Identification of Tet 39, a novel class of tetracycline resistance determinant in Acinetobacter spp. of environmental and clinical origin

Y. Agersø1,* and L. Guardabassi2

1 Danish Institute for Food and Veterinary Research, 1790 Copenhagen V; 2 Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, 1870 Frederiksberg C., Denmark


* Corresponding author. Tel: +45-72-34-6273; Fax: +45-72-34-6001; Email: ya{at}dfvf.dk

Received 8 November 2004; returned 2 December 2004; revised 23 December 2004; accepted 6 January 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A novel tetracycline resistance determinant named Tet 39 was found in unrelated Acinetobacter strains isolated from freshwater trout farms (n=4) and sewage (n=6) in Denmark, and from a clinical specimen in the Netherlands (n=1). The determinant was located on transferable plasmids and consisted of tetA(39), most likely conferring resistance by active efflux, and a putative repressor gene tetR(39).

Keywords: tetracycline-resistant , plasmids , tet(39)


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial resistance to tetracyclines can occur by three distinct mechanisms: active efflux, ribosomal protection and enzyme modification.1 According to the current nomenclature, tetracycline resistance genes are grouped into more than 35 classes, where a new class is defined when there is < 80% amino acid homology to known determinants.1,2 Three classes of genes encoding tetracycline resistance by specific active efflux have been described in Acinetobacter spp.: tet(A), tet(B) and tet(H).3,4 Other tetracycline resistance genes occasionally reported in clinical Acinetobacter baumannii isolates include tet(M), a widespread gene encoding tetracycline resistance by ribosomal protection, and adeB, which confers multi-drug resistance by an unspecific efflux pump mechanism.5,6 The occurrence of unknown tetracycline resistance determinants was reported by Guardabassi et al.3 after screening a heterogeneous collection of tetracycline-resistant Acinetobacter strains of different species and origin.

The aim of this study was to determine the genetic basis of tetracycline resistance in Acinetobacter strains that do not carry known resistance determinants.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The study was conducted using 15 unrelated tetracycline-resistant strains (Table 1) that were previously demonstrated not to carry any of the tetracycline resistance genes most frequently found in Gram-negative bacteria, including tet(A–E), tet(G), tet(M), tet(O), tet(S), tet(P), tet(Q) and tet(Y).3


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Table 1. List of tetracycline-resistant Acinetobacter strains

 
The aquatic tetracycline-resistant Acinetobacter strain LUH5605 has previously been shown to carry transferable plasmids in tetracycline-resistant transconjugants.3 Therefore, plasmid DNA was obtained from the strain as previously described7 and partially digested with HindIII, followed by ligation into the plasmid vector pUC19. The ligation product was electroporated into Escherichia coli, strain HB101, and presumptive clones were selected on Luria–Bertani agar supplemented with 100 mg/L ampicillin and 8 mg/L tetracycline. Plasmids were purified from selected colonies using QIAprep Spin MiniPrep kit (Qiagen, Germany) and electroporated into E. coli, strain DH10B, to verify that the cloned fragment conferred tetracycline resistance.

MICs of various antimicrobial agents (ampicillin, apramycin, ceftiofur, chloramphenicol, ciprofloxacin, co-amoxiclav, colistin, florphenicol, gentamicin, nalidixic acid, neomycin, spectinomycin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim) were determined by SensiTitre panels (Trek Diagnostics Systems, UK) following the NCCLS standards.8 The MIC of minocycline (strains YA5605, LUH5605 and LUH5617) was determined by the agar dilution method according to the NCCLS guidelines.8

The phylogenetic relationship of tetA(39) was revealed by comparing the gene with representative genes of each class of 12-TMS tetracycline efflux pumps (where TMS stands for transmembrane {alpha}-helices). The program Clustal X (version 1.81) was used to perform a multiple alignment. The program TreeView (version 1.6.6) was used to make a tree.

The occurrence of tetA(39) was investigated in the other fourteen tetracycline- resistant Acinetobacter strains (Table 1) using specific primers tet(39)-1 (5'-CTCCTTCTCTATTGTGGCTA-3') and tet(39)-2 (5'-CACTAATACCTCTGGACATCA-3'). Since the putative repressor gene tetR(39) contained a HindIII site, a second PCR was performed targeting the gene on each side of the HindIII site by use of the primers tetR(39)-1 (5'-ATTCACTCCTTGGAGCATGA-3') and tetR(39)-2 (5'-TGGGATGACATGGCAAG-3').

The strains positive for Tet 39 were used as donors in filter mating experiments using methods and three Acinetobacter recipients previously described.3 Southern blotting of plasmid DNA obtained from donors and transconjugants were preformed using a tetA(39) (701 bp) probe labelled with digoxigenin.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Transformants showed an increase in the MIC of tetracycline from 1 mg/L to 128 mg/L. No variations were observed in the MICs of other antimicrobials tested, indicating that the cloned DNA fragment only conferred resistance to tetracyclines. Sequencing of a 3727 bp insert revealed the presence of an open reading frame named tetA(39) (1188 bp) having 50% predicted amino acid identity to TetA(C) in Aeromonas salmonicida (GenBank accession no. AY043298). tetA(39) was flanked upstream by a reverse complementary open reading frame named tetR(39) (642 bp) encoding a putative repressor protein with 41% identity over 202 amino acids to TetR(B) from the transposon Tn10 (GenBank accession no. AP000342). Downstream from tetA(39), there were two reverse complementary open reading frames named Orf1 (188 amino acids) and Orf2 (307 amino acids). Orf1 had 47% amino acid identity to an open reading frame with unknown function found on a broad-host range conjugative plasmid in Citrobacter freundii (GenBank accession no. AF550415). Orf2 had 42% amino acid identity to a plasmid replication protein (RepA AB) described in A. baumannii (Genbank accession no. AY228470).

The genetic structure of Tet 39 is illustrated in Figure 1. Transmembrane helices were predicted from the amino acid sequence of TetA(39) using the program TMHMM Server v. 2.0 (www.cbs.dtu.dk/services/TMHMM/).9,10 The predicted secondary structure of TetA(39) revealed a putative transmembrane protein consisting of 12 TMS as seen for other tetracycline efflux pumps (that have either 12 or 14 TMS) belonging to the major facilitator superfamily (MFS).11 Moreover, the amino acid sequence contained the highly conserved motifs A (GALSDRFGRRPVL, position 35–47), B (LYIGRIFAGITGA, position 70–82), C (GFIAGPVIGGVL, position 114–125) and D2 (VGIGLIMPILP, position 17–27) found in the 12-TMS family of MFS described by Paulsen et al.11 The phylogenetic analysis of 12-TMS tetracycline efflux pumps showed tetA(39) to have the closest evolutionary relationship to tetA(30) found in Agrobacterium tumefaciens originating from soil; the two genes formed a separate branch (Figure 2).



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Figure 1. Schematic representation of the tetracycline resistance determinant Tet 39 and flanking regions from Acinetobacter strain LUH5605. Arrows indicate the direction of transcription. Numbers show transcription start corresponding to the GenBank submission no. AY743590.

 


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Figure 2. Rooted phylogenetic tree based on multiple alignments of representatives for each class of 12-TMS tetracycline efflux proteins (indicated with name of tet class and GenBank accession no.). Multiple alignments were performed by use of ClustalX (version 1.81). Numbers indicate support for the tree nodes obtained from MP bootstrap analysis (1000 replicates). The 12-TMS efflux proteins, NorA and EmrD, were used to root the tree.

 
The occurrence of tet(39) was investigated in the 15 tetracycline-resistant strains. Eleven strains yielded PCR products of the expected size (701 bp) with the primers tet(39)-1 and tet(39)-2. The PCR products from two randomly selected strains (LUH5609 and LUH5617) were sequenced and showed 100% identity to tetA(39) in LUH5605. All tetA(39)-positive strains also contained the putative repressor gene tetR(39). The strains harbouring Tet 39 originated from the environment of Danish freshwater trout farms (n=4), from sewage collected in Denmark (n=6) and from a clinical specimen of urine collected in the Netherlands in 1986. This indicates that this novel tetracycline resistance determinant occurs in different reservoirs and geographical areas.

The 11 strains positive for Tet 39 were used as donors in filter mating experiments to three Acinetobacter recipients. Only one donor did not transfer tetracycline resistance to any of the recipients. In vitro plasmid-mediated transfer of Tet 39 was demonstrated from eight donor isolates to all three recipients and from two donors to one recipient (Table 1). Southern blotting of plasmid DNA obtained from donors and transconjugants using a tetA(39) probe showed that the gene was located on plasmids varying in size from ~25–50 kb (Table 1).

Tet 39 is likely to confer resistance to tetracyclines by an active efflux mechanism. This is suggested by the genetic organization of Tet 39 and the secondary protein structure of TetA(39), which is similar to that of other determinants encoding active efflux of tetracyclines,1 as well as by the homology of tetA(39) and tetR(39) to the corresponding genes in Tet C and Tet B, respectively. Furthermore, similarly to other active efflux determinants, Tet 39 does not confer resistance to minocycline (MIC < 1 mg/L) (strains YA5605, LUH5605 and LUH5617). Although TetR(39) had homology to other repressor genes, no differences in the MICs of tetracycline were observed between cultures pre-grown with or without tetracycline. Thus further investigation is required to determine whether resistance is induced or constitutively expressed.

Tet 39 seems to be more frequent in Acinetobacter spp. from the aquatic environment than in clinical isolates, where Tet A and Tet B have been shown to be the prevalent tetracycline resistance determinants.3 This tetracycline resistance determinant is likely to occur in other Gram-negative genera since: (i) it was shown to be located on transferable plasmids; (ii) it could be expressed in E. coli; and (iii) the open reading frames situated downstream from Tet 39 were homologous to genes located on plasmids of other Gram-negative bacteria, including broad-host range conjugative plasmids. The finding of Tet 39 in Acinetobacter from geographically distinct areas and reservoirs with the clinical strain isolated 18 years ago may suggest the gene is widespread among Acinitobacter strains. The PCR primers described in this study can be usefully employed to investigate the occurrence of Tet 39 in Acinetobacter as well as other bacterial genera.

GenBank submission

The sequence of Tet 39 and flanking sequence described in the present study has been submitted to GenBank (GenBank accession no. AY743590).


    Acknowledgements
 
We would like to thank Jane Larsen for excellent technical assistance and engagement in the project. This study was funded by a grant from the Danish Agricultural and Veterinary Research Council (no. 23-02-1169).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Chopra, I. & Roberts, M. (2001). Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews 65, 232–60.[Abstract/Free Full Text]

2 . Levy, S. B., McMurry, L. M., Barbosa, T. M. et al. (1999). Nomenclature for new tetracycline resistance determinants. Antimicrobial Agents and Chemotherapy 43, 1523–4.[Abstract/Free Full Text]

3 . Guardabassi, L., Dijkshoorn, L., Collard, J.-M. et al. (2000). Distribution and in-vitro transfer of tetracycline resistance determinants in clinical and aquatic Acinetobacter strains. Journal of Medical Microbiology 49, 929–36.[Abstract/Free Full Text]

4 . Miranda, C. D., Kehrenberg, C., Ulep, C. et al. (2003). Diversity of tetracycline resistance genes in bacteria from Chilean salmon farms. Antimicrobial Agents and Chemotherapy 47, 883–8.[Abstract/Free Full Text]

5 . Ribera, A., Ruiz, J. & Vila, J. (2003). Presence of the Tet M determinant in a clinical isolate of Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 47, 2310–2.[Abstract/Free Full Text]

6 . Magnet, S., Courvalin, P. & Lambert, T. (2001). Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrobial Agents and Chemotherapy 45, 3375–80.[Abstract/Free Full Text]

7 . Olsen, J. E. (1990). An improved method for rapid isolation of plasmid DNA from wild-type Gram-negative bacteria for plasmid restriction profile analysis. Letters in Applied Microbiology 10, 209–12.[ISI][Medline]

8 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7–A6. NCCLS, Villanova, PA, USA.

9 . Krogh, A., Larsson, B., von Heijne, G. et al. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of Molecular Biology 305, 567–80.[CrossRef][ISI][Medline]

10 . Sonnhammer, E. L. L., von Heijne, G. & Krogh, A. (1998). A hidden Markov model for predicting transmembrane helices in protein sequences. Proceedings of an International Conference on Intelligent Systems for Molecular Biology 6, 175–82.

11 . Paulsen, I. T., Brown, M. H. & Skurray, R. A. (1996). Proton-dependent multidrug efflux systems. Microbiology and Molecular Biology Reviews 60, 575–608.[Abstract]