All strains used in this study are listed in Table 1 . All human urinary catheter (HUC) clinical isolates, including strains of S. aureus and M. morganii, were isolated from individuals with chronic indwelling urinary catheters as previously described (Walker et al., 2017; Walker et al., 2020). The Escherichia coli strain was isolated from a woman who experienced a UTI (Chen et al., 2009). The K. pneumonia strain was isolated from the respiratory tract of an asymptomatic individual and provided by Drs. Cesar Arias and Blake Hanson (Shropshire et al., 2021). The Proteus spp. strain was isolated from a mouse urinary catheter during experimental urinary catheterization. Brain heart infusion (BHI) broth (BD, cat # 237200) and BHI agar were used to maintain all cultures and prepare overnight cultures for experiments. All transposon mutants were generated using tryptic soy broth (Sigma; cat # C1016-500G) and maintained in BHI supplemented with 10 ug/mL erythromycin (Fisher; cat # AC227330050). The ureC clean deletion was generated in Escherichia coli under selection with 100 ug/ml ampicillin (Sigma, cat #BP1760-25G) in LB (Sigma; cat #BP1426-500) and then maintained in S. aureus under selection with 20 ug/ml chloramphenicol (Sigma; cat # CO37825G) in BHI. Once generated in the UTI MRSA background, the ureC clean deletion was maintained in BHI without antibiotic selection. Christensen Urea Agar (Sigma, cat # 27048-500G-F) plates and slants supplemented with filter sterilized 40% urea solution were used according to the manufactures protocol to determine urease activity. Briefly, slants and plates were inoculated with a single colony from a BHI agar plate and incubated at 37°C for 50 hours. For adapted urease activity broth assays, BHI broth was inoculated with a single colony from a BHI agar plate and grown, shaking at 175 rpm for at least 18 hours at 37°C. Bacterial strains were then either i) spun down at 10,000 g and resuspended or ii) diluted to various concentrations in urease broth. Urease broth consisted of 1 g peptone (BD; cat # 211921), 1 g D(+)-glucose (Sigma; cat #G8270-1KG), 2 g potassium phosphate monobasic (Sigma; cat # P9791-100G), 5 g sodium chloride (Fisher; cat # S671-500), 0.012 g Phenol red (Sigma; cat # 114529-5G), in 1 L of MilliQ water, pH 6.8. Following sterilization via autoclaving, urease broth was either supplemented with filter sterilized 40% urea solution (Fisher; cat # BP169-500) or with an equivalent amount of sterile water (the solution urea was dissolved in). Urease broth with and without urea allowed the determination of urease-specific activity across diverse bacterial species. Potassium phosphate monobasic was a key component in the broth. When lower grade or a different type of phosphate was used, the timing and/or the intensity of the color change of the media was altered. Since this assay relies on a pH change in the media following urea hydrolysis by urease, the buffering capacity of each phosphate may contribute to any difference observed.

The human urinary catheter was previously collected and clinical characteristics were published (catheter #7) (Walker et al., 2020). Scanning electron microscopy (SEM) was performed as previously described (Walker et al., 2017; Walker et al., 2020). Briefly, a 3 cm long piece of the tip of the urinary catheter was fixed in 10% neutral buffered formalin, washed in 0.15 M sodium cacodylate buffer, and then stained with 1% osmium tetroxide. Next, a graded ethanol series (50%, 70%, 90%, 100%, and 100%) was used to dehydrate the sample. A critical point drier (Leica CPD 300, Vienna Austria) was used to dry the sample, which was then sputter-coated with 6 nm iridium (Leica ACE 600, Vienna, Austria). A Zeiss Merlin FE-SEM equipped with a Gemini II electron column (Oberkochen, Germany) was used to image the catheter with secondary electrons detected by the Everhart-Thornley secondary electron detector at an accelerating voltage of 3 keV and a beam current of 200nm.

Bioinformatics analyses were used to determine the percentage of sequenced S. aureus isolates in NCBI that carry ureC, which was used as a marker for the carriage of the urease operon. A total of 10,290 S. aureus assemblies downloaded from NCBI were used. Briefly, open reading frames were identified for each assembly using Prokka v1.14.5 and all open reading frames were clustered using Roary v3.13.0 (Seemann, 2014; Page et al., 2015). The open reading frame encoding ureC was then identified by sequence match to the ureC gene in S. aureus strain JE2, and the presence-absence matrix from Roary was used to calculate prevalence.

To confirm that the S. aureus HUC strains encode urease, genomic DNA was extracted using the Promega Miniprep Kit (Promega; cat # A1330) according to the manufacturer’s guidelines, with the exception that 2 ug/ml of Lysostaphin (Sigma; cat#) was added to the cells resuspended in P1 buffer and incubated for 1 hr at 37°C to digest the staphylococcal cell wall. PCR was then used to confirm the presence of the ureC gene, which encodes the active site within the urease enzyme, in the genome. The ureC-Forward and ureC-Reverse primers shown in Table 2 were created based on the JE2 genome downloaded from the NCBI database. The urease operon of K. pneumonia C699 was confirmed via genomic analysis, previously performed in (Shropshire et al., 2021).

The transposon mutant in ureC (ureC::tn) was obtained from the Nebraska Transposon Mutant Library (BEI Resources). UTI MRSA ureC::tn and JE2 ureC::tn mutants were generated via transduction of the wildtype strains with lysates made from the Nebraska Transposon Mutant Library ureC::tn mutant strain using phage 11, as previously described (Walker et al., 2017). Briefly, a ureC::tn mutant lysate was generated by combining the Nebraska Transposon Mutant Library strain containing the ureC transposon mutation with phage 11 in melted top agar and transferring the solution to a TSA plate containing 5 mM calcium chloride. The top agar was allowed to cool and the plates were incubated at 37°C overnight. Plates containing 30-300 plaque forming units were selected, top agar was removed, and plaques were extracted via centrifugation followed by purification with a 0.22 um syringe filter (Sigma; cat # SLVGM33RS). The resulting phage lysate was then used to introduce the ureC transposon mutation into the UTI MRSA and JE2 background. For this, the UTI MRSA and JE2 strains were streaked on a TSA slant and incubated overnight to create a lawn. The bacteria were then resuspended in 1 mL TSA supplemented with 5 mM calcium chloride. The purified ureC::tn mutant phage lysate and the resuspended bacteria were then incubated, shaking for 20 minutes at 37°C. Cold, sterile 20 mM sodium citrate (Sigma; cat # W302600-1KG-K) was then added to these cultures, which were then spun down, resuspended in 20 mM sodium citrate (Sigma; cat # W302600-1KG-K), and 100 ul was spread on TSA plates containing 20 mM sodium citrate and erythromycin. Colonies that grew were restreaked on TSA with sodium citrate and erythromycin to select against any contaminating phage 11 and to select for the ureC::tn mutation, respectively. DNA was extracted as described above and PCR was performed using the ureC-Forward-Mutant check and Buster primers listed in Table 2 to confirm the presence of the ureC::tn mutation (UTI MRSA ureC::tn and JE2 ureC::tn).

The clean ureC deletion in the UTI MRSA background (UTI MRSA ΔureC) was generated via splicing overlap extension (SOEing) PCR and homologous recombination, as previously described (Walker et al., 2013). Briefly, ~ 500 base pairs upstream and downstream of the ureC gene were amplified from the chromosomal DNA of UTI MRSA using the ureC KO primers listed in Table 2 . Care was taken to ensure the deletion was inframe and did not disrupt the downstream genes of the rest of the urease operon. The upstream and downstream amplicons were then PCR purified using the Qiagen PCR purification kit (Qiagen; cat # 28106) and the ureC KO upstream for and ureC KO downstream rev primers containing the EcoRI and SalI restriction sites, respectively, listed in Table 2 , were used in a SOEing PCR to generate a single amplicon. The single amplicon was then PCR purified and cloned into the TOPO-PCR 2.1 (Thermo Fisher; cat # K45002) according to the manufacturer’s protocol. The TOPO-PCR 2.1 ureC KO construct was then transformed into DH5α chemically competent cells according to the manufacturer’s protocol. The ureC KO construct that was generated was then digested with EcoRI and SalI and ligated into the plasmid pJB38, which was digested with the same restriction enzymes, as previously described (Walker et al., 2013). The ligation (pJB38 ΔureC) was transformed into chemically competent DH5α, selecting on LB ampicillin, and colonies were confirmed for the correct construct using PCR and sanger sequencing. Plasmid pJB38 ΔureC was extracted and transformed into electrocompetent S. aureus cells (RN4220), selecting on BHI chloramphenicol at 30°C. Electrocompetent cells were created by subculturing RN4420 to an OD₆₀₀ of 0.05 in B2 media, which consists of 1% casein hydrolysate (Oxoid; cat # LP0041), 2.5% yeast extract (Fisher; BP1422-500), 0.5% glucose (Sigma; cat #G8270-1KG), 2.5% NaCl (Fisher; cat# S5671-500), and 0.1% K2HPO4 (Sigma; cat # P9791-100G). The subculture was then incubated for 2.5 hours, shaking at 37°C to an OD₆₀₀ of 0.3-0.4. The cells were then harvested via centrifugation (ThermoSientific SORVALL LEGEND X1R) at 5,000 rpm for 10 mins at 4°C and washed with decreasing amounts (1 volume, ½ volume, and ¼ volume) of ice cold, sterile water. The final wash was performed with 1/10 volume ice cold 10% glycerol and the cells were resuspended in 1/250 volume 10% glycerol. Electrocompetent cells were then stored at -80°C until ready for use. Once electrocompetent UTI MRSA cells were created, they were transformed with plasmid pJB38 ΔureC extracted from RN4220. Transformants were selected at 42°C overnight on BHI chloramphenicol. Colonies were screened for double recombination and loss of the plasmid via PCR using the ureC KO upstream forward and ureC KO downstream reverse primers listed in Table 2 and for sensitivity to chloramphenicol, respectively. Sanger sequencing was used to confirm the mutation.

For the adapted urease broth activity assay, strains from overnight cultures were either i) subcultured (1:100 or 1:1000) into urease broth with and without urea or ii) harvested by centrifugation for 5 minutes at 10,000 g, washed with 1 volume of 1X Phosphate Buffered Saline (PBS), and resuspended in urease broth with or without urea. Each strain was transferred to a 96-well plate and tested in triplicate with at least two biological replicates. Purified Jack Bean urease (Sigma; cat # 94281-1G), strains grown in urease broth without urea, urease broth alone with and without urea, and strains with mutants in urease were included as controls. The OD was measured at 415 nm, 560 nm, and 600 nm every 20 mins for 24 hrs, shaking at 37°C using a BioTek Synergy|H1 microtiter plate reader (BioTek, Vermont). The ratio of the OD detected at 560 nm compared to 415 nm is used to calculate the color change that occurs following the increase in the pH of the media, which is due to the urea hydrolysis by urease. Furthermore, the use of urease broth without urea takes into account any color change that is not due strictly to pH changes following urea hydrolysis, such as the release of other byproducts during growth, that occurs during the course of the experiment. Thus, the urease activity was calculated by dividing the OD values at 560 nm by the OD values at 415 nm and the urease activity was then normalized by dividing the OD of each strain grown in urease broth containing urea by the same strain grown in urease broth without urea.

Graphpad Prism 8.4.3 software was used to create all the normalized urease activity graphs. All experiments were performed with at least three replicates and repeated at least twice. Graphs display the mean of the combined replicates and error bars represent the standard deviation from the mean. An additional statistical analysis, using the Mann Whitney U Test, was performed on low urease producers to confirm urease activity detected among strains grown in the presence of urea was significantly different from that strain grown in the absence of urea.

Previous reports indicate that urease detection among S. aureus clinical isolates is low (25%-50%) (Bichler et al., 2002; Gad et al., 2009; Flannigan et al., 2014). Yet a previous report suggested the urease operon was present in a majority (>90%) of sequenced isolates (Konieczna et al., 2012). Our analysis of 10,290 sequenced S. aureus isolates in NCBI found that ureC was present in 93.1% of these strains, supporting the previous report (Konieczna et al., 2012). Furthermore, to determine whether a similar proportion of our S. aureus human urinary catheter (HUC) isolates encoded urease, we used PCR to confirm the presence of the ureC gene, which is a marker for the operon. Amplicons corresponding to an ~1.6 Kb band indicate that the ureC gene was encoded by all HUC isolates (7/7), as well as the urine-associated strain UTI MRSA, and the skin infection isolate JE2 ( Figure 2 ). Thus, all (9/9) S. aureus strains tested encoded ureC.

To determine whether urease could be detected in a diverse set of strains the current clinical test, the Christensen Urea Agar assay, was used ( Figure 3 ). A Proteus spp. strain was used as a positive control and urease activity was rapidly detected, with the entire plate turning pink by 6 hours. E. coli was used as a negative, non-urease producing control and displayed no color change during the course of the experiment. K. pneumonia urease could be detected as early as 6 hours, with complete color change of the agar plate around 24 hours. M. morganii also displayed signs of urease activity as early as 6 hours, but complete color change of the agar plate was delayed compared to K. pneumonia, occurring around 34 hours. For the S. aureus strains, urease production could be detected as early as 24 hours for UTI MRSA, HUC 95, HUC 97-02, HUC 100, and HUC 100-01, 34 hours for JE2 and HUC 86, and 50 hrs for HUC 86-02. Thus, all (9/9) S. aureus strains tested were confirmed to be urease-producers by the Christensen Urea Agar method. All S. aureus ureC mutants remained negative, as they did not display any color change during the course of the experiment.

The adapted urease activity assay was evaluated using the positive controls: Jack Bean urease enzyme ( Figure 4A ) and a Proteus spp. strain ( Figure 4B ). The Jack Bean urease was used to create standard curves to assess activity with decreasing concentrations of the enzyme. The concentrations of pure Jack Bean included 5U, 1U, 0.1U, 0.01U, and 0.001U. Urease activity was detected immediately (within seconds) for the highest concentrations of Jack Bean (5U and 1U) in urea containing media. For the lower concentrations, a dose response was observed, with activity detected around 20 minutes for 0.1U, 40 minutes for 0.01U, and 4 hours for 0.001U ( Figure 4A ). Next, the urease activity of the Proteus spp. strain was assessed at various bacterial concentrations. For the highest concentrations of bacteria – overnight cultures resuspended in urease broth and cultures diluted 1:100 in urease broth – activity was detected around 20 and 40 minutes after the start of the assay, respectively ( Figure 4B ). For the lowest concentrations of bacteria – cultures diluted 1:1000 in urease broth – activity was detected around 1.5 hours after the start of the assay ( Figure 4B ). E. coli was used as a negative control. No urease activity was detected at any concentration over the course of the 18-hour assay ( Figure 4C ).

M. morganii, K. pneumonia, and S. aureus (UTI MRSA and JE2) strains, which are known urease producers, were selected to optimize the urease detection assay for diverse species. Urease activity was detected among various bacterial concentrations for each species and urease activity correlated with bacterial concentration ( Figure 4 ). For M. morganii, urease activity was rapidly detected for the highest bacterial concentrations (overnight resuspensions), ~20 minutes after the start of the assay ( Figure 4D ). For lower bacterial concentrations of M. morganii, diluted 1:100 and 1:1000 in urease broth, enzymatic activity was detected about 40 minutes and 3 hours, respectively, after the start of the assay ( Figure 4D ). For K. pneumonia, urease activity could be detected as early as 40 minutes for the highest bacterial concentration (overnight resuspension) and at 4 hours for both the 1:100 and the 1:1000 dilutions ( Figure 4E ). For UTI MRSA, urease activity could be detected as early as 4 hours for the overnight concentration, while no color change was detected for the lower bacterial concentrations diluted 1:100 and 1:1000 ( Figures 4F , S1 ). Furthermore, the UTI MRSA ureC::tn and UTI MRSA ΔureC strains were used as non-urease producing negative controls and no urease was detected over the 18-hour time course ( Figures 4G, H , S1 ). The well-characterized S. aureus skin infection isolate JE2 was also assessed in the adapted urease assay. Similarly to UTI MRSA, urease activity in JE2 was only detected at the highest bacterial concentration, with detection starting at 3 hours ( Figures 4I , S1 ). JE2 ureC::tn was used as a non-urease producing negative control, and no urease was detected over the course of the 18-hour experiment ( Figures 4J , S1 ).

A panel of clinical catheter-associated S. aureus isolates were selected to further assess the accuracy of the assay on so-called difficult to detect urease producers. For HUC 86, the adapted urease broth assay detected enzymatic activity around 10 hours at the highest bacterial concentration (overnight suspension) ( Figure 4K ). This contrasted with the Christensen Urea Agar plate assay, which detected urease activity around 34 hours. Furthermore, at the 1:100 bacterial concentration, the adapted urease assay detected some enzymatic activity with HUC 86 as early as 3 hours. No urease activity was detected with HUC 86 at the lowest dilution (1:1000) ( Figure S1 ). Additionally, the adapted assay detected urease activity for the serial isolate from the same patient, HUC 86-02, more rapidly, around 2 hours at the highest bacterial concentration (overnight suspension) and at 6 hours for the 1:100 dilution ( Figure 4L ). No urease activity was detected at the lowest bacterial concentration (1:1000) ( Figure S1 ). For the HUC 95 strain, urease activity was detected at 20 hours at the highest bacterial concentration (overnight suspension), and no urease activity was detected for the lower bacterial suspensions (1:100 or 1:1000) ( Figures 4M , S1 ). For the HUC 97-02 strain, urease activity could begin to be detected at 12 hours at the highest bacterial concentration (overnight suspension), but no urease activity could be detected at the lower bacterial concentrations (1:100 or 1:1000) ( Figures 4N , S1 ). For the HUC 100 strain, urease activity was first detected for the 1:100 dilution approximately 3 hours after the start of the assay ( Figure 4O ). Urease activity was next detected at 10 hours for the highest bacterial concentration (overnight suspension). No urease activity was detected at the lowest concentration (1:1000 suspension) ( Figure S1 ). The HUC 100-01 strain, which is the serial isolate from the same patient as HUC 100, displayed the quickest urease activity at the highest bacterial concentration (overnight suspension) and was detected approximately 1 hour after the start of the assay. Notably, the lower bacterial concentration, 1:100, was also detected fairly rapidly, around 4 hours after the start of the assay ( Figure 4P ). No urease activity was detected at the lowest bacterial concentration, 1:1000 dilution ( Figure S1 ). Furthermore, in contrast to the Christensen Urea Agar plate test, urease activity for HUC 111-01C was rapidly detected, as early as 40 minutes after the start of the assay, at the highest bacterial concentration (overnight suspension), with the lower bacterial concentration (1:100 dilution) exhibiting enzymatic activity around 12 hours ( Figure 4Q ). No urease activity was detected at the 1:1000 concentration ( Figure S1 ). Lastly, to provide additional analyses that further differentiate those isolates that display low activity from those that display no activity in the presence of urea, including E. coli, HUC 95, and HUC97-02, statistical analyses comparing the enzymatic activity in the presence and absence of urea were performed ( Figure S2 ). The UTI MRSA strain was used as a positive control and displayed significantly more urease activity at the final time point assessed when grown in the presence of urea compared to the absence, while no difference was detected in the UTI MRSA ΔureC strain between the two conditions ( Figures S2A, B ). Additionally, significantly more urease activity was detected for both HUC 95 and HUC 97-02 when the strains were grown with urea compared to without ( Figures S2C, D ). Lastly, E. coli displayed no difference in detected urease activity when the bacteria were grown in the presence of urea compared to in its absence at the final time point assessed, while the Proteus spp. strain grown with urea had significantly more urease activity detected compared to the condition without urea ( Figures S2E, F ).

To assess whether urease-producing uropathogens could grow in the adapted assay media, bacterial growth was measured at an OD₆₀₀ ( Figure S3 ). Proteus spp, E. coli, M. morganii, and K. pneumonia all displayed similar growth phenotypes, with increasing growth observed within the first few hours of the assay for all bacterial concentrations, before plateauing ( Figures S3A–D ). The UTI MRSA and the isogenic ureC::tn and ΔureC strains displayed slight increased growth during the first few hours at the highest bacterial concentration (overnight suspensions); however, no growth was observed over the time course of the experiment for the 1:100 and 1:1000 bacterial concentrations ( Figures S3E–G ). For the JE2 and the isogenic ureC::tn, no growth was detected for any of the bacterial concentrations over the course of the experiment ( Figures S3H–K ). The HUC 86 and HUC 86-02 displayed slight increased growth when diluted 1:100; however, no growth was observed for the overnight or 1:1000 bacterial concentrations ( Figures S3J, K ). The HUC 95 strain displayed growth at the highest bacterial concentration in the last few hours of the experiment; however, no growth was observed for the 1:100 and 1:1000 bacterial concentrations ( Figures S3L ). Additionally, no growth was detected for HUC 97-02 at any bacterial concentration over the course of the experiment ( Figure S3M ). Steadily increasing growth was detected in the HUC 100 and HUC 100-01 over the course of the experiment for the 1:100 bacterial concentration; however, no growth was detected for overnight and 1:1000 bacterial concentrations ( Figure S3N, O ). Lastly, HUC 111-01C displayed increased growth within the first few hours of the assay at the 1:1000 bacterial concentration, but no growth was detected for overnight and 1:100 bacterial concentrations ( Figure S3P ).