JAC Advance Access originally published online on October 29, 2007
Journal of Antimicrobial Chemotherapy 2008 61(1):46-53; doi:10.1093/jac/dkm397
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Original research |
MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli
1 Wyeth Research, Pearl River, NY, USA 2 Wyeth Research, Cambridge, MA, USA 3 Wyeth Vaccines, Pearl River, NY, USA
* Corresponding author. Tel: +1-845-602-8360; Fax: +1-845-602-5671; E-mail: keeneyd{at}wyeth.com
Received 15 June 2007; returned 20 August 2007; revised 12 September 2007; accepted 24 September 2007
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Abstract |
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Objectives: The purpose of this study was to characterize decreased susceptibility to tigecycline in clinical isolates of Escherichia coli obtained during Phase 3 clinical trials.
Methods: Gene expression was analysed by transcriptional profile analysis and RT-PCR. Transposon mutagenesis with IS903
kan was used for selection of transposon mutants. Transposon insertions were mapped by DNA sequencing and PCR analyses. The MICs were determined by broth microdilution.
Results: Both transcriptional profile analysis and Taqman RT-PCR demonstrated increased expression levels of MarA, a transcriptional activator, and AcrAB, an RND-type efflux pump, in the strains with elevated tigecycline MICs. Transposon mutagenesis generated nine mutants, the majority of which had either marA or acrB inactivated. Sequence analysis revealed a single nucleotide insertion in the open reading frame of the marR gene in less-susceptible strains of E. coli.
Conclusions: This study suggested that a loss of MarR functionality due to a frameshift mutation resulted in constitutive overproduction of MarA and AcrAB and, consequently, in decreased susceptibility to tigecycline in clinical isolates of E. coli.
Keywords: resistance nodulation cell division family , antibiotic resistance , multidrug efflux pump , RT-PCR , transcriptional profile analysis
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Introduction |
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Tigecycline is a novel glycylcycline antibiotic, which has been approved worldwide to treat serious medical conditions such as complicated skin and skin structure infections and complicated intra-abdominal infections.1 This compound is active against a broad range of Gram-negative, Gram-positive, anaerobic and atypical bacteria.2–4 Tigecycline is also active against many antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and extended-spectrum β-lactamase-producing Enterobacteriaceae. Tigecycline overcomes typical tetracycline resistance mechanisms such as the ribosomal protection determinant tet(M) and tetracycline-specific efflux pumps: tet(A), tet(B), tet(C) and tet(D).5
The AcrAB-TolC efflux pump is a tripartite complex containing AcrA, a fusion protein; AcrB, a cytoplasmic membrane transporter protein; and TolC, an outer membrane channel. Previously it was demonstrated that decreased susceptibility to tigecycline is due to an up-regulated expression of the AcrAB efflux pump in several species including Proteus mirabilis,6 Klebsiella pneumoniae,7 Morganella morganii8 and Enterobacter cloacae.9 Overexpression of this pump is often associated with a multidrug resistance (MDR) phenotype.10
Escherichia coli is usually susceptible to tigecycline, however a few less-susceptible clinical isolates were obtained during Phase 3 clinical trials. The purpose of this study was to characterize decreased susceptibility to tigecycline in these isolates.
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Materials and methods |
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Bacterial strains and growth conditions
E. coli strains used in this study are shown in Table 1. Strains G4905, G4906, G4907, G5048, G5049 and G5050 were isogenic clinical isolates from a single patient treated with tigecycline in a clinical trial designed to study resistant Gram-negative pathogens. The strains were propagated at 37°C in Luria–Bertani (LB) broth or agar.
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DNA techniques
Standard DNA manipulations such as restriction digestion and molecular cloning were performed as described previously.11 DNA transformations were performed by electroporation with the Gene Pulser II system (Bio-Rad, Hercules, CA, USA), using the optimal electroporation settings of 2.5 kV, 25 µF, 200 
and 5 ms. E. coli genomic DNA was isolated by using the Puregene tissue kit (Gentra Systems Inc., Minneapolis, MN, USA) and used as a template for PCRs. Primers used for PCR are listed in Table 2. The FailSafe PCR System (EpiCentre, Madison, WI, USA) was used to amplify E. coli acrAB and marRAB DNA sequences in accordance with the manufacturer’s instructions. Oligonucleotide primers were obtained from Genelink (Hawthorne, NY, USA). PCR fragments were gel-purified by using Zymoclean Gel DNA Recovery kit (Zymo Research, Orange, CA, USA). The nucleotide sequence was determined with an automated sequencer ABI 3730 (Applied Biosystems, Foster City, CA, USA).
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Transcriptional profile analysis
RNA was extracted from mid-log phase bacterial cultures using the RNAeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Reverse transcription, cDNA fragmentation and terminal labelling of cDNA fragments with biotin were carried out in accordance with the manufacturer’s (Affymetrix Inc., Santa Clara, CA, USA) instructions for antisense prokaryotic arrays. In brief, hexamer random primers (Invitrogen, Carlsbad, CA, USA) were annealed to 10 µg of total denatured RNA at 25°C for 10 min. cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) following the manufacturer’s instructions. An RNAse inhibitor, SUPERase-In (Ambion, Inc., Austin, TX, USA) was included. Any remaining RNA was degraded by treatment with 1 M NaOH for 30 min at 65°C and neutralized by adding an equal volume of 1 M HCl. cDNA was purified using the DNA Clean and Concentrator kit (Zymo Research) and fragmented with DNase I in One-Phor-All buffer (Amersham Biosciences, Piscataway, NJ, USA) (0.6 U DNase I/µg cDNA). Fragmented cDNA was labelled with biotin on the 3’ terminus using the Enzo BioArray terminal labelling kit with biotin ddUTP (Affymetrix). Labelled, fragmented cDNA (1.5 µg) was hybridized overnight to E. coli Antisense Genome GeneChips (Affymetrix). The ‘antisense’ oligonucleotide array is essentially the same as that described by Selinger et al.,12 except that probe sequences were the same as the coding region sequences. GeneChips were stained, washed and scanned using the Agilent GeneArray laser scanner (Agilent Technologies, Palo Alto, CA, USA) as described previously.13 Affymetrix algorithms calculated signal intensities (average difference values) and made present or absent calls for each gene as detailed by Lockhart et al.14 Signal intensities for elements tiled onto each GeneChip were then normalized to account for loading errors and differences in labelling efficiencies by dividing each signal intensity value by the mean signal intensity for an individual GeneChip. Results were analysed using GeneSpring Version 6.1 software (Silicon Genetics, Redwood City, CA, USA). Changes in gene expression were only considered relevant if there was at least a 2-fold change between the relevant strains, that the genes in the up-regulated condition were considered to be present by Affymetrix algorithms and that differences in expression were significant (t-test with a P value cutoff of at least 0.05) as previously described.15,16
Oligonucleotide primers and probes used for real-time RT-PCR were designed with Primer Express Software version 2.0 (Applied Biosystems) and purchased from Operon Biotechnologies (Huntsville, AL, USA). The probes were labelled by the manufacturer with the reporter dye 6-carboxyfluorescein (6'-FAM) at the 5' end and with the quencher dye 6-carboxytetramethylroda-mine (TAMRA) at the 3' end. DNAse-treated RNA templates were prepared from mid-log phase bacterial cultures by using RNAeasy kit (Qiagen). RT-PCR was performed by using iScript One-Step RT-PCR Kit for Probes (Bio-Rad) on iCycler iQ5TM Real-Time PCR Detection System (Bio-Rad). A typical RT-PCR sample (25 µL) contained: 5 µL of a serial dilution of RNA template (range, 2 ng/mL to 200 mg/L), 6.85 µL of nuclease-free water (Ambion, Austin, Tex.), 12.5 µL of RT-PCR mixture (2x), 0.5 µL of iScript RT enzyme mix (50x), 0.05 µL of 100 µM solutions of both forward and reverse gene-specific primers and 0.05 µL of a 100 µM solution of gene-specific probe. Relative quantification of the target gene expression (acrA or marA) was performed by iCycler iQ5TM software using normalized expression analysis method. The 16S rRNA gene served as a reference gene and G4907 served as a reference condition. Each sample was run in triplicate.
Transposon mutagenesis with IS903kan was performed essentially as described previously.7,17 Briefly, the transposon carrier plasmid, pVJT128, was electroporated into G5049 and transformants were selected on LB plates containing 200 mg/L chloramphenicol. Individual colonies were selected, inoculated into LB broth containing 1 mM IPTG and 200 mg/L chloramphenicol and propagated overnight with shaking to induce transposition. Clones with transposon insertions were selected by plating aliquots of overnight culture onto LB plates containing 50 mg/L kanamycin. Tigecycline-susceptible transposon mutants were isolated by replica plating and selecting for colonies that grew on LB plates containing 50 mg/L kanamycin but not on LB plates containing 2 mg/L tigecycline. The carrier plasmid was cured by serial passage in chloramphenicol-free medium. Transposon insertions were mapped by PCR and sequence analysis of the marRAB and acrAB operons.
Antibiotic susceptibility testing
Tigecycline used in this study was obtained from Wyeth Research (Pearl River, NY, USA). Tetracycline, minocycline, acriflavine, ethidium bromide, erythromycin, chloramphenicol, nalidixic acid, novobiocin, trimethoprim, norfloxacin and kanamycin were obtained from Sigma Chemical Co. (St Louis, MO, USA). The MICs of various antibacterial agents were determined by standard broth microdilution tests.18 Tests for tigecycline susceptibility were performed using fresh Mueller–Hinton broth (<12 h old).19,20
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Results |
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Transcriptional profile analysis of total RNA
Transcriptional profile analysis of total RNA isolated from four isogenic strains, G4907, G5048, G5049 and G5050, was performed to identify genes that were either up- or down-regulated in strains with an increased tigecycline MIC. Two other isolates, G4905 and G4906, were omitted from expression studies because these strains had tigecycline MIC values identical to those for G4907 and G5050. The results are shown in Tables 3 and 4. Genes of the mar operon displayed the greatest changes in expression. Other genes that increased in expression between 2- and 8-fold included acrA, acrB and tolC, which encode components of the AcrAB multidrug efflux pump. Other up-regulated genes are shown in Table 3.
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Several genes were also down-regulated in G5048 and G5049, among them were ompF, fimA and genes from the ymc and ycc clusters. Other down-regulated genes are shown in Table 4.
Analysis of acrA expression by RT-PCR
Transcriptional profile analysis indicated that a decrease in susceptibility to tigecycline might correlate with elevated expression of the AcrAB efflux pump. To confirm the results, RT-PCR analysis of acrA expression was performed. As shown in Figure 1, increased expression of acrA was observed in the less-susceptible strains. Quantitative analysis revealed that expression of acrA increased 2.75- and 3.5-fold in G5048 and G5049, respectively, as compared with tigecycline-susceptible strain, G4907, whereas the expression level in another tigecycline-susceptible strain, G5050, was not elevated relative to G4907. Because acrA and acrB are co-transcribed, this result implied that both genes were overexpressed in the less-susceptible strains and further suggested that overexpression of the AcrAB pump is involved in decreased tigecycline susceptibility in E. coli.
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Analysis of marA expression by RT-PCR
The transcriptional activator MarA was previously shown to up-regulate the production of the AcrAB efflux pump in E. coli.21 To test the hypothesis that MarA could be involved in the overexpression of AcrA in the strains in this study, RT-PCR analysis of marA expression was performed. As shown in Figure 2, marA expression was increased over 30-fold in two strains, G5048 and G5049, compared with both G4907 and G5050. As mentioned above, G5048 and G5049 also displayed elevated levels of acrAB expression as compared with strains G4907 and G5050, suggesting that increased production of the AcrAB pump in G5048 and G5049 is the result of marA overexpression.
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Sequence analysis of marR gene
MarA activates the acr operon by binding to the intergenic region between acrR and acrA thereby lifting the repression caused by the AcrR repressor protein.21 Regulation of the mar operon is in turn, accomplished by the MarR repressor, a dimeric protein that binds to the mar operator region. The nucleotide sequence of marR gene was analysed to determine whether this might contribute to the constitutive overexpression of both mar and acr operons in G5048 and G5049. Both G5048 and G5049 had an insertion of a cytosine residue at position 355 in marR, whereas tigecycline-susceptible strains lacked this mutation. The addition of a cytosine residue at this position causes a frameshift that is likely to result in MarR losing its repressor function, which would lead to constitutive overproduction of MarA and AcrAB and, consequently, to decreased susceptibility to tigecycline in G5048 and G5049.
Transposon mutagenesis and mappingof transposon insertions
To gain an additional insight into the mechanism of the decreased tigecycline susceptibility in E. coli, strain G5049 was subjected to transposon mutagenesis. Mutagenesis of G5049 with IS903kan resulted in the selection of nine tigecycline-susceptible transposon insertion mutants (Table 1). The sites of transposon insertions were mapped by PCR and sequence analyses of the marRAB and acrAB regions. In four of the mutants, transposon was mapped to three different positions within the marA gene: nucleotide 21 in GC7952 and GC7955; nucleotide 30 in GC7953; and nucleotide 87 in GC7954. In the other two mutants, transposon inserted into acrB gene: nucleotide 3102 in GC7956 and nucleotide 3141 in GC7957. In the remaining three mutants, neither marRAB nor acrAB regions contained the transposon.
As shown in Table 5, in addition to decreased susceptibility to tigecycline, G5049 displayed an MDR phenotype, which is consistent with the overexpression of the AcrAB multidrug efflux pump in this strain. All the transposon insertions resulted in a reduction in the MIC of tigecycline; however, they differed in their effect on the MICs of other antibiotics. Insertional inactivation of acrB (strains GC7956 and GC7957) resulted in the most profound effect: resistance decreased to every antibiotic in the panel with the exception of chloramphenicol, nalidixic acid and norfloxacin. Inactivation of marA (strains GC7952 through GC7955) produced less of an effect on the MDR phenotype, which might indicate that the presence of an intact AcrAB pump, although not overexpressed, is sufficient to result in the elevated MICs of erythromycin, ethidium bromide and acriflavine in these strains. Unmapped transposon insertions in strains GC7958 through GC7960 only affected the MICs of tigecycline, tetracycline and minocycline.
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None of the transposon insertions affected the MICs of chloramphenicol, nalidixic acid and norfloxacin, which indicated that strain G5049 might possess specific determinants of resistance to these drugs.
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Discussion |
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Tigecycline is a novel glycylcycline antibiotic developed to overcome bacterial resistance to tetracycline and minocycline. Recently however, our laboratory has reported that increased expression of the multidrug efflux pump AcrAB correlated with decreased susceptibility to tigecycline in P. mirabilis, K. pneumoniae, M. morganii and E. cloacae.6–9
The results of the present study indicate that genes of the mar and acr operons are associated with decreased susceptibility to tigecycline in E. coli. This was suggested by results from transcriptional profile analysis, RT-PCR and by transposon mutagenesis. These results are in agreement with those of Hirata et al.,22 who constructed AcrAB-expressing E. coli strains that showed a 4-fold decrease in susceptibility to tigecycline compared with wild-type strains.
Transcriptional activators such as MarA have been shown to regulate the expression of the AcrAB efflux pump. RamA is a homologue of MarA, which was previously described in K. pneumoniae and Enterobacter aerogenes.23,24 In earlier studies, a correlation between increased overexpression of ramA and acrAB and decreased susceptibility to tigecycline was demonstrated in K. pneumoniae.7 In this study, transcriptional profile analysis and RT-PCR both demonstrated that increased expression of marA in G5048 and G5049 correlated with the overexpression of the acrAB transcript.
MarR has been established as one of the central regulators of the mar operon. DNA sequencing of the marR gene in this study revealed a frameshift mutation in the less-susceptible strains G5048 and G5049, which serves as a likely explanation for the overexpression of both marA and acrAB genes observed in these strains. This is in agreement with previous studies, which described the effect of mutations in marR that increased the MICs of chloramphenicol, tetracycline and cefuroxime.25,26
Transcriptional profile analysis was undertaken in order to determine genes that might play a role in decreased susceptibility to tigecycline in E. coli. Many of the genes that were up-regulated in G5048 and G5049 corresponded to the genes that responded to either constitutive overexpression of marA or stimulation by paraquat as previously reported.27,28 In addition to the mar and acr loci, other up-regulated genes included nfsA (mdaA), rimK and ybjC, genes that have been reported to comprise a single operon whose function is related to oxidative stress.29 The activator of this operon is SoxS, a transcriptional activator belonging to the same XylS/AraC family of activators as MarA. It may not be surprising that genes activated by SoxS can also be up-regulated by MarA, since the two activators share a 41% protein identity.29 It should be noted that, the data presented here did not show an up-regulation of the ORF b0853, which is another putative member of the nfsA, rimK and ybjC operon.
Among the genes that were down-regulated in G5048 and G5049 were ompF, encoding an outer membrane porin, and fimA, a gene associated with adherence to host tissue. A correlation between decreased expression level of ompF and antibiotic resistance has been described previously in E. coli.5,27 The down-regulation of these two genes in response to either the constitutive overexpression of marA or stimulation by paraquat has been described previously.27,28 The altered expression level of several other genes, however, including those of the ymc and ycc gene clusters, has not been noted earlier. These genes may potentially have a role in the modification of the carbohydrate exterior of the cell. A recent study by Peleg et al.30 grouped several of the ymc and ycc genes into the G4C operon and proposed that they have a role in the formation of the O-antigen capsule. In addition, Paulson et al. described a similarity between etk, a tyrosine kinase member of this operon, to other members of the MPA1 family, which may function in the export of complex carbohydrates in bacteria.31,32 Whether the G4C operon is down-regulated in response to an up-regulation of marA remains to be determined. Other studies have noted that MarA may repress the transcription of genes such as purA and hdeA and that modification of the exopolysaccharide layer can occur in association with the efflux mechanism.33,34
It should be noted that because expression analyses of G5048 and G5049 demonstrated an overexpression of genes of the mar and acr operons, targeted mapping of these sites in the IS903kan transposon mutants was accomplished using PCR and DNA sequencing. The majority of transposon insertions mapped to either marA or acrB confirming the involvement of MarA and AcrAB in the decreased tigecycline susceptibility in E. coli. However, three of the transposon insertions did not map to either the marRAB or the acrAB operon. Further studies are in progress to map these mutations and elucidate their effect on tigecycline susceptibility.
In summary, the results suggested that marA overexpression is a key factor that leads to overproduction of the AcrAB multidrug efflux pump and, consequently, to decreased tigecycline susceptibility in E. coli.
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Funding |
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Wyeth Research provided internal funding for this study.
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All authors are employees of Wyeth and own shares in the company.
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Acknowledgements |
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We thank David Figurski for providing plasmid pVJT128. We also thank Jan Kieleczawa and Katarzyna Bajson for DNA sequencing and Peter Petersen for assistance with MIC testing.
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References |
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Visalli MA, Murphy E, Projan SJ, et al. AcrAB multidrug efflux pump is associated with reduced levels of susceptibility to tigecycline (GAR-936) in Proteus mirabilis. Antimicrob Agents Chemother (2003) 47:665–9.
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17
Thomson VJ, Bhattacharjee MK, Fine DH, et al. Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans. J Bacteriol (1999) 181:7298–307.
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