Molecular analysis of the plasmid-borne aacA/aphD resistance gene region of coagulase-negative staphylococci from chickens

Carla C. Lange1,2, Christiane Werckenthin1,3 and Stefan Schwarz1,*

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, 36038–330 Juiz de Fora-MG, Brazil

Received 3 February 2003; returned 20 February 2003; revised 7 March 2003; accepted 13 March 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The aim of this study was to analyse selected coagulase-negative staphylococci from chickens for the genetic basis of plasmid-borne resistance to gentamicin, tobramycin and kanamycin, and also for the presence of Tn4001-like elements.

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 33–43 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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Combined resistance to gentamicin, tobramycin and kanamycin in staphylococci is associated with transposon Tn4001, a non-conjugative, composite transposon of 4568 bp in which the aacA/aphD resistance gene region is flanked by inverted copies of the insertion sequence IS256.1,2 The aacA/aphD gene codes for a single protein of 479 amino acids with acetyltransferase activity in the amino terminal domain and phosphotransferase activity in the carboxy terminal domain.2 This gene occurs on conjugative and non-conjugative plasmids ranging in size from 20.8 to 57 kb.35 Despite numerous reports on the occurrence of aacA/aphD, few studies have focused on the corresponding Tn4001 elements.4,5

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial isolates and antimicrobial susceptibility testing

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 Luria–Bertani (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 16–20-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).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial isolates, resistance phenotypes, plasmid content and hybridization studies

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 gentamicin–tobramycin–kanamycin. 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 pGTK1–pGTK3 (Table 1). Plasmids pGTK1 and pGTK2 were conjugative with transfer frequencies of 1–2 x 10–7. 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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Table 1.  Characteristics of the staphylococcal isolates investigated in this study
 
Molecular analysis of the aacA/aphD resistance gene region on plasmids pGTK1–3

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 Tn4001—as previously found on the S. aureus plasmid pSK15—was not seen in any of the three plasmids pGTK1–3. 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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Figure 1. Structural organization of the aacA/aphD resistance gene area of plasmids pGTK1–3 from avian staphylococci in comparison with Tn4001.1,2 Restriction endonuclease cleavage sites are abbreviated as follows: Bg (BglII), C (ClaI), EV (EcoRV), H (HindIII) and X (XbaI). A distance scale in kb is given below each map. The different complete or truncated IS257 elements as well as the truncated IS256 elements are shown as arrows with the arrowhead indicating their orientation. The aacA/aphD gene and ORF132 are also presented as arrows with the arrowhead indicating their direction of transcription.

 
Comparative analysis of the aacA/aphD resistance gene region present on plasmid pGTK2 from avian S. sciuri matched closely the arrangements of complete IS257 elements, truncated IS256 elements and the aacA/aphD gene on plasmids pSK41, pUW3626 and pSH6.4,5 A comparison of the IS257 sequences confirmed this observation. With the exception of A at position 89 which was G in pSH6 and pSK41, IS257a from pGTK2 showed the same sequence alterations in comparison with IS257L9 as IS257/1 in pSH64 and IS257E in pSK41.5 Moreover, IS257d in pGTK2 exhibited the same bp substitutions, deletions and insertions as IS257/2 in pSH64 and IS257F in pSK41.5 Finally, the IS257f in pGTK2 shared the same bp substitutions with IS257/3 from pSH64 and IS257G from pSK41.5 In contrast, the arrangements of the IS257 elements and IS256 relics on plasmids pGTK3 and pGTK1, both from avian S. warneri isolates, differed from the known arrangements reported on other staphylococcal plasmids.

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 XbaI–ClaI fragments of ~1.2 and 2.8 kb as well as a ClaI fragment of ~2.5 kb of plasmid pGTK2 and the XbaI–ClaI 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 pGTK1–3 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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Figure 2. Comparative analysis of the amino acid sequences of the IS257-associated transposase proteins identified in this study with the amino acid sequence of the transposase of IS257L.10 Amino acids are shown with the one-letter code.

 

    Acknowledgements
 
We wish to thank Erika Nußbeck for her expert technical assistance.


    Footnotes
 
* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Byrne, M. E., Rouch, D. A. & Skurray, R. A. (1989). Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001. Gene 81, 361–7.[CrossRef][ISI][Medline]

2 . Rouch, D. A., Byrne, M. E., Kong, Y. C. & Skurray, R. A. (1987). The aacA-aphD gentamicin and kanamycin resistance determinant of Tn4001 from Staphylococcus aureus: expression and nucleotide sequence analysis. Journal of General Microbiology 133, 3039–52.[ISI][Medline]

3 . Lyon, B. R. & Skurray, R. A. (1987). Antimicrobial resistance of Staphylococcus aureus: genetic basis. Microbiological Reviews 51, 88–134.[ISI]

4 . Byrne, M. E., Gillespie, M. T. & Skurray, R. A. (1990). Molecular analysis of a gentamicin resistance transposon-like element on plasmids isolated from North American Staphylococcus aureus strains. Antimicrobial Agents and Chemotherapy 34, 2106–13.[ISI][Medline]

5 . Berg, T., Firth, N., Apisiridej, S., Hettiaratchi, A., Leelaporn, A. & Skurray, R. A. (1998). Complete nucleotide sequence of pSK41: evolution of staphylococcal multiresistance plasmids. Journal of Bacteriology 180, 4350–9.[Abstract/Free Full Text]

6 . Schwarz, S. & Chaslus-Dancla, E. (2001). Use of antimicrobials in veterinary medicine and mechanism of resistance. Veterinary Research 32, 201–25.[CrossRef][ISI][Medline]

7 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard M31-A. NCCLS, Villanova, PA, USA.

8 . Noble, W. C., Rahman, M., Karadek, T. & Schwarz, S. (1996). Gentamicin resistance gene transfer from Enterococcus faecalis and E. faecium to Staphylococcus aureus, S. intermedius and S. hyicus. Veterinary Microbiology 52, 143–52.[CrossRef][ISI][Medline]

9 . Hesselbarth, J., Werckenthin, C., Liebisch, B. & Schwarz, S. (1995). Insertion elements in Staphylococcus intermedius. Letters in Applied Microbiology 20, 180–3.[ISI][Medline]

10 . Rouch, D. A. & Skurray, R. A. (1989). IS257 from Staphylococcus aureus: member of an insertion sequence superfamily prevalent among gram-positive and gram-negative bacteria. Gene 76, 195–205.[CrossRef][ISI][Medline]

11 . Werckenthin, C., Schwarz, S. & Roberts, M. C. (1996). Integration of pT181-like tetracycline resistance plasmids into large staphylococcal plasmids involves IS257. Antimicrobial Agents and Chemotherapy 40, 2542–4.[Abstract]