Impact of cranberry juice consumption on gut and vaginal microbiota in postmenopausal women

Cranberries have long been purported to provide protection against urinary tract infections.Thereisalineofevidencesuggestingthatcausalpathogensmightbeseeded from the bacteria reservoirs in the intestinal and vaginal tracts. We tested the hypoth-esis whether cranberry intake would reshape bacteria taxa in the gut, as well as the vaginal ecosystem. A total of 25 postmenopausal women were enrolled into a randomized, double-blind, placebo-controlled study. Stool samples and vaginal swabs were collected at baseline and after 15 days of consumption of placebo or cranberry beverages, microbiota analyses were performed by Illumina Miseq sequencing following a double-index 16S rRNA gene amplicon. All baseline stool samples generally fell in the Bacteroides enterotype. Significant increases of Prevotella ( P = 0.04), Clostridium XIVa members ( P = 0.04), Eggerthella ( P = 0.03), and Bifidobacterium ( P = 0.02) were shown following the cranberry juice intervention; this indicates modulation of the gut microbiotabycranberrycomponents.Baselinevaginalmicrobiotasfellinthreedistinct patterns— Lactobacillus dominant, diversified microbiome, and Streptococcus dysbiosis. Compared with the placebo, the cranberry intervention significantly reduced the abundance of pathogenic Streptococcus ( P = 0.04) in the dysbiosis group and increased commensal bacteria Anaerococcus , Finegoldia , Actinomyces , and Corynebacterium in the diversified microbiome and dysbiosis groups. Overall, these data suggest that cranberry consumption may improve vaginal microbiota composition in individuals with dysbiosis. Gut-borne taxa stimulation by the combination of cranberry oligosac-charides and polyphenols present in the cranberry product potentially mediates these beneficial properties.

millions of visits yearly and an estimated cost of $2.8 billion in medical care expenses in the United States (Simmering et al., 2017). In addition, the need for antibiotic treatment is problematic both for the patient's natural microbiota stability and for the community and environment, because antibiotic resistance can result from such treatments (Ianiro et al., 2016;Koutzoumis et al., 2020;Morgun et al., 2015;Stapleton, 2016).
Although pathogenic bacteria, most often Escherichia coli and Streptococcus, are the main UTI causative bacteria, it has been proposed that a reduction in the abundance of commensal vaginal microbes may also contribute to an increased UTI risk in postmenopausal women (Reid, 2018;Stapleton, 2016). It is now well understood that the normal vaginal microbiome is dominated by Lactobacilli, and the vaginal tract is naturally populated with a relatively dense and diverse collection of bacteria (Mendling, 2016;Ravel et al., 2011;White et al., 2011;Witkin & Linhares, 2017). An imbalance of the vaginal microbiome is not only a common cause for bacterial vaginosis (BV) (Stapleton, 2016;White et al., 2011) but may directly affect the potential pathogens to colonize the urethra and/or bladder (Czaja et al., 2009;Stapleton, 2016). Thus, it would appear that asymptomatic changes in the vaginal microbiome may trigger UTI development, either by seeding pathogens or by creating a niche for pathogens to thrive (Czaja et al., 2009;Stapleton, 2016).
The factors shaping the vaginal microbiota composition and stability are still not well known (Braundmeier et al., 2015). In this context, we hypothesize that the vaginal microbiome is strongly influenced by the gut microbiome. There are different lines of evidence to support this hypothesis. First, because of the anatomical proximity, gut microbes in the rectum and distal colon can easily come in contact with the vaginal tract, and indeed several vaginal Lactobacillus species are also found in the digestive tract (Marchesi et al., 2020;Pan et al., 2020).
Second, it has become increasingly evident that metabolites produced by the gut microbiome are highly bioavailable and distributed to any organ/region of the human body and these metabolites may influence the vaginal ecosystem as well (Possemiers et al., 2011). Because the gut microbiome is strongly individualized, shaped by external factors, and naturally evolving to some extent, this would in part explain why the underlying causes of UTI and BV are often challenging to identify. It is plausible that the different responses to cranberry intervention might be the consequence of interindividual differences in gut microbiome composition. During the last decade, reports of potential positive modulation of the human gut microbiota by berries have emerged (Cai et al., 2019;Lavefve et al., 2020;O'Connor et al., 2019;Rodríguez-Morató et al., 2018). Polymeric cranberry phenolic compounds generally have poor bioavailability. However, when they reach the distal intestine, those molecules are broken down and metabolized by microbiota under anaerobic conditions, a process known as fermentation. Compared to their parent compounds, these bacteria-derived metabolites are highly bioavailable and bioactive. Previous studies showed that γ-valerolactone and its sulfate conjugates (Mena et al., 2017) phenylacetic acid, 3,4-dihydroxphenylacetic acid (González de Llano et al., 2015), and hippuric and α-hydroxyhippuric acids (González de Llano et al., 2019) have antibacteria effects under experimental conditions. These findings highlight the critical need to understand how cranberries could affect the possible crosstalk between intestinal and other microbiome ecosystems, thereby modulating the impact of dietary cranberries on UTI prevention. This project aimed to assessing the relationships between the gut microbiome and vaginal microbiome, and how cranberry consumption affecting the gut microbiome may also impact the vaginal microbiome. It is expected that in the long term, nutritional studies using other dietary elements (such as prebiotics) may be designed to optimize and maximize the beneficial effects of cranberries.

Subjects
Twenty-three women volunteered and participated in this study. The eligibility (no menstrual period for at least 12 months) of the participants was determined using a questionnaire. The participants were postmenopausal women between the ages of 50 and 75 years old who met the inclusion criteria including no recent antibiotics or immunosuppressive therapies; surgery of the stomach or small or large intestines; appendectomy, gastric bypass, or gastric banding in the previous 6 months; and diagnosis with any autoimmune diseases.

Study design and sample collection
This study was a randomized, placebo-controlled, crossover, dietary intervention. Ocean Spray Cranberries, Inc. provided the experimental and placebo beverages, which have been validated and used in previous studies (Hsia et al., 2020;Liu et al., 2019;Maki et al., 2016;Zhao et al., 2020). Both beverages were similar in appearance, taste, and aroma and assigned to volunteers according to computer-generated random orders. Both investigators and participants were blind to the assignment and the products. The products were identified by a random three-digit code preprinted on the cap. The participants consumed either cranberry juice or the placebo beverage daily (8 fl oz per day) for 15 days (Table 1). Analytical methods for testing the products were previously described (Maki et al., 2016). Biological samples were collected

Libraries preparation and sequencing
For 16S rRNA gene sequencing analyses, a dual-indexed amplicon strategy was performed using universal primers tagged with Illumina adapters and barcodes as described before (Aljahdali et al., 2017;Kozich et al., 2013;Mayta-Apaza et al., 2018

Statistical analyses
In this study, the Mann-Whitney U test was performed to detect significant differences between groups (by convention, differences were considered significant when P < 0.05). Nonmetric multidimensional scaling and analysis of similarities (ANOSIM) based on count distance metrics (Bray-Curtis similarity index; ANOSIM: P < 0.05) were obtained with Past4 software (Al Othaim et al., 2020;Hammer et al., 2001).

RESULTS AND DISCUSSION
The human study was completed with 23 volunteers, and 21 of which provided the full set of samples. The volunteers' demographics are described in Table 2.

Impact of the cranberry juice on the gut microbiota
Cranberry juice significantly increased the numbers of Bifidobacterium (P = 0.02) and Eggerthella (P = 0.03), two Actinobacteria members that have been shown previously to be able to grow and metabolize dietary polyphenols (Fogliano et al., 2011;Koutsos & Lovegrove, 2015;Morais et al., 2016;Queipo-Ortuño et al., 2012). Bifidobacterium are also known as probiotics and the increased abundance is considered a sign of improved intestinal health (Cuervo et al., 2014;Rajkumar et al., 2014;Singh et al., 2017). Cranberry juice also resulted in a significant increase of Prevotella (P = 0.04), which is considered a beneficial genus associated with polysaccharides and fiber consumption (David et (Figure 2). Butyrate is a short-chain fatty acid shown to offer benefits to not only the local intestinal environment, but also contributes to systemic metabolism O'Keefe, 2016). Akkermansia, a genus that has been associated with a number of health properties, was increased with cranberry consumption in animal models (Anhê et al., 2017;Rodríguez-Daza et al., 2020) and to some extent in humans (Bekiares et al., 2018), it showed a slight but nonsignificant increase between cranberry and placebo beverages (Dao et al., 2016;Png et al., 2010) (Figure 2). It should be noted that all those trends were strongly individualized, and further validation is warranted in more clinical studies with a larger sample size. Overall, cranberry consumption appears to stimulate beneficial genera.

Impact of the cranberry juice on the vaginal microbiota
The vaginal microbiotas were found to be extremely heterogeneous at baseline, and have therefore been stratified into three groups based on the most abundant genera and their known relation to vaginal health ( Figure 3).
Group 1 comprises individuals whose baseline vaginal microbiotas were strongly dominated by Lactobacillus, which is considered the ideal healthy state (Ravel et al., 2011). Group 2 comprises individuals whose baseline vaginal microbiotas were characterized by diverse and relatively well-distributed high number of different taxa, without obvious dominant detrimental genus. Group 3 comprises individuals whose baseline vaginal microbiotas were characterized by high abundance of Streptococcus, which is strongly indicative of microbial dysbiosis that could potentially lead to vaginosis or UTI (Leclercq et al., 2016;Tan et al., 2012;Ulett et al., 2009). Individuals were roughly equally distributed among the three groups, which allowed statistical analyses for each group.

3.2.1
Lactobacillus-dominant vaginal microbiota Consumption of both products appeared to have little impact on the vaginal microbiota of volunteers that have Lactobacillus as the major bacterial taxa (Figure 4).

Diverse baseline vaginal microbiota
As expected with the criterion used to segregate the groups, the vaginal microbiota of these subjects was significantly more diverse ( Figure 5). The number of taxa was significantly decreased after the consumption of cranberry juice, and even more with the placebo.
However, the decrease of the Shannon index was only significant in the placebo group. It can be hypothesized that loss of diversity not associated with Lactobacillus dominance may be detrimental to some Anumba, 2020). To conclude, cranberry juice appears to maintain the diversity of vaginal microbiota, which is a presumably healthier profile for these volunteers.
As suggested by the diversity trends, cranberry juice had a very strong and specific impact on the dysbiosed vaginal microbiota (Figure 7). Most notably, although both beverages reduced the abundance of Streptococcus compared to baseline, the consumption of cranberry juice significantly reduced the relative abundance of Streptococcus to a greater extent, compared to the placebo beverage. Intriguingly, it appeared that Streptococcus has been replaced with a range of different Firmicutes (Anaerococcus and Finegoldia) and Actinobacteria (Actinobaculum, Actinomyces, Varibaculum, and Corynebacterium; Figure 8).

CONCLUSION
These findings suggest a beneficial impact of cranberry juice on vaginal microbiota with dysbiosis, and possibly its contribution to vaginal health. It can be speculated that Streptococcus dysbiosis could be improved directly within the local niche environment by increasing competition of other commensal bacteria or indirectly by decreasing the bacteria migration from the gut reservoir, and thereby restoring a healthier vaginal microbiota. Due to that possible relationship, the beneficial impact of cranberry juice on the gut microbiota is also encouraging for vaginal health. As reported previously, the ageing gut microbiota tends to be less diverse and responsive to dietary changes. Herein, a relatively short-term consumption and daily consumption of cranberry juice shows promising prebiotic effect and may help maintain levels of a wide range of beneficial genera (Prevotella, Bifidobacterium, and butyrate-producing Firmicutes).

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.