Rapid urease test (RUT) for evaluation of urease activity in oral bacteria in vitro and in supragingival dental plaque ex vivo

Abstract

BackgroundUrease is an enzyme produced by plaque bacteria hydrolysing urea from saliva and gingival exudate into ammonia in order to regulate the pH in the dental biofilm. The aim of this study was to assess the urease activity among oral bacterial species by using the rapid urease test (RUT) in a micro-plate format and to examine whether this test could be used for measuring the urease activity in site-specific supragingival dental plaque samples ex vivo.MethodsThe RUT test is based on 2% urea in peptone broth solution and with phenol red at pH 6.0. Oral bacterial species were tested for their urease activity using 100 μl of RUT test solution in the well of a micro-plate to which a 1 μl amount of cells collected after growth on blood agar plates or in broth, were added. The color change was determined after 15, 30 min, and 1 and 2 h. The reaction was graded in a 4-graded scale (none, weak, medium, strong). Ex vivo evaluation of dental plaque urease activity was tested in supragingival 1 μl plaque samples collected from 4 interproximal sites of front teeth and molars in 18 adult volunteers. The color reaction was read after 1 h in room temperature and scored as in the in vitro test.ResultsThe strongest activity was registered for Staphylococcus epidermidis, Helicobacter pylori, Campylobacter ureolyticus and some strains of Haemophilus parainfluenzae, while known ureolytic species such as Streptococcus salivarius and Actinomyces naeslundii showed a weaker, variable and strain-dependent activity. Temperature had minor influence on the RUT reaction. The interproximal supragingival dental plaque between the lower central incisors (site 31/41) showed significantly higher score compared to between the upper central incisors (site 11/21), between the upper left first molar and second premolar (site 26/25) and between the lower right second premolar and molar (site 45/46).ConclusionThe rapid urease test (RUT) in a micro-plate format can be used as a simple and rapid method to test urease activity in bacterial strains in vitro and as a chair-side method for testing urease activity in site-specific supragingival plaque samples ex vivo.

Campylobacter species in chilean patients with chronic periodontitis

“…C. ureolyticus has been detected in the subgingival bacterial plaque. However, it is rarely studied in periodontal disease (DUERDEN et al 1987;DAHLÉN et al, 2018).…”

Presence of Helicobacter pylori and Campylobacter ureolyticus in the oral cavity of a Northern Thailand population that experiences stomach pain

“…It is possible that usage of a more sensitive method, like 16S qPCR, would reveal a higher prevalence of these target bacteria including not only H. pylori and C. ureolyticus but also other urease positive species. Interestingly, another highly urease positive species, H. parainfluenzae [23], was commonly present and may contribute to the alkaline environment in the interproximal sites that was found in the resting plaque. This was in particular found in sites of the lower front region, where some extreme values (>8.0) were noticed for some individuals.…”

“…The urease activity of the dental biofilm was measured by the use of a modified rapid urease test [18,22,23] in interproximal plaque samples from 4 interproximal sites (mesial aspect of 11, 26, 36, and 41). The urea broth used consisted of urea (10%) in distilled water and 0.02 g/L of phenol red; the pH was adjusted to 6.8.…”

“…The color production of the assay was evaluated after 2 h in a temperature of 20–25°C. The color change was divided according to a 4-graded scale [23], where 0 corresponded to no reaction (colorless), 1 corresponded to a pink reaction, 2 to a red reaction, and grade 3 corresponded to a deep purple reaction. Author GD performed the color readings.…”

“…Whole-genomic DNA probes were prepared for common dental plaque bacteria and bacteria with known ureolytic activity [18,23]. The species were Actinomyces oris, Campylobacter gracilis, Campylobacter rectus, C. ureolyticus, Filifactor alocis, Fusobacterium nucleatum, H. pylori, Haemophilus parainfluenzae, Lactobacillus fermentum, Prevotella intermedia, Prevotella tannerae, Streptococcus mutans, Streptococcus salivarius , and Streptococcus sanguinis [18,20].…”