Species level resolution of female bladder microbiota from 16S rRNA amplicon sequencing

Hoffman, Carter; Siddiqui, Nazema Y.; Fields, Ian; Gregory, W. Thomas; Simon, Holly M.; Mooney, Michael A.; Wolfe, Alan J.; Karstens, Lisa

doi:10.1101/2020.10.27.358408

Cited by 4 publications

(6 citation statements)

References 70 publications

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“…We also identified 4 Gardnerella species in healthy FUM, according to recent genus reclassification (15). This demonstrates that the high number of colonies studied and reliable identification of isolates by specific genotypic markers, together with the usage of cutting-edge long-read third generation sequencing of the 16S rRNA gene increase the knowledge on the composition of bacterial community to the species level in microbiome studies (17, 18, 44).…”

Section: Discussionmentioning

confidence: 96%

“…Methodological improvements include also the use of a larger volume sample size (20 ml), compared to previously used urine volume (mostly 1 ml) in DNA extraction protocols, which increased high-quality microbial DNA yield required for high-resolution sequencing, and unveiled detection of species not previously reported in DNA-based studies (e.g., Alistipes putredinis) (1,10,12,34). Another important improvement was the use of a cutting-edge sequencing technique, including near full-length 16S rRNA gene sequencing using PacBio SMRT cell technology (18,(46)(47)(48), and appropriate gene markers to identify cultured isolates at species level, which enable increased taxonomic resolution, as well as validation of several low-read sequencing data (< 0.1% RA) by our extended culturomic protocol.…”

Section: Discussionmentioning

confidence: 99%

“…Current approaches for FUM characterization usually involve culturomics with matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as primary identification method and/or DNA sequencing methodologies targeting individual short hyper-variable regions of the 16S rRNA gene (3, 9, 10, 12, 18). However, some methodological drawbacks concerning the identification at species level of some FUM members can still be recognized.…”

Section: Introductionmentioning

confidence: 99%

“…However, some methodological drawbacks concerning the identification at species level of some FUM members can still be recognized. For instance, culture-based methodologies with limited growth conditions and isolates’ identification of insufficient resolution power do not fully capture bacterial species diversity, while short-read DNA-based methods are often limited to a reliable identification of FUM members only at genus level (18).…”

Section: Introductionmentioning

confidence: 99%

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Extended bacterial diversity of the urinary microbiome of reproductive-age healthy European women captured by culturomics and long-read amplicon sequencing

Perović

Ksiezarek

Rocha

et al. 2022

Preprint

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The recognition of microbiome inhabiting the healthy female bladder engendered the need for comprehensive characterization of the female urinary microbiome (FUM) in health and disease. Although previous studies reported FUM composition at different taxonomic levels, progress towards reliable identification at species level is highly required. The aim of this study was to comprehensively characterize bacterial species of FUM of healthy reproductive-age European women by two complementary methodologies i.e., extended culturomics and long-read third generation sequencing of near full-length 16S rRNA gene. A wide diversity of bacterial species was captured (297 species) with a median of 53 species/sample, including 16 putative uropathogens. Clustering FUM into community structure types revealed high inter-individual differences. Notably, there was not a single species common to all samples, although the Lactobacillus genus was detected in all samples. Lactobacillus crispatus, Lactobacillus iners and Lactobacillus mulieris were observed in high relative abundance in several samples as well as other species (e.g., Streptococcus agalactiae, Atopobium vaginae, Gardnerella vaginalis, Gardnerella swidsinskii), while more prevalent species were often low abundant members (e.g., Finegoldia magna). We captured remarkable richness within Corynebacterium spp. (25 species) and Lactobacillaceae (4 genera, 14 species). While amplicon sequencing allowed detection of more anaerobic species (e.g., 11 Peptoniphilus spp.), culturomics enabled the identification of recently recognized Gardnerella species and putative novel Corynebacterium species. This study provided fine-grained FUM profiling at species level and revealed detailed FUM structure, which is critical to unveil the potential relationship between specific microbiome members and urinary diseases/disorders.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Extended bacterial diversity of the urinary microbiome of reproductive-age healthy European women captured by culturomics and long-read amplicon sequencing

Perović

Ksiezarek

Rocha

et al. 2022

Preprint

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“…We repeated DMM clustering on ASV data from the updated bioinformatic analysis using the same number of clusters that were selected in the original publication. However, since selecting the number of clusters can introduce bias, we also evaluated a nonparametric mixture model called Dirichlet tree multinomial mixture (DTMM) 6 , which automatically adapts the number of clusters based on the complexity of the data. The two methods also differ in the way cross-sample heterogeneity is modeled, resulting in the phenomenon that DMM clusters samples to be highly influenced by the "dominant", or most abundant taxa in a sample.…”

Section: Clustering Into Microbial Communitiesmentioning

confidence: 99%

Updating urinary microbiome analyses to enhance biologic interpretation

Siddiqui

Brubaker

et al. 2021

Preprint

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Objective: An approach for assessing the urinary microbiome is 16S rRNA gene sequencing, where a segment of the bacterial genome is amplified and sequenced. Methods used to analyze these data are rapidly evolving, although the research implications are not known. This re-analysis of an existing dataset aimed to determine the impact of updated bioinformatic and statistical techniques. Methods: A prior Pelvic Floor Disorders Network (PFDN) study compared the urinary microbiome in 123 women with mixed urinary incontinence (MUI) and 84 controls. We used the PFDN unprocessed sequencing data of V1-V3 and V4-V6 16S variable regions, processed operational taxonomic unit (OTU) tables, and de-identified clinical data. We processed sequencing data with an updated bioinformatic pipeline, which used DADA2 to generate amplicon sequence variant (ASV) tables. Taxa from ASV tables were compared to OTU tables generated from the original processing; taxa from different variable regions (e.g., V1-V3 versus V4-V6) after updated processing were also compared. After updated processing, data were analyzed with multiple filtering thresholds. Several techniques were tested to cluster samples into microbial communities. Multivariable regression was used to test for associations between microbial communities and MUI, while controlling for potentially confounding variables. Results: Of taxa identified through updated bioinformatic processing, only 40% were identified originally, though taxa identified through both methods represented >99% of sequencing data in terms of relative abundance. When different 16S rRNA gene regions were sequenced from the same samples, there were differences noted in recovered taxa. When the original clustering methods were applied to reprocessed sequencing data, we confirmed differences in microbial communities associated with MUI. However, when samples were clustered with a different methodology, microbial communities were no longer associated with MUI. Conclusions: Updated bioinformatic processing techniques recover many different taxa compared to prior techniques, though most of these differences exist in low abundance taxa that occupy a small proportion of the overall microbiome. Detection of high abundance taxa are not significantly impacted by bioinformatic strategy. However, there are different biases for less abundant taxa; these differences as well as downstream clustering methodology and filtering thresholds may affect interpretation of overall results.

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Application of Various Techniques to Gain Insights Into the Complex Urinary Tract Microbial Communities of Renal Transplant Recipients

Sathiananthamoorthy

Florman

Richard

et al. 2023

Transplantation Direct

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which seeks novel cures for urinary disorders. S.S. participated in the design, performance of laboratory experiments, data analysis, and write-up. K.F. participated in the data analysis and write-up. D.R. participated in the data analysis and write-up. K.K.C. participated in the performance of laboratory experiments and data analysis. V.T. participated in the performance of laboratory experiments. F.M. participated in the design and Kidney TransplantationBackground. Urinary tract infections (UTIs) are prevalent in renal transplant (RT X ) recipients and associated with worse outcomes. Early detection by sensitive diagnostic tests and appropriate treatment strategies in this cohort is therefore crucial, but evidence has shown that current methods may miss genuine infections. Research has shed light on the urinary tract microbial ecology of healthy individuals and nontransplant patients with UTI, but information on the RTx cohort is scant. We conducted a cross-sectional study to (i) compare the gold standard diagnostic culture with alternative techniques and (ii) characterize RTx patient urinary microbial communities. Methods. Midstream urine specimens were collected from 51 RTx patients attending a renal transplant clinic and 27 asymptomatic controls. Urinary microscopy, dipstick, and routine culture were performed. To improve sensitivity of microbial detection, we cultured the urinary cell sediment and performed 16S rRNA gene sequencing on urine. Uroplakin-positive urothelial cells shed in urine were analyzed by immunofluorescence staining for any bacterial association. Results. Sediment culture and 16S rRNA sequencing confirmed detection deficiencies of diagnostic culture and revealed differences in the urobiomes of RTx patients and controls. Specifically, Gardnerella, Escherichia, and Lactobacillus were most abundant in patients, whereas Lactobacillus, Streptococcus, and Gardnerella were most abundant in controls. The application of both culture and sequencing provided a more nuanced view of the urinary microbial communities. Conclusions. This study provides insight into the potential problems of diagnostic culture within RTx patients and sheds light on their urinary microbial inhabitants. Further work may identify key microbial signatures and facilitate the development of better tools for UTI detection within this cohort, which could allow targeted intervention before an infection leads to serious consequences. http://links.lww.com/TXD/A479

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Species level resolution of female bladder microbiota from 16S rRNA amplicon sequencing

Cited by 4 publications

References 70 publications

Extended bacterial diversity of the urinary microbiome of reproductive-age healthy European women captured by culturomics and long-read amplicon sequencing

Extended bacterial diversity of the urinary microbiome of reproductive-age healthy European women captured by culturomics and long-read amplicon sequencing

Updating urinary microbiome analyses to enhance biologic interpretation

Application of Various Techniques to Gain Insights Into the Complex Urinary Tract Microbial Communities of Renal Transplant Recipients

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