Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18–24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL-1 of vancomycin and 8 μg mL-1 of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation.

The aim of this study was to estimate differences in the prevalence of oral streptococcal species in the subgingival biofilm of patients with aggressive periodontitis and of healthy controls. Thirty-three patients with clinical and radiological proof of aggressive periodontitis and 20 healthy subjects were enrolled in this study. Clinical indices were recorded in a six-point measurement per tooth. Samples of the subgingival biofilm were taken with paper points from four teeth of each individual. Alpha-and non-haemolytic, small and catalase-negative colonies were biochemically identified using a rapid ID 32 STREP system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A total of 118 strains of oral streptococci (11 species) were identified and Streptococcus sanguinis was found significantly more often in healthy subjects (P50.001). Conversely, the absence of S. sanguinis was associated with high values of clinical indices (P50.001-0.002). Aggressive periodontitis seems to be associated with a loss of colonization of S. sanguinis. Whether or not S. sanguinis offers protection against aggressive periodontitis needs to be determined. Otherwise, there were no significant differences in the distribution of oral streptococcal species in patients and healthy subjects. INTRODUCTIONHealthy gingivae are associated with a simple supragingival biofilm composition: a few (1-20) layers of oral streptococci, Gram-positive rods and very few Gram-negative cocci. These bacteria are early colonizers that are able to survive in an aerated environment. In contrast, clinical gingivitis is associated with the development of a more organized dental plaque of 100-300 layers, with anaerobic Gram-negative rods and filaments being predominant. The species involved in biofilm formation may vary depending on local environmental characteristics, but the colonization pattern is always the same (Marsh & Bradshaw, 1999;Marsh, 2004).Bacterial communities from dental biofilms tend to be grouped in clusters (complexes) according to nutritional and atmospheric requirements. The initiation and progression of periodontitis is thought to be caused by several species belonging to 'red' and 'orange' complexes (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, and Prevotella intermedia and Fusobacterium nucleatum, respectively) . However, according to the 'ecological plaque hypothesis ' (Marsh, 1991), the lack of so-called 'protective bacteria' is also thought to play an important role. These are microbial species that can occupy a niche that could shelter pathogenic organisms or that can inhibit some pathogens through metabolic antagonism or by directly inactivating them (Quirynen et al., 2001). Some of the members of the 'yellow' complex of oral streptococci are candidates for this position. Plaque samples from healthy gingival sulci normally contain a large number of oral streptococci (Theilade et al., 1966). Socransky et al. (1998) previously showed that yellow complexes, as a total, were associate...

ObjectiveImmunological processes in the etiopathogenesis of periodontitis, especially the aggressive form, are not well understood. This study examined clinical as well as systemic immunological and local microbiological features in healthy controls and patients with different forms of periodontitis.Materials and Methods14 healthy subjects, 15 patients diagnosed with aggressive periodontitis, and 11 patients with chronic periodontitis were recruited. Periodontal examination was performed and peripheral blood was collected from each patient. Lymphocyte populations as well as the release of cytokines by T-helper cells were determined by flow cytometry and enzyme linked immunosorbent spot assay. Subgingival plaque samples were taken from each individual and immediately cultivated for microbiological examination.ResultsWhen stimulating peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide, a higher IL-1β release was found in patients with moderate chronic periodontitis compared to the other groups (p<0.01). Numbers of B-cells, naïve and transitional B-cells, memory B-cells, and switched memory B-cells were within the reference range for all groups, but patients with chronic periodontitis showed the highest percentage of memory B-cells without class switch (p = 0.01). The subgingival plaque differed quantitatively as well as qualitatively with a higher number of Gram-negative anaerobic species in periodontitis patients. Prevotella denticola was found more often in patients with aggressive periodontitis (p<0.001) but did not show an association to any of the systemic immunological findings. Porphyromonas gingivalis, which was only found in patients with moderate chronic periodontitis, seems to be associated with an activation of the systemic immune response.ConclusionDifferences between aggressive periodontitis and moderate chronic periodontitis are evident, which raises the question of an inadequate balance between systemic immune response and bacterial infection in aggressive periodontitis.

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Our results suggest that MALDI-TOF-MS might become a useful method for the identification of anaerobic bacteria, especially for those that cannot be readily identified by biochemical analysis. It may become an attractive system even for the routine identification of clinical isolates.