BackgroundUrine within the urinary tract is commonly regarded as "sterile" in cultivation terms. Here, we present a comprehensive in-depth study of bacterial 16S rDNA sequences associated with urine from healthy females by means of culture-independent high-throughput sequencing techniques.ResultsSequencing of the V1V2 and V6 regions of the 16S ribosomal RNA gene using the 454 GS FLX system was performed to characterize the possible bacterial composition in 8 culture-negative (<100,000 CFU/ml) healthy female urine specimens. Sequences were compared to 16S rRNA databases and showed significant diversity, with the predominant genera detected being Lactobacillus, Prevotella and Gardnerella. The bacterial profiles in the female urine samples studied were complex; considerable variation between individuals was observed and a common microbial signature was not evident. Notably, a significant amount of sequences belonging to bacteria with a known pathogenic potential was observed. The number of operational taxonomic units (OTUs) for individual samples varied substantially and was in the range of 20 - 500.ConclusionsNormal female urine displays a noticeable and variable bacterial 16S rDNA sequence richness, which includes fastidious and anaerobic bacteria previously shown to be associated with female urogenital pathology.
BackgroundInterstitial Cystitis (IC) is a chronic inflammatory condition of the bladder with unknown etiology. The aim of this study was to characterize the microbial community present in the urine from IC female patients by 454 high throughput sequencing of the 16S variable regions V1V2 and V6. The taxonomical composition, richness and diversity of the IC microbiota were determined and compared to the microbial profile of asymptomatic healthy female (HF) urine.ResultsThe composition and distribution of bacterial sequences differed between the urine microbiota of IC patients and HFs. Reduced sequence richness and diversity were found in IC patient urine, and a significant difference in the community structure of IC urine in relation to HF urine was observed. More than 90% of the IC sequence reads were identified as belonging to the bacterial genus Lactobacillus, a marked increase compared to 60% in HF urine.ConclusionThe 16S rDNA sequence data demonstrates a shift in the composition of the bacterial community in IC urine. The reduced microbial diversity and richness is accompanied by a higher abundance of the bacterial genus Lactobacillus, compared to HF urine. This study demonstrates that high throughput sequencing analysis of urine microbiota in IC patients is a powerful tool towards a better understanding of this enigmatic disease.
BackgroundReduced salivation is considered a major clinical feature of most but not all cases of primary Sjögren’s syndrome (pSS). Reduced saliva flow may lead to changes in the salivary microbiota. These changes have mainly been studied with culture that typically recovers only 65% of the bacteria present.ObjectiveThis study was to use high throughput sequencing, covering both cultivated and not-yet-cultivated bacteria, to assess the bacterial microbiota of whole saliva in pSS patients with normal salivation.MethodsBacteria of whole unstimulated saliva from nine pSS patients with normal salivation flow and from nine healthy controls were examined by high throughput sequencing of the hypervariable region V1V2 of 16S rRNA using the 454 GS Junior system. Raw sequence reads were subjected to a species-level, reference-based taxonomy assignment pipeline specially designed for studying the human oral microbial community. Each of the sequence reads was BLASTN-searched against a database consisting of reference sequences representing 1,156 oral and 12,013 non-oral species. Unassigned reads were then screened for high-quality non-chimeras and subjected to de novo species-level operational taxonomy unit (OTU) calling for potential novel species. Downstream analyses, including alpha and beta diversities, were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. To reveal significant differences between the microbiota of control saliva and Sjögren’s saliva, a statistical method introduced in Metastats www.metastats.cbcb.umd.edu was used.ResultsSaliva of pSS patients with normal salivation had a significantly higher frequency of Firmicutes compared with controls (p=0.004). Two other major phyla, Synergistetes and Spirochaetes, were significantly depleted in pSS (p=0.001 for both). In addition, we saw a nearly 17% decrease in the number of genera in pSS (25 vs. 30). While Prevotella was almost equally abundant in both groups (25% in pSS and 22% in controls), about a twofold increase in pSS of Streptococcus (28% vs. 17%) and Veillonella (26% vs. 12%) was detected. Prevotella melaninogenica was the major species in controls (13%) while Veillonella atypica and the Veillonella parvula groups dominated in patient samples (14 and 14%). The scarcity in bacterial species in pSS compared with controls was also demonstrated by alpha and beta diversity analyses, as well as read abundance depicted in a phylogenetic tree.ConclusionsWhile Firmicutes was significantly higher in pSS patients than in controls, Synergistetes and Spirochaetes were significantly lower. The number of bacterial genera and species was also lower. These data showed that microbial dysbiosis is another key characteristic of pSS whole saliva which can occur independent of hyposalivation.
The present study describes the clinical characteristics of patients with systemic lupus erythematosus (SLE), from the rheumatology service of the two main teaching hospitals in Kuwait. It was a retrospective-cum-prospective clinical study of 108 SLE patients. There were 98 females and 10 males, with a median age of 31.5y. Kuwaitis constituted 69%, while 31% were expatriates. The mean disease duration was 62 months. The main clinical features were: musculoskeletal involvement (87%), photosensitivity (48%), malar rash (43%), discoid lesions (10%), oral ulcers (33%), vasculitic skin lesions (10%), haematological features (53%), constitutional symptoms (51.4%), neuropsychiatric manifestations (23%), renal involvement (37%), serositis (29%), clinical manifestations of antiphospholipid syndrome (21%), cardiac involvement (10%) and pulmonary manifestations (19%). In conclusion, the clinical features of SLE in Kuwait were similar to most major studies from developed countries. Main differences included prominent haematological and mucocutaneous manifestations and possibly a low prevalence of anti-Sm antibodies. Whether these differences are due to the environment or genetic factors, remains to be studied.
Currently, genome sequences of a total of 19 Porphyromonas gingivalis strains are available, including eight completed genomes (strains W83, ATCC 33277, TDC60, HG66, A7436, AJW4, 381, and A7A1-28) and 11 high-coverage draft sequences (JCVI SC001, F0185, F0566, F0568, F0569, F0570, SJD2, W4087, W50, Ando, and MP4-504) that are assembled into fewer than 300 contigs. The objective was to compare these genomes at both nucleotide and protein sequence levels in order to understand their phylogenetic and functional relatedness. Four copies of 16S rRNA gene sequences were identified in each of the eight complete genomes and one in the other 11 unfinished genomes. These 43 16S rRNA sequences represent only 24 unique sequences and the derived phylogenetic tree suggests a possible evolutionary history for these strains. Phylogenomic comparison based on shared proteins and whole genome nucleotide sequences consistently showed two groups with closely related members: one consisted of ATCC 33277, 381, and HG66, another of W83, W50, and A7436. At least 1,037 core/shared proteins were identified in the 19 P. gingivalis genomes based on the most stringent detecting parameters. Comparative functional genomics based on genome-wide comparisons between NCBI and RAST annotations, as well as additional approaches, revealed functions that are unique or missing in individual P. gingivalis strains, or species-specific in all P. gingivalis strains, when compared to a neighboring species P. asaccharolytica. All the comparative results of this study are available online for download at ftp://www.homd.org/publication_data/20160425/.
Introduction: Overactive bladder syndrome (OAB) is described as urgency, with or without urgency incontinence. A range of medical conditions shares the symptoms of OAB, however the diagnosis is contingent on the exclusion of urinary tract infection (UTI). Knowing that urine dipstick and routine culture of bacteria can miss UTI diagnosis caused by low-count bacteriuria or “difficult-to-culture” pathogens, we examined a case of OAB with a culture-independent approach. Case presentation:A 61-year-old Norwegian female with a long history of urinary symptoms and a diagnosis of OAB was selected as a suitable subject for a culture-independent 16S rDNA analysis on the patient´s urine. The patient’s medical records showed no history of recurrent UTI, however, when the urine specimen was sent to routine culture at the time of study it showed a significant bacteriuria caused by a single bacterium, and the patient was prescribed antibiotics. The 16S rDNA analysis revealed not one, but many different bacteria, including a considerable amount of fastidious bacteria, indicating a polymicrobial state. One year later, the subject was still experiencing severe symptoms, and a follow-up analysis was performed. This time the urine-culture was negative, however, the 16S rDNA profile was quite similar to that of the first sample, again displaying a complex bacterial profile. Conclusion: The use of 16S rDNA pyrosequencing and sequence analysis to uncover “difficult-to-culture” bacteria should be considered when examining patients with chronic urinary symptoms. These methods may contribute to further elucidation of the etiology of overactive bladder syndrome and other urinary syndromes.
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