Microbes, including bacteria, viruses and protists, reside both on the surface and deep within numerous sites in the human body. It is estimated that trillions of microorganisms inhabit the average healthy human and that microbial cell counts in and on the human body outnumber the human cells by a factor of 10 [1, 2]. Studies confirm that humans live in a symbiosis with most of these microbes, whose roles span from harmless to important to life and health [1, 3, 4]. However, microorganisms can also be detrimental to their host and cause diseases such as digestive disorders, obesity, skin diseases, oral disease, bacterial vaginosis (BV), sexual transmitted diseases and urinary tract infections (UTI) [2, 5–9].
Urine within the urinary tract has in general been considered sterile [10, 11], based upon a lack of culturable microbial cells present in urine specimens obtained by the clean-catch method and by catheterization [12–15]. Confirmation of a UTI relies on demonstrating significant bacteriuria (or funguria) in a voided midstream urine sample. Traditionally, 105 colony-forming units per ml (CFU/ml) is the threshold for defining a positive (significant) culture result [16, 17]. Conventional culturing techniques favor the fast growing and modest bacteria, whereas fastidious bacteria can evade the standard culture conditions . The presence of intracellular bacteria in uroepithelial cells , and even biofilm formation in the urinary tract has been suggested [20, 21]. Investigation of healthy urine specimens has demonstrated the presence of non-culturable bacterial cells . These findings stress that bacteria present in urine specimens can escape detection by culture-dependent methods, and that the current view of bacterial diversity in urine thus may be incomplete. This leaves a cryptic fraction of bacteria that may be explored by other means.
Culture-independent, 16S ribosomal DNA (rDNA) sequencing has been widely utilized in the past two decades to study bacterial diversity from various habitats since sequencing of PCR-amplified 16S rDNA overcomes the limitations of culture-based bacterial detection . 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 pyrosequencing as developed by 454 Life Sciences , allow for extensive characterization of microbial populations in a high throughput 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.