Université catholique de Louvain (UCL-Bruxelles)
Louvain Drug Research Institute > Cellular and Molecular Pharmacology
Biofilms
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Biofilms can develop both on implanted medical devices and on tissues. They are associated to many human pathologies and are difficult to eradicate.

The efficacy of chemotherapy against bacteria growing in biofilms depends on the capacity of the drug to penetrate within the biofilm and on the bacterial metabolic state within this structure.

Using in vitro models of biofilms, we study the efficacy of antibiotics against bacteria growing in biofilms, the penetration of antibiotcis within these structures, and explore innovative strategies that can improve antibiotic activity agaist these specific forms of infections.


This research program is closely linked to those exploring the chemotherapy of intracellular infection and the novel antibiotic targets

Team

Collaborations

Main current research programs

General principles

Biofilms are defined as communities of sessile bacteria that irreversibly adhere on a support, are embedded in a matrix of polymeric substances produced by the bacteria themselves, and show altered growth rate and profile of gene expression. It is estimated that 60 to 80 % of human infection involve biofilms. Within biofilms, bacteria are exposed to a gradient of oxygen and nutrients, making them adopting dormant lifestyles. Moreover, the polymeric matrix opposed a barrier to the antibiotic diffusion, preventing them from exerting their antimicrobial effects.

Figure 1: Main steps of bacterial biofilm development

After having irrevesibly attached on a support, bacteria start producing polymeric susbstances that form a protective matrix.
In this structure, antibiotic activity is impaired due to

  • poor penetration in the matrix
  • physicochemical conditions (oxygen or pH)
  • altered metabolic state of the bacteria (slow metabolism)

In vitro models for the study of antibiotic pharmacodynamics in biofilms

Using both reference and clinical strains, we study antibiotic activity against young and mature biofilms and try to identify the parameters that can modulate this activity, examining in parallel their effect on the biomass of the biofilm and on the viability of the bacteria within the biofilm.
In a nutshell, we show that bactericidal antibiotics are in general more effective than bacteriostatic molecules, that the activity is higher towards viability than towards biomass, and that it is reduced upon biofilm maturation.

Figure 2: Activity of clarithromycin and moxifloxacin against young (2 days) and mature (11 days) biofilms of Streptococcus pneumoniae ATCC49619.

Concentration-response activity in biofilms incubated with increasing concentrations of antibiotics for 24h. The ordinate shows the change in viability (measured by the decrease in resorufin fluorescence; left panels) or in biofilm mass (measured by the decrease in crystal violet absorbance; right panels) in percentage of the control value (no antibiotic present).

The graph shows that both the maximal efficacy (reduction in signal for an infinitely large antibiotic concentration) and the relative potency (concentration needed to reach a specified effect) are reduced upon biofilm maturation. Moreover, moxifloxacin is more potent and more effective than clarithromycin.

 

adapted from Vandevelde et al, 2014

Parameters affecting antibiotic activity in biofilms

We study the environmental parameters within the biofilm that could affect antibiotic activity, like local pH or oxydant species, the nature of the matrix, and the penetration of antibiotics within the matrix. As an illustrative example, we showed that delafloxacin, an investigational fluoroquinolone characterized by an increased intrinsic activity at acidic pH, was more potent within biofilms formed by clinical isolates of Staphylococcus aureus the pH of which was more acidic (see Figure 3). We also showed that the penetration of antibiotics is highly variable in biofilms formed by different clinical isolates, which is correlated with commensurate changes in antibiotic activity.

Figure 3: Influence of pH on the activity of the fluoroquinolone delafloxacin against clincial isolates of Staphylococcus aureus.

Upper panel: influence of pH on delafloxacin MIC; the drug is more acive at acidic pH (gray squares highlight the zones of pH observed in the corresponding biofilm).

Lower left panel: pH in the deepness of biofilms formed by the same three clinical isolates as assessed by a pH-sensitive fluorescent probe

Lower right panel: correlation between the relative potency (concentration reducing of 25% bacterial viability within biofilms formed by different clinical isolates and Mic fo delafloxacin at the pH found at the surface of each biofilm.

adapted from Siala et al, 2014

Innovative strategies against biofilms

Based on our work exploring the parameters detrimental to antibiotic expression of activity within biofilms, we explore the potential interest of combining antibiotics with other molecules that may affect matrix properties to increase bacterial diffusibility within the biofilm. We were able to show that polycationic substances like norspermidine or norspermine can markedly improve the effect of fluoroquinolones against viability of S. aureus clincial isolates griwng in biofilms by increasing the drug penetration in the deepness of the structure.

Figure 4: Influence of polycationic compounds on the activity (left) or on the penetration (right) of delafloxacin (DFX; top) or of vancomycin (VAN; bottom) towards a biofilm formed by a clincial isolate of S. aureus recalcitrant to all antibotics when used alone.

Left panels: concentration-effect response towards viability within biofilm or antibiotics alone or combined with norspermine or nonspermidine

Right panels: penentration of antibiotic within biofilms in control conditions or in the presence of norspermidine. The graphs on the right show the quantification of antibiotic concentrations based on these images.
TOP: blue: delafloxacin; green, living bacteria and red, dead bacteria (live/dead kit)
BOTTOM: green, bodipy-vancomcyin; red: 5-cyano-2,3-ditolyl tetrazolium chloride (CTC; living bacteria)

adapted from Siala et al, 2014

Selected references (by reverse chronological order; for a full reference list, see our publication list)

Expertise

Additional information:  <tulkens@facm.ucl.ac.be>
Last significant update: Mai 2016