Eveloped. Thereafter, the cell paste was scraped in the surface in the plate applying a plastic inoculating loop or plastic cell scraper, as well as the cells have been transferred to a microcentrifuge tube containing 750 ml of saline (0.9 NaCl). The tubes have been mixed by vortex agitation for 10 min, and the cells had been pelleted by centrifugation. The supernatant was sterilized by passage by means of a 0.22-mm poresize filter, plus the resulting colony biofilm extract was stored at 4uC till use.Table 1. Bacterial strains.Species Acinetobacter lwoffii Acinetobacter haemolyticus Actinobacillus pleuropneumoniae Actinobacillus pleuropneumoniae Actinobacillus pleuropneumoniae Aggregatibacter actinomycetemcomitans Citrobacter freundii Enterobacter aerogenes Enterobacter amnigenus Haemophilus influenzae Klebsiella pneumoniae Lactococcus lactis Pectobacterium carotovorum Proteus vulgaris Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus epidermidisStrain ATCC 15309 ATCC 19002 J45 (wild-type; serotype 5a) J45-100 (J45 Dcps5ABC; capsule-mutant) J45-100+ pJMLCPS5 (genetically-complemented capsule-mutant) CU1000 ATCC 43864 ATCC 35029 ATCC 51816 NJ9725 ATCC BAA-1705 525A ATCC 15713 ATCC 8427 PP SH1000 NJSource or reference* ATCC ATCC [21] [29] [29] [36] ATCC ATCC ATCC [37] ATCC PIC ATCC ATCC Environmental isolate [38] [35]*ATCC, American Kind Culture Collection, Manassas VA, USA; PIC, Presque Isle Cultures, Erie PA, USA. doi:10.1371/journal.pone.0063844.tPLOS 1 | plosone.orgA. pleuropneumoniae Antibiofilm PolysaccharidePurification of A. pleuropneumoniae Serotype 5 Capsular PolysaccharideCapsular polysaccharide was purified from broth cultures of A. pleuropneumoniae strain J45 by Cetavlon precipitation of culture supernatant, extraction on the precipitate with NaCl and aqueous phenol, and Sepharose CL-4B gel filtration chromatography as previously described [21].Results A. pleuropneumoniae Colony Biofilm Extract Exhibits Antibiofilm ActivityWe isolated colony biofilm extracts from 12 distinct bacteria and tested the extracts for their capability to inhibit biofilm formation by S.Methyl 4-hydroxythiophene-3-carboxylate Purity aureus inside a 96-well microtiter plate assay (Fig. 1). The bacteria that were tested comprised a convenience sample of 11 Proteobacteria and Lactococcus lactis (Table 1). Extracts were tested at a concentration of 10 by vol. Beneath these situations, 5 extracts considerably inhibited S. aureus biofilm formation, while seven extracts had no significant effect on biofilm formation. Inhibition of S.Formula of 364385-54-6 aureus biofilm formation by P.PMID:25023702 aeruginosa extract was partially as a consequence of growth inhibition (data not shown). We selected A. pleuropneumoniae IA5 colony biofilm extract for additional analysis for the reason that it exhibited a high degree of biofilm inhibition, however it didn’t inhibit S. aureus development or contain proteases or DNases which might be recognized to inhibit S. aureus biofilm formation (information not shown). A. pleuropneumoniae IA5 colony biofilm extract inhibited biofilm formation by S. aureus, S. epidermidis, and the Gram-negative Aggregatibacter actinomycetemcomitans (Fig. 2). A. pleuropneumoniae colony biofilm extract didn’t inhibit the development of S. aureus, S. epidermidis or even a. actinomycetemcomitans (data not shown).Cell Binding AssayA single-cell suspension of S. aureus (ca. 106?07 CFU/ml) was prepared in fresh broth applying a filtration protocol as previously described [22]. The cell suspension was supplemented with ten A. pleuropneumoniae colony biofilm extract, or ten saline as a handle, after which aliquots.
