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A short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities.

Harrington CR, Lucchini S, Ridgway KP, Wegmann U, Eaton TJ, Hinton JC, Gasson MJ, Narbad A - BMC Microbiol. (2008)

Bottom Line: We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides.Short oligonucleotides consistently gave higher specificity.An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Commensals and Microflora, Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, Norfolk, NR4 7UA, UK. Carl.Harrington@bbsrc.ac.uk

ABSTRACT

Background: The human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract.

Results: We have developed a culture-independent, semi-quantitative, rapid method for detection of gut bacterial populations based on 16S rDNA probes using a DNA microarray. We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides. Short oligonucleotides consistently gave higher specificity. Optimal DNA amplification and labelling, hybridisation and washing conditions were determined using a probe with an increasing number of nucleotide mismatches, identifying the minimum number of nucleotides needed to distinguish between perfect and mismatch probes. An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population. The sensitivity of the microarray was determined to be 8.8 x 104 bacterial cells g-1 faecal sample, which is more sensitive than a number of existing profiling methods. The short oligonucleotide microarray was used to compare the faecal flora from healthy individuals and a patient suffering from Ulcerative Colitis (UC) during the active and remission states. Differences were identified in the bacterial profiles between healthy individuals and a UC patient. These variations were verified by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing.

Conclusion: In this study we demonstrate the design, testing and application of a highly sensitive, short oligonucleotide community microarray. Our approach allows the rapid discrimination of bacteria inhabiting the human GI tract, at taxonomic levels ranging from species to the superkingdom bacteria. The optimised protocol is available at: http://www.ifr.ac.uk/safety/microarrays/#protocols. It offers a high throughput method for studying the dynamics of the bacterial population over time and between individuals.

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GI tract bacterial changes in an Ulcerative Colitis patient during the disease state compared to remission. Selection of bacterial probes showing the fold change in signal intensity during the disease phase compared to remission, in a patient suffering UC. Black bars indicate probes whose signal is greater in the disease state and white bars are those probes which are greater in remission. Probe identities for the black bars are to the left of zero on the x axis and white bar probe identities are to the right of zero. The error bars represent the standard error.
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Figure 4: GI tract bacterial changes in an Ulcerative Colitis patient during the disease state compared to remission. Selection of bacterial probes showing the fold change in signal intensity during the disease phase compared to remission, in a patient suffering UC. Black bars indicate probes whose signal is greater in the disease state and white bars are those probes which are greater in remission. Probe identities for the black bars are to the left of zero on the x axis and white bar probe identities are to the right of zero. The error bars represent the standard error.

Mentions: The short oligonucleotide community microarray was used to investigate the bacterial community of a patient suffering from UC (Figure 4; Table 3; Additional file 5). One sample was taken whilst the patient was suffering from a relapse, and the second six months later during remission. The results indicated a significant difference in the bacterial community from this single individual at two distinct times, and under different circumstances. One of the most noticeable changes in the population during the active disease phase, compared to remission, was the elevated levels of bacteria belonging to Clostridia clusters IV and XIV, particularly E. biforme, E. rectale and the E. cylindroides Clusters. Other Clostridia such as Clostridium Cluster I revealed no significant differences between samples taken during the active disease phase and remission. Of the ruminococci, only the R. flavefaciens and R. albus & R. flavefaciens2 probes demonstrated large increases during the active state. The Roseburia intestinalis sub-cluster exhibited a 6.2-fold elevation in intensity, while Rumin-Eubac-Clost Cluster levels increased by 1.9-fold, the difference probably reflecting the broader specificity of the latter probe. During the disease state, Enterobacteriaceae and the Bifidobacterium longum group were more abundant.


A short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities.

Harrington CR, Lucchini S, Ridgway KP, Wegmann U, Eaton TJ, Hinton JC, Gasson MJ, Narbad A - BMC Microbiol. (2008)

GI tract bacterial changes in an Ulcerative Colitis patient during the disease state compared to remission. Selection of bacterial probes showing the fold change in signal intensity during the disease phase compared to remission, in a patient suffering UC. Black bars indicate probes whose signal is greater in the disease state and white bars are those probes which are greater in remission. Probe identities for the black bars are to the left of zero on the x axis and white bar probe identities are to the right of zero. The error bars represent the standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2628385&req=5

Figure 4: GI tract bacterial changes in an Ulcerative Colitis patient during the disease state compared to remission. Selection of bacterial probes showing the fold change in signal intensity during the disease phase compared to remission, in a patient suffering UC. Black bars indicate probes whose signal is greater in the disease state and white bars are those probes which are greater in remission. Probe identities for the black bars are to the left of zero on the x axis and white bar probe identities are to the right of zero. The error bars represent the standard error.
Mentions: The short oligonucleotide community microarray was used to investigate the bacterial community of a patient suffering from UC (Figure 4; Table 3; Additional file 5). One sample was taken whilst the patient was suffering from a relapse, and the second six months later during remission. The results indicated a significant difference in the bacterial community from this single individual at two distinct times, and under different circumstances. One of the most noticeable changes in the population during the active disease phase, compared to remission, was the elevated levels of bacteria belonging to Clostridia clusters IV and XIV, particularly E. biforme, E. rectale and the E. cylindroides Clusters. Other Clostridia such as Clostridium Cluster I revealed no significant differences between samples taken during the active disease phase and remission. Of the ruminococci, only the R. flavefaciens and R. albus & R. flavefaciens2 probes demonstrated large increases during the active state. The Roseburia intestinalis sub-cluster exhibited a 6.2-fold elevation in intensity, while Rumin-Eubac-Clost Cluster levels increased by 1.9-fold, the difference probably reflecting the broader specificity of the latter probe. During the disease state, Enterobacteriaceae and the Bifidobacterium longum group were more abundant.

Bottom Line: We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides.Short oligonucleotides consistently gave higher specificity.An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Commensals and Microflora, Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, Norfolk, NR4 7UA, UK. Carl.Harrington@bbsrc.ac.uk

ABSTRACT

Background: The human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract.

Results: We have developed a culture-independent, semi-quantitative, rapid method for detection of gut bacterial populations based on 16S rDNA probes using a DNA microarray. We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides. Short oligonucleotides consistently gave higher specificity. Optimal DNA amplification and labelling, hybridisation and washing conditions were determined using a probe with an increasing number of nucleotide mismatches, identifying the minimum number of nucleotides needed to distinguish between perfect and mismatch probes. An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population. The sensitivity of the microarray was determined to be 8.8 x 104 bacterial cells g-1 faecal sample, which is more sensitive than a number of existing profiling methods. The short oligonucleotide microarray was used to compare the faecal flora from healthy individuals and a patient suffering from Ulcerative Colitis (UC) during the active and remission states. Differences were identified in the bacterial profiles between healthy individuals and a UC patient. These variations were verified by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing.

Conclusion: In this study we demonstrate the design, testing and application of a highly sensitive, short oligonucleotide community microarray. Our approach allows the rapid discrimination of bacteria inhabiting the human GI tract, at taxonomic levels ranging from species to the superkingdom bacteria. The optimised protocol is available at: http://www.ifr.ac.uk/safety/microarrays/#protocols. It offers a high throughput method for studying the dynamics of the bacterial population over time and between individuals.

Show MeSH
Related in: MedlinePlus