Growth-inhibitory effects of sulfonamides at different pH: Dissimilar susceptibility patterns of a soil bacterium and a test bacterium used for antibiotic assays

Abstract

The ionic speciation of sulfonamides is pH-driven and this may be crucial for their bioavailability and sorption to soil constituents, as well as for their uptake into bacterial cells. The inhibition behaviour of a bacterial test strain (Pseudomonas aeruginosa; DSM 1117), which was grown in the presence of different concentrations of 8 sulfonamides at pH values from 5 to 8, could be predicted by models that take the speciation of sulfonamides in- and outside of bacterial cells into account. Assuming a pH of 7.5 inside the cells (pH homeostasis), the strongest inhibition was predicted for the lowest external pH and for sulfonamides with the lowest pK_a values. Growth experiments with Ps. aeruginosa basically reflected this predicted behaviour. However, Pantoea agglomerans – a bacterial strain isolated from arable soil – behaved surprisingly different regarding its pH dependency: all sulfonamides showed the strongest effects at pH 7 to 8 instead of being most effective at lowest pH, although the pK_a dependencies followed the same pattern. Experimental and modeling results could be brought into good agreement for P. agglomerans if the cell-internal pH was admitted to approximate the external pH instead of implying pH homeostasis for modeling calculations. Thus, besides the actual concentration of sulfonamides, the pH dependent mode of reaction of different bacteria to sulfonamides may additionally govern the population dynamics in soils.

Introduction

Sulfonamides (SA) are, after tetracyclines, the second largest structural class of antibiotics used in Europe in animal husbandry (Höper et al., 2002). Following administration, substantial amounts of SA are excreted as the parent and active compound (Halling-Sørensen et al., 1998). The animal excrements are either directly released into the environment by grazing animals or indirectly by spreading animal excrements as fertilizer onto agricultural soils. As shown by Lamshöft et al. (2007) for ¹⁴C-labeled sulfadiazine (SDZ), the parent compound SDZ accounted for 44% of the 96% radioactivity excreted. Sulfonamides exert selective pressure on soil microorganisms at environmentally relevant concentrations (Schmitt et al., 2005, Thiele-Bruhn and Beck, 2005). Although a low soil–water partition coefficient suggests a high mobility (Tolls, 2001, Boxall et al., 2002, ter Laak et al., 2006), recent publications demonstrated a fairly strong initial sorption of SA to soil in laboratory (Wehrhan et al., 2007) and field experiments (Stoob et al., 2007). Despite the rather fast initial sorption of SA to soil, substantial amounts should be temporarily bioavailable after manuring. Stoob et al. (2007) found that SA are still present in the soil after a long time period. When estimating the environmental impact and fate of these ionogenic compounds, one has to consider their pH dependent speciation. The pH determines the dissociation of these ionisable antibiotics, which influences their solubility, affinity for soil colloids and organic matter, and uptake or diffusion into microorganisms (Escher and Sigg, 2004, Tomashow et al., 2005). Sulfonamide antimicrobials possess two ionisable functional groups considered relevant to the environmental pH range: the anilinic amine and the amide moiety. The cationic species as SA⁺ dominates at low pH values, the neutral form (SA⁰) is the principal species at pH values between pK_a1 and pK_a2 and the anionic species is the main form at higher pH values. A minor zwitterionic species SA^± is in tautomeric equilibrium with SA⁰ (contribution to overall speciation <0.2% (Gao and Pedersen, 2005)).

Many attempts have been made to relate the antibacterial activity of SA with their physico-chemical properties. Empirically determined relationships pointed out the dominant role of the degree of ionisation on the antimicrobial activity (Mengelers et al., 1997).

Thus, whether the antibacterial activity of a sulfonamide will reach an ecologically significant level depends not only on the absolute concentration but also on the speciation (Fig. 1), which may substantially determine the biologically active fraction. Most SA have pK_a values within the pH range of natural soils and speciation will lead to different sorption interactions in soils (ter Laak et al., 2006, Kahle and Stamm, 2007), and will therefore have an impact on the potential availability of these compounds. Due to the fact that SA enter the soil with manure and the pH of pig manure after anaerobic storage is predominantly alkaline (Canh et al., 1998, Velthof et al., 2005), SA would occur mainly as anionic species, at least temporarily, because it takes a few days before soil pH returns to its previous value (Stoob et al., 2007). It is currently believed that the neutral species is preferably transported across the membrane (Sauermann et al., 2005, Trapp and Horobin, 2005) and the anionic species is the reactive form (Henry, 1943). SA have to pass the bacterial cell membrane to interfere with folate metabolism. Hence, if pH values are higher than pK_a values of the different SA this would result in a decrease of antibacterial activity, as discussed by Henry (1943). Recently, Zarfl et al. (2008) described a mechanistical model to explain substance specific and pH dependent antibiotic effects. For this model, they assumed that differences in accumulation and speciation of SA in bacteria are due to different abilities of bacteria to regulating their internal pH. For an understanding of the impact of SA on soil bacteria it is important to know to what extent sorption to soil and bacterial growth are affected by pH. In the present study we examined the influence of SA on bacterial growth at different pH values. We evaluated the growth dynamics of a commercially available strain (Pseudomonas aeruginosa), recommended for antibiotic assays, and a soil bacterial isolate (Pantoea agglomerans) as a measure for pH dependent inhibitory effects. SA tested were sulfadiazine (SDZ), sulfathiazole (STZ), sulfadimethoxine (SDT), sulfamethazine (=sulfadimidine, SDM), sulfaguanidine (SGD), sulfapyridine (SPY), sulfamethoxazole (SMX), and sulfachloropyridazine (SPZ).