Genetic adaptation of Streptococcus mutans during biofilm formation on different types of surfaces

Abstract

Background: Adhesion and successful colonization of bacteria onto solid surfaces play a key role in biofilm formation. The initial adhesion and the colonization of bacteria may differ between the various types of surfaces found in oral cavity. Therefore, it is conceivable that diverse biofilms are developed on those various surfaces. The aim of the study was to investigate the molecular modifications occurring during in vitro biofilm development of Streptococcus mutans UA159 on several different dental surfaces.

Results: Growth analysis of the immobilized bacterial populations generated on the different surfaces shows that the bacteria constructed a more confluent and thick biofilms on a hydroxyapatite surface compared to the other tested surfaces. Using DNA-microarray technology we identified the differentially expressed genes of S. mutans, reflecting the physiological state of biofilms formed on the different biomaterials tested. Eight selected genes were further analyzed by real time RT-PCR. To further determine the impact of the tested material surfaces on the physiology of the bacteria, we tested the secretion of AI-2 signal by S. mutans embedded on those biofilms. Comparative transcriptome analyses indicated on changes in the S. mutans genome in biofilms formed onto different types of surfaces and enabled us to identify genes most differentially expressed on those surfaces. In addition, the levels of autoinducer-2 in biofilms from the various tested surfaces were different.

Conclusions: Our results demonstrate that gene expression of S. mutans differs in biofilms formed on tested surfaces, which manifest the physiological state of bacteria influenced by the type of surface material they accumulate onto. Moreover, the stressful circumstances of adjustment to the surface may persist in the bacteria enhancing intercellular signaling and surface dependent biofilm formation.

Figure 1

Differentially expressed genes in biofilms formed on different surfaces. Alignments of differentially expressed genes (P < 0.05) of S. mutans biofilms formed on hydroxyapatite, titanuim and composite (vs. polystyrene surfaces), showing the number of overlapping genes between the biofilms on different surfaces. Gene annotations are based on the genome information of S. mutans provided by TIGR.

Figure 2

functional categories of most differentially expressed genes. Most significant (B* > 0) differentially expressed genes of S. mutans, grouped in functional categories, in biofilms formed on hydroxyapatite (A), titanium (B) and composite (C) vs. polystyrene surfaces. Gene annotations are based on information provided by TIGR. *Bayesian test value, i.e. the probability for a gene to be really differentially expressed.

Figure 3

Expression of selected genes analyzed by RT-PCR. Comparison of RT-PCR expression values for selected genes of S. mutans, grown on different surfaces. SMU.81, SMU.82 (dnaK) and SMU.1954 (groEL) are stress-related genes; SMU.574c, SMU.609, and SMU.987 are associated with cell wall proteins. SMU.744 codes for FtsY, while SMU.618 codes for a hypothetical protein. The data are expressed as the means of at least two biologically independent experiments.

Figure 4

Biofilms of S. mutans formed on different surfaces. Biofilms of S. mutans UA159 were grown on different surfaces in BHI, stained with LIVE/DEAD BacLight fluorescent dye and analyzed with CLSM. The panels show cross-section images of biofilms from polystyrene (A), Ti (B), HA (C) and composite (D) materials. Dead cells were stained red, and live cells were stained green.

Figure 5

AI-2 signal secretion by S. mutans biofilms on different surfaces. Biofilms were grown on each material and the resulting conditioned media were exposed to V. harveyi MM77 for AI-2 bioassay. Fold induction in luminescence of each sample was calculated above background luminescence of the negative control (sample without addition of any conditioned medium) and was normalized by the value of total fluorescence of live bacteria within the relevant biofilm detected by CLSM.