Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain
Received 11 May 2005; returned 19 May 2005; revised 26 May 2005; accepted 15 June 2005
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Abstract |
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Results: armA was borne by a complex transposon composite flanked by two direct repeats of IS26. The transposon composite included a class one integron with sul1 for resistance to sulphonamides and ant3''9 conferring resistance to spectinomycinstreptomycin, and a macrolide efflux pump and mefE/mel conferring high-level resistance to erythromycin. We identified in GenBank that another plasmid, pCTX-M3, from a Polish Citrobacter freundii human isolate, bore the same genetic structure, including armA.
Conclusions: armA is present in human and animal isolates within a novel transposon composite. Further spread of armA between bacteria of diverse origin is to be expected.
Keywords: Escherichia coli , animal isolates , 16S rRNA methylase , aminoglycoside resistance , IncN , transposon composite
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Introduction |
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Materials and methods |
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The clinical strain E. coli MUR050 was isolated from a diarrhoeic pig in 2002. The laboratory strains E. coli INVF' [F mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 ara139 (ara-leu)7697 galU galK rpsL (StrR) endA1 nupG] and DH5
[endA1, gyrA96, hsdR17(rk mk+), recA1, relA1, supE44, thi-1, D(lacZYA-argF) U169, f80dl acZDM15, F, lPN25/tetR, Placi q/laci, Spr] were used for cloning purposes and transformed following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).
Susceptibility tests
Bacteria were tested using the Etest (AB Biodisk, Solna, Sweden), and interpretation of susceptibility test results was conducted following the recommendations of the manufacturer. For neomycin and apramycin (Izasa, Madrid, Spain), the MICs were determined by microdilution as previously described.6
DNA sequencing and analysis of pMUR050
Plasmid DNA was extracted from the strain, and re-extracted from an agarose gel to avoid contamination with genomic DNA.7 The purified plasmid DNA (Qiagen, Inc., Chatworth, CA, USA) was digested with Sau3A (Sigma, St Louis, MO, USA) and the resulting fragments ligated with plasmid pBluescript KS+ DNA (Stratagene, La Jolla, CA, USA). The ligation mixture was transformed into E. coli DH5 and plated on agar containing ampicillin (50 mg/L). The inserts of plasmids from 288 random clones, corresponding to approximately three times the estimated size of pMUR050 (50 kbp) giving a mean cloned fragment of 500 bp, were sequenced in both strands with standard oligonucleotides using an ABI Prism DNA Sequencer apparatus (Perkin-Elmer, Foster City, CA, USA). The DNA sequences were assembled using DNASTAR (Madison, WI, USA).
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Results |
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pMUR050 was transformed into the laboratory strain E. coli INVF', in which it conferred high-level resistance to 4,6-disubstituted aminoglycosides. This strain was also resistant to other aminoglycosides, sulphonamides and macrolides, showing that resistance determinants other than armA were encoded in pMUR050 (Table 1).
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armA is located in a complex transposon composite
The structural gene for ArmA was preceded by a putative transposase gene, tnpU, followed by a class one integron formed by intI1-ant3''9-qacE1-sul1-orf513. Downstream of armA, the genes coding for another transposase, tnpD, a mefE/mel macrolide efflux pump and a macrolide phosphotransferase gene, mph, were located. All these genes, the armA locus, were flanked by two copies of IS26. IS26 belongs to the IS6 family of insertion sequences. This family is characterized by the fact that it gives rise exclusively to replicon fusions (cointegrates) in which the donor and target replicons are separated by two directly repeated IS copies. Upon cointegration, mediated by either of the two IS26 elements, the IS element is duplicated in a direct repeat.11 The two IS26 elements at both ends of armA are in direct repeat, forming a putative mobile transposon composite of 15 kbp.
armA transposon composite in other bacterial species
In order to elucidate if this putative mobile genetic element was responsible for the spread of armA, the databases were checked for similar genetic structures. Interestingly, a conjugative plasmid isolated in a Polish hospital from Citrobacter freundii, pCTX-M3, bore the same transposon composite, including armA, with an additional dhfrXII gene and orfF upstream of ant3''9 (Figure 1). Furthermore, the two additional isolates in which armA has been identified to date, a K. pneumoniae in France3 and an E. coli in Japan (GenBank accession number AB117519), are hospital isolates that showed the same genetic organization flanking the armA gene (Figure 1). Overall, our results show that armA has spread within a unique transposon composite both by transposition and conjugation, between different species of Enterobacteriaceae involving animal and human isolates.
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Discussion |
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E. coli MUR050 was isolated from a diarrhoeic pig in south-east Spain.4 The fact that this strain bore armA was already striking, because this gene had only been described in a few human isolates worldwide. In this study, we have further demonstrated that the genetic background for the spread of armA is the same in human and animal strains, indicating that the armA transposon composite will be further detected in Enterobacteriaceae of very diverse origins. This is specially worrisome, as the extended use of sulphonamides or macrolides would select for the presence of the transposon composite, including armA, ant3''9, the melF-mef and mph genes. Extended surveillance of both human and animal isolates should be reinforced, if resistance to aminoglycosides is to be delayed.
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Footnotes |
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Acknowledgements |
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References |
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