However, often the search for microbial agents is performed only after a disease state has been diagnosed. Only a few investigations including urine from healthy persons using 16S rDNA PCR have been reported [12, 24–26]. These studies

had a variable success rate in actually obtaining sequences, resulting in a limited overview of the healthy urine bacterial flora. However, two recent 16S rDNA studies by Nelson et al. (2010) and Dong et al. (2011) [27, 28] have shown that the male urine contains multiple bacterial genera. Advances in sequencing technology, such as massively parallel EGFR targets pyrosequencing as developed by 454 Life Sciences [29], allow for extensive characterization of microbial populations in a high throughput X-396 mouse and cost effective manner [30, 31]. Amplicons of partial 16S

rRNA genes are sequenced on microscopic beads placed separately in picoliter-sized wells, bypassing previously needed cloning and cultivation procedures. Such sequencing has revealed an unexpectedly high diversity within various human-associated microbial communities, e.g. oral-, vaginal-, intestinal- and male first catch urine microbiota [4, 28, 32, 33], but female urine microbial diversity has so far not been studied using high throughput sequencing (HTS) methods. Here, we have investigated the bacterial diversity in urine microbiota from healthy females by means of 16S rDNA amplicon 454 pyrosequencing. This study demonstrates the use of this methodology for investigating bacterial sequence diversity in female urine samples. Our results indicate a diverse spectrum of bacterial profiles associated with healthy, culture negative female urine and provide a resource for further studies in the field of molecular diagnostics of urine specimens. Methods Urine sampling Urine was collected by the clean catch method 6-phosphogluconolactonase in which healthy adult female volunteers (n = 8),

collected midstream urine into a sterile container. Specimens were initially kept at 4°C, and within an hour transported to the laboratory for DNA isolation. All specimens were culture negative, as tested by the Urological Clinic at the University Hospital HF Aker-Oslo. Samples were taken with informed consent and the study was approved by the Regional Committee for Medical Research Ethics East-Norway (REK Øst Prosjekt 110-08141c 1.2008.367). DNA isolation 30 ml urine volume was pelleted by centrifugation at 14000 RCF for 10 min at 4°C. 25 ml of the supernatant was decanted and the pellet was resuspended in the remaining volume. 5 ml of the sample was again pelleted by centrifugation for 10 min at 16000 × g (4°C). The pellet and some supernatant (up to 100 μl) were processed further. DNA was isolated from the urine pellets with DNeasy Blood & Tissue kit (QIAGEN, Germany), following the tissue spin-column protocol with minor modifications.