The outbreak of multidrug resistant bacteria arose from several factors, including the lack of novelty in the pipeline of drug discoveries since the 1980s, and the large and non-adequate administration of antibiotics [7, 36]. To eradicate this problem, explorations of new sources of antimicrobial compounds, and/or finding ways to strengthen the action of well-known antibiotics are needed [31, 37]. For many decades, cranberry juice and extracts have been studied for their antimicrobial potential against bacterial pathogens. Cranberry constituents are known to exert anti-adhesion and antimicrobial activities against several pathogenic bacteria and have been suggested to prevent urinary tract infections caused by uropathogenic P-type Escherichia coli[38–40]. However, the mechanisms supporting the diverse effects of cranberry on microbes are poorly understood. In this study, we produced cranberry extracts by mechanical and ethanol extraction, investigated the antibacterial modes of action of these extracts by transcriptional and macromolecular biosynthesis analyses, and assessed their in vitro and in vivo bactericidal activities.
In an attempt to investigate the mode of action of the cranberry product NC90, we initiated the study by performing an exploratory transcriptional analysis by microarray, which yielded the identification of several bacterial genes known to be up-regulated in the presence of cell-wall acting antibiotics, such as oxacillin, vancomycin and daptomycin [29, 30, 41]. Also, the cell wall stress stimulon we observed correlated well to the list of VraS/R-regulated genes known to be up-regulated in response to the inhibition of cell wall biosynthesis , as reported in Table 1. Interestingly, the cranberry PC extract FC111 showed a slightly lower MIC than the commercial preparation NC90 against S. aureus, indicating enrichment in some antibacterial compounds in FC111. In addition, qPCR analysis of key cell wall stress markers (vraR/S, sgtB, msrA, murZ) revealed similar gene expression modulations by both NC90 and the fraction FC111, indicating that the growth inhibitory compounds remaining in FC111 should be responsible for the cell wall stress observed following exposure of S. aureus to this extract.
Although oxacillin, vancomycin and daptomycin disrupt cell wall peptidoglycan synthesis, these antibiotics act in different ways [29, 43]. In the present study, it was interesting to observe that NC90 and FC111 also modulated the expression of lytM and the mntABC operon reacting to membrane depolarization, as distinctively reported for daptomycin by Muthaiyan et al. . This may indicate that NC90 and the cranberry extract FC111 induce S. aureus membrane depolarization, as that observed after exposure to daptomycin. The gene modulations induced by cranberry also support the S. aureus cell disruption reported by Wu et al. , while, scanning electron microscopy of MRSA after treatment with cranberry proanthocyanidins induced negligible effect on cell morphology .
The action of our cranberry extract on cell wall biosynthesis was also confirmed by macromolecular biosynthesis assays in which incorporation D-Ala into cell wall materials was most affected. Hence, it was therefore tempting to conduct synergy assays with other cell-wall active antibiotics. Because β-lactams are widely used in human and veterinary medicine and because their activity is often impaired by bacterial mechanisms of resistance such as the presence of β-lactamases or the mecA gene in MRSA strains, we estimated that such a class of antibiotic compounds may greatly benefit from the action of cranberry in a variety of clinical applications. Our results showed that the combination of FC111 and amoxicillin or oxacillin used at sub-MICs, is highly bactericidal against either β-lactam-susceptible or -resistant MRSA strains. Moreover, this synergy was proven effective in a murine model of S. aureus-induced mastitis.
Resistance is part of the microbial evolution process and the use of antibiotics in human or animals applies a selective pressure favoring resistance to emerge. We did indeed report the presence of multi-antibiotic-resistant bacteria in animal food production [45, 46]. In order to reduce antibiotic use, which could help break down emergence of antibiotic resistance in conventional and organic animal production, several alternatives to antibiotics have been investigated although none of these alternatives was proven to be as efficient as antibiotics [47, 48]. We previously suggested that cranberry fruit derivatives could be developed to improve health and on-farm food safety while reducing the use of antibiotics as growth promoters . It was recently suggested that fruit pomace can be utilized as a good source of inexpensive antioxidants for improving human health and reducing the risks of some chronic diseases . Recently, we showed that our FC111 fraction from cranberry pomace (press cake) is an excellent natural polyphenolics product with increased antioxidant and vasorelaxant benefits . In the present study, we demonstrated that this FC111 fraction may be developed as a viable alternative to traditional antibiotics or at least to significantly potentiate their activities against multidrug resistant bacteria such as MRSA, and thus, to significantly reduce the amounts of antibiotics required.
The cranberry fruit is rich in polyphenols, specifically anthocyanins, flavonols and flavan3-ols, which often form oligomeric and polymeric structures, along with lesser amounts of benzoic acid derivatives. These natural compounds in cranberry are often collectively referred to as tannins, which are found in abundance among many plants including berries, leaves, bark and roots. Tannins have many biological activities and flavonoids, including anthocyanins and proanthocyanidins, are believed to be the major antimicrobial components . Here, the relative abundances of constituents from fraction FC111 are described in Table 3. The primary anthocyanins present were peonidin-3-O-galactoside, cyanidin-3-O-galactoside and peonidin-3-O-arabinoside. Malvidin-3-O-galactoside and 3-O-arabinoside were also present, consistent with previously reported literature for both fresh cranberry fruit and juice extracts [33, 34]. The dominant flavonol was quercetin-3-O-galactoside (peak 32, 50% abundance), followed by three different quercetin-3-O-pentosides (peaks 34, 35, 36), totaling 25% abundance). While commercial standards were not available for the absolute confirmation of these pentosides, they have been tentatively identified as quercetin-3-O-xyloside, quercetin-3-O-arabinofuranoside, and querecetin-3-O-arabinopyranoside, as previously reported by Borges et al. . Additionally, numerous early eluting peaks (8–16 min) were detected, with absorption wavelength maxima characteristic of benzoic acids (λmax ~280 nm) and hydroxycinnamic acids (λmax ~ 315 nm) (Table 3), also consistent with previously reported literature [33, 34]. Characteristic mass spectra for both parent and fragment ions indicates a number of these simple phenolic acids are linked to hexose sugars, but the absolute identity, galactose versus glucose sugar, or exact O-linkage position could not be determined using HPLC-MS methods alone.
From our analysis of the constituents of laboratory-scale fraction FC111, it is unclear, at this point, whether the observed antibacterial activity stems from iridoids, phenolics or flavonoid components, with further isolation and characterization remaining an active area of research. However, the mechanisms of action of our FC111 fraction seem not to be related to its acidic constituents, which negatively impact bacterial proteins and DNA/RNA syntheses . Bactericidal activity of lab-prepared cranberry proanthocyanidins has been reported against MRSA . Proanthocyanidin peaks could not be conclusively identified, based upon the HPLC-MS-conditions used. It is possible that the ethanolic PC extraction may not have been optimal for the solubilization of proanthocyanidins, which are generally extracted with more apolar solvents such as acetone. Given their low (essentially non-abundance) in the extracts studied herein, proanthocyanidins are not expected to play a major role in the observed biological effects, unless extraordinarily active at minute concentrations. Demonstrating novel antibacterial activity from cranberries not associated with proanthocyanidin components is especially exciting, and warrants further investigation.