ORIGINAL RESEARCH published: 23 June 2021 doi: 10.3389/fmed.2021.667462 Urethral Catheter Biofilms Reveal Plasticity in Bacterial Composition Edited by: Xiaoyun Liu, and Metabolism and Withstand Host Peking University, China Reviewed by: Immune Defenses in Hypoxic Ruibing Chen, Tianjin University, China Environment Xu Zhang, University of Ottawa, Canada Yanbao Yu 1†, Harinder Singh 1†, Tamara Tsitrin 1, Shiferaw Bekele 1‡, Yi-Han Lin 1, *Correspondence: Patricia Sikorski 1‡, Kelvin J. Moncera 2‡, Manolito G. Torralba 2, Lisa Morrow 3, Rembert Pieper Randall Wolcott 3, Karen E. Nelson 1,2 and Rembert Pieper 1*‡ [email protected] 1 J. Craig Venter Institute, Rockville, MD, United States, 2 J. Craig Venter Institute, La Jolla, CA, United States, 3 Southwest † These authors have contributed Regional Wound Care Center, Lubbock, TX, United States equally to this work ‡Present address: Shiferaw Bekele, Biofilms composed of multiple microorganisms colonize the surfaces of indwelling University of Maryland Medical Center, urethral catheters that are used serially by neurogenic bladder patients and cause Baltimore, MD, United States chronic infections. Well-adapted pathogens in this niche are Escherichia coli, Proteus, Patricia Sikorski, Department of Neurology, George and Enterococcus spp., species that cycle through adhesion and multilayered cell Washington University, Washington, growth, trigger host immune responses, are starved off nutrients, and then disperse. DC, United States Kelvin J. Moncera, Viable microbial foci retained in the urinary tract recolonize catheter surfaces. The Western University of Health molecular adaptations of bacteria in catheter biofilms (CBs) are not well-understood, Sciences, College of Osteopathic promising new insights into this pathology based on host and microbial meta-omics Medicine of the Pacific, Pomona, CA, United States analyses from clinical specimens. We examined catheters from nine neurogenic bladder Rembert Pieper, patients longitudinally over up to 6 months. Taxonomic analyses from 16S rRNA gene Janssen Biopharma Inc., South San Francisco, CA, United States sequencing and liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based proteomics revealed that 95% of all catheter and corresponding urinary pellet (UP) Specialty section: samples contained bacteria. CB biomasses were dominated by Enterobacteriaceae This article was submitted to Infectious Diseases–Surveillance, spp. and often accompanied by lactic acid and anaerobic bacteria. Systemic antibiotic Prevention and Treatment, drug treatments of patients resulted in either transient or lasting microbial community a section of the journal perturbations. Neutrophil effector proteins were abundant not only in UP but also Frontiers in Medicine CB samples, indicating their penetration of biofilm surfaces. In the context of one Received: 13 February 2021 Accepted: 06 May 2021 patient who advanced to a kidney infection, Proteus mirabilis proteomic data suggested Published: 23 June 2021 a combination of factors associated with this disease complication: CB biomasses Citation: were high; the bacteria produced urease alkalinizing the pH and triggering urinary salt Yu Y, Singh H, Tsitrin T, Bekele S, Lin Y-H, Sikorski P, Moncera KJ, deposition on luminal catheter surfaces; P. mirabilis utilized energy-producing respiratory Torralba MG, Morrow L, Wolcott R, systems more than in CBs from other patients. The NADH:quinone oxidoreductase II Nelson KE and Pieper R (2021) (Nqr), a Na+ translocating enzyme not operating as a proton pump, and the nitrate Urethral Catheter Biofilms Reveal Plasticity in Bacterial Composition and reductase A (Nar) equipped the pathogen with electron transport chains promoting Metabolism and Withstand Host growth under hypoxic conditions. Both P. mirabilis and E. coli featured repertoires of Immune Defenses in Hypoxic Environment. Front. Med. 8:667462. transition metal ion acquisition systems in response to human host-mediated iron and doi: 10.3389/fmed.2021.667462 zinc sequestration. We discovered a new drug target, the Nqr respiratory system, whose Frontiers in Medicine | www.frontiersin.org 1 June 2021 | Volume 8 | Article 667462 Yu et al. Urethral Catheter Biofilm Composition and Metabolism deactivation may compromise P. mirabilis growth in a basic pH milieu. Animal models would not allow such molecular-level insights into polymicrobial biofilm metabolism and interactions because the complexity cannot be replicated. Keywords: proteomics, catheter biofilm, infection, bacterial metabolism, pathogen, polymicrobial, immune defense INTRODUCTION type of infiltrating immune cells. In this model, E. coli and Enterococcus faecalis caused urothelial barrier disruption and Urethral catheter-associated urinary tract infection (CAUTI) is further immune cell infiltration with pyuria (12). However, a the most common type of complicated UTI. Enhanced risks of proteomic study on samples from catheterized trauma patients recurrence and spread to other organs (e.g., pyelonephritis and was consistent with neutrophil infiltration at a higher level in urosepsis) compared to acute urinary tract infections (UTIs) individuals diagnosed with CAASB or CAUTI as compared to exist in nosocomial environments (1–3). Asymptomatic cases non-bacteriuric patients (13). are diagnosed as catheter-associated asymptomatic bacteriuria The pathogenesis of single-species–associated UTI and (CAASB). The use of nearly 100 million urethral catheters CAUTI has been extensively studied in the context of E. per year worldwide, a 3–10% incidence of bacteriuria over coli and P. mirabilis (4, 14–17). Type I fimbriae are thought 24 h following patient catheterization, and an average bladder to initiate mucosal colonization via their ability to bind to catheter insertion time of 72h (2) suggest that 9 million mannosylated uroplakins that coat the luminal surface of to 27 million CAUTI and CAASB cases occur globally per urothelial umbrella cells (18). P. mirabilis produces several year. Indwelling Foley catheters are used by patients with types of fimbriae of which the best characterized ones are anatomical and neurological urinary tract abnormalities and mannose-resistant Proteus-like (MR/P) fimbriae, uroepithelial typically retained for 1–3 weeks before replacement to minimize cell adhesin (UCA), and proton motive force (PMF) fimbriae microbial colonization. (16). Fimbriae are virulence factors involved in the formation Among the most common etiological agents of CAUTIs are of salt-encrusted (SE) CBs entailing struvite and apatite crystal Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, deposition on catheter surfaces (3, 14, 16). Host proteins such Proteus mirabilis, Enterococcus, and Candida spp. (1, 3, 4). These as fibrinogen are also deposited on catheter surfaces (3, 4). microbes have adapted to form biofilms on catheter surfaces; e.g., Biofilm-embedded bacterial cells embedded disperse, swarm, Proteus and Providencia spp. metabolize urea to NH3 and use and recolonize unoccupied catheter surfaces. Their ascendance it as a nitrogen source (3, 5, 6). Furthermore, urea degradation to the kidneys poses an elevated threat of pyelonephritis and alkalinizes the pH of urine and triggers deposition of phosphate urosepsis (3). Microbial colonization dynamics in CBs over time salt crystals in and on catheters, thus increasing the risks of are influenced by the availability of various nutrients such as luminal occlusion and complications such as urinary stones nitrogen, carbohydrates, transition metal ions (TMIs), oxygen, and kidney infection (3). Unless specific risk factors such as a and antibiotic drug exposure. Iron and zinc ions are specifically compromised immune system exist, clinical guidelines do not sequestered by the innate immune system during infection recommend use of antibiotics in CAASB cases (7). Of major (14, 19, 20). CBs may have a mucoid phenotype derived from concern are acquired and innate resistances of bacteria causing extracellular polysaccharides that encapsulate bacterial cells and CAUTI, many of these belonging to the multidrug-resistant impede phagocytosis. Furthermore, such biofilms have been group of gram-negative pathogens known as ESKAPE pathogens associated with renal complications by blocking urine flow (3). (8). Understanding mechanisms that underlie cooperation and CBs typically harbor more than a single species (5, 6, 21). competition of bacteria and fungi in urethral catheter biofilms Culture-independent metagenomic analyses have highlighted the (CBs) may lead to new approaches to prevent or target considerable microbial diversity in CBs, including fastidious their formation. bacteria, such as Actinomyces, Stenotrophomonas, Finegoldia, and Innate immunity has a key role in defending against bacteria Corynebacterium (22, 23). To what extent the entire microbial invading the urinary tract. They express pathogen-associated community elicits inflammatory responses by the host is unclear. molecular patterns recognized by immunoglobulin A and A few studies have described biofilm profiles related to long- lipopolysaccharide-binding protein and then presented to term catheterization. One study reported high prevalence of Toll-like receptors on urothelial cells. Intracellular signaling E. coli, P. mirabilis, and Providencia stuartii for bacteriuric pathways result in cytokine release followed by leukocyte patients catheterized over 4 or more weeks (24). In a survey infiltration. Evidence from murine studies verified that of hundreds of urine samples