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 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 Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)—Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria

2000 ◽  
Vol 66 (8) ◽  
pp. 3556-3565 ◽  
Author(s):  
Frank Schwieger ◽  
Christoph C. Tebbe
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Diversity ◽  
Sinorhizobium Meliloti ◽  
Chenopodium Album ◽  
Single Strand Conformation Polymorphism ◽  
Rrna Gene ◽  
Field Plots ◽  
Target Plant ◽  
Conformation Polymorphism

ABSTRACT Fourteen weeks after field release of luciferase gene-taggedSinorhizobium meliloti L33 in field plots seeded withMedicago sativa, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of M. sativa plants, S. meliloti L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of M. sativa and Chenopodium album, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the Proteobacteria (α, β, and γ subgroups), members of the Cytophaga-Flavobacterium group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case ofM. sativa, by inoculation. The most abundant isolates were related to Variovorax sp., Arthrobacter ramosus, and Acinetobacter calcoaceticus. In the rhizosphere of M. sativa, inoculation reduced the numbers of cells of A. calcoaceticus and members of the genus Pseudomonas and increased the number of rhizobia. Cultivation-independent PCR–single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except Variovoraxspecies could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation.

Simultaneous Identification of Seven Foodborne Pathogens and Escherichia coli (Pathogenic and Nonpathogenic) Using Capillary Electrophoresis–Based Single-Strand Conformation Polymorphism Coupled with Multiplex PCR

2009 ◽  
Vol 72 (6) ◽  
pp. 1262-1266 ◽  
Author(s):  
MI-HWA OH ◽  
SE-HEE PAEK ◽  
GI WON SHIN ◽  
HAE-YEONG KIM ◽  
GYOO YEOL JUNG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Capillary Electrophoresis ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Pcr Amplification ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Rrna Gene ◽  
Optimal Separation ◽  
Conformation Polymorphism

The objective of this study was to develop a novel technique for parallel analysis of eight important foodborne microbes using capillary electrophoresis–based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex PCR. Specific primers for multiplex PCR amplification of the 16S rRNA gene were designed, corresponding to eight species of bacteria, including Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Salmonella enterica, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus cereus, for the species-specific identification and optimal separation of their PCR products in subsequent analysis by CE-SSCP. Multiplex PCR conditions including annealing temperature, extension time, the number of PCR cycles, and primer concentrations were then optimized for simultaneous detection of all target foodborne bacteria. The diagnostic system using CE-SSCP combined with multiplex PCR developed here can be used for rapid investigation of causative agents of foodborne illness. The simplicity and high sensitivity of the method may lead to improved management of safety and illness related to food.

scholarly journals Molecular Characterization and Identification ofBacillus clausii Strains Marketed for Use in Oral Bacteriotherapy

2001 ◽  
Vol 67 (2) ◽  
pp. 834-839 ◽  
Author(s):  
Sonia Senesi ◽  
Francesco Celandroni ◽  
Arianna Tavanti ◽  
Emilia Ghelardi
Keyword(s):  
Intergenic Spacer ◽  
Single Strand Conformation Polymorphism ◽  
Trna Genes ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Spacer Length ◽  
Dna Reassociation ◽  
Reference Strains ◽  
Physiological And Biochemical ◽  
Conformation Polymorphism

ABSTRACT A substantial number of Bacillus species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the Bacillus strains in Enterogermina, which is made up of aqueous suspensions of viable Bacillusspores, have been partially characterized and aligned with members of the Bacillus alcalophilus subgroup rather than withBacillus subtilis, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four Bacillus strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species Bacillus clausii. Moreover, we provide evidence that in contrast to several reference strains ofB. clausii, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.

scholarly journals A New Approach To Utilize PCR–Single-Strand-Conformation Polymorphism for 16S rRNA Gene-Based Microbial Community Analysis

1998 ◽  
Vol 64 (12) ◽  
pp. 4870-4876 ◽  
Author(s):  
Frank Schwieger ◽  
Christoph C. Tebbe
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Pure Culture ◽  
Community Analysis ◽  
Single Strand Conformation Polymorphism ◽  
Microbial Community Analysis ◽  
Rrna Gene ◽  
Pcr Products ◽  
Conformation Polymorphism

ABSTRACT Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.

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