Biology subjects

Lamanna, O.

Publications and source records attributed to Lamanna, O..

4 recordsLinked to original sources

bioRxiv2025

Evidence for an indigenous female mouse urobiome

Mice have been used as a valuable model of understanding pathophysiological mechanisms of urinary tract infection for almost six decades. Mice offer many advantages including genetic manipulation to test the role of genes and mechanisms, the availability of germ-free mice, and similarities to humans in innate immune defenses and the strain-dependent presence of vesicoureteral reflux. However, like with humans, the mouse bladder urine above the urinary sphincter has generally been assumed to be sterile. Yet, given the presence of urobiomes in other mammals and the emerging role of the human urobiome in the defense of the urinary bladder and upper urinary tract, the existence of a mouse urobiome should be critically examined as indigenous microbiota may influence experimental results. To determine if an indigenous murine urobiome exists, we obtained expressed urine from two sets of female C57BL/6J mice during three different intervals using two different extraction and sequencing methods and analyzed them simultaneously by a single method. For one set, we also obtained urine by suprapubic aspiration, which we compared to the paired expressed urine samples. We conclude that an indigenous murine urobiome exists and that expressed urine contains post-urethral microbes.

microbiology↗

bioRxiv2021

A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli in Vitro and in Vivo

BackgroundPolymerase chain reaction (PCR) is an important means by which to study the urine microbiome and is emerging as possible alternative to urine cultures to identify pathogens that cause urinary tract infection (UTI). However, PCR is limited by its inability to differentiate DNA originating from viable, metabolically active versus non-viable, inactive bacteria. This drawback has led to concerns that urobiome studies and PCR-based diagnosis of UTI are confounded by the presence of relic DNA from non-viable bacteria in urine. Propidium monoazide (PMA) dye can penetrate cells with compromised cell membranes and covalently bind to DNA, rendering it inaccessible to amplification by PCR. Although PMA has been shown to differentiate between non-viable and viable bacteria in various settings, its effectiveness in urine has not been previously studied. We sought to investigate the ability of PMA to differentiate between viable and non-viable bacteria in urine. MethodsVarying amounts of viable or non-viable uropathogenic E. coli (UTI89) or buffer control were titrated with mouse urine. The samples were centrifuged to collect urine sediment or not centrifuged. Urine samples were incubated with PMA and DNA cross-linked using blue LED light. DNA was isolated and uidA gene-specific PCR was performed. For in vivo studies, mice were inoculated with UTI89, followed by ciprofloxacin treatment or no treatment. After the completion of ciprofloxacin treatment, an aliquot of urine was plated on non-selective LB agar and another aliquot was treated with PMA and subjected to uidA-specific PCR. ResultsPMAs efficiency in excluding DNA signal from non-viable bacteria was significantly higher in bacterial samples in phosphate-buffered saline (PBS, dCT=13.69) versus bacterial samples in unspun urine (dCT=1.58). This discrepancy was diminished by spinning down urine-based bacterial samples to collect sediment and resuspending it in PBS prior to PMA treatment. In 3 of 5 replicate groups of UTI89-infected mice, no bacteria grew in culture; however, there was PCR amplification of E. coli after PMA treatment in 2 of those 3 groups. ConclusionWe have successfully developed PMA-based PCR methods for amplifying DNA from live bacteria in urine. Our results suggest that non-PMA bound DNA from live bacteria can be present in urine, even after antibiotic treatment. This indicates that viable but non-culturable E. coli can be present following treatment of UTI, and may explain why some patients have persistent symptoms but negative urine cultures following UTI treatment.

microbiology↗

bioRxiv2020

IPSE, a Parasite-Derived, Host Immunomodulatory Infiltrin Protein, Alleviates Resiniferatoxin-Induced Bladder Pain

The transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor is an important mediator of nociception and its expression is enriched in nociceptive neurons. TRPV1 signaling has been implicated in bladder pain and is a potential analgesic target. Resiniferatoxin is the most potent known agonist of TRPV1. Acute exposure of the rat bladder to resiniferatoxin has been demonstrated to result in pain-related freezing and licking behaviors that are alleviated by virally encoded IL-4. The interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE) is a powerful inducer of IL-4 secretion, and is also known to alter host cell transcription through a nuclear localization sequence-dependent mechanism. We previously reported that IPSE ameliorates ifosfamide-induced bladder pain in an IL-4- and nuclear localization sequence-dependent manner. We hypothesized that pre-administration of IPSE to resiniferatoxin-challenged mice would dampen pain-related behaviors. IPSE indeed lessened resiniferatoxin-triggered freezing behaviors in mice. This was a nuclear localization sequence-dependent phenomenon, since administration of a nuclear localization sequence mutant version of IPSE abrogated IPSEs analgesic effect. In contrast, IPSEs analgesic effect did not seem IL-4-dependent, since use of anti-IL-4 antibody in mice given both IPSE and resiniferatoxin did not dramatically affect freezing behaviors. RNA-Seq analysis of resiniferatoxin- and IPSE-exposed bladders revealed differential expression of TNF/NF-{kappa}b-related signaling pathway genes. In vitro testing of IPSE uptake by urothelial cells and TRPV1-expressing neuronal cells showed uptake by both cell types. Thus, IPSEs nuclear localization sequence-dependent therapeutic effects on TRPV1-mediated bladder pain may act on TRPV1-expressing neurons and/or may rely upon urothelial mechanisms.

neuroscience↗

bioRxiv2018

Profiling of urine bacterial DNA to identify an oncobiome in a mouse model of bladder cancer

IntroductionRecurrent urinary tract infections have been linked to increased risk of bladder cancer, suggesting a potential role of the urinary microbiome in bladder cancer pathogenesis.\n\nObjectiveCompare the urinary microbiomes in mice with and without bladder.\n\nMethodsLongitudinal study of mice exposed to a dilute bladder-specific carcinogen (0.05% n-butyl-n-(4-hydroxybutyl) nitrosamine, BBN mice, n=10), and control mice (n=10). Urine was sampled monthly from individual mice for 4 months. Microbial DNA was extracted from the urine, and the V4 region of the 16S rRNA gene sequenced. Animals were sacrificed and their bladders harvested for histopathology. Bladder sections were graded by a blinded pathologist. The composition and diversity of the urinary microbiome were compared between the BBN and control mice. Metabolic pathway analysis was completed using PICRUST.\n\nResultsBladder histology in the BBN group showed normal tissue with inflammation (BBN-normal, n=5), precancerous pathologies, (BBN-precancerous, n=3), and invasive cancer (BBN-cancer, n=2). Alpha diversity did not differ between the mice exposed to BBN and the control mice at any timepoint. There were no differences in the urinary microbiomes between the BBN and control mice at baseline. At month 4, mice exposed to BBN had higher proportion of both Gardnerella and Bifidobacterium compared to control mice. There were no differences in proportions of specific bacteria between either the BBN-precancer or BBN-cancer and controls at month 4. However, the BBN-normal mice had higher proportions of Gardnerella, Haemophilus, Bifidobacterium, and Ureaplasma Actinobaculum, and lower proportions of Actinomyces, compared to control mice at month 4. Functional pathway analysis demonstrated increases in genes related to purine metabolism, phosphotransferase systems, peptidases, protein folding, and bacterial toxins in the BBN-mice compared to control mice at month 4.\n\nConclusionMice exposed to 4 months of BBN, a bladder-specific carcinogen, have distinct urine microbial profiles compared to control mice.

cancer biology↗