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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.

Since biofilms are important in many clinical, industrial, and environmental settings, reliable methods to quantify these sessile microbial populations are crucial. Most of the currently available techniques do not allow the enumeration of the viable cell fraction within the biofilm and are often time consuming. This paper proposes flow cytometry (FCM) using the single-stain viability dye TO-PRO(®)-3 iodide as a fast and precise alternative. Mature biofilms of Candida albicans and Escherichia coli were used to optimize biofilm removal and dissociation, as a single-cell suspension is needed for accurate FCM enumeration. To assess the feasibility of FCM quantification of biofilms, E. coli and C. albicans biofilms were analyzed using FCM and crystal violet staining at different time points. A combination of scraping and rinsing proved to be the most efficient technique for biofilm removal. Sonicating for 10 min eliminated the remaining aggregates, resulting in a single-cell suspension. Repeated FCM measurements of biofilm samples revealed a good intraday precision of approximately 5 %. FCM quantification and the crystal violet assay yielded similar biofilm growth curves for both microorganisms, confirming the applicability of our technique. These results show that FCM using TO-PRO(®)-3 iodide as a single-stain viability dye is a valid fast alternative for the quantification of viable cells in a biofilm.

The promising anti-adhesion in vitro results could not be confirmed in the mouse model, even after the highest attainable exposure. It is concluded that raw or defatted milk fat globule membrane fractions do not have any prophylactic or therapeutic potential against Helicobacter infection.

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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.

Quantification of bacteria using conventional viable plate counting (VPC) is labor-intensive and time-consuming. Flow cytometry (FCM) can be proposed as a faster alternative. This study aimed to develop a flow cytometric, single-stain approach using TO-PRO ® -3 iodide (TP3) for the quantification of Staphylococcus aureus, Escherichia coli, and Bacillus subtilis cells. Live or dead bacterial suspensions were stained with TP3 and analyzed using a FACSCalibur flow cytometer. After optimization of staining parameters and instrument settings, an excellent separation of viable and dead cells was achieved for all species. The quantitative performance of the technique was assessed by analyzing serial dilutions of bacterial suspensions using FCM and VPC. A highly linear correlation (r 2 > 0.99) was observed between the colony forming units (CFU)/mL as determined by FCM and by VPC over a concentration range of about 10 4 to 10 8 CFU/mL. As such, FCM quantification of viable bacteria using TP3 can be considered as an accurate and reliable alternative for VPC. The monostain procedure is easy to apply and cost-effective, and it allows bacterial enumeration in a broad variety of samples.