Journal of Antimicrobial Chemotherapy Advance Access published April 28, 2015
J Antimicrob Chemother doi:10.1093/jac/dkv105
A novel alanine to serine substitution mutation in SoxS induces overexpression of efflux pumps and contributes to multidrug resistance in clinical Escherichia coli isolates Sherine A. Aly1*, Dawn M. Boothe2 and Sang-Jin Suh3 2
1 Department of Medical Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut 71516, Egypt; Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA; 3 Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA
Received 8 February 2015; returned 2 March 2015; revised 25 March 2015; accepted 27 March 2015 Objectives: The purpose of this study was to describe a putative role for a novel soxS mutation in contributing to multiple-antibiotic resistance in canine fluoroquinolone-associated MDR (FQ-MDR) Escherichia coli. This soxS mutation was discovered in canine faecal E. coli isolates during a study investigating the effect of oral fluoroquinolone administration on faecal E. coli in healthy dogs. Methods: We determined via quantitative real-time RT–PCR that both soxS and acrB were overexpressed in the clinical soxS Ala-12Ser (soxSA12S) mutants and this may account for their FQ-MDR phenotype. We validated the FQ-MDR phenotype of the clinical isolates by reconstructing the WT and the soxSA12S mutation in the E. coli soxS null mutant JW4023 (soxS::kn) via allelic exchange. Results: The JW4023 soxSA12S derivative showed an increase in MICs of ciprofloxacin, enrofloxacin and chloramphenicol compared with the JW4023 derivative in which the WT soxS had been restored. The soxS and acrB genes were overexpressed in the JW4023 soxSA12S mutant compared with JW4023 with WT soxS. A similar overexpression of efflux pump genes and an increase in antibiotic resistance were observed upon stimulation with paraquat to resemble the phenotype of the clinical soxSA12S isolates. Conclusions: Our data suggest that the soxSA12S substitution mutation is selected in clinical isolates when dogs are exposed to a fluoroquinolone and that this mutation contributes to the FQ-MDR phenotype of E. coli isolates. Keywords: fluoroquinolones, acrB, soxS, soxR
Introduction Fluoroquinolone (FQ) resistance has been reported among different members of the family Enterobacteriaceae including Escherichia coli. 1,2 High-level FQ resistance is also associated with multidrug resistance.3,4 Recently, multidrug resistance has been linked to FQ resistance through mutations within the regulatory loci that control the expression of the AcrAB efflux pump.3,5 AcrAB-TolC is one of the most common efflux pumps associated with antimicrobial resistance in E. coli and has a wide substrate specificity to render the organism simultaneously resistant to many antibiotics.6 The expression of the acrAB operon is regulated locally by AcrR and AcrS,7 while globally it is influenced by several transcriptional activators belonging to the AraC/XylS family, especially MarA and SoxS.8 The SoxS protein is a transcription factor that activates the expression of more than 100 genes in the chromosomally
encoded SoxRS regulon.9 In E. coli, SoxR is constitutively expressed and binds to its sole DNA target, a site in the promoter of the soxS gene, without activating its transcription.8 When exposed to oxidative stress, the SoxR protein activates the transcription of soxS,10 which in turn results in an overexpression of the target genes including acrAB.6,7 In both laboratory and clinical strains of E. coli, activation of the soxRS regulon has been shown to contribute to the increased resistance to quinolones and chloramphenicol.11 However, few data exist regarding mutations in soxS and their importance in the acquisition of FQ resistance. We previously demonstrated that the administration of enrofloxacin to dogs in low therapeutic doses was associated with the emergence of FQ-associated MDR (FQ-MDR) faecal E. coli isolates that contained a novel soxS mutation.3 The same soxS mutation has been encountered in clinical FQ-MDR E. coli isolates from the urine of dogs with a suspected urinary tract infection. The purpose of
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*Corresponding author. Tel: +00201097018789; E-mail: [email protected]
Aly et al.
this study was to describe a putative new role for a novel soxS mutation in the contribution to multiple-antibiotic resistance in canine FQ-MDR E. coli.
Materials and methods Bacterial isolates (Table 1) Experimental E. coli isolates
Clinical E. coli isolates FQ-MDR clinical E. coli isolates containing the soxSA12S mutation (n ¼ 3) were drawn from a larger study investigating the mechanisms of FQ-multidrug resistance in companion animals. The three E. coli isolates harboured two mutations in gyrA, a single mutation in gyrB and two mutations in parC and belonged to the B1 phylotype. 6
Sequencing of soxS, soxR and marR from E. coli isolates E. coli isolates were tested for mutations in genes encoding regulators of the AcrAB efflux pump (soxR, soxS and marR) by PCR and DNA sequencing. The primers that were used are listed in Table S1 (available as Supplementary data at JAC Online). Table 1. Bacterial strains and plasmids Strain or plasmid
Properties
Source or reference
Strain CG4468 S61 S71 S81 I78 N81 L94 JW4023 DB0001 DB0002 DB0003 SS0001 SS0002
E. coli K-12 soxS + E. coli clinical isolate E. coli clinical isolate E. coli clinical isolate E. coli clinical isolate E. coli clinical isolate E. coli clinical isolate E. coli K-12 W3110 DsoxS DlacZ pCR2.1 in JW4023 pSAWT in JW4023 pSAMT in JW4023 E. coli WT soxS in JW4023 E. coli mutant-type soxS in JW4023
12 5 5 5 6 6 6 13 this study this study this study this study this study
Plasmid pCR2.1 pGP704 pSAWT pSAMT pSA2000 pSA2001 pSA2002
vector (Apr, Knr) vector (Apr) WT soxS in pCR2.1 soxSA12S mutant in pCR2.1 sacB in pGP704 E. coli WT soxS in pSA2000 E. coli mutant soxS in pSA2000
Invitrogen this study this study this study this study this study this study
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FQ-MDR E. coli isolates were examined by RT – qPCR for the mRNA expression of genes encoding for efflux pump AcrB and its regulators (SoxS and MarA). Overnight bacterial cultures were diluted 1/100 in fresh LB broth and grown with shaking (250 rpm) for 90 min at 378C. At this point, the cultures were divided and the appropriate samples were treated with 250 mM paraquat (PQ), a redox-cycling agent and inducer of the soxRS regulon. PQ-treated cells were further incubated at 378C for another 30 min. RNA was extracted as previously described.6 Reverse transcription of RNA into cDNA was carried out using the iScript Reverse Transcription kit (Bio-Rad, USA). The quantification of cDNA templates by RT – qPCR was performed in a Roche LightCycler 480 using a Roche SYBR Green RT – PCR kit (Roche, USA). The expression of each gene was normalized to that of a ribosomal housekeeping gene (gapA). The relative expression of each target gene was calibrated against the corresponding expression by E. coli ATCC 25922 (expression ¼ 1), which served as the control.
Cloning of mutant soxS from E. coli isolates A 870 bp fragment corresponding to the soxS region (the soxS gene and its promoter) of the WT soxS and soxSA12S was amplified by PCR from the genomic DNA of the E. coli isolates (from both WT E. coli and resistant E. coli). The amplified fragments were cloned into vector pCR2.1 (Apr, Knr) using the pCR2.1 TA cloning kit (Invitrogen, Carlsbad, USA) to generate two plasmids, pSAWT (WT soxS) and pSAMT (soxSA12S).The clones were confirmed by restriction digestion analysis with EcoRI and BamHI and DNA sequencing.
Complementation of a DsoxS strain with cloned soxS alleles for increased antibiotic resistance The pSAWT and pSAMT plasmids were introduced into E. coli K-12: JW4023 (soxS::kn, DlacZ), producing strains DB0002 and DB0003, respectively, and their contribution to the antibiotic resistance phenotype of the recipient strain was assessed by Etest. As a control, the E. coli carrying the vector plasmid alone was also subjected to the Etest. The antibiotics tested were enrofloxacin, ciprofloxacin, doxycycline and chloramphenicol.
Reconstruction of the soxSA12S mutation in the E. coli JW4023 strain (soxS::kn, DlacZ) The soxSA12S mutant allele was inserted into the chromosome via allelic exchange with the soxS::kn insertion allele using the negative selection marker sacB. Plasmid pGP704, an R6K derivative that can be maintained only in E. coli strains that contain the pir gene, was regenerated by removing the Tn5 tnp gene (using the SalI restriction enzyme) from the suicide vector pUT.14 The sacB gene was amplified using PCR from pEX100T15 and cloned into pGP704 to generate the pSA2000 vector, an E. coli suicide plasmid that carries a negative selectable marker. The two soxS (the WT and the soxSA12S mutant allele) were cloned into this pSA2000 vector to generate two plasmids, pSA2001 and pSA2002, respectively, that were used for the allelic exchange. Plasmids pSA2001 and pSA2002 were introduced into the E. coli JW4023 insertion mutant and those in which the plasmid integrated into the genome via homologous recombination (the first cross-over) were selected as Knr Apr resistant colonies. The resulting ampicillin-resistant cells were then grown in the presence of 5% sucrose to force homologous recombination (the second cross-over), which resulted in the loss of the integrated plasmid sequence along with the preexisting soxS::kn sequence, while leaving behind the soxS (WT or soxSA12S) allele in its place. The mutants that had undergone the proper
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Three faecal FQ-MDR E. coli isolates harbouring the soxSA12S mutation were detected during an investigation of the effect of oral FQ administration on canine faecal E. coli. The three isolates contained two mutations in gyrA and two mutations in parC and belonged to three different PFGE pulsotypes.5 Phylogenetic analysis showed that two E. coli isolates belonged to the B1 phylotype (S61 and S71) and one E. coli isolate belonged to the D phylotype (S81).
RNA extraction and relative quantification of gene expression by quantitative real-time RT–PCR (RT– qPCR)
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soxS mutation contributes to multidrug resistance
homologous recombination were identified as sucroser Kns Aps colonies. The presence of the soxSA12S allele was verified via PCR amplification and DNA sequencing. The resulting WT and the soxSA12S mutant (SS0001 and SS0002, respectively) were characterized for antibiotic resistance by Etest and for expression of the acrB gene by RT-qPCR.
Results Mutations in the FQ-MDR E. coli isolates
Expression of acrB, soxS and marA in FQ-MDR E. coli isolates To assess the role of the AcrB pump in contributing to the FQ-MDR phenotype of these isolates, we measured the expression of the
Enrofloxacin
Because the overexpression of WT soxS can result in enhanced resistance to antibiotics, it was imperative to compare the WT and the mutant soxSA12S allele cloned into the same multicopy plasmid to assess the effect of the mutation. Based on the MIC results, the strain expressing the mutant SoxS protein (DB0003) exhibited at least 2-fold higher basal resistance to ciprofloxacin, enrofloxacin, chloramphenicol and doxycycline compared with the strain expressing the WT SoxS (DB0002). PQ treatment further increased the basal antimicrobial resistance of JW4023 derivatives harbouring either pSAWT or pSAMT, but the strain expressing the mutant SoxS still had higher resistance than the strain expressing the WT SoxS for each antibiotic tested (Figure 1).
(b)
Ciprofloxacin 0.256 0.128 0.064 0.032 0.016 0.008 0.004
JW JW 402 40 3 ( 23 Dso + (D xS) DB so 00 xS DB 0 ) 00 1 ( 01 pC + R2 (p .1 CR ) DB 2. 0 1) DB 00 00 2 ( 02 pS + AW (p T SA ) DB W 0 T) DB 00 3 00 ( 03 pS + AM (p T) SA M T)
MIC (mg/L)
0.256 0.128 0.064 0.032 0.016 0.008
JW JW 402 40 3 ( 23 Dso + (D xS) so DB xS 0 ) DB 00 00 1 (p 01 CR + (p 2.1) CR DB 2. 0 1) DB 002 00 (p 02 SA + W (p T SA ) DB W 0 T) DB 00 00 3 ( 03 pS + AM (p T SA ) M T)
MIC (mg/L)
(a)
Antibiotic resistance conferred by cloned mutant soxSA12S gene
E. coli isolate (c)
E. coli isolate (d)
Chloramphenicol
Doxycycline 16
MIC (mg/L)
MIC (mg/L)
16 8 4 2
8 4 2 1
E. coli isolate
23 (Ds + ox (D S) DB so xS 00 DB 0 ) 00 1 ( 01 pC + R2 (p .1 CR ) DB 2. 0 1) DB 00 00 2 ( 02 pS + AW (p T SA ) DB W 00 T) DB 0 00 3 ( 03 pS + AM (p T SA ) M T)
40
23 JW
40 JW
JW JW 402 40 3 ( 23 Dso + (D xS) so DB xS 0 ) DB 00 00 1 (p 01 CR + (p 2.1) CR DB 2. 0 1) DB 00 00 2 ( p 02 S + AW (p T SA ) DB W 00 T) DB 0 00 3 ( 03 pS + AM (p T SA ) M T)
1
E. coli isolate
Figure 1. Antibiotic resistance conferred by mutant soxS genes. Plasmids pCR2.1, pSAWT and pSAMT encoding no soxS, WT soxS and soxSA12S, respectively, were introduced into strain JW4023 (DsoxS) and their contribution to the antibiotic resistance phenotype of the recipient strain was assayed by the broth microdilution method in the presence or absence of 250 mM PQ. (a) Enrofloxacin, 0.008 – 0.512 mg/L. (b) Ciprofloxacin, 0.004 – 0.512 mg/L. (c) Chloramphenicol, 1 – 16 mg/L. (d) Doxycycline, 1– 16 mg/L. + indicates samples containing 250 mM PQ.
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We sequenced soxR, soxS, marR and acrR genes from our E. coli MDR isolates in addition to the entire soxS region to address the possibility that there were other mutations in the soxS gene or its promoter. In all the E. coli isolates that were tested, only a single missense mutation at codon 12 with a GT transversion in codon GCA that changed alanine to serine (Ala-12Ser) was found. This mutation lies outside the helix-turn-helix motif of the SoxS DNA-binding domain. No mutations were found elsewhere in the soxS gene, soxS promoter or other regulators.
genes coding for the pump and its regulators. We determined that basal soxS and acrB were overexpressed (≥2-fold increased expression) in all of the tested E. coli isolates compared with the WT E. coli (CG4468). Upon stimulation with PQ, all the isolates retained the ability to overexpress both soxS and acrB mRNA (Figures S1 and S2). In contrast to soxS expression, all of the isolates exhibited low marA expression (0.653 – 0.875) (data not shown). This suggests that overexpression of the AcrB efflux pump in those mutants was not due to MarA.
Aly et al.
Enrofloxacin
Ciprofloxacin
(b)
0.256
0.128
0.128
0.064
MIC (mg/L)
0.064 0.032 0.016
0.032 0.016 0.008
E. coli isolate
E. coli isolate Chloramphenicol
MIC (mg/L)
8 4 2
4 2 1 2S
JW 23 40 (D 23 sox S + (D ) s ox SS S) 00 01 SS 00 (W 01 T-s o + (W xS) Tso SS xS 00 ) 02 SS ( s 00 ox 02 S A1 + 2S (s ) ox S A1
JW
40
)
2S
JW 23 40 (Ds ox 23 S + (D ) so xS SS ) 00 01 SS ( W 00 01 T-s o + (W xS T- ) so xS SS ) 00 0 2 SS 00 (so x 02 + S A12 S (s ) ox S A1
40
)
MIC (mg/L)
8
1
JW
Doxycycline
(d)
16
E. coli isolate
E. coli isolate
Figure 2. Antibiotic resistance conferred by recombinant mutant soxS genes. E. coli strains JW4023 (DsoxS), SS0001 (WT soxS in JW4023) and SS0002 (soxSA12S in JW4023) were tested for their antibiotic resistance phenotype by the broth microdilution method in the presence or absence of 250 mM PQ. (a) Enrofloxacin, 0.008 – 0.512 mg/L. (b) Ciprofloxacin, 0.004 – 0.512 mg/L. (c) Chloramphenicol, 1 – 16 mg/L. (d) Doxycycline, 1 – 16 mg/L. + indicates samples containing 250 mM PQ.
Antimicrobial resistance conferred by recombinant mutant soxS allele Fold expression
4 3 2 1
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)
2S
00 1 (W SS T00 so 01 xS + ) (W Tso xS ) SS 00 02 (s SS ox 00 S A1 02 2S + ) (s ox S A1
SS 0
JW
40
23
+
(D s
ox
S) (D so x 23 40
E. coli isolate
Activity of the cloned mutant soxS gene We determined whether the SoxSA12S mutant protein affected the expression of the target genes. Interestingly, the soxSA12S mutant (SS0002) exhibited an overexpression of acrB without any need for a prior activation of SoxR. Compared with the E. coli strain expressing the WT SoxS (SS0001), the strain expressing the mutant SoxS (SS0002) overexpressed the acrB gene both under nonstressful experimental conditions and after PQ stimulation (Figure 3).
S)
0
JW
To demonstrate that the soxSA12S mutation contributes to the FQ-MDR phenotype, we introduced the mutation into a WT E. coli strain. The E. coli strain harbouring the soxSA12S mutation (SS0002) showed an increase in the basal resistance to enrofloxacin, ciprofloxacin and chloramphenicol compared with the strain harbouring the WT soxS (SS0001). After PQ stimulation, although both the mutant and the WT derivatives showed increased resistance to antibiotics, the soxSA12S mutant had a 2-fold higher MIC of enrofloxacin, doxycycline and chloramphenicol and a 4-fold increase in the MIC of ciprofloxacin compared with the WT (Figure 2).
5
Figure 3. Relative expression of acrB mRNA in E. coli strains JW4023 (DsoxS), SS0001 (WT-soxS) and SS0002 (soxSA12S) with or without 250 mM PQ treatment. + indicates samples containing 250 mM PQ.
Discussion We previously demonstrated that MDR E. coli experimental isolates harbouring a missense mutation in soxS emerged during
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(c)
)
2S
40
JW
40
23
)
2S
02 (s 02 oxS A1 + 2S (s ox ) S A1
00
SS
SS
00
Tso xS T- ) so xS ) +
(W 01
01
00
00
SS
SS
(W
ox S) ox S) (D s +
(D s 23
23
40
JW
40 JW
(D 23 sox S + (D ) s ox SS S) 00 01 SS 00 (W 01 T-s o + (W xS T- ) so SS xS 00 ) 0 SS 2 00 (s 02 oxS A1 + 2S (s ox ) S A1
0.004
0.008
JW
MIC (mg/L)
(a)
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soxS mutation contributes to multidrug resistance
Acknowledgements We would like to acknowledge Professor Ellen Behrend and Mrs Hollie Lee, College of Veterinary Medicine, Auburn University, for their scientific guidance.
Funding This work was supported by the Auburn University Intramural Grants Program (grant number Boothe AU-IGP 2010). The study was also partially
supported by: the United States Department of Agriculture (grant number 2005-3439415674A) from the Auburn University Detection and Food Safety Peaks of Excellence Program; and the National Science Foundation (grant number EPS 11-58862 to S.-J. S.).
Transparency declarations None of the authors has any financial conflicts of interest to declare. The funding bodies have not played any decision-making role in the preparation of this manuscript.
Supplementary data Table S1, Figure S1 and Figure S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 de Cristo´bal RE, Vincent PA, Salomo´n RA. Multidrug resistance pump AcrAB-TolC is required for high-level, Tet(A)-mediated tetracycline resistance in Escherichia coli. J Antimicrob Chemother 2006; 58: 31– 6. 2 Shaheen BW, Boothe DM, Oyarzabal OA et al. Antimicrobial resistance profiles and clonal relatedness of canine and feline Escherichia coli pathogens expressing multidrug resistance in the United States. J Vet Intern Med 2010; 24: 323–30. 3 Aly SA, Debavalya N, Suh S-J et al. Molecular mechanisms of antimicrobial resistance in fecal Escherichia coli of healthy dogs after enrofloxacin or amoxicillin administration. Can J Microbiol 2012; 58: 1288 –94. 4 Liu X, Boothe DM, Thungrat K et al. Mechanisms accounting for fluoroquinolone multidrug resistance Escherichia coli isolated from companion animals. Vet Microbiol 2012; 161: 159– 68. 5 Swick MC, Morgan-Linnell SK, Carlson KM et al. Expression of multidrug efflux pump genes acrAB-tolC, mdfA and norE in Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance. Antimicrob Agents Chemother 2011; 55: 921–4. 6 Page`s J, Amaral L. Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria. Biochim Biophys Acta 2009; 1794: 826–33. 7 Nikaido H. Multidrug efflux pumps of gram-negative bacteria. J Bacteriol 1996; 178: 5853– 9. 8 Alekshun MN, Levy SB. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother 1997; 41: 2067– 75. 9 Pomposiello PJ, Bennik MH, Demple B. Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate. J Bacteriol 2001; 183: 3890 –902. 10 Ding H, Hidalgo E, Demple B. The redox state of the [2Fe-2S] clusters in SoxR protein regulates its activity as a transcription factor. J Biol Chem 1996; 271: 33173–5. 11 Koutsolioutsou A, Pen˜a-Llopis S, Demple B. Constitutive soxR mutations contribute to multiple-antibiotic resistance in clinical Escherichia coli isolates. Antimicrob Agents Chemother 2005; 49: 2746– 52. 12 Nunoshiba T, DeRojas-Walker T, Tannenbaum SR et al. Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages. Infect Immun 1995; 63: 794–8. 13 Baba T, Ara T, Hasegawa M et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006; 2: 2006.0008.
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oral FQ treatment. This was the first report of an soxS mutation in FQ-MDR E. coli isolates. Furthermore, we found the same soxSA12S mutation in clinical FQ-MDR E. coli isolates from infected dogs. Previous studies have identified mutations in soxS, but these mutations were not linked to antimicrobial resistance.16 In this study, we investigated the possible role of the soxSA12S mutation in contributing to FQ-multidrug resistance in E. coli isolates. To date, the increased basal expression of soxS in E. coli strains has been assumed to be caused by mutations in the soxR gene that activate the SoxR protein in the absence of oxidative stress.11,17 In laboratory experiments, constitutive soxS expression was induced by constructing spacer mutations in the soxS promoter,18 constitutive mutations affecting the C-terminus of SoxR protein19 or increasing the half-life of soxS.20 However, we determined that an increased basal expression of soxS in all the soxSA12S E. coli isolates tested in this study occurred in the absence of any mutation in the soxR gene or the soxS promoter. Additionally, the increased soxS expression was associated with an overexpression of acrB and PQ treatment further increased the expression of both the soxS and acrB genes. These data suggest that the increased resistance of the soxSA12S mutants to several antibiotics might be in part due to an overexpression of the AcrB efflux pump. In E. coli isolates, increased soxS expression has been associated with a 2- to 4-fold increased resistance to nalidixic acid, chloramphenicol and tetracycline.11 Here we demonstrated that a trans complementation of soxS deletion in JW4023 with the mutant soxSA12S resulted in an increase in the basal resistance to ciprofloxacin, enrofloxacin, doxycycline and chloramphenicol with or without induction by PQ. Our results were verified when the soxS deletion in JW4023 was complemented in cis with the soxSA12S mutation. In both cases, the E. coli strains expressing the soxSA12S mutation exhibited an increase in the basal antimicrobial resistance to the same tested antimicrobials. To reveal the mechanism by which the soxSA12S mutation contributes to antimicrobial resistance in E. coli, the expression of the acrB gene was determined in the E. coli strain harbouring the WT soxS (SS0001) and in a strain harbouring the soxSA12S mutation (SS0002). The E. coli strain expressing the mutant SoxSA12S (SS0002) showed an overexpression of acrB mRNA in comparison with the E. coli strain expressing the WT SoxS (SS0001), both under unstressed conditions and after treatment with PQ. Interestingly, the soxSA12S mutants showed an overexpression of acrB without the oxidative stress. This is the first report that directly demonstrates a contribution of the soxSA12S mutation to antibiotic resistance in E. coli. The mechanisms by which this particular mutation catalyses the overexpression of acrB is not yet known. Elucidating such mechanisms will be important for establishing and eventually interfering with the contribution of the soxRS regulon to the emergence of the FQ-MDR phenotype.
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14 Herrero M, de Lorenzo V, Timmis KN. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol 1990; 172: 6557– 67. 15 Schweizer HP, Hoang TT. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 1995; 158: 15–22. 16 O’Regan E, Quinn T, Page`s JM et al. Multiple regulatory pathways associated with high-level ciprofloxacin and multidrug resistance in Salmonella enterica serovar Enteritidis: involvement of RamA and other global regulators. Antimicrob Agents Chemother 2009; 53: 1080 – 7.
17 Hidalgo E, Ding H, Demple B. Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator. Trends Biochem Sci 1997; 22: 207–10. 18 Hidalgo E, Demple B. Spacing of promoter elements regulates the basal expression of the soxS gene and converts SoxR from a transcriptional activator into a repressor. EMBO J 1997; 16: 1056 –65. 19 Nunoshiba T, Demple B. A cluster of constitutive mutations affecting the C-terminus of the redox-sensitive SoxR transcriptional activator. Nucleic Acid Res 1994; 22: 2958 –62. 20 Ding H, Demple B. Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator. Proc Natl Acad Sci USA 2000; 97: 5146– 50.
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J Antimicrob Chemother doi:10.1093/jac/dkv105
A novel alanine to serine substitution mutation in SoxS induces overexpression of efflux pumps and contributes to multidrug resistance in clinical Escherichia coli isolates Sherine A. Aly1*, Dawn M. Boothe2 and Sang-Jin Suh3 2
1 Department of Medical Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut 71516, Egypt; Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA; 3 Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA
Received 8 February 2015; returned 2 March 2015; revised 25 March 2015; accepted 27 March 2015 Objectives: The purpose of this study was to describe a putative role for a novel soxS mutation in contributing to multiple-antibiotic resistance in canine fluoroquinolone-associated MDR (FQ-MDR) Escherichia coli. This soxS mutation was discovered in canine faecal E. coli isolates during a study investigating the effect of oral fluoroquinolone administration on faecal E. coli in healthy dogs. Methods: We determined via quantitative real-time RT–PCR that both soxS and acrB were overexpressed in the clinical soxS Ala-12Ser (soxSA12S) mutants and this may account for their FQ-MDR phenotype. We validated the FQ-MDR phenotype of the clinical isolates by reconstructing the WT and the soxSA12S mutation in the E. coli soxS null mutant JW4023 (soxS::kn) via allelic exchange. Results: The JW4023 soxSA12S derivative showed an increase in MICs of ciprofloxacin, enrofloxacin and chloramphenicol compared with the JW4023 derivative in which the WT soxS had been restored. The soxS and acrB genes were overexpressed in the JW4023 soxSA12S mutant compared with JW4023 with WT soxS. A similar overexpression of efflux pump genes and an increase in antibiotic resistance were observed upon stimulation with paraquat to resemble the phenotype of the clinical soxSA12S isolates. Conclusions: Our data suggest that the soxSA12S substitution mutation is selected in clinical isolates when dogs are exposed to a fluoroquinolone and that this mutation contributes to the FQ-MDR phenotype of E. coli isolates. Keywords: fluoroquinolones, acrB, soxS, soxR
Introduction Fluoroquinolone (FQ) resistance has been reported among different members of the family Enterobacteriaceae including Escherichia coli. 1,2 High-level FQ resistance is also associated with multidrug resistance.3,4 Recently, multidrug resistance has been linked to FQ resistance through mutations within the regulatory loci that control the expression of the AcrAB efflux pump.3,5 AcrAB-TolC is one of the most common efflux pumps associated with antimicrobial resistance in E. coli and has a wide substrate specificity to render the organism simultaneously resistant to many antibiotics.6 The expression of the acrAB operon is regulated locally by AcrR and AcrS,7 while globally it is influenced by several transcriptional activators belonging to the AraC/XylS family, especially MarA and SoxS.8 The SoxS protein is a transcription factor that activates the expression of more than 100 genes in the chromosomally
encoded SoxRS regulon.9 In E. coli, SoxR is constitutively expressed and binds to its sole DNA target, a site in the promoter of the soxS gene, without activating its transcription.8 When exposed to oxidative stress, the SoxR protein activates the transcription of soxS,10 which in turn results in an overexpression of the target genes including acrAB.6,7 In both laboratory and clinical strains of E. coli, activation of the soxRS regulon has been shown to contribute to the increased resistance to quinolones and chloramphenicol.11 However, few data exist regarding mutations in soxS and their importance in the acquisition of FQ resistance. We previously demonstrated that the administration of enrofloxacin to dogs in low therapeutic doses was associated with the emergence of FQ-associated MDR (FQ-MDR) faecal E. coli isolates that contained a novel soxS mutation.3 The same soxS mutation has been encountered in clinical FQ-MDR E. coli isolates from the urine of dogs with a suspected urinary tract infection. The purpose of
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*Corresponding author. Tel: +00201097018789; E-mail: [email protected]
Aly et al.
this study was to describe a putative new role for a novel soxS mutation in the contribution to multiple-antibiotic resistance in canine FQ-MDR E. coli.
Materials and methods Bacterial isolates (Table 1) Experimental E. coli isolates
Clinical E. coli isolates FQ-MDR clinical E. coli isolates containing the soxSA12S mutation (n ¼ 3) were drawn from a larger study investigating the mechanisms of FQ-multidrug resistance in companion animals. The three E. coli isolates harboured two mutations in gyrA, a single mutation in gyrB and two mutations in parC and belonged to the B1 phylotype. 6
Sequencing of soxS, soxR and marR from E. coli isolates E. coli isolates were tested for mutations in genes encoding regulators of the AcrAB efflux pump (soxR, soxS and marR) by PCR and DNA sequencing. The primers that were used are listed in Table S1 (available as Supplementary data at JAC Online). Table 1. Bacterial strains and plasmids Strain or plasmid
Properties
Source or reference
Strain CG4468 S61 S71 S81 I78 N81 L94 JW4023 DB0001 DB0002 DB0003 SS0001 SS0002
E. coli K-12 soxS + E. coli clinical isolate E. coli clinical isolate E. coli clinical isolate E. coli clinical isolate E. coli clinical isolate E. coli clinical isolate E. coli K-12 W3110 DsoxS DlacZ pCR2.1 in JW4023 pSAWT in JW4023 pSAMT in JW4023 E. coli WT soxS in JW4023 E. coli mutant-type soxS in JW4023
12 5 5 5 6 6 6 13 this study this study this study this study this study
Plasmid pCR2.1 pGP704 pSAWT pSAMT pSA2000 pSA2001 pSA2002
vector (Apr, Knr) vector (Apr) WT soxS in pCR2.1 soxSA12S mutant in pCR2.1 sacB in pGP704 E. coli WT soxS in pSA2000 E. coli mutant soxS in pSA2000
Invitrogen this study this study this study this study this study this study
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FQ-MDR E. coli isolates were examined by RT – qPCR for the mRNA expression of genes encoding for efflux pump AcrB and its regulators (SoxS and MarA). Overnight bacterial cultures were diluted 1/100 in fresh LB broth and grown with shaking (250 rpm) for 90 min at 378C. At this point, the cultures were divided and the appropriate samples were treated with 250 mM paraquat (PQ), a redox-cycling agent and inducer of the soxRS regulon. PQ-treated cells were further incubated at 378C for another 30 min. RNA was extracted as previously described.6 Reverse transcription of RNA into cDNA was carried out using the iScript Reverse Transcription kit (Bio-Rad, USA). The quantification of cDNA templates by RT – qPCR was performed in a Roche LightCycler 480 using a Roche SYBR Green RT – PCR kit (Roche, USA). The expression of each gene was normalized to that of a ribosomal housekeeping gene (gapA). The relative expression of each target gene was calibrated against the corresponding expression by E. coli ATCC 25922 (expression ¼ 1), which served as the control.
Cloning of mutant soxS from E. coli isolates A 870 bp fragment corresponding to the soxS region (the soxS gene and its promoter) of the WT soxS and soxSA12S was amplified by PCR from the genomic DNA of the E. coli isolates (from both WT E. coli and resistant E. coli). The amplified fragments were cloned into vector pCR2.1 (Apr, Knr) using the pCR2.1 TA cloning kit (Invitrogen, Carlsbad, USA) to generate two plasmids, pSAWT (WT soxS) and pSAMT (soxSA12S).The clones were confirmed by restriction digestion analysis with EcoRI and BamHI and DNA sequencing.
Complementation of a DsoxS strain with cloned soxS alleles for increased antibiotic resistance The pSAWT and pSAMT plasmids were introduced into E. coli K-12: JW4023 (soxS::kn, DlacZ), producing strains DB0002 and DB0003, respectively, and their contribution to the antibiotic resistance phenotype of the recipient strain was assessed by Etest. As a control, the E. coli carrying the vector plasmid alone was also subjected to the Etest. The antibiotics tested were enrofloxacin, ciprofloxacin, doxycycline and chloramphenicol.
Reconstruction of the soxSA12S mutation in the E. coli JW4023 strain (soxS::kn, DlacZ) The soxSA12S mutant allele was inserted into the chromosome via allelic exchange with the soxS::kn insertion allele using the negative selection marker sacB. Plasmid pGP704, an R6K derivative that can be maintained only in E. coli strains that contain the pir gene, was regenerated by removing the Tn5 tnp gene (using the SalI restriction enzyme) from the suicide vector pUT.14 The sacB gene was amplified using PCR from pEX100T15 and cloned into pGP704 to generate the pSA2000 vector, an E. coli suicide plasmid that carries a negative selectable marker. The two soxS (the WT and the soxSA12S mutant allele) were cloned into this pSA2000 vector to generate two plasmids, pSA2001 and pSA2002, respectively, that were used for the allelic exchange. Plasmids pSA2001 and pSA2002 were introduced into the E. coli JW4023 insertion mutant and those in which the plasmid integrated into the genome via homologous recombination (the first cross-over) were selected as Knr Apr resistant colonies. The resulting ampicillin-resistant cells were then grown in the presence of 5% sucrose to force homologous recombination (the second cross-over), which resulted in the loss of the integrated plasmid sequence along with the preexisting soxS::kn sequence, while leaving behind the soxS (WT or soxSA12S) allele in its place. The mutants that had undergone the proper
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Three faecal FQ-MDR E. coli isolates harbouring the soxSA12S mutation were detected during an investigation of the effect of oral FQ administration on canine faecal E. coli. The three isolates contained two mutations in gyrA and two mutations in parC and belonged to three different PFGE pulsotypes.5 Phylogenetic analysis showed that two E. coli isolates belonged to the B1 phylotype (S61 and S71) and one E. coli isolate belonged to the D phylotype (S81).
RNA extraction and relative quantification of gene expression by quantitative real-time RT–PCR (RT– qPCR)
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soxS mutation contributes to multidrug resistance
homologous recombination were identified as sucroser Kns Aps colonies. The presence of the soxSA12S allele was verified via PCR amplification and DNA sequencing. The resulting WT and the soxSA12S mutant (SS0001 and SS0002, respectively) were characterized for antibiotic resistance by Etest and for expression of the acrB gene by RT-qPCR.
Results Mutations in the FQ-MDR E. coli isolates
Expression of acrB, soxS and marA in FQ-MDR E. coli isolates To assess the role of the AcrB pump in contributing to the FQ-MDR phenotype of these isolates, we measured the expression of the
Enrofloxacin
Because the overexpression of WT soxS can result in enhanced resistance to antibiotics, it was imperative to compare the WT and the mutant soxSA12S allele cloned into the same multicopy plasmid to assess the effect of the mutation. Based on the MIC results, the strain expressing the mutant SoxS protein (DB0003) exhibited at least 2-fold higher basal resistance to ciprofloxacin, enrofloxacin, chloramphenicol and doxycycline compared with the strain expressing the WT SoxS (DB0002). PQ treatment further increased the basal antimicrobial resistance of JW4023 derivatives harbouring either pSAWT or pSAMT, but the strain expressing the mutant SoxS still had higher resistance than the strain expressing the WT SoxS for each antibiotic tested (Figure 1).
(b)
Ciprofloxacin 0.256 0.128 0.064 0.032 0.016 0.008 0.004
JW JW 402 40 3 ( 23 Dso + (D xS) DB so 00 xS DB 0 ) 00 1 ( 01 pC + R2 (p .1 CR ) DB 2. 0 1) DB 00 00 2 ( 02 pS + AW (p T SA ) DB W 0 T) DB 00 3 00 ( 03 pS + AM (p T) SA M T)
MIC (mg/L)
0.256 0.128 0.064 0.032 0.016 0.008
JW JW 402 40 3 ( 23 Dso + (D xS) so DB xS 0 ) DB 00 00 1 (p 01 CR + (p 2.1) CR DB 2. 0 1) DB 002 00 (p 02 SA + W (p T SA ) DB W 0 T) DB 00 00 3 ( 03 pS + AM (p T SA ) M T)
MIC (mg/L)
(a)
Antibiotic resistance conferred by cloned mutant soxSA12S gene
E. coli isolate (c)
E. coli isolate (d)
Chloramphenicol
Doxycycline 16
MIC (mg/L)
MIC (mg/L)
16 8 4 2
8 4 2 1
E. coli isolate
23 (Ds + ox (D S) DB so xS 00 DB 0 ) 00 1 ( 01 pC + R2 (p .1 CR ) DB 2. 0 1) DB 00 00 2 ( 02 pS + AW (p T SA ) DB W 00 T) DB 0 00 3 ( 03 pS + AM (p T SA ) M T)
40
23 JW
40 JW
JW JW 402 40 3 ( 23 Dso + (D xS) so DB xS 0 ) DB 00 00 1 (p 01 CR + (p 2.1) CR DB 2. 0 1) DB 00 00 2 ( p 02 S + AW (p T SA ) DB W 00 T) DB 0 00 3 ( 03 pS + AM (p T SA ) M T)
1
E. coli isolate
Figure 1. Antibiotic resistance conferred by mutant soxS genes. Plasmids pCR2.1, pSAWT and pSAMT encoding no soxS, WT soxS and soxSA12S, respectively, were introduced into strain JW4023 (DsoxS) and their contribution to the antibiotic resistance phenotype of the recipient strain was assayed by the broth microdilution method in the presence or absence of 250 mM PQ. (a) Enrofloxacin, 0.008 – 0.512 mg/L. (b) Ciprofloxacin, 0.004 – 0.512 mg/L. (c) Chloramphenicol, 1 – 16 mg/L. (d) Doxycycline, 1– 16 mg/L. + indicates samples containing 250 mM PQ.
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We sequenced soxR, soxS, marR and acrR genes from our E. coli MDR isolates in addition to the entire soxS region to address the possibility that there were other mutations in the soxS gene or its promoter. In all the E. coli isolates that were tested, only a single missense mutation at codon 12 with a GT transversion in codon GCA that changed alanine to serine (Ala-12Ser) was found. This mutation lies outside the helix-turn-helix motif of the SoxS DNA-binding domain. No mutations were found elsewhere in the soxS gene, soxS promoter or other regulators.
genes coding for the pump and its regulators. We determined that basal soxS and acrB were overexpressed (≥2-fold increased expression) in all of the tested E. coli isolates compared with the WT E. coli (CG4468). Upon stimulation with PQ, all the isolates retained the ability to overexpress both soxS and acrB mRNA (Figures S1 and S2). In contrast to soxS expression, all of the isolates exhibited low marA expression (0.653 – 0.875) (data not shown). This suggests that overexpression of the AcrB efflux pump in those mutants was not due to MarA.
Aly et al.
Enrofloxacin
Ciprofloxacin
(b)
0.256
0.128
0.128
0.064
MIC (mg/L)
0.064 0.032 0.016
0.032 0.016 0.008
E. coli isolate
E. coli isolate Chloramphenicol
MIC (mg/L)
8 4 2
4 2 1 2S
JW 23 40 (D 23 sox S + (D ) s ox SS S) 00 01 SS 00 (W 01 T-s o + (W xS) Tso SS xS 00 ) 02 SS ( s 00 ox 02 S A1 + 2S (s ) ox S A1
JW
40
)
2S
JW 23 40 (Ds ox 23 S + (D ) so xS SS ) 00 01 SS ( W 00 01 T-s o + (W xS T- ) so xS SS ) 00 0 2 SS 00 (so x 02 + S A12 S (s ) ox S A1
40
)
MIC (mg/L)
8
1
JW
Doxycycline
(d)
16
E. coli isolate
E. coli isolate
Figure 2. Antibiotic resistance conferred by recombinant mutant soxS genes. E. coli strains JW4023 (DsoxS), SS0001 (WT soxS in JW4023) and SS0002 (soxSA12S in JW4023) were tested for their antibiotic resistance phenotype by the broth microdilution method in the presence or absence of 250 mM PQ. (a) Enrofloxacin, 0.008 – 0.512 mg/L. (b) Ciprofloxacin, 0.004 – 0.512 mg/L. (c) Chloramphenicol, 1 – 16 mg/L. (d) Doxycycline, 1 – 16 mg/L. + indicates samples containing 250 mM PQ.
Antimicrobial resistance conferred by recombinant mutant soxS allele Fold expression
4 3 2 1
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)
2S
00 1 (W SS T00 so 01 xS + ) (W Tso xS ) SS 00 02 (s SS ox 00 S A1 02 2S + ) (s ox S A1
SS 0
JW
40
23
+
(D s
ox
S) (D so x 23 40
E. coli isolate
Activity of the cloned mutant soxS gene We determined whether the SoxSA12S mutant protein affected the expression of the target genes. Interestingly, the soxSA12S mutant (SS0002) exhibited an overexpression of acrB without any need for a prior activation of SoxR. Compared with the E. coli strain expressing the WT SoxS (SS0001), the strain expressing the mutant SoxS (SS0002) overexpressed the acrB gene both under nonstressful experimental conditions and after PQ stimulation (Figure 3).
S)
0
JW
To demonstrate that the soxSA12S mutation contributes to the FQ-MDR phenotype, we introduced the mutation into a WT E. coli strain. The E. coli strain harbouring the soxSA12S mutation (SS0002) showed an increase in the basal resistance to enrofloxacin, ciprofloxacin and chloramphenicol compared with the strain harbouring the WT soxS (SS0001). After PQ stimulation, although both the mutant and the WT derivatives showed increased resistance to antibiotics, the soxSA12S mutant had a 2-fold higher MIC of enrofloxacin, doxycycline and chloramphenicol and a 4-fold increase in the MIC of ciprofloxacin compared with the WT (Figure 2).
5
Figure 3. Relative expression of acrB mRNA in E. coli strains JW4023 (DsoxS), SS0001 (WT-soxS) and SS0002 (soxSA12S) with or without 250 mM PQ treatment. + indicates samples containing 250 mM PQ.
Discussion We previously demonstrated that MDR E. coli experimental isolates harbouring a missense mutation in soxS emerged during
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(c)
)
2S
40
JW
40
23
)
2S
02 (s 02 oxS A1 + 2S (s ox ) S A1
00
SS
SS
00
Tso xS T- ) so xS ) +
(W 01
01
00
00
SS
SS
(W
ox S) ox S) (D s +
(D s 23
23
40
JW
40 JW
(D 23 sox S + (D ) s ox SS S) 00 01 SS 00 (W 01 T-s o + (W xS T- ) so SS xS 00 ) 0 SS 2 00 (s 02 oxS A1 + 2S (s ox ) S A1
0.004
0.008
JW
MIC (mg/L)
(a)
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soxS mutation contributes to multidrug resistance
Acknowledgements We would like to acknowledge Professor Ellen Behrend and Mrs Hollie Lee, College of Veterinary Medicine, Auburn University, for their scientific guidance.
Funding This work was supported by the Auburn University Intramural Grants Program (grant number Boothe AU-IGP 2010). The study was also partially
supported by: the United States Department of Agriculture (grant number 2005-3439415674A) from the Auburn University Detection and Food Safety Peaks of Excellence Program; and the National Science Foundation (grant number EPS 11-58862 to S.-J. S.).
Transparency declarations None of the authors has any financial conflicts of interest to declare. The funding bodies have not played any decision-making role in the preparation of this manuscript.
Supplementary data Table S1, Figure S1 and Figure S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 de Cristo´bal RE, Vincent PA, Salomo´n RA. Multidrug resistance pump AcrAB-TolC is required for high-level, Tet(A)-mediated tetracycline resistance in Escherichia coli. J Antimicrob Chemother 2006; 58: 31– 6. 2 Shaheen BW, Boothe DM, Oyarzabal OA et al. Antimicrobial resistance profiles and clonal relatedness of canine and feline Escherichia coli pathogens expressing multidrug resistance in the United States. J Vet Intern Med 2010; 24: 323–30. 3 Aly SA, Debavalya N, Suh S-J et al. Molecular mechanisms of antimicrobial resistance in fecal Escherichia coli of healthy dogs after enrofloxacin or amoxicillin administration. Can J Microbiol 2012; 58: 1288 –94. 4 Liu X, Boothe DM, Thungrat K et al. Mechanisms accounting for fluoroquinolone multidrug resistance Escherichia coli isolated from companion animals. Vet Microbiol 2012; 161: 159– 68. 5 Swick MC, Morgan-Linnell SK, Carlson KM et al. Expression of multidrug efflux pump genes acrAB-tolC, mdfA and norE in Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance. Antimicrob Agents Chemother 2011; 55: 921–4. 6 Page`s J, Amaral L. Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria. Biochim Biophys Acta 2009; 1794: 826–33. 7 Nikaido H. Multidrug efflux pumps of gram-negative bacteria. J Bacteriol 1996; 178: 5853– 9. 8 Alekshun MN, Levy SB. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother 1997; 41: 2067– 75. 9 Pomposiello PJ, Bennik MH, Demple B. Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate. J Bacteriol 2001; 183: 3890 –902. 10 Ding H, Hidalgo E, Demple B. The redox state of the [2Fe-2S] clusters in SoxR protein regulates its activity as a transcription factor. J Biol Chem 1996; 271: 33173–5. 11 Koutsolioutsou A, Pen˜a-Llopis S, Demple B. Constitutive soxR mutations contribute to multiple-antibiotic resistance in clinical Escherichia coli isolates. Antimicrob Agents Chemother 2005; 49: 2746– 52. 12 Nunoshiba T, DeRojas-Walker T, Tannenbaum SR et al. Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages. Infect Immun 1995; 63: 794–8. 13 Baba T, Ara T, Hasegawa M et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006; 2: 2006.0008.
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oral FQ treatment. This was the first report of an soxS mutation in FQ-MDR E. coli isolates. Furthermore, we found the same soxSA12S mutation in clinical FQ-MDR E. coli isolates from infected dogs. Previous studies have identified mutations in soxS, but these mutations were not linked to antimicrobial resistance.16 In this study, we investigated the possible role of the soxSA12S mutation in contributing to FQ-multidrug resistance in E. coli isolates. To date, the increased basal expression of soxS in E. coli strains has been assumed to be caused by mutations in the soxR gene that activate the SoxR protein in the absence of oxidative stress.11,17 In laboratory experiments, constitutive soxS expression was induced by constructing spacer mutations in the soxS promoter,18 constitutive mutations affecting the C-terminus of SoxR protein19 or increasing the half-life of soxS.20 However, we determined that an increased basal expression of soxS in all the soxSA12S E. coli isolates tested in this study occurred in the absence of any mutation in the soxR gene or the soxS promoter. Additionally, the increased soxS expression was associated with an overexpression of acrB and PQ treatment further increased the expression of both the soxS and acrB genes. These data suggest that the increased resistance of the soxSA12S mutants to several antibiotics might be in part due to an overexpression of the AcrB efflux pump. In E. coli isolates, increased soxS expression has been associated with a 2- to 4-fold increased resistance to nalidixic acid, chloramphenicol and tetracycline.11 Here we demonstrated that a trans complementation of soxS deletion in JW4023 with the mutant soxSA12S resulted in an increase in the basal resistance to ciprofloxacin, enrofloxacin, doxycycline and chloramphenicol with or without induction by PQ. Our results were verified when the soxS deletion in JW4023 was complemented in cis with the soxSA12S mutation. In both cases, the E. coli strains expressing the soxSA12S mutation exhibited an increase in the basal antimicrobial resistance to the same tested antimicrobials. To reveal the mechanism by which the soxSA12S mutation contributes to antimicrobial resistance in E. coli, the expression of the acrB gene was determined in the E. coli strain harbouring the WT soxS (SS0001) and in a strain harbouring the soxSA12S mutation (SS0002). The E. coli strain expressing the mutant SoxSA12S (SS0002) showed an overexpression of acrB mRNA in comparison with the E. coli strain expressing the WT SoxS (SS0001), both under unstressed conditions and after treatment with PQ. Interestingly, the soxSA12S mutants showed an overexpression of acrB without the oxidative stress. This is the first report that directly demonstrates a contribution of the soxSA12S mutation to antibiotic resistance in E. coli. The mechanisms by which this particular mutation catalyses the overexpression of acrB is not yet known. Elucidating such mechanisms will be important for establishing and eventually interfering with the contribution of the soxRS regulon to the emergence of the FQ-MDR phenotype.
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14 Herrero M, de Lorenzo V, Timmis KN. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol 1990; 172: 6557– 67. 15 Schweizer HP, Hoang TT. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 1995; 158: 15–22. 16 O’Regan E, Quinn T, Page`s JM et al. Multiple regulatory pathways associated with high-level ciprofloxacin and multidrug resistance in Salmonella enterica serovar Enteritidis: involvement of RamA and other global regulators. Antimicrob Agents Chemother 2009; 53: 1080 – 7.
17 Hidalgo E, Ding H, Demple B. Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator. Trends Biochem Sci 1997; 22: 207–10. 18 Hidalgo E, Demple B. Spacing of promoter elements regulates the basal expression of the soxS gene and converts SoxR from a transcriptional activator into a repressor. EMBO J 1997; 16: 1056 –65. 19 Nunoshiba T, Demple B. A cluster of constitutive mutations affecting the C-terminus of the redox-sensitive SoxR transcriptional activator. Nucleic Acid Res 1994; 22: 2958 –62. 20 Ding H, Demple B. Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator. Proc Natl Acad Sci USA 2000; 97: 5146– 50.
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