Use of 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis for analysis of the microfloras of healing and non-healing chronic venous leg ulcers

Davies, Charlotte E., Hill, Katja E. ORCID: https://orcid.org/0000-0002-8590-0117, Wilson, Melanie ORCID: https://orcid.org/0000-0002-8834-8268, Stephens, Philip ORCID: https://orcid.org/0000-0002-0840-4996, Hill, C. Michael, Harding, Keith Gordon and Thomas, David William ORCID: https://orcid.org/0000-0001-7319-5820 2004. Use of 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis for analysis of the microfloras of healing and non-healing chronic venous leg ulcers. Journal of Clinical Microbiology 42 (8) , pp. 3549-3557. 10.1128/JCM.42.8.3549-3557.2004

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Abstract

The bacterial microfloras of 8 healing and 10 nonhealing chronic venous leg ulcers were compared by using a combination of cultural analysis and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene products. Cultural analysis of the microflora revealed that the majority of both wound types carried the aerobes Staphylococcus and Pseudomonas spp. (89 and 80%, respectively). Sequencing of 16S ribosomal DNAs selected on the basis of DGGE profiling allowed the identification of strains not detected by cultural means. Of considerable interest was the finding that more than 40% of the sequences represented organisms not cultured from the wound from which they were amplified. DGGE profiles also revealed that all of the wounds possessed one apparently common band, identified by sequencing as Pseudomonas sp. The intensity of this PCR signal suggested that the bacterial load of nonhealing wounds was much higher for pseudomonads compared to healing wounds and that it may have been significantly underestimated by cultural analysis. Hence, the present study shows that DGGE could give valuable additional information about chronic wound microflora that is not apparent from cultural analysis alone.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Dentistry Medicine Systems Immunity Research Institute (SIURI)
Subjects:	R Medicine > R Medicine (General)
Additional Information:	Pdf uploaded in accordance with publisher's policy at http://www.sherpa.ac.uk/romeo/issn/0095-1137/ (accessed 25/02/2014)
Publisher:	American Society for Microbiology
ISSN:	0095-1137
Date of First Compliant Deposit:	30 March 2016
Last Modified:	06 Nov 2024 22:27
URI:	https://orca.cardiff.ac.uk/id/eprint/700

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