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Claudia Schabereiter-Gurtner, Saskia Maca, Sabine Rölleke, Karl Nigl, Julius Lukas, Alexander Hirschl, Werner Lubitz, Talin Barisani-Asenbauer; 16S rDNA-Based Identification of Bacteria from Conjunctival Swabs by PCR and DGGE Fingerprinting. Invest. Ophthalmol. Vis. Sci. 2001;42(6):1164-1171.
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purpose. Establishment of a new molecular biology technique for the
identification of multiple bacteria from the ocular environment, which
can be applied supplementarily to cultivation in cases of severe
methods. From 60 human conjunctivae (29 with purulent and 31 with nonpurulent
conjunctivitis), swabs were taken and DNA was extracted. Fragments of
200 bp, spanning the V3 region of the eubacterial 16S rDNA, were
amplified by polymerase chain reaction (PCR) and separated by
denaturing gradient gel electrophoresis (DGGE). For phylogenetic
identification, DGGE bands were excised and directly sequenced, or 16S
rDNA clone libraries were constructed and clones were screened by DGGE.
Sequences were compared with sequences of known bacteria listed in the
EMBL database. Furthermore, the results were compared with results
obtained from conventional cultivation.
results. 16S rDNA could be amplified from 25 of 29 investigated swabs taken from
purulent conjunctivitis eyes and from 2 of 31 investigated swabs taken
from nonpurulent conjunctivitis eyes. Sixteen samples showed
monomicrobial and 11 samples showed polymicrobial infections. The
following genera (n is number of samples) were detected: Staphylococcus (n = 8), Corynebacterium (n = 7),
Propionibacterium (n = 7),
Streptococcus (n = 6), Bacillus (n = 2),
Acinetobacter (n = 3),
Pseudomonas (n = 3), Proteus (n = 1), and Brevundimonas (n = 1). Four sequences could not be identified to
the genus level. They had highest sequence similarities both to
sequences of Pantoea and Enterobacter (n = 1), Kingella and Neisseria (n = 1), Serratia and Aranicola (n = 1), and Leuconostoc and Weissella (n = 2), respectively.
Culture was only positive for coagulase-negative staphylococci
(n = 9), Corynebacteria (n = 3), Staphylococcus aureus (n = 1), Streptococcus sp.
(n = 1), Proteus sp.
(n = 1), Klebsiella oxytoca (n = 1), and Pseudomonas aeruginosa (n = 1). In total, 45% of the 60 analyzed
conjunctival swabs were PCR positive, whereas only 22% were culture
positive. No sample positive by culture gave negative results by PCR.
conclusions. 16S rDNA sequence analyses and DGGE fingerprinting are appropriate
methods for the detection and identification of monomicrobial as well
as polymicrobial ocular infections of bacteria that might not be
detected by conventional cultivation.
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