scholarly journals Rapid Identification of Bacteria from Positive Blood Cultures by Fluorescence-Based PCR–Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene

2000 ◽  
Vol 38 (2) ◽  
pp. 513-520 ◽  
Author(s):  
Christine Y. Turenne ◽  
Evelyn Witwicki ◽  
Daryl J. Hoban ◽  
James A. Karlowsky ◽  
Amin M. Kabani
Keyword(s):  
Blood Culture ◽  
16S Rrna ◽  
Single Strand Conformation Polymorphism ◽  
Rapid Identification ◽  
Blood Cultures ◽  
Rrna Gene ◽  
Pathogen Identification ◽  
Reference Collection ◽  
The 16S Rrna Gene ◽  
Conformation Polymorphism

Bacteremia continues to result in significant morbidity and mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impose significant delays in and limitations to pathogen identification. To address the limitations of growth-based identification, the sequence variability of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluorescence-based PCR–single-strand conformation polymorphism (SSCP) protocol. Species-specific SSCP patterns were determined for 25 of the most common bacterial species isolated from blood cultures; these isolates subsequently served as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns distinct from those in the reference collection. Time to identification from blood culture positivity ranged from 1 to 8 days with biochemical testing, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect mixed cultures as well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identification would result in a timely diagnosis with possible implications for patient management costs and the mortality and morbidity of infections.

scholarly journals Capillary Electrophoresis–Single-Strand Conformation Polymorphism Analysis for Rapid Identification of Pseudomonas aeruginosa and Other Gram-Negative Nonfermenting Bacilli Recovered from Patients with Cystic Fibrosis

1999 ◽  
Vol 37 (10) ◽  
pp. 3374-3379 ◽  
Author(s):  
Rafiaa Ghozzi ◽  
Philippe Morand ◽  
Agnes Ferroni ◽  
Jean-Luc Beretti ◽  
Edouard Bingen ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Capillary Electrophoresis ◽  
16S Rrna ◽  
Single Strand Conformation Polymorphism ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Gram Negative ◽  
Reference Strains ◽  
Conformation Polymorphism

We used capillary electrophoresis–single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF). Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes. The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer. Data were analyzed with GeneScan 672 software. This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers. Thirty-four reference strains belonging to the genera Pseudomonas,Brevundimonas, Burkholderia,Comamonas, Ralstonia,Stenotrophomonas, and Alcaligenes were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism. The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (Escherichia coli 16S rRNA positions 1173 to 1389 [M. N. Widjojoatmodjo, A. C. Fluit, and J. Verhoef, J. Clin. Microbiol. 32:3002–3007, 1994]). This primer set differentiated the main CF pathogens from closely related species but did not distinguishP. aeruginosa from Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes and Alcaligenes xylosoxidans from Alcaligenes denitrificans. Two hundred seven CF clinical isolates (153 of P. aeruginosa, 26 of Stenotrophomonas maltophilia, 15 ofBurkholderia spp., and 13 of A. xylosoxidans) were tested with P11P-P13P. The CE-SSCP patterns obtained were identical to those for the corresponding reference strains. Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains. This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF.

scholarly journals Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis

2003 ◽  
Vol 69 (7) ◽  
pp. 3840-3848 ◽  
Author(s):  
Frédérique Duthoit ◽  
Jean-Jacques Godon ◽  
Marie-Christine Montel
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
Gram Positive ◽  
Leuconostoc Pseudomesenteroides ◽  
The 16S Rrna Gene ◽  
Conformation Polymorphism

ABSTRACT Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products.

Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene

2006 ◽  
Vol 66 (1) ◽  
pp. 156-164 ◽  
Author(s):  
Inge Vliegen ◽  
Jan A. Jacobs ◽  
Erik Beuken ◽  
Cathrien A. Bruggeman ◽  
Cornelis Vink
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Identification Of Bacteria ◽  
Time Amplification ◽  
The 16S Rrna Gene

scholarly journals Direct Rapid Identification from Positive Blood Cultures by MALDI-TOF MS: Specific Focus on Turnaround Times

2021 ◽  
Author(s):  
Hazan Zengin Canalp ◽  
Banu Bayraktar
Keyword(s):  
Blood Culture ◽  
Rapid Identification ◽  
Blood Cultures ◽  
Pathogen Identification ◽  
Short Term ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Solid Media ◽  
Time Required ◽  
Tof Ms

Using MALDI-TOF MS directly from blood culture bottles reduces the time required for pathogen identification, and the turnaround times for final identification have been compared with overnight incubation from solid media in previous studies. However, identification from a short incubation of agar plates has been increasingly accepted and successfully implemented in routine laboratories, but there is no data comparing direct MALDI-TOF MS with the short-term, incubated agar plates.

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