Genetic characterization of the fusidic acid and cadmium resistance determinants of Staphylococcus aureus plasmid pUB101

Frances G. O’Brien1, Christopher Price1, Warren B. Grubb1 and John E. Gustafson1,2,*

1 School of Biomedical Sciences and Gram-positive Bacteria Typing and Research Unit, Curtin University of Technology, Perth 6845, Western Australia, Australia; 2 Microbiology Department, College of Osteopathic Medicine, Midwestern University, Downers Grove, IL 60515, USA

Received 7 March 2002; returned 7 May 2002; revised 14 June 2002; accepted 20 June 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We report the cloning of the fusidic acid and cadmium resistance determinants from Staphylococcus aureus plasmid pUB101. The pUB101 fusidic acid resistance determinant was located on a 2.9 kb HindIII fragment. Sequencing of this fragment revealed three putative open reading frames (ORFs) of 213 (far1), 152 (orf152) and 170 amino acids (orf170), which are flanked by the right-hand end of insertion sequence IS431/257 (IS431/257RH) and a partial ORF. Far1 and Orf152 demonstrated homology with a chromosomally encoded fibronectin-binding protein of Listeria monocytogenes and the putative protein YosT, found on the SPßc2 prophage of Bacillus subtilis, respectively. Transformation of S. aureus with a construct containing a 949 bp far1-specific amplicon led to the isolation of a fusidic acid-resistant transformant, thereby identifying the pUB101 fusidic acid resistance structural gene. Between orf152 and far1 we identified a unique 113 bp symmetrical element and other repeat elements that may be involved with the control of orf152 and/or far1 expression. Hybridization of Southern blots revealed that far1 was not located on the chromosome or plasmid content of a limited number of Australian, UK and Hong Kong fusidic acid-resistant isolates. The pUB101 cadmium resistance determinant was located on a 3.6 kb HindIII fragment that carried a cadDX operon, remnants of two putative plasmid replication protein genes and IS431/257RH. Sequence analysis also demonstrated the presence of a single-stranded origin of replication, normally found on rolling circle replicating plasmids, within the putative promoter region of the cadDX operon.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fusidic acid is an effective component of antibiotic combinations used to treat infections caused by Staphylococcus aureus.1 Fusidic acid binds to a complex of elongation factor G (EF-G)–GTP/GDP and the ribosome of susceptible bacteria, inhibiting the release of EF-G–GDP after the translocation step of peptide synthesis.2,3 This denies access of the complex of aminoacyl-tRNA-elongation factor Tu-GTP to the ribosome, thus blocking further rounds of protein synthesis.4 Certain mutations in fusA, the chromosomally located gene encoding EF-G in S. aureus, presumably lead to fusidic acid resistance by reducing the affinity of the drug for the protein synthesis machinery.5,6

Plasmid-encoded fusidic acid resistance in S. aureus was first reported in 1966.7 This initial report was followed by additional articles.810 In these early studies, plasmid-mediated resistance represented the predominant mechanism of fusidic acid resistance in clinical isolates of S. aureus.1

The S. aureus fusidic acid resistance plasmid pUB101 (21.9 kb) has been partially characterized5,1114 and is reported to encode a fusidic acid permeability barrier.5,11 Plasmid pUB101 also encodes a ß-lactamase and resistance to cadmium.14 The pUB101 gene for ß-lactamase production, blaZ, resides on a 1.3 kb HindIII fragment and has been cloned and characterized previously.13 In an effort to further understand the mechanisms of resistance encoded by pUB101, the fusidic acid and cadmium resistance determinants have been cloned and sequenced.


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

Strains and plasmids used for cloning of the fusidic acid and cadmium resistance determinants are described in Table 1. Escherichia coli DH5{alpha} (Bethesda Research Laboratories) was used in general cloning procedures and for maintenance of S. aureusE. coli shuttle vector pLI5015 constructs. Strains of DH5{alpha} were grown at 35°C in Luria-Bertani (LB) broth (Life Technologies, Paisley, Scotland) or on LB agar plates and, when appropriate, ampicillin was added to these media (final concentration 100 mg/L; Sigma Chemical Company, St Louis, MO, USA). S. aureus strains were grown in brain–heart infusion (BHI) broth (Gibco Diagnostics, Gaithersberg, MD, USA) or maintained on BHI agar.


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Table 1.  Strains (with their MICs) and plasmids used in this study
 
Population analyses and susceptibility testing

Population analyses were performed by microdilution plating as described previously.16 Resistance to fusidic acid was tested by disc diffusion using a 10 µg fusidic acid disc (Oxoid, Basingstoke, UK) according to published guidelines17 and resistance to cadmium was tested using a 10 mmol cadmium acetate disc (Ajax Chemicals, Sydney, Australia) as described previously.18 MICs of fusidic acid (Leo Pharmaceutical Products, Ballerup, Denmark) and cadmium were determined by agar dilution in accordance with NCCLS guidelines.19

Electroporation and plasmid curing

Electroporation of S. aureus was performed essentially as described by Augustin & Gotz,20 with cells for electroporation cultured at 35°C in B2 broth (1% casein hydrolysate, 2.5% yeast extract, 0.1% K2PO4, 0.5% glucose, 2.5% NaCl, pH 7.5) and electrotransformants selected on NYE agar (1% casein hydrolysate, 0.5% yeast extract, 0.5% sodium chloride)21 containing 20 mg/L chloramphenicol (Sigma). Plasmid curing was performed at 43.5°C as previously described.18 Isolated colonies were screened by replication onto BHI agar and BHI agar containing chloramphenicol (20 mg/L).

General molecular biological techniques

Fusidic acid resistance plasmid pUB101 was isolated from S. aureus strain WBG1576 (Table 1) using the cetyltrimethylammonium bromide method as described previously.22 Restriction endonucleases, calf intestinal alkaline phosphatase and T4 DNA ligase were used according to the manufacturer’s directions (Promega Corporation, Madison, WI, USA). DNA fragments used in cloning procedures were purified using the BRESAclean DNA gel purification kit (Geneworks, Adelaide, South Australia). Plasmid DNA for sequencing and subcloning was isolated from E. coli using the alkaline lysis method and purified by precipitation with polyethylene glycol.23 Sequencing was performed on pUB101 HindIII fragments that were cloned into pBluescript SK+ (Stratagene, La Jolla, CA, USA) or pGEM-T Easy Vector (Promega) using the SequiTherm Excel II DNA Sequencing Kit-LC (Epicentre Technologies, Madison, WI, USA) according to the manufacturer’s instructions. Forward and reverse primers were supplied with the sequencing kit and additional primers were designed as required and supplied by MWG-Biotech GmbH, Ebersberg, Germany. Reaction products were separated on a LI-COR Long Readir 4200 DNA sequencer and sequences were collected using Base Imagir software (LI-COR, Inc., Lincoln, NE, USA). Sequences were then stored and assembled with the program AssemblyLIGN and analysed using MacVector 6.0 (Accelrys, Cambridge, UK). Protein alignments were determined with AlignX of the Vector NTI Suite V.6 (Informax, Bethesda, MD, USA) and DNA alignments were determined using MacVector 6.0. PCR primers were synthesized and supplied by Geneworks. The sequences of the 2.9 and 3.6 kb HindIII pUB101 fragments have been deposited in the Genbank database under the accession numbers AF468686 and AF468685, respectively.

Cloning of fusidic acid and cadmium resistance determinants from plasmid pUB101

Plasmid pUB101 was isolated from S. aureus strain WBG1576, digested with HindIII (Promega), ligated into pBluescript SK+ and transformed into DH5{alpha} followed by selection for ampicillin resistance. Single colonies of the DH5{alpha} transformants were then picked and their respective plasmid constructs were isolated and analysed following HindIII restriction and the viewing of digests on standard agarose gels.23 Eight of the 10 possible pUB101 HindIII fragments (those of 5.9, 3.6, 2.9, 2.4, 2.0, 1.3, 0.6 and 0.2 kb) had been successfully cloned; the 2.2 and 0.3 kb fragments were not cloned in the library. All cloned pUB101 fragments were then sequenced. The 2.9 and 3.6 kb HindIII fragments were then subcloned into the S. aureusE. coli shuttle vector pL150 and transformed into DH5{alpha}, followed by selection for ampicillin resistance. Both pUB101::pL150 constructs were then isolated and electroporated into S. aureus strain RN4220,24 followed by selection for chloramphenicol resistance. S. aureus RN4220 transformants FO52, containing plasmid pFO69 (pL150::2.9 kb HindIII pUB101 fragment) and FO59, containing plasmid pFO47 (pL150::3.6 kb HindIII pUB101 fragment), demonstrated resistance to fusidic acid and cadmium, respectively.

Primers (5'-ACTTGTCCGTGTGCTAAC-3' and 5'-AGTTCGGGAGGTGATGATG-3') were designed to amplify a 949 bp region (bp 1503–2451 of the 2.9 kb HindIII pUB101 fragment) covering far1 and its flanking regions, using undigested pUB101 DNA as template (annealing temperature 55°C). This amplicon was purified and ligated into pGEM-T Easy vector to give construct pFO73, and transformed into DH5{alpha} (Table 1). The authenticity of the cloned amplicon was confirmed by sequencing the pFO73 cloned insert. The amplicon was recovered from pFO73 by EcoRI digestion, gel purified and subcloned into the EcoRI site of pL150. This construct (pFO82) was then transformed into RN4220 to produce strain FO88 (Table 1). Relevant MICs for all strains are presented in Table 1.

Southern blotting and hybridization with the far1 amplicon

Attempts were made to detect the far1 sequence on the chromosome or plasmid content of known fusidic acid-resistant strains of S. aureus from the well characterized strain collection of the Gram-positive Bacteria Typing and Research Unit (GPTRU, Perth, Western Australia). Australian strains not referenced below were obtained by the GPTRU within Australia between 1997 and 2001. These strains included: Australian community strains: CN105N, N83T, NG5T, WBG8343,25 WBG9456, WBG940925 and 912952;26 Australian epidemic strains: 17708, 16880, AH10, AH53, AH73, RPAH22 and 912180;26 UK epidemic strains: UK EMRSA-1227 and UK EMRSA-13;27 and Hong Kong strains 91132826 and 911329.26 Strains WBG1576 (pUB101) and WBG448328 (fusidic acid susceptible) were used as positive and negative controls, respectively, for the plasmid hybridization experiment, and S. aureus NCTC832529 was the negative control for the chromosomal hybridization experiment. Initially, chromosomal and plasmid DNA was isolated as described previously.22,25 Chromosomal DNA was restricted with SmaI and separated by PFGE and plasmid preparations were separated in agarose gels as described previously.25 DNA from the plasmid and chromosomal gels was then transferred onto Hybond-N+ nylon (Amersham Pharmacia Biotech UK Ltd, Little Chalfont, UK). Purified far1 amplicon was labelled with digoxigenin-11-dUTP by the random-primed labelling technique using the DIG DNA Labelling and Detection Kit (Boehringer Mannheim, Mannheim, Germany) and was hybridized with the membrane-bound DNA as directed by the label manufacturers. Hybridization signals were detected on Hyperfilm MP autoradiography film (Amersham Pharmacia Biotech UK Ltd).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Characterization of the pUB101 fusidic acid resistance determinant

S. aureus transformant FO52, containing the shuttle vector construct pFO69, demonstrated an MIC of fusidic acid of 64 mg/L, which was higher than the pUB101-containing parent strain, WBG1576 (MIC 16 mg/L) (Table 1). Fusidic acid resistance population analyses also demonstrated that more cells of the fusidic acid-resistant transformant FO52 were surviving at concentrations of 2–16 mg/L fusidic acid, compared with WBG1576 (Figure 1). The increase in fusidic acid resistance in FO52 compared with WBG1576 may be due to differential expression of the pUB101 fusidic acid resistance determinant on pFO69 compared with pUB101, or to a copy number effect of the recombinant shuttle vector construct compared with the wild-type plasmid.



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Figure 1. Fusidic acid resistance population analyses of fusidic acid-resistant WBG1576 (pUB101) (circles), fusidic acid-resistant transformants, FO52 (pFO69) (squares) and FO88 (pFO82) (triangles), and fusidic acid-susceptible FO66 (pLI50) (diamonds).

 
Sequence analysis of the 2951 bp HindIII fragment (Figure 2) harbouring the fusidic acid resistance determinant revealed three intact putative open reading frames (ORFs): orf213 (bp 1651–2292), orf152 (bp 927–1385) and orf170 (bp 257–769). At one end, a partial ORF (60 aa) is divergently transcribed from the above ORFs, while the other end consists of the right-hand end of insertion sequence IS431/25730,31 (IS431/257RH) (Figure 2). The 60 aa sequence of the flanking partial ORF did not demonstrate homology with proteins currently in the public databases and, since only a portion of the ORF was present, it probably is not required for fusidic acid resistance expression.



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Figure 2. Cartoon representing ORFs within the 2951 bp pUB101 HindIII fragment. Base pair (bp) or nucleotide (nt) positions representing relevant coding sequences and other sequences within the fragment are displayed. The start codon of orf152 and the stop codon of far1 are boxed. The putative orf152 ribosomal binding site is in bold type and underlined. The pUB101 symmetrical element is indicated by a thickly emboldened line above the sequence. Direct repeats (DR) and inverted repeats (IR) and their respective nucleotide lengths are indicated; arrows represent repeat orientation and nucleotides that differ between imperfect repeat elements are in lower case. The symmetrical element central nt (T) is circled.

 
Orf213 (213 aa) shared greatest identity (34.1%) along the entire length of a 215 aa fibronectin-binding protein (Fbp; GenBank accession number CAB76365) of Listeria monocytogenes.32 Orf213 also demonstrated strong homology with a putative 218 aa protein (GenBank accession number AAK06272) found in the Lactococcus lactis subsp. lactis genome (Figure 3).33 It is possible that both Orf213 and the putative 218 aa protein identified in the L. lactis genome have similar functions.



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Figure 3. Protein alignment of Far1, the 213 aa fibronectin-binding protein of L. monocytogenes (GenBank accession number CAB76365) and a putative 218 aa protein of L. lactis subsp. lactis (accession number AAK06272). Residues coloured white on a black background are completely conserved at that position. Residues coloured black on a dark grey background represent conserved consensus residues at that position. Residues coloured black on a light grey background represent similar consensus residues at a given position.

 
Cloning of a 949 bp amplicon covering orf213 and 148 bp downstream and 159 bp upstream of orf213 into pLI50 resulted in plasmid pFO82 (Table 1). Transformation of RN4220 with pFO82 resulted in the isolation of a fusidic acid-resistant transformant (FO88) with an MIC of fusidic acid (8 mg/L) lower than strains WBG1576 (16 mg/L) and FO52 (64 mg/L) (Table 1). This result was mirrored in the population analysis (Figure 1), which showed subpopulations of FO88 cells surviving only up to 10 mg/L fusidic acid, whereas subpopulations of FO52 and WBG1576 cells survived up to 16 mg/L fusidic acid. This demonstrated that other sequences on the 2.9 kb HindIII fragment are required in addition to orf213 for the full expression of fusidic acid resistance. Curing strain FO88 of pFO82 led to the loss of fusidic acid and chloramphenicol resistance, as expected (data not shown). Since orf213 is required for fusidic acid resistance expression, we have named orf213, far1 (for fusidic acid resistance gene). Hybridization experiments revealed that far1 was not present in the chromosome and plasmid contents of 18 recently isolated Australian, UK and Hong Kong fusidic acid-resistant S. aureus strains.

The amino acid sequence of Orf152 (152 aa) demonstrated greatest identity (38.2%) along the entire length of the putative 149 aa YosT (GenBank accession number NP_046718) found on the SPßc2 prophage of Bacillus subtilis.34 Two additional putative proteins of 117 aa and a large protein of 299 aa (GenBank accession numbers BAB07728 and BAB04120, respectively) located on the Bacillus halodurans genome35 also demonstrated homology to Orf152 (data not shown). The larger putative protein demonstrated homology with Orf152 at its N-terminus, suggesting that this protein has evolved with an Orf152-like module. A Kyte–Doolittle hydrophobicity plot of Orf152 demonstrated that the protein is predominantly hydrophilic in nature (data not shown), suggesting that it is a cytoplasmic protein. The functions of Orf152 and the Orf152 homologues are presently unknown.

orf170 begins with the less common staphylococcal start codon, GTG, and its 3' end extends into IS431/257RH, which provides a TAA stop codon. Orf170 does not possess homology with any other proteins currently in the public databases.

The fusidic acid resistance mechanism of pUB101 has previously been reported to be a fusidic acid-inducible system.5 The start codon of orf152 and the stop codon of far1 are separated by 265 bp (bp 1386–1650) (Figure 2). In between these two ORFs we have identified two sets of imperfect direct repeat sequences (DR11 and DR12, and DR21 and DR22) and two sets of imperfect inverted repeat sequences (IR11 and IR12, and IR21 and IR22) (Figure 3). The manner in which the DR1 (11 bp), DR2 (11 bp) and IR1 (12 bp) sets present themselves is symmetrical. DR11 and DR12, and DR21 and DR22 are both separated by 13 bp. In addition, the end of DR12 and beginning of DR21 are both equidistant (9 bp) from IR11 and IR12, respectively. Lastly, IR11 and IR12 are separated by 1 bp, making this symmetrical element 113 nt in total length. Interestingly, what we have termed the pUB101 symmetrical element does not share significant homology with any other DNA sequence currently in the databases. The IR2 set (Figure 2) appears 30 bp downstream of the far1 stop codon, and 28 bp upstream of the far1 start codon there is another 11 bp imperfect IR set (IR3) (Figure 2). It has previously been reported that repeat elements in DNA sequences upstream of start codons and downstream of stop codons play roles in bacterial transcriptional regulation.3639 We hypothesize that the pUB101 symmetrical element and the IR2 and IR3 sets are involved with the inducibility of fusidic acid resistance by pUB101.

Characterization of the pUB101 cadmium resistance determinant

S. aureus RN4220 transformant FO59, containing the recombinant shuttle vector construct pFO47, demonstrated a cadmium MIC of 256 µM, which is equivalent to the cadmium MIC for the pUB101 containing parent strain WBG1576 (Table 1). The 3620 bp HindIII fragment harboured two contiguous ORFs, making up a cadDX cadmium resistance operon (bp 1351–2346), two truncated genes (rep1 and rep2) and a flanking IS431/257RH on one end of the fragment. The cadDX operon of pUB101 encodes the cadmium resistance protein CadD (95.7% identity with S. aureus CadD, GenBank accession number AAB51227, 209 aa)40 and downstream of cadD, the positive regulator of cadDX, CadX (98.3% identity with S. aureus CadX, GenBank accession number NP_395542.1, 115 aa).41

In the 209 bp sequence downstream of cadDX, including the cadD start codon, we found two sets of perfect inverted repeats (IR4 and IR6), an imperfect inverted repeat set (IR51 and IR52) and a set of perfect direct repeats (DR3) (Figure 4). A portion of this sequence is highly homologous with a conserved region of single-stranded origins of replication (SSO) that are required to initiate replication of the lagging strand in rolling circle replicating plasmids (Figure 4).42



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Figure 4. The cadDX promoter region (nt 2344–2552) of the 3620 bp pUB101 HindIII fragment covering the conserved SSO sequence described by Kramer et al.47 is shown. Nucleotides completely conserved in staphylococcal SSO sequences47 are shown in bold type and are underlined. The start codon of cadD is boxed and direct repeats (DR) and inverted repeats (IR) and their respective nucleotide length are indicated; arrows represent repeat orientation and nucleotides that differ between imperfect repeat elements are in lower case.

 
Conceptual translation of rep1 (bp 697–897) and rep2 (bp 3321–3497) produced truncated proteins (66 and 58 aa, respectively) that are homologous with numerous examples of staphylococcal plasmid replication proteins. Since both Rep1 and Rep2 are truncated, it is unlikely that they act as pUB101 replication proteins. Truncated Rep1 demonstrated the greatest identity (83% identity over a 54 aa sequence overlap) with the C-terminal end of the Staphylococcus epidermidis 287 aa putative plasmid replication protein (GenBank accession number AAC18950).43 Truncated Rep2 demonstrated the greatest identity (86% identity over 57 aa sequence overlap) to the C-terminal end of a 334 aa putative plasmid replication protein (GenBank accession number AAA60377) of S. epidermidis.44 At the end of rep2 (including the rep2 stop codon, bp 3260–3325) was a 25 nt perfect IR set (IR6) separated by 16 nt that can form a single-stranded cruciform structure. It is possible that this IR set is involved in the transcriptional termination of rep2 in its untruncated form, if it exists, or could be involved in plasmid replication. Database searches failed to reveal any sequences significantly similar to this IR set, which, together with the pUB101 symmetrical unit, makes another unusual pUB101 sequence. The TTG start codon of rep1 is within a 15 bp pUB101 sequence of 3'-CCAAGACAAGTTTC-5', which is identical to the first 15 nt of the left hand IR of IS431/257.30 This suggests that movement of an IS431/257 element was probably involved with the event that led to the truncation of rep1.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The pUB101 fusidic acid resistance mechanism does not involve drug modification and does not protect cell-free protein synthesis machinery directly from the action of fusidic acid.5 Using this historical evidence it is still logical to suggest that the pUB101 far1-mediated mechanism does not allow fusidic acid to reach its target site.5,11 It was initially demonstrated that the pUB101 mechanism leads to a decrease in the molar ratio of phosphatidylglycerol to lysylphosphatidylglycerol, suggesting that this membrane alteration in a pUB101-containing strain led to the formation of a fusidic acid permeability barrier.5 However, in a later study using a strain that harboured pUB101 and another containing a pUB101 derivative that lacked the fusidic acid resistance determinant, no alterations in membrane composition were observed.11 In addition, no membrane protein profile alteration has been demonstrated in pUB101-containing strains.5,11 Therefore, further experimentation will be required to determine how Far1, a fibronectin-binding protein homologue, mediates fusidic acid resistance in S. aureus, or if Far1 possesses fibronectin-binding activity as well.

far1 was not present in the other fusidic acid-resistant strains investigated, suggesting that the fusidic acid resistance in these strains may result from mutations in the fusA gene. Interestingly, a recent study demonstrated fusA mutations in only five of 20 Danish clinical fusidic acid resistant isolates,6 suggesting that, in Denmark at least, another fusidic acid resistance mechanism is evolving. More fusidic acid-resistant strains from other geographical locations need to be screened with far1 before conclusions can be drawn as to the prevalence of the far1-mediated fusidic acid resistance mechanism among S. aureus isolates.

The fact that both the pUB101 2951 bp and the 3620 bp HindIII fragments contain an IS431/257RH, argues for the existence of two IS431/257 copies on pUB101. IS431/257 is thought to play a role in the capture of various drug resistance genes by the chromosome and plasmids of staphylococci.45,46 It is possible that IS431/257 has played a role in the capture of the fusidic acid and/or the cadmium resistance determinants by pUB101. We have also shown that the pUB101 cadDX promoter region shares similarity to staphylococcal SSOs, described previously, suggesting that the cadDX operon/promoter evolved from an episome that replicates via the rolling circle mechanism.

In summary, a limited sequence analysis of pUB101 revealed a novel fusidic acid resistance mechanism and additional DNA sequence that is probably involved with its induction. Further analysis of the pUB101 sequence will undoubtedly provide more details on the evolution of this ß-lactamase plasmid.


    Acknowledgements
 
The authors would like to acknowledge Julie Pearson of the GPBTRU for assistance in the preparation of chromosomal DNA for some of the fusidic acid-resistant S. aureus strains used in the far1 hybridization experiment. J.E.G. thanks Saleem Khan of the University of Pittsburgh School of Medicine for valuable discussions on SSO sequences. We also thank the Australian Research Council and Leo Pharmaceutical Products Ltd for funding this research project, and the Central Public Health Laboratory, Collindale for the gift of strains UK EMRSA-12 and UK EMRSA-13. C.P. is paid by an Australian Postgraduate Stipend.


    Footnotes
 
* Correspondence address. Department of Biology MSC 3AF, New Mexico State University, PO Box 30001, Las Cruces, NM 88003-8001, USA. Tel: +1-505-646-3611; Fax: +1-505-646-5660; E-mail: jgustafs{at}nmsu.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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