Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm.

INTRODUCTION: Our previous studies showed that Streptococcus mutans and Veillonella parvula dual-species biofilms have a different acid production profile and a higher resistance to chlorhexidine than their single-species counterparts. The aim of the current study was to test whether the susceptibility of S. mutans grown in the presence of V. parvula is also decreased when it is exposed to various other antimicrobials. Furthermore, the aim was to identify other changes in the physiology of S. mutans when V. parvula was present using transcriptomics. METHODS: Susceptibility to antimicrobials was assessed in killing experiments. Transcript levels in S. mutans were measured with the help of S. mutans microarrays. RESULTS: When V. parvula was present, S. mutans showed an increase in survival after exposure to various antimicrobials. Furthermore, this co-existence altered the physiology of S. mutans. The expression of genes coding for proteins involved in amino acid synthesis, the signal recognition particle-translocation pathway, purine metabolism, intracellular polysaccharide synthesis, and protein synthesis all changed. CONCLUSION: Growing in a biofilm together with a non-pathogenic bacterium like V. parvula changes the physiology of S. mutans, and gives this bacterium an advantage in surviving antimicrobial treatment. Thus, the study of pathogens implicated in polymicrobial diseases, such as caries and periodontitis, should be focused more on multispecies biofilms. In addition, the testing of susceptibility to currently used and new antimicrobials should be performed on a multispecies microbial community rather than with single pathogens.

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.

The effect of the growth phase of Staphylococcus aureus on resistance to disinfectants in a suspension test.

The influence of growth phase on the resistance of Staphylococcus aureus to the surface-active agents benzalkonium chloride and dodecylbenzyl sulfonic acid and the oxidizing agents sodium hypochlorite and hydrogen peroxide was studied. The resistances of cells in different growth phases were compared to those of solid medium cells grown according to the European phase I suspension test. Using cells from different growth phases (+/- 3 x 10(7) CFU ml(-1)), we found that decline-phase cells were the most resistant cells. However, the decline-phase cell suspension contained more than 90% dead cells. A 10-fold-diluted suspension with a total concentration of cells equal to that of the other cell suspensions still revealed decline-phase cells to be generally the most resistant cell type. However, the resistance was drastically reduced, indicating that the large proportion of dead cells provided significant protection to the viable decline-phase cells. Hydrogen peroxide resistance could be partly explained by the high catalase activity in the dead-cell fraction. Exponential-phase cells were less resistant than decline-phase cells, and, surprisingly, stationary-phase cells were the least resistant of the three. Cells grown according to the European phase 1 suspension test were never the most resistant cells. Their survival was 1 to 3 log units lower than that of the most resistant cells. These findings show that the solid-medium cells currently used in disinfectant tests are not the most resistant cells that can be used.

Selection of fluorescent probes for flow cytometric viability assessment of Listeria monocytogenes exposed to membrane-active and oxidizing disinfectants.

The aim of this study was to select fluorescence methods for use as alternatives to plate counting to assess the viability of Listeria monocytogenes cells exposed to benzalkonium chloride (BAC) and hydrogen peroxide, two disinfectants with different mechanisms of action. A further aim of this study was to determine whether growth phase influences fluorescence labeling and whether it is possible to predict whether a probe will be a good viability indicator for cells exposed to a certain disinfectant on the basis of the mechanism of action of the disinfectant and the target of the fluorescent probe. The fluorescence methods used were labeling with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC; dehydrogenase activity), labeling with TOTO-1 iodide (TOTO; membrane-impermeant probe), and assessment of pH gradient maintenance in a low-pH buffer after labeling with the pH-sensitive probe 5-(and 6)-carboxyfluorescein succinimidyl ester (CFSE) (the pH(in) method). Growth phase influenced fluorescent labeling. However, the cutoff value for distinction between viable and nonviable cells was the same for both growth phases. The viability (determined by plate counts) of BAC-exposed cells correlated well with CTC labeling and TOTO exclusion. For both BAC-exposed and hydrogen peroxide-exposed cells, the pH(in) method gave a good qualitative indication of viability, sublethal damage, and cell death. CTC labeling and TOTO exclusion did not correlate with the viability of hydrogen peroxide-exposed cells. Our results demonstrate that even if the mechanism of action of a disinfectant is known, in some cases it is still difficult to predict whether a certain fluorescent probe is suitable for viability assessment. Thus, the proper selection of fluorescent probes for the assessment of the efficacy of antimicrobial agents is essential.