Rather, previous investigations have been largely restricted to endpoint susceptibility determinations in dispersed, pure cultures or have inferred effects from individuals with defined HDP deficiencies (Dale & Fredericks, 2005). The aim of the current investigation therefore was to evaluate the effect of representatives of the four classes of HDPs (HNP 1, HNP 2, hβD 1, hβD 2, hβD 3, His 5, His 8 and LL37), selected on the basis their in situ predominance, using a previously validated in vitro plaque ecosystem (Ledder & McBain, 2011). Since nascent plaque communities are arguably the dominant mode of bacterial growth in the mouth (Marsh & Martin, 1999) and
are more amenable to compositional modification than mature plaques (Pham Erlotinib in vivo et al.,
2006; Madhwani & McBain, 2011), salivary ecosystems were developed upon hydroxyapatite surfaces in the presence of various peptides. These were applied singly and in various combinations, and effects on consortial composition and bacterial aggregation, which is reportedly an important process in plaque development (Kolenbrander et al., 1989; Palmer et al., 2004), were assessed. Chemicals and formulated bacteriological media were obtained from Sigma (Dorset, UK) and Oxoid (Basingstoke, UK), respectively. Hydoxyapatite discs used for the establishment of in vitro plaques were obtained from Clarkson Chromatography Inc. (Philadelphia, PA). This was used to support oral bacteria in nutritional PS-341 mouse conditions similar to human saliva. Composition was as follows (g L−1 in distilled water): mucin (porcine type II), 2.5; tryptone, 2.0; bacteriological peptone, 2.0; yeast
extract, 1.0; NaCl, 0.35; KCl, 0.2; CaCl2 0.2; cysteine hydrochloride, 0.1; haemin, 0.001; Vitamin K1, 0.0002 (McBain et al., 2003). These were set up using 2-mm (diameter) hydroxyapatite discs. Double-strength of artificial saliva (100 μL) supplemented with 0.4% sucrose was added to each well of a 96-well microtitre plate. Physiological saline or a double-strength salivary HDP in saline (concentrations detailed in Table 1; 100 μL) was added to each well. Presterilized hydroxyapatite discs were transferred aseptically to each well of the plate which was then mounted on an orbital shaker (144 oscillations min−1) for 1 h to allow conditioning of the discs. For inoculation, unstimulated saliva samples (c. 5 mL) were obtained by expectoration from a healthy human donor who had no extant periodontal disease and who had not used antibiotics for at least 1 year. The transfer of endogenous HDPs from the salivary inoculum to the growing cultures was minimized by centrifugation (2 mL) at 13 000 g for 5 min. and resuspension in physiological saline (200 μL). This resuspended pellet (10 μL per well) was then used to inoculate the HDMs.