Novel Techniques to Unravel Causative Bacterial Ecological Shifts in Chronic Urinary Tract Infection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Processing and Sorting into Urinary White Blood Cell and Epithelial Cell Fractions
2.2. PNA FISH and Immunofluorescence
2.3. Bacterial Colony Counts of WBC and EPC Fractions Grown on Chromogenic Agar
2.4. Bacterial DNA Extraction and 16s rRNA Sequencing
2.5. Sequencing Data Processing and Clustering Analysis
2.6. Statistical Analyses
3. Results
3.1. PNA FISH and Confocal Microscopy Showed a Diversity of Bacteria Associated with Urinary White Blood Cells and Epithelial Cells
3.2. The Three Study Groups Were Distinguished by Urinary White Blood Cell Counts and Lower Urinary Tract Symptoms
3.3. Gold Standard Diagnostic Tests for UTI Were Unable to Reliably Distinguish Between the Three Study Groups
3.4. Enriched Urine Culture Following CD45 Sorting into WBC and EPC Fractions Uncovered Bacteria Uniquely Associated with White Blood Cell and Epithelial Cell in All Study Groups
3.5. 16S rRNA Sequencing Showed Lactobacillus as the Predominant Genus Across All Study Groups and Differences Between WBC and EPC Fractions
3.6. Bacterial Signatures in the Urine Were Related to Lower Urinary Tract Symptoms in Chronic UTI
3.7. Clustering Analysis of Mean Rank Changes in Relative Bacterial Abundance Revealed Significant Ecological Shifts Amidst a Stable Bacterial Community Between the Study Groups
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Vos, T.; Lim, S.S.; Abbafati, C.; Abbas, K.M.; Abbasi, M.; Abbasifard, M.; Abbasi-Kangevari, M.; Abbastabar, H.; Abd-Allah, F.; Abdelalim, A.; et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2020, 396, 1204–1222. [Google Scholar] [CrossRef] [PubMed]
- Yang, X.; Chen, H.; Zheng, Y.; Qu, S.; Wang, H.; Yi, F. Disease burden and long-term trends of urinary tract infections: A worldwide report. Front. Public Health 2022, 10, 888205. [Google Scholar] [CrossRef]
- Ikähelmo, R.; Siitonen, A.; Heiskanen, T.; Kärkkäinen, U.; Kuosmanen, P.; Lipponen, P.; Mäkelä, P.H. Recurrence of urinary tract infection in a primary care setting: Analysis of a I-year follow-up of 179 women. Clin. Infect. Dis. 1996, 22, 91–99. [Google Scholar] [CrossRef] [PubMed]
- Swamy, S.; Barcella, W.; De Iorio, M.; Gill, K.; Khasriya, R.; Kupelian, A.S.; Rohn, J.L.; Malone-Lee, J. Recalcitrant chronic bladder pain and recurrent cystitis but negative urinalysis: What should we do? Int. Urogynecol. J. 2018, 29, 1035–1043. [Google Scholar] [CrossRef]
- Ciaccio, L.; Fountain, H.; Beech, E.; Brown, C.S.; Demirjian, A.; Gerver, S.; Muller-Pebody, B.; Bou-Antoun, S. Trends in urine sampling rates of general practice patients with suspected lower urinary tract infections in England, 2015–2022: A population-based study. BMJ Open 2024, 14, e084485. [Google Scholar] [CrossRef] [PubMed]
- Mambatta, A.K.; Jayarajan, J.; Rashme, V.L.; Harini, S.; Menon, S.; Kuppusamy, J. Reliability of dipstick assay in predicting urinary tract infection. J. Fam. Med. Prim. Care 2015, 4, 265–268. [Google Scholar] [CrossRef]
- Sathiananthamoorthy, S.; Malone-Lee, J.; Gill, K.; Tymon, A.; Nguyen Trang, K.; Gurung, S.; Collins, L.; Kupelian Anthony, S.; Swamy, S.; Khasriya, R.; et al. Reassessment of routine midstream culture in diagnosis of urinary tract infection. J. Clin. Microbiol. 2019, 57, e01452-18. [Google Scholar] [CrossRef]
- Chieng, C.C.Y.; Kong, Q.; Liou, N.S.Y.; Khasriya, R.; Horsley, H. The clinical implications of bacterial pathogenesis and mucosal immunity in chronic urinary tract infection. Mucosal Immunol. 2023, 16, 61–71. [Google Scholar] [CrossRef]
- Collins, L.; Sathiananthamoorthy, S.; Rohn, J.; Malone-Lee, J. A revalidation and critique of assumptions about urinary sample collection methods, specimen quality and contamination. Int. Urogynecol. J. 2020, 31, 1255–1262. [Google Scholar] [CrossRef]
- Horsley, H.; Malone-Lee, J.; Holland, D.; Tuz, M.; Hibbert, A.; Kelsey, M.; Kupelian, A.; Rohn, J.L. Enterococcus faecalis subverts and invades the host urothelium in patients with chronic urinary tract infection. PLoS ONE 2013, 8, e83637. [Google Scholar] [CrossRef]
- Lifshitz, E.; Kramer, L. Outpatient urine culture: Does collection technique matter? Arch. Intern. Med. 2000, 160, 2537–2540. [Google Scholar] [CrossRef] [PubMed]
- Choi, H.W.; Bowen, S.E.; Miao, Y.; Chan, C.Y.; Miao, E.A.; Abrink, M.; Moeser, A.J.; Abraham, S.N. Loss of bladder epithelium induced by cytolytic mast cell granules. Immunity 2016, 45, 1258–1269. [Google Scholar] [CrossRef]
- Khasriya, R.; Barcella, W.; De Iorio, M.; Swamy, S.; Gill, K.; Kupelian, A.; Malone-Lee, J. Lower urinary tract symptoms that predict microscopic pyuria. Int. Urogynecol. J. 2018, 29, 1019–1028. [Google Scholar] [CrossRef] [PubMed]
- Kupelian, A.S.; Horsley, H.; Khasriya, R.; Amussah, R.T.; Badiani, R.; Courtney, A.M.; Chandhyoke, N.S.; Riaz, U.; Savlani, K.; Moledina, M.; et al. Discrediting microscopic pyuria and leucocyte esterase as diagnostic surrogates for infection in patients with lower urinary tract symptoms: Results from a clinical and laboratory evaluation. BJU Int. 2013, 112, 231–238. [Google Scholar] [CrossRef] [PubMed]
- Mulvey, M.A.; Schilling, J.D.; Hultgren, S.J. Establishment of a persistent Escherichia coli reservoir during the acute phase of a bladder infection. Infect. Immun. 2001, 69, 4572–4579. [Google Scholar] [CrossRef]
- Mysorekar, I.U.; Mulvey, M.A.; Hultgren, S.J.; Gordon, J.I. Molecular regulation of urothelial renewal and host defenses during infection with uropathogenic Escherichia coli. J. Biol. Chem. 2002, 277, 7412–7419. [Google Scholar] [CrossRef]
- Siddiqui, H.; Nederbragt, A.J.; Lagesen, K.; Jeansson, S.L.; Jakobsen, K.S. Assessing diversity of the female urine microbiota by high throughput sequencing of 16S rDNA amplicons. BMC Microbiol. 2011, 11, 244. [Google Scholar] [CrossRef]
- Wolfe, A.J.; Toh, E.; Shibata, N.; Rong, R.; Kenton, K.; Fitzgerald, M.; Mueller, E.R.; Schreckenberger, P.; Dong, Q.; Nelson, D.E.; et al. Evidence of uncultivated bacteria in the adult female bladder. J. Clin. Microbiol. 2012, 50, 1376–1383. [Google Scholar] [CrossRef]
- Khasriya, R.; Sathiananthamoorthy, S.; Ismail, S.; Kelsey, M.; Wilson, M.; Rohn, J.L.; Malone-Lee, J. Spectrum of bacterial colonization associated with urothelial cells from patients with chronic lower urinary tract symptoms. J. Clin. Microbiol. 2013, 51, 2054–2062. [Google Scholar] [CrossRef]
- Fouts, D.E.; Pieper, R.; Szpakowski, S.; Pohl, H.; Knoblach, S.; Suh, M.J.; Huang, S.T.; Ljungberg, I.; Sprague, B.M.; Lucas, S.K.; et al. Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury. J. Transl. Med. 2012, 10, 174. [Google Scholar] [CrossRef]
- Pearce, M.M.; Hilt, E.E.; Rosenfeld, A.B.; Zilliox, M.J.; Thomas-White, K.; Fok, C.; Kliethermes, S.; Schreckenberger, P.C.; Brubaker, L.; Gai, X.; et al. The female urinary microbiome: A comparison of women with and without urgency urinary incontinence. mBio 2014, 5, e01283-14. [Google Scholar] [CrossRef]
- Hilt, E.E.; McKinley, K.; Pearce, M.M.; Rosenfeld, A.B.; Zilliox, M.J.; Mueller, E.R.; Brubaker, L.; Gai, X.; Wolfe, A.J.; Schreckenberger, P.C. Urine is not sterile: Use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder. J. Clin. Microbiol. 2014, 52, 871–876. [Google Scholar] [CrossRef]
- Du, J.; Khemmani, M.; Halverson, T.; Ene, A.; Limeira, R.; Tinawi, L.; Hochstedler-Kramer, B.R.; Noronha, M.F.; Putonti, C.; Wolfe, A.J. Cataloging the phylogenetic diversity of human bladder bacterial isolates. Genome Biol. 2024, 25, 75. [Google Scholar] [CrossRef]
- Pearce, M.M.; Zilliox, M.J.; Rosenfeld, A.B.; Thomas-White, K.J.; Richter, H.E.; Nager, C.W.; Visco, A.G.; Nygaard, I.E.; Barber, M.D.; Schaffer, J.; et al. The female urinary microbiome in urgency urinary incontinence. Am. J. Obstet. Gynecol. 2015, 213, 347.e1–347.e11. [Google Scholar] [CrossRef]
- Karstens, L.; Asquith, M.; Davin, S.; Stauffer, P.; Fair, D.; Gregory, W.T.; Rosenbaum, J.T.; McWeeney, S.K.; Nardos, R. Does the urinary microbiome play a role in urgency urinary incontinence and its severity? Front. Cell Infect. Microbiol. 2016, 6, 78. [Google Scholar] [CrossRef]
- Thomas-White, K.J.; Kliethermes, S.; Rickey, L.; Lukacz, E.S.; Richter, H.E.; Moalli, P.; Zimmern, P.; Norton, P.; Kusek, J.W.; Wolfe, A.J.; et al. Evaluation of the urinary microbiota of women with uncomplicated stress urinary incontinence. Am. J. Obstet. Gynecol. 2017, 216, 55.e1–55.e16. [Google Scholar] [CrossRef]
- Fok, C.S.; Gao, X.; Lin, H.; Thomas-White, K.J.; Mueller, E.R.; Wolfe, A.J.; Dong, Q.; Brubaker, L. Urinary symptoms are associated with certain urinary microbes in urogynecologic surgical patients. Int. Urogynecol. J. 2018, 29, 1765–1771. [Google Scholar] [CrossRef]
- Price, T.K.; Lin, H.; Gao, X.; Thomas-White, K.J.; Hilt, E.E.; Mueller, E.R.; Wolfe, A.J.; Dong, Q.; Brubaker, L. Bladder bacterial diversity differs in continent and incontinent women: A cross-sectional study. Am. J. Obstet. Gynecol. 2020, 223, 729.e1–729.e10. [Google Scholar] [CrossRef]
- Horwitz, D.; McCue, T.; Mapes, A.C.; Ajami, N.J.; Petrosino, J.F.; Ramig, R.F.; Trautner, B.W. Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference. J. Infect. 2015, 71, 358–367. [Google Scholar] [CrossRef]
- Morand, A.; Cornu, F.; Dufour, J.-C.; Tsimaratos, M.; Lagier, J.-C.; Raoult, D. Human bacterial repertoire of the urinary tract: A potential paradigm shift. J. Clin. Microbiol. 2019, 57, e00675-18. [Google Scholar] [CrossRef]
- Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods 2012, 9, 676–682. [Google Scholar] [CrossRef]
- Berg, S.; Kutra, D.; Kroeger, T.; Straehle, C.N.; Kausler, B.X.; Haubold, C.; Schiegg, M.; Ales, J.; Beier, T.; Rudy, M.; et al. ilastik: Interactive machine learning for (bio)image analysis. Nat. Methods 2019, 16, 1226–1232. [Google Scholar] [CrossRef]
- Rath, S.; Heidrich, B.; Pieper, D.H.; Vital, M. Uncovering the trimethylamine-producing bacteria of the human gut microbiota. Microbiome 2017, 5, 54. [Google Scholar] [CrossRef]
- Ewels, P.A.; Peltzer, A.; Fillinger, S.; Patel, H.; Alneberg, J.; Wilm, A.; Garcia, M.U.; Di Tommaso, P.; Nahnsen, S. The nf-core framework for community-curated bioinformatics pipelines. Nat. Biotechnol. 2020, 38, 276–278. [Google Scholar] [CrossRef]
- Straub, D.; Blackwell, N.; Langarica-Fuentes, A.; Peltzer, A.; Nahnsen, S.; Kleindienst, S. Interpretations of environmental microbial community studies are biased by the selected 16S rRNA (gene) amplicon sequencing pipeline. Front. Microbiol. 2020, 11, 550420. [Google Scholar] [CrossRef]
- Delbeke, H.; Casteels, I.; Joossens, M. DNA extraction protocol impacts ocular surface microbiome profile. Front. Microbiol. 2023, 14, 1128917. [Google Scholar] [CrossRef]
- dos Anjos Borges, L.G.; Pastuschek, J.; Heimann, Y.; Dawczynski, K.; PEONS study group; Schleussner, E.; Pieper, D.H.; Zollkau, J. Vaginal and neonatal microbiota in pregnant women with preterm premature rupture of membranes and consecutive early onset neonatal sepsis. BMC Med. 2023, 21, 92. [Google Scholar] [CrossRef]
- Salter, S.J.; Cox, M.J.; Turek, E.M.; Calus, S.T.; Cookson, W.O.; Moffatt, M.F.; Turner, P.; Parkhill, J.; Loman, N.J.; Walker, A.W. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 2014, 12, 87. [Google Scholar] [CrossRef]
- Chaux, C.; Crepy, M.; Xueref, S.; Roure, C.; Gille, Y.; Freydiere, A.M. Comparison of three chromogenic agar plates for isolation and identification of urinary tract pathogens. Clin. Microbiol. Infect. 2002, 8, 641–645. [Google Scholar] [CrossRef]
- Rigaill, J.; Verhoeven Paul, O.; Mahinc, C.; Jeraiby, M.; Grattard, F.; Fonsale, N.; Pozzetto, B.; Carricajo, A. Evaluation of new bioMérieux chromogenic CPS media for detection of urinary tract pathogens. J. Clin. Microbiol. 2015, 53, 2701–2702. [Google Scholar] [CrossRef]
- Stefaniuk, E.M. The usefulness of chromogenic media for qualitative and semi-quantitative diagnostic of urinary tract infections. Pol. J. Microbiol. 2018, 67, 213–218. [Google Scholar] [CrossRef]
- Yarbrough Melanie, L.; Wallace Meghan, A.; Marshall, C.; Mathias, E.; Burnham Carey-Ann, D. Culture of urine specimens by use of chromID CPS Elite medium can expedite Escherichia coli identification and reduce hands-on time in the clinical laboratory. J. Clin. Microbiol. 2016, 54, 2767–2773. [Google Scholar] [CrossRef]
- Carnes, M.U.; Siddiqui, N.Y.; Karstens, L.; Gantz, M.G.; Dinwiddie, D.L.; Sung, V.W.; Bradley, M.; Brubaker, L.; Ferrando, C.A.; Mazloomdoost, D.; et al. Urinary microbiome community types associated with urinary incontinence severity in women. Am. J. Obstet. Gynecol. 2024, 230, 344.e1–344.e20. [Google Scholar] [CrossRef]
- Price, T.K.; Hilt, E.E.; Thomas-White, K.; Mueller, E.R.; Wolfe, A.J.; Brubaker, L. The urobiome of continent adult women: A cross-sectional study. BJOG Int. J. Obstet. Gynaecol. 2020, 127, 193–201. [Google Scholar] [CrossRef]
- Horsley, H.; Dharmasena, D.; Malone-Lee, J.; Rohn, J.L. A urine-dependent human urothelial organoid offers a potential alternative to rodent models of infection. Sci. Rep. 2018, 8, 1238. [Google Scholar] [CrossRef]
- Mulvey, M.A.; Lopez-Boado, Y.S.; Wilson, C.L.; Roth, R.; Parks, W.C.; Heuser, J.; Hultgren, S.J. Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science 1998, 282, 1494–1497. [Google Scholar] [CrossRef]
- Anderson, G.G.; Palermo, J.J.; Schilling, J.D.; Roth, R.; Heuser, J.; Hultgren, S.J. Intracellular bacterial biofilm-like pods in urinary tract infections. Science 2003, 301, 105–107. [Google Scholar] [CrossRef]
- Justice, S.S.; Hung, C.; Theriot, J.A.; Fletcher, D.A.; Anderson, G.G.; Footer, M.J.; Hultgren, S.J. Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis. Proc. Natl. Acad. Sci. USA 2004, 101, 1333–1338. [Google Scholar] [CrossRef]
- Dempsey, E.; Corr, S.C. Lactobacillus spp. for gastrointestinal health: Current and future perspectives. Front. Immunol. 2022, 13, 840245. [Google Scholar] [CrossRef]
- Chee, W.J.Y.; Chew, S.Y.; Than, L.T.L. Vaginal microbiota and the potential of Lactobacillus derivatives in maintaining vaginal health. Microb. Cell Fact. 2020, 19, 203. [Google Scholar] [CrossRef]
- Verstraelen, H.; Verhelst, R.; Claeys, G.; De Backer, E.; Temmerman, M.; Vaneechoutte, M. Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora. BMC Microbiol. 2009, 9, 116. [Google Scholar] [CrossRef] [PubMed]
- Gaston Jordan, R.; Johnson Alexandra, O.; Bair Kirsten, L.; White Ashley, N.; Armbruster Chelsie, E. Polymicrobial interactions in the urinary tract: Is the enemy of my enemy my friend? Infect. Immun. 2021, 89, IAI.00652-20. [Google Scholar] [CrossRef] [PubMed]
- Jiang, L.; Wang, H.; Luo, L.; Pang, X.; Liu, T.; Sun, L.; Zhang, G. Urogenital microbiota-driven virulence factor genes associated with recurrent urinary tract infection. Front. Microbiol. 2024, 15, 1344716. [Google Scholar] [CrossRef]
- Wu, C.; Wei, X.; Huang, Z.; Zheng, Z.; Zhang, W.; Chen, J.; Hong, H.; Li, W. Urinary microbiome dysbiosis is associated with an inflammatory environment and perturbed fatty acids metabolism in the pathogenesis of bladder cancer. J. Transl. Med. 2024, 22, 628. [Google Scholar] [CrossRef]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Chieng, C.C.Y.; Kong, Q.; Liou, N.S.Y.; Neira Rey, M.; Dalby, K.L.; Jones, N.; Khasriya, R.; Horsley, H. Novel Techniques to Unravel Causative Bacterial Ecological Shifts in Chronic Urinary Tract Infection. Pathogens 2025, 14, 299. https://doi.org/10.3390/pathogens14030299
Chieng CCY, Kong Q, Liou NSY, Neira Rey M, Dalby KL, Jones N, Khasriya R, Horsley H. Novel Techniques to Unravel Causative Bacterial Ecological Shifts in Chronic Urinary Tract Infection. Pathogens. 2025; 14(3):299. https://doi.org/10.3390/pathogens14030299
Chicago/Turabian StyleChieng, Catherine C. Y., Qingyang Kong, Natasha S. Y. Liou, Mariña Neira Rey, Katie L. Dalby, Neil Jones, Rajvinder Khasriya, and Harry Horsley. 2025. "Novel Techniques to Unravel Causative Bacterial Ecological Shifts in Chronic Urinary Tract Infection" Pathogens 14, no. 3: 299. https://doi.org/10.3390/pathogens14030299
APA StyleChieng, C. C. Y., Kong, Q., Liou, N. S. Y., Neira Rey, M., Dalby, K. L., Jones, N., Khasriya, R., & Horsley, H. (2025). Novel Techniques to Unravel Causative Bacterial Ecological Shifts in Chronic Urinary Tract Infection. Pathogens, 14(3), 299. https://doi.org/10.3390/pathogens14030299