Infection and disinfection of transmucosal implant surfaces "in vitro"

Peri-implantitis is an inflammatory process, which is initiated by a biofilm-induced
pathway causing a reversible inflammatory reaction in the soft tissues surrounding the
implant; however, it may progress into irreversible damage in the surrounding alveolar
bone and result in loss of attachment. Modelling any kind of oral biofilm is challenging as
up to 700 species have been identified on various surfaces within the mouth. Thus, also
data on bacterial colonization and biofilm formation on dental implant surfaces is limited
and mostly based on identification of species. However, knowledge about this process, in
particular in its early stages, is essential for the development of strategies to prevent and
control microbial adherence and biofilm formation.
The aims of this study were (i) to develop and validate a flow chamber model to assess
peri-implantitis related multispecies biofilm formation; (ii) to test antimicrobial
susceptibility of this biofilm; (iii) to evaluate the reliability of commonly used methods
and novel isothermal microcalorimetry (IMC) analyses for vitality testing of adherent
microorganisms in vitro.
An in vitro flow chamber model was developed to study single- and multispecies
adhesion to protein-coated surface in reduced nutrient conditions that mimic the clinical
situation of peri-implant disease.
Adherent single-species model with S. sanguinis was used for antimicrobial susceptibility
testing (Original study 1 in Chapter 4) to answer two questions: firstly, how efficient is
disinfectant treatment and secondly, to identify discrepancies between the results revealed
by different vitality detection methods (commonly applied: staining by Live/dead
Baclight kit, conventional culturing; and a novel approach by IMC). Chlorhexidine has
been considered for many years the standard in oral infection control, however, the three
other disinfectants (povidone-iodine, octenidine dihydrochloride, polyhexanide) showed
comparable efficacy against the adherent microorganism. The interpretation of the results
of staining and traditional culturing is difficult as major discrepancies were present. The
disagreement between the results of the methods can be attributed to different aspects: (i)
staining is based on the destruction or integrity of the cell wall; (ii) conventional culturing
requires bacteria to be re-suspended prior to cultivation and subsequent quantification of
colony-forming units. However, several factors – such as physical damage to the cells by
re-suspension or lack of growth due to suboptimal culture conditions – may cause false
negative results; (iii) the population of cells that are viable but not cultivable (VBNC), but
are still present by staining and microscopy.
The IMC is a very sensitive method, which detects heat-flow from all metabolic processes
in any living microorganism. The principle of the method is that all living systems
produce heat and heat release can be measured calorimetrically without any interference
with the processes. In other words, IMC measures heat production or consumption, which
is proportional to the rate at which any given chemical, physical or biological process is
taking place (i.e., metabolic heat from bacterial growth). Our results on adherent
disinfectant-treated S. sanguinis indicate that IMC is a useful tool to avoid the problem
with VBNC cells and provides a novel approach in oral microbiology that detects reliably
all the cells that are able to resuscitate after disinfectant treatment and thereby may lead to
the expression of virulence factors in the colonized sites.
In order to study antimicrobial efficacy on a more complex system, a 72-hour biofilm
model including S. sanguinis, F. nucelatum and P. gingivalis was created (Original study
2 in Chapter 5). After 72 h, the three species were always detected with similar
proportions throughout experiments by microscopic methods. Even, when such stability
in the proportions and structure was found, the metabolic activity of biofilms in IMC
revealed high heterogeneity. Thus, this heterogeneity suggests that bacteria appear to
have intra-species variance affecting their behaviour in biofilms, which is dependent on
different factors that are hitherto not well understood.
IMC analysis of the behaviour of the three-species biofilm provided a novel opportunity
to observe how an intact biofilm acts as an entity when exposed to antibiotics (Original
study 3 in Chapter 6). The clinical efficacy of adjuvant antibiotic therapy in the periimplantitis
treatment is unclear; in order to gain better understanding the most frequently
used antibiotics: amoxicillin, metronidazole and their combination were tested.
Additionally, in order to obtain comparable data to the previously published information
(Chapter 4), the vitality of adherent single-species of S. sanguinis and P. gingivalis after
antibiotic treatment was assessed by vitality staining, conventional culturing and IMC.
The results of staining and culturing correlated poorly. This can be due to reasons
mentioned earlier, such as limitations of the methods and VBNC cells, but can also be
due to the bacteriostatic effects of antibiotics that cannot be reliably detected by staining.
Although amoxicillin and metronidazole are considered bactericidal, IMC results suggest
that when applied alone, the antibiotics seem to work primarily through bacteriostatic
effects; when combined, the efficacy is increased by their synergistic bactericidal action.
Furthermore, this indicates that it might not be possible to strictly divide the effects of
antibiotics into bacteriostatic and bactericidal, as the results here imply that the effect
seems to be dependent on their concentration and combination plus the nature of targeted
cells.
In conclusion, this study emphasises the need to critically evaluate the results of live/dead
staining and conventional culturing as many aspects can affect the outcome of these
methods leading to miscalculations on the proportion of cells that are able to resuscitate
and express virulence factors on treated sites.
Thus, as a novel alternative in oral biofilm research IMC gives new insights helping to
monitor the efficacy and dynamics of biofilms and their antimicrobial susceptibility in
vitro.