JAC Advance Access originally published online on March 15, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):1001-1004; doi:10.1093/jac/dkm058
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AdeABC multidrug efflux pump is associated with decreased susceptibility to tigecycline in Acinetobacter calcoaceticus–Acinetobacter baumannii complex
Department of Infectious Disease, Wyeth Research, Pearl River, NY 10965, USA
* Corresponding author. Tel: +1-845-602-4592; Fax: +1-845-602-5671; E-mail: ruzina{at}wyeth.com
Received 25 October 2006; returned 5 December 2006; revised 21 December 2006; accepted 5 February 2007
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Abstract |
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Objectives: To investigate the role of the AdeABC multidrug efflux pump in the decreased susceptibility of clinical isolates of Acinetobacter calcoaceticus–Acinetobacter baumannii complex to tigecycline.
Methods: Gene expression was analysed by Taqman RT-PCR. A single cross-over achieved insertional inactivation of the adeB gene with a suicide plasmid construct carrying an adeB fragment obtained by PCR. Analysis of the adeRS locus was performed by PCR and sequencing. Ribotyping was performed with the RiboPrinter system. MICs were determined by Etest.
Results: Expression analysis revealed constitutive overexpression of adeABC in less-susceptible clinical isolates G5139 and G5140 (tigecycline MIC = 4 mg/L) when compared with the isogenic clinical isolates G4904 and G5141 (MIC = 1.5 mg/L). Insertional mutants GC7945 (adeB knockout in G5139) and GC7951 (adeB knockout in G5140) were obtained, which resulted in tigecycline MICs of 0.5 mg/L. As reported previously, the expression of adeABC is regulated by the two-component signalling system encoded by the adeR and adeS genes. PCR and sequencing analyses revealed an insertion of an ISABA-1 element in the adeS gene of G5139 and G5140.
Conclusions: The results of this study suggest that decreased susceptibility to tigecycline in the A. calcoaceticus–A. baumannii complex is associated with the overexpression of the AdeABC multidrug efflux pump.
Keywords: Acinetobacter , antibiotic resistance , efflux pump expression , RT-PCR , gene inactivation
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Introduction |
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Tigecycline is a novel expanded broad-spectrum glycylcycline antibiotic. Unlike the tetracyclines, the activity of tigecycline is not affected by classical tetracycline resistance mechanisms including ribosomal protection and efflux by tetracycline-specific pumps.1 Earlier studies showed that decreased tigecycline susceptibility is associated with constitutive overexpression of the components of multidrug efflux systems such as MexXY in Pseudomonas aeruginosa2 and AcrAB in Proteus mirabilis,3 Klebsiella pneumoniae,4 Morganella morganii5 and Enterobacter cloacae.6 These pumps are often associated with multidrug resistance (MDR).
The Acinetobacter calcoaceticus–A. baumannii complex is commonly resistant to multiple antibiotics including ß-lactams, aminoglycosides and quinolones. These bacterial species are generally susceptible to tigecycline; however, a few clinical strains with decreased tigecycline susceptibility have been isolated.
The AdeABC efflux pump of A. baumannii confers resistance to various classes of antibiotics.7–9 This pump belongs to the resistance–nodulation–division family, which also includes other multidrug efflux transporters such as MexXY and AcrAB, which have previously been shown to be associated with tigecycline resistance in Enterobacteriaceae and P. aeruginosa. As reported previously, sequence analysis of the adeB gene has a potential to be used for typing of MDR A. baumannii strains.10 This study was performed to investigate the role of the AdeABC pump in decreased susceptibility to tigecycline in clinical isolates of the A. calcoaceticus–A. baumannii complex.
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Materials and methods |
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Bacterial strains and growth conditions
Bacterial strains used in this study are shown in Table 1. Strains G4904, G5139, G5140 and G5141 are clinical isolates that were obtained from a single patient from Ohio, USA, with an intra-abdominal infection. They were determined to represent an isogenic series by ribotyping. Strains GC7945 and GC7951 are adeB-insertional knockout mutants of G5139 and G5140, respectively. The strains were propagated at 37°C in Luria–Bertani (LB) broth or agar. The identification of the clinical isolates was performed with the Vitek system (Biomerieux). Phenotypic identification systems such as this cannot distinguish between A. baumannii and A. calcoaceticus. Therefore, the strains in this study are designated as the A. calcoaceticus–A. baumannii complex.
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Ribotyping
Ribotyping was performed by using the RiboPrinter microbial characterization system (Qualicon, Wilmington, DE, USA) according to the manufacturer's instructions. Each isolate was analysed with two restriction enzymes, EcoRI and PvuII.
Antibiotic susceptibility testing
The MICs of tigecycline, gentamicin, chloramphenicol and levofloxacin were determined by Etest (AB BIODISK, Piscataway, NJ, USA) according to the manufacturer's instructions. Bacterial suspensions equivalent to a 0.5 McFarland standard were prepared by using a BBL Prompt inoculation system (Becton Dickinson, Sparks, MD, USA), plated on Mueller–Hinton agar plates (Becton Dickinson) and overlaid with Etest strips. MIC values were determined after incubation of the plates at 35°C for 16 h.
Standard DNA manipulations such as restriction enzyme 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. The A. calcoaceticus–A. baumannii complex genomic DNA was isolated by using the Puregene tissue kit (Gentra Systems Inc., Minneapolis, MN, USA) and used as a template for PCR reactions. The following primers were used for PCR amplifications: (i) cloning of adeB fragment, forward, 5'-CTAGGTACCGTATGAATTGATGCTGC-3' and reverse, 5'-CTAGGTACCACTCGTAGCCAATACC-3'; (ii) insertion analysis of adeB, forward, 5'-TTACGCTGGTATTGGC-3' and reverse, 5'-CTTTCATAGAGTGCAGCC-3'; (iii) sequence analysis of adeRS, forward, 5'-AAGACAGCTTGGGATCAGG-3' and reverse, 5'-ACACTGACTTTAGCCG; (iv) cloning of tet(M), forward, 5'-TACAAATATGCTCTTACG-3' and reverse, 5'-TTTCATCTTATTTAATAAGAAACC-3'. PCR fragments were gel-purified by using a Zymoclean Gel DNA Recovery kit (Zymo Research, Orange, CA, USA). The nucleotide sequence was determined with an ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA).
Insertional inactivation of adeB gene
A DNA fragment containing a functional tet(M) gene was amplified from Staphylococcus aureus Mu3 strain12 by PCR and was ligated into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA). The resulting plasmid, pCLL3468, was modified by cloning a PCR-amplified 981 bp internal fragment of the adeB gene into the KpnI site of pCLL3468. The resulting plasmid, pCLL3469, was transformed into G5139 and G5140, and the transformants were selected on LB plates containing 50 mg/L kanamycin and 8 mg/L minocycline. Insertion of pCLL3469 into the adeB gene was confirmed by PCR analysis and sequencing. The PCR analysis was done as described previously using M13 reverse and M13 (–20) forward primers and two internal primers specific for adeB.7
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 (Huntsville, AL, USA). The following primers and probes were used: (i) adeA-specific, forward, 5'-TTGATCGTGCTTCTATTCCTCAAG-3' and reverse, 5'-GGCTCGCCACTGATATTACGTT-3'; fluorescent probe, 5'-CTATTGGTTCCGGCGCAAGCGA-3'; (ii) 16S rRNA gene-specific, forward, 5'-TCGCTAGTAATCGCGGATCA-3' and reverse, 5'-GACGGGCGGTGTGTACAAG-3'; fluorescent probe, 5'-TGCCGCGGTGAATACGTTCC-3'. 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-carboxytetramethylrhodamine (TAMRA) at the 3' end. DNase-treated RNA templates were prepared from the mid-log phase bacterial cultures by using an RNAeasy kit (Qiagen, Valencia, CA, USA). RT-PCR was performed by using an iScript One-Step RT-PCR Kit for Probes (Bio-Rad) on an 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–200 mg/L), 6.85 µL of nuclease-free water (Ambion, Austin, TX, USA), 12.5 µL of RT-PCR reaction 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 (adeA) was performed by iCycler iQ5TM software using a normalized expression analysis method; the 16S rRNA gene served as a reference gene and G5141 served as a reference condition. Each sample was run in duplicate.
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Results and discussion |
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To investigate whether the AdeABC pump plays a role in decreased susceptibility to tigecycline in clinical isolates of the A. calcoaceticus–A. baumannii complex, the expression of adeA was analysed by Taqman RT-PCR. As shown in Figure 1, increased expression of adeA was observed in the less-susceptible strains. Quantitative analysis revealed that expression of adeA increased 27-fold and 37-fold in G5140 and G5139, respectively, when compared with G4904 and G5141. Because adeA, adeB and adeC genes are co-transcribed,8 the RT-PCR analysis suggested that all of the components of the AdeABC pump were overexpressed in the less-susceptible strains.
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As decreased susceptibility of the A. calcoaceticus–A. baumannii complex to tigecycline correlated with overexpression of the adeABC locus, insertional inactivation of the adeB gene was performed to further assess the role of the AdeABC pump. The strategy for insertional inactivation was based on two factors: (i) the inability of the A. calcoaceticus–A. baumannii complex to support the replication of E. coli plasmid vectors and (ii) the susceptibility of both G5139 and G5140 to minocycline (MIC = 0.75 mg/L). A suicide plasmid construct pCLL3469 carrying an internal fragment of the adeB gene and a functional tet(M) gene was assembled and transformed into both G5139 and G5140, resulting in the isolation of the insertional mutants GC7945 and GC7951, respectively. The effect of inactivation of the adeB gene was assessed by antibiotic susceptibility tests. As shown in Table 1, insertional inactivation of the adeB gene in G5139 and G5140 resulted in a decrease in the MICs of tigecycline, gentamicin, chloramphenicol and levofloxacin, confirming the previously reported broad-substrate specificity of the AdeABC pump7,8 and further suggesting that the AdeABC pump is involved in decreased susceptibility of the A. calcoaceticus–A. baumannii complex to tigecycline. Additional studies on the prevalence of such a mechanism in other clinical isolates of the A. calcoaceticus–A. baumannii complex are required.
In addition to the pump components, the ade gene cluster encodes the AdeRS two-component system that regulates the expression of AdeABC.7,8 Analyses of the adeRS locus were performed to determine the reason for constitutive overexpression of adeABC in G5139 and G5140. In G5139 and G5140, the adeS gene was disrupted by an insertion sequence ISABA-1, whereas it remained intact in tigecycline-susceptible strains G4904 and G5141. Besides the presence of the insertion element, no other sequence changes were identified in the PCR-amplified adeRS fragment in G5139 and G5140 when compared with G4904 and G5141. ISABA-1 was previously identified in Acinetobacter spp. and is thought to affect the expression of antibiotic resistance genes that are adjacent to the site of insertion.13 Further experiments are required to identify the exact mechanism leading to the increased expression of adeABC in G5139 and G5140 and to determine whether ISABA-1 insertion has any effect on the regulation of adeABC expression in these strains.
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None to declare.
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Acknowledgements |
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We thank Guy Singh for ribotyping, Jan Kieleczawa for nucleotide sequencing and Peter Petersen and Tasha Weaver-Sands for help with susceptibility testing.
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References |
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1 Petersen PJ, Jacobus NV, Weiss WJ, et al. (1999) In vitro and in vivo antimicrobial activities of a novel glycylcycline, the 9-t-butylglycylamido derivative of minocycline (GAR-936). Antimicrob Agents Chemother 43:738–44.
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4 Ruzin A, Visalli MA, Keeney D, et al. (2005) Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in. Klebsiella pneumoniae. Antimicrob Agents Chemother 49:1017–22.[CrossRef]
5 Ruzin A, Keeney D, Bradford PA. (2005) AcrAB efflux pump plays a role in decreased susceptibility to tigecycline in. Morganella morganii. Antimicrob Agents Chemother 49:791–3.[CrossRef]
6 Keeney D, Ruzin A, Bradford PA. (2007) RamA, a transcriptional regulator, and AcrAB, a RND-type efflux pump, are associated with decreased susceptibility to tigecycline in. Enterobacter cloacae. Microb Drug Resist 13: in press.
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Magnet S, Courvalin P, Lambert T. (2001) Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrob Agents Chemother 45:3375–80.
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Marchand I, Damier-Piolle L, Courvalin P, et al. (2004) Expression of the RND-type efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS two-component system. Antimicrob Agents Chemother 48:3298–304.
9
Higgins PG, Wisplinghoff H, Stefanik D, et al. (2004) Selection of topoisomerase mutations and overexpression of adeB mRNA transcripts during an outbreak of Acinetobacter baumannii.. J Antimicrob Chemother 54:821–3.
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Huys G, Cnockaert M, Nemec A, et al. (2005) Sequence-based typing of adeB as a potential tool to identify intraspecific groups among clinical strains of multidrug-resistant Acinetobacter baumannii.. J Clin Microbiol 43:5327–31.
11 Sambrook J, Fritsch EF, Maniatis T. (1989) Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor).
12 Hiramatsu K, Aritaka N, Hanaki H, et al. (1997) Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350:1670–3.[CrossRef][ISI][Medline]
13 Segal H, Garny S, Elisha BG. (2005) Is IS(ABA-1) customized for Acinetobacter? FEMS Microbiol Lett 243:425–9.[CrossRef][ISI][Medline]
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