• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
Antimicrob Agents Chemother. Oct 2010; 54(10): 4389–4393.
Published online Aug 9, 2010. doi:  10.1128/AAC.00155-10
PMCID: PMC2944555

Overexpression of Resistance-Nodulation-Cell Division Pump AdeFGH Confers Multidrug Resistance in Acinetobacter baumannii[down-pointing small open triangle]

Abstract

Acinetobacter baumannii is a major nosocomial pathogen which frequently develops multidrug resistance by acquisition of antibiotic resistance genes and overexpression of intrinsic efflux systems, such as the RND efflux pumps AdeABC and AdeIJK. A third RND system was characterized by studying spontaneous mutants BM4663 and BM4664, which were selected in the presence of chloramphenicol and norfloxacin, respectively, from the AdeABC- and AdeIJK-defective derivative A. baumannii BM4652. They exhibited enhanced resistance to fluoroquinolones, tetracycline-tigecycline, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, sodium dodecyl sulfate, and dyes such as ethidium bromide, safranin O, and acridine orange. Comparison of transcriptomes of mutants with that of their parental strain, using a microarray technology, demonstrated the overexpression of three genes that encoded an RND efflux system, named AdeFGH. Inactivation of AdeFGH in BM4664 restored an antibiotic susceptibility profile identical to that of BM4652, indicating that AdeFGH was cryptic in BM4652 and responsible for multidrug resistance in its mutants. RNA analysis demonstrated that the three genes were cotranscribed. The adeFGH operon was found in 36 out of 40 A. baumannii clinical isolates, but none of the 22 isolates tested overexpressed the pump genes. Spontaneous MDR mutant BM4684, overexpressing adeFGH, was obtained from clinical isolate BM4587, indicating that adeFGH can be overexpressed in a strain harboring adeABC-adeIJK. An open reading frame, coding a LysR-type transcriptional regulator, named adeL, was located upstream from the adeFGH operon and transcribed in the opposite direction. Mutations in adeL were found in the three adeFGH-overexpressing mutants, suggesting that they were responsible for overexpression of AdeFGH.

Multidrug resistance by overexpression of chromosomally encoded efflux systems is common in Gram-negative bacteria, most often associated with members of the resistance-nodulation-cell division (RND) superfamily (20). These pumps consist of an efflux membrane transporter (RND), which interacts with a major fusion protein (MFP) and an outer membrane factor (OMF) for drug export through both the inner and the outer membranes. The RND systems are involved in various functions, including homeostasis of the cell, export of virulence determinants, and extrusion of toxic compounds (19). They generally exhibit a broad substrate spectrum, and their overexpression can diminish the activity of several unrelated drug classes.

Acinetobacter baumannii is typically associated with hospital-acquired infections and possesses an extraordinary propensity to develop multiresistance (6). In addition to acquired resistance determinants, carried by plasmids, transposons, or resistance islands (18), two RND efflux systems, AdeABC (15) and AdeIJK (3), have been reported to confer a multidrug-resistant (MDR) phenotype to the species. The adeABC operon is found in ca. 80% of clinical isolates and is naturally cryptic due to tight regulation of its expression by the two-component system AdeRS (16). Overexpression of AdeABC can result from point mutations in AdeR-AdeS (16) or from the insertion of an ISAba1 copy upstream from the operon (22). AdeIJK is constitutively expressed at low levels, leading to a weak decrease in activity of some antibiotics (3), but its overexpression can contribute to multidrug resistance (2). Three additional efflux systems belonging to other superfamilies have been described in A. baumannii: CraA, a pump of the major facilitator superfamily that confers intrinsic chloramphenicol resistance (21), AbeS, a small drug resistance efflux pump mostly involved in resistance to detergents and dyes (26), and AbeM, a system of the multidrug and toxic compound extrusion family (27). However, the role of AbeM in antibiotic resistance remains to be determined.

An oligonucleotide-based DNA microarray has been developed to both quantify expression of chromosomal genes for efflux systems and detect acquired resistance determinants in A. baumannii (2). This powerful tool has been successfully used to detect overexpression of AdeABC and AdeIJK efflux pumps in isogenic mutants and allows study of clinical MDR strains by comparative analysis of their transcriptome relative to that of a reference strain. This is of particular interest because of the difficulty in evaluating the contribution of efflux to antibiotic resistance in clinical isolates. The microarray also enabled us to identify the role of a new RND efflux pump, AdeFGH, in conferring multidrug resistance in a single-step mutant obtained from the AdeABC- and AdeIJK-defective strain BM4652 (2). The aim of this work was to characterize the AdeFGH pump and the involvement of AdeL, a LysR-type transcriptional regulator, in its overexpression.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and antibiotic susceptibility testing.

The bacterial strains used in this study are listed in Table Table1.1. Cells were grown at 37°C in brain heart infusion (BHI) broth and agar (Difco Laboratories, Detroit, MI). Antibiotic susceptibility was tested by disk diffusion on Mueller-Hinton agar (Bio-Rad, Marnes-la-Coquette, France), and MICs were determined by the Etest procedure (AB Biodisk, Solna, Sweden). Susceptibility to ethidium bromide, safranin O, acridine orange, and sodium dodecyl sulfate (SDS) was screened for on BHI agar plates containing gradients of each drug (13).

TABLE 1.
Bacterial strains and plasmids used in this study

Selection of MDR mutants.

BM4652, in which both the AdeABC and AdeIJK systems are inactivated (3), and clinical isolate BM4587 (2) were grown on plates containing a gradient of cefotaxime, chloramphenicol, ciprofloxacin, erythromycin, imipenem, norfloxacin, rifampin, tetracycline, ticarcillin, or trimethoprim (28). Mutants were tested for antibiotic susceptibility by agar diffusion, and those resistant to several drug classes were selected for further analysis. MDR mutants BM4663 and BM4664 were obtained from BM4652 on chloramphenicol and norfloxacin, respectively, and BM4684 from BM4587 on chloramphenicol (Table (Table11).

DNA manipulations.

A. baumannii genomic DNA was extracted as described previously (24). DNA amplification was performed in a GeneAmp PCR system 9700 (Perkin-Elmer Cetus, Norwalk, CT) with Taq (MPbio, Illkirch, France) or Phusion (Finnzymes, Espoo, Finland) DNA polymerase. PCR elongation times and temperatures were adjusted according to the expected size of the PCR products and the nucleotide sequences of the primers (Table (Table2),2), respectively. Sequence determination was carried out with a CEQ 2000 DNA analysis system automatic sequencer (Beckman Instruments, Inc., Palo Alto, CA).

TABLE 2.
Oligonucleotides used in this study

RNA analysis.

A. baumannii total RNA was isolated from exponentially growing cells (optical density at 600 nm [OD600], 0.8 to 1.2) as described previously (2). cDNA synthesis was performed with avian myeloblastosis virus (AMV) reverse transcriptase (Roche Diagnostic GmbH, Mannheim, Germany) and real-time PCR using the SYBR PCR master kit (Roche) and gene-specific primers, 0.5 mM each (Table (Table2),2), according to the manufacturer's instructions. Amplification and detection of specific products were performed using the LightCycler sequence detection system (Roche) with the following cycle profile: 1 cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 58°C for 10 s, and 72°C for 30 s. Each experiment was performed in triplicate.

Microarray experiment.

A microarray developed to quantify the expression of genes for putative efflux systems in A. baumannii was used for transcriptomic study (2). cDNAs of BM4664 and BM4652 were synthesized from 10 μg of total RNA, labeled with Cy3 or Cy5 cyanin (GE Healthcare, Uppsala, Sweden) using the SuperScript indirect cDNA labeling system (Invitrogen), mixed at equal concentrations (250 pmol of cyanin), and hybridized on the microarray. Slides were scanned (GenePix 4000 A scanner; Axon), and data were analyzed as described previously (2). Three independent biological replicates and a dye swap were performed, leading to six hybridizations per experiment. A spot was considered differentially expressed when the Bonferroni method-adjusted P value was lower than 0.05.

Insertion-inactivation of adeFGH.

Fragments internal to the adeF and adeH genes from BM4652 were amplified with primers F1/F2 and H1/H2, respectively (Table (Table2).2). The first 27 and 25 bp of F2 and H1, respectively, are complementary to an ant(3″)-Ia streptomycin-spectinomycin resistance cassette so that the adeF and adeH fragments when mixed with the ant(3″)-Ia cassette allow amplification of an adeF-ant(3″)-Ia-adeH sequence using primers F1 and H2 (Fig. (Fig.1).1). F1 and H2 possess a BamHI and an EcoRI restriction site, respectively. The adeF-ant(3″)-Ia-adeH fragment digested by both enzymes was ligated into BamHI-EcoRI-linearized pUC18, generating plasmid pAT520, which was introduced into competent cells of strain BM4664 by electrotransformation. Since pAT520 does not stably replicate in A. baumannii, the transformants selected for streptomycin-spectinomycin resistance and screened for ticarcillin susceptibility should result from a double recombination event leading to partial deletion-inactivation of adeFGH. The genetic construction was confirmed by PCR and sequencing of the BM4579 transformant.

FIG. 1.
Schematic representation of the adeFGH operon of A. baumannii BM4664 and derivative BM4679. Open arrows indicate coding sequences and direction of transcription. The streptomycin-spectinomycin ant(3")-Ia resistance gene is hatched. Black arrows indicate ...

RESULTS AND DISCUSSION

Substrate specificity of AdeFGH pump.

Spontaneous MDR BM4663 and BM4664 were selected on chloramphenicol and norfloxacin, respectively, from BM4652 (ΔadeABC ΔadeIJK), which had been derived from BM4454 by inactivation of RND efflux systems AdeABC and AdeIJK (3). The mutants had a similar phenotype, i.e., high-level resistance to chloramphenicol, clindamycin, fluoroquinolones, and trimethoprim and decreased susceptibility to tetracycline-tigecycline and sulfonamides (Table (Table3);3); susceptibility to β-lactams, erythromycin, and rifampin was not affected. The impact against most aminoglycosides could not be assessed due to the presence of a kanamycin or an apramycin resistance cassette to inactivate the adeABC and adeIJK gene clusters (3). However, the activity of amikacin, which is not affected by the corresponding APH(3′)-I and AAC(3)-VI enzymes, remained unchanged, suggesting that aminoglycosides are not substrates for the pump. BM4663 and BM4664 also demonstrated, compared with parental strain BM4652, increased resistance to ethidium bromide, safranin O, acridine orange, and SDS (data not shown). Furthermore, the mutants exhibited a higher level of resistance to trimethoprim and sulfamethoxazole than BM4454, which possesses AdeIJK and overexpresses AdeABC (15).

TABLE 3.
Antibiotic susceptibility of A. baumannii strains

Overexpression of AdeFGH in MDR mutants.

Multidrug resistance resulting from in vitro single-step selection can be due either to a decrease in membrane permeability or, more likely, to increased efflux. In addition, diminished susceptibility to tigecycline is known to result from overexpression of RND efflux systems in A. baumannii (22), Pseudomonas aeruginosa (4), and enterobacteria (8, 23). Comparison of the transcriptome of BM4664 with that of BM4652, using a microarray which enables quantification of the expression of all putative efflux genes in A. baumannii (2), revealed overexpression in BM4664 of three adjacent open reading frames (ORFs) annotated as encoding putative MFP (CT025800) (2.9-fold increase), RND efflux protein (CT025801) (3.8-fold), and OMF (CT025802) (3.1-fold) (7). The microarray did not detect any other gene differentially expressed. Overexpression of the three ORFs, respectively named adeF, adeG, and adeH, was confirmed in BM4663 and BM4664 by quantitative reverse transcriptase PCR (RT-PCR) using primers F3/F4, G2/G3, and H3/H4 (Table (Table2).2). Increases of more than 300-fold and 200-fold in adeFGH expression in BM4663 and BM4664, respectively, were observed. The adeFGH operon was thus considered responsible for the MDR phenotype of the mutants.

A third spontaneous mutant, BM4684, was selected on chloramphenicol from clinical strain BM4587, which harbors functional adeABC and adeIJK efflux gene clusters (2). BM4684 had an MDR phenotype similar to those of BM4663 and BM4664 (Table (Table3),3), and quantitative RT-PCR indicated overexpression of more than 200-fold of the adeFGH genes whereas expression of adeABC or adeIJK remained unchanged. These data indicate that AdeFGH-mediated resistance can be selected in a strain containing adeABC and adeIJK.

Relationship of AdeFGH with other efflux systems.

Comparison of the 407-, 1,060-, and 484-amino acid sequences of AdeF, AdeG, and AdeH, respectively, with those in the GenBank database revealed a low degree of identity with AdeABC and AdeIJK (<40%) and a high degree of identity with putative efflux transporters and already-characterized RND systems, in particular BpeEF-OprC from Burkholderia pseudomallei (55, 79, and 53% for the MFP, RND, and OMF proteins, respectively) and CeoAB-OpcM from Burkholderia cenocepacia (54, 79, and 50%). BpeEF-OprC extrudes chloramphenicol and trimethoprim (11), whereas CeoAB-OpcM exports, in addition, ciprofloxacin (17). These substrate ranges are consistent with the phenotypes of BM4663, BM4664, and BM4684, all of which were highly resistant to chloramphenicol, trimethoprim, and ciprofloxacin. Transmembrane topology of the AdeG transporter was predicted with the TMHMM program of the ExPASy server. As expected, AdeG contained 12 transmembrane domains (TMDs) and two large periplasmic loops between TMDs 1 and 2 and TMDs 7 and 8, typical of RND proteins.

Inactivation of adeFGH gene cluster in BM4664.

To confirm the role of AdeFGH overexpression in multidrug resistance of BM4664, the pump was inactivated by partial replacement of the structural genes for the system with an ant(3")-Ia streptomycin-spectinomycin resistance cassette following homologous recombination (Fig. (Fig.1).1). The double crossing-over was confirmed by PCR and sequencing of derivative BM4679 (ΔadeABC ΔadeIJK ΔadeFGH) (Fig. (Fig.1).1). BM4679 had restored susceptibility to drugs and dyes that are substrates for AdeFGH, confirming the contribution of AdeFGH to multidrug resistance in BM4664 and the broad-substrate spectrum of the pump (Table (Table3).3). Furthermore, BM4679 (ΔadeABC ΔadeIJK ΔadeFGH) had the same level of resistance as BM4652 (ΔadeABC ΔadeIJK), indicating that AdeFGH is not expressed in BM4652 and thus does not contribute to intrinsic resistance of A. baumannii (Table (Table3),3), an observation confirmed by quantitative RT-PCR (data not shown).

Transcriptional analysis of the adeFGH gene cluster.

Transcription of the adeFGH gene cluster was analyzed by RT-PCR and Northern hybridization. Products with the expected size were obtained by amplification of adeFG, adeGH, and adeFGH overlapping fragments from cDNA of BM4664, using primers F3/G3, G2/H4, and F3/H2, respectively, whereas no amplification was obtained for the adeLF overlapping fragment using primers L2/F4 (Table (Table2).2). Genes of RND efflux systems are typically organized in operons, and our data indicate that the adeFGH genes were cotranscribed.

Involvement of adeL in expression of the adeFGH operon.

An ORF for a putative LysR-type transcriptional regulator (LTTR) and named adeL was located upstream from the adeFGH operon and transcribed in the opposite direction (Fig. (Fig.1).1). The 337-amino-acid deduced sequence of AdeL exhibited homology with those of other LTTRs, in particular the CeoR regulator (56% identity). Thus, the AdeL-AdeFGH system appears to have an organization similar to that of the CeoR-CeoAB-OpcM efflux system from B. cenocepacia (17). The HelixTurnHelix program (http://mobyle.pasteur.fr/) predicted the presence of a helix-turn-helix (HTH) DNA-binding motif between residues 11 and 32, typical of the LTTR family. Sequence analysis of the adeL-adeF intergenic region using BProm software (Softberry, Inc., Mt. Kisco, NY) predicted the presence of overlapping promoters for adeL and adeFGH expression (Fig. (Fig.2).2). This region includes a TTA-N7-TAA motif typical of a LTTR box which is implicated in DNA binding by LTTR (14).

FIG. 2.
Analysis of the adeL-adeF intergenic region. Start codons of adeL and adeF genes are in bold; arrows represent transcripts and indicate direction of transcription; promoters for adeL and adeFGH, predicted by the BProm software, are indicated on each strand. ...

The sequences of adeL and of the putative promoter region of adeFGH in the three mutants were compared with those of the parental strain. No mutations were detected in the putative promoter region or in the LTTR box upstream from the adeFGH operon, but mutations in adeL were found in both strains. In BM4663, a thymidine was inserted at position 981 of adeL, introducing a premature stop codon resulting in deletion of the 11 C-terminal residues, whereas an A-to-C mutation leading to a valine-to-glycine substitution at position 139 of AdeL was present in BM4664. A threonine-to-lysine substitution was detected at position 319 of AdeL in BM4684. Mutations in the C-terminal region of LTTRs have been reported to confer a constitutive phenotype (10, 12) by alteration of oligomerization and interaction with the RNA polymerase (5). Thus, deletion of the 11 C-terminal residues and a threonine-to-lysine substitution in BM4684 of AdeL could be responsible for overexpression of adeFGH in BM4663 and BM4684, respectively. In BM4664, the V139G substitution occurred in the putative signal recognition domain of AdeL and probably conferred, as already reported for several LTTRs (14), a signal-independent phenotype.

Distribution and expression of the adeFGH operon in clinical isolates.

The structural genes for the pump and the putative regulator are present and conserved in the genomes of the seven sequenced A. baumannii strains (1, 9, 25, 29). The adeG gene was detected by PCR using primers G1/G3 in 40 out of 44 A. baumannii strains from our laboratory collection. In the four adeG-negative strains, PCR using L2/L1, F1/F4, and H 1/H4 (Table (Table22 and Fig. Fig.1)1) showed the presence of adeL and adeF whereas adeH was lacking. These data suggest that the AdeFGH system is intrinsic to the A. baumannii species, although adeGH has been lost for some.

AdeFGH conferred high-level resistance to clindamycin when overexpressed. Quantitative RT-PCR was performed to measure the level of expression of adeG in isolates exhibiting high-level resistance to clindamycin. None of the 22 selected clinical strains expressed the efflux gene, indicating that AdeFGH is cryptic in A. baumannii. However, the number of isolates studied is too low to determine the incidence of AdeFGH-mediated multidrug resistance in A. baumannii.

Increased expression of AdeFGH in A. baumannii is an additional mechanism for high-level resistance to fluoroquinolones and decreased susceptibility to tigecycline and is therefore of a clinical relevance. The presence of the adeFGH operon in ca. 90% of the strains studied and overexpression likely due to point mutation in adeL suggest that this event could occur in clinical strains. Molecular and biochemical studies of the transcriptional regulator AdeL should allow better understanding of the mechanism of AdeFGH expression in A. baumannii.

Acknowledgments

We thank G. Guigon for assistance with the microarray, S. Brémont for construction of pAT520, and P. E. Reynolds for reading the manuscript.

S.C. was the recipient of a fellowship from the Fondation pour la Recherche Médicale.

Footnotes

[down-pointing small open triangle]Published ahead of print on 9 August 2010.

REFERENCES

1. Adams, M. D., K. Goglin, N. Molyneaux, K. M. Hujer, H. Lavender, J. J. Jamison, I. J. MacDonald, K. M. Martin, T. Russo, A. A. Campagnari, A. M. Hujer, R. A. Bonomo, and S. R. Gill. 2008. Comparative genome sequence analysis of multidrug-resistant Acinetobacter baumannii. J. Bacteriol. 190:8053-8064. [PMC free article] [PubMed]
2. Coyne, S., G. Guigon, P. Courvalin, and B. Périchon. 2010. Screening and quantification of the expression of antibiotic resistance genes in Acinetobacter baumannii with a microarray. Antimicrob. Agents Chemother. 54:333-340. [PMC free article] [PubMed]
3. Damier-Piolle, L., S. Magnet, S. Bremont, T. Lambert, and P. Courvalin. 2008. AdeIJK, a resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter baumannii. Antimicrob. Agents Chemother. 52:557-562. [PMC free article] [PubMed]
4. Dean, C. R., M. A. Visalli, S. J. Projan, P. E. Sum, and P. A. Bradford. 2003. Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1. Antimicrob. Agents Chemother. 47:972-978. [PMC free article] [PubMed]
5. Deghmane, A. E., and M. K. Taha. 2003. The Neisseria meningitidis adhesion regulatory protein CrgA acts through oligomerization and interaction with RNA polymerase. Mol. Microbiol. 47:135-143. [PubMed]
6. Dijkshoorn, L., A. Nemec, and H. Seifert. 2007. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5:939-951. [PubMed]
7. Fournier, P. E., D. Vallenet, V. Barbe, S. Audic, H. Ogata, L. Poirel, H. Richet, C. Robert, S. Mangenot, C. Abergel, P. Nordmann, J. Weissenbach, D. Raoult, and J. M. Claverie. 2006. Comparative genomics of multidrug resistance in Acinetobacter baumannii. PLoS Genet. 2:e7. [PMC free article] [PubMed]
8. Hirata, T., A. Saito, K. Nishino, N. Tamura, and A. Yamaguchi. 2004. Effects of efflux transporter genes on susceptibility of Escherichia coli to tigecycline (GAR-936). Antimicrob. Agents Chemother. 48:2179-2184. [PMC free article] [PubMed]
9. Iacono, M., L. Villa, D. Fortini, R. Bordoni, F. Imperi, R. J. Bonnal, T. Sicheritz-Ponten, G. De Bellis, P. Visca, A. Cassone, and A. Carattoli. 2008. Whole-genome pyrosequencing of an epidemic multidrug-resistant Acinetobacter baumannii strain belonging to the European clone II group. Antimicrob. Agents Chemother. 52:2616-2625. [PMC free article] [PubMed]
10. Kim, J., J. Oh, O. Choi, Y. Kang, H. Kim, E. Goo, J. Ma, T. Nagamatsu, J. S. Moon, and I. Hwang. 2009. Biochemical evidence for ToxR and ToxJ binding to the tox operons of Burkholderia glumae and mutational analysis of ToxR. J. Bacteriol. 191:4870-4878. [PMC free article] [PubMed]
11. Kumar, A., K. L. Chua, and H. P. Schweizer. 2006. Method for regulated expression of single-copy efflux pump genes in a surrogate Pseudomonas aeruginosa strain: identification of the BpeEF-OprC chloramphenicol and trimethoprim efflux pump of Burkholderia pseudomallei 1026b. Antimicrob. Agents Chemother. 50:3460-3463. [PMC free article] [PubMed]
12. Lang, G. H., and N. Ogawa. 2009. Mutational analysis of the inducer recognition sites of the LysR-type transcriptional regulator TfdT of Burkholderia sp. NK8. Appl. Microbiol. Biotechnol. 83:1085-1094. [PubMed]
13. Lebel, S., S. Bouttier, and T. Lambert. 2004. The cme gene of Clostridium difficile confers multidrug resistance in Enterococcus faecalis. FEMS Microbiol. Lett. 238:93-100. [PubMed]
14. Maddocks, S. E., and P. C. Oyston. 2008. Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 154:3609-3623. [PubMed]
15. Magnet, S., P. Courvalin, and T. Lambert. 2001. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrob. Agents Chemother. 45:3375-3380. [PMC free article] [PubMed]
16. Marchand, I., L. Damier-Piolle, P. Courvalin, and T. Lambert. 2004. Expression of the RND-type efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS two-component system. Antimicrob. Agents Chemother. 48:3298-3304. [PMC free article] [PubMed]
17. Nair, B. M., K. J. Cheung, Jr., A. Griffith, and J. L. Burns. 2004. Salicylate induces an antibiotic efflux pump in Burkholderia cepacia complex genomovar III (B. cenocepacia). J. Clin. Invest. 113:464-473. [PMC free article] [PubMed]
18. Perez, F., A. M. Hujer, K. M. Hujer, B. K. Decker, P. N. Rather, and R. A. Bonomo. 2007. Global challenge of multidrug-resistant Acinetobacter baumannii. Antimicrob. Agents Chemother. 51:3471-3484. [PMC free article] [PubMed]
19. Piddock, L. J. 2006. Multidrug-resistance efflux pumps—not just for resistance. Nat. Rev. Microbiol. 4:629-636. [PubMed]
20. Poole, K. 2004. Efflux-mediated multiresistance in Gram-negative bacteria. Clin. Microbiol. Infect. 10:12-26. [PubMed]
21. Roca, I., S. Marti, P. Espinal, P. Martinez, I. Gibert, and J. Vila. 2009. CraA: an MFS efflux pump associated with chloramphenicol resistance in Acinetobacter baumannii. Antimicrob. Agents Chemother. 53:4013-4014. [PMC free article] [PubMed]
22. Ruzin, A., D. Keeney, and P. A. Bradford. 2007. AdeABC multidrug efflux pump is associated with decreased susceptibility to tigecycline in Acinetobacter calcoaceticus-Acinetobacter baumannii complex. J. Antimicrob. Chemother. 59:1001-1004. [PubMed]
23. Ruzin, A., M. A. Visalli, D. Keeney, and P. A. Bradford. 2005. Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae. Antimicrob. Agents Chemother. 49:1017-1022. [PMC free article] [PubMed]
24. Sambrook, J., and D. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
25. Smith, M. G., T. A. Gianoulis, S. Pukatzki, J. J. Mekalanos, L. N. Ornston, M. Gerstein, and M. Snyder. 2007. New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. Genes Dev. 21:601-614. [PMC free article] [PubMed]
26. Srinivasan, V. B., G. Rajamohan, and W. A. Gebreyes. 2009. Role of AbeS, a novel efflux pump of the SMR family of transporters, in resistance to antimicrobial agents in Acinetobacter baumannii. Antimicrob. Agents Chemother. 53:5312-5316. [PMC free article] [PubMed]
27. Su, X. Z., J. Chen, T. Mizushima, T. Kuroda, and T. Tsuchiya. 2005. AbeM, an H+-coupled Acinetobacter baumannii multidrug efflux pump belonging to the MATE family of transporters. Antimicrob. Agents Chemother. 49:4362-4364. [PMC free article] [PubMed]
28. Szybalski, W., and V. Bryson. 1952. Genetic studies on microbial cross resistance to toxic agents. I. Cross resistance of Escherichia coli to fifteen antibiotics. J. Bacteriol. 64:489-499. [PMC free article] [PubMed]
29. Vallenet, D., P. Nordmann, V. Barbe, L. Poirel, S. Mangenot, E. Bataille, C. Dossat, S. Gas, A. Kreimeyer, P. Lenoble, S. Oztas, J. Poulain, B. Segurens, C. Robert, C. Abergel, J. M. Claverie, D. Raoult, C. Medigue, J. Weissenbach, and S. Cruveiller. 2008. Comparative analysis of Acinetobacters: three genomes for three lifestyles. PLoS One 3:e1805. [PMC free article] [PubMed]
30. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119. [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...