Investigation of an anaerobic microbial community associated with a corneal ulcer by denaturing gradient gel electrophoresis and 16S rDNA sequence analysis

Abstract

The bacterial community manifested in a corneal ulcer was investigated with culture-independent techniques. DNA was extracted from the eye swab, 200-bp fragments spanning the hypervariable V3 region of the 16S rRNA gene (16S rDNA) were amplified by broad-range PCR and genetic fingerprinting of the total bacterial community was performed by denaturing gradient gel electrophoresis (DGGE). Additionally, 16S rDNA clone libraries containing 1500-bp fragments were constructed, clones were screened by DGGE and sequenced. Microorganisms were phylogenetically most closely related to the Cytophaga/Flavobacterium/Bacteroides phylum (eight clones), Fusobacteria (four clones), spirochetes (three clones) and to the low G+C Gram-positive bacteria (two clones). Low sequence similarity values less than 93% to sequences of known bacteria indicated that some bacteria belonged to hitherto unknown genera. Bacteria which were detected in the healthy eye of the same patient, were phylogenetically related to the low G+C and high G+C Gram-positive bacteria (two clones) and to the Proteobacteria (one clone). To our knowledge, this is the first time that such a complex and anaerobic bacterial community normally found in subgingival crevices is reported to play a role in corneal ulceration. Previous treatment of the ulcer with several topical antibiotics had shown no effect for six months. The followed culture-independent identification of spirochetes and Gram-negative, anaerobic bacilli facilitated the appropriate treatment with topical penicillin G, which stopped further destruction of the eye. Results demonstrated that 16S rDNA genotyping in combination with DGGE fingerprinting are appropriate molecular methods for the investigation of severe bacterial infections which might not be detected by conventional cultivation.