16S rDNA-Based Identification of Bacteria from Conjunctival Swabs by PCR and DGGE Fingerprinting

Abstract

Bacterial ocular infections are common. Although many cases show a benign course, some can be associated with sight-threatening ocular complications. Identification of the causative pathogens in these cases is mandatory but often difficult because some bacteria have special growth requirements. Furthermore, sample size from ocular tissues is usually small, leading to unreliable cultivation results. Initiation of proper therapy can then be delayed with possible devastating visual consequences.

Molecular approaches to the identification of bacteria show promising results. The amplification of 16S rDNA of any bacterial species is possible without prior cultivation when broadrange PCR primers targeted to highly conserved regions are applied. The comparison of amplified and sequenced 16S rDNA sequences with sequences of known bacteria in 16S rDNA databases facilitates a subsequent phylogenetic identification. In ophthalmology, the 16S rDNA-based identification of pathogens is still at its beginning and, except in a few studies, is rarely applied. Hykin et al.1 and Therese et al.2 used eubacterial primers and Propionibacterium-specific primers to detect bacterial DNA in vitreous samples of patients who had endophthalmitis. Lohmann et al.3 and Knox et al.4 detected and identified bacteria in corneal scrapings and in vitreous samples of patients who had keratitis and endophthalmitis by amplification and subsequent direct sequencing of 16S rDNA. These studies allowed the simple detection of eubacterial DNA or the identification of monomicrobial infections, whereas pathogens of polymicrobial infections could not be identified by direct sequencing.

Nevertheless, bacterial infections of the eye are sometimes polymicrobial. In the studies of Ormerod et al.5 and Kunimoto et al.,6 it was shown that ocular infections such as endophthalmitis were polymicrobial in up to 32%. Concerning polymicrobial communities, the direct sequencing of mixed 16S rDNAfragments fails, and sequence information can only be obtained through 16S rDNA clone libraries.7,8 To avoid the sequencing of clones containing identical sequences, clone libraries can be screened by restriction fragment length polymorphism analysis (RFLP) or by denaturing gradient gel electrophoresis (DGGE). By applying RFLP, 16S rDNA amplicons are digested with a set of different restriction endonucleases, and DNA fragments are separated in agarose gels, leading to different RFLP profiles of individual 16S rDNA sequences.9,10 DGGE facilitates profiling of monomicrobial as well as polymicrobial communities in polyacrylamide gels because of the sequence-specific separation of 16S rDNA amplicons of same length.11 During gel electrophoresis, short 16S rDNA amplicons migrate toward increasing denaturing concentrations, leading to a partial melting of the DNA helix and to a decrease and subsequent ending of electrophoretic migration. As a consequence, a band pattern is produced in which each band theoretically represents a bacterial taxon. In the present study, a method is proposed that combines 16S rDNA genotyping with DGGE fingerprinting. Figure 1 shows a scheme of the applied experimental procedure. The microbial communities of conjunctival swabs were investigated by amplifying, cloning, and sequencing of 16S rDNA.