1 Servei de Microbiologia, Institut Clinic Infeccions i Immunologia, IDIBAPS, Facultat de Medicina, Universitat de Barcelona, Villarroel, 170, 08036 Barcelona; 2 Laboratori de Genètica Molecular Bacteriana, Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
Received 10 May 2002; returned 12 December 2002; revised 16 January 2003; accepted 18 May 2003
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Abstract |
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Keywords: tetracycline resistance, Acinetobacter baumannii
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Introduction |
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Tetracycline is one of the antibiotics to which A. baumannii has developed resistance. This antibiotic acts by binding to the 30S ribosomal subunit, resulting in the inhibition of protein synthesis.2 Tetracycline-resistant bacteria generally express one of two different mechanisms: an efflux pump or a ribosomal protection system. Tetracycline resistance determinants A to E, G and H among Enterobacteriaceae and other Gram-negative bacilli and determinants K and L among Gram-positive bacteria specify efflux pumps for tetracyclines that enable the bacteria to grow in the presence of therapeutic levels of tetracycline. Unlike Gram-positive bacteria, efflux determinants from Gram-negative bacteria have a common genetic organization, with all containing a structural gene and a repressor gene in opposing orientations and expressed from overlapping operator regions.2 The Gram-negative tet efflux genes are found on transposons inserted into a diverse group of plasmids from a variety of incompatibility groups, most of which are conjugative.2
Guardabassi et al.3 have reported the mechanisms of resistance to tetracycline in A. baumannii, by finding the Tet(A) and Tet(B) determinants in clinical and aquatic strains. However, little is known about the genetic context of these determinants.
The aim of this study was to analyse the molecular mechanisms of resistance to tetracycline in a clinical isolate of A. baumannii and estimate the prevalence of the genetic construct containing the tet(A) gene in other isolates of this microorganism.
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Materials and methods |
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Clinical isolates of A. baumannii, recovered from respiratory secretions, were submitted to the Clinical Laboratory of Microbiology of the Hospital Clinic of Barcelona. Isolates were identified as A. baumannii by standard biochemical testing and by amplified ribosomal DNA restriction analysis (ARDRA).1 Escherichia coli DH5-MDR (Gibco-BRL, Life Technologies Inc., Gaithersburg, MD, USA) was used as host strain in transformation experiments. All cloning procedures were carried out with the phagemid vector pBluescript(SK(+/) [high-copy-number cloning vector encoding resistance to ampicillin (Apr)] (Stratagene Cloning Systems, La Jolla, CA, USA). When plasmids were to be maintained in E. coli strains, agar was supplemented with 18 mg/L of tetracycline and 100 mg/L of ampicillin.
DNA methodology
Basic DNA procedures, including restriction endonuclease digestions, ligations, transformations and agarose gel electrophoresis were carried out as described previously.4 To isolate plasmids, an alkaline lysis method was used.4 Genomic DNA was extracted using the Wizard Genomic DNA purification kit (Promega Corporation, Madison, WI, USA). DNA fragments obtained from the cloning procedures were purified from agarose gels using the Concert Rapid Purification System according to the manufacturers instructions (Gibco-BRL, Life Technologies Inc., Gaithersburg, MD, USA).
Construction of the genomic library and DNA sequencing
The genomic DNA of the clinical isolate A. baumannii (A5-22) was partially digested with Sau3AI.4 Fragment patterns were examined in 0.7% agarose gels and fragments of 4 to 9 kb were recovered and purified from the gel as noted. The recovered fragments were then directly cloned into the pBSK phagemid vector previously linearized with BamHI and treated with calf intestinal alkaline phosphatase.4 Thereafter, the pool of recombinant plasmids with different inserts was introduced into E. coli DH5-MDR by heat-shock transformation and plasmid-containing DH5
clones were recovered on LB agar containing tetracycline 18 mg/L. Random screening of plasmids isolated from the transformants revealed the presence of inserts of
7 kb.
The whole insert from one representative recombinant plasmid was sequenced using the dRhodamine Terminator Cycle Sequencing kit and an automatic DNA sequencer (Abi Prism 377, Perkin Elmer, Emeryville, USA). DNA sequencing was carried out either by using M13 universal primers or by primer walking using custom-designed primers.
Computer analysis of sequence data
Nucleotide and amino acid sequences were analysed at the website of the National Center for Biotechnology Information (www.ncbi.nih.gov/gorf/gorf.html). The GenBank and protein databases were screened for sequence similarities. The EMBL accession number of our sequence (7077 bp) is AY196695.
Antimicrobial susceptibility assay
MICs of tetracycline and minocycline were determined on MuellerHinton agar by Etest (AB Biodisk, Sölna, Sweden), according to the manufacturers instructions.
PCR amplification
The presence of the tet(A) gene in 22 epidemiologically unrelated A. baumannii strains and in E. coli DH5 strains (wild-type and transformed) was established by PCR amplification of a 954 bp fragment using primers specific for the gene3 and using the cycling conditions previously described by Vila et al.5 PCR products were resolved in agarose gel (2% w/v) and stained with ethidium bromide. In all cases, the products obtained were recovered and sequenced to establish the accuracy of the PCR. In the same manner, the amplification of a 212 bp starting in tnpA and terminating in the IS element was achieved using the primers: (1) 5'-CGCTGGACGACACATTG-3' and (2) 5'-TCCGAATGAAA- GCCTGTCC-3' and the same cycling conditions.
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Results |
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Discussion |
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Tn1721-like elements have also been found in Aeromonas spp. and Salmonella spp.9,10 The presence of a Tn1721-like transposon in our isolate, not previously described in A. baumannii, together with the high similarity of the nucleotide sequences of the tnpA and the IS element with those present in other Gram-negative bacteria, suggest horizontal transfer between microorganisms sharing the same ecological niches.
Several events that could give rise to the situation with the IS element may have occurred. It could be a result of a recombination with the IRRI found in the Tn1721.6 Some of these genetic elements are thought to be responsible for the incorporation of genes of antibiotic resistance, including tetracycline determinants, in bacterial chromosomes.11 It is known that the dissemination of the IS elements is mediated by plasmids, and that posterior integration of the IS within the chromosome can occur.11
The results of the PCR amplification of the 212 bp sequence containing a fragment of tnpA and the IS, suggest that this arrangement (tnpAIS) is common in the tetracycline-resistant isolates of A. baumannii investigated and probably indicate a common structure to that in A. baumannii A5-22 (Table 1).
Very little is known about the genetic basis of tetracycline resistance in A. baumannii. This article provides novel information about the resistance of this microorganism to tetracycline. We found a Tn1721-like transposon carrying the Tet(A) determinant in a clinical isolate of A. baumannii, suggesting horizontal transfer among different genera of Gram-negative bacteria sharing the same ecological niche.
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Acknowledgements |
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Footnotes |
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References |
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2
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3
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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, 92936.
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9 . Frech, G. & Schwarz, S. (1999). Plasmid-encoded tetracycline resistance in Salmonella enterica subsp. enterica serovars choleraesuis and typhimurium: identification of complete and truncated Tn1721 elements. FEMS Microbiology Letters 176, 97103.[CrossRef][ISI][Medline]
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Rhodes, G., Huys, G., Swings, J. et al. (2000). Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Applied and Environmental Microbiology 66, 388390.
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Simpson, A. E., Skurray, R. A. & Firth, N. (2000). An IS257-derived hybrid promoter directs transcription of a tetA(K) tetracycline resistance gene in the Staphylococcus aureus chromosomal mec region. Journal of Bacteriology 182, 334552.