16S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE fingerprinting.
OBJECTIVE
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 bacterial infections.


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.
Schabereiter-Gurtner
C
C
Institute of Microbiology and Genetics, University of Vienna, Währingergürtel 18-20, 1090 Vienna, Austria.
Maca
S
S
Rölleke
S
S
Nigl
K
K
Lukas
J
J
Hirschl
A
A
Lubitz
W
W
Barisani-Asenbauer
T
T
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D015373:Bacterial Typing Techniques; D003228:Conjunctiva / Q000382:microbiology*; D003234:Conjunctivitis, Bacterial / Q000382:microbiology*; D016172:DNA Fingerprinting / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis*; D004275:DNA, Ribosomal / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S