Detection of a mixed infection in a culture-negative brain abscess by broad-spectrum bacterial 16S rRNA gene PCR.
Ontology highlight
ABSTRACT:
SUBMITTER: Keller PM
PROVIDER: S-EPMC2884506 | biostudies-literature | 2010 Jun
REPOSITORIES: biostudies-literature
Publications
Detection of a mixed infection in a culture-negative brain abscess by broad-spectrum bacterial 16S rRNA gene PCR.
Keller Peter M PM Rampini Silvana K SK Bloemberg Guido V GV
Journal of clinical microbiology 20100414 6
We describe the identification of two bacterial pathogens from a culture-negative brain abscess by the use of broad-spectrum 16S rRNA gene PCR. Simultaneous detection of Fusobacterium nucleatum and Porphyromonas endodontalis was possible due to a 24-bp length difference of their partially amplified 16S rRNA genes, which allowed separation by high-resolution polyacrylamide gel electrophoresis. ...[more]
Similar Datasets
Project description:While 16S rRNA PCR-Sanger sequencing has paved the way for the diagnosis of culture-negative bacterial infections, it does not provide the composition of polymicrobial infections. We aimed to evaluate the performance of the Nanopore-based 16S rRNA metagenomic approach, using both partial and full-length amplification of the gene, and to explore its feasibility and suitability as a routine diagnostic tool for bacterial infections in a clinical laboratory. Thirty-one culture-negative clinical samples from mono- and polymicrobial infections based on Sanger-sequencing results were sequenced on MinION using both the in-house partial amplification and the Nanopore dedicated kit for the full-length amplification of the 16S rRNA gene. Contamination, background noise definition, bacterial identification, and time-effectiveness issues were addressed. Cost optimization was also investigated with the miniaturized version of the flow cell (Flongle). The partial 16S approach had a greater sensitivity compared to the full-length kit that detected bacterial DNA in only 24/31 (77.4%) samples. Setting a threshold of 1% of total reads overcame the background noise issue and eased the interpretation of clinical samples. Results were obtained within 1 day, discriminated polymicrobial samples, and gave accurate bacterial identifications compared to Sanger-based results. We also found that multiplexing and using Flongle flow cells was a cost-effective option. The results confirm that Nanopore technology is user-friendly as well as cost- and time-effective. They also indicate that 16S rRNA targeted metagenomics is a suitable approach to be implemented for the routine diagnosis of culture-negative samples in clinical laboratories.
| S-EPMC9329087 | biostudies-literature
Project description:Clostridium sordellii is usually associated with skin and soft tissue infections. We describe the first case to our knowledge of a Clostridium sordellii-associated brain abscess, diagnosed by 16S rRNA gene sequencing, expanding the microbiological spectrum of brain abscesses, with emphasis on the role of 16S rRNA gene PCR in their etiologic diagnosis.
| S-EPMC2937712 | biostudies-literature
Project description:The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture ("16S Capture"). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 Staphylococcus aureus genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA.
| S-EPMC7685877 | biostudies-literature
Project description:OBJECTIVE:Microbial community profiling using 16S rRNA gene has provided invaluable insights into diverse microbial communities. Recently a few studies have attempted to use 16S rRNA gene microbiota profiling in combination with the conventional culture methods to explore bacterial communities. In this "culture-enriched microbiota profiling" approach, microbes in a sample are cultured on solid media, and the resulting colonies are combined and subjected to 16S rRNA gene microbiota profiling. Here we investigated the effect of cell densities as determined by varying levels of sample dilution on the culture-enriched microbiota profiles using De Man, Rogosa and Sharpe (MRS) agar medium as a model system. RESULTS:Cecal samples collected from 10 healthy chickens were serially diluted to 102 fold (M-LOW), 104 fold (M-MEDIUM), and 106 fold (M-HIGH), and the dilutions were plated on MRS agar. 16S rRNA gene profiling showed that the relative abundance of certain genera showed gradual increase (Pediococcus and Enterococcus) or decrease (Lactobacillus and Turicibacter) with higher dilutions, though it was significant only for Pediococcus (p?<?0.05). The result indicates that the dilution levels of the samples can alter the resulting microbiota profiles via unknown density-dependent mechanisms and thus should be considered for designing experiments using culture-enriched microbiota profiling.
| S-EPMC7268277 | biostudies-literature
Project description:According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.
| S-EPMC4319838 | biostudies-literature
Project description:Forty-one stored samples from cases of spontaneous brain abscess were investigated to gain insight into the natural history, causative agents, and relevant laboratory diagnostics of a rare infection. Samples from a larger collection were selected based on retrospective analysis of patient records. All samples were subjected to amplicon sequencing of 16S rRNA gene fragments. Supplementary culture on selected media was performed as suggested by bioinformatics analysis. For three cases, no microorganism was disclosed, while Toxoplasma gondii, Aspergillus fumigatus, and various bacteria were the cause of 1, 2, and 35 cases, respectively. Bacterial infections were monomicrobial in 20 cases and polymicrobial in 15; the microorganisms of the latter cases were restricted to residents of cavum oris. Amplicon sequencing did not further enhance the importance of the Streptococcus anginosus group, which was involved in 17 cases, and the single primer set used may be suboptimal for amplification of Actinomyces and Nocardia. But, amplicon-based sequencing unquestionably expanded the number of polybacterial infections, with focus on the Fusobacterium nucleatum group, Parvimonas, and Porphyromonas. Culture on selective media confirmed the presence of F. nucleatum group bacteria, which attained a prominence in spontaneous brain abscess similar to the S. anginosus group. Metagenomics is a powerful tool to disclose the spectrum of agents in polymicrobial infections, but a reliable cutoff value for substantial detection is complex. Commercial media for isolation of F. nucleatum group bacteria from mixed infections are available, and these pathogens should be carefully characterized. Isolation of Parvimonas and Porphyromonas in polymicrobial infections has not been resolved. IMPORTANCE Polymicrobial brain abscess is a challenge to the clinical microbiology laboratory due to the aggregative nature of the dental and oral microbiota. Because polymicrobial infections may escape detection by conventional culture methods, directed therapy toward a single detected bacterium is problematic. Amplicon-based sequencing provides important clues to these infections, but only cultured microorganisms can be fully characterized, subjected to antimicrobial susceptibility testing, and formally named. By use of specific selective culture plates, we successfully isolated bacteria of the Fusobacterium nucleatum group, and these bacteria rose to the same prominence as the widely recognized pathogen, the Streptococcus anginosus group. Named and unnamed members of the Fusobacterium nucleatum group must be further investigated to gain insight into a rare but grave disease.
| S-EPMC9045185 | biostudies-literature
Project description:Here we report 16S rRNA data in gut microbiota of autism spectrum disorders compared with healthy volunteers. A total of 1322 operational taxonomic units (OTUs) were identified in the sequence data. The Bacteroidetes and Firmicutes were both dominated phylum in ausitic subjects and healthy controls. Phylum level analysis showed a clear alteration of the bacterial gut community in ASD characterized by a higher Firmicutes (P < 0.05), Proteobacteria (P < 0.001), and Actinobacteria (P < 0.001) than that in healthy controls. However, Bacteroidetes were significantly decreased in ASD patients (P < 0.001).
2020-09-30 | GSE113690 | GEO
Project description:This article contains data on the bacterial communities and its diversity associated with Anadara granosa. The A. granosa samples were obtained from two major estuaries in Penang, Malaysia using a culture dependent and 16S rRNA Illumina sequencing approaches. A. granosa, a commercial blood cockles and popular seafoods, is fragile to the surrounding environments. Thus, our research focused to better understand the bacterial communities and it diversity in the A. granosa, as well as on the generation of a metagenomic library from A. granosa to further understanding on it diversity. The bacteria Vibrionaceae (34.1%) was predominant in the A. granosa from both environments followed by Enterobacteriaceae (33.3%) and Bacillaceae (16.75%). Vibrio sp., Klebsiella sp., and Bacillus subtilis were the most abundant species present. The data generated in this research is the first metagenomic examination of A. granosa and will provide as a baseline to understand the bacterial communities associated with A. granosa and its surrounding natural environments.
| S-EPMC5997914 | biostudies-literature
Project description:Effect of ingestion of different types oils in the microbial diversity of overweight women
| PRJNA542277 | ENA
Project description:Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and -negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.
| S-EPMC3490953 | biostudies-literature