Urinary tract infections (UTIs) are a widespread problem [1, 2] whose recurrence is common in women, resulting in considerable morbidity, multiple antibiotic treatments and increased expenditures. Recently, uropathogenic Escherichia coli (UPEC), the major pathogen involved in these infections, acquired new resistance mechanisms against β-lactams and fluoroquinolones usually used to treat UTIs, leading to therapeutic deadlock of these frequent infections [3–10]. Therefore, new strategies to prevent or treat UTIs are essential.

Recent evidence suggested that ingestion of cranberries (Vaccinium macrocarpon Ait.) has been used for prevention of UTI [11–13]. A recent systematic review concluded that there is some positive clinical evidence that consumption of cranberry juice can reduce the number of symptomatic UTIs in women over a 12-month period [12]. Research showed that consuming cranberry products may prevent adhesion of E. coli strains to the uroepithelium [14–16], notably multidrug resistant strains [17], thus interfering with this important initial step in the infection process [18]. The proanthocyanidins (PACs) in cranberry, in particular the A-type linkages, have been implicated as important inhibitors of primarily P-fimbriated E. coli adhesion to uroepithelial cells in vitro [13, 19–21] and ex vivo [22, 23].

Only cranberry juice with A-type PACs prevented bacterial adhesion, and molecular weight of PACs could potentially impact the bacterial anti-adhesion activity. Indeed, Foo et al. [20] showed that the active compounds consisted predominantly of epicatechin units with mainly degree of polymerization of 4 and 5 containing at least one A-type linkage. Thus, the active components in cranberry should be type-A oligomeric procyanidins.

Propolis is a resinous material collected by bees from exudates and buds of plants, then mixed with wax and bee enzymes. Propolis's antimicrobial activities are well documented against different bacteria [24]. In vitro propolis may act directly on microorganisms, and in vivo it may stimulate the immune system, activating the killing of bacteria. Propolis may show additional effects with antimicrobial drugs (fluoroquinolones and β-lactams).

The objective of this study was to evaluate the association of proanthocyanidins and propolis concerning bacterial anti-adhesion activity, to evaluate additional dosage regimes and collection time-periods following ingestion of the propolis and PACs-standardized cranberry powder.

The increasing resistance of E. coli, bacteria most frequently isolated during UTI to diverse antibiotics is an international public health problem [7–10]. The very common recurrence of UTI in women requires new antibiotic treatments, promotes the emergence of new mechanisms of resistance, inducing a vicious circle. Finding new ways to prevent and/or treat these infections is essential. A number of strategies minimizing antibiotic exposure have been used attempting to prevent recurrent UTI. This study shows the effect of propolis and cranberry (Vaccinium macrocarpon) in ex vivo and in vivo anti-adhesion assays.

When analysing different results obtained after cranberry intake by our team [17, 23], we established that the minimal PACs dose with an observed anti-adhesion effect is 36 mg. Moreover, we could see an anti-adherent effect dose and time-dependent inversely proportional to the PACs dose. This study shows that the dose-dependent and time-dependent effects are also observed when propolis was added to PACs (Table 1).

Moreover, an additional effect of propolis with PACs was clearly noted. At 60 mg PACs and 170 mg of propolis, no supplementary effect was observed on AI compared to the consumption of 60 mg PACs alone. However, with 192 mg of propolis, a significant anti-adhesion effect is observed at 12 h (p < 0.001) for the same PACs content. With 120 mg of PACs and 340 mg of propolis a nycthemeral effect was clearly noted: the AI observed at 24 h is significantly higher than the AI detected for 144 mg PACs alone (p < 0.001). An additional anti-adherence action of PACs plus propolis is observed, especially since the amount of absorbed PACs is important.

We could speculate that in addition to the anti-adhesion activity of PACs, propolis killed the bacteria, decreasing the number of adherent bacteria and facilitating the activity of PACs. The PACs contained in cranberry inhibit E. coli adhesins notably against P-fimbriae [15, 20]. Adhesion is the first step of bacterial virulence, ensuring survival and establishing replication. To demonstrate the effect of PACs on bacterial virulence, we used an in vivo killing nematode model validated by different teams [17, 26, 27]. This nematode lives in soil and eats environmental bacteria. It is a simple and easy to handle model that can be used to evaluate the virulence of bacterial pathogens. It is increasingly being used to study host-pathogen interactions and has helped identify basic evolutionarily conserved pathways associated with microbial pathogenesis. In particular, this has revealed important factors of the host response with remarkable parallels in higher organisms. This organism's short (2-3-weeks) life span facilitates host-bacteria interaction analysis, offering an ideal compromise between complexity and tractability. We determined that the reduced ability of E. coli strain to kill worms correlates with the consumption of cranberry plus propolis capsules. Bacteria grown in the urine of individuals consuming cranberry capsules was not able to adhere to worms, thus leading to a reduced killing of the worms despite the presence and proliferation of E. coli strain in the C. elegans intestine. E. coli were localized within the nematode gut, but could not exert any virulent effect due to an inability to establish infection and loss of virulence. In our in vivo experiment we again demonstrated the additional action of PACs and propolis: a significant decrease of LT50 with E. coli grown in urine samples collected after the intake of 120 mg PACs and 340 mg of propolis observed after 24 h compared to all other regimens over the same time, even for 144 mg PACs.

In our study we evaluated the presence of the main propolis flavonoids (galangin and pinocembrin) and PACs in capsules and urine samples by HPLC-PDA-MS. Currently there is no universally accepted standard method for quantification of cranberry PACs in products [28]. Quantification of PACs using colorimetric methods (DMAC reagent, European Phamacopeae, Bate-Smith, vanillin reagent) can lead to erroneous and unreproducible results, absolutely not specific to the type of linkage of proanthocyanidins units (A-or B-type). Since many of these methods were used to determine the total PACs content in the capsules used in the published clinical studies, little is known about the specific A-type PACs content. In the present study, we analysed the metabolites of the administrated cranberry and propolis in urine before and after treatment with β-glucuronidase/sulfatase. No PACs were detected. This result, in agreement with literature [29], lacks evidence that PACs may be biotransformed to unique biologically active urinary metabolites, or even reach the urine intact as dimers or trimers. Indeed, little is known about the metabolic route of A-type PACs in humans, especially if they are degraded in the gut, and/or not assimilated in any quantity, and do not reach the urinary tract intact. Because the putative active proanthocyanidins are possibly not absorbed and/or may be metabolized before reaching the urinary tract, it is probable that other cranberry compounds or their metabolites are active. Conversely, we demonstrated the presence of metabolites of propolis in urine samples collected at various times after administration of capsules containing cranberry and propolis. Pinocembrin and galangin were present in urine, which suggests that these two compounds are absorbed in the blood and excreted as glucuronide and/or sulfo-conjugates. Their high concentrations in urine collected 24 h after capsule administration could be responsible for the observed effects. The antibacterial activities of these two metabolites are part of our ongoing research. A recent study showed that galangin isolated from Alpinia officinarum Hance have the potential to reverse bacterial resistance to β-lactam antibiotics against penicillin-resistant Staphylococcus aureus[30].

The main limitation of this study is the use of ex vivo experiments to observe the effect. Further human trials are needed to correlate the level of ex vivo anti-adhesion activity with the prevention of clinical UTI.

Administration of PACs-standardized cranberry plus propolis powder could offer a protection against bacterial adhesion in the urinary tract and could represent an additional means to help prevent infections.