JAC Advance Access originally published online on October 23, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1254-1256; doi:10.1093/jac/dkl418
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Fusidic acid resistance, mediated by fusB, in bovine coagulase-negative staphylococci
1 Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science Oslo, Norway 2 TINE Norwegian Dairies BA, R&D Centre Oslo, Norway 3 Section of Bacteriology, National Veterinary Institute Oslo, Norway
*Correspondence address. Norwegian Scientific Committee for Food Safety, Norwegian Institute of Public Health, PO Box 4404 Nydalen, N-0403 Oslo, Norway. Tel: +47-21-62-28-09; Fax +47-21-62-28-01; E-mail: siamak.yazdankhah{at}fhi.no
Received 22 June 2006; returned 25 July 2006; revised 18 September 2006; accepted 18 September 2006
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Abstract |
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Objectives: The aim of this study was to determine the occurrence of fusidic acid resistance, mediated by the fusB gene, among coagulase-negative staphylococci (CoNS) isolated from bovine mastitis.
Methods: A total of 113 CoNS isolates were screened for susceptibility to fusidic acid by using a disc diffusion method. The fusB gene was detected by using PCR and subsequent DNA sequencing. The localization of fusB was determined by hybridization.
Results: The fusB gene was detected in 3 of 11 fusidic acid-resistant bovine CoNS isolates. The organization of the fusB downstream region on a 40 kb plasmid in a Staphylococcus haemolyticus isolate (288/96) was highly similar to the previously reported organization of fusB on plasmid pUB101 from a Staphylococcus aureus isolate of human origin. The fusB gene was chromosomally located in the remaining two isolates.
Conclusions: Fusidic acid resistance mediated by fusB is not the dominant resistance mechanism in fusidic acid-resistant CoNS studied in this work. The similarity between the organization of the fusB downstream region in S. haemolyticus (isolate 288/96) and on plasmid pUB101 from an S. aureus isolate of human origin indicates a common ancestral origin of these genes.
Keywords: bovine mastitis , plasmid , S. haemolyticus
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Introduction |
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Fusidic acid is an effective antistaphylococcal agent, mainly used for topical treatment of skin infections. Fusidic acid inhibits the synthesis of bacterial proteins by interfering with the elongation factor, EF-G.1 EF-G is an important factor in bacteria involved in movement of tRNA on the ribosome and thereby advancement of the translation cycle by one codon.
In staphylococci, fusidic acid resistance is mainly caused by two mechanisms. One mechanism is based on reduced affinity of the protein synthesis system for fusidic acid.2 This is caused by point mutations in the chromosomal gene encoding EF-G (fusA). The point mutations occur mainly in two different domains of the fusA gene in Staphylococcus aureus.3,4 The other mechanism refers to reduced permeability, which was suggested to be plasmid-associated.5 Recently, plasmid-associated fusidic acid resistance has been characterized as a transposon-like element encoding a small (25 kDa) cytoplasmic protein.6 However, it is not known if this protein is involved in reduced permeability in fusidic acid-resistant staphylococci.
The S. aureus fusidic acid resistance plasmid pUB101 has been studied and a gene named ‘fusidic acid resistance gene’ has been characterized.7 This genetic determinant, fusB, was subsequently identified in fusidic acid-resistant S. aureus strains isolated from humans with impetigo bullosa in Scandinavia.8 The fusB gene was chromosomally located in all examined isolates.
According to the Norwegian monitoring programme for antimicrobial resistance in bacteria from humans (NORM) (www.zoonose.no), a marked increase in isolation frequencies of fusidic acid-resistant S. aureus associated with impetigo bullosa among children has been recognized. The Norwegian monitoring programme for antibiotic resistance in bacteria from feed, food and animals (NORM-VET) (www.zoonose.no) has registered a relatively high percentage of fusidic acid-resistant staphylococci isolated from animals.
In this study we report the occurrence of fusidic acid resistance, mediated by the fusB gene among coagulase-negative staphylococci (CoNS) isolated from bovine mastitis. We have determined the genetic organization of the fusB gene on a large plasmid in a Staphylococcus haemolyticus isolate.
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Materials and methods |
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Bacterial isolates and susceptibility testing
Staphylococci, resistant to one or more of the antimicrobial agents penicillin, tetracycline, streptomycin and the combination of sulphonamide and trimethoprim, isolated from bovine mastitis at the National Veterinary Institute in 1996 were stored frozen at –80°C. A disc diffusion test with semi-confluent growth on Mueller–Hinton agar plates was used in accordance with the manufacturer's instructions (Neo-Sensitabs®, Rosco Diagnostica, Taastrup, Denmark) for testing of susceptibility to the antimicrobial agents listed above. In total, 1389 staphylococcal isolates were collected (776 S. aureus and 613 CoNS). From this collection, 113 CoNS isolates were selected arbitrarily and screened for fusidic acid resistance with a disc diffusion test as described. The inhibition zones used to classify the strains as resistant or susceptible (resistant, 
31 mm; susceptible, 
32 mm) were those recommended by the Norwegian AFA Group (described in ‘User's guide’ NEO-SENSITABS®). For determination of fusidic acid MICs the Etest was used (AB Biodisk, Solna, Sweden). Strains with an MIC above 0.5 mg/L were classified as resistant (breakpoints recommended by the Norwegian AFA group). Two susceptible strains (S. aureus ATCC 29213 and S. aureus ATCC 25923) were included as quality controls when susceptibility testing was carried out. ß-Lactamase production was detected by using the ‘clover-leaf’ method.9 Fusidic acid-resistant CoNS species were identified using the Staph-Zym kit (Rosco Diagnostica).
Molecular methods
PCR amplification of the fusB gene was carried out using the primer pair FB(iii) (5'-ATTCAATCGGAAACCTATAATGATA-3') and FB(iv) (5'-TTATATATTTCCGATTTGATGCAAG-3'), as described previously.8 A possible genetic linkage between fusB and the structural gene (blaZ) of the bla operon was examined using either primers blaZ4F (5'-TTGATAAGTGAAACCGCC-3') (indicated in Figure 1), and fusB primers FB(iii) or FB(iv). In addition, the blaIR primer 5'-CTATGGCTGAATGGGAT-3' (indicated in Figure 1), and the fusB primers FB(iii) or FB(iv) were used for amplification of a possible linkage between the fusB gene and the repressor of the bla operon, blaI. The primer sequences (blaZ4F and blaIR) were based on nucleotide sequence data available under accession numbers X52734 [GenBank] and X53818 [GenBank] , respectively.
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PCR primers for amplification of the structural gene (blaZ) of the bla operon and insertion sequence IS257 was based on sequence data available under accession numbers X52734 [GenBank] and X53951 [GenBank] , respectively.
Plasmid DNA was purified from CoNS isolates using a miniprep kit (QIAGEN, Hilden, Germany). The staphylococcal cells (
1010) were lysed prior to plasmid purification using 100 µg of lysostaphin (Sigma, St Louis, MO, USA). Southern blotting and subsequent hybridization of HindIII-digested whole-cell DNA, HindIII-digested plasmid and undigested plasmid DNA was carried out using standardized methodology. DNA sequencing was performed on a model 3100-Avant genetic analyzer (Applied Biosystems, CA, USA). Plasmid transformation to a plasmid-free S. aureus RN4220 was performed using the protoplast method of Chang and Cohen.10
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Results and discussion |
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Eleven (of the 113) isolates were classified as resistant to fusidic acid. The MICs for these isolates are presented in Table 1. Ten (of 11 fusidic acid-resistant CoNS) were resistant to penicillin G, whereas resistance to tetracycline and streptomycin was observed in six isolates each (Table 1).
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All 11 isolates were screened for fusB by PCR amplification of the gene. Three of the 11 fusidic acid-resistant CoNS contained the fusB gene (Table 1). The genetic basis of resistance to fusidic acid in the remaining eight isolates remains to be determined.
Plasmid profiles of the fusB-harbouring isolates, Staphylococcus epidermidis 1708/96, S. haemolyticus 288/96 and Staphylococcus hominis 1505/96, were found to be distinct (results not shown). Hybridization showed that fusB was located on a plasmid of 40 kb in S. haemolyticus 288/96 and was chromosomally located in the two other isolates. In S. haemolyticus 288/96, the fusB gene was located on HindIII fragments of equal size when whole-cell DNA and plasmid DNA was digested.
PCR amplification revealed the presence of the structural gene (blaZ) of the bla operon and IS257 in all three isolates. Close association between fusB and the bla operon has been reported in S. aureus plasmid pUB101.7 A genetic linkage between blaZ and fusB was demonstrated in S. haemolyticus 288/96 [a PCR product of 2050 bp was generated with the primer combination blaZ4F and FB(iv)]. The amplicon was subsequently sequenced. Sequence data showed that the PCR product contained IS257, an orf152–orf170 and the fusB gene as illustrated in Figure 1. No close association was found between fusB and the bla operon in the two other isolates.
We have previously studied the isolate S. haemolyticus 288/96 and have identified the penicillin resistance gene cluster (blaZ, blaR and blaI) on a plasmid of 40 kb.11 Aiming to characterize the flanking region of the bla operon, the plasmid was transformed to a plasmid-free S. aureus. The transformant produced ß-lactamase and was resistant to fusidic acid with an MIC of 12 mg/L. The plasmid was designated pNVH96 and a DNA sequence of 13 kb from this plasmid has been determined (accession number AJ302698
[GenBank]
).
Comparison of the DNA sequences of plasmids pNVH96 from S. haemolyticus and pUB101 from S. aureus showed that the area containing the fusidic acid resistance determinant (fusB, bla genes, IS257, orf152 and orf170) are organized in a similar manner (Figure 1). However, the orf152–170 on pNVH96 was identified as a fusion of orf152 and orf170 on pUB101. The plasmid size differed between pUB101 (21.845 kb) and pNVH96 (40 kb).
Use of fusidic acid in dairy cattle in Norway is very limited, while penicillin G is the first choice drug in bovine mastitis therapy. In this study 10 of 11 fusidic acid-resistant CoNS were resistant to penicillin G (Table 1). Penicillin selection pressure may contribute to a further spread of fusidic acid resistance within the staphylococcal species.
The fusA gene is a housekeeping gene and in S. aureus specific fusA mutations are supposed to result in increased MICs of fusidic acid. So far, such mutations have not been mapped for the various CoNS species. A considerable amount of fusA sequence data for the various CoNS species must be provided for analysis of mutations possibly involved in fusidic acid resistance.
This is the first report of fusB in staphylococci other than S. aureus. Bovine CoNS harbouring fusB might represent a reservoir of fusidic acid resistance genes that may be transmitted to other staphylococci, including those pathogenic to humans and other animals.
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None to declare.
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Acknowledgements |
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The technical help of Hanne Tharaldsen (National Veterinary Institute) is highly appreciated.
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References |
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