Ellen Lanckacker

Importance of biofilm formation and dipeptidyl peptidase IV for the pathogenicity of clinical Porphyromonas gingivalis isolates

The ability of Porphyromonas gingivalis to cause adult periodontitis is determined by its arsenal of virulence factors. Here, we investigated the importance of biofilm formation and bacterial dipeptidyl peptidase IV (DPPIV) for the pathogenicity of clinical P. gingivalis isolates. In our study, the isolates with biofilm-forming capacity also showed high DPPIV activity in vitro. Moreover, DPPIV activity increased in P. gingivalis biofilms compared to planktonic cells. In a murine subcutaneous abscess model, the biofilm-forming isolates with high DPPIV activity proved to be pathogenic, while the nonbiofilm formers with low DPPIV activity did not induce abscesses. The biofilm-forming ATCC 33277 strain with low DPPIV activity was not pathogenic in mice either. Our results suggest that biofilm formation and DPPIV activity contribute to the pathogenic potential of P. gingivalis. Furthermore, we show that biofilm formation may enhance P. gingivalis virulence through an increased DPPIV activity. Because of their importance for bacterial colonization and growth, biofilm formation and DPPIV activity could present interesting therapeutic targets to tackle periodontitis.

Artemisinins, new miconazole potentiators resulting in increased activity against Candida albicans biofilms

ABSTRACT Mucosal biofilm-related fungal infections are very common, and the incidence of recurrent oral and vulvovaginal candidiasis is significant. As resistance to azoles (the preferred treatment) is occurring, we aimed at identifying compounds that increase the activity of miconazole against Candida albicans biofilms. We screened 1,600 compounds of a drug-repositioning library in combination with a subinhibitory concentration of miconazole. Synergy between the best identified potentiators and miconazole was characterized by checkerboard analyses and fractional inhibitory concentration indices. Hexachlorophene, pyrvinium pamoate, and artesunate act synergistically with miconazole in affecting C. albicans biofilms. Synergy was most pronounced for artesunate and structural homologues thereof. No synergistic effect could be observed between artesunate and fluconazole, caspofungin, or amphotericin B. Our data reveal enhancement of the antibiofilm activity of miconazole by artesunate, pointing to potential combination therapy consisting of miconazole and artesunate to treat C. albicans biofilm-related infections.

Comparison of viable plate count, turbidity measurement and real‐time PCR for quantification of Porphyromonas gingivalis

The viable plate count (VPC) is considered as the reference method for bacterial enumeration in periodontal microbiology but shows some important limitations for anaerobic bacteria. As anaerobes such as Porphyromonas gingivalis are difficult to culture, VPC becomes time‐consuming and less sensitive. Hence, efficient normalization of experimental data to bacterial cell count requires alternative rapid and reliable quantification methods. This study compared the performance of VPC with that of turbidity measurement and real‐time PCR (qPCR) in an experimental context using highly concentrated bacterial suspensions. Our TaqMan‐based qPCR assay for P. gingivalis 16S rRNA proved to be sensitive and specific. Turbidity measurements offer a fast method to assess P. gingivalis growth, but suffer from high variability and a limited dynamic range. VPC was very time‐consuming and less repeatable than qPCR. Our study concludes that qPCR provides the most rapid and precise approach for P. gingivalis quantification. Although our data were gathered in a specific research context, we believe that our conclusions on the inferior performance of VPC and turbidity measurements in comparison to qPCR can be extended to other research and clinical settings and even to other difficult‐to‐culture micro‐organisms.

Development of a novel in vitro onychomycosis model for the evaluation of topical antifungal activity

A novel in vitro onychomycosis model was developed to easily predict the topical activity potential of novel antifungal drugs. The model encompasses drug activity and diffusion through bovine hoof slices in a single experimental set-up. Results correspond well with the antifungal susceptibility assay and Franz cell diffusion test.

Characterizing the in vitro biofilm phenotype of Staphylococcus epidermidis isolates from central venous catheters

Central venous catheter (CVC)-related infections are commonly caused by Staphylococcus epidermidis that is able to form a biofilm on the catheter surface. Many studies involving biofilm formation by Staphylococcus have been published each adopting an own in vitro model. Since the capacity to form a biofilm depends on multiple environmental factors, direct comparison of results obtained in different studies remains challenging. This study characterized the phenotype (strong versus weak biofilm-producers) of S. epidermidis from CVCs in four different in vitro biofilm models, covering differences in material type (glass versus polymer) and nutrient presentation (static versus continuous flow). A good correlation in phenotype was obtained between glass and polymeric surfaces independent of nutrient flow, with 85% correspondence under static growth conditions and 80% under dynamic conditions. A 80% correspondence between static and dynamic conditions on polymeric surfaces could be demonstrated as well. Incubation time had a significant influence on the biofilm phenotype with only 55% correspondence between the dynamic models at different incubation times (48h versus 17h). Screening for the presence of biofilm-related genes only revealed that ica A was correlated with biofilm formation under static but not under dynamic conditions. In conclusion, this study highlights that a high level of standardization is necessary to interpret and compare results of different in vitro biofilm models.

Evaluation of topical antifungal products in an in vitro onychomycosis model

Many topical commercial products are currently available for the treatment of onychomycosis. However, limited data are available concerning their antifungal activity. Using an in vitro onychomycosis model, the daily application of seven nail formulations was compared to the antifungal reference drug amorolfine (Loceryl®) and evaluated for inhibitory activity against Trichophyton mentagrophytes using an agar diffusion test. Of all commercial nail formulations, only Excilor® and Nailner® demonstrated inhibitory activity, which was much lower compared to the daily application of Loceryl®. However, Excilor® showed similar efficacy compared to the conventional weekly application of Loceryl®. These results suggest a role for organic acids in the antifungal effect of Excilor® (acetic acid, ethyl lactate) and Nailner® (lactic acid, citric acid, ethyl lactate) as all tested formulations without organic acids were inactive.

Optimization and validation of an existing, surgical and robust dry eye rat model for the evaluation of therapeutic compounds.

The aim of this research was to optimize and validate an animal model for dry eye, adopting clinically relevant evaluation parameters. Dry eye was induced in female Wistar rats by surgical removal of the exorbital lacrimal gland. The clinical manifestations of dry eye were evaluated by tear volume measurements, corneal fluorescein staining, cytokine measurements in tear fluid, MMP-9 mRNA expression and CD3(+) cell infiltration in the conjunctiva. The animal model was validated by treatment with Restasis(®) (4 weeks) and commercial dexamethasone eye drops (2 weeks). Removal of the exorbital lacrimal gland resulted in 50% decrease in tear volume and a gradual increase in corneal fluorescein staining. Elevated levels of TNF-α and IL-1α have been registered in tear fluid together with an increase in CD3(+) cells in the palpebral conjunctiva when compared to control animals. Additionally, an increase in MMP-9 mRNA expression was recorded in conjunctival tissue. Reference treatment with Restasis(®) and dexamethasone eye drops had a positive effect on all evaluation parameters, except on tear volume. This rat dry eye model was validated extensively and judged appropriate for the evaluation of novel compounds and therapeutic preparations for dry eye disease.