A mechanistical model for the uptake of sulfonamides by bacteria
Introduction
Antibiotics are defined as “low molecular weight microbial metabolites” that at low concentrations inhibit the capability of microorganisms to reproduce and thus its growth (Lancini and Parenti, 1982, Thiele-Bruhn, 2003). They are used in pharmacotherapy to act very effectively against bacterial or protozoal infections even at low doses. One of the largest and most frequently applied groups of antibiotics is the sulfonamides that have been discovered as early as 1939 (Vree and Hekster, 1987, Thiele-Bruhn and Aust, 2004). Their total use volume is unknown (Mellon et al., 2001) and can only be estimated from production and sales data. Sulfonamides have two acid/base dissociation constants, pKa, the protonating one in the acid (pKa1) and the deprotonating one in the neutral or basic pH range (pKa2). Thus, depending on the actual pH and the pKa of the individual substance sulfonamides are present in different species, namely in cationic (protonated), neutral, zwitterionic or anionic (deprotonated) form. Sulfonamides interfere with the folic acid cycle in microorganisms by competing for one of the key enzymes, dihydropteroate synthase (DHPS). As a result the production of folic acid is reduced, which leads to a complete inhibition of gram-positive as well as gram-negative bacterial cell reproduction (Brown, 1962). Therefore, sulfonamides are characterized as broadband antibiotics in pharmacotherapy. Use of growth regulators in livestock feeding is interdicted since 2006 according to the EC directive 1831/20031, but sulfonamides are still applied in large amounts in veterinary medicine (Thiele-Bruhn, 2003, Kreuzig et al., 2003).
Sulfonamides have to be transported into the cell to compete with p-aminobenzoate for the enzyme DHPS. It is known that the antibiotic effect of sulfonamides on cell activity differs depending on substance properties and pH conditions. Mengelers et al. (1997) investigated the effect of several sulfonamides on Actinobacillus pleuropneumoniae, a pathogenic bacterium causing pig respiratory disease (Marsteller and Fenwick, 1999), and observed that the minimal inhibitory concentration (MIC) varied with the pKa2 of the substance and the extracellular pH. Thiele-Bruhn (2005) showed that effective doses for the suppression of the microbial iron(III)-reduction in soils also vary depending on the speciation of antibiotics influenced indirectly by the soil pH. Therefore, the pH-dependent uptake and accumulation of sulfonamides in bacterial cells is of particular interest not only for human and veterinary chemotherapy but also for the assessment of fate and effects of pharmaceuticals in the environment.
In this paper, we investigate the influence of the speciation on the potential intracellular accumulation of sulfonamides. For this purpose we have adapted and supplemented a recently published mechanistic model for the transport of chemicals into cells (Trapp and Horobin, 2005). The model is based on the assumption that not only neutral molecules but also the dissociated species are able to pass the cell envelope. Critical model parameters are identified and their sensitivity on model results is tested within realistic parameter ranges. The aim of our investigation is to find out in how far the observed substance-specific and pH-dependent antibiotic effects can be explained by a different accumulation of sulfonamides in bacterial cells.
Section snippets
Theory
The lower pKa of sulfonamides (pKa1) describes the protonation of the amino group, whereas the other one (pKa2) signifies deprotonation of the SO2NH moiety, which is the antibiotic active part (Ingerslev and Halling-Sørensen, 2000). Sulfonamide speciation in solution depends on their individual pKa values and the actual pH of the medium. The actual species distribution can be calculated from the dissociation constants (pKa) for the proton transfer reactions (for details see the Appendix):
Results and discussion
Dynamic simulations revealed that steady state is reached within a few minutes for all compounds, if permeabilities are calculated for −1.2 < log Kow < 1.6 according to Eqs. (13), (14). Assuming that the reaction kinetics of sulfonamides in the cell is much slower than the uptake, the steady-state ratio of the total sulfonamide concentration in the cell (Ccell) to the applied sulfonamide dose (Cenv) (potential accumulation factor AF) can be used as a measure for the maximum accumulation of
Conclusions
Potential accumulation of sulfonamides in bacterial cells is sensitive to the pH values in- and outside the cells if cell metabolic reactions are too slow to affect the steady-state concentration in the cell. The ratio of intra- to extracellular pH and the absolute pH values determine the total sulfonamide accumulation in dependence of pKa2. Intracellular pH directly affects the concentration of the effective anionic species and thus, the antibiotic inhibition of DHPS. The model allows
Acknowledgements
We thank Andreas Focks, Hildgund Schrempf as well as our colleagues from the Research Centre Jülich, especially Wolfgang Tappe, Joost Groeneweg and Sirgit Kummer for helpful discussions. Financial support of the German Research Foundation (DFG) and the Hans Mühlenhoff Foundation is gratefully acknowledged.
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