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Antimicrobial Agents and Chemotherapy, October 2004, p. 4054-4055, Vol. 48, No. 10
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.10.4054-4055.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Novel Resistance-Nodulation-Cell Division Efflux System AdeDE in Acinetobacter Genomic DNA Group 3

Sze-Lok Chau,1 Yiu-Wai Chu,1* and Elizabeth T. S. Houang1

Department of Microbiology, The Chinese University of Hong KongHong Kong Special Administrative Region, People's Republic of China

Received 15 December 2003/ Returned for modification 3 March 2004/ Accepted 26 June 2004


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ABSTRACT
 
Resistance-nodulation-cell division type efflux pump AdeDE was identified in acinetobacters belonging to genomic DNA group 3. Inactivation of adeE showed that it may be responsible for reduced susceptibility to amikacin, ceftazidime, chloramphenicol, ciprofloxacin, erythromycin, ethidium bromide, meropenem, rifampin, and tetracycline. However, unlike what was found for other similar efflux systems, the open reading frame for the outer membrane component was not found downstream of the adeDE gene cluster.


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TEXT
 
Acinetobacter species, especially genomic DNA group (GDG) 3, are important nosocomial pathogens in Hong Kong (5). The therapy of Acinetobacter infections can be complicated by multidrug resistance to a range of antimicrobial agents (1). Resistance-nodulation-cell division (RND) type efflux pumps can be responsible for a wide substrate specificity in gram-negative bacteria. AdeABC, shown to contribute to multidrug resistance in Acinetobacter baumannii (GDG 2) by Magnet et al., is the only published efflux system in Acinetobacter (2). The three clustering genes adeA, adeB, and adeC were identified as encoding the membrane fusion, multidrug transporter, and outer membrane components characteristic of the RND efflux pump family, respectively (2). We used the degenerate primers described by these authors to detect adeB-like sequence in acinetobacters isolated from clinical specimens at Prince of Wales Hospital, Hong Kong. An amplicon (1.2 kb) was generated from a 1997 blood culture isolate (strain 4365) belonging to GDG 3. The amplicon was then sequenced, and the theoretical translation revealed 50% amino acid identity to AdeB and 46 to 62% amino acid identity (BlastP; European Bioinformatics Institute, Cambridge, United Kingdom) to other members of the RND efflux protein family. Genome walking utilizing the commercial kit GenomeWalker (BD Biosciences Clontech) was employed to clone the flanking regions of the amplicon. After six rounds of walking with primers based on the sequences obtained in the previous round, 11 contigs were generated. Each round of walking was performed at least twice with amplicons obtained from independent PCRs, and at least two clones were picked from each transformation for sequencing in both directions. Alignment of the 11 contigs yielded two complete open reading frames (ORFs) of 1,125 and 3,111 bp, theoretically encoding two peptides with 375 and 1,037 residues, respectively. While the nucleotide sequences failed to match those of any known genes in GenBank, the two peptide sequences have 28 to 53% identity (ClustalW; EBI) to 10 RND efflux systems (Table 1). The theoretical peptides appeared to resemble MexAB and AcrEF most, and the four known conserved motifs of RND proteins were readily aligned and identifiable (6). The ORFs were designated adeD and adeE (Acinetobacter drug efflux), and the products were designated AdeD and AdeE. The ORF immediately downstream of adeE was partially sequenced and, interestingly, the theoretical translation of this partial ORF resulted in a peptide (400 residues) with 66% identity to a nitroreductase in Pseudomonas aeruginosa.


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  		TABLE 1. Peptide sequence identity of the AdeDE theoretical peptides to members of the RND efflux protein family

To demonstrate the involvement of AdeE in multidrug resistance, a 460-bp fragment (bp 79 to 538) of adeE was subcloned into the Acinetobacter suicide vector pUC18 and electrotransformed into another adeE-harboring GDG 3 strain, 8108, in an attempt to disrupt the adeE sequence and hence its function. Strain 8108 was chosen over 4365 because it is sensitive to the transformant-selecting agent ticarcillin. The transformant 8108-15 was obtained by following procedures described previously (2). The successful adeE disruption and genetic identity of wild-type 8108 and transformant 8108-15 were proven by PCR of the targeted site and pulsed-field gel electrophoresis, respectively (data not shown).

A standard disk diffusion test (4) initially showed that mutant 8108-15 was more susceptible (>6-mm zone diameter difference) to ciprofloxacin, ofloxacin, nalidixic acid, imipenem, netilmicin, amikacin, gentamicin, and neomycin than its parent isolate 8108. Six beta-lactams were also tested, namely, methicillin, piperacillin, ceftazidime, cefotaxime, cefuroxime, and cefepime. Interestingly, only with ceftazidime was the zone diameter for 8108-15 larger than that for 8108 (7-mm difference), while all the other agents gave similar zone diameters (<2-mm difference). MICs of nine agents were further determined by the standard agar dilution method (4). By comparing MICs for wild-type 8108 and 8108-15, it was found that, for the mutant, there were more-than-fourfold decreases in the MICs of amikacin (2 to <0.06 mg/liter), ceftazidime (1 to <0.125 mg/liter), chloramphenicol (128 to <32 mg/liter), ciprofloxacin (0.06 to <0.02 mg/liter), erythromycin (4 to <0.125 mg/liter), ethidium bromide (128 to <32 mg/liter), meropenem (0.25 to <0.004 mg/liter), rifampin (2 to <0.06 mg/liter), and tetracycline (2 to <0.06 mg/liter). The substrate specificity of AdeE appeared different from that of the other Acinetobacter RND protein, AdeB, as inactivation of the latter had no effect on MICs of ceftazidime and rifampin (2).

By PCR, the adeE gene was detected in 75 of 83 GDG 3 acinetobacters in our culture collection (1997 to 2000), and the gene has not been found in our A. baumannii (GDG 2) strains (0 of 56). It is thought that AdeE may be a major efflux protein in GDG 3.

Unlike what was found for gene clusters encoding other common RND tripartite protein complexes, the sequence downstream to adeDE was not an ORF encoding the outer membrane component. The ORF encoding the outer membrane protein may not be clustered with the other two components and perhaps is not regulated as an operon, like many described RND systems. On the other hand, AdeDE may complex with other outer membrane proteins not specific to itself, such as MexXY in Pseudomonas aeruginosa (3). Moreover, studies have demonstrated that outer membrane protein OprM can be expressed independently and interplay with several RND pumps in the same organism (7). Further characterization of the function of the components and clinical significance studies of this newly identified efflux system are under way.

Nucleotide sequence accession number. The 5,420-bp sequence of adeDE has been deposited in the GenBank database under the accession no. AY147867.


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ACKNOWLEDGMENTS
 
S.L.C. was supported by a CUHK studentship. This study was partly supported by a grant (4290/99 M) from the Research Grants Council, Hong Kong SAR, People's Republic of China.

We thank Thierry Lambert and Patrice Courvalin (Institut Pasteur, France) for their invaluable advice and Norman Lo for his technical support.


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FOOTNOTES
 
* Corresponding author. Mailing address: Public Health Laboratory Centre, Department of Health, Hong Kong Special Administrative Region, People's Republic of China. Phone: 852 2319 8685. Fax: 852 2776 1446. E-mail: so_micro4{at}dh.gov.hk. Back


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Antimicrobial Agents and Chemotherapy, October 2004, p. 4054-4055, Vol. 48, No. 10
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.10.4054-4055.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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