PMID- 11328723
OWN - NLM
STAT- MEDLINE
DCOM- 20010510
LR  - 20100324
IS  - 0146-0404 (Print)
IS  - 0146-0404 (Linking)
VI  - 42
IP  - 6
DP  - 2001 May
TI  - 16S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE 
      fingerprinting.
PG  - 1164-71
AB  - 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 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.
FAU - Schabereiter-Gurtner, C
AU  - Schabereiter-Gurtner C
AD  - Institute of Microbiology and Genetics, University of Vienna, Wahringergurtel 
      18-20, 1090 Vienna, Austria.
FAU - Maca, S
AU  - Maca S
FAU - Rolleke, S
AU  - Rolleke S
FAU - Nigl, K
AU  - Nigl K
FAU - Lukas, J
AU  - Lukas J
FAU - Hirschl, A
AU  - Hirschl A
FAU - Lubitz, W
AU  - Lubitz W
FAU - Barisani-Asenbauer, T
AU  - Barisani-Asenbauer T
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PL  - United States
TA  - Invest Ophthalmol Vis Sci
JT  - Investigative ophthalmology & visual science
JID - 7703701
RN  - 0 (DNA Primers)
RN  - 0 (DNA, Bacterial)
RN  - 0 (DNA, Ribosomal)
RN  - 0 (RNA, Bacterial)
RN  - 0 (RNA, Ribosomal, 16S)
SB  - IM
MH  - Bacteria/*genetics/isolation & purification
MH  - Bacterial Typing Techniques
MH  - Conjunctiva/*microbiology
MH  - Conjunctivitis, Bacterial/*microbiology
MH  - DNA Fingerprinting/methods
MH  - DNA Primers/chemistry
MH  - DNA, Bacterial/*analysis
MH  - DNA, Ribosomal/*analysis
MH  - Electrophoresis, Agar Gel
MH  - Humans
MH  - Polymerase Chain Reaction/methods
MH  - RNA, Bacterial/genetics
MH  - RNA, Ribosomal, 16S/*genetics
MH  - Sequence Analysis, DNA
EDAT- 2001/05/01 10:00
MHDA- 2001/05/22 10:01
CRDT- 2001/05/01 10:00
PHST- 2001/05/01 10:00 [pubmed]
PHST- 2001/05/22 10:01 [medline]
PHST- 2001/05/01 10:00 [entrez]
PST - ppublish
SO  - Invest Ophthalmol Vis Sci. 2001 May;42(6):1164-71.