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Identification and characterisation of Pseudomonas 16S ribosomal DNA from ileal biopsies of children with Crohn's disease.

ABSTRACT: Molecular analysis of bacterial 16S rRNA genes has made a significant contribution to the identification and characterisation of bacterial flora in the human gut. In particular, this methodology has helped characterise bacterial families implicated in the aetiology of inflammatory bowel disease (IBD). In this study we have used a genus specific bacterial 16S PCR to investigate the prevalence and diversity of Pseudomonas species derived from the ileum of children with Crohn's disease (CD), and from control children with non-inflammatory bowel disease (non-IBD) undergoing their initial endoscopic examination. Fifty eight percent of CD patients (18/32) were positive using the Pseudomonas PCR, while significantly fewer children in the non-IBD group, 33% (12/36), were PCR positive for Pseudomonas (p<0.05, Fischer's exact test). Pseudomonas specific 16S PCR products from 13 CD and 12 non-IBD children were cloned and sequenced. Five hundred and eighty one sequences were generated and used for the comparative analysis of Pseudomonas diversity between CD and non-IBD patients. Pseudomonas species were less diverse in CD patients compared with non-IBD patients. In particular P.aeruginosa was only identified in non-IBD patients.

SUBMITTER: Wagner J

PROVIDER: S-EPMC2572839 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Identification and characterisation of Pseudomonas 16S ribosomal DNA from ileal biopsies of children with Crohn's disease.

Wagner Josef J Short Kirsty K Catto-Smith Anthony G AG Cameron Don J S DJ Bishop Ruth F RF Kirkwood Carl D CD

PloS one 20081031 10

Molecular analysis of bacterial 16S rRNA genes has made a significant contribution to the identification and characterisation of bacterial flora in the human gut. In particular, this methodology has helped characterise bacterial families implicated in the aetiology of inflammatory bowel disease (IBD). In this study we have used a genus specific bacterial 16S PCR to investigate the prevalence and diversity of Pseudomonas species derived from the ileum of children with Crohn's disease (CD), and fr ...[more]

PMID: 18974839

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Project description:BACKGROUND: Chronic wounds affect millions of people and cost billions of dollars in the United States each year. These wounds harbor polymicrobial biofilm communities, which can be difficult to elucidate using culturing methods. Clinical molecular microbiological methods are increasingly being employed to investigate the microbiota of chronic infections, including wounds, as part of standard patient care. However, molecular testing is more sensitive than culturing, which results in markedly different results being reported to clinicians. This study compares the results of aerobic culturing and molecular testing (culture-free 16S ribosomal DNA sequencing), and it examines the relative abundance score that is generated by the molecular test and the usefulness of the relative abundance score in predicting the likelihood that the same organism would be detected by culture. METHODS: Parallel samples from 51 chronic wounds were studied using aerobic culturing and 16S DNA sequencing for the identification of bacteria. RESULTS: One hundred forty-five (145) unique genera were identified using molecular methods, and 68 of these genera were aerotolerant. Fourteen (14) unique genera were identified using aerobic culture methods. One-third (31/92) of the cultures were determined to be < 1% of the relative abundance of the wound microbiota using molecular testing. At the genus level, molecular testing identified 85% (78/92) of the bacteria that were identified by culture. Conversely, culturing detected 15.7% (78/497) of the aerotolerant bacteria and detected 54.9% of the collective aerotolerant relative abundance of the samples. Aerotolerant bacterial genera (and individual species including Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis) with higher relative abundance scores were more likely to be detected by culture as demonstrated with regression modeling. CONCLUSION: Discordance between molecular and culture testing is often observed. However, culture-free 16S ribosomal DNA sequencing and its relative abundance score can provide clinicians with insight into which bacteria are most abundant in a sample and which are most likely to be detected by culture.

Project description:BackgroundClinical microbiology laboratories have to accurately identify clinical microbes. However, some isolates are difficult to identify by the automated biochemical text platforms, which are called "difficult-to-identify" microbes in this study. Therefore, the ability of 16S ribosomal DNA (16S rDNA) and internal transcribed spacer 2 (ITS2) sequencing to identify these "difficult-to-identify" bacteria and fungi was assessed in this study.MethodsSamples obtained from a teaching hospital over the past three years were examined. The 16S rDNA of four standard strains, 18 clinical common isolates, and 47 "difficult-to-identify" clinical bacteria were amplified by PCR and sequenced. The ITS2 of eight standard strains and 31 "difficult-to-identify" clinical fungi were also amplified by PCR and sequenced. The sequences of 16S rDNA and ITS2 were compared to reference data available in GenBank by using the BLASTN program. These microbes were identified according to the percentage of similarity to reference sequences of strains in GenBank.ResultsThe results from molecular sequencing methods correlated well with automated microbiological identification systems for common clinical isolates. Sequencing results of the standard strains were consistent with their known phenotype. Overall, 47 "difficult-to-identify" clinical bacteria were identified as 35 genera or species by sequence analysis (with 10 of these identified isolates first reported in clinical specimens in China and two first identified in the international literature). 31 "difficult-to-identify" clinical fungi tested could be identified as 15 genera or species by sequence analysis (with two of these first reported in China).ConclusionsOur results show the importance of 16S rDNA and internal ITS2 sequencing for the molecular identification of "difficult-to-identify" bacteria and fungi. The development of this method with advantages of convenience, availability, and cost-effectiveness will make it worth extending into clinical practice in developing countries.

Dataset Information

Identification and characterisation of Pseudomonas 16S ribosomal DNA from ileal biopsies of children with Crohn's disease.

Publications

Identification and characterisation of Pseudomonas 16S ribosomal DNA from ileal biopsies of children with Crohn's disease.

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