Low prevalence of multi-resistant bacteria in undergraduate dental students; an observational case-control multi-centre study in Europe.

Objective: This study assessed the prevalence of MRSA, ESBL and VRE in students from four dental schools in Europe. Methods: The hand, tongue and nostrils of the students who treated patients (study group) and who did not treat patients (control group) were sampled. After incubation in TSB and subculturing in the presence of 4 µg/ml oxacillin, positive cultures were identified for Staphylococcus aureus by Mannitol salt agar and agglutination tests. The presence of MRSA was confirmed by specific PCR on the species and on the SSCmec genes. ESBL and VRE were isolated using specific CHROMagar and confirmed using antibiotic sensitivity tests. Results: Of the 879 students who participated in this study (454 students which treated patients, 425 controls) a total of 50 students (5.7%) tested positive for a multi-drug resistant bacterium (MDRB); 13 (1.5%) students tested positive for MRSA, 26 (3.0%) for ESBL and 12 (1.4%) for VRE. No statistically significant differences were found between the students who treated patients compared to the control group for any of the MDRB and study centres, excluding MRSA carriage in the Italian student population. The use of antibiotics the year before sampling, was positively associated with the presence of an MDRB (OR 2.0; 95% Confidence Interval 1.10-3.68; p = 0.02). Conclusion: The risk for MDRB carriage and sequential transmission of MDRB for dental health care students and their patients were acceptably low.

Antimicrobial activity of vanadium chloroperoxidase on planktonic Streptococcus mutans cells and Streptococcus mutans biofilms.

The aim of this study was to investigate the antimicrobial activity of vanadium chloroperoxidase (VCPO) reaction products on planktonic and biofilm cells of Streptococcus mutans C180-2. Planktonic and biofilm cells were incubated in a buffered reaction mixture containing VCPO, halide (either chloride or bromide) and hydrogen peroxide, and the killing efficacy was assessed by CFU counts. The enzymatic products formed by VCPO significantly reduced the viability of planktonic and biofilm cells compared to their negative controls and the effect on the biofilm cells was more effective than a 0.2% chlorhexidine digluconate treatment. We conclude that VCPO and its reaction products form a potent antimicrobial system against S. mutans.

Effects of different kinds of fluorides on enolase and ATPase activity of a fluoride-sensitive and fluoride-resistant Streptococcus mutans strain.

Enolase and ATPase are sensitive to fluoride. It is unclear whether this sensitivity differs for F-sensitive and F-resistant cells or for different types of fluoride. Permeabilized cells of the fluoride-sensitive strain Streptococcus mutans C180-2 and its fluoride-resistant mutant strain C180-2 FR were preincubated at pH 7 or 4 with NaF, the amine fluorides Olaflur and Dectaflur and amine chloride controls. After preincubations, enolase and ATPase activities of the cells were assessed. Enolase activity was more inhibited after preincubation at pH 7 with NaF than with Olaflur. Amine chloride stimulated, although not with statistical significance, the enolase activity of both strains. After preincubation at pH 4 the enolases were strongly inactivated, but the fluoride-resistant strain's enolase to a lesser extent. The results suggested that amine acts to protect enolase activity against the detrimental low pH effect. Gene sequencing showed that the enolase genes of the fluoride-resistant and fluoride-sensitive strain were identical. ATPase activity was not reduced after NaF preincubation at either pH 7 or pH 4. The amine fluorides and their chloride controls in the preincubation mixture reduced the ATPase activity significantly at both pH values. In conclusion, our results showed that preincubation with amine fluoride did not inhibit enolase activity more effectively than NaF. The amine part of the molecule may protect enolase activity against preincubations at low pH. ATPase activity was not inhibited by NaF preincubation but was significantly inhibited after preincubation with amine fluorides and amine chlorides.

Effect of Psidium cattleianum leaf extract on Streptococcus mutans viability, protein expression and acid production.

Plants naturally produce secondary metabolites that can be used as antimicrobials. The aim of this study was to assess the effects of Psidium cattleianum leaf extract on Streptococcus mutans. The extract (100%) was obtained by decoction of 100 g of leaves in 600 ml of deionized water. To assess killing, S. mutans biofilms were treated with water (negative control) or various extract dilutions [100, 50, 25% (v/v) in water] for 5 or 60 min. To evaluate the effect on protein expression, biofilms were exposed to water or 1.6% (v/v) extract for 120 min, proteins were extracted and submitted to 2-dimensional difference gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry. The effect of 1.6% (v/v) extract on acid production was determined by pH measurements and compared to a water control. Viability was similar after 5 min of treatment with the 100% extract or 60 min with the 50% extract (about 0.03% survival). There were no differences in viability between the biofilms exposed to the 25 or 50% extract after 60 min of treatment (about 0.02% survival). Treatment with the 1.6% extract significantly changed protein expression. The abundance of 24 spots was decreased compared to water (p < 0.05). The extract significantly inhibited acid production (p < 0.05). It is concluded that P. cattleianum leaf extract kills S. mutans grown in biofilms when applied at high concentrations. At low concentrations it inhibits S. mutans acid production and reduces the expression of proteins involved in general metabolism, glycolysis and lactic acid production.

Determination of arginine catabolism by salivary pellet.

To determine the formation of ammonium from arginine by oral bacteria residing in saliva and dental plaque, an arginolytic activity assay based on the work described by Nascimento et al. [2] was developed. Following the original methodology, insufficient ammonium production could be determined. To improve the method for our research goal, the following modifications were made to the original protocols:•The following changes were made to the arginine catabolism assay resulting in a 1000-fold increase in sensitivity: (i) the salivary pellet was washed and concentrated five times resulting in the removal of low density compounds interfering with the assay, (ii) the pH of the Tris-maleate buffer was increased from 6.0 to 7.5 resulting in a better conversion of arginine to ammonium and (iii) the incubation time was increased to 3 h to ensure that non-responders and salivary pellets low in cell numbers could yield detectable levels of ammonium.•Removal of a centrifuge step from the protein determination resulted in a higher protein yield improving the accuracy of the assay.•Changing from the use of the toxic, environmentally hazardous, mercury containing Nessler's reagent to a colorimetric enzyme assay achieved a safer and greener determination of ammonium concentration.

Novel metabolic activity indicator in Streptococcus mutans biofilms.

Antimicrobial resistance of micro-organisms in biofilms requires novel strategies to evaluate the efficacy of caries preventive agents in actual biofilms. Hence we investigated fluorescence intensity (FI) in Streptococcus mutans biofilms constitutively expressing green fluorescent protein (GFP). Upon addition of glucose FI in these biofilms increased significantly to steady state levels. FI-increase could be inhibited by oral care products in a dose-responsive manner. Lactic acid produced in these biofilms was measured at the end of the FI-recording. A linear correlation with a coefficient of 0.96 (p<0.01) was observed between FI-increase and lactate production, irrespective of the inhibitor used. The viability of biofilm cells after chlorhexidine (CHX) titration was also examined. Reduction of FI-increase was observed at low concentrations of CHX whereas a loss in viability was only seen at high concentrations. In conclusion, GFP synthesis can be used as a metabolic activity indicator in S. mutans biofilms.