address: Laboratorio Microbiología, Hospital Las Higueras, Avenida Alto
*Corresponding authors’ mailing address:
Departamento
de Biotecnología
11
Microbiana, Centro Nacional de Biotecnología (CSIC), Darwin 3, Cantoblanco, 28049
12
Madrid, Spain. Tel: +34-91-5854542; Fax: +34-91-5854506;
13
MBS e-mail: [email protected]
14
JLM e-mail: [email protected]
15 16
Keywords: Quinolone resistance; Smqnr; MDR efflux pumps; topoisomerases;
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SmeDEF; intrinsic resistance; resistome; bacterial evolution; Stenotrophomonas
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maltophilia; Qnr; mutation frequency.
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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12408
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1
Accepted Article 1
Summary
2
To analyze whether the mutation-driven resistance-acquisition potential of a given
3
bacterium might be a function of its intrinsic resistome, quinolones were used as
4
selective agents and Stenotrophomonas maltophilia was chosen as a bacterial model. S.
5
maltophilia has two elements - SmQnr and SmeDEF - that are important in intrinsic
6
resistance to quinolones. Using a battery of mutants in which either or both of these
7
elements had been removed, the apparent mutation frequency for quinolone resistance,
8
and the phenotype of the selected mutants were found to be related to the intrinsic
9
resistome and also depended on the concentration of the selector. Most mutants had
10
phenotypes compatible with the overexpression of multidrug efflux pump(s); SmeDEF
11
overexpression was the most common cause of quinolone resistance. Whole genome
12
sequencing showed that mutations of the SmeRv regulator, which result in the
13
overexpression of the efflux pump SmeVWX, are the cause of quinolone resistance in
14
mutants not overexpressing SmeDEF. These results indicate that the development of
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mutation-driven antibiotic resistance is highly dependent on the intrinsic resistome,
16
which, at least for synthetic antibiotics such as quinolones, did not develop as a
17
response to the presence of antibiotics in the natural ecosystems in which S. maltophilia
18
evolved.
19
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Accepted Article 1
Introduction
2
Antibiotic resistance is a worrisome problem for human health, and is also one of the
3
few evolution processes that can be experimentally addressed. Due to its clinical
4
relevance, most works on antibiotic resistance focus on clinical settings. However, an
5
increasing number of articles propose that natural, non-clinical ecosystems are relevant
6
elements in the evolution of resistance (Alonso et al., 1999; Baquero et al., 2008;
7
Martinez, 2008; Aminov, 2009; Martinez, 2009; Walsh, 2013). In favour of this concept
8
are the facts that many antibiotics are produced by environmental microorganisms
9
(Waksman and Woodruff, 1940; Linares et al., 2006; Fajardo et al., 2009) and that most
10
(if not all) genes that human pathogens have acquired by horizontal gene transfer (HGT)
11
have their origin in environmental bacteria (Martinez, 2008; Allen et al., 2010; Davies
12
and Davies, 2010). It was early thought that such antibiotic resistance genes originated
13
from antimicrobial producers where they have an auto-protective role (Benveniste and
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Davies, 1973). However, the only resistance determinants for which an origin has been
15
tracked, namely QnrA and CTXM, are originated in the non-producers Shewanella
16
algae (Poirel et al., 2005) and Kluyvera (Humeniuk et al., 2002) respectively. This
17
suggests that some elements capable of conferring resistance may have evolved for
18
playing a different role in non-clinical ecosystems. Also in favour of this hypothesis is
19
the finding that opportunistic pathogens with an environmental origin as Pseudomonas
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aeruginosa and Stenotrophomonas maltophilia are among those presenting lower
21
susceptibility to antibiotics, despite they are not antibiotic producers.
22
Since the elements involved in intrinsic resistance in these pathogens may be
23
relevant for acquiring high-level resistance to antibiotics, we wondered whether the
24
evolution, out of clinical settings, of a given determinant, not necessarily related with
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Accepted Article 1
antibiotic resistance in natural ecosystems, may modulate the emergence of antibiotic
2
resistant mutants. If that holds true, this will mean that bacterial adaptation for
3
colonising non-clinical environments can be of relevance for its success in causing
4
infections in a treated patient and puts the focus on the role that non-clinical natural
5
ecosystems may have in the evolution of antibiotic resistance. For addressing this topic,
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we used as models the synthetic antibiotics quinolones, not present in natural
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ecosystems before 1960s, and hence not influencing bacterial evolution, and S.
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maltophilia a free-living microorganism commonly found in soil and water (Ribbeck-
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Busch et al., 2005; Turrientes et al., 2010). Different strains of this microorganism have
10
been studied for their potential use in, biotechnological processes, including the
11
production of proteases (Dunne et al., 2000) and antifungal compounds (Jakobi et al.,
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1996), the biodegradation of pollutants such as aromatic hydrocarbons (Lee et al., 2002)
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and trinitrotoluene (Lee et al., 2009), and the control of plant infections (Ryan et al.,
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2009). In addition to its biotechnological applications, S. maltophilia is an opportunistic
15
pathogen, and its prevalence in hospitalized patients has increased in recent years
16
(Looney et al., 2009; Brooke, 2012). One worrisome characteristic of this organism is
17
their low susceptibility to the antibiotics currently in clinical use (Quinn, 1998;
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McGowan, 2006; Sanchez et al., 2009).
19
It is important to remark that S. maltophilia intrinsic resistance was acquired in the
20
field long before the dawn of antibiotic therapy (Baquero et al., 2009; Martinez, 2009;
21
Martinez et al., 2009b). It can be attributed to the low permeability of the cell
22
membranes, the carriage of genes coding for different antibiotic-modifying enzymes
23
(beta lactamases, aminoglycoside-inactivating enzymes) (Avison et al., 2002; Li et al.,
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2003; Okazaki and Avison, 2007), the possession of efflux pumps such as SmeYZ,
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Accepted Article 1
SmeIJK and SmeDEF (Alonso and Martinez, 2000; Zhang et al., 2001; Crossman et al.,
2
2008), and the existence of a chromosomally encoded quinolone resistance determinant
3
belonging to the Qnr family known as SmQnr (Sanchez et al., 2008; Shimizu et al.,
4
2008; Sanchez and Martinez, 2010). For some of these elements, as SmeYZ and
5
SmeIJK, function has been inferred by sequence homology when the whole sequence of
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S. maltophilia was obtained (Crossman et al., 2008). However, for others as SmQnr and
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SmeDEF a role in intrinsic and acquired resistance to quinolones, as well as other
8
antibiotics as tetracycline, chloramphenicol and erythromycin, has been described
9
(Alonso and Martinez, 2001; Zhang et al., 2001; Sanchez et al., 2004; Gould and
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Avison, 2006; Sanchez et al., 2008; Sanchez and Martinez, 2010). Together, these
11
elements allow S. maltophilia to avoid the activity of antibiotics, but they might also be
12
important in this bacterium’s evolutionary potential. Just like any other bacterium, S.
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maltophilia can also attain increased levels of resistance via the acquisition of resistance
14
genes through HGT, or by mutations in chromosomally-encoded determinants. Indeed,
15
the presence of integrons containing antibiotic resistance genes has been reported in
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different isolates of S. maltophilia (Barbolla et al., 2004; Chang et al., 2004; Toleman et
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al., 2007), and mutations in the transcriptional regulators of efflux pumps that lead to
18
the latters’ overexpression - and therefore to a subsequent increase in antibiotic
19
resistance - have been described (Alonso and Martinez, 2000, 2001; Sanchez et al.,
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2002, 2004; Hernandez et al., 2009).
21
Generally speaking, mutation-driven acquired resistance might be due to the
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increased expression of detoxifying elements such as the aforementioned efflux pumps
23
or antibiotic-inactivating enzymes, or to mutations in genes coding for the antibiotic
24
target. The latter certainly affects resistance to the quinolones. It was suggested that
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Accepted Article 1
since these are synthetic antibiotics, there should be no quinolone resistance genes; the
2
only cause of such resistance, therefore, should be mutations in the genes coding for
3
their targets (DNA gyrase and topoisomerase IV) and the transporters involved in the
4
entrance into bacterial cells of these antimicrobials (Verbist, 1986; Hirai et al., 1987;
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Mouton, 1987; Hooper and Wolfson, 1989). However, the finding that MDR efflux
6
pumps are capable of extruding quinolones (Martinez et al., 2009a), plus the description
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of plasmid-encoded (Gomez-Gomez et al., 1997) quinolone resistance determinants in
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different bacterial pathogens (Martinez-Martinez et al., 1998), showed this thinking to
9
be wrong (Martinez et al., 1998; Hernandez et al., 2011a). Despite this, the mutation of
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the topoisomerase genes remains the most important mechanism leading to high-level
11
quinolone resistance in most bacteria (Hooper, 1999). Opposite to this general rule, in S.
12
maltophilia, the analysis of quinolone-resistant clinical isolates has shown no mutations
13
in the genes coding for topoisomerases (Ribera et al., 2002; Valdezate et al., 2002;
14
Valdezate et al., 2005). One explanation for this issue is the presence in the S.
15
maltophilia genome of genes coding for several proteins capable of detoxifying
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antibiotics, quinolones included (Crossman et al., 2008); they are therefore involved in
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intrinsic resistance. Although their original functional role in the field is unlikely
18
conferring resistance to quinolones, their overexpression may indeed contribute towards
19
acquired resistance to this family of antimicrobials.
20
The present work studies the contribution of the best-known determinants of intrinsic
21
resistance to quinolones in S. maltophilia (SmeDEF (Zhang et al., 2001) and SmQnr
22
(Sanchez and Martinez, 2010)) to the development of mutation-driven acquired
23
resistance. SmQnr belongs to the Qnr family and is just involved in resistance to
24
quinolones (Sanchez et al., 2008). The Qnr proteins protect bacterial cells from the
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Accepted Article 1
action of quinolones by binding the targets of these antimicrobials, the bacterial
2
topoisomerases (Tran et al., 2005a, b). SmeDEF is a tripartite complex formed by an
3
efflux pump located on the inner membrane (SmeE), an outer membrane protein
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(SmeF), and a periplasmic membrane fusion protein (SmeD) (Alonso and Martinez,
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2000). It is involved in resistance to antibiotics of different structural families,
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quinolones included (Alonso and Martinez, 1997, 2000). Its expression is regulated by
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the SmeT repressor (Sanchez et al., 2002; Hernandez et al., 2009); its overexpression is
8
due to mutations of smeT (Sanchez et al., 2002, 2004). To determine the contribution
9
of SmeDEF and SmQnr to the development of quinolone resistance in S. maltophilia,
10
comparisons were made of the mutation frequencies and the types of mutant selected by
11
quinolones in the wild-type S. maltophilia strain D457 and in mutants lacking either
12
SmQnr, SmeDEF or both.
13
Mutation frequency is a fixed value for each bacterial strain. However, in the case of
14
antibiotic resistance, the number of mutants that can be selected at different
15
concentrations of a selective agent can vary if several mutations confer the phenotype
16
(Martinez and Baquero, 2000). In the present article, the mutation frequency observed at
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a given concentration of antibiotic is termed the 'apparent mutation frequency'. It has
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been theoretically discussed that the apparent mutation frequencies will depend on the
19
concentration of the selective agent and on the number of genes that can confer
20
resistance upon their mutation (Martinez and Baquero, 2000). The present results
21
support this hypothesis and indicate that the potential to acquire increased resistance
22
depends on the elements involved in intrinsic resistance to antibiotics encoded by the
23
bacterial genome. It is important to notice that, at least for synthetic antibiotics as
24
quinolones, the determinants involved in intrinsic resistance to these drugs did not
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Accepted Article 1
evolve for this purpose in nature. Rather, in the case of efflux pumps, they play an
2
arrange of different functions including interaction with plant or animal hosts,
3
intercellular communication or extrusion of toxic metabolites among others (Ma et al.,
4
1995; Lee and Shafer, 1999; Llama-Palacios et al., 2002; Barabote et al., 2003;
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Rosenberg et al., 2003; Maggiorani Valecillos et al., 2006; Piddock, 2006; Martinez et
6
al., 2009a; Olivares et al., 2012). This supports that if we want to understand in depth
7
the processes governing the emergence and spread of antibiotic resistance, we have to
8
take into consideration the role that natural ecosystems have played in such evolution.
9
Results
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Construction of mutants deficient in smeE
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The tripartite efflux pump SmeDEF is known to contribute to the intrinsic resistance
12
of S. maltophilia to different antibiotics, including quinolones, fluoroquinolones,
13
tetracycline, chloramphenicol and erythromycin (Zhang et al., 2001). Mutations in
14
smeT, which encodes the transcriptional repressor of smeDEF, lead to SmeDEF
15
overexpression (Sanchez et al., 2002, 2004). As shown in (Alonso and Martinez, 2000),
16
this allows a phenotype of high-level resistance to different antibiotics, including
17
quinolones (Table 1). To study the effect of the different intrinsic resistance
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mechanisms in acquired resistance, a set of isogenic deletion mutants, all of them
19
derived from the D457 wild-type strain, was generated that included an already
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characterized mutant lacking Smqnr (Sanchez and Martinez, 2010), a mutant lacking
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smeE, and a double mutant lacking both smeE and Smqnr. The deletion of smeE
22
removes a key component of the SmeDEF tripartite complex - the efflux pump, thus
23
preventing antibiotics being extruded from the cell. Markerless chromosomal in-frame
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deletion mutants were constructed in S. maltophilia D457 (wild-type strain) and MBS82
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Accepted Article 1
(Smqnr defective mutant) by homologous recombination, and the deletion of smeE
2
confirmed by PCR as described in Experimental Procedures. Whole genome sequence
3
(see below) further confirmed the deletions. The deletion of smeE reduced the minimum
4
inhibitory concentrations (MICs) of norfloxacin, ofloxacin, nalidixic acid and
5
ciprofloxacin in the mutant strains between 2 and 8 fold (Table 1), confirming this
6
efflux pump to be involved in the intrinsic resistance of S. maltophilia to quinolones.
7 8
Elimination of determinants involved in intrinsic resistance reduces the apparent
mutation frequencies of quinolone resistance.
9
To determine whether the elements involved in intrinsic resistance modulate the
10
emergence of mutants (acquired resistance), the apparent mutation frequencies for
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ofloxacin resistance were measured in the wild-type strain S. maltophilia D457, the
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mutant MBS82 (which lacks Smqnr), the mutant MBS411 (defective in smeE), and the
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double mutant GGL199 (which lacks both Smqnr and smeE). Resistance can be
14
achieved by mutations or by transient, non-inheritable, phenotypic changes via the
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induction of an adaptive transient response (Levin and Rozen, 2006). The stability of
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the mutations conferring quinolone resistance was therefore confirmed. For this, a
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representative number of mutants (between 9 and 16, typically 12) obtained under each
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of the conditions shown in Figure 1 were then cultured by two passages in a medium
19
without antibiotics, and susceptibility to quinolones then tested. All the mutants
20
analyzed were stable, indicating the change in susceptibility to quinolones not to be a
21
transient phenotypic change but the result of mutation. To analyze in detail all the
22
potential phenotypes, we examined all the selected mutants independently of whether or
23
not their MICs reached the breakpoint defining resistance from a clinical viewpoint.
24
This definition of resistance, based on changes in the MIC compared to the wild-type
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Accepted Article 1
population, has recently been used to define epidemiological breakpoints (Kronvall,
2
2010).
3
In agreement with an earlier hypothesis (Martinez and Baquero, 2000), the deletion
4
of the intrinsic quinolone resistance determinants, smeE and Smqnr, reduced the
5
apparent mutation frequency towards ofloxacin resistance at any given concentration,
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the effect being greater when both determinants were deleted (Figure 1). To address
7
whether this effect on the apparent mutation frequencies was specific for quinolones or
8
a more general effect on the overall mutation frequency of S. maltophilia, the mutation
9
frequencies of each of the strains towards rifampin resistance was measured as
10
described in Experimental Procedures. The values were 1.2x10-8± 3.4x10-9 for the wild-
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type strain D457, 2.5x10-8± 8.1x10-9 for the MBS82 ∆Smqnr mutant, 5.5x10-8± 1.7x10-8
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for the MBS411 ∆smeE mutant, and 1.6x10-8± 2.1x10-9 for the GGL199 ∆Smqnr∆smeE
13
double mutant. The observed differences were minor and within the error range of the
14
technique. By contrast, the differences in the apparent mutation rates towards quinolone
15
resistance among these strains where some orders of magnitude higher, in occasions
16
close to 104 fold (Figure 1). This indicates that removing the elements of intrinsic
17
resistance to quinolones does not produce a general, non-specific relevant effect on S.
18
maltophilia mutation rates.
19 20
The type of quinolone-resistant mutants selected differs depending on the selective
pressure exerted by quinolones
21
It was predicted that the type of mutations selected at different concentrations of the
22
antibiotic would be different. At low concentrations, all the potential mutants, whether
23
conferring low-level or high-level resistance, would be selected. However, at high
24
concentrations, only a subset of mutants (those with high-level resistance) would be
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Accepted Article 1
selected. To test this hypothesis, the susceptibility to different antibiotics of several
2
spontaneous, stable, ofloxacin-resistant mutants isolated in the mutation frequency
3
assay (see above) of S. maltophilia D457, was analyzed. Four different types of
4
phenotypes were observed (Table 1SI, supplemental information): A, increased
5
resistance to tetracycline, erythromycin, chloramphenicol and quinolones (a phenotype
6
compatible with the overexpression of SmeDEF); B, high-level resistance to
7
chloramphenicol and quinolones; C, resistance to quinolones only, with no change in
8
susceptibility to the other tested antibiotics; and D, resistance to erythromycin and
9
quinolones. Figure 2A shows that the distribution of the mutant phenotypes is a function
10
of the concentration of the antibiotic used for selection. It is noteworthy that the
11
percentage of mutants in which only the susceptibility to quinolones changed
12
(phenotype C, white boxes in Figure 2) was low. Most mutants showed changes in the
13
susceptibility to antibiotics belonging to different structural families; a phenotype
14
consistent with efflux pump overproduction.
15 16
The type of quinolone-resistant mutants differs depending on the presence of
determinants involved in intrinsic resistance to quinolones.
17
If different resistant mutants are selected at different concentrations, it is conceivable
18
that the removal of elements involved in intrinsic resistance might also modify the
19
phenotypic profile of the mutants selected. Susceptibility to different antibiotics was
20
therefore tested as above in mutants derived from the strains lacking either smeE or
21
Smqnr and the double mutant lacking both smeE and Smqnr. In agreement with the data
22
obtained for the wild-type strain, the distribution of mutant phenotypes for each strain
23
and at each concentration differed (Figure 2). As found for the wild-type strain, the
24
percentage of mutants showing changes just in their susceptibility to quinolones was
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11
Accepted Article 1
low, and this phenotype was not selectable upon inactivation of the SmeDEF efflux
2
pump.
3 4
Overexpression of SmeDEF is an important mechanism of acquired resistance to
quinolones in S. maltophilia.
5
The overexpression of the multidrug efflux pump SmeDEF confers increased
6
resistance to tetracycline, erythromycin, chloramphenicol and quinolones (Alonso and
7
Martinez, 1997, 2000), Table 1. The most prevalent phenotype of the mutants selected
8
for strains D457 and MBS82 was consistent with the overproduction of this efflux pump
9
(Table 1SI, Figure 2). The mutants derived from strains MBS411 and GGL199 did not
10
present this phenotype, in agreement with the lack of smeE in these strains. Previous
11
work has shown that the amount of SmeF is a good marker for measuring the expression
12
of SmeDEF, which correlates well with the level of expression of the other components
13
of the tripartite efflux pump (Alonso and Martinez, 2001). Consequently, in order to
14
confirm that SmeDEF was overproduced in the mutants showing increased resistance to
15
tetracycline, erythromycin, chloramphenicol and quinolones, the level of expression of
16
the porin of the pump, SmeF, was estimated by Western blotting in a set of 14 mutants
17
presenting a phenotype compatible with SmeDEF overexpression. Two mutants in
18
which only the susceptibility to quinolones change and the wild-type strain D457 were
19
used as negative controls of SmDEF overexpression, whereas the SmeDEF
20
overexpressing mutant D457R (Alonso and Martinez, 1997, 2000) was used as positive
21
control of such overexpression. As shown in Figure 3, all the analyzed mutants with a
22
phenotype compatible with SmeDEF overexpression did indeed produce large amounts
23
of this efflux pump, indicating that SmeDEF is an important determinant in the
24
acquisition of quinolone resistance in S. maltophilia.
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12
Accepted Article 1
Quinolone resistance in S. maltophilia is not associated with mutations in the
2
quinolone resistance-determining regions of the genes encoding the bacterial
3
topoisomerases, nor with Smqnr overexpression, even in the absence of smeE.
4
It has been reported that clinical quinolone-resistant isolates of S. maltophilia do not
5
harbour mutations in the genes coding for bacterial topoisomerases (Ribera et al., 2002;
6
Valdezate et al., 2002; Valdezate et al., 2005). Since SmeDEF overexpression is a major
7
element for the acquisition of quinolone resistance in this bacterial species (see above),
8
it was deemed possible that the removal of smeE might allow for the selection of
9
quinolone resistant mutants with altered topoisomerases. Nevertheless, all the mutants
10
selected from the strains MBS411 and GGL199 showed reduced susceptibility to
11
different antibiotics as well as quinolones, indicating that the basis of the resistance is
12
not a mutation in the genes coding for the topoisomerases. The observed phenotypes are
13
compatible with the overexpression of efflux pump(s) despite these strains’ lack of
14
smeE.
15
Some resistant mutants derived from D457 and from MBS82, however, were only
16
quinolone-resistant; a phenotype compatible with mutations in the genes coding for the
17
bacterial topoisomerases. The quinolone resistance-determining regions (QRDRs) of
18
gyrA, gyrB, parC and parE, where the mutations conferring quinolone resistance in
19
bacteria are located, were amplified and sequenced in six representative strains showing
20
this phenotype (MBS123, MBS135 and MBS143 derived from D457, and MBS116,
21
MBS161 and MBS162 derived from MBS82). No mutations were identified in any of
22
the mutants analyzed, indicating that the observed phenotype was not due to mutations
23
in the QRDRs of the topoisomerases. It has been described that SmQnr overexpression
24
can confer resistance to quinolones in S. maltophilia (Sanchez and Martinez, 2010).
This article is protected by copyright. All rights reserved.
13
Accepted Article 1
While this mechanism cannot contribute to resistance in the strain MBS82, which lacks
2
Smqnr, it was reasoned that in S. maltophilia D457 the mechanism of resistance in
3
mutants in which only the susceptibility to quinolones changes might involve SmQnr
4
overexpression. The expression of Smqnr was thus analyzed by real time RT-PCR in
5
the mutants MBS123, MBS135 and MBS143 (all derived from S. maltophilia D457),
6
none of which have QRDR mutations. No significant changes in the expression of
7
Smqnr were observed (Figure 4) compared to the original strain S. maltophilia D457 or
8
the mutant MBS130 which overexpresses SmeDEF. This indicates that the quinolone
9
resistance of these mutants is not due to the overexpression of SmQnr.
10 11
The overexpression of the SmeVWX efflux pump contributes to quinolone resistance
in the absence of SmeDEF overexpression
12
To address in greater detail the molecular basis of the quinolone resistance in those
13
strains not overexpressing SmeDEF, the whole genomes of five different, one-step,
14
selected mutants, with different phenotypes were sequenced using Illumina technology
15
and following a single read 1x75 protocol. Of the five sequenced strains, two mutants
16
derived from D457, one from MBS82, one from MBS411, and one from GGL199. A
17
total of 4.3 ± 15% million pass-filter sequences were obtained for each of the different
18
strains, which approximately corresponds to 325 Mb of sequence and a genome
19
coverage of 60x. In all cases, 100% of each genome was sequenced without any gap,
20
except for the deleted genes smeE and Smqnr in the corresponding mutants.
21
The genome sequences of these mutants were compared to their respective parental
22
strains to detect mutations that might be responsible for the observed phenotypes. Each
23
mutant strain showed just one mutation in its genome. Insertions were not detected and
24
the only deletions found correspond to those previously generated at smeE and Smqnr.
This article is protected by copyright. All rights reserved.
14
Accepted Article 1
In all cases, these mutations were in smeRv (Table 4), which codes for the repressor of
2
the MDR efflux system SmeVWX (Chen et al., 2011). The presence of these mutations
3
in each of the mutants was confirmed by amplifying and sequencing the corresponding
4
DNA region. To ascertain whether the mutations caused the overexpression of
5
SmeVWX, the expression of smeW was analyzed by real time RT-PCR. Table 4 shows
6
that, in agreement with the finding of mutations in the gene coding for the SmeVWX
7
regulator, all the mutants overexpressed this efflux pump (although the level of
8
expression varied). Given that the five sequenced genomes contained only one mutation
9
each, that all these mutations laid in the same transcriptional repressor SmeRv and that
10
in all of them the expression of the SmeRv-regulated efflux pump SmeVWX is de-
11
repressed, our data strongly support the idea that quinolone resistance was acquired in
12
these mutants via the overexpression of SmeVWX.
13
Discussion
14
This work explores the idea that the development of acquired resistance is modulated
15
by the variability of elements conferring intrinsic resistance encoded in the genomes of
16
bacterial pathogens. In other words, the intrinsic resistome – understood to be the
17
ensemble of genes that contribute to the 'natural' or intrinsic resistance of bacterial
18
pathogens (Fajardo et al., 2008), which has evolved, long before human use of
19
antibiotics, in natural ecosystems - defines mutation-driven acquired resistance to
20
antibiotics. This was examined by studying resistance to quinolones in S. maltophilia.
21
Quinolones were chosen since these drugs are synthetic, and as such could not have
22
contributed to the natural evolution of antibiotic resistance. S. maltophilia was chosen
23
since it is an opportunistic pathogen with an environmental origin and the quinolone-
24
resistant mutants isolated in clinical settings do not show mutations in the genes coding
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15
Accepted Article 1
for bacterial topoisomerases (Ribera et al., 2002; Valdezate et al., 2002; Valdezate et al.,
2
2005) – unlike that seen in all other studied bacterial species. This feature suggest that
3
S. maltophilia may harbour elements, which have evolved in the field and allow this
4
bacterial species to counteract the activity of quinolones. In this regard, an interesting
5
feature of S. maltophilia is the presence in its genome of a gene coding for the
6
quinolone resistance protein SmQnr (Shimizu et al., 2008; Sanchez and Martinez, 2010)
7
as well as operons coding for several multidrug efflux pumps. The latter might also
8
contribute towards resistance to these drugs when overexpressed (Crossman et al.,
9
2008).
10
Using a set of S. maltophilia mutants lacking the best-known elements involved in
11
intrinsic resistance to quinolones, the capacity to acquire resistance (apparent mutation
12
frequency) was found to be a function of the intrinsic resistome and the concentration of
13
the challenging antibiotic. Since Qnr proteins bind bacterial topoisomerases (Tran et al.,
14
2005b, a), Smqnr deletion could potentially alter the overall mutation frequency of S.
15
maltophilia. However, the fact that the mutation frequencies did not change when using
16
rifampin as the selective agent goes against this hypothesis, and indicates that the
17
apparent mutation frequency for a given antibiotic depends on the mechanisms of
18
intrinsic resistance against it. Inhibiting elements involved in intrinsic resistance, such
19
as SmeDEF or SmQnr, might thus preclude the emergence of quinolone-resistant
20
mutants in S. maltophilia (Garcia-Leon et al., 2012; Martinez, 2012). Further, the
21
deletion of these intrinsic resistance elements not only altered the number of mutants
22
selectable at a given concentration of antibiotic, but changed the phenotypic profile of
23
the mutants selected. This might help define novel mechanisms of resistance that in the
24
presence of those elements would not be detectable. Notably, the deep genome
This article is protected by copyright. All rights reserved.
16
Accepted Article 1
sequencing of mutants with slightly different phenotypes showed all to have mutations
2
at the regulator of SmeVWX expression, SmeRv, which led to the overexpression of
3
SmeVWX. Previous work has shown SmeVWX not to contribute to intrinsic resistance
4
to antibiotics in S. maltophilia, most likely because its level of expression is very low in
5
the wild-type strain (Chen et al., 2011). Deep sequencing analysis of the S. maltophilia
6
transcriptome shows that this is indeed the case (not shown). However, other authors
7
reported a chloramphenicol-selected mutant that overexpressed SmeVWX to acquire
8
resistance to different antibiotics (Chen et al., 2011). Notably, the SmeVWX-
9
overexpressing mutant analyzed in the latter work had no mutations in smeRv,
10
indicating that other elements besides this local regulator participate in the regulation of
11
the expression of this efflux pump. SmeRv is a LysR-type regulator, and this family
12
contains both activators and repressors that respond to external signals. Among the
13
mutants analyzed in the present work, three showed mutations at Gly266 and one at
14
Glu256, and both these positions are inside the predicted effector-binding motif of the
15
protein. Binding of the effector alters the structure of the regulator and consequently its
16
ability to bind to its operator DNA. Mutations in the effector-binding region might thus
17
also alter the DNA-binding capability of the regulator (Hernandez et al., 2011b). The
18
strength of the structural modification, and hence the level of overexpression achieved,
19
depending on the mutation involved. Indeed, the present results show that different
20
mutations in smeRv can retrieve smeVWX repression to different degrees; this is
21
reflected in the level of expression of this efflux pump and therefore in the degree of
22
resistance achieved.
23
Two factors can thus explain the different phenotypes of the mutants. The first is the
24
level of expression achieved by SmeVWX. The MBS287 mutant showed resistance to
This article is protected by copyright. All rights reserved.
17
Accepted Article 1
quinolones and chloramphenicol and high levels of expression of smeW, while
2
MBS123, which expressed less smeW, showed only quinolone resistance. These
3
different phenotypes probably occur because SmeVWX extrudes chloramphenicol less
4
efficiently than quinolones. Consequently the differences in susceptibility to this
5
antibiotic cannot be detected at the level of expression shown by MBS123. In contrast,
6
they are detectable at the level of SmeVWX expression shown by MBS287. Secondly,
7
the genomic context of the mutations is also important in terms of the phenotype
8
observed. A phenotype of increased resistance to erythromycin is only detectable in the
9
absence of smeE since, in S. maltophilia, SmeDEF is a major contributor to intrinsic
10
resistance to this antibiotic. Indeed, the removal of smeE reduces the erythromycin MIC
11
from 128 µg/ml to 32 µg/ml (Table 1). This agrees with previously published work
12
showing that, on occasion, the full profile of an efflux pump can only be determined
13
when other efflux pumps that extrude the same antibiotics more efficiently are removed
14
(Chuanchuen et al., 2002; Mima et al., 2005; Mima et al., 2009).
15
The present results indicate that the most prevalent cause of acquired quinolone
16
resistance in S. maltophilia is the overproduction of multidrug efflux pumps, the most
17
important of which is SmeDEF. However, even when this pump is removed, the
18
phenotype of the mutants obtained is compatible with the overexpression of efflux
19
pump(s) such as SmeVWX, but not with mutations in the genes coding for
20
topoisomerases. Indeed, in agreement with data obtained using clinical quinolone
21
resistant isolates (Ribera et al., 2002; Valdezate et al., 2002; Valdezate et al., 2005), no
22
mutant with mutations in the genes coding for topoisomerases was detected. This might
23
be the consequence of unaffordable fitness costs (Martinez et al., 2011) or of the
24
presence in the genome of a large number of elements capable of efficiently conferring
This article is protected by copyright. All rights reserved.
18
Accepted Article 1
resistance to these drugs (Crossman et al., 2008). Although no evidence is available to
2
support either statement, the fact that mutations in topoisomerases are easily selectable
3
in any other tested microorganism favours the second hypothesis. Our work also has
4
some practical implications for predicting the emergence of resistance to quinolones in
5
S. maltophilia and the consequences of such emergence. Our results support that the
6
probability that S. maltophilia quinolone resistant mutants presenting mutations at the
7
genes encoding bacterial topoisomerases appear in the future is likely low, unless
8
efficient inhibitors of multidrug efflux pumps are developed. Main causes of resistance
9
will be overproduction of SmeDEF, an issue already addressed in some articles (Alonso
10
and Martinez, 2001; Chang et al., 2004; Sanchez et al., 2004; Sanchez et al., 2005;
11
Gould and Avison, 2006) and SmeVWX overexpression. In this respect is worth
12
mentioning that we have found that a clinical quinolone resistant S. maltophilia isolate
13
overexpresses SmeVWX and presents the same Gly266Ser amino acid change at
14
SmeRv that we found in the in vitro obtained mutants MBS300 and GGL231 (MBS et
15
al. to be published). Finally, since most quinolone resistant mutants overproduce efflux
16
pumps that have other antibiotic substrates, this means that treating S. maltophilia
17
infections with quinolones may select resistant mutants, not just to quinolones, but to
18
several other antibiotics also, which can compromise the therapy.
19
The present findings support the idea that the capacity to acquire resistance by means
20
of mutations is strongly dependent on the mechanisms of intrinsic resistance that
21
bacteria have acquired over their evolution in non-clinical, natural ecosystems. In the
22
present work we studied the acquisition of resistance to quinolones, a family of
23
synthetic drugs, which has had no role in the selection of the elements of intrinsic
24
resistance present in the bacterial genome. In the case of S. maltophilia, the most
This article is protected by copyright. All rights reserved.
19
Accepted Article 1
important elements dealing with resistance are efflux pumps. These elements are
2
encoded in the genomes of all organisms and, in addition of being involved in
3
resistance, they have several other roles in natural ecosystems (Piddock, 2006; Martinez
4
et al., 2009a), including intercellular signalling, detoxification from metabolic toxic
5
intermediates, resistance to solvents and interaction with plant hosts among others
6
(Kohler et al., 2001; Rojas et al., 2001; Aendekerk et al., 2005; Maggiorani Valecillos et
7
al., 2006; Ravirala et al., 2007; Olivares et al., 2012). In this regard, it is worth
8
mentioning that preliminar evidences suggest that the primary role of SmeDEF might
9
actually be in the interaction of S. maltophilia with the plant host (GGL to be
10
published).
11
From an ecological and evolutionary standpoint, our results indicate that the capacity
12
to acquire antibiotic resistance might depend on the capabilities of bacteria to confront
13
other types of insult via systems that evolved before the use of antibiotics by humans
14
(Martinez, 2008). This non-antibiotic selection of resistance might still be at work. A
15
better comprehension of the role that environmental (non-clinical) ecosystems play in
16
the evolution of resistance is needed if we are to understand the emergence of resistance
17
in bacterial pathogens.
18
Experimental procedures
19
Bacterial strains, plasmids and growth conditions
20
Table 2 shows the bacterial strains and plasmids used in the present work. Table 1SI shows
21
the spontaneous mutants derived from these strains. All strains were grown in LB medium
22
(Atlas, 1993) at 37ºC, unless indicated otherwise.
23
Construction of the smeE deletion mutants
This article is protected by copyright. All rights reserved.
20
Accepted Article 1
To generate smeE deletion mutants in D457 and MBS82, two DNA fragments homologous
2
to the 5’ end (X fragment) and the 3’ end (Y fragment) of the smeE gene were obtained from
3
pAS2 using the Expand Long Template PCR System (Roche) and 0.5 µM of the primers X1-
4
X2 and Y1-Y2 respectively. The fragments were designed not to break the reading frame. The
5
reactions consisted of one denaturation step at 94ºC for 2 min and 15 amplification cycles of
6
94ºC for 10 s, 50ºC for 30 s, and 68ºC for 1 min, followed by a further 25 amplification cycles
7
of 94ºC for 10 s, 55ºC for 30 s, and 68ºC for 1 min, plus a final extension step at 68ºC for 7
8
min. The PCR products were cloned into pGEMT-Easy, generating pGG1 (X fragment) and
9
pGG2 (Y fragment). To check the fragments, they were sequenced with the universal primers
10
F24 and R24 by Secugen (www.secugen.es). pGG1 and pGG2 were digested with EcoRI-
11
XbaI and XbaI-HindIII respectively, and the X and Y fragments cloned into the same
12
restriction sites in the suicide vector pEX18Gm, yielding pGG8, which was introduced into
13
Escherichia coli TG1, then into E. coli CC118lpir, and finally into S. maltophilia D457 and
14
MBS82 by tripartite mating (de Lorenzo and Timmis, 1994). In both cases, the
15
transconjugants containing pGG8 were selected on LB agar containing 80 µg/ml gentamycin
16
and 20 µg/ml imipenem after 48 h of incubation. To check the insertion of the plasmid,
17
selected transconjugants were streaked onto LB agar containing 80 µg/ml gentamycin and 20
18
µg/ml imipenem, and after 24 h of incubation PCR with primers pair X3/Y3 was performed
19
with gentamycin-resistant colonies (colonies with the insertion gave two fragments of 2269
20
and 226 bp length). Colonies containing the insert were streaked onto 10% sucrose-LB agar,
21
and sucrose-resistant colonies arising after 48 h of incubation at room temperature (RT) were
22
screened for the presence of the smeE deletion by PCR. For this, two sets of primers were
23
used. The pair Z3/Z4 locates to the region to be deleted. Amplification with these primers
24
rendered a 643 bp product in the strains D457 and MBS82, which contain smeE, whereas
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21
Accepted Article 1
amplification was negative for the strains in which smeE had been deleted, i.e., MBS411
2
(derived from D457) and GGL199 (derived from MBS82). Further confirmation of the
3
deletion was obtained using the primers pair X3/Y3, which locates to outside the region to be
4
deleted. The primers amplified a region of 2269 bp in the strains D457 and MBS82, and of
5
226 bp in MBS411 and GGL199, thus confirming smeE deletion
6
Determination of mutation frequencies
7
Each tube containing LB was inoculated with one isolated colony and incubated overnight.
8
100 µl of different dilutions (100 to 10-7) of the overnight cultures was seeded onto Mueller
9
Hinton agar plates (MHA)(Pronadisa) without antibiotic, or containing either ofloxacin (2, 4,
10
8 or 16 µg/ml) or rifampin (250 µg/ml). Colonies were counted and mutation frequencies
11
estimated as the ratio between colonies growing in the presence and absence of antibiotics.
12
Antibiotic susceptibility assays
13
The MICs of the different antibiotics were determined on MHA plates using the two-fold
14
agar dilution method. The antibiotics used were tetracycline, erythromycin, chloramphenicol,
15
norfloxacin, ofloxacin, nalidixic acid and ciprofloxacin. The results were recorded after 24 h
16
of incubation at 37ºC.
17
Detection of SmeDEF by Western blotting
18
To determine the amount of SmeDEF in the different mutants, the expression of SmeF was
19
determined by Western blotting as previously described (Alonso and Martinez, 2000). Whole
20
cell extracts from overnight cultures of the tested strains were electrophoresed in 12% SDS–
21
PAGE gels (Sambrook and Russell, 2001). Proteins were transferred to Immobilon-P
22
(Millipore) and the presence of SmeF was determined using a polyclonal anti-SmeF antibody
23
(Alonso and Martinez, 2000) at a final dilution of 1:5000. A secondary goat anti-rabbit HRP
This article is protected by copyright. All rights reserved.
22
Accepted Article 1
conjugate (Bio-Rad) was used at a final dilution of 1:40000. Bands were detected by
2
chemiluminiscence with Immobilon™ Western (Millipore). The A600 of the overnight cultures
3
was measured and in all cases an extract of around 2-3x108 cells from each strain was loaded
4
in each well.
5
Sequencing of the topoisomerase gene QRDRs
6
The QRDRs of gyrA, gyrB, parC and parE were amplified by PCR using the Expand Long
7
Template PCR System (Roche). Purified PCR products were sequenced by Macrogen
8
(www.macrogen.com). The primers used for PCR and sequencing were those described by
9
Valdezate (Valdezate et al., 2002).
10
Real time PCR
11
Total bacterial RNA was isolated from 25 ml mid-log-phase cultures (A600 = 0.6-0.7) using
12
the Qiagen RNeasy Mini Kit (Qiagen, Inc.). To eliminate genomic DNA, samples were
13
treated with RNase-free TURBO DNase (Ambion, Inc.), and RNA purified using the Qiagen
14
RNeasy Mini Kit’s RNA cleanup protocol. This RNA was then used as a template for reverse
15
transcription using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
16
Real-time PCR reactions were performed using Power SYBR Green PCR Master Mix and a
17
7300 Real-Time PCR System (Applied Biosystems). Primer pairs RTqnr1/2 (Sanchez and
18
Martinez, 2010) and SmeWQ5/6 were used to analyze the expression of the Smqnr and smeW
19
genes respectively. The expression of the ftsZ gene (primers FtsZ1/2), a house keeping gene
20
used as reference for quantifying RNA in different species (Delogu et al., 2006; Schwan et al.,
21
2006; Duquenne et al., 2010), was used to normalize the data. The relative amount of mRNA
22
for Smqnr and smeW was calculated following the 2–∆∆Ct method (Livak and Schmittgen,
This article is protected by copyright. All rights reserved.
23
Accepted Article 1
2001). The absence of genomic DNA was verified by real-time PCR using total RNA without
2
reverse transcription. The results are the means of three independent experiments.
3
Whole genome sequencing and determination of mutations
4
DNA from the different samples was analyzed for integrity in agarose gels and quantified
5
by PicoGreen®; a total amount of 3 µg was used for library preparation.
6
DNA was mechanically fragmented and libraries prepared using the TruSeq DNA Sample
7
Preparation v.2 Kit (Illumina). Briefly, the DNA was made blunt ended, phosphorylated, and
8
an adenine added in the 5´-end of the fragments so that Illumina adapters could be ligated.
9
Libraries including adapters were gel-purified and PCR-amplified using common primers so
10
that a sufficiently large library could be subjected to sequencing. Final libraries were titrated
11
by quantitative PCR using external controls as a reference, and showed appropriate
12
concentrations ranging from 95 nM to 434 nM. The libraries were then adjusted to a final
13
concentration of 10 pM, denatured, seeded on a flowcell (Illumina) using a Cluster Station,
14
and sequenced at the Unidad de Genómica Antonia Martín Gallardo, Parque Científico de
15
Madrid, Spain, following a single read 1x75 protocol and using an Illumina GAIIx apparatus.
16
Quality filtering was automatically performed according to Illumina specifications and
17
individual reads were demultiplexed using the CASAVA pipeline. The percentage of pass-
18
filter reads was 89-95%; the mean quality was >Q33. Finally, fastq files were used for
19
mapping. The mutations were identified by the Computational Genomics Service of the CNB.
20
Single-end 75 nt reads were aligned against the S. maltophilia D457 (Lira et al., 2012)
21
genome (HE798556) with the Burrows-Wheeler Alignment tool (Li and Durbin, 2009) using
22
the
23
samtools/mpileup/bcftools/vcfutils (Li et al., 2009). Final SNP candidates were selected by
default
settings.
Single
nucleotide
This article is protected by copyright. All rights reserved.
polymorphisms
were
detected
using
24
Accepted Article 1
removing those with a read depth of >100 (varFilter -D100) and those in which the SNP was
2
present in
Interplay between intrinsic and acquired resistance to quinolones in
Stenotrophomonas maltophilia1
3
Guillermo García-León, Fabiola Salgado1, Juan Carlos Oliveros, María Blanca
4 5
Sánchez*, José Luis Martínez*
6
Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología,
7
CSIC, Darwin 3, Cantoblanco, 28049 Madrid, Spain.
8
1
9
Horno 777, Talcahuano, Chile.
10
Present address: Laboratorio Microbiología, Hospital Las Higueras, Avenida Alto
*Corresponding authors’ mailing address:
Departamento
de Biotecnología
11
Microbiana, Centro Nacional de Biotecnología (CSIC), Darwin 3, Cantoblanco, 28049
12
Madrid, Spain. Tel: +34-91-5854542; Fax: +34-91-5854506;
13
MBS e-mail: [email protected]
14
JLM e-mail: [email protected]
15 16
Keywords: Quinolone resistance; Smqnr; MDR efflux pumps; topoisomerases;
17
SmeDEF; intrinsic resistance; resistome; bacterial evolution; Stenotrophomonas
18
maltophilia; Qnr; mutation frequency.
19
20
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12408
This article is protected by copyright. All rights reserved.
1
Accepted Article 1
Summary
2
To analyze whether the mutation-driven resistance-acquisition potential of a given
3
bacterium might be a function of its intrinsic resistome, quinolones were used as
4
selective agents and Stenotrophomonas maltophilia was chosen as a bacterial model. S.
5
maltophilia has two elements - SmQnr and SmeDEF - that are important in intrinsic
6
resistance to quinolones. Using a battery of mutants in which either or both of these
7
elements had been removed, the apparent mutation frequency for quinolone resistance,
8
and the phenotype of the selected mutants were found to be related to the intrinsic
9
resistome and also depended on the concentration of the selector. Most mutants had
10
phenotypes compatible with the overexpression of multidrug efflux pump(s); SmeDEF
11
overexpression was the most common cause of quinolone resistance. Whole genome
12
sequencing showed that mutations of the SmeRv regulator, which result in the
13
overexpression of the efflux pump SmeVWX, are the cause of quinolone resistance in
14
mutants not overexpressing SmeDEF. These results indicate that the development of
15
mutation-driven antibiotic resistance is highly dependent on the intrinsic resistome,
16
which, at least for synthetic antibiotics such as quinolones, did not develop as a
17
response to the presence of antibiotics in the natural ecosystems in which S. maltophilia
18
evolved.
19
This article is protected by copyright. All rights reserved.
2
Accepted Article 1
Introduction
2
Antibiotic resistance is a worrisome problem for human health, and is also one of the
3
few evolution processes that can be experimentally addressed. Due to its clinical
4
relevance, most works on antibiotic resistance focus on clinical settings. However, an
5
increasing number of articles propose that natural, non-clinical ecosystems are relevant
6
elements in the evolution of resistance (Alonso et al., 1999; Baquero et al., 2008;
7
Martinez, 2008; Aminov, 2009; Martinez, 2009; Walsh, 2013). In favour of this concept
8
are the facts that many antibiotics are produced by environmental microorganisms
9
(Waksman and Woodruff, 1940; Linares et al., 2006; Fajardo et al., 2009) and that most
10
(if not all) genes that human pathogens have acquired by horizontal gene transfer (HGT)
11
have their origin in environmental bacteria (Martinez, 2008; Allen et al., 2010; Davies
12
and Davies, 2010). It was early thought that such antibiotic resistance genes originated
13
from antimicrobial producers where they have an auto-protective role (Benveniste and
14
Davies, 1973). However, the only resistance determinants for which an origin has been
15
tracked, namely QnrA and CTXM, are originated in the non-producers Shewanella
16
algae (Poirel et al., 2005) and Kluyvera (Humeniuk et al., 2002) respectively. This
17
suggests that some elements capable of conferring resistance may have evolved for
18
playing a different role in non-clinical ecosystems. Also in favour of this hypothesis is
19
the finding that opportunistic pathogens with an environmental origin as Pseudomonas
20
aeruginosa and Stenotrophomonas maltophilia are among those presenting lower
21
susceptibility to antibiotics, despite they are not antibiotic producers.
22
Since the elements involved in intrinsic resistance in these pathogens may be
23
relevant for acquiring high-level resistance to antibiotics, we wondered whether the
24
evolution, out of clinical settings, of a given determinant, not necessarily related with
This article is protected by copyright. All rights reserved.
3
Accepted Article 1
antibiotic resistance in natural ecosystems, may modulate the emergence of antibiotic
2
resistant mutants. If that holds true, this will mean that bacterial adaptation for
3
colonising non-clinical environments can be of relevance for its success in causing
4
infections in a treated patient and puts the focus on the role that non-clinical natural
5
ecosystems may have in the evolution of antibiotic resistance. For addressing this topic,
6
we used as models the synthetic antibiotics quinolones, not present in natural
7
ecosystems before 1960s, and hence not influencing bacterial evolution, and S.
8
maltophilia a free-living microorganism commonly found in soil and water (Ribbeck-
9
Busch et al., 2005; Turrientes et al., 2010). Different strains of this microorganism have
10
been studied for their potential use in, biotechnological processes, including the
11
production of proteases (Dunne et al., 2000) and antifungal compounds (Jakobi et al.,
12
1996), the biodegradation of pollutants such as aromatic hydrocarbons (Lee et al., 2002)
13
and trinitrotoluene (Lee et al., 2009), and the control of plant infections (Ryan et al.,
14
2009). In addition to its biotechnological applications, S. maltophilia is an opportunistic
15
pathogen, and its prevalence in hospitalized patients has increased in recent years
16
(Looney et al., 2009; Brooke, 2012). One worrisome characteristic of this organism is
17
their low susceptibility to the antibiotics currently in clinical use (Quinn, 1998;
18
McGowan, 2006; Sanchez et al., 2009).
19
It is important to remark that S. maltophilia intrinsic resistance was acquired in the
20
field long before the dawn of antibiotic therapy (Baquero et al., 2009; Martinez, 2009;
21
Martinez et al., 2009b). It can be attributed to the low permeability of the cell
22
membranes, the carriage of genes coding for different antibiotic-modifying enzymes
23
(beta lactamases, aminoglycoside-inactivating enzymes) (Avison et al., 2002; Li et al.,
24
2003; Okazaki and Avison, 2007), the possession of efflux pumps such as SmeYZ,
This article is protected by copyright. All rights reserved.
4
Accepted Article 1
SmeIJK and SmeDEF (Alonso and Martinez, 2000; Zhang et al., 2001; Crossman et al.,
2
2008), and the existence of a chromosomally encoded quinolone resistance determinant
3
belonging to the Qnr family known as SmQnr (Sanchez et al., 2008; Shimizu et al.,
4
2008; Sanchez and Martinez, 2010). For some of these elements, as SmeYZ and
5
SmeIJK, function has been inferred by sequence homology when the whole sequence of
6
S. maltophilia was obtained (Crossman et al., 2008). However, for others as SmQnr and
7
SmeDEF a role in intrinsic and acquired resistance to quinolones, as well as other
8
antibiotics as tetracycline, chloramphenicol and erythromycin, has been described
9
(Alonso and Martinez, 2001; Zhang et al., 2001; Sanchez et al., 2004; Gould and
10
Avison, 2006; Sanchez et al., 2008; Sanchez and Martinez, 2010). Together, these
11
elements allow S. maltophilia to avoid the activity of antibiotics, but they might also be
12
important in this bacterium’s evolutionary potential. Just like any other bacterium, S.
13
maltophilia can also attain increased levels of resistance via the acquisition of resistance
14
genes through HGT, or by mutations in chromosomally-encoded determinants. Indeed,
15
the presence of integrons containing antibiotic resistance genes has been reported in
16
different isolates of S. maltophilia (Barbolla et al., 2004; Chang et al., 2004; Toleman et
17
al., 2007), and mutations in the transcriptional regulators of efflux pumps that lead to
18
the latters’ overexpression - and therefore to a subsequent increase in antibiotic
19
resistance - have been described (Alonso and Martinez, 2000, 2001; Sanchez et al.,
20
2002, 2004; Hernandez et al., 2009).
21
Generally speaking, mutation-driven acquired resistance might be due to the
22
increased expression of detoxifying elements such as the aforementioned efflux pumps
23
or antibiotic-inactivating enzymes, or to mutations in genes coding for the antibiotic
24
target. The latter certainly affects resistance to the quinolones. It was suggested that
This article is protected by copyright. All rights reserved.
5
Accepted Article 1
since these are synthetic antibiotics, there should be no quinolone resistance genes; the
2
only cause of such resistance, therefore, should be mutations in the genes coding for
3
their targets (DNA gyrase and topoisomerase IV) and the transporters involved in the
4
entrance into bacterial cells of these antimicrobials (Verbist, 1986; Hirai et al., 1987;
5
Mouton, 1987; Hooper and Wolfson, 1989). However, the finding that MDR efflux
6
pumps are capable of extruding quinolones (Martinez et al., 2009a), plus the description
7
of plasmid-encoded (Gomez-Gomez et al., 1997) quinolone resistance determinants in
8
different bacterial pathogens (Martinez-Martinez et al., 1998), showed this thinking to
9
be wrong (Martinez et al., 1998; Hernandez et al., 2011a). Despite this, the mutation of
10
the topoisomerase genes remains the most important mechanism leading to high-level
11
quinolone resistance in most bacteria (Hooper, 1999). Opposite to this general rule, in S.
12
maltophilia, the analysis of quinolone-resistant clinical isolates has shown no mutations
13
in the genes coding for topoisomerases (Ribera et al., 2002; Valdezate et al., 2002;
14
Valdezate et al., 2005). One explanation for this issue is the presence in the S.
15
maltophilia genome of genes coding for several proteins capable of detoxifying
16
antibiotics, quinolones included (Crossman et al., 2008); they are therefore involved in
17
intrinsic resistance. Although their original functional role in the field is unlikely
18
conferring resistance to quinolones, their overexpression may indeed contribute towards
19
acquired resistance to this family of antimicrobials.
20
The present work studies the contribution of the best-known determinants of intrinsic
21
resistance to quinolones in S. maltophilia (SmeDEF (Zhang et al., 2001) and SmQnr
22
(Sanchez and Martinez, 2010)) to the development of mutation-driven acquired
23
resistance. SmQnr belongs to the Qnr family and is just involved in resistance to
24
quinolones (Sanchez et al., 2008). The Qnr proteins protect bacterial cells from the
This article is protected by copyright. All rights reserved.
6
Accepted Article 1
action of quinolones by binding the targets of these antimicrobials, the bacterial
2
topoisomerases (Tran et al., 2005a, b). SmeDEF is a tripartite complex formed by an
3
efflux pump located on the inner membrane (SmeE), an outer membrane protein
4
(SmeF), and a periplasmic membrane fusion protein (SmeD) (Alonso and Martinez,
5
2000). It is involved in resistance to antibiotics of different structural families,
6
quinolones included (Alonso and Martinez, 1997, 2000). Its expression is regulated by
7
the SmeT repressor (Sanchez et al., 2002; Hernandez et al., 2009); its overexpression is
8
due to mutations of smeT (Sanchez et al., 2002, 2004). To determine the contribution
9
of SmeDEF and SmQnr to the development of quinolone resistance in S. maltophilia,
10
comparisons were made of the mutation frequencies and the types of mutant selected by
11
quinolones in the wild-type S. maltophilia strain D457 and in mutants lacking either
12
SmQnr, SmeDEF or both.
13
Mutation frequency is a fixed value for each bacterial strain. However, in the case of
14
antibiotic resistance, the number of mutants that can be selected at different
15
concentrations of a selective agent can vary if several mutations confer the phenotype
16
(Martinez and Baquero, 2000). In the present article, the mutation frequency observed at
17
a given concentration of antibiotic is termed the 'apparent mutation frequency'. It has
18
been theoretically discussed that the apparent mutation frequencies will depend on the
19
concentration of the selective agent and on the number of genes that can confer
20
resistance upon their mutation (Martinez and Baquero, 2000). The present results
21
support this hypothesis and indicate that the potential to acquire increased resistance
22
depends on the elements involved in intrinsic resistance to antibiotics encoded by the
23
bacterial genome. It is important to notice that, at least for synthetic antibiotics as
24
quinolones, the determinants involved in intrinsic resistance to these drugs did not
This article is protected by copyright. All rights reserved.
7
Accepted Article 1
evolve for this purpose in nature. Rather, in the case of efflux pumps, they play an
2
arrange of different functions including interaction with plant or animal hosts,
3
intercellular communication or extrusion of toxic metabolites among others (Ma et al.,
4
1995; Lee and Shafer, 1999; Llama-Palacios et al., 2002; Barabote et al., 2003;
5
Rosenberg et al., 2003; Maggiorani Valecillos et al., 2006; Piddock, 2006; Martinez et
6
al., 2009a; Olivares et al., 2012). This supports that if we want to understand in depth
7
the processes governing the emergence and spread of antibiotic resistance, we have to
8
take into consideration the role that natural ecosystems have played in such evolution.
9
Results
10
Construction of mutants deficient in smeE
11
The tripartite efflux pump SmeDEF is known to contribute to the intrinsic resistance
12
of S. maltophilia to different antibiotics, including quinolones, fluoroquinolones,
13
tetracycline, chloramphenicol and erythromycin (Zhang et al., 2001). Mutations in
14
smeT, which encodes the transcriptional repressor of smeDEF, lead to SmeDEF
15
overexpression (Sanchez et al., 2002, 2004). As shown in (Alonso and Martinez, 2000),
16
this allows a phenotype of high-level resistance to different antibiotics, including
17
quinolones (Table 1). To study the effect of the different intrinsic resistance
18
mechanisms in acquired resistance, a set of isogenic deletion mutants, all of them
19
derived from the D457 wild-type strain, was generated that included an already
20
characterized mutant lacking Smqnr (Sanchez and Martinez, 2010), a mutant lacking
21
smeE, and a double mutant lacking both smeE and Smqnr. The deletion of smeE
22
removes a key component of the SmeDEF tripartite complex - the efflux pump, thus
23
preventing antibiotics being extruded from the cell. Markerless chromosomal in-frame
24
deletion mutants were constructed in S. maltophilia D457 (wild-type strain) and MBS82
This article is protected by copyright. All rights reserved.
8
Accepted Article 1
(Smqnr defective mutant) by homologous recombination, and the deletion of smeE
2
confirmed by PCR as described in Experimental Procedures. Whole genome sequence
3
(see below) further confirmed the deletions. The deletion of smeE reduced the minimum
4
inhibitory concentrations (MICs) of norfloxacin, ofloxacin, nalidixic acid and
5
ciprofloxacin in the mutant strains between 2 and 8 fold (Table 1), confirming this
6
efflux pump to be involved in the intrinsic resistance of S. maltophilia to quinolones.
7 8
Elimination of determinants involved in intrinsic resistance reduces the apparent
mutation frequencies of quinolone resistance.
9
To determine whether the elements involved in intrinsic resistance modulate the
10
emergence of mutants (acquired resistance), the apparent mutation frequencies for
11
ofloxacin resistance were measured in the wild-type strain S. maltophilia D457, the
12
mutant MBS82 (which lacks Smqnr), the mutant MBS411 (defective in smeE), and the
13
double mutant GGL199 (which lacks both Smqnr and smeE). Resistance can be
14
achieved by mutations or by transient, non-inheritable, phenotypic changes via the
15
induction of an adaptive transient response (Levin and Rozen, 2006). The stability of
16
the mutations conferring quinolone resistance was therefore confirmed. For this, a
17
representative number of mutants (between 9 and 16, typically 12) obtained under each
18
of the conditions shown in Figure 1 were then cultured by two passages in a medium
19
without antibiotics, and susceptibility to quinolones then tested. All the mutants
20
analyzed were stable, indicating the change in susceptibility to quinolones not to be a
21
transient phenotypic change but the result of mutation. To analyze in detail all the
22
potential phenotypes, we examined all the selected mutants independently of whether or
23
not their MICs reached the breakpoint defining resistance from a clinical viewpoint.
24
This definition of resistance, based on changes in the MIC compared to the wild-type
This article is protected by copyright. All rights reserved.
9
Accepted Article 1
population, has recently been used to define epidemiological breakpoints (Kronvall,
2
2010).
3
In agreement with an earlier hypothesis (Martinez and Baquero, 2000), the deletion
4
of the intrinsic quinolone resistance determinants, smeE and Smqnr, reduced the
5
apparent mutation frequency towards ofloxacin resistance at any given concentration,
6
the effect being greater when both determinants were deleted (Figure 1). To address
7
whether this effect on the apparent mutation frequencies was specific for quinolones or
8
a more general effect on the overall mutation frequency of S. maltophilia, the mutation
9
frequencies of each of the strains towards rifampin resistance was measured as
10
described in Experimental Procedures. The values were 1.2x10-8± 3.4x10-9 for the wild-
11
type strain D457, 2.5x10-8± 8.1x10-9 for the MBS82 ∆Smqnr mutant, 5.5x10-8± 1.7x10-8
12
for the MBS411 ∆smeE mutant, and 1.6x10-8± 2.1x10-9 for the GGL199 ∆Smqnr∆smeE
13
double mutant. The observed differences were minor and within the error range of the
14
technique. By contrast, the differences in the apparent mutation rates towards quinolone
15
resistance among these strains where some orders of magnitude higher, in occasions
16
close to 104 fold (Figure 1). This indicates that removing the elements of intrinsic
17
resistance to quinolones does not produce a general, non-specific relevant effect on S.
18
maltophilia mutation rates.
19 20
The type of quinolone-resistant mutants selected differs depending on the selective
pressure exerted by quinolones
21
It was predicted that the type of mutations selected at different concentrations of the
22
antibiotic would be different. At low concentrations, all the potential mutants, whether
23
conferring low-level or high-level resistance, would be selected. However, at high
24
concentrations, only a subset of mutants (those with high-level resistance) would be
This article is protected by copyright. All rights reserved.
10
Accepted Article 1
selected. To test this hypothesis, the susceptibility to different antibiotics of several
2
spontaneous, stable, ofloxacin-resistant mutants isolated in the mutation frequency
3
assay (see above) of S. maltophilia D457, was analyzed. Four different types of
4
phenotypes were observed (Table 1SI, supplemental information): A, increased
5
resistance to tetracycline, erythromycin, chloramphenicol and quinolones (a phenotype
6
compatible with the overexpression of SmeDEF); B, high-level resistance to
7
chloramphenicol and quinolones; C, resistance to quinolones only, with no change in
8
susceptibility to the other tested antibiotics; and D, resistance to erythromycin and
9
quinolones. Figure 2A shows that the distribution of the mutant phenotypes is a function
10
of the concentration of the antibiotic used for selection. It is noteworthy that the
11
percentage of mutants in which only the susceptibility to quinolones changed
12
(phenotype C, white boxes in Figure 2) was low. Most mutants showed changes in the
13
susceptibility to antibiotics belonging to different structural families; a phenotype
14
consistent with efflux pump overproduction.
15 16
The type of quinolone-resistant mutants differs depending on the presence of
determinants involved in intrinsic resistance to quinolones.
17
If different resistant mutants are selected at different concentrations, it is conceivable
18
that the removal of elements involved in intrinsic resistance might also modify the
19
phenotypic profile of the mutants selected. Susceptibility to different antibiotics was
20
therefore tested as above in mutants derived from the strains lacking either smeE or
21
Smqnr and the double mutant lacking both smeE and Smqnr. In agreement with the data
22
obtained for the wild-type strain, the distribution of mutant phenotypes for each strain
23
and at each concentration differed (Figure 2). As found for the wild-type strain, the
24
percentage of mutants showing changes just in their susceptibility to quinolones was
This article is protected by copyright. All rights reserved.
11
Accepted Article 1
low, and this phenotype was not selectable upon inactivation of the SmeDEF efflux
2
pump.
3 4
Overexpression of SmeDEF is an important mechanism of acquired resistance to
quinolones in S. maltophilia.
5
The overexpression of the multidrug efflux pump SmeDEF confers increased
6
resistance to tetracycline, erythromycin, chloramphenicol and quinolones (Alonso and
7
Martinez, 1997, 2000), Table 1. The most prevalent phenotype of the mutants selected
8
for strains D457 and MBS82 was consistent with the overproduction of this efflux pump
9
(Table 1SI, Figure 2). The mutants derived from strains MBS411 and GGL199 did not
10
present this phenotype, in agreement with the lack of smeE in these strains. Previous
11
work has shown that the amount of SmeF is a good marker for measuring the expression
12
of SmeDEF, which correlates well with the level of expression of the other components
13
of the tripartite efflux pump (Alonso and Martinez, 2001). Consequently, in order to
14
confirm that SmeDEF was overproduced in the mutants showing increased resistance to
15
tetracycline, erythromycin, chloramphenicol and quinolones, the level of expression of
16
the porin of the pump, SmeF, was estimated by Western blotting in a set of 14 mutants
17
presenting a phenotype compatible with SmeDEF overexpression. Two mutants in
18
which only the susceptibility to quinolones change and the wild-type strain D457 were
19
used as negative controls of SmDEF overexpression, whereas the SmeDEF
20
overexpressing mutant D457R (Alonso and Martinez, 1997, 2000) was used as positive
21
control of such overexpression. As shown in Figure 3, all the analyzed mutants with a
22
phenotype compatible with SmeDEF overexpression did indeed produce large amounts
23
of this efflux pump, indicating that SmeDEF is an important determinant in the
24
acquisition of quinolone resistance in S. maltophilia.
This article is protected by copyright. All rights reserved.
12
Accepted Article 1
Quinolone resistance in S. maltophilia is not associated with mutations in the
2
quinolone resistance-determining regions of the genes encoding the bacterial
3
topoisomerases, nor with Smqnr overexpression, even in the absence of smeE.
4
It has been reported that clinical quinolone-resistant isolates of S. maltophilia do not
5
harbour mutations in the genes coding for bacterial topoisomerases (Ribera et al., 2002;
6
Valdezate et al., 2002; Valdezate et al., 2005). Since SmeDEF overexpression is a major
7
element for the acquisition of quinolone resistance in this bacterial species (see above),
8
it was deemed possible that the removal of smeE might allow for the selection of
9
quinolone resistant mutants with altered topoisomerases. Nevertheless, all the mutants
10
selected from the strains MBS411 and GGL199 showed reduced susceptibility to
11
different antibiotics as well as quinolones, indicating that the basis of the resistance is
12
not a mutation in the genes coding for the topoisomerases. The observed phenotypes are
13
compatible with the overexpression of efflux pump(s) despite these strains’ lack of
14
smeE.
15
Some resistant mutants derived from D457 and from MBS82, however, were only
16
quinolone-resistant; a phenotype compatible with mutations in the genes coding for the
17
bacterial topoisomerases. The quinolone resistance-determining regions (QRDRs) of
18
gyrA, gyrB, parC and parE, where the mutations conferring quinolone resistance in
19
bacteria are located, were amplified and sequenced in six representative strains showing
20
this phenotype (MBS123, MBS135 and MBS143 derived from D457, and MBS116,
21
MBS161 and MBS162 derived from MBS82). No mutations were identified in any of
22
the mutants analyzed, indicating that the observed phenotype was not due to mutations
23
in the QRDRs of the topoisomerases. It has been described that SmQnr overexpression
24
can confer resistance to quinolones in S. maltophilia (Sanchez and Martinez, 2010).
This article is protected by copyright. All rights reserved.
13
Accepted Article 1
While this mechanism cannot contribute to resistance in the strain MBS82, which lacks
2
Smqnr, it was reasoned that in S. maltophilia D457 the mechanism of resistance in
3
mutants in which only the susceptibility to quinolones changes might involve SmQnr
4
overexpression. The expression of Smqnr was thus analyzed by real time RT-PCR in
5
the mutants MBS123, MBS135 and MBS143 (all derived from S. maltophilia D457),
6
none of which have QRDR mutations. No significant changes in the expression of
7
Smqnr were observed (Figure 4) compared to the original strain S. maltophilia D457 or
8
the mutant MBS130 which overexpresses SmeDEF. This indicates that the quinolone
9
resistance of these mutants is not due to the overexpression of SmQnr.
10 11
The overexpression of the SmeVWX efflux pump contributes to quinolone resistance
in the absence of SmeDEF overexpression
12
To address in greater detail the molecular basis of the quinolone resistance in those
13
strains not overexpressing SmeDEF, the whole genomes of five different, one-step,
14
selected mutants, with different phenotypes were sequenced using Illumina technology
15
and following a single read 1x75 protocol. Of the five sequenced strains, two mutants
16
derived from D457, one from MBS82, one from MBS411, and one from GGL199. A
17
total of 4.3 ± 15% million pass-filter sequences were obtained for each of the different
18
strains, which approximately corresponds to 325 Mb of sequence and a genome
19
coverage of 60x. In all cases, 100% of each genome was sequenced without any gap,
20
except for the deleted genes smeE and Smqnr in the corresponding mutants.
21
The genome sequences of these mutants were compared to their respective parental
22
strains to detect mutations that might be responsible for the observed phenotypes. Each
23
mutant strain showed just one mutation in its genome. Insertions were not detected and
24
the only deletions found correspond to those previously generated at smeE and Smqnr.
This article is protected by copyright. All rights reserved.
14
Accepted Article 1
In all cases, these mutations were in smeRv (Table 4), which codes for the repressor of
2
the MDR efflux system SmeVWX (Chen et al., 2011). The presence of these mutations
3
in each of the mutants was confirmed by amplifying and sequencing the corresponding
4
DNA region. To ascertain whether the mutations caused the overexpression of
5
SmeVWX, the expression of smeW was analyzed by real time RT-PCR. Table 4 shows
6
that, in agreement with the finding of mutations in the gene coding for the SmeVWX
7
regulator, all the mutants overexpressed this efflux pump (although the level of
8
expression varied). Given that the five sequenced genomes contained only one mutation
9
each, that all these mutations laid in the same transcriptional repressor SmeRv and that
10
in all of them the expression of the SmeRv-regulated efflux pump SmeVWX is de-
11
repressed, our data strongly support the idea that quinolone resistance was acquired in
12
these mutants via the overexpression of SmeVWX.
13
Discussion
14
This work explores the idea that the development of acquired resistance is modulated
15
by the variability of elements conferring intrinsic resistance encoded in the genomes of
16
bacterial pathogens. In other words, the intrinsic resistome – understood to be the
17
ensemble of genes that contribute to the 'natural' or intrinsic resistance of bacterial
18
pathogens (Fajardo et al., 2008), which has evolved, long before human use of
19
antibiotics, in natural ecosystems - defines mutation-driven acquired resistance to
20
antibiotics. This was examined by studying resistance to quinolones in S. maltophilia.
21
Quinolones were chosen since these drugs are synthetic, and as such could not have
22
contributed to the natural evolution of antibiotic resistance. S. maltophilia was chosen
23
since it is an opportunistic pathogen with an environmental origin and the quinolone-
24
resistant mutants isolated in clinical settings do not show mutations in the genes coding
This article is protected by copyright. All rights reserved.
15
Accepted Article 1
for bacterial topoisomerases (Ribera et al., 2002; Valdezate et al., 2002; Valdezate et al.,
2
2005) – unlike that seen in all other studied bacterial species. This feature suggest that
3
S. maltophilia may harbour elements, which have evolved in the field and allow this
4
bacterial species to counteract the activity of quinolones. In this regard, an interesting
5
feature of S. maltophilia is the presence in its genome of a gene coding for the
6
quinolone resistance protein SmQnr (Shimizu et al., 2008; Sanchez and Martinez, 2010)
7
as well as operons coding for several multidrug efflux pumps. The latter might also
8
contribute towards resistance to these drugs when overexpressed (Crossman et al.,
9
2008).
10
Using a set of S. maltophilia mutants lacking the best-known elements involved in
11
intrinsic resistance to quinolones, the capacity to acquire resistance (apparent mutation
12
frequency) was found to be a function of the intrinsic resistome and the concentration of
13
the challenging antibiotic. Since Qnr proteins bind bacterial topoisomerases (Tran et al.,
14
2005b, a), Smqnr deletion could potentially alter the overall mutation frequency of S.
15
maltophilia. However, the fact that the mutation frequencies did not change when using
16
rifampin as the selective agent goes against this hypothesis, and indicates that the
17
apparent mutation frequency for a given antibiotic depends on the mechanisms of
18
intrinsic resistance against it. Inhibiting elements involved in intrinsic resistance, such
19
as SmeDEF or SmQnr, might thus preclude the emergence of quinolone-resistant
20
mutants in S. maltophilia (Garcia-Leon et al., 2012; Martinez, 2012). Further, the
21
deletion of these intrinsic resistance elements not only altered the number of mutants
22
selectable at a given concentration of antibiotic, but changed the phenotypic profile of
23
the mutants selected. This might help define novel mechanisms of resistance that in the
24
presence of those elements would not be detectable. Notably, the deep genome
This article is protected by copyright. All rights reserved.
16
Accepted Article 1
sequencing of mutants with slightly different phenotypes showed all to have mutations
2
at the regulator of SmeVWX expression, SmeRv, which led to the overexpression of
3
SmeVWX. Previous work has shown SmeVWX not to contribute to intrinsic resistance
4
to antibiotics in S. maltophilia, most likely because its level of expression is very low in
5
the wild-type strain (Chen et al., 2011). Deep sequencing analysis of the S. maltophilia
6
transcriptome shows that this is indeed the case (not shown). However, other authors
7
reported a chloramphenicol-selected mutant that overexpressed SmeVWX to acquire
8
resistance to different antibiotics (Chen et al., 2011). Notably, the SmeVWX-
9
overexpressing mutant analyzed in the latter work had no mutations in smeRv,
10
indicating that other elements besides this local regulator participate in the regulation of
11
the expression of this efflux pump. SmeRv is a LysR-type regulator, and this family
12
contains both activators and repressors that respond to external signals. Among the
13
mutants analyzed in the present work, three showed mutations at Gly266 and one at
14
Glu256, and both these positions are inside the predicted effector-binding motif of the
15
protein. Binding of the effector alters the structure of the regulator and consequently its
16
ability to bind to its operator DNA. Mutations in the effector-binding region might thus
17
also alter the DNA-binding capability of the regulator (Hernandez et al., 2011b). The
18
strength of the structural modification, and hence the level of overexpression achieved,
19
depending on the mutation involved. Indeed, the present results show that different
20
mutations in smeRv can retrieve smeVWX repression to different degrees; this is
21
reflected in the level of expression of this efflux pump and therefore in the degree of
22
resistance achieved.
23
Two factors can thus explain the different phenotypes of the mutants. The first is the
24
level of expression achieved by SmeVWX. The MBS287 mutant showed resistance to
This article is protected by copyright. All rights reserved.
17
Accepted Article 1
quinolones and chloramphenicol and high levels of expression of smeW, while
2
MBS123, which expressed less smeW, showed only quinolone resistance. These
3
different phenotypes probably occur because SmeVWX extrudes chloramphenicol less
4
efficiently than quinolones. Consequently the differences in susceptibility to this
5
antibiotic cannot be detected at the level of expression shown by MBS123. In contrast,
6
they are detectable at the level of SmeVWX expression shown by MBS287. Secondly,
7
the genomic context of the mutations is also important in terms of the phenotype
8
observed. A phenotype of increased resistance to erythromycin is only detectable in the
9
absence of smeE since, in S. maltophilia, SmeDEF is a major contributor to intrinsic
10
resistance to this antibiotic. Indeed, the removal of smeE reduces the erythromycin MIC
11
from 128 µg/ml to 32 µg/ml (Table 1). This agrees with previously published work
12
showing that, on occasion, the full profile of an efflux pump can only be determined
13
when other efflux pumps that extrude the same antibiotics more efficiently are removed
14
(Chuanchuen et al., 2002; Mima et al., 2005; Mima et al., 2009).
15
The present results indicate that the most prevalent cause of acquired quinolone
16
resistance in S. maltophilia is the overproduction of multidrug efflux pumps, the most
17
important of which is SmeDEF. However, even when this pump is removed, the
18
phenotype of the mutants obtained is compatible with the overexpression of efflux
19
pump(s) such as SmeVWX, but not with mutations in the genes coding for
20
topoisomerases. Indeed, in agreement with data obtained using clinical quinolone
21
resistant isolates (Ribera et al., 2002; Valdezate et al., 2002; Valdezate et al., 2005), no
22
mutant with mutations in the genes coding for topoisomerases was detected. This might
23
be the consequence of unaffordable fitness costs (Martinez et al., 2011) or of the
24
presence in the genome of a large number of elements capable of efficiently conferring
This article is protected by copyright. All rights reserved.
18
Accepted Article 1
resistance to these drugs (Crossman et al., 2008). Although no evidence is available to
2
support either statement, the fact that mutations in topoisomerases are easily selectable
3
in any other tested microorganism favours the second hypothesis. Our work also has
4
some practical implications for predicting the emergence of resistance to quinolones in
5
S. maltophilia and the consequences of such emergence. Our results support that the
6
probability that S. maltophilia quinolone resistant mutants presenting mutations at the
7
genes encoding bacterial topoisomerases appear in the future is likely low, unless
8
efficient inhibitors of multidrug efflux pumps are developed. Main causes of resistance
9
will be overproduction of SmeDEF, an issue already addressed in some articles (Alonso
10
and Martinez, 2001; Chang et al., 2004; Sanchez et al., 2004; Sanchez et al., 2005;
11
Gould and Avison, 2006) and SmeVWX overexpression. In this respect is worth
12
mentioning that we have found that a clinical quinolone resistant S. maltophilia isolate
13
overexpresses SmeVWX and presents the same Gly266Ser amino acid change at
14
SmeRv that we found in the in vitro obtained mutants MBS300 and GGL231 (MBS et
15
al. to be published). Finally, since most quinolone resistant mutants overproduce efflux
16
pumps that have other antibiotic substrates, this means that treating S. maltophilia
17
infections with quinolones may select resistant mutants, not just to quinolones, but to
18
several other antibiotics also, which can compromise the therapy.
19
The present findings support the idea that the capacity to acquire resistance by means
20
of mutations is strongly dependent on the mechanisms of intrinsic resistance that
21
bacteria have acquired over their evolution in non-clinical, natural ecosystems. In the
22
present work we studied the acquisition of resistance to quinolones, a family of
23
synthetic drugs, which has had no role in the selection of the elements of intrinsic
24
resistance present in the bacterial genome. In the case of S. maltophilia, the most
This article is protected by copyright. All rights reserved.
19
Accepted Article 1
important elements dealing with resistance are efflux pumps. These elements are
2
encoded in the genomes of all organisms and, in addition of being involved in
3
resistance, they have several other roles in natural ecosystems (Piddock, 2006; Martinez
4
et al., 2009a), including intercellular signalling, detoxification from metabolic toxic
5
intermediates, resistance to solvents and interaction with plant hosts among others
6
(Kohler et al., 2001; Rojas et al., 2001; Aendekerk et al., 2005; Maggiorani Valecillos et
7
al., 2006; Ravirala et al., 2007; Olivares et al., 2012). In this regard, it is worth
8
mentioning that preliminar evidences suggest that the primary role of SmeDEF might
9
actually be in the interaction of S. maltophilia with the plant host (GGL to be
10
published).
11
From an ecological and evolutionary standpoint, our results indicate that the capacity
12
to acquire antibiotic resistance might depend on the capabilities of bacteria to confront
13
other types of insult via systems that evolved before the use of antibiotics by humans
14
(Martinez, 2008). This non-antibiotic selection of resistance might still be at work. A
15
better comprehension of the role that environmental (non-clinical) ecosystems play in
16
the evolution of resistance is needed if we are to understand the emergence of resistance
17
in bacterial pathogens.
18
Experimental procedures
19
Bacterial strains, plasmids and growth conditions
20
Table 2 shows the bacterial strains and plasmids used in the present work. Table 1SI shows
21
the spontaneous mutants derived from these strains. All strains were grown in LB medium
22
(Atlas, 1993) at 37ºC, unless indicated otherwise.
23
Construction of the smeE deletion mutants
This article is protected by copyright. All rights reserved.
20
Accepted Article 1
To generate smeE deletion mutants in D457 and MBS82, two DNA fragments homologous
2
to the 5’ end (X fragment) and the 3’ end (Y fragment) of the smeE gene were obtained from
3
pAS2 using the Expand Long Template PCR System (Roche) and 0.5 µM of the primers X1-
4
X2 and Y1-Y2 respectively. The fragments were designed not to break the reading frame. The
5
reactions consisted of one denaturation step at 94ºC for 2 min and 15 amplification cycles of
6
94ºC for 10 s, 50ºC for 30 s, and 68ºC for 1 min, followed by a further 25 amplification cycles
7
of 94ºC for 10 s, 55ºC for 30 s, and 68ºC for 1 min, plus a final extension step at 68ºC for 7
8
min. The PCR products were cloned into pGEMT-Easy, generating pGG1 (X fragment) and
9
pGG2 (Y fragment). To check the fragments, they were sequenced with the universal primers
10
F24 and R24 by Secugen (www.secugen.es). pGG1 and pGG2 were digested with EcoRI-
11
XbaI and XbaI-HindIII respectively, and the X and Y fragments cloned into the same
12
restriction sites in the suicide vector pEX18Gm, yielding pGG8, which was introduced into
13
Escherichia coli TG1, then into E. coli CC118lpir, and finally into S. maltophilia D457 and
14
MBS82 by tripartite mating (de Lorenzo and Timmis, 1994). In both cases, the
15
transconjugants containing pGG8 were selected on LB agar containing 80 µg/ml gentamycin
16
and 20 µg/ml imipenem after 48 h of incubation. To check the insertion of the plasmid,
17
selected transconjugants were streaked onto LB agar containing 80 µg/ml gentamycin and 20
18
µg/ml imipenem, and after 24 h of incubation PCR with primers pair X3/Y3 was performed
19
with gentamycin-resistant colonies (colonies with the insertion gave two fragments of 2269
20
and 226 bp length). Colonies containing the insert were streaked onto 10% sucrose-LB agar,
21
and sucrose-resistant colonies arising after 48 h of incubation at room temperature (RT) were
22
screened for the presence of the smeE deletion by PCR. For this, two sets of primers were
23
used. The pair Z3/Z4 locates to the region to be deleted. Amplification with these primers
24
rendered a 643 bp product in the strains D457 and MBS82, which contain smeE, whereas
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21
Accepted Article 1
amplification was negative for the strains in which smeE had been deleted, i.e., MBS411
2
(derived from D457) and GGL199 (derived from MBS82). Further confirmation of the
3
deletion was obtained using the primers pair X3/Y3, which locates to outside the region to be
4
deleted. The primers amplified a region of 2269 bp in the strains D457 and MBS82, and of
5
226 bp in MBS411 and GGL199, thus confirming smeE deletion
6
Determination of mutation frequencies
7
Each tube containing LB was inoculated with one isolated colony and incubated overnight.
8
100 µl of different dilutions (100 to 10-7) of the overnight cultures was seeded onto Mueller
9
Hinton agar plates (MHA)(Pronadisa) without antibiotic, or containing either ofloxacin (2, 4,
10
8 or 16 µg/ml) or rifampin (250 µg/ml). Colonies were counted and mutation frequencies
11
estimated as the ratio between colonies growing in the presence and absence of antibiotics.
12
Antibiotic susceptibility assays
13
The MICs of the different antibiotics were determined on MHA plates using the two-fold
14
agar dilution method. The antibiotics used were tetracycline, erythromycin, chloramphenicol,
15
norfloxacin, ofloxacin, nalidixic acid and ciprofloxacin. The results were recorded after 24 h
16
of incubation at 37ºC.
17
Detection of SmeDEF by Western blotting
18
To determine the amount of SmeDEF in the different mutants, the expression of SmeF was
19
determined by Western blotting as previously described (Alonso and Martinez, 2000). Whole
20
cell extracts from overnight cultures of the tested strains were electrophoresed in 12% SDS–
21
PAGE gels (Sambrook and Russell, 2001). Proteins were transferred to Immobilon-P
22
(Millipore) and the presence of SmeF was determined using a polyclonal anti-SmeF antibody
23
(Alonso and Martinez, 2000) at a final dilution of 1:5000. A secondary goat anti-rabbit HRP
This article is protected by copyright. All rights reserved.
22
Accepted Article 1
conjugate (Bio-Rad) was used at a final dilution of 1:40000. Bands were detected by
2
chemiluminiscence with Immobilon™ Western (Millipore). The A600 of the overnight cultures
3
was measured and in all cases an extract of around 2-3x108 cells from each strain was loaded
4
in each well.
5
Sequencing of the topoisomerase gene QRDRs
6
The QRDRs of gyrA, gyrB, parC and parE were amplified by PCR using the Expand Long
7
Template PCR System (Roche). Purified PCR products were sequenced by Macrogen
8
(www.macrogen.com). The primers used for PCR and sequencing were those described by
9
Valdezate (Valdezate et al., 2002).
10
Real time PCR
11
Total bacterial RNA was isolated from 25 ml mid-log-phase cultures (A600 = 0.6-0.7) using
12
the Qiagen RNeasy Mini Kit (Qiagen, Inc.). To eliminate genomic DNA, samples were
13
treated with RNase-free TURBO DNase (Ambion, Inc.), and RNA purified using the Qiagen
14
RNeasy Mini Kit’s RNA cleanup protocol. This RNA was then used as a template for reverse
15
transcription using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
16
Real-time PCR reactions were performed using Power SYBR Green PCR Master Mix and a
17
7300 Real-Time PCR System (Applied Biosystems). Primer pairs RTqnr1/2 (Sanchez and
18
Martinez, 2010) and SmeWQ5/6 were used to analyze the expression of the Smqnr and smeW
19
genes respectively. The expression of the ftsZ gene (primers FtsZ1/2), a house keeping gene
20
used as reference for quantifying RNA in different species (Delogu et al., 2006; Schwan et al.,
21
2006; Duquenne et al., 2010), was used to normalize the data. The relative amount of mRNA
22
for Smqnr and smeW was calculated following the 2–∆∆Ct method (Livak and Schmittgen,
This article is protected by copyright. All rights reserved.
23
Accepted Article 1
2001). The absence of genomic DNA was verified by real-time PCR using total RNA without
2
reverse transcription. The results are the means of three independent experiments.
3
Whole genome sequencing and determination of mutations
4
DNA from the different samples was analyzed for integrity in agarose gels and quantified
5
by PicoGreen®; a total amount of 3 µg was used for library preparation.
6
DNA was mechanically fragmented and libraries prepared using the TruSeq DNA Sample
7
Preparation v.2 Kit (Illumina). Briefly, the DNA was made blunt ended, phosphorylated, and
8
an adenine added in the 5´-end of the fragments so that Illumina adapters could be ligated.
9
Libraries including adapters were gel-purified and PCR-amplified using common primers so
10
that a sufficiently large library could be subjected to sequencing. Final libraries were titrated
11
by quantitative PCR using external controls as a reference, and showed appropriate
12
concentrations ranging from 95 nM to 434 nM. The libraries were then adjusted to a final
13
concentration of 10 pM, denatured, seeded on a flowcell (Illumina) using a Cluster Station,
14
and sequenced at the Unidad de Genómica Antonia Martín Gallardo, Parque Científico de
15
Madrid, Spain, following a single read 1x75 protocol and using an Illumina GAIIx apparatus.
16
Quality filtering was automatically performed according to Illumina specifications and
17
individual reads were demultiplexed using the CASAVA pipeline. The percentage of pass-
18
filter reads was 89-95%; the mean quality was >Q33. Finally, fastq files were used for
19
mapping. The mutations were identified by the Computational Genomics Service of the CNB.
20
Single-end 75 nt reads were aligned against the S. maltophilia D457 (Lira et al., 2012)
21
genome (HE798556) with the Burrows-Wheeler Alignment tool (Li and Durbin, 2009) using
22
the
23
samtools/mpileup/bcftools/vcfutils (Li et al., 2009). Final SNP candidates were selected by
default
settings.
Single
nucleotide
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polymorphisms
were
detected
using
24
Accepted Article 1
removing those with a read depth of >100 (varFilter -D100) and those in which the SNP was
2
present in
