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

BACKGROUND: Urease 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. METHODS: The 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. RESULTS: The 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 scores 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). CONCLUSION: The 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.

Antimicrobial Effect of a Single Dose of Amoxicillin on the Oral Microbiota.

PURPOSE: Amoxicillin is commonly used in oral surgery for antimicrobial prophylaxis against surgical-site infection and bacteremia because of its effect on oral streptococci. The aim of this study was to determine whether amoxicillin reaches the break-point concentrations in saliva and has any effect on the salivary microbiota, colonizing bacteria on mucosal membranes and on the gingival crevice after a single dose of amoxicillin. MATERIAL AND METHODS: Twenty subjects received 2 g of amoxicillin, per os. The facultative and strictly anaerobic microflora, as well as the streptococcal microflora specifically, were followed from baseline and after 1, 4, and 24 hours. Samples were taken for microbial analysis from saliva, the dorsum of the tongue, and the gingival crevice, and were inoculated and cultured. Plasma samples and saliva samples were analyzed for amoxicillin concentrations (free and protein bound) using liquid chromatography and mass-spectrometry. RESULTS: Amoxicillin was detected in concentrations over the break-point (>2 µg/mL) of amoxicillin in plasma after 1 and 4 hours but not after 24 hours. The dose had a significant effect on the streptococci in the gingival crevice. CONCLUSION: A single dose given as prophylaxis to prevent a surgical-site infection results in a significant reducing effect on the oral streptococcal microflora in the gingival crevice and may have an impact on bacteria spreading into tissues and the bacteremia of streptococci.

Estimation of bacterial hydrogen sulfide production in vitro.

Oral bacterial hydrogen sulfide (H2S) production was estimated comparing two different colorimetric methods in microtiter plate format. High H2S production was seen for Fusobacterium spp., Treponema denticola, and Prevotella tannerae, associated with periodontal disease. The production differed between the methods indicating that H2S production may follow different pathways.

Phenotype, genotype, and antibiotic susceptibility of Swedish and Thai oral isolates of Staphylococcus aureus.

OBJECTIVE: The present study investigated phenotypes, virulence genotypes, and antibiotic susceptibility of oral Staphylococcus aureus strains in order to get more information on whether oral infections with this bacterium are associated with certain subtypes or related to an over-growth of the S. aureus variants normally found in the oral cavity of healthy carriers. MATERIALS AND METHODS: A total number of 157 S. aureus strains were investigated. Sixty-two strains were isolated from Swedish adults with oral infections, 25 strains were from saliva of healthy Swedish dental students, and 45 strains were from tongue scrapings of HIV-positive subjects in Thailand, and 25 Thai strains from non-HIV controls. The isolates were tested for coagulase, nitrate, arginine, and hemolysin, and for the presence of the virulence genes: hlg, clfA, can, sdrC, sdrD, sdrE, map/eap (adhesins) and sea, seb, sec, tst, eta, etb, pvl (toxins). MIC90 and MIC50 were determined by E-test against penicillin V, oxacillin, amoxicillin, clindamycin, vancomycin, fusidic acid, and cefoxitin. RESULTS: While the hemolytic phenotype was significantly (p<0.001) more common among the Thai strains compared to Swedish strains, the virulence genes were found in a similar frequency in the S. aureus strains isolated from all four subject groups. The Panton-Valentine leukocidin (PVL) genotype was found in 73-100% of the strains. More than 10% of the strains from Swedish oral infections and from Thai HIV-positives showed low antibiotic susceptibility, most commonly for clindamycin. Only three methicillin-resistant S. aureus (MRSA) strains were identified, two from oral infections and one from a Thai HIV patient. CONCLUSIONS: S. aureus is occasionally occurring in the oral cavity in both health and disease in Sweden and Thailand. It is therefore most likely that S. aureus in opportunistic oral infections originate from the oral microbiota. S. aureus should be considered in case of oral infections and complaints and the antibiotic susceptibility (including MRSA) should regularly be checked. The frequent presence of S. aureus, although in low numbers among students and staff, emphasizes the importance of standard infection control precautions and of using diagnostic test in the dental clinic.

Virulence factors and antibiotic susceptibility in enterococci isolated from oral mucosal and deep infections.

OBJECTIVE: This study evaluates the presence of virulence factors and antibiotic susceptibility among enterococcal isolates from oral mucosal and deep infections. METHODS: Forty-three enterococcal strains from oral mucosal lesions and 18 from deep infections were isolated from 830 samples that were sent during 2 years to Oral Microbiology, University of Gothenburg, for analysis. The 61 strains were identified by 16S rDNA, and characterized by the presence of the virulence genes efa A (endocarditis gene), gel E (gelatinase gene), ace (collagen binding antigen gene), asa (aggregation substance gene), cyl A (cytolysin activator gene) and esp (surface adhesin gene), tested for the production of bacteriocins and presence of plasmids. MIC determination was performed using the E-test method against the most commonly used antibiotics in dentistry, for example, penicillin V, amoxicillin and clindamycin. Vancomycin was included in order to detect vancomycin-resistant enterococci (VRE) strains. RESULTS: Sixty strains were identified as Enterococcus faecalis and one as Enterococcus faecium. All the virulence genes were detected in more than 93.3% (efa A and esp) of the E. faecalis strains, while the presence of phenotypic characteristics was much lower (gelatinase 10% and hemolysin 16.7%). Forty-six strains produced bacteriocins and one to six plasmids were detected in half of the isolates. CONCLUSIONS: Enterococcal strains from oral infections had a high virulence capacity, showed bacteriocin production and had numerous plasmids. They were generally susceptible to ampicillins but were resistant to clindamycin, commonly used in dentistry, and no VRE-strain was found.

Oral microflora in infants delivered vaginally and by caesarean section.

BACKGROUND. Early in life, vaginally delivered infants exhibit a different composition of the gut flora compared with infants delivered by caesarean section (C-section); however, it is unclear whether this also applies to the oral cavity. AIM. To investigate and compare the oral microbial profile between infants delivered vaginally and by C-section. DESIGN. This is a cross-sectional case-control study. Eighty-four infants delivered either vaginally (n = 42) or by C-section (n = 42) were randomly selected from the 2009 birth cohort at the County Hospital in Halmstad, Sweden. Medically compromised and premature children (<32 weeks) were excluded. The mean age was 8.25 months (range 6-10 months), and parents were asked to complete a questionnaire on socioeconomic factors, lifestyle, and hygiene habits. Saliva was collected and analysed using checkerboard DNA-DNA hybridization. RESULTS. A higher prevalence of salivary Streptococcus salivarius, Lactobacillus curvata, Lactobacillus salivarius, and Lactobacuillus casei was detected in infants delivered vaginally (P < 0.05). The caries-associated bacteria Streptococcus mutans and Streptococcus sobrinus were detected in 63% and 59% of all children, respectively. CONCLUSION. A significantly higher prevalence of certain strains of health-related streptococci and lactobacilli was found in vaginally delivered infants compared with infants delivered by C-section. The possible long-term effects on oral health need to be further investigated.

Microbiological profiles in saliva and supragingival plaque from caries-active adolescents before and after a short-term daily intake of milk supplemented with probiotic bacteria - a pilot study.

PURPOSE: The aim of the present pilot study was to investigate the microbial profile in saliva and supragingival plaque samples collected from caries-active adolescents before and after a daily short-term intake of milk supplemented with the probiotic bacteria. MATERIALS AND METHODS: The present study group consisted of 18 caries-active adolescents of both sexes who volunteered for participation giving an informed consent. The study has a randomised placebo-controlled double-blind pilot design with two parallel arms. After a 2-week run-in period, the subjects were instructed to drink 2.5 dl of milk supplemented with Lactobacillus rhamnosus LB21 (107 CFU/ml) (test) or standard control milk (placebo) once daily for a period of 2 weeks (intervention period). Samples of stimulated whole saliva and supragingival plaque were collected at baseline (after run-in) and immediately after the end of the intervention period (follow-up). The salivary levels of mutans streptococci and lactobacilli were estimated by conventional culturing on selective agar plates. The presence and level of 19 oral species associated with the caries process were determined using the checkerboard DNA-DNA hybridisation technique. Differences between the groups were assessed using the non-parametric Wilcoxon­Mann­Whitney and chi-square tests. RESULTS: The mean caries experience was high with an average of 7.0 ± 3.8 proximal enamel lesions. The most prevalent dominating species in the plaque samples were Streptococcus mitis, Veillonella parvula and Streptococcus gordonii. The saliva samples displayed a more mixed profile, with Streptococcus mitis, Rothia dentocariosa, Lactobacillus casei and Lactobacillus curvata being frequently identified species. All of the subjects harboured mutans streptococci in their saliva, with 61% of them colonised with salivary lactobacilli. No statistically significant differences in the microbial profiles or the estimated counts between the baseline and follow-up samples, or between the two study groups, were observed. CONCLUSIONS: The present study showed that a short-term daily intake of milk supplemented with the probiotic bacterium L. rhamnosus LB21 did not significantly affect the microbial profiles or the levels of caries-associated bacteria in saliva and supragingival plaque samples collected from caries-active adolescents.