Use of denaturing gradient gel electrophoresis for analysis of the stool microbiota of hospitalized patients

Donskey, Curtis J.; Hujer, Andrea M.; Das, Sarbani M.; Pultz, Nicole J.; Bonomo, Robert A.; Rice, Louis B.

doi:10.1016/s0167-7012(03)00059-9

Cited by 103 publications

(69 citation statements)

References 15 publications

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“…Thus they inhibit susceptible organisms and select for resistant ones. A number of different molecular fingerprinting techniques have previously been used to analyse the impact of antibiotics on the faecal microbiota such as denaturing or temperature gradient gel electrophoresis (DGGE or TGGE) (Donskey et al, 2003;Harmoinen et al, 2004), temporal TGGE (TTGE) (De La Cochetiere et al, 2005), terminal restriction fragment length polymorphism (T-RFLP) (Jernberg et al, 2005;Engelbrektson et al, 2006) and denaturing high-performance liquid chromatography (DHPLC) (Goldenberg et al, 2006). These techniques all bypass the necessity for cultivation, enabling comparative analyses of community fingerprints and the ability to monitor relative shifts in specific populations.…”

Section: Introductionmentioning

confidence: 99%

Long-term ecological impacts of antibiotic administration on the human intestinal microbiota

et al. 2007

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Antibiotic administration is known to cause short-term disturbances in the microbiota of the human gastrointestinal tract, but the potential long-term consequences have not been well studied. The aims of this study were to analyse the long-term impact of a 7-day clindamycin treatment on the faecal microbiota and to simultaneously monitor the ecological stability of the microbiota in a control group as a baseline for reference. Faecal samples from four clindamycin-exposed and four control subjects were collected at nine different time points over 2 years. Using a polyphasic approach, we observed highly significant disturbances in the bacterial community that persisted throughout the sampling period. In particular, a sharp decline in the clonal diversity of Bacteroides isolates, as assessed by repetitive sequence-based PCR (rep-PCR) and long-term persistence of highly resistant clones were found as a direct response to the antibiotic exposure. The Bacteroides community never returned to its original composition during the study period as assessed using the molecular fingerprinting technique, terminal restriction fragment length polymorphism (T-RFLP). Furthermore, using real-time PCR we found a dramatic and persistent increase in levels of specific resistance genes in DNA extracted from the faeces after clindamycin administration. The temporal variations in the microbiota of the control group were minor compared to the large and persistent shift seen in the exposed group. These results demonstrate that long after the selection pressure from a short antibiotic exposure has been removed, there are still persistent long term impacts on the human intestinal microbiota that remain for up to 2 years post-treatment.

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Section: Introductionmentioning

confidence: 99%

Long-term ecological impacts of antibiotic administration on the human intestinal microbiota

et al. 2007

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“…from the Day 28 faecal samples (five samples for each group) was extracted according to the bead-beating method (Donskey et al, 2003). After extraction, the total DNA was precipitated with 80% …”

Section: Dna Extraction and Pcr Amplification Total Genomic Dnamentioning

confidence: 99%

The Effects of Rebaudioside A on Microbial Diversity in Mouse Intestine

Chen

Dong

et al. 2014

FSTR

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Rebaudioside A (RA), a natural high potency sweetener, is isolated from the leaves of the Stevia rebaudiana plant, and has potential for wide use in our diet today. To investigate the effect of RA on the changes of microbial diversity in animal intestinal tract, the viable cell count and denaturing gradient gel electrophoresis (DGGE) method were used to monitor the microbial number and species in vivo, and before that, we also evaluated its influence on the growth of some selected bacteria in vitro. Our results indicated that the RA had little or no effect on the growth of E. coli O157 H7, S. typhimurium, L. monocytogenes and B. longum, while could promote the growth of L. plantarum and inhibit that of S. aureus in vitro; moreover, the viable cell count and DGGE results confirmed that the RA posed little pressure on the composition of total bacteria, enterobacteria and lactobacilli in vivo. In conclusion, RA posed little pressure on the growth and the composition of microbes, suggesting it is safe for gut microbes.

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“…Techniques such as denaturing gradient gel electrophoresis (DGGE) (8,20,21,27), terminal restriction fragment length polymorphism (15,22,28,38), length heterogeneity PCR (29, 34), automated rRNA intergenic spacer analysis (13), and ribosomal intergenic spacer length polymorphism (9, 40) are now widely used and reported in the literature. In the in silico analyses of 41 completely sequenced bacterial genomes, DGGE appeared to be one of the best molecular community fingerprinting techniques in terms of predicting the actual Shannon-Wiener diversity index, richness, and evenness (3).…”

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confidence: 99%

Comparisons of Different Hypervariable Regions ofrrsGenes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis

Morrison

2004

Appl Environ Microbiol

433

300

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Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-meltingtemperature (T m(L) ) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to T m(L) . The average T m(L) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.The inherent limitations associated with cultivation-based approaches to characterizing microbial communities are widely recognized, and a number of cultivation-independent, molecular methods have emerged in recent years to improve our understanding of this aspect of microbial ecology. Techniques such as denaturing gradient gel electrophoresis (DGGE) (8,20,21,27), terminal restriction fragment length polymorphism (15,22,28,38), length heterogeneity PCR (29, 34), automated rRNA intergenic spacer analysis (13), and ribosomal intergenic spacer length polymorphism (9, 40) are now widely used and reported in the literature. In the in silico analyses of 41 completely sequenced bacterial genomes, DGGE appeared to be one of the best molecular community fingerprinting techniques in terms of predicting the actual Shannon-Wiener diversity index, richness, and evenness (3). Additionally, DGGE supports the identification of community members because the amplification products can be recovered and sequenced (4,6,20,31). This may explain why DGGE has become the most frequently used method of molecular community fingerprinting.In PCR-DGGE, either a single hypervariable (V) region or a combinatio...

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Use of denaturing gradient gel electrophoresis for analysis of the stool microbiota of hospitalized patients

Cited by 103 publications

References 15 publications

Long-term ecological impacts of antibiotic administration on the human intestinal microbiota

Long-term ecological impacts of antibiotic administration on the human intestinal microbiota

The Effects of Rebaudioside A on Microbial Diversity in Mouse Intestine

Comparisons of Different Hypervariable Regions ofrrsGenes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis

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