JultikaUniversity of Oulu repository

Abstract

Cranberry, Vaccinium oxycoccos, and lingonberry, Vaccinium vitis-idaea, have long been known to provide many health benefits as a source of nutrition. Similar to other berries, these traditional herbs contain flavonoids and vitamins, but also have a special benefit to prevent urinary tract infection (UTI). Previous studies have shown that A-type proanthocyanins (PAC) of cranberries may influence the adhesion of bacteria causing urinary tract infection. UTI pathogens originate from the gut and earlier studies have shown that there is a connection between urinary tract and gut microbiome UTI causing pathogens. However, the cranberry mechanism of action on the gut and urinary tract microbiome is not yet elucidated.

This study aimed to investigate the Cranberry-Lingonberry juice (CLJ) effect on the gut and urinary tract bacterial communities. The hypothesis assumed that the metabolism of proanthocyanidins in the gut alters bacterial communities and reduces the amount of E. coli and possibly other proteobacteria in the urine. The research was done by examining urine and fecal samples from children with urinary tract infection for three (urine) to twelve (fecal samples) months. The samples were collected by Oulu University Hospital Child Health and Maternity Clinic from 77 patients who drank CLJ or flavonoid-free control juice in randomized trial. Total of 206 samples, including 40 urine and 166 fecal samples, were collected for study.

DNA was extracted from samples using two different DNA extraction protocols of QIAGEN, USA and quantified using Nanodrop spectrophotometer. The bacterial 16S rRNA was amplified by using Polymerase chain reaction (PCR), which also attached unique barcodes for each sample. Agarose gel electrophoresis was used to ensure amplification of PCR. All amplified PCR products were prepared for sequencing by Ion Torrent next generation sequencing.

The QIIME 2 next-generation microbiome bioinformatics platform was used to analyze the sequence data and metadata information. Greengenes 16S rRNA, Silva gene databases and Human oral microbiome database (HOMD) were used as alignment reference databases. Metadata information about sample material and collection time was used for grouping. The alpha and beta diversity, as well as differential abundances between treatments, were analyzed using QIIME 2 platform and R-statistical program. Compliance data was used to limit the data to patients who used more than 80% probability of CLJ or control juice in the second round of statistical analysis.

Altogether 183 samples were amplified, of which 150 was fecal and 40 urine, for downstream analysis. From the samples, 18 different phyla and 511 genera were identified, most of them even at species level.

Statistical analysis showed no significant differences in alpha- or beta diversity between CLJ and controls in any groups. Different abundances between treatment groups were found, but in the end none of them were statistically significant. By using HOMD-database, E. coli and other UTI-related species were identified from compliance 80% limited data. Statistical analyses showed a significant decrease of these bacteria in the urinary tract and gut microbiomes of CLJ group patients. In the future, chemical studies about microbial metabolism products could be done from the samples to get a more specific view about CLJ treatment effect on gut and microbial communities, and cranberry juice polyphenols effect on the body.