1 Institut für Tierzucht der Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee; 3 Institut für Medizinische Mikrobiologie, Seuchen- und Infektionsmedizin, Ludwig-Maximilians-Universität München, Veterinärstr. 13, 80539 München, Germany; 2 Empresa Brasileira de Pesquisa Agropecuária, Rua Eugênio do Nascimento 610, 36038330 Juiz de Fora-MG, Brazil
Received 3 February 2003; returned 20 February 2003; revised 7 March 2003; accepted 13 March 2003
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Abstract |
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Methods: Three staphylococcal strains, two Staphylococcus warneri, and one Staphylococcus sciuri, were included in this study. The gene aacA/aphD coding for a bifunctional enzyme that mediates resistance to gentamicin, tobramycin and kanamycin was detected by hybridization with a specific probe. Plasmid location of this gene was also confirmed by hybridization and conjugation. The resistance gene and its adjacent regions were cloned and sequenced.
Results: Three different types of Tn4001-like elements in which the IS256 elements were largely truncated and replaced by IS257 elements were identified on large conjugative and non-conjugative plasmids of 3343 kb in the staphylococcal strains from chickens. Seven different types of IS257-analogous insertion sequences were identified.
Conclusions: Resistance to gentamicin, tobramycin and kanamycin in three staphylococcal strains from chickens was mediated by plasmid-borne aacA/aphD genes located on structurally modified Tn4001-like elements. In one of the three plasmids studied, the arrangement of the elements in the aacA/aphD resistance gene area closely resembled that on plasmids pSH6, pSK41 and pUW3626 from Staphylococcus aureus of human origin.
Keywords: aminoglycoside resistance, aacA-aphD gene, coagulase-negative staphylococci, chicken, plasmid
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Introduction |
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To date, very little is known about the presence of the aacA/aphD gene in staphylococci from animals, particularly in those from avian sources. Tobramycin and gentamicin are not licensed for therapeutic use in poultry in the EU, and kanamycin is only licensed for use in poultry in Greece.6 However, the aminocyclitol apramycin is licensed for use in poultry in Italy, Spain, Portugal, Greece, the UK and Ireland and selects for cross-resistance to gentamicin, but not to kanamycin and tobramycin.6 Moreover, chicken eggs for hatching are often dipped in a gentamicin solution to prevent contaminating bacteria from invading the egg. This treatment is commonly applied before the eggs are transferred into a hatcher and might represent a high selective force for staphylococci resident on the egg shell to become resistant to gentamicin.
In this study, we investigated three independent staphylococcal strains from chickens for the molecular basis of resistance to gentamicin, tobramycin and kanamycin with particular reference to the detection of complete or truncated Tn4001 elements.
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Materials and methods |
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During April/May 1996, nasal swabs from 65 broiler chickens with no known contact to antimicrobial agents were taken at day 21 after hatching and streaked on sheep blood agar plates (blood agar base, Oxoid, Wesel, Germany, supplemented with 5% sheep blood). Confirmation of the staphylococcal species was done with the ID32 STAPH kit (bioMérieux, Nürtingen, Germany). Resistance patterns were determined by agar disc diffusion and were evaluated according to NCCLS criteria.7 In addition, MICs of gentamicin and kanamycin were determined by the macrodilution broth method.7
DNA preparation, hybridization experiments, conjugation and cloning
Plasmid DNA was prepared according to a Staphylococcus-specific modification8 of the alkaline lysis method with subsequent purification of the plasmids by affinity chromatography on Qiagen midi columns (Qiagen, Hilden, Germany). The reference plasmids of Escherichia coli V517 served as size standards.
For hybridization experiments, the uncleaved plasmids and plasmid fragments obtained after HindIII-, BamHI-, ClaI-, EcoRI- and XbaI-digests were electrophoretically separated, transferred to a nitrocellulose membrane and subjected to hybridization with specific gene probes for the aacA/aphD gene and the insertion sequences IS256 and IS257.8,9 The gene probes were labelled with the non-radioactive enhanced chemiluminescence system (ECL, Pharmacia-Biotech, Freiburg, Germany). Labelling and signal detection followed the specifications given by the manufacturer.
Conjugation via filter mating with Staphylococcus aureus strain B111 as recipient followed a previously described protocol8 and was repeated up to eight times at independent occasions. Transconjugants were selected on triple selective sheep blood agar plates containing 30 mg/L gentamicin, 60 mg/L rifampicin and 25 mg/L fusidic acid.
HindIII, BamHI, ClaI, EcoRI and XbaI fragments of the aacA/aphD-carrying plasmids were cloned into the corresponding sites of pBluescript II SK+ (Stratagene, Amsterdam, The Netherlands). E. coli JM107 transformants that grew on LuriaBertani (LB) agar plates (Oxoid) supplemented with 30 mg/L kanamycin were analysed for their plasmid content and subjected to sequence analysis using an automated sequencer (ALF DNA analysis system, Pharmacia-Biotech). For this, the commercially available standard primers T3, T7, M13 universal and M13 reverse (Stratagene) were initially used. Another twelve 1620-mer oligonucleotide primers, derived either from the sequence obtained with the standard primers or from the Tn4001 or IS257 sequences, were designed to complete the sequence analysis. The sequences of the three plasmids analysed in this study have been deposited with the EMBL database under the following accession numbers: pGTK1 (AJ536196), pGTK2 (AJ536193, AJ 536194), and pGTK3 (AJ536195).
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Results and discussion |
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A total of 68 staphylococcal isolates representing seven different species were isolated from the nasal swabs of 65 broiler chickens. Seventeen of these isolates showed resistance to gentamicintobramycinkanamycin. Based on their plasmid profiles, species identification and their animal origin, two Staphylococcus warneri (isolates 1 and 3), and one Staphylococcus sciuri (isolate 2), which appeared to be non-related were chosen for further analysis. Resistance phenotypes and plasmid content of these isolates are given in Table 1. Hybridization experiments with the aacA/aphD probe revealed that this gene was located on large plasmids of 33 to 43 kb, which were designated pGTK1pGTK3 (Table 1). Plasmids pGTK1 and pGTK2 were conjugative with transfer frequencies of 12 x 107. Further hybridization experiments were conducted to identify aacA/aphD-carrying XbaI, HindIII, BamHI and ClaI restriction fragments suitable for cloning (Table 1).
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Analysis of the aacA/aphD resistance gene regions of the three plasmids identified three different types of Tn4001-like elements (Figure 1). However, a complete copy of Tn4001as previously found on the S. aureus plasmid pSK15was not seen in any of the three plasmids pGTK13. In all three cases, the aacA/aphD gene was indistinguishable from that of Tn4001. However, the flanking regions differed from one another in the three plasmids and revealed the presence of largely truncated IS256 elements of 99, 262 and 425 bp as well as nine different types of complete and incomplete IS257 elements. The arrangements of the different genes and insertion sequences are displayed in Figure 1.
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Comparisons of the IS elements and their flanking regions
Sequences complementary to those of the IS256 probe were not present in the 262-bp and the 99-bp IS256 relics or only rudimentarily available in the 425-bp IS256 relics. This might explain the lack of hybridization signals when probing the three plasmids.
Probing of XbaI/ClaI digested DNA from the three plasmids with a specific gene probe for IS257 showed three to five hybridizing fragments ranging between 1.2 and >12 kb. Some of these fragments, such as XbaIClaI fragments of 1.2 and 2.8 kb as well as a ClaI fragment of
2.5 kb of plasmid pGTK2 and the XbaIClaI fragments of
2.0 and 3.9 kb of plasmid pGTK3 could be mapped in the aacA/aphD resistance gene area of the respective plasmids (Figure 1).
None of the IS257 elements detected on plasmids pGTK13 had sequences that matched exactly that of IS257L;10 a variety of nucleotide exchanges, insertions and deletions were detected. Most of these structural alterations also caused changes in the deduced amino acid sequence of the transposase proteins of the respective IS257 elements (Figure 2). Although IS257 commonly produces 8 bp direct repeats at the integration site,10,11 no directly repeated sequences adjacent to the IS257 elements identified in this study were detected. Therefore, the structural alterations of the Tn4001-like elements seen in plasmids pGTK2 and pGTK3 cannot be explained by simple insertion of IS257 elements at various sites in the Tn4001-associated IS256 elements. Analysis of the sequences upstream of the inserted IS257 elements showed no identity to IS256 sequences suggesting the loss of the major part of the IS256 sequences. The absence in pGTK2 and pGTK3 of the typical 8 bp direct repeats usually found adjacent to the complete IS257 elements requires another explanation. Possible scenarios are either the transfer of the truncated Tn4001-IS257 structure by IS257-mediated transposition or by recombination with other IS257 copies located on the same or different plasmids.4 The latter explanation appears to be more likely since IS257 elements have been detected on a wide variety of plasmids from human and animal staphylococci35,11 and no direct repeats were detectable adjacent to the truncated Tn4001-IS257 structures on plasmids pGTK2 and pGTK3.
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Acknowledgements |
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Footnotes |
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References |
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