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Aerococcus Urinae Isolated from Women with Lower Urinary Tract Symptoms: In Vitro Aggregation and Genome Analysis - Beyond the Abstract

Since its first classification, Aerococcus urinae has emerged as a human pathogen capable of causing urinary tract infections (UTI), as well as more invasive infections, including bacteremia,1,2 endocarditis,3,4 septicemia 5 and necrotizing soft tissue infections.6-8 We became particularly interested in A. urinae as a member of the urinary microbiota—the polymicrobial collection of bacteria detected in urine. We detected A. urinae using Next Generation Sequencing technology and an expanded urine culture method called EQUC. By detecting fastidious or lower abundance organisms, these methods considerably outperform the standard clinical microbiology urine culture method.9,10 In the past, we have detected A. urinae more frequently in urine collected from women with urgency urinary incontinence (UUI) and UTI compared to healthy controls.10,11 In fact, in all our studies combined (844 women), we have cultured A. urinae from an asymptomatic woman only twice. Consistent with our studies, Coorevits et al. never detected A. urinae from urines from asymptomatic controls (86 females, 15 males) using their own expanded urine culture technique.12

Prior to our study,13 in which we sequenced the genomes of 24 urinary isolates of A. urinae, five A. urinae genome sequences were available in GenBank®, all from A. urinae clinical isolates from cases of UTI or bacteremia. Another 63 genome sequences have been reported but not deposited in GenBank®. Carkaci et al. (2017)14 reported the genomic characterization of 40 A. urinae strains isolated from men and women experiencing episodes of UTI, bacteremia or infective endocarditis, and Senneby et al. (2019)15 sequenced an additional 23 A. urinae blood isolates. Our study included one additional A. urinae UTI isolate, two isolates from asymptomatic controls and the remainder came from women with lower urinary tract symptoms (UUI, overactive bladder, or stress urinary incontinence). We expect that the number of A. urinae genomes available from a wide range of diseases from around the globe will increase, permitting comparative genomics to unravel the relationship between genotype and virulence potential.

In addition to genome sequencing, we also examined the ability of A. urinae strains to display two in vitro autoaggregative phenotypes. Autoaggregation is often the first step in biofilm formation.16 A previous study showed that A. urinae forms biofilms on plastic in vitro.17 Recent studies detected A. urinae biofilms in heart valve tissue from patients with infective endocarditis patients18 and on the surface of urinary catheters,19 suggesting that A. urinae biofilms are important during human infections. We are very interested in understanding whether the aggregative phenotypes we observe in vitro are related to the virulence potential of each A. urinae strain. Is the self-aggregation phenotype a proxy for the ability of a strain to adhere to the epithelium, to form biofilms on abiotic surfaces such as catheters, to aggregate with other members of the urinary microbiome, and/or to survive in the bloodstream? Currently, the lack of a reverse genetic system hampers our ability to directly test the autoaggregation role of candidate genes, such as those encoding the recently described ASP proteins that are abundant on the A. urinae cell surface.15 However, the observation that the flocking phenotype of certain A. urinae strains could be manipulated by growth in different media conditions creates a kind of inducible system for investigating the association between aggregation and other phenotypes of interest. Ultimately, the development of both reverse genetic systems in A. urinae and small animal models for investigating pathogenesis in the urinary tract and bloodstream are key for continuing to move our understanding of this emerging pathogen forward.

Written by: Evann E Hilt, Catherine Putonti, Krystal Thomas-White, Amanda L Lewis, Karen L Visick, Nicole M Gilbert, Alan J Wolfe

Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA., Center for Women's Infectious Disease Research, Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO USA., Center for Women's Infectious Disease Research, Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO USA ., Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA .

References:

  1. Senneby, Erik, L. Göransson, S. Weiber, and Magnus Rasmussen. "A population-based study of aerococcal bacteraemia in the MALDI-TOF MS-era." European Journal of Clinical Microbiology & Infectious Diseases 35, no. 5 (2016): 755-762.
  2. Colakoglu, S., T. Turunc, M. Taskoparan, H. Aliskan, E. Kizilkilic, Y. Z. Demiroglu, and H. Arslan. "Three cases of serious infection caused by Aerococcus urinae: a patient with spontaneous bacterial peritonitis and two patients with bacteremia." Infection 36, no. 3 (2008): 288.
  3. Kristensen, B., and G. Nielsen. "Endocarditis caused byAerococcus urinae, a newly recognized pathogen." European Journal of Clinical Microbiology and Infectious Diseases 14, no. 1 (1995): 49-51.
  4. Skov, R. L., M. Klarlund, and S. Thorsen. "Fatal endocarditis due to Aerococcus urinae." Diagnostic microbiology and infectious disease 21, no. 4 (1995): 219-221.
  5. Heilesen, Aina Møller. "Septicaemia due to Aerococcus urinae." Scandinavian journal of infectious diseases 26, no. 6 (1994): 759-760.
  6. Schuur, P. M. H., L. Sabbe, A. J. Van Der Wouw, G. J. Montagne, and A. G. M. Buiting. "Three cases of serious infection caused by Aerococcus urinae." European Journal of Clinical Microbiology and Infectious Diseases 18, no. 5 (1999): 368-371.
  7. Senneby, E., A. C. Petersson, and Magnus Rasmussen. "Clinical and microbiological features of bacteraemia with Aerococcus urinae." Clinical Microbiology and Infection 18, no. 6 (2012): 546-550.
  8. Forsvall, Andreas, Magnus Wagenius, and Magnus Rasmussen. "Perigenital necrotizing soft tissue infection caused by Aerococcus urinae." IDCases 18 (2019): e00590.
  9. Hilt, Evann E., Kathleen McKinley, Meghan M. Pearce, Amy B. Rosenfeld, Michael J. Zilliox, Elizabeth R. Mueller, Linda Brubaker, Xiaowu Gai, Alan J. Wolfe, and Paul C. Schreckenberger. "Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder." Journal of clinical microbiology 52, no. 3 (2014): 871-876.
  10. Pearce, Meghan M., Evann E. Hilt, Amy B. Rosenfeld, Michael J. Zilliox, Krystal Thomas-White, Cynthia Fok, Stephanie Kliethermes et al. "The female urinary microbiome: a comparison of women with and without urgency urinary incontinence." MBio 5, no. 4 (2014): e01283-14.
  11. Price, Travis K., Tanaka Dune, Evann E. Hilt, Krystal J. Thomas-White, Stephanie Kliethermes, Cynthia Brincat, Linda Brubaker, Alan J. Wolfe, Elizabeth R. Mueller, and Paul C. Schreckenberger. "The clinical urine culture: enhanced techniques improve detection of clinically relevant microorganisms." Journal of clinical microbiology 54, no. 5 (2016): 1216-1222.
  12. Coorevits, Liselotte, Stefan Heytens, Jerina Boelens, and Geert Claeys. "The resident microflora of voided midstream urine of healthy controls: standard versus expanded urine culture protocols." European Journal of Clinical Microbiology & Infectious Diseases 36, no. 4 (2017): 635-639.
  13. Hilt, Evann E., Catherine Putonti, Krystal Thomas-White, Amanda L. Lewis, Karen L. Visick, Nicole M. Gilbert, and Alan J. Wolfe. "Aerococcus urinae isolated from women with lower urinary tract symptoms: in vitro aggregation and genome analysis." Journal of Bacteriology (2020).
  14. Carkaci, Derya, Katrine Højholt, Xiaohui Chen Nielsen, Rimtas Dargis, Simon Rasmussen, Ole Skovgaard, Kurt Fuursted, Paal Skytt Andersen, Marc Stegger, and Jens Jørgen Christensen. "Genomic characterization, phylogenetic analysis, and identification of virulence factors in Aerococcus sanguinicola and Aerococcus urinae strains isolated from infection episodes." Microbial pathogenesis 112 (2017): 327-340.
  15. Senneby, Erik, Torgny Sunnerhagen, Björn Hallström, Rolf Lood, Johan Malmström, Christofer Karlsson, and Magnus Rasmussen. "Identification of two abundant Aerococcus urinae cell wall-anchored proteins." International Journal of Medical Microbiology 309, no. 7 (2019): 151325.
  16. Trunk, Thomas, Hawzeen S. Khalil, and Jack C. Leo. "Bacterial autoaggregation." AIMS microbiology 4, no. 1 (2018): 140.
  17. Shannon, Oonagh, Matthias Mörgelin, and Magnus Rasmussen. "Platelet activation and biofilm formation by Aerococcus urinae, an endocarditis-causing pathogen." Infection and immunity 78, no. 10 (2010): 4268-4275.
  18. Yaban, Berrin, Judith Kikhney, Michele Musci, Annett Petrich, Julia Schmidt, Maria Hajduczenia, Felix Schoenrath, Volkmar Falk, and Annette Moter. "Aerococcus urinae–A potent biofilm builder in endocarditis." Plos one 15, no. 4 (2020): e0231827.
  19. Yu, Yanbao, Tamara Tsitrin, Shiferaw Bekele, Vishal Thovarai, Manolito G. Torralba, Harinder Singh, Randall Wolcott et al. "Aerococcus urinae and Globicatella sanguinis Persist in Polymicrobial Urethral Catheter Biofilms Examined in Longitudinal Profiles at the Proteomic Level." Biochemistry insights 12 (2019): 1178626419875089.
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