Semi-Quantitative Assay to Measure Urease Activity by Urinary Catheter-Associated Uropathogens

Abstract

Catheter-associated urinary tract infections (CAUTIs) are one of the most common healthcare-associated infections in the US, accounting for over 1 million cases annually and totaling 450 million USD. CAUTIs have high morbidity and mortality rates and can be caused by a wide range of pathogens, making empiric treatment difficult. Furthermore, when urease-producing uropathogens cause symptomatic CAUTI or asymptomatic catheter colonization, the risk of catheter failure due to blockage increases. The enzyme urease promotes catheter blockage by hydrolyzing urea in urine into ammonia and carbon dioxide, which results in the formation of crystals that coat the catheter surface. If CAUTI is left untreated, the crystals can grow until they block the urinary catheter. Catheter blockage and subsequent failure reduces the quality of life for the chronically catheterized, as it requires frequent catheter exchanges and can promote more severe disease, including dissemination of the infection to the kidneys or bloodstream. Thus, understanding how urease contributes to catheter blockages and/or more severe disease among the broad range of urease-producing microbes may provide insights into better prevention or treatment strategies. However, clinical assays that detect urease production among clinical isolates are qualitative and prioritize the detection of urease from Proteus mirabilis, the most well-studied uropathogenic urease producer. While urease from other known urease producers, such as Morganella morganii, can also be detected with these methods, other uropathogens, including Staphylococcus aureus and Klebsiella pneumonia, are harder to detect. In this study, we developed a high throughput, semiquantitative assay capable of testing multiple uropathogens in a rapid and efficient way. We validated the assay using Jack Bean urease, the urease producing species: Proteus spp., M. morganii, K. pneumonia, and S. aureus strains, and the non-urease producer: Escherichia coli. This modified assay more rapidly detected urease-producing strains compared to the current clinical test, Christensen Urea Agar, and provided semiquantitative values that may be used to further investigate different aspects of urease regulation, production, or activity in these diverse species. Furthermore, this assay can be easily adapted to account for different environmental stimuli affecting urease production, including bacterial concentration, aeration, or addition of anti-urease compounds.

Keywords: CAUTI; Klebsiella spp.; Morganella spp.; Proteus spp.; Staphylococcus aureus; urease; urease detection assay; urease-producing uropathogens.

Figure 1

Catheter Encrustation. Scanning electron microscope (SEM) image of crystalline precipitates forming a catheter encrustation (green) resulting from asymptomatic catheter-associated bacteriuria caused by Staphylococcus aureus (pink). The encrustation and bacteria are surrounded by host immune cells (gray).

Figure 2

S. aureus ureC carriage. PCR using the ureC-Forward and ureC-Reverse primers ( Table 2 ) was performed to confirm the presence of the ureC gene within clinical S. aureus isolates. The presence of an amplicon around 1.6 Kb indicates all the strains tested encoded ureC. Numbers denote lanes: (1) Ladder, (2) JE2, (3) UTI MRSA, (4) HUC 86, (5) HUC 86-02, (6) HUC 95, (7) HUC 97-02, (8) HUC 100, (9) HUC 100-01, (10) HUC 111-01C.

Figure 3

Representative assay of the Christensen Urea Agar urease activity test. Christensen Urea Agar plates were streaked with a single colony of each bacterial strain: Proteus spp., Escherichia coli, Klebsiella pneumonia, Morganella morganii, and Staphylococcus aureus strains including JE2, JE2 ureC::tn, UTI MRSA, UTI MRSA ΔureC, UTI MRSA ureC::tn, HUC 86, HUC 86-02, HUC 95, HUC 97-02, HUC 100, HUC 100-01, and HUC 111-01C. Urease activity was detected by 6 hours in Proteus spp., while no urease activity was detected in Escherichia coli. Urease activity was detected by 6 hours for both Klebsiella pneumonia and Morganella morganii. For Staphylococcus aureus strains, urease activity in was detected by 24 hours in UTI MRSA, HUC 95, HUC 97-02, HUC 100, and HUC 100-01, 34 hours in JE2, HUC 86, and HUC 111-01C, 50 hours in HUC 86-02. No urease activity was detected in JE2 ureC::tn, UTI MRSA ΔureC, or UTI MRSA ureC::tn.

Figure 4

High throughput, semi-quantitative urease activity detection assay. The urease activity detection assay was validated with (A) Jack Bean enzyme, (B) Proteus spp., and (C) Escherichia coli. A dose response curve (5U, 1U, 0.1U, and 0.001U) of Jack Bean urease was assessed. Enzymatic activity was detected immediately for 5U and 1U, at 20 minutes for 0.1U, at 40 minutes for 0.01U, and at 4 hours for 0.001U. For the bacterial strains, increasing concentrations of bacteria were assessed including 1:1000 and 1:100 dilutions of overnight cultures, as well as overnight cultures resuspended in urease broth, with urease activity detected at 1.5 hours, 40 minutes, and 20 minutes for the Proteus spp. strain, respectively. The E coli strain did not display urease activity. Additional, diverse urease-producing uropathogens at 1:1000 and 1:100 dilutions of overnight cultures, as well as overnight cultures resuspended in urease broth were assessed for urease activity, including (D) Morganella morganii detected at 3 hours, 40 minutes, and 20 minutes, respectively; and (E) Klebsiella pneumonia detected at 4, 4, and 0.5 hours, respectively. For S. aureus strains urease activity was assessed at 1:100 dilutions of overnight cultures, as well as overnight cultures resuspended in urease broth, with (F) UTI MRSA detected at 4 hours only at the highest bacterial concentration (overnight suspension). The (G) UTI MRSA ureC::tn and (H) UTI MRSA ΔureC, were used as negative controls. No urease was detected over the course of the 18-hour experiment. Furthermore, (I) JE2 urease was detected at 4 hours only in the highest bacterial concentration tested (overnight suspension), while no urease was detected for (J) JE2 ureC::tn across the course of the 18-hour experiment. Lastly, additional S. aureus strains were assessed for urease activity to determine the assay’s accuracy in detecting variable urease producers. Urease activity was detected for (K) HUC 86 at 10 hours and 3 hours for the 1:100 dilution and overnight culture suspension, respectively; (L) HUC 86-02 at 6 hours and 2 hours for the 1:100 and overnight suspension, respectively; (M) HUC 95 at 20 hours only in overnight suspensions; (N) HUC 97-02 at 12 hours only in overnight suspensions; (O) HUC 100 at 10 hours and 3 hours for the 1:100 dilution and overnight suspension, respectively; (P) HUC 100-01 at 4 hours and 1.5 hours for the 1:100 dilution and overnight suspension, respectively; and (Q) HUC 111-01C at 12 hours and 0.5 hour for the 1:100 dilution and overnight suspension, respectively. Points represent the mean of three technical replicates and two biological replicates and error bars represent standard deviation from the mean.