Abstract
Cranberry (Vaccinium macrocarpon) is a low bush fruit cultivated throughout northern United States and Canada and is well-known for its ability to alleviate the symptoms and duration of urinary tract infections. Recent studies, however, have shown promising antimicrobial activity against several other organisms responsible for different infections. These studies have reported inhibition of bacterial adhesion, co-aggregation and biofilm formation, and have shown that several constituents including proanthocyanidins, flavonols and polysaccharides may work synergistically to reduce E. coli adhesion forces and co-aggregation of oral bacteria. The rise in antibiotic-resistance has driven investigations for alternative methods to treat infectious diseases. Inhibiting or disrupting the formation of biofilms is a potential method to prevent infectious disease without promoting the development of new antibiotic-resistant strains of bacteria. It is, therefore, important to explore the mechanisms by which cranberry phytochemicals prevent certain infectious processes by isolating, characterizing and assaying antimicrobial properties of phytochemicals found in cranberry fruit. The aims of this study were to first identify phytochemicals derived from cranberry fruit and then to compare their effects on the biofilm formation of different bacteria both alone or in combination. Whole cranberry fruit was extracted and then further separated into different fractions based on phytochemical types by open column chromatography. Composition of these extracts were determined by HPLC-PDA, UPLC-QTOF-MS, and MALDI-TOF-MS methods. These fractions were categorized as phenolic acid-containing, flavonol-containing, anthocyanin-containing, and proanthocyanidin-containing extracts. Phenolic acid-containing extracts were determined to contain organic acids such as chlorgenic acid. Flavonol-containing extracts were predominately composed of glycosylated myricetin, quercetin, and kaempeferol. Anthocyanin-containing extracts were mainly cyanidin and peonidin derivatives. Proanthocyanidin-containing extracts were composed of different proanthocyanindin oligomers ranging from monomer units to nine degrees of polymerization with varying numbers of A-type and B-type linkages. Several Enterococcus and Streptococcus strains were selected for further evaluation based on their clinical relevance in prostate infections, dental caries, and throat infections; information on cranberry’s efficacy against these organisms is lacking. Isolates from whole cranberry fruit were evaluated against five organisms for their biofilm inhibitory capability alone and in combination, using a crystal violet stain biofilm assay. The effects were varied based on phytochemical class and organism. Several fractions inhibited biofilm formation in a dose-dependent manner. A fraction containing primarily A-type proanthocyanidin oligomers ranging from 2 to 7 (epi)catechin units, was a potent biofilm inhibitor against all organisms tested. These fractions were able to prevent biofilm formation by all organisms tested at low concentrations. Other fractions were effective in reducing biofilm formation in specific cases. Anthocyanin-containing fractions showed greater selectivity for Enterococcus spp. while fractions containing primarily flavonols were more effective on Streptococcus spp. Phenolic acid-containing fractions showed no consistent effect against biofilm formation. The synergistic effect of fractions was also evaluated by combining flavonol-containing fractions and proanthocyanidin-containing fractions. In most cases, this combination was able to reduce biofilm formation better than the flavonol-containing and proanthocyanidin-containing fractions alone. These findings suggest there could be an ideal composition of cranberry phytochemicals that would be highly effective in reducing biofilm formation. Based on these results, cranberry may help reduce infection by these pathogens by interfering with the initial steps of adhesion and biofilm formation without promoting the development of antibiotic-resistant strains.