Partial characterization of a transposon containing the tet(A) determinant in a clinical isolate of Acinetobacter baumannii

Anna Ribera1, Ignasi Roca2, Joaquim Ruiz1, Isidre Gibert2 and Jordi Vila1,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A genomic library from one clinical isolate of Acinetobacter baumannii was obtained to find genes responsible for tetracycline resistance. Escherichia coli DH5{alpha}-MDR transformants, selected on Mueller–Hinton agar supplemented with tetracycline 18 mg/L, were thoroughly characterized. In one clone, with an insert of 7070 bp, it was found that the resistance to tetracycline was mediated by the tet(A) gene (1200 bp) which encodes a tetracycline efflux pump. This gene was recovered together with tetR(A) (651 bp), the tet(A) repressor. Moreover, the partial sequence (2008 bp) of a transposase gene, tnpA, and 1316 bp corresponding to an IS, similar to that described in one strain of Salmonella typhi (IS4321), were found. In this A. baumannii clinical isolate, the tet(A) gene is located in a transposon. The structure of this transposon is similar to that of Tn1721, with the tet(A), tetR(A) and the regions between these genes, being closely related to those of Tn1721. The data indicate horizontal transfer of tetracycline resistance genes between A. baumannii and other genera sharing the same ecological niches.

Keywords: tetracycline resistance, Acinetobacter baumannii


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Acinetobacter baumannii is a Gram-negative bacillus that is usually commensal, but in the past few decades has emerged as an important opportunistic pathogen, especially in the hospital setting.1 This microorganism has unique characteristics among nosocomial Gram-negative bacteria, such as resistance to desiccation, that favours its persistence in the hospital environment. This fact, together with a facility for developing resistance to most of the antibiotics currently available,1 explains in part at least the propensity of A. baumannii to cause extended outbreaks, mainly located in intensive care units.1

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains, plasmids and growth conditions

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{alpha}-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 manufacturer’s 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{alpha}-MDR by heat-shock transformation and plasmid-containing DH5{alpha} 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 Mueller–Hinton agar by Etest (AB Biodisk, Sölna, Sweden), according to the manufacturer’s instructions.

PCR amplification

The presence of the tet(A) gene in 22 epidemiologically unrelated A. baumannii strains and in E. coli DH5{alpha} 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From a genomic library of the A. baumannii isolate, one clone of E. coli DH5{alpha} resistant to tetracycline was obtained. Analysis of the DNA sequence revealed that tetracycline resistance was due to the presence of the tet(A) gene. Together with tet(A), tetR(A), the tet(A) repressor gene, was also cloned. The partial sequence of a Tn3-like transposase gene, tnpA, and an IS element similar to that described in Salmonella typhi (IS4321) were also found at the same locus. The insert was 7077 bp in length (Figure 1), consisting of 2008 bp contributed by tnpA (partial sequence), 1316 bp by the IS element, 1200 bp by the tet(A) gene, 651 bp by tetR(A) and 75 bp by another transposase (partial sequence of a truncated and non-functional transposase, tnpA'). The tet(A) and the tetR(A) genes are adjacent, orientated divergently and share a central regulatory region (105 bp) with overlapping promoters and operators. The tnpA and the IS element are separated by 18 bp and the IS element and tetR(A) by 324 bp. Following tet(A), there is a region of 1380 bp in which no ORFs could be identified. The tet gene organization coincides with that of Tn1721. The nucleotide sequences of the tet(A) and the tetR(A) genes, those of the non-coding regions (324 and 1380 bp), and tnpA', are 99.41% similar to those of Tn1721. In contrast, the tnpA nucleotide sequence differs markedly from the tnpA of Tn1721, despite being in the same position and orientation. The sequence of our tnpA showed 100% identity with a tnpA found in the genome of one strain of Salmonella typhi and one strain of Pseudomonas aeruginosa (EMBL accession numbers AL513383 and U12338, respectively) (Figure 1). Furthermore, instead of the IRRI (inverted repeat right I) between the tnpA and the tetR(A) genes found in the Tn1721, a whole IS element was inserted between these two genes in the A. baumannii transposon. Surprisingly, the nucleotide sequence of this IS element shows 99.55% homology with IS4321 described in the S. typhi strain CT18 (EMBL accession number AL513383).



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Figure 1. Gene arrangement of the partial transposon found in a clinical isolate of A. baumannii (A5-22) in comparison with the gene arrangement of Tn1721. The direction of the arrows shows the direction of transcription. The relative orientations of the three 38 bp inverted repeats, IRL, IRRI and IRRII, are symbolized by black arrowheads. tnpA' is a 5' truncated tnpA.

 
The MIC of tetracycline was determined for various strains (Table 1). The MIC of tetracycline for the clinical isolate of A. baumannii was >256 mg/L. The MIC of tetracycline for E. coli DH5{alpha} was 0.75 mg/L, and the transformed strain had a tetracycline MIC of 256 mg/L.


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Table 1.  MICs of tetracycline and minocycline for 22 strains of A. baumannii and for E. coli DH5{alpha} wild-type and tetracycline-resistant transformants, and results of the PCR amplification of either tet(A) or tnpA-IS.
 
PCR amplifications of the internal segment of the tet(A) gene and of the fragment containing part of tnpA and the IS were carried out directly from colonies of the 22 isolates of A. baumannii, and the E. coli DH5{alpha} wild-type and transformed strains. The results (Table 1) show that in 12 strains of A. baumannii (54.54%) and in the transformed E. coli DH5{alpha} strain, the PCR was positive for the tet(A) gene. Moreover, in 11 of these 12 strains (91.67%), and the E. coli transformant, a second PCR detected the tnpA followed by the IS element, suggesting that the genetic context in these isolates is the same as in A. baumannii A5-22.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The presence of the Tet(A) determinant is herein described in one clinical isolate of A. baumannii. The gene responsible for tetracycline resistance appears to be part of a transposon, but whether the transposon remains functional has yet to be determined. However, its gene organization, transcription polarity and the nucleotide sequences of the genes responsible for tetracycline resistance, show striking similarity to those of the well-known Tn1721,6 strongly suggesting the presence of a Tn1721-like transposon in our clinical isolate of A. baumannii. However, despite significant similarity to Tn21, the nucleotide sequence of the tnpA gene differs markedly from its counterpart in Tn1721, being 100% identical with the tnpA described in S. typhi strain CT187 and in P. aeruginosa.8 Moreover, the nucleotide sequence of the IS has 99% homology with that of strain CT18 (IS4321).7 It is interesting to note that despite having the same transposase gene as that found in A. baumannii, and an almost identical IS element, the relative position of these genes in the CT18 strain differs from that in A. baumannii A5-22.

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 (tnpA–IS) 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.


    Acknowledgements
 
This study was supported by grants from Fondo de Investigaciones Sanitarias (FIS 00/0997), Spain; the Spanish Dirección General de Enseñanza Superior e Investigación Científica (PB97–0196), and partially by Fundació Maria Francesca de Roviralta, Spain. A.R. has a fellowship from the Ministerio de Educación y Ciencia, Spain and I.R. has a predoctoral fellowship from the Direcció General d’Universitats de la Generalitat de Catalunya.


    Footnotes
 
* Corresponding author. Tel: +34-93-2275522; Fax: +34-93-2275454; E-mail: vila{at}medicina.ub.es Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Bergogne-Berezin, E. & Towner, K. J. (1996). Acinetobacter species as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clinical Microbiology Reviews 9, 148–65.[Free Full Text]

2 . 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]

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 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.

5 . Vila, J., Ruiz, J., Marco, F. et al. (1994). Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and minimal inhibitory concentration. Antimicrobial Agents and Chemotherapy 38, 2477–9.[Abstract]

6 . Allmeier, H., Cresnar, B., Greck, M. et al. (1992). Complete nucleotide sequence of Tn1721: gene organization and a novel gene product with features of a chemotaxis protein. Gene 111, 11–20.[CrossRef][ISI][Medline]

7 . Parkhill, J., Dougan, G., James, K. D. et al. (2001). Complete genome sequence of a multiple drug resistance Salmonella enterica serovar Typhi CT18. Nature 413, 848–52.[CrossRef][ISI][Medline]

8 . Partridge, S. R., Brown, H. J., Strokes, H. W. et al. (2001). Transposons Tn1696 and Tn21 and their integrons In4 and In2 have independent origins. Antimicrobial Agents and Chemotherapy 45, 1263–70.[Abstract/Free Full Text]

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, 97–103.[CrossRef][ISI][Medline]

10 . 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, 3883–90.[Abstract/Free Full Text]

11 . 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, 3345–52.[Abstract/Free Full Text]