Single Clinical Isolates from Acute Uncomplicated Urinary Tract Infections Are Representative of Dominant
            <i>In Situ</i>
            Populations

Willner, Dana; Low, Serene; Steen, Jason A.; George, Narelle; Nimmo, Graeme R.; Schembri, Mark A.; Hugenholtz, Philip

doi:10.1128/mbio.01064-13

Cited by 47 publications

(28 citation statements)

References 82 publications

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“…A recent comparison of 16s sequencing to urine culture in patients with UTI also found that the dominant uropathogen identified by culture was the most abundant organism identified by sequencing. 30 Thus, the clinical relevance of the additional species picked up by 16s rRNA analysis is not clear.…”

Section: Discussionmentioning

confidence: 99%

Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference

Horwitz

McCue

Mapes

et al. 2015

Journal of Infection

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Background Patients with long-term indwelling catheters are at high risk of catheter-associated urinary tract infection (CAUTI). We hypothesized that colonizing the bladder with a benign E. coli strain (E. coli HU2117, a derivative of E. coli 83972 would prevent CAUTI in older, catheterized adults. Materials and Methods Adults with chronic, indwelling urinary catheters received study catheters that had been pre-coated with E. coli HU2117. We monitored the cultivatable organisms in the bladder for 28 days or until loss of E. coli HU2117. Urine from 4 subjects was collected longitudinally for 16S rRNA gene profiling. Results Eight of the ten subjects (average age 70.9 years) became colonized with E. coli HU2117, with a mean duration of 57.7 days (median: 28.5, range 0-266). All subjects also remained colonized by uropathogens. Five subjects suffered invasive UTI, 3 febrile UTI and 2 urosepsis/bacteremia, all associated with overgrowth of a urinary pathogen. Colonization with E. coli HU2117 did not impact bacterial bladder diversity, but subjects who developed infections had less diverse bladder microbiota. Conclusions Colonization with E. coli HU2117 did not prevent bladder colonization or subsequent invasive disease by uropathogens. Microbial diversity may play a protective role against invasive infection of the catheterized bladder.

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Section: Discussionmentioning

confidence: 99%

Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference

Horwitz

McCue

Mapes

et al. 2015

Journal of Infection

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“…Metaproteomic methods have enabled a deeper characterization of the inflammatory response towards uropathogens in cases of UTI and asymptomatic bacteriuria [5,6]. Several studies have characterized the urinary microbiome by 16S rRNA sequencing to study adults with a cumulative number of 253 subjects investigated across these studies (UTI, n=50; other urinary manifestations, n=154; sexually transmitted diseases, n=20; renal transplant samples, n=60) [4,[7][8][9][10][11][12][13][14][15][16][17][18][19]. However, a complete view of the microbiome, including eukarya and viruses, as well as an unbiased characterization of abundance and the identification of virulence factors as presented in this study can only be achieved by comprehensive metagenome analyses using whole genome shotgun sequencing (WGS).…”

Section: Introductionmentioning

confidence: 99%

“…Nextera XT libraries were prepared manually following the manufacturer's protocol (Illumina). Briefly, samples were normalized to 0.2 ng/μl DNA material per library using a Quant-iT picogreen assay system (Life Technologies) on an AF2200 plate reader (Eppendorf), then fragmented and tagged via16 tagmentation. Amplification was performed by Veriti 96 well PCR (Applied Biosystems) followed by AMPure XP bead cleanup (Beckman Coulter).…”

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confidence: 99%

Microbial metagenome of urinary tract infection

Moustafa

Singh

et al. 2017

Preprint

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Urine culture and microscopy techniques are used to profile the bacterial species present in urinary tract infections. To gain insight into the urinary flora in infection and health, we analyzed clinical laboratory features and the microbial metagenome of 121 clean-catch urine samples. 16S rDNA gene signatures were successfully obtained for 116 participants, while whole genome shotgun sequencing data was successfully generated for samples from 49 participants. Analysis of these datasets supports the definition of the patterns of infection and colonization/contamination. Although 16S rDNA sequencing was more sensitive, whole genome shotgun sequencing allowed for a more comprehensive and unbiased representation of the microbial flora, including eukarya and viral pathogens, and of bacterial virulence factors. Urine samples positive by whole genome shotgun sequencing contained a plethora of bacterial (median 41 genera/sample), eukarya (median 2 species/sample) and viral sequences (median 3 viruses/sample). Genomic analyses revealed cases of infection with potential pathogens (e.g., Alloscardovia sp, Actinotignum sp, Ureaplasma sp) that are often missed during routine urine culture due to species specific growth requirements. We also observed gender differences in the microbial metagenome. While conventional microbiological methods are inadequate to identify a large diversity of microbial species that are present in urine, genomic approaches appear to comprehensively and quantitatively describe the urinary microbiome. 5 Results Clinical and laboratory data representation.To support an unbiased assessment of the clinical nature of the specimens, we approached the urine sample laboratory and microbiology data using dimensionality reduction, and K-mer clustering analysis. The PCA representation of the clinical laboratory data is presented in Fig 1. The PCA analysis showed that the first two components (PC1, PC2) explained 65% of the variance in the clinical laboratory dataset. PC1 was driven by the vaginal contamination score (VCO), PC2 was contributed primarily by neutrophil activation and degranulation score (NAD), and secondarily by the erythrocyte and vascular injury score (ERY) and the presence of red blood cells (RBC) and leukocytes (WBC) (Fig 1B). The partitioning around medoids clustering resulted in three Clusters, with 9 individuals in Cluster #1, 63 individuals in Cluster #2, and 49 individuals in Cluster #3 (Fig 1C). From these data, we established a preliminary definition of Cluster #1 as likely representing urine from non-infected individuals, while Clusters #2 and #3 are consistent with separate manifestations of infectious and inflammatory processes of the urinary tract. The performance of 16S rDNA and whole genome shotgun sequencing across clinical laboratory clusters is presented in Table 1. 16S rDNA sequencing. 16S rDNA sequencing was successful for 116 (96%) samples ( Table 1). The median (range) number of genera identified per individual was 38 (6-220). The median (range) number of genera varied acro...

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“…The 16S rRNA PCR assay was performed using the previously published primers, 803F (5′-TTA GAT ACC 119 CTG GTA GTC -3′) and 1392R (5′-ACG GGC GGT GTG TRC -3′) and PCR cycling conditions (Willner et al, 120 2014). Fusion primers with 454 adaptor sequences were ligated to the 803F and 1392R primers to 121 amplify the V5 and V8 regions of the 16S rRNA gene (Willner et al, 2014). PCR reactions were 122 performed as previously described (Pelzer et al, 2018).…”

Section: Next-generation Sequencing 118mentioning

confidence: 99%

Amplification of the V5 – V8 region of the 16S rRNA gene effectively speciates medically important genital tractLactobacillusspecies in the upper female genital tract

Willner

Buttini

et al. 2019

Preprint

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Amplification of the V5 -V8 region of the 16S rRNA gene effectively speciates medically important 2 genital tract Lactobacillus species in the upper female genital tract 3 4 AUTHORS 5 ABSTRACT 31Background: The endometrial cavity is an upper genital tract site largely heralded as sterile, however, 32 advances in culture-independent, next generation sequencing technology have revealed that this site 33 harbours a rich microbial community which includes multiple Lactobacillus species. These bacteria are 34 considered to be the most common non-pathogenic genital tract commensals. Next-generation 35 sequencing of the female lower genital tract has revealed significant variation amongst microbial 36 community composition with respect to Lactobacillus sp. in samples collected from healthy and 37 diseased women. The aim of this study was to evaluate the ability of the 16S rRNA gene to 38 characterize genital tract lactobacilli to species-level taxonomy. 39Methods: Samples were interrogated for the presence of microbial DNA using two-step next 40 generation sequencing technology to exploit the V5-V8 regions of the 16S rRNA gene and compared 41 to standard speciation using qPCR. 42Results: The V5-V8 region of the 16S rRNA gene has sufficient sequence variation within frequently 43 encountered genital tract lactobacilli to allow accurate determination of relative abundance within 44 the community, and speciation for several key community members without completing additional 45 experimentation. 46 Conclusions: Next-generation sequencing of clinical genital tract isolates is an effective method for 47 high throughput identification to species-level of key Lactobacillus sp. 48 KEYWORDS 49 Lactobacillus sp.; genital tract; 16S rRNA; pyrosequencing; qPCR; speciation 50 51 IMPORTANCE 52

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Single Clinical Isolates from Acute Uncomplicated Urinary Tract Infections Are Representative of Dominant In Situ Populations

Cited by 47 publications

References 82 publications

Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference

Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference

Microbial metagenome of urinary tract infection

Amplification of the V5 – V8 region of the 16S rRNA gene effectively speciates medically important genital tractLactobacillusspecies in the upper female genital tract

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