Characterization of vaginal microflora of healthy, nonpregnant women by chaperonin-60 sequence-based methods.
OBJECTIVE
The purpose of this study was to use a novel method that was based on the application of chaperonin-60 sequencing to describe the vaginal microflora of 16 healthy women.


METHODS
Asymptomatic women consented for vaginal swabs to be collected at the time of a clinical pelvic examination. Total genomic DNA was isolated from the vaginal swabs. Degenerate, universal polymerase chain reaction primers were used to amplify an approximately 555 base pair region of the universal chaperonin-60 gene, which is found in all eubacteria and eukaryotes, from the total genomic DNA and libraries of cloned polymerase chain reaction products were constructed. Library clones were sequenced, and the resulting sequences were assigned to taxonomic groups on the basis of similarity to reference sequence data. Presence of Chlamydophila psittaci sequences in the samples was confirmed by species-specific polymerase chain reaction.


RESULTS
Sixteen of the 23 women who were enrolled had normal flora by Nugent's score of <4 and had adequate polymerase chain reaction product for assessment. Vaginal flora libraries were dominated by a variety of sequences with similarity to Lactobacillus spp L. crispatus, L. iners, L. gasseri, L. jensenii, and L. buchneri. Other sequences that were identified included representatives of Gardnerella spp, sequences with similarity to Porphyromonas spp and Megasphaera spp and sequences identical to C psittaci.


CONCLUSIONS
Culture-independent, chaperonin-60 sequence-based molecular methods can lead to the identification of greater diversity within defined taxa compared with those that are identified by standard culture-based methods and to the identification of novel organisms that were not previously associated with vaginal flora.
Hill
Janet E
JE
National Research Council of Canada, Plant Biotechnology Institute, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Goh
Swee Han
SH
Money
Deborah M
DM
Doyle
Melissa
M
Li
Andra
A
Crosby
William L
WL
Links
Matthew
M
Leung
Amy
A
Chan
Debbie
D
Hemmingsen
Sean M
SM
D000328:Adult; D001644:Bifidobacterium / Q000235:genetics; D018834:Chaperonin 60 / Q000235:genetics*; D002691:Chlamydophila psittaci / Q000235:genetics; D005260:Female; D006194:Gardnerella vaginalis / Q000235:genetics; D015723:Gene Library; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017422:Sequence Analysis, DNA; D014621:Vagina / Q000382:microbiology*
D018834:Chaperonin 60