Microbiology Group, School of Biomedical Sciences, Curtin University of Technology, GPO Box U1987, Perth 6845, Western Australia
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Abstract |
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Introduction |
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The Gram-negative organisms Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa and Pseudomonas cepaciaexhibit phenotypic multiple antibiotic resistance, following growth in sub-inhibitory concentrations of salicylate. 8 ,9 ,10 ,11 ,12
Salicylate can also be applied topically in creams as an antimicrobial. 13 Aspirin is often prescribed to prevent heart attacks and cerebral thromboses. 14 Ingesting aspirin 1-3 g/day can reduce pain and fever. The ingestion of 4-8 g/day can reduce redness and swelling of joints due to rheumatic fever, gout and rheumatoid arthritis. 14 Once ingested by humans, aspirin and salicylate can be detected at the skin surface. 15 Fusidic acid is an anti-staphylococcal agent administered systemically and topically. 16 Fusidic acid and aspirin, or salicylate, can be present in patients undergoing anti-staphylococcal therapy. Therefore, a study of the effects of salicylate and related compounds on staphylococcal fusidic acid resistance is warranted.
Fusidic acid is a steroid antibiotic, effective in combating infections caused by Gram-positive organisms, in particular Staphylococcus aureus. 17 Fusidic acid inhibits the translocation step in protein synthesis by binding to elongation factor G (EF-G). 18 Resistance to fusidic acid in S. aureus is mediated by chromosomal mutations occurring in the gene encoding EF-G (fusA), leading to the production of an altered EF-G with decreased fusidic acid binding, 19 and by plasmid pUB101, which encodes a fusidic acid permeability barrier. 20
This study shows that salicylate and related compounds increase fusidic acid resistance levels in fusidic acid-resistant and -susceptible S. aureus, representing at least six lineages.
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Materials and methods |
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The strains in this study are described in Table I.
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Stock solutions of sodium salicylate (1 M), sodium acetate (1 M) and sodium benzoate (0.5 M) (BDH Laboratory Supplies, Poole, UK) and sodium ibuprofen (0.5 M) (Sigma Chemical Company, St Louis, MO, USA) were made up in H 2O. Acetaminophen (0.5 M) and acetyl salicylic acid (0.5 M) (ICN Pharmaceuticals, Costa Mesa, CA, USA) and saligenin (Sigma Chemical Company) stocks were made up in ethanol. The pH of all stock solutions was adjusted to 7, and when required, filter sterilized. All stock solutions were stored in dark containers at 4°C. Sodium fusidate (Leo Pharmaceuticals, Ballerup, Denmark) was dissolved in H 2O, filter sterilized and stored at -20°C until needed.
Phage typing and pulsed-field gel electrophoresis
Phage typing was performed according to the method of Blair & Williams, 21 using the International basic set of phages and the Australian supplementary set of phages. 22,23
Pulsed-field gel electrophoresis was performed by the contour-clamped homogeneous electric field (CHEF) method. 24,25 Chromosomal DNA was digested with SmaI (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Electrophoresis was performed in 1% w/v molecular grade agarose (BioRad Laboratories, Richmond, CA, USA) using the CHEF-DRIII system (BioRad Laboratories). Initial pulse time was 1 s and final pulse time was 40 s at 12°C for 20 h in 0.5 x TBE buffer (45 mM Tris, 45 mM Boric acid, 1 mM EDTA, pH 8.0) at 200 V. The chromosomal banding patterns were scanned with a Fluor-S MultiImager (BioRad Laboratories) and analysed on Multi-Analyst/PC, version 1.1 (BioRad Laboratories).
MIC determination
Fusidic acid MIC determinations were performed either by agar dilution on Mueller-Hinton agar (MHA) (Oxoid, Basingstoke, UK) according to NCCLS guidelines, or using the gradient plate method essentially as described by Szybalski & Bryson. 26
Gradient plates were prepared in square plates (120 mm x 120 mm) with Luria Base agar (LBA) (Gibco Diagnostics) and fusidic acid gradients of 0-32 mg/L and 0-0.3 mg/L fusidic acid for fusidic acid-resistant and -susceptible strains, respectively. Salicylate or related substances were added to a final concentration of 2 mM to both agar layers. Overnight Luria broth (LB) (Gibco Diagnostics) cultures, inolculated with single colonies, were diluted to an OD 625 of 0.1 with LB and streaked on to freshly prepared gradient plates with sterile cotton swabs. Inoculated plates were incubated at 35°C and read following 48 h incubation. MICs were calculated as follows:
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MICs were taken as the point at which confluent bacterial growth halted. All experiments were performed in triplicate.
Population analyses
Single colonies were grown overnight in 5 mL LB. Population analyses were performed as described previously, 27 with LBA plates containing different concentrations of fusidic acid, with and without the addition of 2 mM salicylate. Colonies of BB255 and WBG8287 picked from fusidic acid-containing media, with and without the addition of 2 mM salicylate, were isolated and grown in three passages of drug-free LB overnight before fusidic acid MICs were determined.
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Results |
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Phage types and fusidic acid MICs are presented in Table I. Fusidic acid
MIC levels vary from 0.03125 mg/L to 32 mg/L. Strains with fusidic acid MICs 2 mg/L
are considered resistant.
28,29 Eight phage
types are represented by the strains used in this study (Table I). Using the
Multi-Analyst/PC, version 1.1, a dendrogram of % relatedness (Figure 1b) was constructed by comparing the SmaI banding patterns in Figure 1a. Analyses of the dendrogram revealed that the strains used in this study
stem from two major SmaI restricted chromosomal clusters.
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WBG1576 contains the fusidic acid resistance plasmid pUB101 and is therefore fusidic acid-resistant. BE8 is a high-level fusidic acid-resistant derivative of WBG1576, which contains pUB101 and a chromosomal mutation which leads to increased fusidic acid resistance. 30 BE8 and WBG1576 possess the same phage type (Table I) and cluster closely together within the SmaI dendrogram (Figure 1b), but differ in fusidic acid MIC, as expected.
BB270 is a methicillin-resistant transducant of BB255. 31 BB255 and BB270 possess the same phage types (Table I) and cluster closely together on the SmaI dendrogram (Figure 1b).
The following groups of strains can thus be considered related: WBG8287, WBG8289, WBG8324 and WBG8375; WBG1576 and BE8; and BB255 and BB270.
WBG9007, WBG9229 and WBG8397 did not show any close relationship to the other clustered groups of strains, or to one another, by phage typing or SmaI restricted chromosomal analysis.
Strains used in this study therefore represent six different genetic lineages.
Effect of salicylate and related compounds on fusidic acid gradient plate MICs
When added to the media, salicylate, acetyl-salicylate, benzoate or ibuprofen at a final concentration of 2 mM, increased fusidic acid gradient plate MICs in all fusidic acid-resistant and -susceptible strains studied (Tables II and III). The degree to which of these substances increased the level of induced fusidic acid resistance varied from strain to strain. The addition of acetate and acetaminophen did not alter the fusidic acid gradient plate MICs for any strains studied (Price, C. T. D., O'Brien, F. G., Shelton, B. P. and Gustafson, J. E., unpublished observations). The presence of saligenin, the alcohol of salicylate, greatly reduced growth of all fusidic acid-resistant strains on 0-32 mg/L fusidic acid gradients and -susceptible strains on 0-0.3 mg/L gradients.
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Figure 2 represents the results of representative population analysis for unrelated fusidic acid-resistant strains WBG8287 and WBG1576. The addition of 2 mM salicylate increased the number of cells surviving on media containing 16 mg/L fusidic acid by 4 log units for WBG8287 and 2 log units for WBG1576.
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Three WBG8287 high-level fusidic acid-resistant mutants (CP41-CP43) picked from media containing 24 mg/L fusidic acid had MIC values four to eight times that of the parent strain. Eight WBG8287 high-level fusidic acid-resistant mutants (CP54-CP62) picked from media containing 24 mg/L fusidic acid and 2 mM salicylate, had MICs two to eight times that of the parent strain. High-level fusidic acid-resistant WBG8287 mutants appeared on media containing 24 mg/L fusidic acid, and media containing 24 mg/L fusidic acid and 2 mM salicylate, at mutation frequencies of 3.33 x 10- 8 and 3.33 x 10-7, respectively.
Figure 3 represents the results of a representative population analysis for unrelated fusidic acid-susceptible strains BB255 and WBG9007, performed in the presence of increasing fusidic acid concentrations with and without 2 mM salicylate. The addition of 2 mM salicylate increased the number of surviving cells on media containing 0.1 mg/L of fusidic acid by 4 log units for BB255 and 5 log units for WBG9007.
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Ten BB255 fusidic acid-resistant mutants (CP1-CP10) picked from media containing 0.30 mg/L fusidic acid had MICs 32 to 64 times that of the parent, with the exception of CP8 which retained the MIC for the parent strain. Five BB255 fusidic acid-resistant mutants (CP26-CP30) picked off media containing 0.30 mg/L fusidic acid and 2 mM salicylate, had MICs eight to 128 times that for the parent strain. BB255 fusidic acid-resistant mutants appeared on media containing 0.30 mg/L fusidic acid, and on media containing 0.30 mg/L fusidic acid and 2 mM salicylate, at similar mutation frequencies of 6.1 x 10-6 and 1.67 x 10-6, respectively.
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Discussion |
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Growth of E. coli in the presence of salicylate, acetyl-salicylate or benzoate induces a phenotypic multiple antibiotic resistance mechanism which is partially dependent on the induction of the multiple antibiotic resistance (mar) operon. 32 The mar operon controls intrinsic-resistance levels to chloramphenicol, tetracycline, ampicillin, cephalosporins, naladixic acid and quinolones 32 and is reliant on the AcrAB multidrug efflux system. 34
It is possible that a mar-like system is present in S. aureus, and induction of this system by salicylate and related substances leads to an increase in fusidic acid resistance levels.
Media containing 2 mM salicylate produced a dramatic increase in the number of cells surviving on inhibitory concentrations of fusidic acid for two unrelated fusidic acid-resistant strains, WBG8287 and WBG1576, and two unrelated fusidic acid-susceptible strains, BB255 and WBG9007.
It has been previously demonstrated that increased fusidic acid resistance emerges in vitroin fusidic acid-resistant and -susceptible strains of S. aureus. 30 ,34 ,35 The increase in fusidic acid resistance is attributed to mutations occurring in the gene encoding EF-G, fusA. 19 ,30 Mutants of BB255 or WBG8287 expressing increased fusidic acid resistance levels picked off plates containing 0.10 mg/L or 16 mg/L fusidic acid respectively, had fusidic acid MICs greater than their parent strains. Colonies of these strains picked off plates containing the same relative fusidic acid concentrations described above, and 2 mM salicylate, did not show any increase in fusidic acid MICs compared with parent strains. This demonstrates that the salicylate-inducible fusidic acid resistance mechanism is also phenotypically expressed in S. aureus, and does not require the occurrence of a stable mutation leading to fusidic acid resistance. The ability of salicylate to increase phenotypic fusidic acid resistance occurred only at low fusidic acid concentrations, relative to the fusidic acid resistance levels of the strains. At high fusidic acid concentrations both BB255 (0.3 mg/L) and WBG8287 (24 mg/L) mutated to higher fusidic acid resistance levels. Salicylate-inducible multiple antibiotic resistance in E. coli is also phenotypic. 10
The addition of 2 mM salicylate to media containing 24 mg/L fusidic acid also caused an increase in the number of high-level fusidic acid-resistant WBG8287 mutants. This demonstrates that salicylate enables WBG8287 to mutate to higher fusidic acid resistance levels more readily in the presence of otherwise inhibitory concentrations of fusidic acid.
Salicylate (pKa 3.0), acetyl-salicylate (pKa 3.5), benzoate (pKa 4.2), ibuprofen (pKa 4.5) and acetate (pKa 4.8) are all weak acids. Acetate did not affect fusidic acid resistance levels in any of the strains studied. This suggests that the ability of salicylate and related compounds to increase fusidic acid resistance is not solely due to their ability to act as weak acids. The alcohol of salicylate, saliginen, decreased fusidic acid MICs for all strains studied, while acetaminophen had no effect on fusidic acid resistance levels. It is therefore probable that the ability of salicylate and related compounds to increase fusidic acid resistance in S. aureus is due to their aromatic ring structure, and the presence of a functional carboxylic acid group.
The compounds used in this study are frequently ingested for therapeutic reasons, or as part of food preparations. 13,36 A blood level of 2 mM for salicylate and acetyl-salicylate is recommended for the treatment of rheumatic fever. 37 Relatively high concentrations of aspirin are prescribed to reduce pain and fever, and redness and swelling of joints due to rheumatic fever, gout and rheumatoid arthritis. 14 At these salicylate concentrations it is possible that the anti-staphylococcal activity of fusidic acid is reduced. Since salicylate and related compounds have been shown to induce multiple antibiotic resistance in other bacteria, further investigations of their effects on S. aureus resistance to other anti-staphylococcal agents are warranted.
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Acknowledgments |
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Notes |
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References |
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Received 3 August 1998; returned 5 January 1999; revised 25 January 1999; accepted 15 March 1999