Amy B. Rosenfeld

Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder

ABSTRACT Our previous study showed that bacterial genomes can be identified using 16S rRNA sequencing in urine specimens of both symptomatic and asymptomatic patients who are culture negative according to standard urine culture protocols. In the present study, we used a modified culture protocol that included plating larger volumes of urine, incubation under varied atmospheric conditions, and prolonged incubation times to demonstrate that many of the organisms identified in urine by 16S rRNA gene sequencing are, in fact, cultivable using an expanded quantitative urine culture (EQUC) protocol. Sixty-five urine specimens (from 41 patients with overactive bladder and 24 controls) were examined using both the standard and EQUC culture techniques. Fifty-two of the 65 urine samples (80%) grew bacterial species using EQUC, while the majority of these (48/52 [92%]) were reported as no growth at 103 CFU/ml by the clinical microbiology laboratory using the standard urine culture protocol. Thirty-five different genera and 85 different species were identified by EQUC. The most prevalent genera isolated were Lactobacillus (15%), followed by Corynebacterium (14.2%), Streptococcus (11.9%), Actinomyces (6.9%), and Staphylococcus (6.9%). Other genera commonly isolated include Aerococcus, Gardnerella, Bifidobacterium, and Actinobaculum. Our current study demonstrates that urine contains communities of living bacteria that comprise a resident female urine microbiota.

The Female Urinary Microbiome: a Comparison of Women with and without Urgency Urinary Incontinence

ABSTRACT Bacterial DNA and live bacteria have been detected in human urine in the absence of clinical infection, challenging the prevailing dogma that urine is normally sterile. Urgency urinary incontinence (UUI) is a poorly understood urinary condition characterized by symptoms that overlap urinary infection, including urinary urgency and increased frequency with urinary incontinence. The recent discovery of the urinary microbiome warrants investigation into whether bacteria contribute to UUI. In this study, we used 16S rRNA gene sequencing to classify bacterial DNA and expanded quantitative urine culture (EQUC) techniques to isolate live bacteria in urine collected by using a transurethral catheter from women with UUI and, in comparison, a cohort without UUI. For these cohorts, we demonstrated that the UUI and non-UUI urinary microbiomes differ by group based on both sequence and culture evidences. Compared to the non-UUI microbiome, sequencing experiments revealed that the UUI microbiome was composed of increased Gardnerella and decreased Lactobacillus. Nine genera (Actinobaculum, Actinomyces, Aerococcus, Arthrobacter, Corynebacterium, Gardnerella, Oligella, Staphylococcus, and Streptococcus) were more frequently cultured from the UUI cohort. Although Lactobacillus was isolated from both cohorts, distinctions existed at the species level, with Lactobacillus gasseri detected more frequently in the UUI cohort and Lactobacillus crispatus most frequently detected in controls. Combined, these data suggest that potentially important differences exist in the urinary microbiomes of women with and without UUI, which have strong implications in prevention, diagnosis, or treatment of UUI. IMPORTANCE New evidence indicates that the human urinary tract contains microbial communities; however, the role of these communities in urinary health remains to be elucidated. Urgency urinary incontinence (UUI) is a highly prevalent yet poorly understood urinary condition characterized by urgency, frequency, and urinary incontinence. Given the significant overlap of UUI symptoms with those of urinary tract infections, it is possible that UUI may have a microbial component. We compared the urinary microbiomes of women affected by UUI to those of a comparison group without UUI, using both high-throughput sequencing and extended culture techniques. We identified statistically significant differences in the frequency and abundance of bacteria present. These differences suggest a potential role for the urinary microbiome in female urinary health. New evidence indicates that the human urinary tract contains microbial communities; however, the role of these communities in urinary health remains to be elucidated. Urgency urinary incontinence (UUI) is a highly prevalent yet poorly understood urinary condition characterized by urgency, frequency, and urinary incontinence. Given the significant overlap of UUI symptoms with those of urinary tract infections, it is possible that UUI may have a microbial component. We compared the urinary microbiomes of women affected by UUI to those of a comparison group without UUI, using both high-throughput sequencing and extended culture techniques. We identified statistically significant differences in the frequency and abundance of bacteria present. These differences suggest a potential role for the urinary microbiome in female urinary health.

Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans

Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20°C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25°C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856>N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856>N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear-mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism.

Replication of early and recent Zika virus isolates throughout mouse brain development

Significance Zika virus (ZIKV) infection has been associated with multiple pathologies of the central nervous system (CNS) including microcephaly, Guillain-Barré syndrome, lissencephaly, the loss of white and gray matter volume and acute myelitis. Using organotypic brain slice cultures, we determined that ZIKV replicates across different embryonic developmental stages, and viral infection can disrupt proper brain development leading to congenital CNS complications. These data illustrate that all lineages of ZIKV tested are neurotropic, and that infection may disrupt neuronal migration during brain development. The results expand our understanding of neuropathologies associated with congenital Zika virus syndrome. Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus-associated injury. Therefore, a critical aspect of ZIKV-induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and midgestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly elongated basal processes of the radial glial progenitor cells and impairment of postmitotic neuronal migration, were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.

Genomes of Gardnerella Strains Reveal an Abundance of Prophages within the Bladder Microbiome

Bacterial surveys of the vaginal and bladder human microbiota have revealed an abundance of many similar bacterial taxa. As the bladder was once thought to be sterile, the complex interactions between microbes within the bladder have yet to be characterized. To initiate this process, we have begun sequencing isolates, including the clinically relevant genus Gardnerella. Herein, we present the genomic sequences of four Gardnerella strains isolated from the bladders of women with symptoms of urgency urinary incontinence; these are the first Gardnerella genomes produced from this niche. Congruent to genomic characterization of Gardnerella isolates from the reproductive tract, isolates from the bladder reveal a large pangenome, as well as evidence of high frequency horizontal gene transfer. Prophage gene sequences were found to be abundant amongst the strains isolated from the bladder, as well as amongst publicly available Gardnerella genomes from the vagina and endometrium, motivating an in depth examination of these sequences. Amongst the 39 Gardnerella strains examined here, there were more than 400 annotated prophage gene sequences that we could cluster into 95 homologous groups; 49 of these groups were unique to a single strain. While many of these prophages exhibited no sequence similarity to any lytic phage genome, estimation of the rate of phage acquisition suggests both vertical and horizontal acquisition. Furthermore, bioinformatic evidence indicates that prophage acquisition is ongoing within both vaginal and bladder Gardnerella populations. The abundance of prophage sequences within the strains examined here suggests that phages could play an important role in the species’ evolutionary history and in its interactions within the complex communities found in the female urinary and reproductive tracts.

Suppression of cellular transformation by poly (A) binding protein interacting protein 2 (Paip2).

Controlling translation is crucial for the homeostasis of a cell. Its deregulation can facilitate the development and progression of many diseases including cancer. Poly (A) binding protein interacting protein 2 (Paip2) inhibits efficient initiation of translation by impairing formation of the necessary closed loop of mRNA. The over production of Paip2 in the presence of a constitutively active form of hRasV12 can reduce colony formation in a semi-solid matrix and focus formation on a cell monolayer. The ability of Paip2 to bind to Pabp is required to suppress the transformed phenotype mediated by hRasV12. These observations indicate that Paip2 is able to function as a tumor suppressor.

Neurotropism of enterovirus D68 isolates is independent of sialic acid and is not a recently acquired phenotype

Acute flaccid myelitis /acute flaccid paralysis (AFM/AFP) is a rare but serious illness of the nervous system, specifically affecting the grey matter of the spinal cord, motor controlling regions of the brain and the cranial nerve. Most cases of AFM/AFP are pathogen associated, typically with poliovirus and enterovirus infections, and occur in children under the age of 6 years old. Enterovirus D68 (EV-D68) was first isolated from children with pneumonia in 1962, but an association with AFM/AFP was not observed until the 2014 outbreak. Organotypic mouse brain slice cultures generated from postnatal day 1 to 10 mice were used to determine if neurotropism of EV-D68 is shared among virus isolates. Six of the seven EV-D68 isolates examined, including two from 1962 and four from the 2014 outbreak, replicated in neurons, and all replicated in astrocytes. Furthermore, a putative viral receptor, sialic acid, is not required for neurotropism of EV-D68, as both sialic acid dependent and independent viruses replicated within neurons. These observations demonstrate that EV-D68 is neurotropic independent of its genetic lineage, can infect both neurons and astrocytes, and that neurotropism is not a recently acquired characteristic as has been suggested. Significance Recently there has been an increase in the number of children infected with enterovirus D68 (EV-D68). Most infections are associated with mild flu-like symptoms, but neurological dysfunction may develop in a small number of children. How the biochemical and genetic differences among EV-D68 isolates relates to development of neurological disease remains an unanswered question. Assessing infection of multiple viral isolates in organotypic brain slice cultures from postnatal day 1 to 10 mice revealed that multiple isolates are neurotropic. Both neuraminidase sensitive and resistant viruses infected neurons, indicating that sialic acid binding does not play a role in EV-D68 neuropathogenesis. Establishment of a genetically and pharmacologically amenable system using organotypic brain slice cultures will provide insight into how EV-D68 neuropathologies develop.