Urinary tract infections (UTI) are the most common bacterial infection in humans, and have a higher rate

of incidence in women than men . Between 40-50% of women will experience at least one UTI, and 1 in 3 will have a UTI by the age of 24 (Nicolle, 2008). Many women also experience chronic UTIs throughout their lifetime. Though prompt treatment of UTIs can lead to rapid recovery with no lasting damage, in certain populations, e.g. children and pregnant women, there can be devastating long-term effects. The majority of UTIs have been attributed to infections by E. Coli, but other micro-organisms have been implicated in the disease (Nicolle, 2008; Ulett et al., 2009). Traditionally, urine samples from UTI patients have been cultured in the laboratory to determine the underlying pathogen. A recent study demonstrated that culturing alone misses many bacteria present in urine, and may not provide a complete picture of the infection (Imirzalioglu, Hain, Chakraborty, & Domann, 2008). Moreover, as bacterial resistance to antibiotics increases, knowing the full extent and true nature of UTI pathogens will be critical to prescribing effective treatments (Gupta, Sahm, Mayfield, & Stamm, 2001). Culture-independent microbial community profiling has traditionally been challenging due to technical limitations. Methods such as pulsed field gel-electrophoresis, DGGE and terminal restriction fragment length polymorphism profiling are time-consuming and expensive, making them impractical for clinical use on a large number of samples. Advances in molecular techniques and the advent of high-throughput sequencing technologies has allowed for rapid, inexpensive characterization of environmental microbial communities from a full spectrum of environments including the ocean, soil, coral reefs, and the human gut. High-throughput sequencing has also been used to determine causative agents of disease in outbreak situations in humans and other animals, as well as plants. In this study, I propose to use a high-throughput approach which allows for rapid large-scale characterization of bacteria in the urine of individuals with and without UTIs. The first phase of the study will characterize bacterial communities in infected urine samples, and samples from healthy individuals (controls) catheterized for other conditions. DNA will be isolated from 120 samples, and the 16S rRNA gene will be specifically amplified and sequenced using 454 pyrosequencing. This will provide approximately 4,000 sequences per sample. The 16S gene is carried universally by bacteria, and allows for discrimination down to the genus and in some case species level. By comparing communities in affected and non-affected individuals, I will be able to identify bacterial populations uniquely associated with or differentially abundant in urinary tract infections. The community profiling approach will not only identify known pathogens, but also other members of the bacterial consortium, and even new bacterial species. The approach will also provide information on approximately how abundant each type of bacteria is in infected urine. In many diseases, changes in abundance of the constituents of the microbial community can be more representative of the disease state than the presence or absence of specific community members. These results will be compared to microbiological cultures obtained in the lab to determine how efficacious culturing is as a diagnostic technique. The second phase of this study will focus more specifically on microbes identified as possible pathogens by community profiling. By amplifying genes which evolve more rapidly than the 16S rRNA, bacteria in urine samples can be typed down to the strain level using the high-throughput pyrosequencing approach. In this way, it can be determined if infections are due to one strain of a bacterium (a true monoculture) or the activities of many strains. Previous studies have implicated specific virulence factors in the ability of bacteria to cause UTI (Allsopp et al., 2010; Watts et al., 2010). I will also be able to screen for these virulence determinants using the same approach. The proposed research is the only study of its kind, and will provide valuable clnical information for the prophylaxis and treatment of UTIs, with particular relevance to women's health. Additionally, the results will have a broader impact in the scope of the NIH Human Microbiome Project, which identified the urogenital tract as a key body area for microbial profiling. At the University of Queensland, I will have the opportunity to work under the guidance of Dr. Phil

Hugenholtz who pioneered the field of microbial community profiling, and is considered an international expert in the field. Dr. Mark Schembri, who has an extensive track record in UTI research and genetic studies of uropathogenic bacteria, will also be a collaborator in the study. Samples will be obtained through an existing collaboration with the Royal Women's Hospital of Brisbane, and laboratory work and sequencing will be carried out under the auspices of the newly founded Australian Centre for Ecogenomics, which has its own 454 pyrosequencing facility. The major financial burden of this project lies in sequencing, however, as the sequencing can be done in house the cost is dramatically reduced, which would allow for many more samples to be sequenced. As this project represents a joint effort between research scientists and physicians, the results will be presented directly to clinicians, who can work collaboratively with us to determine the clinical applications and implications of our results. Allsopp, L. P., Totsika, M., Tree, J. J., Ulett, G. C., Mabbett, A. N., Wells, T. J., Kobe, B., et al. (2010). UpaH is a newly identified autotransporter protein that contributes to biofilm formation and bladder colonization by uropathogenic Escherichia coli CFT073. Infection and Immunity, 78(4), 1659-1669. doi:10.1128/IAI.01010-09 Gupta, K., Sahm, D. F., Mayfield, D., & Stamm, W. E. (2001). Antimicrobial resistance among uropathogens that cause community-acquired urinary tract infections in women: a nationwide analysis. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, 33(1), 89-94. doi:10.1086/320880 Imirzalioglu, C., Hain, T., Chakraborty, T., & Domann, E. (2008). Hidden pathogens uncovered: metagenomic analysis of urinary tract infections. Andrologia, 40(2), 66-71. doi:10.1111/j.14390272.2007.00830.x Nicolle, L. E. (2008). Uncomplicated urinary tract infection in adults including uncomplicated pyelonephritis. The Urologic Clinics of North America, 35(1), 1-12, v. doi:10.1016/j.ucl.2007.09.004 Ulett, K. B., et al. (2009). Journal of Clinical Microbiology, 47(7), 2055-2060. doi:10.1128/JCM.00154-09 Watts, R. E., , et al. (2010). Journal of Clinical Microbiology, 48(7), 2449-2458. doi:10.1128/JCM.01611-09