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
Received 8 November 2004; returned 2 December 2004; revised 23 December 2004; accepted 6 January 2005
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Abstract |
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Keywords: tetracycline-resistant , plasmids , tet(39)
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Introduction |
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The aim of this study was to determine the genetic basis of tetracycline resistance in Acinetobacter strains that do not carry known resistance determinants.
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Materials and methods |
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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 -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.
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Results and discussion |
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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 3547), B (LYIGRIFAGITGA, position 7082), C (GFIAGPVIGGVL, position 114125) and D2 (VGIGLIMPILP, position 1727) 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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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 2550 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).
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Acknowledgements |
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References |
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