Synergistic inhibition of ureolytic activity and growth of Klebsiella pneumoniae in vitro suggests cobinding of fluoride and acetohydroxamic acid at the urease active site and provides a novel strategy to combat ureolytic bacteria

Abstract

The ability of ureolytic bacteria to break down stable urea to alkaline ammonia leads to several environmental and health challenges. Ureolytic bacteria such as Helicobacter pylori, Klebsiella pneumoniae, and Proteus mirabilis can become pathogenic and cause persistent infections that can be difficult to treat. Inhibiting urease activity can reduce the growth and pathogenicity of ureolytic bacteria. In the present in vitro study, we investigated the synergistic effects of tannic acid (TA) and the urease inhibitors fluoride (F^-) and acetohydroxamic acid (AHA). The concentration of AHA needed for efficient inhibition of the ureolytic activity of K. pneumoniae can be significantly reduced if AHA is coapplied with tannic acid and sodium fluoride (NaF). Thus, only 1.20 μmol l^-1 AHA in combination with 0.30 mmol l^-1 tannic acid and 0.60 mmol l^-1 NaF delayed the onset of ureolytic pH increase by 95.8 % and increased the growth lag phase by 124.3 % relative to untreated K. pneumoniae. At these concentrations, without AHA, TA and NaF increased the onset of the ureolytic pH change by only 37.0 % and the growth lag phase by 52.5 %. The strong inhibition obtained with low concentrations of AHA in triple-compound treatments suggests cobinding of F^- and AHA at the urease active site and could reduce the side effects of AHA when it is employed as a drug against e.g. urinary tract infections (UTIs) and blocked catheters. This study reports the basis for a promising novel therapeutic strategy to combat infections caused by ureolytic bacteria and the formation of urinary tract stones and crystalline biofilms on catheters.

Keywords: Acetohydroxamic acid; Fluoride; Klebsiella pneumoniae; Tannic acid; Urease inhibition; Ureolytic; Urinary tract infection.

Graphical abstract

Fig. 1

Anti-ureolytic effects of NaF and AHA on K. pneumoniae in the presence of TA. a) Ureolytic activity (A557-A630) and b) growth (OD630) of untreated K.pneumoniae (o) and K.pneumoniae treated with 0.30 mmol l⁻¹ TA and 0.60 mmol l⁻¹ NaF (▲); 0.30 mmol l⁻¹ TA and 10.0 μmol l⁻¹ AHA (■); and 0.30 mmol l⁻¹ TA, 0.60 mmol l⁻¹ NaF, and 10.0 μmol l⁻¹ AHA (♦). The data are from Replica 1. K. pneumoniae treated with 0.30 mmol l⁻¹ TA and 0.60 mmol l⁻¹ NaF had significantly later onset of the pH increase and growth than the untreated control. In contrast, treatment with 10.0 μmol l⁻¹ AHA in the presence of 0.30 mmol l⁻¹ TA did not affect the ureolytic activity or growth of K. pneumoniae. However, the addition of 10.0 μmol l⁻¹ AHA in the presence of both 0.30 mmol l⁻¹ TA and 0.60 mmol l⁻¹ NaF dramatically delayed the onset of the pH increase and growth. Thus, in the presence of TA, strong synergistic effects of NaF and AHA were observed. Plots of the ureolytic activity and growth of biological replicates from Replica 2 and Replica 3 are presented in the Supporting Information (Fig. S4 a-d).

Fig. 2

NaF and AHA synergistically inhibited cell-free urease activity. a) Average ureolytic activity (A557) of untreated jack bean urease (JBU) (x), JBU with 0.60 mmol l⁻¹ NaF (▲), 10.0 μmol l⁻¹ AHA (■), or 0.60 mmol l⁻¹ NaF and 10.0 μmol l⁻¹ AHA (♦). The activity of JBU was not affected by 10.0 μmol l⁻¹ AHA (measured initial reaction rate in the period 0–95 min), whereas 0.60 mmol l⁻¹ NaF decreased the initial reaction rate significantly compared to that of the untreated JBU (measured in the period 75–133 min). The combination of 0.60 mmol l⁻¹ NaF with 10.0 μmol l⁻¹ AHA slightly decreased the initial reaction rate further, albeit not significantly (at 75–245 min). The maximum absorbance related to the ureolytic pH increase decreased by 23.3 % with 0.60 mmol l⁻¹ NaF and 44.3 % with 0.60 mmol l⁻¹ NaF and 10.0 μmol l⁻¹ AHA after 20 h compared to the final A557 for the uninhibited urease reaction. b) The initial rates and final absorption at 557 nm obtained from the data shown in a).

Fig. 3

Binding modes of urea, fluoride, and AHA in the active site of urease. Binding modes in the active site of a) native urease [36], b) urease cobinding urea and fluoride [32], c) fluoride-inhibited urease [33], and d) AHA-inhibited urease [34] according to crystal structures. e) Currently accepted binding mechanism of AHA in the active site of urease under acidic and neutral conditions [34]. g) Proposed mechanism and binding modes for the simultaneous binding of fluoride and deprotonated AHA in the active site of urease under alkaline conditions.