The lower genital tract microbiota in relation to cytokine-, SLPI- and

endotoxin levels: application of checkerboard DNA-DNA

hybridization (CDH).
Nikolaitchouk N1, Andersch B, Falsen E, Strmbeck L, Mattsby-Baltzer I.
Author information
APMIS. 2008 Apr;116(4):263-77. doi: 10.1111/j.16000463.2008.00808.x.

Abstract
In the present study the lower genital tract microbiota in asymptomatic
fertile women (n=34) was identified and quantified by culturing vaginal
secretions. Also, vaginal and cervical samples were analyzed by a
semiquantitative checkerboard DNA-DNA hybridization technique (CDH)
based on genomic probes prepared from 13 bacterial species (Bacteroides
ureolyticus, Escherichia coli, Fusobacterium nucleatum, Gardnerella
vaginalis, Mobiluncus curtisii ss curtisii, Prevotella bivia, Prevotella disiens,
Prevotella melaninogenica, Atopobium vaginae, Lactobacillus iners,
Staphylococcus aureus ss aureus, Streptococcus anginosus, and
Streptococcus agalactiae). The bacterial species found by either culture or
CDH were correlated with proinflammatory cytokines (IL-1 alpha, IL-1 beta,
IL-6, IL-8), secretory leukocyte protease inhibitor (SLPI), and endotoxin in the
cervicovaginal samples. Grading the women into healthy, intermediate, or
bacterial vaginosis (BV) as based on Gram staining of vaginal smears, the
viable counts of lactobacilli (L. gasseri) and of streptococci-staphylococci
combined were highest in the intermediate group. In BV, particularly the
high concentrations of Actinomyces urogenitalis, Atopobium vaginae, and
Peptoniphilus harei were noted (>or=10(11) per ml). The total viable counts
correlated with both cervical IL-1 alpha and IL-1 beta. A strong negative
correlation was observed between L. iners and total viable counts, G.
vaginalis, or cervical IL-1 alpha, while it correlated positively with SLPI.
Analysis of vaginal and cervical samples from 26 out of the 34 women by
CDH showed that anaerobic bacteria were more frequently detected by CDH
compared to culture. By this method, A. vaginae correlated with G.
vaginalis, and L. iners with S. aureus. With regard to cytokines, B.
ureolyticus correlated with both cervical and vaginal IL-1 alpha as well as
with cervical IL-8, while F. nucleatum, S. agalactiae, S. anginosus, or S.
aureus correlated with vaginal IL-1 alpha. Furthermore, all Gram-negative
bacteria taken together, as measured by CDH, correlated with vaginal
endotoxin and inversely with vaginal SLPI. The significance of the results is
discussed. In summary, mapping of the identity and quantity of vaginal
bacterial species and their association with locally produced host innate
immune factors will help in defining various types of abnormal vaginal
microbiota, developing new ways of assessing the risk of ascending

subclinical infections, and in treating them. CDH appears to be a suitable

tool for future analyses of large numbers of clinical samples with an
extended number of bacterial probes.
abstracto
En el presente estudio se identific la microbiota del tracto genital inferior
en las mujeres frtiles asintomticos (n = 34) y se cuantifica mediante el
cultivo de las secreciones vaginales. Tambin, las muestras vaginales y
cervicales fueron analizados mediante una tcnica de hibridacin ADN-ADN
de tablero de ajedrez semicuantitativa (CDH), basado en sondas genmicas
preparadas a partir de 13 especies bacterianas (Bacteroides ureolyticus,
Escherichia coli, Fusobacterium nucleatum, Gardnerella vaginalis,
Mobiluncus curtisii curtisii ss, Prevotella bivia, disiens Prevotella,
melaninogenica Prevotella, Atopobium vaginas, Iners Lactobacillus,
Staphylococcus aureus ss aureus, Streptococcus anginosus, y Streptococcus
agalactiae). Las especies bacterianas que se encuentran ya sea por cultivo o
CDH se correlacionaron con citoquinas proinflamatorias (IL-1 alfa, IL-1 beta,
IL-6, IL-8), inhibidor de proteasa secretoria de leucocitos (SLPI), y la
endotoxina en las muestras cervicovaginales. La clasificacin de las mujeres
en la vaginosis saludable, intermedio o bacteriana (VB) como basado en la
tincin de Gram de los frotis vaginales, los recuentos viables de lactobacilos
(L. gasseri) y de estreptococos estafilococos combinado fueron ms altas en
el grupo intermedio. En BV, especialmente las altas concentraciones de
Actinomyces urogenitalis, Atopobium vaginas, y Peptoniphilus harei se
observaron (> o = 10 (11) por ml). Los recuentos de viables totales
correlacionados tanto con cervical IL-1 alfa e IL-1 beta. Se observ una
fuerte correlacin negativa entre L. Iners y recuentos viables totales, G.
vaginalis, o cervical IL-1 alfa, mientras que una correlacin positiva con SLPI.
Anlisis de muestras vaginales y cervicales de 26 de las 34 mujeres por CDH
mostr que las bacterias anaerobias se detectaron con mayor frecuencia por
la CDH en comparacin con la cultura. Mediante este mtodo, A. Vaginas
correlacionada con G. vaginalis, y L. Iners con S. aureus. Con respecto a las
citoquinas, B. ureolyticus correlacionado tanto con los de cuello uterino y
vaginal IL-1 alfa, as como con IL-8 cervical, mientras que F. nucleatum, S.
agalactiae, S. anginosus, o S. aureus se correlacion con IL-1 vaginal alfa.
Adems, todas las bacterias Gram-negativas tomados juntos, medida por
CDH, correlacionada con endotoxina vaginal e inversamente con SLPI
vaginal. Se discute la importancia de los resultados. En resumen, la
cartografa de la identidad y la cantidad de especies de bacterias vaginales
y su asociacin con factores de produccin local de acogida inmune innata
le ayudar en la definicin de los distintos tipos de la microbiota vaginal
anormal, el desarrollo de nuevas formas de evaluar el riesgo de infecciones
subclnicas ascendente, y en el tratamiento de ellos . CDH parece ser una
herramienta adecuada para futuros anlisis de grandes nmeros de
muestras clnicas con un mayor nmero de sondas bacterianas.